Global Cardiology Science and Practice (2013) 1

VOLUME 2013

ISSN 2305-7823

The zebrafish in cardiovascular research: A tiny fish with mighty prospects

Global Cardiology Science and Practice A Qatar Foundation Academic Journal Aims and Scope Global Cardiology Science and Practice is an international peer reviewed journal dedicated to keeping cardiologists abreast of advances in the influence of basic science on clinical practice.

The journal will also publish original material and case studies judged to have important influence on practice in all aspects of cardiology, particular those relating to cardiology and global health.

ISSN: 2305-7823 Editor-in-chief Magdi Yacoub, Imperial College, UK & Qatar Cardiovascular Research Center Consulting Editor Eugene Braunwald, Brigham and Women's Hospital, Boston, USA Managing Editors Mark Radford, Qatar Cardiovascular Research Center Maria Rogers, Qatar Cardiovascular Research Center Deputy Editors Robert Bonow, Northwestern University, Chicago, USA Iacopo Olivotto, Careggi Hospital, Florence, Italy Associate Editor Jassim Al-Suwaidi, Hamad Medical Corporation, Doha, Qatar Editorial Board David Antoniucci, Careggi Hospital, Florence, Italy Christian Bollensdorff, Qatar Cardiovascular Research Center, Qatar Blase Carabello, Mount Sinai Medical Center, New York, USA Franco Cecchi, Careggi Hospital, Florence, Italy Ken Chien, Harvard Stem Cell Institute, Boston, USA Ahmed El Guindy, Aswan Heart Centre, Cairo, Egypt Volkmar Falk, University of Zurich, Zurich, Switzerland Marc de Leval, International Congenital Cardiac Center, London, UK Leslie Miller, University of South Florida, Tampa, USA Joachim Miro, Sainte-Justine Hospital, Montreal, Canada Raad Mohiaddin, Imperial College, London, UK Friedrich Mohr, University of Leipzig, Leipzig, Germany Nadia Rosenthal, Imperial College, London, UK Karim Said, Aswan Heart Centre, Aswan, Egypt Pravin Shah, Hoag Hospital, Newport Beach, USA Hans Sievers, Universit채tsklinikum Schleswig-Holstein, Germany Gaetano Thiene, University of Padua Medical School, Padova, Italy Abdul Al Wael, Kasr-el-Aini Medical School, Cairo, Egypt Gavin Wright, Wellcome Trust Sanger Institute, Cambridge, UK FRONT COVER: An image of the zebrafish Danio rerio, a tropical freshwater fish, belongs to the family of cyprinidae, which in the last 30 years has developed into a very popular model organism for studies of embryonic development and human diseases. Its use in CV disease is explored in this issue by Poon and Brand. Image courtesy of Kar Lai Poon.

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Editorial

Editors’ page Robert B Bonow, Iacopo Olivotto, Magdi H Yacoub*

*Email: m.yacoub@imperial.ac.uk

In this issue of the Journal we introduce three additional new features. The first relates to inclusion of engineering and materials science articles, which is felt to be a growing area with relevance to cardiology. The second is important registries detailing regional variations in cardiac conditions and their treatment. This we believe is complementary to the section ‘Lessons from the trials’. In this issue, the registries from the Gulf region are discussed by Jassim Al Suwaidi.

The third new feature is the use of animal models in cardiovascular research. We launch this feature in this issue with an in-depth analysis, by Thomas Brand and his colleagues, of the rapidly-expanding use of the zebrafish for this purpose. We believe this will be of interest, not only to researchers, but also to clinicians.

The trials that we highlight in the current issue include three recent early studies of cardiac-derived stem cells in humans. These trials in particular have stimulated considerable interest and controversy.

We look forward to publishing review articles, clinical trials, as well as original research articles on regenerative therapies and stem cell biology.

The journal continues to evolve and we welcome any comments from our readers regarding the topics presented in this journal.

http://dx.doi.org/ 10.5339/gcsp.2013.1 Submitted: 6 March 2013 Accepted: 6 March 2013 q 2013 Bonow, Olivotto, Yacoub, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Bonow RB, Olivotto I, Yacoub MH. Editors’ page, Global Cardiology Science & Practice 2013:1 http://dx.doi.org/10.5339/gcsp.2013.1

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Review article

Acute coronary syndrome in the Middle East: The importance of registries for quality assessment and plans for improvement Jassim Al Suwaidi* Department of Adult Cardiology, Heart Hospital, Hamad Medical Corporation, Doha, Qatar *Email: jalsuwaidi@hotmail.com

ABSTRACT Acute coronary syndrome (ACS) represents one of the most common causes of death worldwide.

Several practice guidelines have been developed in Europe and North America to improve outcome of ACS patients through implementation of the recommendations into clinical practice. It is well know that there is wide gap between guidelines and implementation in real practice as was demonstrated in registry findings mainly conducted in the developed world. Here in we review main gaps in the management of ACS patients observed from two recent registries conducted in the Middle East.

Keywords: acute coronary syndrome, ST-elevation myocardial infarction, Non-ST-elevation acute coronary syndrome, thrombolytic therapy, primary percutaneous coronary intervention

http://dx.doi.org/ 10.5339/gcsp.2013.2 Submitted: 16 December 2012 Accepted: 9 February 2013 q 2013 Al Suwaidi, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Al Suwaidi J. Acute coronary syndrome in the Middle East: The importance of registries for quality assessment and plans for improvement, Global Cardiology Science & Practice 2013:2 http://dx.doi.org/10.5339/gcsp.2013.2

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ACS IN THE MIDDLE EAST Heart disease is the major cause of death worldwide. Many individuals with heart disease present with acute coronary syndrome (ACS); this puts them at significant risk of morbidity and mortality. This significant burden necessitates ongoing improvements in patient management to minimize these complications. These improvements in outcome are promoted by an evidence-based approach shaped by comprehensive clinical guidelines.

The Gulf Heart Association (GHA) has launched two multicenter multinational registries of ACS: The Gulf Registry of Acute Coronary Events (Gulf RACE),1 which was conducted in 2007 and included 8,169 patients with ACS from six adjacent Middle eastern countries (Bahrain, Kuwait, Qatar, Oman, the United Arab Emirates, and Yemen), and the Gulf-RACE-2, which was conducted in 2009 and included 7,939 patients with ACS from Middle eastern countries (Bahrain, Saudi Arabia, Qatar, Oman, United Arab Emirates and Yemen) with one-year follow-up.2 These two registries provided valuable information to health care officials. Whereas some aspects of the care provided were comparable to that of the developed countries, other aspects where clearly suboptimal. These main suboptimal practices are summarized in this commentary.

One of the most striking findings was the under-utilization of emergency medical services (EMS).

Only 17% of patients in Gulf RACE were presented to the emergency department by EMS,3 with the remaining patients arriving by private cars. When compared to reports in the developed world this is extremely low rate. Canto et al.4 reported 53.4% use of EMS in the 2nd National Registry of Myocardial Infarction, which was conducted between June 1994 and March 1998 in the United States; this rate increased only to 60% a decade later as was documented in the National Cardiovascular Data Registry Acute Coronary Treatment and Intervention Outcomes Network Registry – Get With the Guidelines (2007 – 2009).5 Nevertheless it is much higher than that demonstrated in our two registries. We also observed that the frequency of EMS utilization was similarly low in patients presenting with ST-segment elevation myocardial infarction (18%) and non-ST elevation ACS (17%). Moreover, the utilization of EMS was low among patients presenting with typical chest pain (16%), pulmonary edema (32%), cardiogenic shock (30%), or cardiac arrest (29%).3 However, when patients with ST-elevation myocardial infarction (STEMI) were transported by EMS, they were significantly less likely to exhibit major delay in presentation and were significantly more likely to receive favorable processes of care, including shorter door-to-ECG time and more frequent reperfusion therapy emphasizing the importance of using EMS services.3 These findings have significant implications for improving care and outcome of ACS patients for Gulf countries and may suggest redirecting emphasis in improvement of pre-hospital care. The improvement in inpatient care is reflected in relatively low in-hospital mortality rates among patients with ACS in the region, as was documented in the 2 registries.2,3 The second issue is the reperfusion therapy used for STEMI patients in the Gulf countries. In many randomized clinical trials, primary percutaneous coronary intervention (PCI) has been shown to be superior to thrombolytic therapy (TT).6 This benefit is related to a much higher early mechanical reperfusion rate in comparison to TT. Indeed, the vast majority of acute cardiac centers in North America and Europe use primary PCI as the main modality of reperfusion therapy.6 In a recent analysis of 30 European countries, primary PCI was the main modality of treatment.7 The striking finding in Gulf RACE registries was the use of TT as the primary reperfusion modality. Among 2,155 STEMI patients in Gulf RACE, 84% underwent thrombolytic therapy and only 8% underwent primary PCI. This low overall use was present in small as well as larger countries, and in poor as well as well rich countries.8 Thirdly, there is an overall under-utilization of cardiac catheterization for patients admitted with acute coronary syndrome. The overall rate of in-hospital cardiac catheterization for ACS patients was only 20% with some variability among the various Gulf countries,9 which is considerably low when compared to previous studies. In the multinational GRACE (Global Registry of Acute Coronary Events) registry,10 catheterization use was about 60%, as was the case in in the CRUSADE (Can Rapid Stratification of Unstable Angina Patients Suppress Adverse Outcomes with Early Implementation of the ACC/AHA Guidelines) registry11 for Non-STE-ACS. Sixty-five percent of patients with ACS in the Canadian registry underwent cardiac catheterization.12 Furthermore, we observed that low-risk patients were more likely to undergo cardiac catheterization when compared with intermediate and high-risk patients. This is consistent with many studies reported from Western countries, suggesting the urgent need to implement guidelines that risk-stratify patients more appropriately. Moreover, there is lack of cardiac catheterization facilities in significant number of

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hospitals involved in the region, which undoubtedly contributes to this overall low use. These two ACS registries suggested the need for current and future expansion of cardiac catheterization laboratories in many hospitals in the Gulf. This need obviously varies among the different countries involved. Finally, there is urgent need to implement ways to target patients for catheterization who would benefit most from this procedure.

The current review suggests three major gaps in the management of ACS in the Gulf; which are underuse of EMS, primary PCI and in-hospital cardiac catheterization. In Qatar, plans are underway to launch a nationwide primary PCI program which will require educating the public of the need to use EMS, close and coordinated work between EMS personal, emergency room and cardiology staff for expedited process of ECG evaluation and transfer for cardiac catheterization laboratory at the Heart Hospital for primary PCI or early invasive therapies, with the hope of further improvement of outcome in these high risk patients.

REFERENCES [1] Zubaid M, Rashed WA, Almahmeed W, Al-Lawati J, Sulaiman K, Al-Motarreb A, Amin H, Al Suwaidi J, Alhabib K. Management and outcomes of middle Eastern patients admitted with acute coronary syndromes in the Gulf Registry of Acute Coronary Events (Gulf RACE). Acta Cardiol. 2009;64(4):439–446.

[2] AlHabib K, Sulaiman K, Al-Motarreb A, Almahmeed W, Asaad N, Amin H, Hersi A, Al-Saif S, AlNemer K, Al-Lawati J, Al-Sagheer NQ, AlBustani N, Al Suwaidi J; Gulf RACE-2 investigators. Baseline characteristics, management practices, and long-term outcomes of middle Eastern patients in the second Gulf Registry of Acute Coronary Events (Gulf RACE-2). Ann Saud Med. 2012;32(1):9–18.

[3] Fares S, Zubaid M, Al-Mahmeed W, Ciottone G, Sayah A, Al Suwaidi J, Amin H, Al-Atawna F, Ridha M, Sulaiman K, Alsheikh-Ali AA. Utilization of emergency medical services by patients with acute coronary syndromes in the Arab Gulf states. J Emerg Med. 2011;41(3):310–316.

[4] Canto JG, Zalenski RJ, Ornato JP, Rogers WJ, Kiefe CI, Magid D, Shlipak MG, Frederick PD, Lambrew CG, Littrell KA, Barron HV; National Registry of Myocardial Infarction 2 Investigators. Use of emergency medical services in acute myocardial infarction and subsequent quality of care: observations from the National Registry of Myocardial Infarction 2. Circulation 2002;106(24):3018–3023.

[5] Mathews R, Peterson ED, Li S, Roe MT, Glickman SW, Wiviott SD, Saucedo JF, Antman EM, Jacobs AK, Wang TY. Use of emergency medical service transport among patients with ST-segment-elevation myocardial infarction: findings from the national cardiovascular data registry acute coronary treatment intervention outcomes network registry-get with the guidelines. Circulation 2011;124:154–163.

[6] Keeley EC, Boura JA, Grines CL. Comparison of primary angioplasty and intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet 2003;361:13–20.

[7] Widimsky P, Wijns W, Fajadet J, de Belder M, Knot J, Aaberge L, Andrikopoulos G, Baz JA, Betriu A, Claeys M, Danchin N, Djambazov S, Erne P, Hartikainen J, Huber K, Kala P, Klinceva M, Kristensen SD, Ludman P, Ferre JM, Merkely B, Milicic D, Morais J, Noc M, Opolski G, Ostojic M, Radovanovic D, De Servi S, Stenestrand U, Studencan M, Tubaro M, Vasiljevic Z, Weidinger F, Witkowski A, Zeymer U European association for percutaneous cardiovascular interventions.

Reperfusion therapy for ST elevation acute myocardial infarction in Europe: description of the current situation in 30 countries. Eur Heart J. 2010;31(8):943–957.

[8] Zakwani I, Zubaid M, Al-Riyami A, Alanbaie M, Suliman K, Almahmeed W, Al-Motarreb A, Al Suwaidi J, Amin H. Primary coronary intervention versus thrombolytic therapy in ST-segment elevation myocardial infarction patients in six Middle Eastern countries: data from Gulf Registry of Acute Coronary Events. Int J Clin Pharm. 2012;50(6):418–425.

[9] Panduranga P, Sulaiman K, Al-Zakwani I, Zubaid M, Rashed W, Al-Mahmeed W, Al-Lawati J, Al-Motarreb A, Haitham A, Suwaidi J, Al-Habib K. Utilization and determinants of in-hospital cardiac catheterization in patients with acute coronary syndrome from the middle east. Angiology 2010;61(8):744–750.

[10] Fox KA, Goodman SG, Anderson FA, Granger CB, Moscucci M, Flather MD, Spencer F, Budaj A, Dabbous OH, Gore JM, GRACE Investigators. From guidelines to clinical practice: the impact of hospital and geographical characteristics on temporal trends in the management of acute coronary syndromes. The Global Registry of Acute Coronary Events (GRACE). Eur Heart J. 2003;24(15):1414–1424.

[11] Bhatt DL, Roe MT, Peterson ED, Li Y, Chen AY, Harrington RA, Greenbaum AB, Berger PB, Cannon CP, Cohen DJ, Gibson CM, Saucedo JF, Kleiman NS, Hochman JS, Boden WE, Brindis RG, Peacock WF, Smith SC Jr, Pollack CV Jr, Gibler WB, Ohman EM, CRUSADE Investigators. Utilization of early invasive management strategies for high-risk patients with non-ST- segment elevation acute coronary syndromes: results from the CRUSADE Quality Improvement Initiative. JAMA 2004;292(17):2096– 2104.

[12] Lee CH, Tan M, Yan AT, Yan RT, Fitchett D, Grima EA, Langer A, Goodman SG, Canadian Acute Coronary Syndromes (ACS) Registry II Investigators. Use of cardiac catheterization for non-ST-segment elevation acute coronary syndromes according to initial risk: reasons why physicians choose not to refer their patients. Arch Intern Med. 2008;168(3): 291– 296.

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Lessons from the trials

CADUCEUS, SCIPIO, ALCADIA: Cell therapy trials using cardiac-derived cells for patients with post myocardial infarction LV dysfunction, still evolving Magdi H Yacoub1, John Terrovitis2,* 1

Qatar Cardiovascular Research Center, Doha, Qatar University of Athens School of Medicine, Athens, Greece 2

*Email: john.terrovitis@gmail.com

http://dx.doi.org/ 10.5339/gcsp.2013.3 Submitted: 12 December 2012 Accepted: 19 February 2013 q 2013 Yacoub, Terrovitis, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

CADUCEUS The early results of the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction study were recently published in the Lancet.1 This study is a phase 1 prospective randomised study, performed at two centres. The study was designed to test the hypothesis that intracoronary infusion of autologous cardiac-derived cells following myocardial infarction can reduce the size of the infarct and increase the amount of viable myocardium. The eligible patients were randomised in a 2:1 ratio to receive CDCs or standard care. In all, 17 patients were randomised to cell therapy and 8 to standard care. The cell therapy consisted of an infusion of 25 million cells into the infarct related artery, 1.5– 3 months after successful primary angioplasty in patients who developed LV dysfunction (EF less than 37 per cent). The cells were derived from RV endomyocardial biopsies performed within the previous 37 days. The number of cells was determined from previous experimental studies of the maximum number of cells which can be injected without inducing infarction. The study was not blinded because of ethical considerations regarding performing right ventricular biopsy on the controls. The exclusion criteria included patients who had evidence of right ventricular infarction, or could not have an MRI examination because of claustrophobia or prior insertion of devices. There was no death, myocardial infarction or serious arrhythmia reported in either group during the period of follow up, which was between 6– 12 months. Serious adverse events were observed in 24 percent of the intervention group versus 12 per cent in the controls (p not significant).

Although the study was not powered to determine efficacy, the authors observed significant reduction in the size of the infarct (2 7.7 per cent at 6 month and 2 12.3 per cent at 12 months) and importantly an increase in the amount of viable myocardium. In addition there was a significant increase in the thickness and rate of thickening in the peri-infarction zone, however there was no change in ejection fraction of left ventricular volumes.

The authors concluded that injection of a specific number of CDCs into the infarct related artery is safe and should be tried further.

The strong points in this trial are the fact that it has shown for the first time that injection of CDCs in humans is feasible and safe, and that this might increase the amount of viable myocardium and therefore influence longer-term outcome. The study was started after extensive preclinical research by the authors, to justify moving into the clinic. In addition the choice of MRI to assess results enhances the ultimate value of the study.

Cite this article as: Yacoub MH, Terrovitis J. CADUCEUS, SCIPIO, ALCADIA: Cell therapy trials using cardiac-derived cells for patients with post myocardial infarction LV dysfunction, still evolving, Global Cardiology Science & Practice 2013:3 http://dx.doi.org/10.5339/gcsp.2013.3

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What have we learned?

In spite of the fact that the results of the study are potentially very exciting, they are not sufficient to influence practice, because of the small number of patients, the short period of follow up, the putative nature of the results, and the limit imposed on the number of cells which can be safely injected into the coronary arteries because of the large size of the CDCs (20 microns in diameter) which is considerably larger than the myocardial capillaries (7 microns).

In addition, although the CDCs are cardiac derived and have been labelled progenitor cells, there is no evidence that they can differentiate into myocardial cells, and therefore the mechanisms involved in the beneficial actions observed remain unknown. Recent studies using carbon-14 or multi-isotope imaging mass spectrometry (MIMS), suggest that myocardial regeneration following injury results from division of mature myocardial cells. Future reports about the longer term results from the same group in phase 2 trials are awaited with great interest, while other strategies to enhance division of adult cardiomyocytes2 – 6 should be actively explored.

SCIPIO The early results of the phase 1 trial, cardiac Stem Cell In Patients with Ischemic cardiomyopathy (SCIPIO) were recently published in The Lancet.7 This trial appears to have raised expectations to an unrealistic degree with the hope that “it will transform cardiac cell therapy that its namesake, Scipio Africanus achieved in Roman Military Campaigns”.8 The trial is an open label randomised trial of patients with post infarction left ventricular dysfunction (EF less than 45 per cent) requiring surgical revascularisation. At the time of operation, performed at one of two centres in Kentucky, the atrial appendage was excised and sent to Dr Piero Anversa’s lab in Boston. Following cell culture, cells expressing the cell surface marker c-kit, which are also lineage negative, were extracted using magnetic beads and thoroughly characterised and tested for lack of senility and ability to proliferate. The cells were then shipped to another lab in Kentucky to be prepared for clinical use. For the patients randomised to the cell therapy arm, intra-coronary injection of the specified number of cells were injected into the graft or vessel supplying the infarction area about 120 days after operation. For patients with large anterior infarcts, one million cells were injected, while for patients with smaller posterior infarcts one or more injections of 500,000 cells were used. The primary end point was short-term safety and the secondary end point was efficacy as determined by clinical, quality of life questionnaire and MRI at 1 and 4 months. Patient recruitment utilised a complex system with the first nine consecutive eligible patients assigned to cell therapy and the next four conseutivec patients acting as controls. After that patients were randomised in a 3:2 ratio to treatment and controls. In all, 16 patients received cells and 7 were controls.

The authors report no mortality or major adverse events following this form of therapy. In addition, although the study was not powered to address efficacy, in the patients who could be investigated by MRI, there was improvement in EF by 8 and 12 percent at one and four-year time points in the cell therapy group, as opposed to no improvement in the controls. There was also evidence of diminution in the size of the infarct in the cell therapy group. These results are superior to those described after infusion of bone marrow derived cells. However the numbers are small, and the potential confounding effect of myocardial revascularisation cannot be excluded.

What have we learned?

The results of this phase 1 trial are unlikely to influence practice because of the putative nature of the findings and the small number of patients. The results of the phase 2 trial will be eagerly awaited.

Similarly, studies to define the mechanism of changes observed would be of great value. In the meantime, the major expectations have to wait (Figure 1).

ALCADIA Results of the ALCADIA (AutoLogous Human CArdiac-Derived Stem Cell To Treat Ischemic cArdiomyopathy) trial were presented during the American Heart Association Scientific Sessions in November 2012.9 The study evaluated a hybrid cell therapy application, namely the administration of autologous cardiac stem cells together with a controlled release formulation of basic Fibroblast Growth Factor (bFGF) in patients with ischemic cardiomyopathy and heart failure.

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Figure 1. Scipio Africanus The Elder was a general in the Second Punic War and statesman of the Roman Republic.

He was best known for defeating Hannibal at the final battle of the Second Punic War at Zama, a feat that earned him the agnomenAfricanus, the nickname “the Roman Hannibal�, as well as recognition as one of the finest commanders in military history. Picture credit: http://upload.wikimedia.org/wikipedia/commons/3/38/Scipio _Africanus_the_Elder.jpg

This is one of the first human trials, together with CADUCEUS and SCIPIO, where cardiac derived cells are used to treat heart failure. ALCADIA is the smallest and most preliminary of them and the final results have not been published yet.

ALCADIA was an open label, non-randomized, phase 1 safety/feasibility study of autologous cardiac derived stem cell combined with bFGF administration in advanced heart failure.9 Six patients (55.5 ^ 10.8 years old, 5 men and one woman) with ischemic cardiomyopathy (left ventricular ejection fraction between 15 and 45%), symptomatic heart failure (NYHA class III or IV), myocardial viability and indication for coronary artery bypass surgery, were enrolled.

Cardiac-derived stem cells were grown from endomyocardial biopsies and expanded for a period of approximately one month. They were delivered to the heart during the subsequent CABG surgery, by 20 intramyocardial injections (total number 0.5million/kg). Finally, a biodegradable gelatin hydrogel sheet containing 200mg of bFGF was implanted on the epicardium, covering the injection sites areas.

The primary safety end point was the occurrence of Major Cardiac Event (MACE) during the one year follow up period. Efficacy end points included changes in Left Ventricular Ejection Fraction (LVEF, assessed by echocardiography and MRI), infarct volume (assessed by MRI) and symptoms (NYHA class and exercise capacity) at 6 months.

One patient experienced acute occlusion of a graft 3 weeks after surgery and was excluded from further evaluation. From the remaining five patients, one experienced a worsening heart failure episode during the follow up. No other serious adverse events were observed. At 6 months, there was an increase in LVEF measured by both imaging modalities (9% and 12%, by echocardiography and MRI respectively). Infarct size decreased by 3.3% of the total LV volume and maximal aerobic exercise capacity (VO2peak) increased by 4.5 ml/kg/min.

What have we learned?

The ALCADIA trial is a small, preliminary Phase 1 study without a control group. No conclusions about the efficacy of this hybrid therapy can be derived. The favorable trends observed in LV function and symptom severity can also be attributed to the effects of revascularization. In the small number of patients (five) who were followed for 12 months, there was only one serious adverse event, related to worsening heart failure; therefore, no concerns about safety were raised.

The concept is interesting since poor cell survival is currently one of the major hurdles limiting the effectiveness of cell therapies, irrespective of the type of the cell used. Tissue engineering approaches may offer a solution to this problem. In this study, a sustained release bFGF gelatin hydrogel sheet was used, in order to augment the effect exerted by the cells. The investigators have previously shown the efficacy of this method in a preclinical animal model.10 FGF has many attractive properties, since it

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promotes cell proliferation and angiogenesis. The cardiac derived cells used in ALCADIA were autologous and were grown from endomyocardial biopsies. The cells have surface markers characteristic of mesenchymal cells (CD105 and CD90), but also express transcription factors characteristic of progenitor cells. It should be noted however that there is no compelling evidence that these cells can differentiate into cardiomyocytes in vivo and most experimental studies suggest a paracrine mechanism of action, leading to augmentation of endogenous repair mechanisms (both cardiomyocyte proliferation and recruitment of resident cardiac stem cells).11 In conclusion, the ALCADIA trial has demonstrated the safety of the combined administration of autologous cardiac derived stem cells together with a sustained bFGF gelatin hydrogel in patients with ischemic cardiomyopathy. Larger, prospective, randomized, placebo control clinical trials are required in order to investigate the effectiveness of this approach.

REFERENCES [1] Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LE, Berman D, Czer LS, Marba´n L, Mendizabal A, Johnston PV, Russell SD, Schuleri KH, Lardo AC, Gerstenblith G, Marba´n E. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet.

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2011;378(9806):1847–1857. Available at: http://dx.doi.org/10.1016/s0140-6736(11)61590-0 [8] Heusch G. SCIPIO brings new momentum to cardiac cell therapy. Lancet. 2011;378(9806):1827–1828. Available at: http://dx.doi.org/10.1016/S0140-6736(11)61648-6 [9] Takehara N, Nagata M, Ogata T, Nakamura T, Matoba S, Gojo S, Sawada T, Yaku H, Matsubara H. The ALCADIA (Autologous Human Cardiac-derived Stem Cell To Treat Ischemic Cardiomyopathy) trial. AHA 2012; LBCT-20032.

[10] Takehara N, Tsutsumi Y, Tateishi K, Ogata T, Tanaka H, Ueyama T, Takahashi T, Takamatsu T, Fukushima M, Komeda M, Yamagishi M, Yaku H, Tabata Y, Matsubara H, Oh H. Controlled delivery of basic fibroblast growth factor promotes human cardiosphere-derived cell engraftment to enhance cardiac repair for chronic myocardial infarction. J Am Coll Cardiol. 2008;52(23):1858–1865. Available at: http://dx.doi.org/10.1016/j.jacc.2008.06.052 [11] Malliaras K, Zhang Y, Seinfeld J, Galang G, Tseliou E, Cheng K, Sun B, Aminzadeh M, Marba´n E. Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart. EMBO Mol Med. 2013;5(2):191–209. Available at: http://dx.doi.org/10.1002/emmm.201201737

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Review article

The zebrafish model system in cardiovascular research: A tiny fish with mighty prospects Kar Lai Poon, Thomas Brand* Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, Hill End Road, Harefield, Middlesex, UB9 6JH, United Kingdom *Email: t.brand@imperial.ac.uk

http://dx.doi.org/ 10.5339/gcsp.2013.4 Submitted: 30 October 2012 Accepted: 29 January 2013 q 2013 Poon, Brand, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

1. INTRODUCTION The zebrafish Danio rerio, a tropical freshwater fish, belongs to the family of cyprinidae, which in the last 30 years has developed into a very popular model organism for studies of embryonic development and human diseases. Initially the zebrafish species has been selected on the basis of its small size of approximately 3– 5 cm, its transparency during development and its high fertility, qualities first identified by George Stresinger, the founding father of zebrafish research.1 The ability to house thousands of small fishes and the ease of screening mutations in the translucent embryos made it feasible to perform large-scale forward genetic screens in a vertebrate model organism. The abundance of eggs obtained, approximately 200 eggs per female per week, is ideal for genetic and statistical analysis. The mutagenesis screens performed in the early 1990s have led to the identification of genes important in vertebrate organogenesis in an unbiased fashion.2,3 Many of the isolated mutants have now been fully characterized and the mutated genes mapped, as the zebrafish genome sequencing completes. The knowledge derived has led to a better understanding of the underlying genetic networks governing vertebrate development. More sophisticated phenotype-based screens have since been developed to screen for mutations in defined biological processes.4 The development of the zebrafish is very fast and by 24 hours post fertilization (hpf), the fertilized egg has developed into an embryo that has established most organ primordia including the nervous system and a contracting heart tube. This is followed by the commencement of blood circulation.

The embryo hatches after about 2 days post fertilization (dpf) and reaches the larval stage at 3 dpf when most internal organs have matured. At 30 dpf, the zebrafish metamorphose and after 90 days has reached adulthood.

Zebrafish are easily maintained and are relatively economical in comparison to mammalian species.

About 5 adult fishes are kept in 1 liter and therefore a small laboratory aquarium facility can easily house more than 1000 animals. Non-invasive imaging can follow the entire phase of embryonic development while the embryo is kept in a petri dish, which allows cell biology to be performed in an intact organism.5,6 In fact, by introducing pigments and scale mutations that renders the fish transparent, tumors formation can be visualized and thus enable transplantation experiments to be performed in adult zebrafish.7 The small size of the embryo allows it to be placed into 384 well plates for small molecule screenings.8 There are however also drawbacks that restrict the utility of the zebrafish. These include the currently limited biological resources for this model organism in comparison to the mouse. For example antibodies used in mammals often do not cross-react in the zebrafish limiting the utilization of this model organism for protein biochemistry. Likewise during teleost evolution, genome duplication took place,9 which resulted in the presence of many duplicated genes. On one hand, this complicates genetic analysis but it allows the examination of multiple functions of a gene in separate mutants since the duplicated genes display subfunctionalization.10 The lack of embryonic stem cells, which is required for reverse genetic approaches such as the

Cite this article as: Poon KL, Brand T. The zebrafish model system in cardiovascular research: A tiny fish with mighty prospects, Global Cardiology Science & Practice 2013:4 http://dx.doi.org/10.5339/gcsp.2013.4

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generation of knockout strains, has hampered the progress to utilize this organism in biomedical research. Nonetheless, recent technological advances have greatly improved this situation.11 – 13 In this review, we will outline the knowledge of cardiac development in the zebrafish. Moreover, the current state of knowledge in studying development and function of the cardiac conduction system in zebrafish will be discussed. The zebrafish as a novel disease model in cardiac hypertrophy and heart failure will be introduced. We will also discuss the role of the zebrafish as a premier model to study cardiac regeneration. Since no single review is able to cover all aspects of this rapidly growing field, the reader is also referred to some other excellent reviews in the field, which have been recently published.14 – 17 2. HEART DEVELOPMENT Apart from sharing well-conserved genetic pathways that govern heart formation, zebrafish heart development proceeds through very similar steps as amniotes, and begins with the specification of precardiac cells in the anterior lateral plate mesoderm followed by fusion of the bilateral heart fields at the embryonic midline (Table 1). The mechanism of this seemingly simple morphogenetic event at the very early stages of embryonic development became better understood largely due to the powerful genetic amenability and high-resolution bioimaging of zebrafish embryos as illustrated below.18 – 20

Table 1. Milestones of early heart development shared by different organisms. (modified from57).

Cardiac Events

Human

Mouse

Zebrafish

Migration of precardiac cells from epiblast First evident assembly of myocardial plate Generation of single heart tube initiated Tubular heart starts contraction Looping Endocardial cushions form

15–16 days

7 dpc (primitive streak)

50% epiboly (5.5 hpf)

18 days

7 dpc (late primitive streak)

8–10 somites (, 13 hpf)

22 days (4 –10 somites)

8 dpc (5–10 somites)

20 somites (,19 hpf)

23 days

8.5 dpc (8–10 somites)

26 somites (22 hpf)

23 days 28 days (30 –38 somites)

8.5 dpc 9.5 dpc

33 hpf 48 hpf

In the zebrafish, cardiac progenitors are specified as early as 5 hpf,21 just before gastrulation, a developmental phase lasting nearly 5 hours, which results in the formation of the three primary germ layers: ectoderm, mesoderm and the endoderm. Fusion of the bilateral heart fields by 16 hpf produces a cardiac cone, which expresses cardiac differentiation markers such as the sarcomeric genes tropomyosin and troponin.22 The cardiac cone then extends anteriorly and is gradually remodeled into a primitive heart tube.

By 24 hpf, the early heart tube consists of an outer myocardial and an inner endocardial layer separated by extracellular matrix, known as the “cardiac jelly”. Shortly after its formation, the linear heart tube is capable of contracting rhythmically in a peristaltic manner with a conduction velocity of 1 mms21.23 The heart tube loops at 33 hpf, into an S-shaped configuration, displacing the ventricle to the right of the atrium. The transition from slow peristaltic wave to sequential chamber contraction by 36 hpf suggests the onset of cardiac conduction system (CCS) function.

Fate mapping studies have shown that chamber identities are established as early as 5 hpf,21 attributing to differential gene expression that governs later differences in size, morphology and contractile properties conferred by different sets of sarcomeric proteins (atrial myosin heavy chain, amhc, in the atrium versus ventricular myosin heavy chain, vmhc, in the ventricle). By 2 dpf, the ventricular chamber balloons into a natriuretic peptide precursor A (nppa) – labeled outer curvature (OC) and an inner curvature (IC), which forms the future apex and base of the mature heart respectively.

Myocardial cells of the OC are elongated, bigger and conduct electrical activity thrice faster than cells located at the IC.23,24

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2.1. Heart muscle growth As the heart develops, the atrium remains a thin layer of myocardial cells whereas the ventricle thickens to form trabeculae (myocardial projections), which are necessary to generate sufficient contractile force to propel circulation in the growing embryo. At the same time myofibrillogenesis is completed whereby thin and thick filaments, M line, and Z discs are assembled into uniform arrays of cardiac sarcomeres.25 Studies in mice have shown that trabeculation of the ventricular myocardium is dependent on endocardial-derived signals such as neuregulin, BMP10, and Brg1 (a chromatin remodeling protein) as well as a supportive microenvironment provided by the cardiac jelly.26 – 29 The requirement for the neuregulin signaling pathway in trabeculation is resonated in the zebrafish where embryos mutated in erbB2 encoding the neuregulin receptor fail to undergo trabeculation and hence show aberrant electrical conduction in the ventricular myocardium.30 Starting with 150 cardiac myocytes at 24 hpf, the cell number has doubled at 3 dpf. However the embryonic heart does not rely on cell proliferation, which is negligible at this stage in development.

Addition of cells at both arterial and venous poles of the heart followed by waves of cardiac myocyte differentiation is found to be the major mechanism for increasing cell numbers.31 The adult zebrafish heart grows to a size with a ventricular length of 1 mm or larger, its atrium is lined internally with pectinate muscle and the ventricle consists of a compact layer enveloping the inner trabeculae, which in contrast to the mammalian heart persists into adulthood (Fig. 1). To investigate the

Figure 1. Anatomy of the adult zebrafish heart. (A) Schematic representation of the adult zebrafish heart. (B) Trichrome stained histological section through the ventricle and bulbus arteriosus, which labels myocardium in red whereas nonmuscle tissue and extracellular matrix in blue. Note the trabecular layer, which is extensive in the adult zebrafish heart, while the compact layer makes only a minor fractor of the total mass of the ventricular wall. Rarely coronary vessels are observed in the compact layer mocardium.

complexity of ventricular wall formation, Gupta and Poss recently employed a highly sophisticated multicolor lineage tracing tool, termed Brainbow, which allows after modification and optimization of the technique, to label approximately 20 cardiac myocytes with different colors at embryonic stage and to trace their descendants in the adult heart.32 These experiments not only uncovered an “inside-out” mechanism for the formation of the outer compact layer of the heart where the source of surface myocardium originates from inner muscle fibers, but also has led to the identification of the amazing properties of a few ‘clonally dominant’ cardiac myocytes, which display proliferative ability in a stem cell-like manner to give rise to the entire heart wall. Hence, further investigation into the properties of this specific cardiac myocyte population, in particular its expression profile, which underlies its unique abilities, is necessary and may provide an insight to rebuild the damaged heart.

2.2. Endocardium and valves Cardiac valves in the form of leaflets develop between the chambers to ensure unidirectional blood flow.33,34 Valve formation is initiated by the restriction of bmp4, tbx2b, and vcana expression to the AV myocardium at 37 hpf35,36 followed by the endocardial expression of notch1b, has2 and neuregulin.37,38 These changes in gene expression are accompanied by changes in endocardial cell morphology. The endocardial cells are initially cuboidal in shape and by 72 hpf form primitive valve leaflets, which serve to block retrograde blood flow.34 In zebrafish, the process of valve maturation, which also involves

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epithelial – mesenchymal transition (EMT) proceeds into the larval stages of development and is completed by 16 dpf.39 In amniotes, endocardial cells also participate in septum formation in the atria, ventricles and outflow tract (OFT). The significance of proper valve formation, illustrated in congenital valve defects and adult onset congenital valve disease (CVD), renders it one of the most extensively studied aspects of heart development. Studies in vertebrate embryos and in vitro with the help of EMT collagen gel assays have demonstrated the involvement of multiple signaling pathways, including VEGF,40 Notch,41,42 ErbB,43,44 Bmp,45,46 TGFb,47 Wnt48 and NFAT49 (for reviews50,51).

Zebrafish mutants with defective heart valves usually display symptoms of blood regurgitation, which is used as an indicator in a forward genetic screen for valvular defects.52 The importance of extracellular matrix, heparan sulfate proteoglycan production, in valve specification is highlighted in the Jekyll mutant that encodes UDP-glucose dehydrogenase,35 which has also been recently linked to CVD in humans.53 Another ECM protein, nephronectin, regulates AV valve differentiation acting through a Bmp4-Has2 pathway.54 In addition, zebrafish mutants defective in WNT signaling pathway components – axin (mbl), adenomatous polyposis coli (apc) and glycogen synthase kinase 3 (gsk3) do not form proper valves.48 – 55 The endocardium, which is connected to the vasculature, is constantly exposed to hemodynamic forces. Experimental changes of cardiac hemodynamics have a strong impact on the formation and growth of the valve leaflets.52 With the help of high speed video imaging and blood flow pattern analysis, Vermot et al. demonstrated recently the requirement of reverse blood flow in patterning the atrioventricular valve in addition to the identification of endothelium-derived klf2a acting as a sensor of hemodynamic force and possibly as an indicator of defective valve formation.56 Blood flow also has its impact on cardiac trabeculation, myocyte growth and chamber maturation, most likely via the endocardium.57 – 59 In the mature heart two valves are present, the atrioventricular (AV) and the bulboventricular (BV) valves (Fig. 1).60 Some significant morphological differences exist between the valvular apparatus in fish and the mammalian heart. For example, the AV valve lacks papillary muscles and chordae tendinae. Moreover, the BV valve, homologous to the aortic valve, has only two leaflets instead of three, which is found in the mammalian heart.60 The outflow tract in the zebrafish also shows a significant divergence form the mammalian heart in that is has an additional chamber-like structure, the bulbus arteriosus (BA), which is highly distensible due to the presence of large amounts of elastic fibers (Fig. 1). The BA is believed to be a vascular adaptation, which is required to accommodate the short distance between the BA and the gill apparatus and to maintain constant blood perfusion.61,62 The endocardium also plays a non-cell autonomous role in myocardial wall formation. The zebrafish mutants heart of glass (heg), santa (san) and valentine (vtn) display a dilatation of the ventricular chamber during early development.63,64 While the cell number is maintained, the ventricular myocardium is unable to develop into a multilayered wall. As a consequence, the chambers are massively dilated and dysfunctional. Heg1, which is the gene mutated in heart of glass, encodes a transmembrane protein that directly associates with the proteins Ccm1 and Ccm2, which are defective in the santa and valentine mutants and also display ventricular dilatation due to insufficient wall formation.64,65 CCM1, CCM2 and CCM3 have been genetically linked to a common neurological disease, cerebral cavernous malformation, which causes a dilatation of blood vessels in the brain and ultimately causes cerebral hemorrhage.65 The CCM proteins form with Heg1 a plasma membrane-associated complex that affects endothelial cell-cell-interaction through the modulation of the actin cytoskeleton.66 Presently, it is unclear how defective endocardial cell-cell interaction affects ventricular wall formation, but secretion of an endocardium-derived signaling molecule is a likely scenario. In the liver, the CCM complex in the vascular endothelium affects apicobasal polarity of hepatocytes in a non-cell autonomous manner67 and it can be envisioned that a similar activity is required at least transiently in the early heart. It will be interesting to find out whether such a signaling pathway is still active in the adult myocardium and possibly involved in the pathogenesis of dilated cardiomyopathy.

2.3. Secondary heart field Recent advances in understanding heart development in mouse and chick embryos has led to the identification of two different sources of cells that give rise to the early embryonic heart. A first heart field lineage contributes mainly to the linear heart tube and subsequently develops into the left ventricle, whereas the second heart field lineage develops into the right ventricle and both the outflow

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and inflow tract of the heart.68 – 70 The second heart field cells are located dorsal to the primitive heart tube in the pharyngeal mesoderm. Mutants of genes that are expressed in the second heart field, such as Isl1, are missing outflow tract, right ventricle and most of the atria.71 Interestingly, second heart field cells also give rise to a subset of craniofacial muscles.72,73 The presence of only two non-septated heart chambers and a primitive OFT prompts the question of the presence of the second heart field in the zebrafish. Recent work verified that there exist secondary heart field-like cells that contribute to specific lineages of the outflow tract and distal ventricular myocardium, which acts under latent TGFb?signaling.74 Interestingly, isl1, which is essential for second heart field development in the mouse is dispensable for zebrafish OFT development.31 2.4. Epicardium and neural crest cells In amniotes, epicardial cells are derived from a transient structure, the proepicardium (PE) located adjacent to the sinus venosus at the venous pole; they migrate to the surface of the myocardium, proliferate rapidly, and envelop most of the heart except the OFT, which is covered by epicardium from an arterial source.75 It has been shown that morphogenetic signaling from the epicardium is necessary for proper heart morphogenesis as well as development of coronary vasculature and cardiac fibroblasts (reviewed in76 – 78). Only a few marker genes, namely wt1, tbx18 and tcf21 have been linked to the formation of the PE and its transformation into the epicardium. As a result, the epicardium remains the least understood cell lineage of the heart. In zebrafish, the presence of the PE and its derivative, the epicardium, has been documented,11,79,80 but very little research has been done. This is partly due to the lack of useful live markers. Tbx5 and Bmp signaling in PE specification, which has been previously demonstrated both in the chicken and mouse embryo, are also essential for PE development in the zebrafish.79 In contrast, zebrafish PE induction does not require signals from the liver primordium as proposed in higher vertebrates.79,81 Epicardium – derived cells (EPDC) were initially reported to also contribute to the myocardial lineage.82 However, it was subsequently demonstrated to be due to the endogenous expression of epicardial marker genes in a subset of cardiac myocytes.83 Similarly in zebrafish, the non-epicardial expression of wt1 and tbx18 rendered these genes unsuitable for lineage studies.84 The cardiac neural crest cells (NCC) is another extra-cardiac cell population, which in the amniote embryo migrates into the forming pharyngeal arches and is involved in re-patterning the initially bilaterally symmetrical pharyngeal arch arteries to form the asymmetric great arteries of the thorax.85 The NCC will form the smooth muscle tunica media of the arteries. A subpopulation of the cardiac NCC in the caudal pharyngeal arches migrates into the cardiac outflow tract and forms the aorticopulmonary septum dividing the common arterial outflow into the aorta and pulmonary trunk.86 A combination of lineage tracing and ablation experiments have demonstrated Wnt-dependent contribution of cardiac neural crest to myocardium in all regions of the embryonic fish heart87 – 89; this is in contrast to higher vertebrates, where neural crest cells contribute mostly to the OFT and do not appear to invade deeply into the developing heart tube. However, further analysis of the functional importance of the NCC in zebrafish heart development has yet to be performed.

3. MUTANT SCREENING, TRANSGENICS AND DRUGS In the 1990s, the first large-scale chemical mutagenesis screens using ENU in Tu¨bingen and Boston has generated hundreds of mutants.2,3 Mark Fishman, a cardiologist who initiated the Boston screen envisioned the strength of the zebrafish as model to study cardiovascular diseases. As described throughout the review, many heart mutants are derived from these two screens.57 Due to a lack of embryonic stem cells in the zebrafish, targeted knockout by genetic recombination has not been possible. Hence, the zebrafish community has been using antisense technology mainly morpholinos, which are modified oligonucleotides that block translation or mRNA splicing leading to a knockdown of specific genes of interest.90 However, some morpholinos can cause undesirable nonspecific side effects such as apoptosis, edema of the fourth brain ventricle and the pericardial sac.

Careful experimental design that includes stringent controls and rescue experiments is required when using morpholinos.91,92 Regardless, the effect of morpholinos is short-lived and therefore analysis of gene function is confined to the embryonic and early larval stages. Attempts to generate mutants include targeting induced local lesions in genomes (TILLING) and insertional mutagenesis. Tilling is slow and tedious, however it has generated a significant number of mutations in important genes.93 Insertional

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mutagenesis, which aims to create mutants by insertion of a foreign DNA into the zebrafish genome, has been carried out using a number of different constructs with varying results. Large-scale screens were performed with a mouse retroviral vector94 and transposons. Tol2 transposon95,96 and the sleeping beauty transposon97 have been used in enhancer and gene trap screenings. The use of Tol2 transposon to mutagenize genes has not been successful, but hundreds of transgenic marker lines have been produced instead that serve as markers for studying various biological process or organ structures (Fig. 2).11,95,98 A promising novel approach involves the use of an in vivo protein-trap mutagenesis system, which can be used to assess spatiotemporal protein expression dynamics and gene function.99

Figure 2. Transgenic labeling of tissues and cell types in the zebrafish heart using GFP reporter genes. (A) The different cell types of the zebrafish heart are GFP-labeled by transposon mediated insertional transgenesis. (A) Schematic depiction of a zebrafish heart indicating the route of blood flow from the sinus venous, through the atrium, ventricle and exiting from the bulbus arteriosus. GFP expression present in the three cardiac layers - (B,E) endocardium, (C) myocardium and (D, F) epicardium as well as examples of transgenes with a region-specific expression pattern including the (G) ventricle, (H) atrium, (I) bulbus arteriosus and (J-L) valves. Expression patterns are shown in the (B-D, I) embryonic, or (E-F,J-L) adult heart. Abbreviations: A, atrium; AV, atrioventricular valve; BA, bulbus arteriosus; BV, bulboventricular valve; V, ventricle. From11 with permission.

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The plan to saturate the zebrafish genome or to generate a mutant for every gene is becoming possible with the arrival of zinc-finger based mutagenesis tools - zinc finger technology100 and TALEN (transcription activator-like effector nucleases).101 Zinc-finger nucleases (ZFNs) induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions.102 The major drawback for the use of ZFNs is the cost involved and the requirement of laborious screening efforts. TALEN however seems to be a technology with potential to become the method of choice in creating mutants of any gene of interest. TALEN proteins have a simpler mode of DNA base recognition than zinc fingers, with an individual base recognized by only two amino acids in an individual TALE repeat unit.101 Due to this property, gene-specific TALENs are rapidly assembled even for small laboratories, thus making it likely that this technology will find wide distribution. Initial reports suggest that mutagenized genes are germline transmitted, and therefore novel mutant lines are rapidly and efficiently produced.103 A recent report suggests that not only indel mutations are possible but also site-directed integration of loxP sites, which is a prerequisite for Cre-mediated conditional mutagenesis. Likewise, the DNA repair following TALEN nuclease treatment can also be modified so that single point mutations can be introduced in any gene, which will be useful to model gene mutations associated with cardiovascular disease in the zebrafish model.104 Another important area, in which the zebrafish model may find its use, is chemical genetics, i.e. the search for novel cardiovascular drugs.105 The zebrafish larvae can be kept in 384 well plates and chemical compounds are directly added to its aquatic environment, and are, therefore, readily absorbed avoiding the need for time-consuming injections. Zebrafish embryos can be maintained for a few days in 96-well plates with several embryos per well or singly in 384 well plates. Compounds can be added and the plates can be screened either by eye or with the help of high-throughput video techniques. Numerous chemical libraries exist, from small collections of characterized compounds to larger libraries consisting of tens of thousands of compounds of uncharacterized function. In a recent report, larval zebrafish were used to screen chemical compounds that modulate heart failure-specific gene expression patterns.106 The authors made use of the promoter of the zebrafish nppb gene, which appears to respond to pathological signaling pathways in a very similar way as in mammals. The long QT (LQT) phenotype, which develops in the zebrafish kcnh2 mutant, was utilized in a large-scale screening approach to search for chemical compounds with a LQT suppressive ability.107 A screen for molecules that enhances FGF signaling resulted in the identification of a novel compound, which increased the size of the embryonic zebrafish heart.108 This compound may have the potential to enhance cardiac regeneration in vivo or to improve the efficiency of generating cardiac myocytes from stem cell cultures. These few examples demonstrate the power of the zebrafish model for chemical genetic approaches.

4. CARDIAC CONDUCTION SYSTEM (CCS) Even with only a two-chambered heart, a functional CCS is necessary to initiate, maintain and coordinate heart rhythm so that synchronized contraction can take place. A large-scale screen for gene mutations affecting the CCS was recently conducted which helped to define four stages of CCS development in zebrafish and furthermore lead to the identification of 17 mutants with defects in one of these stages.109 Stage 1 occurs between 20– 24 hpf, when a linear activation wave travels across the heart tube from the sinus venosus to the OFT; in stage 2 (36 – 48 hpf), a significant AV conduction delay develops; in stage 3 (72 – 96 hpf), an immature fast conduction network develops within the ventricle; and in stage 4, this fast conduction network fully matures resulting in appearance of an apex-to-base activation pattern.109 The presence of the pacemaker in zebrafish had been enigmatic for many years with only two publications describing a mutant, slow mo, with defective pacemaker current.110,111 However, pacemaker activity is clearly present in fishes and its function is similar to that of mammals.112 By recording action potentials from the hearts of a variety of fishes, Saito (1969) concluded that the pacemaker site is at the sinoatrial valve.113 Consistent with this, a recent study showed that knockdown of shox2 resulted in pronounced sinus bradycardia.114 Finally, with a combination of optogenetics, which utilizes transgenic expression of light-controlled ion channels and light sheet microscopy, the cardiac pacemaker of the zebrafish has been localized to a few cells in the sinoatrial region.115 The Isl1 genes in zebrafish, human and mouse heart is expressed in sinoatrial pacemaker cells.116 While in mammals Isl1 labels a subset of sinoatrial node cells, in zebrafish, isl1 expression demarcates the entire pacemaker population, which forms a ring-like structure at the border between the sinus

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venosus and the atrium.116 The isl1-positive cells also express tbx2b and are characterized by the presence of primitive action with a pacemaker potential.

The AV conduction system is responsible for delaying the electrical impulse between the chambers so as to achieve coordinated contractions. By calcium mapping with calcium green dyes, a retardation of calcium waves at the AV junction is observed at 40 hpf in the zebrafish. Interestingly, the specification of zebrafish AV conduction is mediated by the endocardial signals notch1b and neuregulin.117 In addition, foxn4, which drives the expression of tbx2b, is involved in AV canal specification and mutants of both genes display AV conduction defects.36 Recently, one member of the Popeye domain containing gene family, popdc2 has been shown to exhibit AV conduction abnormalities, which reflects its role in the regulation of the cardiac conduction system (Fig. 3).118

Figure 3. Analysis of cardiac arrhythmias in zebrafish popdc2 morphants. (A) Ventricular contractions are regular in control animals (CTR) but display 2:1 and 3:1 secondary AV block in popdc2 morphants (MO1). (B, C) Optical mapping of calcium waves in control and morphant hearts reveal aberrant calcium waves in Popdc2 morphants.

From111 with permission.

The apex-to-base ventricular activation in both embryonic and adult zebrafish has been optically mapped by individual groups.109,119 This electrical activity is most likely transmitted by the gap junction protein Cx40, which immunoreactivity has been detected in the embryo at 4 dpf, supporting the presence of a fast CCS at this stage in development.109 In the absence of morphologically defined His-Purkinje bundles in zebrafish, it has been proposed that the impulse travels through ventricular trabeculae, which are the evolutionary precursors of Purkinje fibers in amniotes. In fact, the CCS does not mature in a mutant that is devoid of trabeculae.30 Studies in embryonic zebrafish with an aberrant cardiac conduction revealed the importance of cardiac conduction in influencing chamber morphogenesis.120 The authors furthermore uncovered a novel connexin gene, cx46, in conduction of electrical impulse which results in CCS defects in mutant mouse that resemble human heart failure patients.

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5. CARDIAC ELECTROPHYSIOLOGY Despite the small size of the zebrafish embryonic heart (100– 150 mm), an electrocardiogram (ECG) of the zebrafish heart can be recorded using micropipette electrodes from as early as 5 dpf with apparent P and R waves that reflect atrial and ventricular depolarization, respectively.121 As the larvae develop further and the heart matures, the ECG pattern changes accordingly with the emergence of T waves at 2 weeks post fertilization followed by the shortening of QRS interval and finally distinct P waves, QRS complexes, and T waves, which are detectable by 35 dpf.122,123 The surprisingly close resemblance of the zebrafish ECG morphology to that of the human heart probably reflects the crucial importance of electrical activity for governing the heartbeat and is therefore evolutionary conserved.

The relative simple design of the zebrafish heart, containing only the essential elements of the cardiac conduction system, offers an advantage to study fundamental questions of cardiac electrophysiology.

The fish heart beats at a rate of 120 – 180 beats per minute (bpm), which is closer to the human heart rate (60 – 100 bpm) than the mouse (300– 600 bpm). As a result, the cardiac repolarization phase, a diagnostic feature for possible arrhythmias represented by the length of the corrected QT (QTc) interval, is more similar between human (300– 450 msec) and zebrafish (300 – 440 msec), than mouse (83 – 96 msec).124,125 Furthermore, both the human and the zebrafish generate a repolarizing current during the cardiac action potential (AP) using a similar rapidly activating delayed rectifier potassium current (IKr), which is in contrast to the mouse that uses a different set of potassium channels.126 The similarity in cardiac repolarization between humans and mice is also evident in the phenotypes of the breakdance and reggae mutants, which are defective in the ERG potassium channel and exhibiting either long QT syndrome or short QT syndrome.127 – 129 In fact, QT syndrome mutations have been successfully modeled in the zebrafish but not in mice.125 Specific ion channels, mainly gating sodium, potassium and/or calcium ions generate unique currents that shape the different phases of the cardiac AP. Knowledge of the set of ion channels that contribute to the zebrafish cardiac AP are derived from fish mutants defective in ion channels as well as with the help of pharmacological experiments.126 By detailed comparison of the AP between human, mouse and zebrafish cardiac myocytes, it was found that the AP parameters - AP amplitude and the resting membrane potential were highly similar between zebrafish and humans, whereas the fish has a lower maximum depolarization velocity (dV/dtmax) (Fig. 4). This is possibly due to less competent or fewer fast sodium channel that drives INA. The AP profile of both atrial and ventricular adult zebrafish cardiac myocytes are comparable to that of the humans, especially the long plateau phase 2 and the resulting action potential duration (APD) except for the lack of the sharp early repolarization phase 1, which is probably due to a non-functional Iks current in zebrafish cardiac myocytes.119 Sodium channels blocker (tetrodotoxin, TTX) decelerate the AP upstroke velocity confirming the role of INA in establishing the steep slope of the rapid depolarization phase 0 of the AP. Interestingly zebrafish cardiac myocytes are 10 – 100x more sensitive to TTX than mammalian cardiac myocytes due to the presence of an aromatic amino acid at a critical position, which is thought to increase the TTX sensitivity.130 The L-type calcium channel (LTCC) contributes to the AP plateau phase indicated by the result that LTCC blocker, nifedipine, shortens both atrial and ventricular AP. Moreover, the LTCC mutant island beat embryos exhibit an absence of the QRS complex causing atrial fibrillation and a silent ventricle.131 Unexpectedly, apart from LTCC, functional T-type calcium channel (TTCC) also contribute to the calcium current in adult zebrafish cardiac myocytes,126 which can be considered as an indication of an immature state of the adult zebrafish heart in comparison to mammals, where the TTCC is only present in the embryonic heart and in pacemaker tissue.

Although this has not yet been confirmed, the zebrafish heart most likely employs a similar pacemaking mechanism as mammals and probably depends on the activity of the hyperpolarizationactivated cyclic nucleotide-gated channel. Unfortunately, the slow mo mutation, which has a defective pacemaker current, has not been molecularly characterized until now.110,111 In terms of establishing ion homeostasis, cardiac myocytes utilize the sodium/calcium exchanger (Ncx1) and calcium ATPase at the plasma membrane (PMCA) and sarcoplasmic reticulum (SERCA) to regulate the cytosolic calcium concentration and the Na/K-ATPase for maintaining a proper sodium/potassium gradient. The mouse Ncx1 mutant is embryonic lethal due to a vascular function of the Ncx1 gene whereas a cardiac-specific Ncx1 null mutant reaches adulthood with cardiac myocytes exhibiting normal calcium transients.132,133 In contrast, the cardiac-specific ncx1 mutant (tremblor, tre) displays atrial fibrillation and arrhythmia whereas the ventricle is noncontractile due to calcium

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Figure 4. Atrial and ventricular action potentials in the zebrafish, human and mouse. Representative shapes of action potentials in (A) zebrafish, (B) human, and (C) mouse. Note the similarity between the ventricular action potentials in humans and zebrafish. From119 with permission.

overload.134,135 The difference in phenotype severity of the mutants is most likely based on differences in the calcium homeostasis and genetic buffering mechanisms in these species.

One interesting observation is that some channel proteins do have non-electrical related functions in early embryonic development. The heart and mind (had), which is defective in the Na/K-ATPase a1B1 isoform, causes severe abnormalities in primitive heart tube extension, cardiac myocyte differentiation and compromised cardiac function. These phenotypic anomalies are found to be direct consequences of defective pump activity resulting in ionic imbalances that affect the maintenance of proper myocardial cell junctions in the early embryonic heart.136,137 The morpholino-mediated knockdown of the voltage-gated sodium channel scn5a caused a reduction in the expression of the cardiac transcription factors and had an negative impact on proliferation and differentiation of cardiac myocyte resulting in abnormal cardiac morphogenesis.138 These phenotypes were not reproduced by the administration of TTX, suggesting a novel non-electrogenic function of scn5a.

6. CARDIOMYOPATHY Zebrafish has an established track record in uncovering the molecular mechanisms of human cardiovascular diseases (CVD), which includes prevalent forms of cardiomyopathy - dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). It was observed that some mutants, generated from forward genetic screens, exhibit a lack of blood circulation together with pericardial edema. By high-speed video imaging of the embryonic heart, standard non-invasive assessments of cardiac performance were made and revealed compromised cardiac contractility, shortening fraction

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(sometimes presented in M mode), stroke volume and/or cardiac output. Positional cloning of these mutants showed that many of the mutated genes were cardiac sarcomeric genes, which have been previously implicated in human cardiomyopathy such as titin (ttn),139 tropomyosin (tpm4),140 troponin 2 (tnnt2),141 myosin light chain genes (cmlc1, myl7)142 – 144 and myosin heavy chain (myh6).145 Moreover, these mutants also exhibit myofibrillar disarray, one of the hallmarks of cardiomyopathy as revealed by ultrastructural examination of sarcomeric organization.146 Cardiac myofibrillogenesis in the zebrafish has been characterized but not completely understood.

Non-striated actin filaments are the first to assemble at the plasma membrane. Sarcomeric myosin independently assembles into thick filaments before being integrated into the thin filament network.25 Next, M-lines of fixed width and Z-discs, which elongate from Z bodies, are recruited to the contractile units. Phenotypic differences of different mutations of sarcomeric genes also provide an opportunity to understand their unique and specific roles in myofibrillogenesis. This becomes apparent in the case of titin isoforms and has also been observed for the alkali and regulatory myosin light chains, which have distinct roles in the process of myofibril assembly.143,147 The zebrafish is also instrumental to unravel the function of novel myofibrillar genes. Kindlin2, an integrin-binding protein first identified in Caenorhabditis elegans localizes to the intercalated disc. Morpholino-mediated knockdown abolishes sarcomere assembly at the plasma membrane and causes contractile dysfunction.148,149 Myozap, another intercalated disc protein, which directly interacts with desmoplakin and zonula occludens-1 participates in the Rho-signaling pathway and activates serum response factor dependent transcription.150 Consistent with its role in myofibrillar-nuclear communication is the fact that the knockdown of Myozap in zebrafish causes a severe cardiomyopathy-like phenotype. Chap is another newly identified Z-Disks proteins and the loss-of-function phenotype includes severely impaired heart function.151 The efficient and cost-effective zebrafish system is also used to study the physiological roles of mutations found in patients with heart failure such as in TNNT2146 and especially non-sarcomeric genes, including transcriptional coactivator EYA4152 and RNA-binding protein RBM24 (Fig. 5).153 Cardiac

Figure 5. Contractile dysfunction in the zebrafish heart. Morpholino-mediated knockdownn of the gene encoding the RNA-binding protein Rbm24a causes contractile dysfunction as evidenced by M mode analysis (left panels) which shows significant difference between end-diastolic (Dd) and end-systolic (Sd) diameter of the rbm24a morphant (lower panel) as compared to control embryo (upper panel). Furthermore, sarcomeric defects are observed in the thin filament (visualized by anti-Tnnt2 immunohistochemistry) and Z-Disc (visualized by anti-aActinin immunohistochemistry) that most likely cause the contractile dysfunction. From146 with permission.

myosin light chain kinase (mlck) was identified in a microarray comparison of healthy and failing human hearts. Its crucial role in cardiac function is suggested by its morphants, which develop a dilated cardiac ventricle with immature sarcomere organization.154 Nexilin, a novel Z disk protein, functions to stabilize the Z-disks in zebrafish and is a candidate in causing DCM in patients.155 Interestingly, more evidence has substantiated the effect of cardiac mechanical stretch sensing on regulation of heart function hence resulting in cardiomyopathy. In zebrafish, the first clue comes from the positional cloning of the contractility-deficient mutant main squeeze (msq), which fails to express stretch-responsive genes.156 The extracellular matrix protein integrin-linked kinase (ilk) is mutated in msq. ILK, found to localize to Z-Disks interacts with integrin and b-parvin together with many other proteins forming a complex at the cell membrane, which acts as a mechanosensor and signals into cardiac myocytes. PINCH, which is also part of the mechanosensor complex, has been shown to regulate contraction in the zebrafish heart.157 More importantly, human patients with DCM were also found to bear mutations in ILK or laminin-alpha4 (Lama4), yet another of ILK interaction partner.156,158 Collectively, these genetic and biochemistry data emphasize the importance of the mechanosensor

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complex in the regulation of cardiac function. In addition, a M-band protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein) has been shown to regulate stretch responsive genes and hence cardiac stretch sensing via an alternative pathway that involves serum response factor (SRF)-dependent signaling.159 To investigate myocardial function in greater detail, the micromechanical properties of zebrafish myofibril has been evaluated and proven to be a feasible contractile model where functional studies at subcellular level can be carried out.160 This might serve as another step forward in reaching a detailed molecular understanding of the etiology of cardiomyopathy. On another note, Werdich et al. tested the stretch response of embryonic zebrafish heart exploring the feasibility of using the zebrafish as novel model to study the mechano-electrical feedback of the heart.161 One main limitation of the zebrafish embryonic models of cardiomyopathy is that it does not go through the progressive stages of cardiac remodeling as in patients due to the fact that so far most of the studies utilized zebrafish embryos or larvae. Hence, developing an adult cardiomyopathy model in zebrafish, which more closely mimics the human pathological process would be an important improvement. Recently, two adult models of cardiomyopathy have indeed been developed. The first is anemia-induced either by a band3 mutant that inflicts chronic stress in the heart or through the use of the anemia-inducing drug phenylhydrazine.162 Encouragingly, the mutant hearts displayed myocyte hypertrophy and hyperplasia as well as several features of human cardiomyopathy, comprising myocyte disarray, fetal gene expression reactivation, and severe arrhythmia. The second model was generated by cardiomyopathy-inducing doxorubicin (DOX) injection.163 These two zebrafish cardiomyopathy models used in combination with a target of rapamycin (ztor) mutant fish, served to provide genetic evidence for a cardioprotective function of target of rapamycin (TOR) signaling.

7. HEART REGENERATION The zebrafish heart demonstrates the surprising capability of myocardial regeneration within months after experimental induction of myocardial injury. Experimental procedures to induce myocardial regeneration include 20% ventricular resection,164,165 or cryocautherization, which induces localized injury of 25% of myocardium166,167 or diphtheria toxin A chain (DTA)-induced cytoxicity that kills up to 60% of cardiac myocyte.168 Apart from demonstrating a similar capacity for cardiac regeneration in neonatal mice,169 this trait of regeneration is absent or possibly turned off in adult mammals. This prompted the zebrafish to become the key model for elucidating the molecular machinery of heart regeneration, which would fuel the development of regenerative medicine for human cardiovascular diseases. Newts and some other lower vertebrates also exhibit a similar level of cardiac regenerative capability170,171 but are not as favorable to work on due to practical reasons relating to the lack of genetic infrastructure and tools as compared to the zebrafish model.

Within hours of ventricular resection, the endocardium undergoes extensive changes in morphology and gene expression, most notably the upregulation of raldh2, which is a key enzyme, involved in the synthesis of retinoic acid (RA) and promotes cardiac myocyte proliferation.172 The stimulation and prolongation of retinoic acid signaling is supported by epicardial cells and it is thought that these combined efforts of the endocardium and epicardium, may serve as the major trigger of cardiac myocyte proliferation.

The activated epicardial cells (3 days post amputation) invade the wound site in the zebrafish heart and give rise to coronary vessels via epithelial – mesenchymal transition (EMT), regulated by snail2 and twist1b.173 This process is reminiscent of coronary arteriogenesis during embryonic heart development, which is regulated by FGF and PDGF signaling.174 – 176 FGFRs ( fgfr2 and fgfr4) in the epicardial cells respond to their ligand, fgf17b, which is transcribed in the myocardium, to promote epicardial EMT and further neovascularisation.173 This process when inhibited will results in a block in regeneration. A hint for an involvement of PDGF signaling stems from an earlier transcriptome analysis of regenerated heart tissues, which identified an upregulation of pdgfa.177 Blocking PDGF using an inhibitor demonstrates the requirement of epicardial-derived pdgfrb for epicardial proliferation and coronary vessel formation.

In experiments where zebrafish epicardial cells are cultured on fibrin gel in vitro, PDGF is found to induce stress fiber formation and loss of cell– cell contacts, mediated by Rho-associated protein kinase, ROCK.178 Cardiac revascularization, inherent to the zebrafish, may account for the difference in regeneration abilities, between fish and mammals. Therefore, elucidation of the process of epicardial EMT and subsequent neovascularisation may help to shed light on new developments for regenerative medicine.

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The origin of the new myocardium that replaced the injured myocardium has been a case of speculation and epicardial cells with their ability to undergo cell type switch is suspected of contributing to new myocardium. However, the studies described above indicate that the epicardium does not contribute to new cardiac myocytes, but instead serves as a source of coronary vasculature, which is essential for the growth of new cardiac myocytes. Most recently, Kikuchi et al. confirmed that tcf21positive epicardial cells are restricted to non-myocardial cell fates by Cre-based fate-mapping experiments.84 The quest for the origin of new myocardium has lead to several genetic cell lineage studies that finally revealed that the source is not cardiac progenitors but pre-existing cardiac myocytes.179,180 In response to injury, these cardiac myocytes undergo dedifferentiation and is accompanied by sarcomeric disassembly and aberrant electrical phenotypes including a reduction in conduction velocity and breaking of the depolarization wavefront, which are signs of functional uncoupling of cardiac myocytes. Dedifferentiated cardiac myocytes can then re-enter the cell cycle and proliferate to supply new functional cardiac myocytes. This intriguing finding suggests the possibility of harnessing the potential of pre-existing cardiac myocytes in failing human hearts.

Cardiac myocyte proliferation is thus an important step, and if blocked, as in the cell cycle mutants mitotic checkpoint kinase (mps1) and polo-like kinase 1 (plk1)181,182 or by overexpression of the microRNA miR-133,183 cardiac regeneration is retarded. miR-133 is one of the microRNAs, which is downregulated during the course of cardiac regeneration. Similarly, if injured zebrafish heart is blocked from entering into the cell cycle by activating the well-known mammalian cell cycle inhibitors p38a MAPK, the ability of cardiac regeneration is compromised.182 It is exciting to note that the zebrafish heart employs similar molecular mechanisms for the regulation of cardiac myocyte proliferation as in mammals, substantiating the relevance of the zebrafish model to develop regenerative therapies for the human heart.

Recently, the question of how proliferating cardiac myocytes actually reach the wounded myocardium was addressed. Expression screening using the regenerating zebrafish heart identified the genes cxcl12a, encoding a chemokine ligand (also known as sdfa1), and its receptor cxcrb4 to be expressed during heart regeneration.184 The CXCL12-CXCR4 system is known to regulate directed cell migration during embryonic development.185 Interestingly, cxcrb4 was expressed in proliferating cardiac myocytes, while the ligand was found in activated epicardium. Loss-of-function experiments clearly demonstrated that blocking Cxcr4 function only affects the migration of proliferating cardiac myocytes to the wounded area, while other aspects like the level of cell proliferation or gene expression were not affected. Thus an epicardium-mediated guidance of proliferating cardiac myocytes via CXCL12-CXCR4 signaling to the wounded area is essential for zebrafish heart regeneration.

As mentioned earlier, cryoinjury was also applied as technique to cause damage to zebrafish cardiac cells.166,186 This method appears to be superior to ventricular resection, since it better resembles myocardial infarction in patients, which is characterized by irreversible replacement of damaged myocardium by a fibrotic collagenous scar that lacks the functional contractile properties of healthy myocardial cells. Despite scar formation, the teleost is able to rapidly regenerate its injured heart by elimination of the scar through apoptosis and triggering proliferation of cardiac myocytes. In fact, it has been recognized that transient scar formation is advantageous for cardiac regeneration serving as a transitional support required by the dynamic beating heart, before being removed and the space is populated by cardiac myocytes.187 Chablais and Jaz´win´ska further elucidated that Smad3-dependent TGFb/activin signaling mediates multiple important steps: inflammatory response of infiltrating immune cells during cardiac cells apoptosis; ECM production by fibrotic cells to form scar and subsequent cardiac myocyte proliferation. The coordination of this highly complex process remains to be clarified.

In a short span of ten years since the discovery of the regenerative potential of the zebrafish heart, our understanding of this process has greatly advanced and further research promises more answers and mechanistic insights into the amazing property of the zebrafish heart, which potentially will also lead to novel therapeutic options to treat heart failure patients.

8. FUTURE PROSPECTS The zebrafish has seen a surprisingly rapid and extremely successful career as a model organism in cardiovascular research. As an affordable in vivo model, the zebrafish will continue to be used to assess candidate genes (from microarray or genome wide association studies) that may have a cardiac

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phenotype. There are only a few areas in cardiovascular research in which the potential of the zebrafish model has not yet been tested. The challenge for the zebrafish community will be to model human cardiovascular pathology in this organism even closer than it is now. Recent technical improvements allow the generation of mutants for any gene. Thus it is likely that in the not too far future, mutants for nearly every gene will be available. Genetic interactions between genes, gene-environment interactions, or the role of modifier genes, which are important determinants of the phenotype severity of a particular mutation, can be analyzed at ease and with significant lower costs than in mice. There is exciting progress in the increasingly sophisticated means of genetic manipulation in zebrafish that parallels that of mice. Certainly the zebrafish will not be able to fully substitute the mouse, however it will reduce the number of experiments that need to be performed in mammals. Moreover, recent improvements in mass screening approaches such as chemical genetics will be a prime area for the zebrafish model. In conclusion, with well-constructed thoughtful experimental designs, a bright future is assured for the small but mighty zebrafish model.

Acknowledgements The drawing of the adult zebrafish heart and the histological section are the courtesy of Dr. Jan Schlu¨ter, Harefield Heart Science Centre, which is herewith gratefully acknowledged. Work in the authors’ lab is funded through the MRC (MR/J010383/1) and the Magdi Yacoub Institute.

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OPEN ACCESS

Review article

Bioengineering and the cardiovascular system Robert M Nerem* Institute Professor Emeritus, Parker H. Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology, Atlanta, GA 30332-0363, USA *Email: robert.nerem@ibb.gatech.edu

ABSTRACT The development of the modern era of bioengineering and the advances in our understanding of the cardiovascular system have been intertwined over the past one-half century. This is true of bioengineering as an area for research in universities. Bioengineering is ultimately the beginning of a new engineering discipline, as well as a new discipline in the medical device industry.

http://dx.doi.org/ 10.5339/gcsp.2013.5 Submitted: 3 August 2012 Accepted: 12 March 2013 q 2013 Nerem, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Nerem RM. Bioengineering and the cardiovascular system, Global Cardiology Science & Practice 2013:5 http://dx.doi.org/10.5339/gcsp.2013.5

Page 2 of 8 Nerem. Global Cardiology Science and Practice 2013:5

HISTORICAL PERSPECTIVE As discussed by both Citron and Nerem1 and also Bergman and Nerem,2 the medical device industry evolved in the second half of the 20th century. First, there was kidney dialysis, a technology accredited to the pioneering work of a Dutch physician, Willem Kolff. Beyond the treatment of chronic kidney failure, Kolff’s technologies formed the foundation to the membrane oxygenator. In turn, this led to cardiopulmonary bypass being performed safely for extended periods of time. There followed the development of prosthetic heart valves with the first implant being performed in 1952. This was followed by the first successful open heart surgery in 1953.

Clinically-practical electrical stimulation therapies for cardiac rhythm disorders began to emerge in the 1950s. In 1958, Dr. C. Walton Lillehei collaborated with Earl Bakken, an electrical engineer, in the use of the world’s first transistorized battery-powered cardiac pacemaker that was externally powered.

However, a few years later, in 1960, William Chardack implanted the first pacemaker that was completely internal, i.e. within the body. This was possible because of the mercury/zinc battery that had been designed by Wilson Greatbach, an engineer who passed away in 2011. This was the start of the medical device/implant industry. This industry grew in the last 50 years to become a major industry with $200 billion in annual sales, with a workforce of 300,000, and an industry that invests seven percent of revenues back into R&D. Bioengineering in universities has developed in parallel to the development of the medical device/implant industry. Back in the 1950s and 1960s, bioengineering involved the application of the traditional engineering disciplines to problems in medicine and biology. However, this all began to change in the 1970s with the establishment of academic departments, called either biomedical engineering or bioengineering. This development grew slowly until the 1990s when there was an acceleration in the formation of such academic units. This was due to many factors; chief among these was the impact of the biological revolution and the significant investments made by the Whitaker Foundation. Worldwide, there are now more than 100 departments of this type, and the field of biomedical engineering/bioengineering is now recognized as being its own academic discipline, an engineering discipline based on the science of biology.

Cardiovascular research and orthopaedic research have been two major areas of focus in the evolving discipline of bioengineering. Three areas of cardiovascular research in bioengineering will be discussed in this article. Each section focuses on a certain area of cardiovascular research in which this author has been personally involved.

HEMODYNAMICS AND ATHEROSCLEROSIS An important research area in the historical development of bioengineering is the role of hemodynamics in the disease atherosclerosis, particularly in the early atherogenic stage of the disease. In the 1960s, there was already evidence that the pattern of atherogenesis appeared to correlate with the pattern of blood flow. As studies developed, it became clear that there was greater predilection of the disease in low-shear stress regions. The vascular endothelium, which was in contact with the flowing blood and the associated shear stress environment, became a focus on the effects of the hemodynamic environment on vascular endothelial biology.3 These studies included the effects of flow, the effects of cyclic stress and in some cases even pressure; however, the major focus was on the study of the influence of flow and the associated shear stress environment. Much of this research over the years has been done in vitro with cultured vascular endothelial cells exposed to a variety of flow environments either using a parallel-plate flow chamber4 or a cone-plate device.5 The flow environments studied have included steady laminar flow, a purely oscillating flow, and pulsatile type flows, either with a reversing or a non-reversing waveform.6,7 Although a key indicator of the influence of flow is the change in morphology, as illustrated in Figure 1, and the reorganization of the cytoskeletal network,8 there are also changes in gene expression and protein expression. For a flow environment where there is a non-zero mean flow component, vascular endothelial cells elongate and align their major axis parallel to the direction of flow. There is reorganization of F-actin, such that the fibers also are aligned with the flow; and if the actin cytoskeleton is disrupted, then the vascular endothelial cells do not elongate and align. This thus indicates that the change in morphology is due to a reorganization of the F-actin. Interestingly, there is no morphological change (no elongation nor alignment of the vascular endothelial cells) for a purely oscillatory flow.9 With respect to gene expression, some genes are upregulated and some downregulated. Obviously, much has been learned about vascular

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Figure 1. In vitro images of morphology and F-actin for bovine aortic endothelial cells for static conditions (A and B) and after 24 hours of laminar shear stress (C and D) with flow left to right; scale of F-actin images a factor of ten different from the morphology images.

endothelial biology from these in vitro studies and later confirmed with in vivo studies using a variety of animal models.

Interestingly, some life scientists studying atherosclerosis in the 1970s did not believe that the physical forces associated with the hemodynamic environment could have any influence at all on cell behavior and the cellular processes involved in the initiation of the disease. Today, the concept of an important role for hemodynamics is widely accepted. In fact, it now is clear that the function of a cell is determined by the signals associated with the microenvironment in which the cell resides. This “symphony� of signals, what I call Nature’s Orchestra, is made up of the soluble molecules to which the cell is exposed, the other cells which are in contact, the substrate to which it is adhered (extracellular matrix and/or some type of synthetic material), and the mechanical environment in which the cell resides, i.e. the physical forces to which it is exposed.

One may speculate that the reason much was learned about vascular endothelial function from in vitro studies was because these monolayer studies mimicked the fact that the vascular endothelium is a monolayer in vivo. As the environment in cell culture is not physiologic, there have been efforts to engineer a more physiologic in vitro environment.10 This has included using a co-culture of vascular endothelial cells with vascular smooth muscle cells.11 It also included using a three dimensional architecture. It is now clear that smooth muscle cells in a three-dimensional environment have different characteristics than such cells in a two-dimensional environment.12 Even with the advances made, much due to the involvement of engineers in the study of vascular biology, there is more to be done. This includes continuing to develop in vitro models that are more physiologic and better at simulating in vivo environments. It should also be noted that, although there were many engineers who learned biology to be leaders in this field, there were also life scientists who became more like engineers in their approach in the study of vascular biology. Thus, the bioengineering community that emerged from studies in this area has been very much an interdisciplinary one, and this provided the foundation for the emergence of bioengineering as a discipline in its own right, a discipline based on the science of biology, a discipline in which biology and engineering are very much integrated.

HEART VALVE ENGINEERING Heart valves engineering is another area, in which engineers have been involved and which has fostered the growth of bioengineering. Initially, much of the engineering effort was focused on the development of improved prosthetic valves to be used as defective valve replacements. This certainly was important to the emerging medical device/implant industry in the 1970s and 1980s, and it was an

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area in which university researchers became very active. A key issue was to optimize the fluid mechanical characteristics of such heart valves implants.13 Engineers have begun to make a contribution to the area of heart valves engineering in another way, in order to learn more about the basic biology of heart valves, a tissue that resides in an extremely dynamic mechanical environment.

Our knowledge about the biology of blood vessels has increased considerably over the last few decades; this was largely driven by studies aimed at achieving a better understanding of the biology and pathobiology associated with atherosclerosis. However, the same situation is not paralleled in the area of heart valves. Although there were clinical problems associated with heart valves, there seemingly was not satisfactory motivation to study the basic biology of heart valves. However, this has changed now. Several bioengineering laboratories are participating in this, and the area of heart valves has became an important part of bioengineering research.

One example is the heart valve team from Georgia Tech and Emory University School of Medicine in Atlanta, Georgia. This team includes the laboratories of Professors Hanjoong Jo and Ajit Yoganathan, and Robert M. Nerem. An issue being investigated is whether there is any difference between endothelial cells on the different sides of the aortic valve leaflets, i.e. the ventricularis side as compared to the fibrosa side. Whereas the hemodynamic environment on the ventricularis side may be characterized as a unidirectional, time varying laminar flow, on the fibrosa side it is a reversing pulsatile flow (Figure 2). The question thus is whether there are differences between these two sides? If so, are these differences due to the very different hemodynamic environments on each side, or alternatively due to fundamental differences between the endothelial cells? Is the difference between the endothelial cells on the two sides genetic or is it environmental? We found over 700 genes downregulated and over 300 genes upregulated by oscillatory flow as compared to steady laminar flow. However, no significant difference has been found when fibrosa side and ventricularis side endothelial cells are exposed to the same shear stress conditions. There is no side-dependency and no apparent difference other than differences in their respective hemodynamic environment.14 Also, miRNA array analysis yielded 30 shear-sensitive miRNAs and three side-specific miRNAs. Moreover, miRNA validation confirmed four of 17 shear-sensitive and one of three side-dependent miRNAs. Although there is clearly much more to do, we are slowly beginning to better understand the biology of heart valves.

Figure 2. Illustration of difference in the hemodynamic environment for the fibrosa side versus the ventricularis side of the aortic heart valve and the influence on calcification and sclerosis.

The above is only an example; however, the biology of heart valves including their biomechanical properties has become a major topic with several sessions at virtually every bioengineering conference.

It thus has become very much a part of the evolution of bioengineering as a field and the intertwining of this field with cardiovascular research.

TISSUE ENGINEERING AND REGENERATIVE MEDICINE Tissue engineering is another research area where bioengineering has had a significant involvement, and thus has been intertwined with the growth of this new engineering discipline. It was only in 1987 that the term “tissue engineering” was created; and it was in 1988 that a conference called “tissue engineering” was first held at Lake Tahoe, California. The focus was on fabricating replacement tissues

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and organs outside of the body using cells and scaffolds for later implantation into the body.15 This field of engineering was driven largely by clinicians and engineers. However, a much broader interdisciplinary effort evolved in the 1990s as stem cells received more interest. Furthermore, the field of tissue engineering broadened into what now is called regenerative medicine and includes, in addition to replacement, also repair and regeneration.16 Clinical targets being pursued include targets in the cardiovascular system. Chief among these are the development of a small-diameter blood vessel substitute for the use in coronary bypass surgery, repair of a damaged myocardial wall following a heart attack and the development of a valvular substitute for use in defective heart valve replacement.

The tissue engineering of a heart valve substitute is in fact what has stimulated much of the interest in the biology of heart valves.17 Furthermore, the pediatric population is one of the main targeted patient populations. A young child that has a heart valve with a congenital defect will need a larger sized replacement every few years. If one had a replacement valve made of living cells that would grow over time as the child grows, then only a single surgery would be needed. To date there has been some success with the two major efforts of the laboratories of John Mayer in Boston Sacks et al.18 and Simon Hoerstrup in Zurich, Switzerland,19 with both laboratories having engineers as part of their teams. The successful tissue engineering of a heart valve requires a combination of the right cells, a scaffold to provide the initial architecture, and the signals necessary to drive the process. Although none of the current efforts has progressed to human studies yet, large animal experiments have been conducted.

Furthermore, from this one can see that there is a real role for engineering.

The tissue engineering of a small-diameter blood vessel substitute in many ways may be viewed as one of the field’s “holy grails”.20 This is because there are many patients who need the coronary bypass procedure but do not have native vessels available for use. Here again success depends on the right combination of cells, a scaffold either biologic or a synthetic material, and the necessary signals. In this area some progress has been made, and at least three efforts have been able to move into clinical trials. First is the work of Shinoka and colleagues, who reported in 2001 the first clinical use of a tissue-engineered blood vessel (TEVB), based on an autologous cell-seeded biodegradable scaffold, to repair cardiac defects in the low-pressure circulation of children.36 Although the cells initially were taken from excised tissue and cultured in vitro, later studies were performed with cells isolated from the bone marrow and then directly seeded into the scaffold in the operating room.21 This approach was used in more than 40 patients with considerable success.22 Dr. Shinoka moved to Yale University, where he received FDA clearance to start a clinical trial.a Another important effort has been that of Dr. Laura Niklason and her co-workers. This was based on the 1999 report of the use of a tubular synthetic, biodegradable scaffold composed of polyglycolic acid and seeded with smooth muscle cells as a vascular graft.23 In a recently published study by by Niklason’s startup company, Humacyte,24 the TEBVs showed good patency in both coronary and carotid bypass models. Perhaps the most important achievement in that study was to produce a small diameter human TEBV with a burst pressure in excess of 3000 mmHg. These vessels, which were produced in 10 weeks in a pulsating bioreactor and decellularized, showed better compliance than that of a human saphenous vein, but still considerably less than that of human arteries.

The third promising approach has as its foundation in the research conducted in 1990s by the laboratory of Dr. Francois Auger in Quebec, Canada. The innovation here was to create sheets from the matrix secreted by cells, with these “self-assembled” sheets then rolled into the many distinct layers that compose a natural blood vessel. This method was used to construct the first tissue-engineered human blood vessel that was truly biological and that displayed physiological mechanical properties.25 In 2000, Cytograft Tissue Engineering, Inc. was founded with the purpose of developing this new technology and bringing it to the clinic. The company aimed to simplify the complex in vitro model that was developed in Auger’s laboratory, while retaining its many biological advantages. This was done by eliminating the medial layer of SMCs. The argument for eliminating this layer was based on contractility having little effect on the patency of a TEBV substitute. However, by eliminating the medial layer, the mechanical properties of this TEBV substitute were significantly altered. Preliminary results from initial human trials of this type of graft were reported a few years ago.26 This trial involved the use of the graft as an arterio-venous (AV) shunt for hemodialysis access. Biomechanical testing indicated an average burst pressure in excess of 3000 mmHg. Furthermore, production of the graft proved to be

a

http://news.yale.edu/2012/04/13/rebuilding-heart-saving-life

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reproducible. More recently an expanded study was published.27,28 Cytograft is focusing its clinical trials in Europe and Asia, and the Phase III clinical studies for hemodialysis access employs both autologous and allogeneic grafts. Of interest is that the new allogeneic model has the potential of reducing the overall production time to 3 weeks. Furthermore, it allows 1000 of grafts to be fabricated from a single master cell line.

The more recent approaches of Niklason and her co-workers, L’Heureux and the Cytograft team involve creating a biological scaffold made up of extracellular matrix components. Still, a major issue is cells’ source. If one is to use an autologous cell approach, then the concept of a TEBV off-the-shelf availability is not possible to implement. On the other hand, the use of an allogeneic cell approach could lead to off-the-shelf availability. Such approach might use allogeneic smooth muscle cells and/or fibroblasts. However, one must again emphasize that, if allogeneic endothelial cells are to be used, then one must incorporate some type of immune-suppressive strategy. Alternatively one could possibly recruit endothelial cells from the patient into a non-endothelialized TEBV which has been implanted. For all the progress in the last decade, it still appears that we are a few years away from having a TEBV substitute achieving FDA approval.

Another important area is that of the heart itself 29 and the use of a cell-based therapy for myocardial repair. Although there have been a variety of studies, they have not been particularily encouraging. This is because there has been only modest improvements in left ventricular function no matter what cell type is used. Also, cell engraftment has been poor, and it is very unlikely that significant repair/regeneration actually occurred. The fact that the use of different cell types leads to very similar results suggests that it is a paracrine effect, not one of cell replacement. A review of this area was published in 2011.30 One important question is “what is the best method for delivering the cells to effect cardiac repair”? This is an area where engineers can contribute with one example being the work of Simpson et al.31 A critical issue in tissue engineering and regenerative medicine is that of cell source. Because of this, the field of stem cell technology has taken on an important role in the field of tissue engineering and regenerative medicine.32 Engineers are also contributing to the advances being made in this field.33 Here again there have been studies of the role of physical forces, in this case in the modulation of stem cell behavior. One example is the use of flow and the associated shear stress to influence the differentiation of mouse embryonic stem cells to endothelial cells.34 There is an emerging tissue engineering and regenerative medicine industry.35 In the translation of the benchtop research to patient therapies, there is a real role for engineers. There also is a recognition that there is a need for the further development of bioprocessing systems for stem cell biomanufacturing. These systems will need to provide for the scaleup in cell numbers needed for a patient therapy and also the systematic assessment of the cell population required to provide for quality control and thus regulatory approval.

CONCLUDING DISCUSSION Over the past half century, bioengineering has emerged as an engineering discipline in its own right. At the same time, there have been major advances in our understanding of cardiovascular disease and in the development of therapeutic approaches. As described here, there has been an intertwining of these advances; and although one might argue that cardiovascular research would have advanced without the involvement of bioengineers, at the same time engineers have made major contributions.

The participation and contribution of bioengineers to cardiovascular research has been illustrated here using three specific areas: hemodynamics and atherosclerosis, heart valve engineering, and tissue engineering. These are areas in which the biomechanical aspects of the problem proved to be important. This was true in terms of the role of flow and the associated shear stress in the development of atherosclerosis, the fluid mechanic characteristics of prosthetic heart valves and more recently sidespecific differences in the function of valvular endothelial cells, and in the development of innovative therapies using tissue engineering and regenerative medicine approaches.

There have been other contributions by bioengineers that have led to advancements in our understanding of the cardiovascular system and disease processes. This includes imaging, which engineers have contributed to the advancement of technologies that range from ultrasound to computerized tomography to magnetic resonance imaging to position emission tomography. This resulted in lower usage of surgical biopsy as a diagnostic. Engineers also have contributed to our understanding of the electrophysiological characteristics of the heart and to cardiac rhythm therapies.

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After all, it was only over half century ago that critical contributions of engineers led to the implantable pacemaker and to the establishment of the medical device industry.

The story told here, however, has been based on biomechanics and its role in various aspects of the cardiovascular system. Furthermore, it may be argued that it was individuals with an engineering background that recognized the importance of biomechanics in biology and in physiology. As noted earlier, the mechanical environment to which cells are exposed is part of the symphony of signals that orchestrates function, both normal and pathological. The result is that biomechanics has not only contributed to our understanding of the cardiovascular system, but it also has contributed to the establishment of the discipline of bioengineering. Looking into the future, there will be the continuing involvement of bioengineers including biomechanicians in cardiovascular research, and with this there will be continuing advances in our understanding of the cardiovascular system, including the basic biology and pathobiological aspects of disease as well as the development of new therapeutic approaches.

REFERENCES [1] Citron P, Nerem RM. Bioengineering: 25 years of progress, but still only a beginning. Technol Soc. 2004;26 (2– 3):415– 431.

[2] Bergman RM, Nerem RM. The cardiovascular technology industry: past, present, and future. Cardiovasc Eng Technol.

2010;1(1):19– 24.

[3] Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6:16.

[4] Levesque MJ, Nerem RM. The elongation and orientation of cultured endothelial cells in response to shear stress.

ASME J Biomech Eng. 1985;107(4):341–347.

[5] Dewey CF Jr, Bussolari SR, Gimbrone MA Jr, Davies PF. The dynamic response of vascular endothelial cells to fluid shear stress. ASME J Biomech Eng. 1981;103:177 –185.

[6] Conway DE, Williams MR, Eskin SG, McIntire LV. Endothelial cell responses to atheroprone flow are driven by two separate flow components: low time-average shear stress and fluid flow reversal. Am J Physiol Heart Circ Physiol.

2010;298:H367.

[7] Chiu JJ, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives.

Physiol Rev. 2011;91:327.

[8] Girard PR, Nerem RM. Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins. J Cell Physiol. 1995;163:179 –193.

[9] DeKeulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res. 1998;82:1094– 1101.

[10] Johnson TL, Barabino GA, Nerem RM. Engineering more physiologic in vitro models for the study of vascular biology.

Prog Pediatr Cardiol. 2006;21:201–210.

[11] Ziegler T, Alexander RW, Nerem RM. An endothelial cell-smooth muscle cell co-culture model for use in the investigation of flow effects on vascular biology. Ann Biomed Eng. 1995;23:216 –225.

[12] Stegemann JP, Nerem RM. Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three- dimensional culture. Exp Cell Res. 2003;283:146–155.

[13] Dasi LP, Simon HA, Sucosky P, Yoganatha AP. Fluid mechanics of artificial heart valves. Clin Exp Pharmacol Physiol.

2009;36(2):225– 237.

[14] Holliday CJ, Ankeny RF, Jo H, Nerem RM. Discovery of shear-and side-specific mRNAs and miRNAs in human aortic valvular cells. AJP Heart Circ Physiol. 2011;301(3):H856–H867.

[15] Nerem RM, Sambanis A. Tissue engineering: from biology to biological substitute. Tissue Eng. 1995;1:3–13.

[16] Badylak SF, Nerem RM. Progress in tissue engineering and regenerative medicine. Proc Natl Acad Sci USA. 2010;107(8):3285– 3286.

[17] Butcher J, Nerem RM. Valvular endotelial cells and the mechanoregulation of valvular pathology. Phil Trans R Soc Lond Part B. 2007;362(1484):1445–1457.

[18] Sacks MS, Schoen FJ, Mayer JE. Bioengineering challenges for heart valve tissue engineering. Ann Rev Biomed Eng. 2009;11:289– 313.

[19] Schmidt D, Dikman PE, Driessen-Mol A, Stenger R, Mariani C, Puolakka A, Rissanen M, Deichmann T, Odermatt B, Weber B, Emmert MY, Zund G, Baaijens F, Hoerstrup S. Minimally-invasive implantation of living tissue engineered heart valves: a comprehensive approach from autologous vascular cells to stem cells. J Am Cell Cardiol.

2010;56(6):510 – 520.

[20] Nerem RM, Ensley AE. The tissue engineering of blood vessels and the heart. Am J Transplant. 2004;4(Supplement 6):36–42.

[21] Shin’oka T, Matsumura G, Hibino N, Naito Y, Watanabe M, Konuma T, Sakamoto T, Nagatsu M, Kurosawa H. Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells. J Thorac Cardiovasc Surg. 2005;129:1330.

[22] Hibino N, McGillicuddy E, Matsumura G, Ichihara Y, Naito Y, Breuer C, Shinoka T. Late-term results of tissue-engineered vascular grafts in humans. J Thorac Cardiovasc Surg. 2010;139(2):431–436.

[23] Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, Langer R. Functional arteries grown in vitro. Science.

1999;284(5413):489–493.

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[24] Dahl SL, Kypson AP, Lawson JH, Blum JL, Strader JT, Li Y, Manson RJ, Tente WE, Dibernardo L, Hensley MT, Carter R, Williams TP, Prichard HL, Dey MS, Begeleman KG, Niklason LE. Readily available tissue-engineered vascular grafts. Sci Transl Med. 2011;3(68):1–11.

[25] L’Heureux N, Paquet S, Labbe R, Germain L, Auger F. A completely biological tissue-engineered human blood vessel.

FASEB J. 1998;12(1):47–56.

[26] L’Heureux N, Dusserre N, Konig G, Victor B, Keire P, Wight TN, Chronos NA, Kyles AE, Gregory CR, Hoyt G, Robbins RC, McAllister TN. Human tissue-engineered blood vessels for adult arterial revascularization. Nat Med. 2006;12(3):361 – 365.

[27] Garrido S, McAllister T, Dusserre N, Marini A, L’Heureux N. Haemodialysis access via tissue-engineered vascular graft.

Lancet. 2009;374(9685):20.

[28] McAllister TN, Maruszewski M, Garrido SA, Wystrychowski W, Dusserre N, Marini A, Zagalski K, Fiorillo A, Avila H, Manglano X, Antonelli J, Kocher A, Zembala M, Cierpka L, de la Fuente LM, L’Heureux N. Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study. Lancet.

2009;373(9673):1440–1446.

[29] Yacoub MY, Nerem RM. Introduction: bioengineering of the heart. Philos Trans R Soc Lond Part B. 2007;362(1484):1253–1255.

[30] Malliaras K, Kreke M, Marban E. The stuttering progress of cell therapy for heart disease. Clin Pharmacol Ther.

2011;90(4):532 –541.

[31] Simpson D, Liu H, Hwang T, Fan M, Nerem RM, Dudley SC. A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling. Stem Cells. 2007;25:2350–2357.

[32] Vats A, Bielby RC, Tolley NS, Nerem R, Polak JM. Stem cells. Lancet. 2005;366:592–602.

[33] McCloskey KE, Lyons I, Rao RR, Stice SL, Nerem RM. Purified and proliferating endothelial cells derived and expanded in vitro from embryonic stem cells. Endothelium. 2003;10:329–336.

[34] Ahsan T, Nerem RM. Fluid shear stress promotes an endothelial phenotype during early differentiation of embryonic stem cells. Tissue Eng Part A. 2010;16(11):3547–3553.

[35] Nerem RM. Regenerative medicine: the emergence of an industry. J R Soc Interface. 2010;7:S771–S775.

[36] Shin’oka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonary artery. N Engl J Med. 2001;344(7):532– 533.

OPEN ACCESS

Review article

Self-Assembled Metal-Organic Polyhedra (MOPs): Opportunities in Biomedical Applications Mohamed H Alkordi* Zewail University of Science and Technology, 6th of October, Egypt *Email: malkordi@zewailcity.edu.eg

ABSTRACT Self-assembly is a powerful synthetic tool that has enabled chemists to construct numerous, structurally complex, supermolecules of various shapes, functionality, and dimensions from relatively simple precursors. Metal-organic polyhedra (MOPs) are an emerging family of self-assembled supermolecules that have intriguing structures and tailored functionality. During the last decade, research in this area have rapidly evolved and interest is now directed towards fine tuning and tailoring such structures targeting applications in sensing, catalysis, and most recently, in the biomedical field.

Three examples of MOPs of interest showing promising potentials for biomedical applications are described.

http://dx.doi.org/ 10.5339/gcsp.2013.6 Submitted: 11 November 2012 Accepted: 1 March 2013 q 2013 Alkordi, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Alkordi M. Self-assembled metal-organic polyhedra (MOPs): opportunities in biomedical applications, Global Cardiology Science & Practice 2013:6 http://dx.doi.org/10.5339/gcsp.2013.6

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INTRODUCTION Self-assembly is defined by George Whitesides as “a process where pre-designed components assemble in a determined structure without the intervention of human operators”.1 This broad but carefully stated definition calls for three essential characteristics for a process to be termed self-assembly. This includes utilization of carefully designed building blocks where information encoded within the building blocks will dictate a specific way of their interaction, generating determined structures of higher complexity and by default functionality, as compared to the simpler building blocks, and finally the process will commensurate without intervention of a human operator. Working with chemical self-assembled systems entails close familiarity with the concepts and tools of supramolecular chemistry. The term supramolecular chemistry was coined by Jean-Marie Lehn who shared the 1987 Nobel Prize in chemistry with Donald J. Cram and Charles J. Pederson for “their development and use of molecules with structure-specific interactions of high specificity”.2 In supramolecular chemistry, chemistry beyond the molecule as defined by Lehn,3 strong emphasis is given to intermolecular forces dominated by reversible, non-covalent interactions that range from Van der Waals, dipole-induced dipole, dipole-dipole, hydrogen and halogen bonding to even, more recently utilized, coordination bonds. Intermolecular interactions of the types mentioned here involve molecular recognition processes that can be designed, and further tailored, to dictate specific modes of binding to construct particular ordered structures.

WHY SELF-ASSEMBLY? Self-assembly is an efficient synthetic pathway to construct relatively complex, multi-component systems that would be extremely difficult to attain otherwise. In step-wise syntheses, the reaction intermediates are commonly isolated and further purified prior to further elaboration. This enables synthetic chemists to devise synthetic pathways utilizing what is commonly referred to as a retrosynthetic approach, armed with an arsenal of reagents, protecting groups, established synthesis conditions, and rigorous procedures developed over the last decades.

In contrast, self-assembly is not amenable to retrosynthetic analysis as the reaction intermediates are present transiently and no isolation/purification steps are carried out. Although this imposes considerable challenges to successfully construct chemical species, it simultaneously provides an unmatched powerful tool in construction of complex, multi-component systems in high yield and with minimal intervention from the chemist.

Merging the powerful tools of synthetic chemistry, the knowledge of types and nature of intermolecular interactions, and the underlying principles of crystal engineering and supramolecular chemistry open the doors for constructing tailor-made structures for demanding applications.

Examples of self-assembled systems are numerous of which the self-assembled DNA double helix and the lipid bi-layer membranes in cell walls are outstanding examples. In the DNA double helix, Figure 1, specific and complimentary hydrogen bond interactions between the base pairs dictates very specific mode of binding that results almost invariably into the observed structure.

It is because of the reversible nature of hydrogen bond interactions that pairing and un-pairing of the two DNA molecules are feasible, providing room for correction of mismatches. In the lipid bilayer membrane, reversible intermolecular interactions between the phospholipids molecules derive their self-assembly into the bilayer structure. It is again due to the reversible nature of such intermolecular interactions that the lipid bi-layer membranes express their remarkable physical and physiological properties.

A relatively recently developed family of self-assembled supermolecules is the metal-organic polyhedra (MOPs). An MOP is typically composed of organic molecules coordinating metal ions. The organic molecules commonly referred to as the linkers, contain functional groups like carboxylic acids, phenols, or heterocycles (e.g. pyridine, imidazole, pyrimidine, etc) acting as Lewis-bases towards wide range of metal ions, acting as Lewis-acids.

As synthetic chemistry is capable of delivering an ever-increasing number of designer organic linkers, and as the number of metal ions available for utilization from the periodic table is relatively large, the number of structures accessible from rational reaction permutations is nearly innumerable. However, design strategies that rely on rational and judicial selection of reaction components, conditions, etc can be devised to guide the synthesis of targeted structures.

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Figure 1. Self-assembled double helix DNA supermolecule.

METAL-ORGANIC POLYHEDRA (MOPS) The early examples of self-assembled supermolecules containing organic and inorganic parts connected through coordination bonds came to existence through the works of Fujita et al.4 and Stang et al.5 in 1993 and 1994, respectively. Both authors reported independently the synthesis and characterization of self-assembled macrocyclic complexes composed of four metal ions – Pd(II) or Pt(II) – where two of the available coordination sites on each metal ion are occupied by capping phosphine or amine ligands and the other two sites coordinated to nitrogen-donor organic linkers. The earlier organic linkers were commonly selected as linear ditopic linkers (containing two opposing nitrogen atoms that can coordinate metal ions). It is due to geometric complimentarity between linear linkers and the available coordination sites around each metal ion (oriented at 908 angle) that the 8-component system (4 linkers and 4 metal ions) can self-assemble into a supermolecule with a square-like geometry, Figure 2.

The intriguing symmetry of the resulted square-like supermolecules and the almost quantitative yield of the syntheses, along with the relatively straightforward reaction conditions, stimulated many research groups to explore this area more actively. It was clear from those seminal publications that skepticism towards the ability to generate such beautiful assemblies with relative ease is no longer

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Figure 2. Syntheses of the self-assembled Pt(II) or Pd(II) macrocycles.

well-founded. Such skepticism was rooted in the chemists’ perception of the many ways such molecular or ionic components can come together to yield a large number of possible structures in absence of directing forces. Although such “directing forces” were not fully defined in the early days, it is becoming clearer to scientists in the field that a plethora of complex factors in subtle balance holds the key to realize one of chemists’ most sought dreams, to make molecules to order and at will. Those directing forces can be very broadly classified into two categories: 1) information encoding at the molecular level and 2) realization of proper reaction conditions.

Maintaining structural and functional complimentarity between the building blocks, along with placing interaction sites on each of the reacting species at correct relative disposition, is the first and foremost design element. This step, if done properly, ensures feasibility to construct the targeted metalorganic polyhedra (MOP), given that a proper set of experimental reaction conditions is formulated.

Reaction conditions of interest include; mixing stoichiometry, reaction temperature and time, solvent system, ionic strength and concentration, addition of weak acids or bases, nature of counterions, among others. It becomes evident from the large number of reaction conditions that need to be explored and then optimized that a trial-and-error process is essential to arrive at a proper reaction setup for each system. Although initial screening trials can be time and effort consuming, relying on good chemist’s intuition can cut down the number of trials to manageable size and accumulation of knowledge over the past decade have contributed positively towards this goal.

One fascinating example of such MOPs is the MOP constructed from 24 Cu(II) ions and 24 isophthalate ions, Figure 3.6 This MOP (known as nanoball or cuboctahedron) is constructed through coordination interactions between Cu(II) ions and carboxylate groups on benzene rings. The 1208 angle between two carboxylate groups on each benzene ring of the isophthalate dictate the orientation between two coordination clusters in a way that results in formation of a ball-like structure with c.a.

Figure 3. Synthesis of the self-assembled Cu(II) nanoball from Cu(II) and isopthalate ions. Cu (green), O (red), C (gray), H (white). Yellow sphere represents guest-accessible void inside the nanoball (,1 nm diameter).

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2.6 nm outer diameter. Experimentally, the nanoball can successfully be constructed by heating at 858C for 12 h a mixture of copper nitrate and isopthalic acid in 1:1 ratio in N, N’-dimethylformamide as the major solvent and methanol as a co-solvent.7 Several research groups started to explore the potential for MOPs in various applications soon after chemists were able to generate isostructural variants of the same MOP,8 those sharing same underlying structure but with larger dimensions and/or with peripheral functionalities. Among the applications of special interest are applications in bio-nanotechnology. One of the earliest examples was presented in 2008 when Kim et al.10 reported a synthetic ion channel based on a tailor-made MOP, the nanoball bearing 24 saturated hydrocarbon arms (C12 chains), Figure 4.9 The lipophilic arms

Figure 4. Self-assembled MOP with 24 lipophilic, saturated aliphatic, chains decorating its surface (to the left is crystal structure of the MOP).5 This MOP with diameter of ,5 nm was utilized as synthetic ion channel when situated in-between a lipid bi-layer membrane.8

protruding from the MOP surface facilitated its incorporation in-between a model lipid bi-layer membrane. Due to the voids inside the nanoball that ion conductivity was observed. Furthermore, the authors reported that the recorded behavior in terms of the ion transport activity (Liþ . . Naþ . Kþ . Rbþ . Csþ) following Eisenman sequence XI. The authors suggested10 that one possible explanation is that binding of the cations to the synthetic channel (the MOP) is more crucial than dehydration of the cations in the ion transport process.

Figure 5. Self-assembly of M12L24 complexes with 24 saccharide moieties at the periphery. When combined with lectins, they form aggregates because of the cluster effect of the saccharides on the spheres. Reprinted with permission from (N. Kamiya; M. Tominaga; S. Sato; M. Fujita. J. Am. Chem. Soc. 2007, 129, 3816–3817).

Copyright (2007) American Chemical Society.

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In 2007 Fujita and co-workers11 reported a fascinating example of the molecular recognition process involving MOP supermolecule (a supermolecule being composed of a number of smaller molecular and/or ionic species held together through non-covalent interactions). In this example, the authors synthesized a MOP from Pd(II) ions and pyridine-containing linkers where the MOP surface was uniformly functionalized by 24 saccharide molecules, Figure 5. Molecular recognition between the saccharide-functionalized MOPs and saccharide-binding proteins induced cross-linking of the soluble proteins and formation of aggregates followed. The authors studied the interactions of the family of saccharide functionalized MOPs with concanavalin A (ConA), a well-studied lectin from Canavalia ensiformis. ConA is known to selectively recognize a-mannopyranoside and a-glucopyranoside at its four binding sites. In accordance, the MOP bearing 24 a-mannopyranoside units at its periphery served as a cross-linker of ConA to form aggregates in solution, Figure 6.

0.5

Abs. at 500 nm

0.4 2a + ConA 0.3 0.2 0.1

Inhibtor additon

2b + ConA

0 0

5

10

15

20

25

30

Time (min) Figure 6. Turbidity changes, monitored by the absorbance at 500 nm, on addition of MOPs 2a and 2b to (a) ConA. A large excess of (a) a-methyl mannopyranoside or (b) a -galactose was added as an inhibitor at 20 min.

Reprinted with permission from (N. Kamiya; M. Tominaga; S. Sato; M. Fujita. J. Am. Chem. Soc. 2007, 129, 3816– 3817). Copyright (2007) American Chemical Society.

Another example of surface-functionalized MOP capable of molecular recognition of nucleic bases was later reported in 2010.12 In this report, well-defined, perfectly monodisperse short DNA strands on the surface of a self-assembled coordination MOP was presented, Figure 7. The MOP served as a nanoparticle template to control the number, spacing, and alignment of the peripheral DNA strands.

Figure 7. Self-assembly of DNA-displaying coordination nanospheres. (a) Structures of ligands 1a2c. (b) Selfassembly of DNA-conjugated molecular sphere 2c from 12 Pd(II) ions and 24 1c ligands. Ligands 1a and 1b also give the corresponding M12L24 spheres (consisting of 12 Pd(II) ions and 24 ligands). Reprinted with permission from (T. Kikuchi; S. Sato; M. Fujita. J. Am. Chem. Soc. 2010, 132, 15930–15932). Copyright (2010) American Chemical Society.

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Figure 8. The values of downfield shift of thymine proton signals of 2a after the addition of mono-A (3a), G (4), C(5) (red line: 2a þ 3a; blue line: 2a þ 4; green line: 2a þ 5) ([2] ¼ 0.167 mM, DMSO-d6/CDCl3 ¼ 1:4, 500 MHz, 300 K). Reprinted with permission from (T. Kikuchi; S. Sato; M. Fujita. J. Am. Chem. Soc. 2010, 132, 15930– 15932). Copyright (2010) American Chemical Society.

The investigated system was deoxythymine (T) based MOP that showed specific binding to the complimentary nucleotide deoxyadenosine (A), Figure 8. The binding of MOP (T) nucleotides to the added (A) was monitored in solution through measurement of changes in proton chemical shifts using solution NMR spectroscopy.

CONCLUSION Although practical utilization of self-assembled MOPs is still in its infancy, interest is growing in exploring their potentials as novel materials to address some particular challenges in biomedical applications. The wide versatility of accessible MOPs starting from a wide range of organic linkers and metal ions along with the arsenal of synthetic pathways that chemists can utilize to construct made-toorder supermolecules calls for collaborative efforts from chemists, molecular biologists and clinicians to fully explore and exploit such material in current demanding applications.

REFERENCES [1] Whitesides GM. Self-assembly at all scales. Science 2002;295(5564):2418–2421. Available at: http://dx.doi.org/10.1126/science.1070821 [2] The Nobel Prize in Chemistry 1987. Nobelprize.org. Retrieved 28 Aug 2012 http://www.nobelprize.org/nobel_prizes/ chemistry/laureates/1987/ [3] Lehn J-M, Atwood JL, Davies JED, MacNicol DD, Vogtel F, eds. Comprehensive Supramolecular Chemistry. Oxford: Pergamon; 1996.

[4] Fujita M, Yazaki J, Kuramochi T, Ogura K. Self-assembly of a Macrocyclic dinuclear Pd(II)-phosphine complex. Bull Chem Soc Jpn. 1993;66(6):1837–1839. Available at: http://dx.doi.org/10.1246/bcsj.66.1837 [5] Stang PJ, Cao DH. Transition metal based cationic molecular boxes. Self-assembly of macrocyclic platinum(II) and palladium(II) tetranuclear complexes. J Am Chem Soc. 1994;116(11):4981–4982. Available at: http://dx.doi.org/10.

1021/ja00090a051 [6] Eddaoudi M, Kim J, Wachter JB, Chae HK, O Keeffe M, Yaghi OM. Porous metal2organic polyhedra: 25 A˚ cuboctahedron constructed from 12 Cu2(CO2)4 paddle-wheel building blocks. J Am Chem Soc. 2001;123(18):4368– 4369. Available at: http://dx.doi.org/10.1021/ja0104352 [7] Moulton B, Lu J, Mondal A, Zaworotko MJ. Nanoballs: nanoscale faceted polyhedra with large windows and cavities.

Chem Commun. 2001;(9):863 –864. Available at: http://dx.doi.org/10.1039/b10,2714j [8] Tranchemontagne D, Ni Z, O’Keeffe M, Yaghi O. Reticular chemistry of metal–organic polyhedra. Angewandte Chem Int Ed. 2008;47(28):5136–5147. Available at: http://dx.doi.org/10.1002/anie.200705008 [9] Furukawa H, Kim J, Plass KE, Yaghi OM. Crystal Structure, dissolution, and deposition of a 5 nm functionalized metal2organic great rhombicuboctahedron. J Am Chem Soc. 2006;128(26):8398–8399. Available at: http://dx.doi. org/10.1021/ja062491e [10] Jung M, Kim H, Baek K, Kim K. Synthetic ion channel based on metal–organic polyhedra. Angewandte Chem Int Ed. 2008;47(31):5755–5757. Available at: http://dx.doi.org/10.1002/anie.200802240 [11] Kamiya N, Tominaga M, Sato S, Fujita M. Saccharide-coated M12L24 molecular spheres that form aggregates by multiinteraction with proteins. J Am Chem Soc. 2007;129(13):3816–3817. Available at: http://dx.doi.org/10.1021/ja0693082 [12] Kikuchi T, Sato S, Fujita M. Well-defined DNA Nanoparticles templated by self-assembled M12L24 molecular spheres and binding of complementary oligonucleotides. J Am Chem Soc. 2010;132(45):15930–15932. Available at: http://dx.doi.org/10.1021/ja108334g

OPEN ACCESS

Review article

Temperature management in cardiac surgery Hesham Saad1,*, Mostafa Aladawy2 1

Aswan Heart Centre, Aswan, Egypt Ain Shams university, Cairo, Egypt

2

*Email: dr.heshamsaad@gmail.com

http://dx.doi.org/ 10.5339/gcsp.2013.7 Submitted: 13 January 2012 Accepted: 6 March 2013 q 2013 Saad, Aladawy, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION Maintaining a constant core body temperature of 378C is essential for survival, through maintaining optimal organ, tissue and cellular function. Tight temperature regulation is achieved through a complex, integrated system of thermogenesis and heat loss. Different levels of hypothermia for defined periods of time can be protective. Temperature management during and after cardiac surgery, as well as after accidental hypothermia can have major effects on both immediate and long-term outcome.

Through knowledge of the mechanisms of temperature regulation, we can understand its influence of on different vital organ functions in modern cardiac anaesthesia. We here describe mechanisms of thermoregulation, the protective effect of induced hypothermia, detailed techniques and risks of temperature changes during cardiac surgeries.

THERMOREGULATION Thermoregulation involves an extremely sophisticated system of balancing heat production by several organs, and heat loss. Heat production may be classified into shivering and non-shivering components, with each component playing a more dominant role during different physiological and pathological conditions. The different mechanisms of thermoregulation are summarized in Table 1.

Heat loss occurs primarily from the skin of a patient to the environment through several processes, including; radiation, conduction, convection and evaporation. Of these processes, radiation is the most significant and accounts for approximately 60% of total heat loss. Radiation is emitted in the form of infrared rays. Heat from core body tissues is transported in blood to subcutaneous vessels, where heat is lost to the environment through radiation. Radiation is the major source of heat loss in most surgical patients.

Conduction refers to loss of kinetic energy from molecular motion in skin tissues to surrounding air.

Water absorbs far more conducted heat than air, and this accounts for more rapid hypothermia during accidental drowning, as well as the efficacy of water baths to cool hyperthermic patients. For this to be effective, warmed air or water must be moved away from the skin surface by currents in a process called convection. This accounts for the cooling effect of wind and laminar air flow in many surgical suites. Conduction and convection account for , 15% of body heat loss.

Approximately 22% of heat loss occurs by evaporation, as energy in the form of heat is consumed during the vaporization of water. Water evaporates from the body even when the body is not sweating, but mechanisms that enhance sweating increase evaporation. As long as the skin temperature is greater than its surroundings, radiation and conduction provide heat loss. At very high environmental temperatures, these processes cannot work and evaporation is the only manner in which heat can be dissipated. This generally is not the case in clinical settings.

Skin temperature rises and falls with the temperature of a patient’s surroundings. However, the core temperature remains relatively constant. This is due to a remarkable thermoregulatory system that is conventionally organized into three components: afferent sensing, central control and efferent responses (Figure 2).1

Cite this article as: Saad H, Aladawy M. Temperature management in cardiac surgery, Global Cardiology Science and Practice 2013:7 http://dx.doi.org/10.5339/gcsp.2013.7

Muscles contract causing vasoconstriction. Less heat is carried from the core to the surface of the body, maintaining core temperature. Extremities can turn blue and feel cold, and can even be damaged (frostbite).

No sweat produced.

Smooth muscles in arterioles in the skin.

Glands secrete sweat onto surface of skin, where it evaporates.

Since water has a high latent heat of evaporation, it takes heat from the body. High humidity, and tight clothing made of manmade fibres reduce the ability of the sweat to evaporate and causes discomfort in hot weather. Transpiration from trees has a dramatic cooling effect on surrounding air temperature.

Muscles relax, lowering the skin hairs and allowing air to circulate over the skin, encouraging convection and evaporation.

Muscles relax causing vasodilation. More heat is carried from the core to the surface, where it is lost by convection and radiation (conduction is generally low, except when in water). Skin turns red.

Response to high temperature

Muscles contract, raising skin hairs and trapping an insulating layer of warm air next to the skin. Not very effective in humans, other than causing “goosebumps”.

Skeletal muscles Shivering: Muscles contract and relax repeatedly, generating heat by No shivering. friction and from metabolic reactions respiration is only 40% efficient: 60% of increased respiration thus generates heat).

Adrenal and thyroid glands Glands secrete adrenaline and thyroxine, which generate heat and Glands stop secreting adrenaline and thyroxine. increase the metabolic rate in different tissues, especially the liver.

Behaviour Curling up, huddling, finding Stretching out, finding shade, Stretching out, finding shade, swimming, removing clothes. shelter, putting on more clothes.

Nonshivering thermogenesis (NST) from The cold environment centrally activates the sympathetic muscle and brown adipose tissue (BAT) nervous system (SNS), which releases norepinephrine (NE) to activate the Ca2 þ pump SERCA, which is under control of sarcolipin (SLN) and phospholamban (PLN) in muscle.

Sarcolipin uncouples SERCA-mediated ATP hydrolysis from ‘work’ (that is, Ca2 þ pumping), resulting in the liberation of energy in the form of heat. Simultaneously, brown adipocyte formation is stimulated in white adipose tissue (WAT) by the SNS and by irisin and naturietic peptide, which are secreted by skeletal and cardiac muscle, respectively.

This results in an increase of the uncoupling protein 1 (UCP1) in the mitochondria, which induces heat production. A program of enriched physical activity is also proposed to increase brown adipocyte formation in WAT via the SNS. Accordingly, the concerted action of a cold environment and physical activity generates heat from muscle and white fat to reduce systemic obesity. b-AR, b-androgenic receptor; RyR, ryanodine receptor10 (Figure 1).

Erector pili muscles in skin (attached to skin hairs)

Sweat glands

Response to low temperature

Effectors

Table 1. Response of different organs to changes in temperature.

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Figure 1. Nonshivering thermogenesis (NST) from muscle and brown adipose tissue (BAT).

Afferent sensing and central control: Some integration and temperature regulation may occur at the spinal cord level. However, the hypothalamus is the primary control center for thermoregulatory as it integrates most afferent input and coordinates the various efferent outputs required to maintain a normothermic level.

Efferent response: As temperature receptors transmit information to the hypothalamus, this information is integrated and compared with threshold settings. Values above or below these thresholds determine the generated efferent response. Efferent outputs from the hypothalamus regulate body temperature by altering subcutaneous blood flows, sweating, skeletal muscle tone and overall metabolic activity. Heat loss is promoted by vasodilatation and sweating, while heat is conserved by inhibiting these processes.

Production of heat (thermogenesis) is promoted by shivering and increases the overall metabolic rate (Figure 3).

Temperature inputs to the hypothalamus are integrated and compared with threshold temperatures that trigger appropriate thermoregulatory responses. Normally these responses are initiated at as little as 0.18C above and below normal body temperature of 37 8C. Therefore, the difference between temperatures that initiate sweating versus those initiating vasoconstriction is only 0.28C. This is defined as the ‘interthreshold range’ and represents the narrow range at which the body does not initiate thermoregulatory efforts.

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Core temperature in blood decreased

increased Hypothalamus Heat loss centre

Heat conversion centre

vasoconstriction

shivering increased metabolic rate

sweating

vasodilation hair raised

decreased metabolic rate

hair lowered

Figure 2. Cerebral temperature control.

General anesthesia and thermogenesis: Causes for inadvertent hypothermia include patients’ exposure to a cold environment and the inability to initiate behavior responses. Volatile anesthetics, propofol, and older opioids such as morphine and meperidine promote heat loss through vasodilation. This process is compounded further by the fact that these drugs, as well as fentanyl and its derivatives, directly impair hypothalamic thermoregulation in a dose-dependent manner. Opioids also depress overall sympathetic outflow, which further inhibits any attempts at thermoregulation. The depressant effect on the hypothalamus results in an elevated threshold for heat response, along with a diminished threshold for cold response such as vasoconstriction and shivering. Therefore, opioids widen the normal interthreshold range from ,0.28C to as much as 48C,2 and patients are unable to adjust to cold environments and heat loss resulting from vasodilation.1 It is notable that nitrous oxide depresses thermoregulation to a lesser extent than equipotent concentrations of the volatiles, and midazolam has minimal or no influence.3 This should be true for other benzodiazepines as well.2,3 Following induction of general anesthesia, the decline in body temperature occurs in three phases.

The greatest decline occurs during the first half hour or phase 1. Normally body heat is maintained in an unevenly distributed manner; the temperature of core tissues is 28C to 48C greater than skin temperature. Following anesthesia induction, however, vasodilation combined with a lowered cold threshold in the hypothalamus allows a redistribution of body heat from core tissues to skin, where

Figure 3. Temperature homeostasis.

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heat is lost primarily through radiation. Phase 2 commences after approximately 1 h, as core temperature decreases at a slower rate and proceeds in a linear manner as heat lost from the body exceeds heat production. Finally, after 3 to 5 h, phase 3 commences, as equilibrium is reached where heat loss is matched by heat production and thermoregulated vasoconstriction commences to function (Figure 4).1 – 3

Figure 4. Temperature drop during anesthesia.

Perioperative temperature monitoring: Temperature monitoring devices vary according to the type of transducer used and the site to be monitored. The most commonly used transducers are thermistors and thermocouples. A more recent development is the use of monitors that emit infrared to measure temperature; these monitors are commonly found in aural canal thermometers, which are often referred to as tympanic membrane thermometers. Liquid crystal sensors also can be used to measure skin temperature.

Core temperature is the best single indicator of body temperature. Therefore, all noncore temperature-monitoring sites need to be judged by their ability to accurately assess core temperature.

Core temperature monitoring is appropriate for most patients undergoing general anesthesia, to facilitate detection and treatment of fever, malignant hyperthermia, and hypothermia.

Monitoring sites: 1- Pulmonary artery catheter (PAC): PAC thermistor is located at the tip of the distal end allowing measurement of central blood temperature. Although it is the gold standard for core temperature measurement,4 it is not used routinely due to numerous drawbacks including invasiveness and cost effectiveness.

2- Oesophageal temperature: This is usually monitored with a thermistor or thermocouple that is incorporated into an esophageal stethoscope. Oesophageal temperature accurately reflects core temperature in almost all conditions.

These readings, however, can be artificially affected during general anesthesia by the use of humidified gases if the probe is not inserted far enough.5,6 The optimal position for the sensor in adults is approximately 45 cm from the nose, which is 12 to 16 cm distal from where the heart and breath sounds are heard best.7 More proximal positioning can result in falsely decreased temperatures as a result of the proximity to the trachea and the impact of cold, dry gases on the site.6 Esophageal temperature probes are used frequently for their ease of placement and relatively minimal risk, and because the site is reliable.

3- Nasopharyngeal temperature: Nasopharyngeal temperature can be measured with an esophageal probe positioned above the palate, and it is close to brain and core temperature.8

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4- Tympanic membrane temperature: Because the eardrum is close to the carotid artery and the hypothalamus, tympanic membrane temperature is a reliable measure of core temperature and often is used as a reference for other sites.

This measurement requires that a transducer be placed in contact with the tympanic membrane.

5- Bladder temperature: Bladder temperature is measured with a Foley catheter and attached temperature thermistor or thermocouple. Although bladder temperature is a close approximation of core temperature, the accuracy of this site decreases with low urine output and during surgical procedures of the lower abdomen.9 6- Rectal temperature: Rectal temperature measurement is another site that approximates core temperature, but these readings may be affected by the presence of stool and of bacteria that generate heat.10 Consequently, rectal temperature tends to exceed core temperature. Rectal and bladder temperatures lag behind other central monitoring sites during conditions in which the temperature changes rapidly such as cardiopulmonary bypass surgery.

7- Skin temperature: Intraoperative skin temperature monitoring is confounded by several factors; Core-to-peripheral redistribution which may be seen on anesthetic induction, thermoregulatory changes in vasomotor tone triggered when sufficient core hypothermia initiates intraoperative cutaneous vasoconstriction which can lead to a reduction in skin blood flow and temperature, and finally changes in ambient temperature.

8- Axillary temperature: Axillary temperatures are relatively close to core temperature and may be a reasonable choice in selected patients. However, its accuracy is questionable unless the probe is positioned carefully over the axillary artery and the arms positioned at the patient’s side.11 HYPOTHERMIA AS A CYTOPROTECTIVE STRATEGY Hypothermia had demonstrated potential benefits in myocardial infarction, organ transplantation, cardiopulmonary bypass (CPB), spinal cord injury (SCI), intestinal ischemia, and neonatal hypoxic ischemia.12 Understanding how hypothermia can be of benefit is not simple as its action is mediated through complex systemic and cellular changes; moreover, such changes can vary widely depending on underlying pathological and metabolic condition. However, examples from nature have indicated that physiological hypothermia could be observed in species that normally hibernate. In these animals, body temperature and metabolism drops in a manner as that observed when intentional hypothermia is applied.12 Hypothermia can be classified based on the depth of cooling from a normal body temperature of 37388C: mild hypothermia (32-358C), moderate hypothermia (28-328C) and deep hypothermia (, 288C).

While deep hypothermia has been embraced by various surgical subspecialties, the neuroprotective effect of mild to moderate hypothermia appears to be similar to deep.13 Thus, less drastic decreases in body temperature have been favored by other disciplines, as it is easier to cool nonsurgical patients to these target temperatures, and the risks of medical complications such as infection, arrhythmia, hypokalemia, coagulopathies and even heart failure are lower.13 HISTORICAL PERSPECTIVE OF THERAPEUTIC HYPOTHERMIA Therapeutic hypothermia was first used in 1960 in patients with acute neurological insults. The publication “Management of the comatose patient”14 was released in 1965. This early experience was instrumental in the evolution of the current recommendation by the American Heart Association and the International Liaison Committee on Resuscitation.15 In addition, in 1964, the historic “first ABCs of resuscitation” recommended that hypothermia could be used in patients who remain comatose after successful restoration of spontaneous circulation.16 This recommendation is still followed to the present day.15

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However, therapeutic hypothermia began to fall out of favor in the 1980s. This resulted, in part, from overzealous application in some patients, who were treated for durations longer than a week and at temperatures in the moderate (288C to 328C) rather than mild (338C to 358C) range which resulted in complications.17 CYTOPROTECTIVE MECHANISMS OF INDUCED HYPOTHERMIA There are many mechanisms by which hypothermia may be cytoprotective. Earlier literature has focused on its ability to preserve metabolic stores and thus render the tissue or organism in a state of ‘suspended animation’.18 Suspended animation is defined as therapeutic induction of a state of tolerance to temporary complete systemic ischemia.

The objectives of suspended animation include: a) To help save victims of temporarily uncontrollable exsanguination, followed by delayed resuscitation; b) To help save some non-traumatic cases of sudden death, seemingly non-resuscitable before definite repair; and c) To enable performing selected (elective) surgical procedures, which are only feasible in a bloodless field.

Hypothermia has generally been viewed as a means by which protein synthesis can be suppressed.19 However, the widely held notion that hypothermia protects because lower temperatures slow metabolism is not completely accurate. Recent research has shown that hypothermia can alter a plethora of cell death and cell survival pathways including gene regulation resulting in the inhibition of apoptosis and inflammation19 and the up-regulation of anti-apoptotic20 or trophic factors.21 In some systems, hypothermia has been shown to up-regulate a family of ‘cold shock proteins’ at temperatures between 25-338C. This up-regulation could potentially regulate cell survival at a proximal point in the cell death pathways. These cold shock proteins include cold inducible RNA binding protein (CIRP) and RNA-binding motif protein 3 (RBM3). CIRP has been speculated to protect and restore native RNA conformations during stress, and protects against apoptosis by up-regulating extracellular signalregulated kinase (ERK), which is involved in a cell survival pathway.22 RBM3 may also protect cells from death by acting in a manner similar to the X-linked inhibitor of apoptosis (XIAP).23 Approximately 5% of the oxygen and glucose consumption is reduced per degree centigrade cooling of the brain.

Hypothermia is also thought to couple both the brain energy and blood flow to a lower rate for cerebral tissue.24 Injury triggers the release of excitatory amino acids (probably a specific phenomenon in brain). Glutamate is now recognized to potentiate brain injury by binding to its ionotropic receptors and allow entry of toxic levels of calcium. A key mechanism in hypothermic brain protection is to prevent this toxic increase.25 – 27 Activation of peripheral leukocytes and brain resident microglia also occurs after brain injury, and mild hypothermia has been shown by multiple investigators to inhibit this activation and thus provide protection against ischemic injury.28,29 Suppression of this activation could be explained by the observation that hypothermia inhibits the pro-inflammatory transcription factor NFkB.30 – 32 Anti-apoptotic effects of hypothermia have also been documented by several investigators.

Hypothermia decreases numbers of apoptotic cells28,33,34 and reduces expression of pro-apoptotic genes35 while increasing anti-apoptotic genes such as Bcl-2.36,37 Hypothermia for neuroprotection: Prior work has shown that the extent of neuroprotection by hypothermia is not related to the amount of cooling, but the time when cooling is begun and its duration.38 Similar amounts of neuroprotection have been observed where cooling to 30-348C and that observed at 258C.13 Clinically, cooling to 358C appears to be as beneficial as 338C while causing fewer complications.39 Timing of the initiation of cooling is also important, in that the likelihood of a better outcome seems to depend on earlier initiation.40 Prolonged cooling (24-48 h) is essential for long term and robust protection.41,42 In such context, hypothermia had been used in various clinical situations aiming for neuroprotection: 1- Cardiac arrest: Clinical studies now show that mild hypothermia improves neurological outcome from cardiac arrest.20 A multi-center clinical trial in Europe studied 274 patients with cardiac arrest due to ventricular

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fibrillation, and showed that cooling to 328C to 348C over a period of 24 h had a significant protection.

As a consequence of therapeutic hypothermia, the 6 month mortality was reduced from 76/138 (normothermia) to 56/137 (hypothermia, P , 0.05). The cooled patients also had improved neurological outcome 6 months later, compared to those who were not cooled.43 Another clinical trial in Australia included 77 patients who had cardiac arrest and randomly treated patients with either hypothermia (338C within 2 h after the return of spontaneous circulation and maintained for 12 h) or remained normothermic. In this second study, 49% of the patients who received hypothermia survived with a good neurological outcome, while only 26% of the patients survived in the normothermia group (P , 0.05).44 2- Brain trauma: Hypothermia has been used in brain trauma for many years, and results of several small clinical trials have been published.45,46 One study showed modest but transient benefit in patients with head trauma, provided the Glasgow Coma Scale (GCS) was between 5– 7,47 but further studies in adults are ongoing to clarify whether earlier intervention might result in a favorable outcome.

3- Neonatal hypoxic encephalopathy: Clinical trials of therapeutic hypothermia in neonates with hypoxic encephalopathy also suggest a benefit in this patient population. A recently published study in 2008, Total Body Hypothermia for Neonatal Encephalopathy Trial (TOBY), showed similar benefits in newborns with perinatal asphyxia that received whole body cooling within a similar timeframe.48 The primary outcome at 18 months of age showed that cooled infants had better survival and neurological outcomes than those that were not cooled. However, defining the optimal candidate for hypothermia has yet to be defined, and subsequent studies are ongoing.

4- Ischemic stroke: Experimental stroke studies have shown the neuroprotective effects of hypothermia when cooling was applied in the range of 248C to 338C,49 with temperatures in the mild to moderate range being better tolerated. The timing of cooling is also important, in that hypothermia should be initiated within 2– 3 h of ischemia onset.

A significant challenge in applying hypothermia to stroke patients is that unlike other neurological conditions, stroke patients are generally awake and do not tolerate cooling. Like that encountered in the cardiac arrest and brain injury studies, attaining target temperature and maintaining it, especially in adult humans, is challenging.

5- Hemorrhagic stroke: Models of ICH are frequently studied where bacterial collagenase was instilled into the striata to cause hemorrhage due to proteolytic disruption of the basal lamina, or autologous blood is directly injected into the brain.50 Unlike that observed in ischemic brain ischemia models, studies of therapeutic hypothermia in brain hemorrhage are less consistent. While a few studies have shown beneficial effects of hypothermia in ICH, others have not.22 A reason why the response of hemorrhagic stroke is so varied, is that cooling can also affect endogenous coagulant and thrombolytic systems, and may predispose to bleeding with potential worsening.

Hypothermia for Cardiac procedures: Cardiopulmonary bypass (CPB), with its potential for creating temperature fluctuations, imposes enormous challenges to cerebral oxygenation and perfusion. Two physiologic principles, functional and structural cerebral metabolic rate of oxygen (CMRO2), are vital to understanding the importance of temperature in patients undergoing CPB.51 In humans, the CMRO2 is approximately 20% at basal metabolism: approximately 3.5 mL/100g21/min21. Of the total CMRO2, 60% is used to support cerebral electrophysiological function (functional CMRO2), and 40% is used for maintenance of cellular integrity (structural CMRO2). Temperature is the only agent known to affect both functional and structural CMRO2; total CMRO2 decreases 6%– 7% per degree centigrade reduction in temperature. Anesthetic drugs, on the other hand, alter only functional CMRO2.52 Intraoperative hypothermia is used to varying degrees in surgical procedures requiring CPB or circulatory arrest. Relatively small degrees of hypothermia, e.g., 2– 38C, can offer significant brain

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protection.53,54 The unique and beneficial effects of more profound hypothermia consist of reduction in both functional and structural metabolic rates, making it superior to any neuroprotective drugs.

In addition to the effects on metabolism, other mechanisms of therapeutic hypothermia include suppression of free radicals, inhibition of destructive enzymatic reactions, reduction in metabolic requirements in low-flow regions, and inhibition of the biosynthesis, release, and uptake of excitatory neurotransmitters.44,55,56 Through these mechanisms, hypothermia provides a favorable balance between oxygen supply and demand, slows the onset of ischemic depolarization, decreases the release of ischemic-induced intracellular calcium influx, and suppresses nitric oxide synthase activity.

Cerebral blood flow (CBF) decreases as temperature decreases during CPB57 – 59 with the electroencephalogram tracing becoming isoelectric at approximately 188C. The use of IV anesthetic drugs, thiopental in particular, reduces cerebral metabolic requirements responsible for brain function and synaptic activity, conditions achieved during the isoelectric electroencephalogram state.60 Hypothermia is widely used in open cardiac surgery not only to protect against perioperative brain ischemia that could potentially develop61 but also the myocardium. Following coronary artery occlusion, myocardial tissue consequently endures oxygen and energy depletion and thus cell death if not rescued. For several decades, cardiac surgeons have been using hypothermia to protect the non-working heart during open heart surgeries.62 In the ischemic working heart, mild hypothermia (348C) preserves microvascular flow and maintains cardiac output.21 In addition to maintaining myocardial contractility after ischemia, studies in a rabbit model of acute myocardial infarction showed that regional myocardial hypothermia (328C), initiated 10 min before reperfusion and maintained for 2 h, significantly improved the reflow, reduced the “no-flow� phenomenon, myocardial necrosis and infarct size.63 Like the brain, timing of hypothermia is a major concern in order to rescue the heart from ischemia.

Several studies have shown salutary effects of hypothermia in cardioprotection when cooling was initiated prior to the onset of ischemia.64 Post insult hypothermia has also been shown to benefit experimental myocardial ischemia (MI). Whether initiated before or after ischemia onset, mild hypothermia resulted in protection provided the target temperature (left atrial temperature 2-2.58C lower than normal) was reached at the time of coronary artery reperfusion resulted in a marked protection.65 Possible mechanisms of protection seem to parallel those described for brain ischemia. Studies have shown correlations between protection and reduced energy demand,66 delayed ATP depletion,67 induced HSP70 expression68 decreased apoptotic cell death through reduced expression of tumor suppressor gene p53 translational product66 and improved blood flow to the myocardial microvasculature.69 Clinical application of hypothermia for cardioprotection has largely been used in the intraoperative setting; however, some smaller studies have been conducted in patients with acute myocardial infarction. Considerations to an extent specific to the heart include compromised cardiac function with cooling.

Other indications for hypothermia: 1) Spinal cord injury (SCI): Mild hypothermia also decreased astrocyte and capillary proliferation which could affect regeneration of axons. However, laboratory studies seem to mostly suggest that hypothermia improves axon recovery.70 Studies to define optimal cooling methods, depth and duration of cooling, timing of cooling and ideal candidates for cooling are still needed.

2) Hepatic encephalopathy: Severe liver failure may increase toxic metabolite accumulation in the body with portal blood being shunted into the systemic circulation. Mild hypothermia can improve detrimental effects of liver failure by improving ammonia metabolism, suppressing inflammation, normalizing brain osmolarity and cerebral blood flow.71 3) Hypotensive bleeding trauma patients: Severe bleeding is a frequent cause of hypotension and shock in trauma patients. Since hypotension does not develop until loss of more than 30-40% blood volume, these injuries are often fatal and must be managed rapidly to avoid death. Mild to moderate hypothermia decreases heart rate and increases systemic vascular resistance while maintaining stroke volume and blood pressure. Hence, hypothermia decreases cardiac metabolic demands while sustaining cardiac output and myocardial perfusion.72

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In a model of uncontrolled hemorrhage, mild to moderate hypothermia induced by surface cooling delayed the onset of cardiac arrest and significantly improved survival in rats.73 – 76 POTENTIAL RISKS ASSOCIATED WITH THERAPEUTIC HYPOTHERMIA Combination of anesthetic induced impairment of thermoregulatory control and exposure to a cool operating room environment makes most surgical patients hypothermic. Several prospective, randomized trials have demonstrated various hypothermia-induced complications.

1- Myocardial ischemia: Evidence connecting perioperative hypothermia with myocardial complications was initially based on a retrospective analysis of data collected prospectively for a different purpose.77 These data indicated that patients becoming hypothermic were more likely to experience myocardial ischemia and ventricular arrhythmias.

The mechanism by which mild hypothermia triggers myocardial events remains unclear. Coldinduced hypertension in the elderly is associated with a three-fold increase in plasma norepinephrine concentrations,78 which may augment cardiac irritability and facilitate development of ventricular arrhythmias. Hypothermia also causes hypertension in elderly patients and those at high risk for cardiac complications.79 2- Coagulopathy: Surgeons have long suspected that hypothermia produces coagulopathy and increases perioperative blood loss. Other surgeons believed that hypothermia “thickened the blood” and reduced bleeding.

Schmied and colleagues80 showed that mild hypothermia increases blood loss. In their study, patients were randomly assigned to normothermia or mild hypothermia during elective primary hip arthroplasty.

Just 1.68C core hypothermia increased blood loss by 500 ml (30%) and significantly augmented allogeneic transfusion requirement. The same group subsequently confirmed the haemostatic benefits of maintaining intraoperative normothermia in a retrospective analysis.81 In contrast, another study of blood loss during hip arthroplasty failed to identify temperature dependence to blood loss.82 Three general mechanisms contribute to temperature-related coagulation disorders: platelet function, clotting factor enzyme function, and fibrinolytic activity.

Hypothermia and platelet function: Platelet numbers remains normal during mild hypothermia. However, Valeri and colleagues83 demonstrated that mild perioperative hypothermia seriously impaired platelet function. Inhibition was a strictly local phenomenon: bleeding time was comparably increased by systemic or local hypothermia. Subsequent work indicated that the defect resulted from reduced release of thromboxane A2.84,85 Hypothermia and clotting factor enzyme function: One feature of hypothermic coagulopathy is that standard coagulation tests, including the prothrombin time and the partial thromboplastin times, remain normal.86 The reason is that the tests are normally performed at 378C, regardless of what the patient’s temperature is. These sometimes are prolonged by hypothermia when they are performed at the patient’s actual core temperature. Rohrer et al demonstrated that series of enzymatic reactions of the coagulation cascade are strongly inhibited by hypothermia, as demonstrated by the dramatic prolongation of prothrombin time and partial thromboplastin time tests at hypothermic deviations from normal temperature in a situation where factor levels were all known to be normal.87 Hypothermia and fibrinolytic activity: Fibrin is a major structural element in formed clots but is subject to degradation by plasmin, the activated enzymatic form of plasminogen. The conversion of plasminogen to plasmin is at the core of the fibrinolytic mechanism. Preliminary data suggest that fibrinolysis remain normal during mild hypothermia but is significantly increased during hyperthermia, suggesting that hypothermia-induced coagulopathy does not result from excessive clot lysis. The corresponding effects of thermal disturbances on plasminogen activator are yet to be determined, but thromboelastographic data suggest that hypothermia impairs clot formation rather than facilitates clot degeneration.88 – 90

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3- Wound infection and healing: Hypothermia primarily impairs wound infection and healing through thermoregulatory vasoconstriction and impairment of immune function. Hypothermia triggers thermoregulatory vasoconstriction,91 vasoconstriction decreases subcutaneous oxygen tension and incidence of wound infection correlates with subcutaneous oxygen tension.92 Evidence indicating that mild core hypothermia directly impairs immune function, including T-cell—mediated antibody production and nonspecific oxidative bacterial elimination by neutrophils, was demonstrated over 30 years ago.93 – 95 4- Thermal discomfort: It had been indicated that feeling cold in the immediate postoperative period is the worst part for patients during hospitalization, with some patients even rating the sensation as worse than surgical pain.96,97 5- Other consequences: Although their clinical significance remains trivial, there are several consequences associated with hypothermia: Hypokalemia,98,99 increased cardiotoxicity of bupivacaine,100 mild affection of somatosensory evoked potentials,101 and obliteration of the oxymeter signal by sufficient vasoconstriction.102 PHARMACOKINETICS AND PHARMACODYNAMICS WITH HYPOTHERMIA: The enzymes that moderate organ function and metabolize most drugs are highly temperaturesensitive. It is therefore not surprising that drug metabolism is temperature-dependent. Curiously however, the pharmacokinetics of only few anesthetic drugs had been evaluated. Hypothermia also alters the pharmacodynamics of various drugs, especially volatile anesthetics.

Muscle relaxants: The duration of action of vecuronium bromide is doubled in patients with a 28C reduction in core temperature.103 Hypothermic prolongation of vecuronium action results from a pharmacokinetic effect, as pharmacodynamics of muscle relaxants are essentially unchanged by mild hypothermia.104 Volatile anesthetics: The tissue solubility of volatile anesthetics increases with hypothermia. At a given steady-state plasma partial pressure, body anesthetic content increases at subnormal temperatures. This does not alter anesthetic potency, because potency is determined by partial pressure rather than anesthetic concentration. However, it may slow recovery from anesthesia because larger amounts of anesthetic eventually need to be exhaled.96 Intravenous anesthetics: During a constant infusion of propofol, plasma concentration is approximately 30% greater than normal when individuals are 38C hypothermic. The increase apparently results from a reduced intercompartmental clearance between the central and peripheral compartments. Hypothermia also increases steady state plasma concentrations of fentanyl by approximately 5%/8C. The effects of mild hypothermia on the metabolism and pharmacodynamics of most other drugs has yet to be reported.

Evidence suggests that hypothermia was associated with delayed discharge of adult patients from the postanesthesia care unit.105 TEMPERATURE MANAGEMENT DURING CARDIAC SURGERY Intraoperative temperature regimen is usually planned preoperatively by the anesthesiologist, surgeon and the perfusionist. Selecting and understanding the impact of the temperature regimen (normothermia, or mild or moderate or severe hypothermia) is usually related to the type of the cardiac surgery (using CPB circulatory arrest or beating heart surgery).

Temperature monitoring during cardiopulmonary bypass: Cardiopulmonary bypass constitutes a challenging situation for monitoring temperature because of the rapid and extraordinary degree of heat transferred through the bypass circuit during heating and

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cooling. The core compartment undergoes the fastest temperature changes because of the rapid rate of blood reinfused into the mediastinal organs. The thermal changes from the core compartment then are dissipated slowly to the periphery because of the slower nature of heat passing through tissue as opposed to the cardiopulmonary bypass circuit.

During bypass, core-monitoring sites are useful for temperature monitoring. However, the pulmonary artery catheter may not give an accurate temperature reading because of diminished flow in the central circulation. Bladder and rectal temperatures often are considered intermediate temperature monitoring sites during bypass, because they lag behind the core sites but change faster than peripheral tissues.

During cardiac surgery, bladder temperature equals rectal temperature when urine flow is low, but equals the pulmonary artery temperature when urine flow is high.106 Because urine flow is such an important determinant of temperature in the cardiac surgery, it is best to consider both core and intermediate sites when judging adequacy of rewarming or cooling. Skin temperature, although unrelated to core temperature, it may be helpful in evaluating heat distribution between compartments at the end of bypass.107 Management of temperature during cardiopulmonary bypass: The clinical technique of temperature management during extracorporeal circulation (ECC) in open heart surgery is divided into three groups according to the nasopharyneal temperature: mild hypothermia group (32-358C), moderate hypothermia group (26-318C) and deep hypothermia group (258C).

In hypothermic CPB, patients are cooled to 31-328C after the beginning of CPB. Rewarming begins 1015 min before release of aortic cross-clamp. The gradient between heat-exchanger and nasopharynx during rewarming will be maintained at 2-38C. ln normothermic CPB, patients are kept at normothermia throughout the procedure (.368C).

Hypothermic versus normothermic CPB: Hypothermic CPB had become an established practice for adult cardiac surgery by the late 1960s, and constituted the largest part of the surgical practice at most institutions until the reintroduction of warm CPB in early 1990s.108 A systematic review of benefits and risks of maintaining normothermia during CPB among adults undergoing cardiac surgery had been published109 and demonstrated no benefit of hypothermia during CPB in regard to mortality, risk of stroke, cognitive decline, atrial fibrillation, use of inotropic support or intra-aortic balloon pump, myocardial infarction, all cause infections, and acute kidney injury after cardiac surgery were comparable. Moreover, hypothermic bypass was associated with an increased risk of allogeneic red blood cells, fresh frozen plasma, and platelet transfusion. The authors concluded that “maintaining normothermia during cardiopulmonary bypass in adult cardiac surgery is as safe as that of hypothermic surgery, and associated with a reduced risk of allogeneic blood transfusion�.109 This systemic review supports earlier trials in such field where normothermic CPB and warm cardioplegia were concomitantly used during adult cardiac surgery.110 – 112 On the other hand, arguments for hypothermic CPB techniques are still favored due to its effectiveness in reducing O2 demand and in increasing ischemic tolerance: these arguments have been established a long time ago.62,113 Moreover, other argued that the reduction in metabolic rate associated with hypothermia would also allow for the use of reduced CPB flows.51 Finally, as hypothermia requires haemodilution as hypothermia with whole blood prime results in hypertension during CPB, the introduction of hypothermia is of crucial importance in decreasing strain on blood bank resources.114 Total aortic arch replacement (TARCH) is generally performed with hypothermic circulatory arrest (HCA) at 15-228C plus selective cerebral perfusion (SCP) in an attempt to minimize neuropsychological morbidities associated with this type of surgery. However, the cooling and rewarming phases of HCA are time-consuming, and the complications due to prolonged cardiopulmonary bypass (CPB) remain a serious problem. Moreover, there is great controversy regarding optimal perfusion temperature and flow for SCP and the safe time limit for HCA. In a recent trial, the safety and efficacy of TARCH with deep HCA (at the lowest rectal temperatures of 20-258C) was compared with TARCH with tepid HCA (32 8C), retrospectively. Twenty-seven patients (group C) who underwent TARCH with deep hypothermia at the lowest rectal temperatures of 20-258C were compared with 23 patients (group W), who underwent TARCH with 328C tepid hypothermia.

Circulatory arrest time, cardiopulmonary bypass time, operating time, amount of blood transfused

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and postoperative neurological complications were significantly reduced in group W compared with group C.115 Temperature management during off pump surgeries: In OPCAB surgery, a patient’s temperature is influenced by the same environmental sources of heat loss that many other non-CPB surgical patients encounter. Additionally, because of an open thorax and extremities exposed for vascular conduit harvest, maintaining normothermia can be difficult.

Although the optimal temperature during OPCAB is not clearly known, efforts to maintain normothermia are generally instituted and normothermia has been considered a goal, particularly to aid in the timely extubation of these patients. These efforts include maintaining an elevated ambient operating room temperature, warming ventilated gases and intravenous fluids, water blankets, and forcing warm convective air over non exposed portions of the body.

Potential benefits of maintaining normothermia had been thoroughly discussed and can be summarized as: maintenance of normothermia, rather than hypothermia, may facilitate early tracheal extubation. Hypothermia alters the distribution and decreases the metabolism of most drugs, including anesthetic drugs and muscle relaxants, thus prolonging recovery. Postoperative shivering increases metabolic rate and potentially lead to myocardial ischemia; coagulopathies, increased incidence of surgical wound infection, and perioperative cardiac morbidity are other potential risk factors.116 Normothermia is proved to be associated with better cardiac and vascular conditions, a lower cardiac injury rate, and a lower inflammatory response. The close correlation between the increased interleukin-6 and troponin-I levels indicates a potential deleterious effect of lowered temperature on the patient’s outcome.117 During off-pump coronary artery bypass grafting, hypothermia increases vasoconstriction, myocardial after load, coagulopathy and postoperative bleeding.118 Woo and colleagues118 demonstrated that during off-pump coronary artery bypass grafting, hypothermia increases vasoconstriction, myocardial after load, coagulopathy and postoperative bleeding. On the other hand, a prospective randomized study to evaluate the effect of fluid warming using Hotlinee system during off-pump coronary artery bypass (OPCAB) surgery demonstrated no significant differences between the 2 thermal management groups in hemodynamic parameters, serum catecholamine concentrations, duration of intensive care unit stay, or duration of ward stay though warming IV fluids with Hotlinee system was capable in preventing hypothermia.119 Again, it is obvious that the optimal temperature during OPCAB is not known. Efforts to maintain normothermia are generally appreciated, however no insights regarding whether temperature management during such procedure can positively influence patient outcome.

ACTIVE THERMAL MANIPULATIONS It is well known that initial 0.5 – 1.58C reduction in core temperature is difficult to prevent because it results from redistribution of heat from the central thermal compartment to cooler peripheral tissues.120 Even the most effective clinical warmers do not prevent hypothermia during the first hour of anesthesia.120,121 Although redistribution cannot effectively be treated, it can be prevented.

Redistribution results when anesthetic induced vasodilatation allows heat to flow peripherally down the normal temperature gradient. Skin surface warming before induction of anesthesia does not significantly alter core temperature (which remains well regulated), but it does increase body heat content. When peripheral tissue temperature is sufficiently increased, little redistribution hypothermia occurs.122,123 Airway heating and humidification: Simple thermodynamic calculations indicate that less than 10% of metabolic heat production is lost via the respiratory tract. Because little heat is lost via respiration, even active airway heating and humidification minimally influence core temperature.124 Consequently, airway heating and humidification are even less effective than usual in patients in most need of effective warming. Airway heating and humidification are more effective in infants and children than in adults,125 but cutaneous warming also is more effective in these patients and transfers more than ten times as much heat. Hygroscopic condenser humidifiers and heat-and-moisture exchanging filters (“artificial noses”) retain substantial amounts of moisture and heat within the

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respiratory system. In terms of preventing heat loss, these passive devices are about half as good as active systems, however their costs are much affordable compared to other active systems.

Warm intravenous fluids: It is not possible to warm patients by administering heated fluids, because the fluids cannot (much) exceed body temperature. On the other hand, heat loss resulting from cold IV fluids becomes significant when large amounts of crystalloid solution or blood are administered. One unit of refrigerated blood or one litre of crystalloid solution administered at room temperature decreases mean body temperature more than 0.258C. Fluid warmers minimize these losses and should be used when large amounts of IV fluid or blood are administered. Most warmers allow fluid to warm in the tubing between the heater and the patient.

Cutaneous warming: Operating room temperature is the most critical factor influencing heat loss, because it determines the rate at which metabolic heat is lost by radiation and convection from the skin and by evaporation from within surgical incisions. Consequently, increasing room temperature is one way to minimize heat loss.

However, room temperatures exceeding 238C are generally required to maintain normothermia in patients undergoing all but the smallest procedures126 as most operating room personnel find such temperatures uncomfortably warm. Infants may require ambient temperatures exceeding 268C to maintain normothermia. Such temperatures are sufficiently high to impair performance of operating room personnel.

The easiest method of decreasing cutaneous heat loss is to apply passive insulation to the skin surface. Insulators readily available in most operating rooms include cotton blankets, surgical drapes, plastic sheeting, and reflective composites (space blankets). A single layer of insulators reduces heat loss by approximately 30 percent; moreover, there are no clinically important differences among the insulation types.127 The reduction in heat loss from all commonly used passive insulators is similar because the layer of still air trapping beneath the covering provides most of the insulation.

Consequently, adding additional layers of insulation further reduces heat loss only slightly.128 These data indicate that simply adding additional layers of passive insulation or warming the insulation before application usually is insufficient in patients who become hypothermic while covered with a single layer of insulation.

Cutaneous heat loss is roughly proportional to surface area throughout the body.129 Consequently, the amount of skin covered is more important than which surfaces are insulated. Passive insulation alone rarely is sufficient to maintain normothermia in patients undergoing large operations. Active warming is required in those cases. Because about 90% of metabolic heat is lost via the skin surface, only cutaneous warming transfers sufficient heat to prevent hypothermia. Consequently, for intraoperative use, circulating-water and forced-air devices are the two major systems requiring consideration.

Studies consistently report that circulating-water mattresses are nearly ineffective.130 Circulating water is more effective, and safer, when placed over patients rather than under them, and in that position, it can almost completely eliminate metabolic heat loss.131 The forced air warming device is the most effective method to maintain temperature during most surgical procedures.120,132,133 These devices are able to maintain normothermia even in patients undergoing large surgical procedures, and if employed in the intraoperative period, they increase central temperature by almost 0.758C/hour.120 MALIGNANT HYPERTHERMIA IN CARDIAC SURGERY The underlying mechanism for malignant hyperthermia (MH) is uncontrolled release of calcium from the sarcoplasmic reticulum triggered by volatile anesthetics and depolarizing muscle relaxants.134 This exaggerated intracellular calcium release in MH-susceptible skeletal muscle leads to large increases in aerobic and anaerobic metabolism as the muscle cells attempt to re-establish homeostasis by sequestering unbound calcium.135 However, in MH-susceptible muscle, the rise in calcium caused by the triggering agents overwhelms the cellular capacity to re-establish homeostasis. The pathologically enhanced intracellular calcium rise eventually reaches the threshold levels for myofibrillar contraction and muscular rigidity begins. This leads to increased oxygen consumption and increased carbon

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dioxide production. Heat is generated with rising lactic acid levels. Rhabdomyolysis ensues with leakage of muscle cell contents into the circulation.134 – 136 In case an MH episode is suspected, all triggering agents should be discontinued immediately and anesthesia should be changed to total intravenous anesthesia.137 Help should be called as early as possible. Hyperventilation with a high fresh gas flow (. 10 l/min) should be started and treatment with a bolus of dantrolene at 2.5 mg/kg initiated.138 The initial bolus of dantrolene (2.5 mg/kg) should be repeated until clinical signs and acidosis subside.139 Cooling measures need to be started (e.g., placing ice packs on the groin, axillae and neck) and maintained until the temperature of the patient is below 38.58C.135 Urine output of at least 2 ml/kg/h should be targeted.136 There are only a few reports of surgery with CPB and MH. Those reports suggest the use of nontriggering agents and careful management of temperature changes in the intra- and postoperative period, specifically during rewarming.140,141 In patients with a presumptive history of MH undergoing cardiac surgery, normothermic CPB and off-pump surgery are alternatives that should be considered individually.

CONCLUSION: Temperature is a physiologic variable that can be manipulated to suit the requirements of a particular management strategy according to patients’ preoperative risk factors. Because of its profound physiologic and pathophysiologic implications, temperature is a crucial homeostatic variable, particularly in the setting of cardiac surgery during which significant changes in temperature can occur.

A debate has emerged in recently published studies about the optimum cardiopulmonary bypass temperature for good outcome (normothermic vs. hypothermic). The ideal temperature for CPB is probably an indeterminate value that varies with the physiological goals. The choice of CPB temperature will always be a compromise between competing goals.

To date, there has been a lack of evidence regarding the optimal temperature management strategy during cardiopulmonary bypass. Thus, temperature management strategies during CPB rely primarily on personal or institutional preference, rather than a solid scientific basis.

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OPEN ACCESS

Review article

Science and practice of arrhythmogenic cardiomyopathy: A paradigm shift Mohamed ElMaghawry1,2*, Federico Migliore2, Nazar Mohammed4, Despina Sanoudou3, Mohammed Alhashemi4 1

Aswan Heart Centre, Aswan, Egypt Division of Cardiology, Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy 3 Department of Pharmacology, Medical School, University of Athens, Greece 4 The Heart Hospital, Hamad Medical Corporation, Doha, Qatar 2

*Email: Maghawry79@gmail.com

http://dx.doi.org/ 10.5339/gcsp.2013.8 Submitted: 29 December 2012 Accepted: 6 March 2013 q 2013 ElMaghawry, Migliore, Mohammed, Sanoudou, Alhashemi, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial license CC BY 3.0, which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

“A paradigm shift is a change in basic assumptions (paradigms) within the frame work of the theories of sciences�1

Thomas Kuhn, The structure of scientific revolutions, 1962.

INTRODUCTION The clinical, genetic, and molecular paradigm of arrhythmogenic right ventricular cardiomyopathy (ARVC) has markedly progressed through the last three decades, shifting from the classical ARVC as a progressive condition characterized by fibrofatty replacement of the right ventricle,2,3,4 into a wider spectrum of arrhythmogenic cardiomyopathy (AC),5 which covers ARVC with its various clinical phases (occult, electric, right heart failure and late stage biventricular heart failure), biventricular arrhythmic cardiomyopathy, left dominant arrhythmic cardiomyopathy, Naxos and Carvajal syndromes.

Epidemiologically, the disease was first associated with the Mediterranean basin (mainly Italy and France), however further studies have reported AC in many races and ethnic backgrounds.6,7,8 Moreover, with regard to the pathoitiology of the disease, dysplasia was originally assumed as the disease mechanism. Other mechanisms were later postulated, such as inflammation and transdifferentiation. However, more recent animal models have established that dystrophy, either by myocyte necrosis or apoptosis, is the founding pathological process of AC. In addition, in 1994 when the first genetic locus mutation was described, the researchers were investigating chromosome 14, as it was thought that ARVC and hypertrophic cardiomyopathy (HCM) may have a similar genetic background.9 The paradigm, however, shifted towards desmosome mutations as the genetic basis of AC in 2000 with the discovery that mutations in plakoglobin and desmoplakin cause the cardio-cutaneous autosomal recessive forms of the disease, i.e., Naxos and Carvajal syndromes.10,11 In this work, we will shed some light on the progress of the many faces of AC: pathology, molecular, genetics, clinical, electrophysiology, imaging, risk stratification and management.

INCIDENCE AC incidence is estimated to be 1 in 2,500 to 5,000 in the general population, with male predominance.12 It is considered one of the major causes of sudden cardiac death (SCD) in the young and young athletes.3 Athletes affected with AC represent an especially high risk SCD group.13 PATHOLOGY The pathological diagnosis of AC has been traditionally based on the gross and histological evidence of transmural myocardial loss with fibrofatty replacement of the right ventricle (RV) free wall, extending from the epicardium toward the endocardium.3 Gross morphological findings of AC include focal areas

Cite this article as: ElMaghawry M, Migliore F, Mohammed N, Sanoudou D, Alhashemi M. Science and practice of arrhythmogenic cardiomyopathy: A paradigm shift, Global Cardiology Science & Practice 2013:8 http://dx.doi.org/10.5339/gcsp.2013.8

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of severe muscle thinning that may transilluminate under a light source, local or global ventricular cavity enlargement, and ventricular wall aneurysms. Aneurysms are only present in 20 to 50% of autopsy cases of AC.14,15 RV aneurysms, located in the triangle of dysplasia (inflow, apex, outflow tract), are considered pathognomonic for AC.2 However, autoptic features of AC may range from grossly normal hearts, in which only a careful histopathological investigation can reveal AC features, up to massive RV and/or LV involvement. Therefore, the existence of cases with biventricular involvement or predominantly with LV or RV involvement suggest the use of the more comprehensive term AC.16,17 Histological examination reveals islands of surviving myocytes interspersed within fibrous and fatty tissue. Clusters of dying myocytes provide evidence of the acquired nature of myocardial atrophy, and are frequently associated with inflammatory infiltrates (Figure 1).16 Rather than being a continuous

Figure 1. Arrhythmic cardiomyopathy pathology. A: Cross section of a heart of a 17 year old male who died suddenly during a football match. Note the right ventricular dilatation, fibro-fatty replacement and anterior and posterior wall aneurysms. B: Histology of the right ventricular free wall of the same patient showing transmural fibrofatty replacement. C: Histology of the left ventricular free wall showing focal subepicardial left ventricular involvement. (Modified from Thiene, G., Corrado, D. & Basso, C. Arrhythmogenic right ventricular cardiomyopathy/dysplasia. Orphanet journal of rare diseases 2, 45 (2007).

process, disease progression may occur in periodic bursts. Environmental factors, such as exercise or inflammation, may facilitate onset and progression of myocyte loss and fibrofatty replacement.18 The deposition of adipose tissue is a peculiarity of AC, however its specificity has been controversial.

Significant fat infiltration of the RV is reported in more than 50% of normal hearts in the elderly.19 It has been recently suggested that isolated adipose replacement of the RV myocardium should only be considered pathological if observed in association with myocytes at various stages of cell death.20 PATHOGENESIS Many theories have been postulated to explain the mechanism of the loss of the ventricular myocardium and its substitution by fibrous and fatty tissue: dysplasia, myocarditis, transdifferentiation, and dystrophy. The original concept of the disease as congenital abnormality (dysplasia, aplasia, or hypoplasia) characterized with maldevelopment of RV myocardium.2 This led to the historical confusion in literature about AC and Uhl’s anomaly. Henry Uhl, at the Johns Hopkins Hospital in Baltimore, in 1952, reported a case of an almost total absence of the myocardium of the RV in a 7-month-old infant. The epicardium and endocardium lay adjacent to each other, with no intervening cardiac muscle and no fibrofatty tissue observed in the RV free wall.21 On contrary, in AC, myocardial death occurs after birth, usually during childhood, and is progressive with time. Another theory was the inflammatory process; with myocardial loss due to infective or immune mechanisms. Cardiotropic viruses, such as adenovirus, hepatitis C virus, and parvovirus B19, have been reported in the myocardium of some AC patients.22 However, the viral agent might be just an innocent bystander or play a secondary role to the progression of myocardial loss. Transdifferentiation of myocytes into fibrocytes and/or adipocytes has also been proposed.23 This theory is questionable because of the limited de-differentiaton capabilities of adult cardiomyocytes. Myocyte dystrophy remains the most likely explanation to the AC pathological process. Similar to skeletal muscle dystrophy observed in

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Duchenne or Becker diseases, a progressive and acquired myocardial atrophy – with replacement by exuberant fatty and fibrous tissue– occurs in the hearts of AC patients. This dystrophy, either by apoptosis or necrosis, could account for a genetically determined loss of myocardium.15,18,24 Transgenic animal models have been recently developed, supporting the dystrophy theory. A transgenic mouse with cardiac-restricted overexpression of the C-terminal mutant (R2834H) desmoplakin has been shown to develop increased cardiomyocte apoptosis, myocardial fibrosis, and lipid accumulation as well as biventricular dilatation/dysfunction.25 In another seminal study of a transgenic mouse model (Tg-NS) with cardiac overexpression of desmoglein-2 gene mutation N271S, clinical features of AC, as well as, myocyte necrosis were obsereved in all Tg-NS hearts.26 ROLE OF DESMOSOMES IN ARRHYTHMIC CARDIOMYOPATHY PATHOGENESIS Desmosomes are a specific type of cell junction within intercalated discs, the specialized intercellular junctions of cardiomyocytes (Figure 2). They form membrane anchorage sites for intermediate

Figure 2. The desmosome consists of three families of proteins: the desmosomal cadherins, desmocollin and desmoglein, members of the armadillo family of proteins, plakoglobin and plakophillin and the plakins. Binding of these proteins tethers desmin intermediate filaments to the plasma membrane in cardiac myocytes and adheres adjacent myocytes together.

filaments, and the resulting complex is thought to impart tensile strength and resilience. The cardiac desmosomes have been proposed to support structural stability through cell-cell adhesion, to regulate adipogenesis and apoptosis related genes, and to maintain proper electrical conductivity through the regulation of gap junctions and Ca2Ăž homeostasis. Functionally, desmin forms intermediate filaments in mature striated muscle that surround the Z discs and link the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and nucleus. Desmoplakin (DSP) and junctional plakoglobin (JUP) are constituents of the submembranous plaques of the desmosomes, along with plakophillin (PKP), and they form part of the link between the intermediate filament cytoskeleton and the cytoplasmic tail of cadherins. The desmosomal cadherins are calcium-dependent cell adhesion

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glycoproteins, divided in two classes namely, the desmoglein (DSG) and the desmocollin (DSC), and they mediate lateral and transcellular desmosomal adhesion (Figures 2 and 3).

Figure 3. Electron microscopic view of cardiac myocyte intercalated discs. (A) Normal case control. Regular cell membrane (arrows) and intercalated disc between adjacent myocytes. (B) ARVC patient with desmoplakin gene splice site mutation. Note the abnormal position of long desmosomes (arrowhead) and the widened gap of facia adherens (arrow). Original magnification: X15 000. Adapted from Basso C. et al. Ultrastructural evidence of intercalated disc remodelling in arrhythmogenic right ventricular cardiomyopathy: an electron microscopy investigation on endomyocardial biopsies. Eur Heart J 27, 1847–1854 (2006).

A desmosomal protein alteration may compromise either cell-to-cell adhesion and/or intermediate filament (desmin) function. The right ventricle with its thinner wall and higher distensibility is particularly vulnerable to the impaired cell adhesion. In contrast, disruption of intermediate filament (desmin) binding, such as in desmoplakin mutation since it is directly interacting with desmin, may result in dominant and/or severe left ventricular involvement. In either case, this mechanical disintegration will eventually lead to significant ventricular myocyte loss, especially during higher volume state loads, as for example, during sports activity. Because the regenerative capacity of the myocardium is limited, repair by fibrous or fibrofatty replacement takes place. This fibrofatty islands are the substrate for macro-reentry ventricular arrhythmias. Re-entrant tachyarrhythmias circle around the fibrous tissue and into an isthmus of surviving myocytes, in a figure-of-8 fashion similar to that occurring around ischemic myocardial scars. Moreover, disruption of desmosomal integrity per se can alter the electric stability of the myocytes, regardless of the extent of myocyte loss. For example, it has been recently demonstrated that PKP2 associates with sodium voltage gated channels Na(V)1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes.27 HERITABILITY AC cases have been shown to have a genetic component, with approximately one-third to one-half of them being familial. The inheritance pattern is autosomal dominant, i.e. both mutation homozygotes as well as heterozygotes can develop AC, although rare autosomal recessive cases – where only mutation homozygotes can present with the disease– have also been reported.4,28 AC-causing mutations have variable expressivity, with highly-variable phenotypes, ranging from severe disease with early death, to individuals who were completely asymptomatic late in life, even among family members carrying the same gene mutation.29,30 Penetrance is incomplete (20-30% or higher), with a significant percentage of the mutation carriers not presenting with an unaffected, normal phenotype.31 Interestingly, gender may have an influence on penetrance, with male mutation carriers more likely to develop specific phenotypic manifestations of this disease.14 The reduced penetrance along with variable expressivity, suggest that other genetic modifiers and/or environmental factors are implicated in disease

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pathogenesis. At the genetic counseling level, these characteristics make it difficult to trace the disease along a family line and to identify the members at risk of carrying a mutation.

GENETICS AC-causative mutations have been identified in ten different genes, although two of these (TGFB3 and RYR2) are rarely associated with AC. Four additional genes associated with autosomal dominant AC have been mapped but not identified (locus names ARVD3, ARVD4, ARVD6, and ARVD7). Molecular genetic testing is clinically available for eight of the ten known genes (Table 1). Since AC is emerging a Table 1. Arrhythmogenic right ventricular dysplasia (ARVD) gene loci Disease locus

Gene Name

Gene Symbol

Chromosome location

Frequency

ARVD1 ARVD2 ARVD5 ARVD8 ARVD9 ARVD10 ARVD11 ARVD12

transforming growth factor beta-3 ryanodine receptor 2 transmembrane protein 43 desmoplakin plakophilin-2 desmoglein-2 desmocollin-2 junction plakoglobin (or gamma catenin)

TGFB3 RYR2 TMEM43 DSP PKP2 DSG2 DSC2 JUP

14q24 1q42 3p25 6p24 12p11 18q12 18q12 17q21

Rare Rare Unknown 6-16% 11-43% 12-40% Rare Rare

desmosome related disease (eight of ten genes are desmosome related), desmosome gene mutations accounting for approximately 50% of symptomatic individuals,32 and compound and digenic heterozygosity being often encountered, screening of all desmosomal AC related genes is now recommended.33 Among the different desmosome genes, mutations have been identified in desmoplakin (DSP), plakophilin-2 (PKP-2), desmoglein-2 (DSG-2), desmocollin-2 (DSC-2), junction plakoglobin (or gamma catenin) (JUP), and more recently in plakophilin-4 and desmin.17,34

Figure 4. Deramological phenotype and genetic screening of a case of Cardiocutaenous syndrome (Carvajal). A: Dystrophic nail plates. B: Striate keratoderma of the palms. C: Plantar keratoderma, C: Erythemato-squamous skin lesions of the knee. E: curly wooly hair. F: Electropherograms of the desmoplakin mutation and of the wild type: a heterozygous variant in DSP, c.1748 T . C, was identified, resulting in the missense mutation p.Leu583Pro at a heterozygous state. From: Keller, D. et al. De novo heterozygous desmoplakin mutations leading to Naxos-Carvajal disease. Swiss medical weekly 142, (2012).

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Table 2. Revised 2010 Task Force critiria for diagnosis of ARVC. Definite diagnosis: 2 major or 1 major and 2 minor criteria or 4 minor from different categories; borderline: 1 major and 1 minor or 3 minor criteria from different categories; possible: 1 major or 2 minor criteria from different categories.

Adapted from Marcus F et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/ dysplasia. Proposed Modification of the Task Force Criteria. Eur Heart J 31, 806 – 814 (2010) 1-Right ventricular structural and functional abnormalities by: A- Echocardiography

B- Cardiac magnetic resonance

C- Right ventricular angiogram.

2-Characteristics of right ventricular wall tissue by Endomyocardium biopsy.

3- Electrocardiographic repolarization abnormalities.

4-Electrocardiographic depolarization abnormalities.

5- Arrhythmias

Major:- Regional wall akinesia, dyskinesia, or aneurismal dilatation of the RV Plus one of the following :- Right ventricular out flow tract of 19 mm/m2 in parasternal long axis view at the end diastole, 21 mm/m2 in parasternal short axis view at the end diastole - Change of fractional area by less than 33%.

Minor:-Regional wall akinesia or dyskinesia Plus one of the following :- Right ventricular out flow tract of 16 to19 mm/m2 in parasternal short axis view at the end diastole or 18 to 21 mm/m2 in parasternal short axis view at the end diastole - Change of fractional area more than 33% but less than 40%.

Major:- Regional wall akinesia or dyskinesia or dyssynchronous contraction plus one of the following: - Ejection fraction less than 40%, - End-diastolic volume t more than 110 mL/m2 ,or more 100 mL/m2 in males and females, respectively.

Minor:- Regional wall akinesia or dyskinesia or dyssynchronous contraction plus one of the following: - Ejection fraction more than 40% but less than 45%.

-end-diastolic volume more than 100 to but less than 110 mL/m2 or more than 90% but less than 100% in males and females, respectively Major:- Regional wall akinesia, dyskinesia, or aneurysmal dilatation Major:- The total amount of the residual myocytes are less than 60% by morphometric analysis (or less than 50% if estimated), and the remaining of the free wall myocardium are replaced by fibrous tissue with or with out fatty changes in more than one sample.

Minor:- The total amount of the residual myocytes are 60% to 75% by morphometric analysis (or 50% to 65% if estimated), and the remaining of the free wall myocardium are replaced by fibrous tissue with or with out fatty changes in more than one sample.

Major:-Inverted T waves in right precordial leads (V1, V2, and V3) or beyond in individuals >14 years of age (in the absence of complete right bundle-branch block QRS > 120 ms).

Minor:-Inverted T waves in leads V1 and V2 in individuals > 14 years of age (in the absence of complete right bundle-branch block) or in V4, V5, or V6 - Inverted T waves in leads V1, V2, V3, and V4 in individuals > 14 years of age in the presence of complete right bundle-branch block.

Major:-Epsilon wave (reproducible low-amplitude signals between end of QRS complex to onset of the T wave) in the right precordial leads (V1 to V3).

Minor:- Late potentials by Signal Avereged ECG in > 1 of 3 parameters in the absence of a QRS duration of >110 ms on the standard ECG. - Filtered QRS duration (fQRS) >114 ms.

- Duration of terminal QRS,40 mV (low-amplitude signal duration) > 38 ms -Root-mean-square voltage of terminal 40 ms < 20 mV - Terminal activation duration of QRS >55 ms measured from the nadir of the S wave to the end of the QRS, including R0, in V1, V2, or V3, in the absence of complete right bundle-branch block.

Major:- Nonsustained or sustained ventricular tachycardia of left bundle-branch morphology with superior axis (negative or indeterminate QRS in leads II, III, and aVF and positive in lead aVL).

Minor:- Nonsustained or sustained ventricular tachycardia of RV outflow configuration, left bundle-branch block morphology with inferior axis (positive QRS in leads II, III,and aVF and negative in lead aVL) or of unknown axis.

- > 500 ventricular extra systoles per 24 hours (Holter).

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6-Family history of ARVC.

Major:- Positive family history of first-degree relative confirmed by current task force criteria.

- Pathological confirmation of the disease in first-degree relative either by autopsy or surgery.

- Discovering of a DNA pathogenic mutation that has been recognized to be associated or probably associated with ARVC in the patient who has been evaluated for ARVC. Minor:-Positive family history of first-degree relative in whom the diagnosis is not feasible to be confirmed by current Task Force criteria.

-Positive family history of young (< 35 years) first degree relative with sudden death due to suspected ARVC. - Positive family history of disease in second degree relative who has been confirmed to have the disease either by current Task Force Criteria or pathologically.

Of note, mutations in some of these genes have been associated with genetically related (allelic) disorders. Specifically, RYR2 mutations have been identified in individuals with catecholaminergic polymorphic ventricular tachycardia (CPVT),35,36 as well as patients with “atypical” or “borderline” long QT syndrome (LQTS) who did not have mutations identified in the five genes associated with LQTS.37 DSP mutations can lead to Carvajal syndrome, an autosomal recessive disease characterized by ventricular dilated cardiomyopathy associated with keratoderma and woolly hair38,11,39 (Figure 4), while JUP mutations can be causative of Naxos disease, an autosomal recessive form of ARVD characterized by palmoplantar keratoderma and peculiar woolly hair that was first observed on the island of Naxos, Greece.10,40 Historically, it was the phenotype of woolly hair and palmoplantar keratoderma in those two syndromes that pointed scientists towards screening desmosomal genes for pathogenetic mutations.

Interestingly, as opposed to other AC subgroups, Naxos disease has full penetrance by adolescence.41 Ongoing efforts are aiming to identify genotype-phenotype correlations, with interesting preliminary.

For example, compared with those without a desmosome gene mutation, individuals with a desmosome gene mutation had earlier-onset AC and were more likely to have ventricular tachycardia.42 Recent studies have also suggested that for rare desmosome gene mutations (namely, DSG2 and DSC2), the presence of multiple mutations simultaneously may be required to manifest the AC phenotype43 and may be associated with increased disease severity, namely higher frequency of sudden death.28 CLINICAL PICTURE AND TASK FORCE CRITERIA Clinically, the patient usually presents with palpitations, due to premature ventricular complexes (PVCs) or nonsustained ventricular tachycardia. Other presentations are sustained ventricular tachycardia, syncope, resuscitated cardiac arrest, right heart failure or late stage biventricular heart failure.12 Multiple criteria are needed to diagnose AC, as there is no single gold standard criterion sufficiently specific to establish the diagnosis.41 Even the presence of desmosomal genetic abnormality is not sufficient as there is variable penetrance. However, it is important to note that the recessive form of AC (Naxos disease) presents full penetrance by adolescence, being associated with cutaneous abnormalities consisting of woolly hair and palmoplantar keratoderma.44 This diagnostic challenge led to the formation of an expert Task Force that in 1994 proposed major and minor criteria to aid in the diagnosis.45 The report achieved its goal of standardizing diagnostic criteria. With the growing international experience, an updated modified task force criteria (TFC) were published in 2010.46 The modified criteria include structural alterations observed by echocardiogram, cardiac magnetic resonance imaging and/or angiography.

They also include tissue characterization of RV wall, repolarization abnormalities, depolarization abnormalities, arrhythmias, and family history (Table 2). The modified criteria are also based on more quantitative analysis rather than the 1994 TFC qualitative nature.

ELECTROCARDIOGRAM The 12-lead electrocardiogram (ECG) is one of the most important tools for the diagnosis, follow-up and SCD risk stratification of AC (Figure 5). Depolarization abnormalities due to activation delay as a result of cellular uncoupling and fibrofatty alteration include the epsilon wave, widening of the QRS complex (. 110 msec) in leads V1 to V3 and evidence of late potentials by signal averaged ECG (SAECG). Epsilon wave is a defined as reproducible low amplitude signals between the end of the QRS complex to

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Figure 5. 12 Lead ECG of arrhythmogenic cardiomyopathy. Note the â&#x20AC;&#x153;epsilon waveâ&#x20AC;? in V3, the right pericardial leads localized QRS prolongation which is mainly due to a terminal activation delay (from nadir of S to the end of QRS complex), and T wave inversion in V1-3.

the onset of the T wave in the right precordial leads (V123). Although the epsilon wave is very specific for AC, Cox et al, showed that this parameter had a very low sensitivity (10%). Widening of the QRS complex was a criterion in 1994 TFC, but it was deleted in the 2010 TFC due to possible confusion especially in the presence of a right bundle branch block (RBBB). To overcome this confusion, many studies have proposed new ECG markers that focus on delayed RV activation in precordial leads. These include the presence of partial block,47 delayed S wave upstroke in V123 $ 55 msec,48 increased ratio of QRS duration in the right versus the left precordial leads,49 and a prolonged terminal activation duration $ 55 msec.50 Right precordial QRS prolongation and QRS dispersion have been significantly associated with an increase of the arrhythmic risk in patients with AC. In an important study, Turrini et al., showed a greater QRS prolongation (125 msec) in V1 to V2/V3 in AC patients with SCD, in comparison with living AC patients (113 msec). They also demonstrated that QRS dispersion of more than 40 msec (between the longest and shortest QRS intervals) was a strong predictor of SCD in AC.51 Abnormalities in repolarization in AC are represented as inverted T wave. Due to its high sensitivity, inverted T waves in V1-V3 or beyond in the absence of RBBB and in .12 year old individuals was upgraded from a minor criterion in the 1994 TFC to a major criterion in the 2010 TFC, for individuals older than 14 years and in absence of complete RBBB.46 The morphology of recorded ventricular tachycardia (VT) reflects its site of origin. In AC, affected areas in the triangle of dysplasia usually produce a VT with a left bundle branch block morphology and a superior axis, defined from 2 308 to 2 1508. Because of the variable extension of the disease, multiple VT morphologies are usually recorded in a single patient. Studies showed the mean number of different VT morphologies per patient ranges from 1.8 to 3.8.52,53

ECHOCARDIOGRAPHY Echocardiography is of paramount importance in the initial evaluation and follow up of AC patients because of its availability, ease of performance and interpretation, cost effectiveness and non invasive advantages (Figure 6).54 The multidisciplinary Study of Right Ventricular Dysplasia demonstrated that the diagnostic performance of transthoracic echocardiography was superior to MRI with 80% accuracy in affected individuals with AC and 40% accuracy in borderline individuals, compared with 49% and 15% for MRI, respectively.55,6 RV outflow tract dilatation (in parasternal long axis view . 32 mm or in parasternal short axis view . 36 mm) coupled with localized anuerysms (akinesia or dyskinesia) or global dysfunction (fractional area change ,33%) is now considered a major criterion for the diagnosis of AC.46 Other echocardiographic features to assess RV anatomic alteration include the ratio between the RVOT/aorta in parasternal short axis view (abnormal if . 1.2), longitudinal and transverse RV axes in apical four chamber and subcostal views, visualization of RV apical trabeculation in the subcostal view. Emerging echocardiographic techniques being currently evaluated include 3-dimensional echocardiography,56 RV free wall myocardial velocity, strain and strain rate by Doppler or speckle tracking (Figure 7).57,58

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Figure 6. Echocardiography of a patient with arrhythmic cardiomyopathy. Upper panel shows an apical 4 chamber view with dilated anuerysmal right ventricle. Lower panel shows a modified short axis left paraternal view to visualize the subtricuspid area which shows a localized aneurysm. (Images courtesy of MP Marra, MD, University of Padua, Italy).

RIGHT VENTRICULOGRAPHY RV ventriculography remains an integral imaging modality and reference technique in the diagnosis and evaluation of patients with AC. This technique should be performed in all patients with suspected or definite AC and may be combined with electrophysiological study and/or RV endomyocardial biopsy. The angiographic diagnosis of AC is based on segmental abnormalities rather than diffuse RV enlargement or hypokinesia. Dedicated computer software for the evaluation of RV volume and regional wall motion, have been developed and provide a convenient and reproducible method for quantitative assessment of global and regional RV contraction and relaxation.59,60 MAGNETIC RESONANCE IMAGING Among the current cardiac magnetic resonance imaging (MRI) applications in cardiomyopathies, the greatest potentials and challenges are in the diagnosis of AC. Routinely used imaging planes are suboptimal for RV evaluation, and the technique of AC imaging involves unconventional imaging planes. Furthermore, the lack of familiarity of the MRI interpreters with RV contraction pattern and the normal epicardial fat distribution pose challenges for accurate and reproducible reporting. Also, it requires a high degree of expertise to accurately differentiate AC from alternative diseases with similar MRI picture -especially late gadolinium enhancement (LGE)- such as myocarditis and sarcoidosis.61 Finally, over-reliance on the presence of intramyocardial fat has resulted in a high frequency of misdiagnosis of AC.62 Despite of these limitations, cardiac MRI have emerged as a robust tool to evaluate AC patients (Figure 8). It has the ability to noninvasively provide tissue characterization for

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Figure 7. Apical 4-chamber view with 2D speckle imaging of the LV septum and RV free wall in a normal older patient. The images display longitudinal velocity (A), strain (B), strain rate (SR) (C), and displacement (D) curves.

A’, Late diastolic waveform; AR, apical right ventricle; AS, apical septum; BR, basal right ventricle; BS, basal septum; E’, early diastolic waveform; MR, mid right ventricle; MS, mid septum; S’, systolic waveform. Adapted from Horton K, et al. Assessment of the right ventricle by echocardiography: a primer for cardiac sonographers.

Journal of the American Society of Echocardiography 22, 776-92 (2009)

detection of fat and more importantly fibrosis in the RV, and also the LV. Quantitative data on ventricular volumes, functions, and regional contraction abnormalities are useful in the diagnosis and follow up of patients with AC.63 MRI studies have also helped in genotype-phenotype correlation of AC; LV involvement is rare in PKP 2 mutation but more common in desmoplakin and plakoglobin mutation carriers.17,64 ELECTROPHYSIOLOGICAL MAPPING AND ABLATION Three-dimensional electro-anatomic mapping has helped in understanding the substrate and mechanisms underlying VT in AC patients. Electrical activation through normal RV myocardium was defined in patients with no structural heart disease with the use of the CARTO electro-anatomic mapping system and the Navistar catheter (Biosense Webster, Diamond Bar, CA, USA), which has a 4mm distal tip electrode, and a 1-mm inter-electrode distance. Normal RV endocardium is characterized by bipolar signals displaying 3 or fewer deflections from baseline, with peak-to-peak amplitude greater than 1.5 mV, whereas areas of bipolar voltage less than 0.5 mV correspond to dense scar, i.e., electrovoltage scar (EVS). RV bipolar EVS was demonstrated to correlate with the histopathologic finding of fibrofatty myocardial replacement at endomyocardial biopsy in AC patients.65,66 Corrado et al demonstrated that electrovoltage mapping enhances the diagnostic specificity of AC by distinguishing between pure genetically-determined AC, which is characteristically associated with EVS involvement, and acquired RV inflammatory cardiomyopathy, mimicking AC but showing a preserved electrogram voltage and a better prognosis.65,66 Moreover, electrovoltage mapping has been proven to increase diagnostic sensitivity for early/minor form of AC underlying apparently idiopathic RVOT tachycardias, by detecting otherwise concealed segmental RV EVS areas in the RVOT, which are associated with a worse arrhythmic outcome.66 Finally, EVM has been recently reported to be significantly more sensitive than contrast-enhancement-cardiac magnetic resonance in identify RV scar lesion (Figure 9).67 In accordance with the pathological findings concerning the progress of the fibrofatty dystrophy from the epicardium towards the endocardium; Garcia et al demonstrated that most patients with AC have a far

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Figure 8. Cardiac magnetic resonance imaging of a patient with extensive AC. Short axis view from base (upper left) to apex (lower right) demonstrating extensive fibrosis as evidenced by delayed gadolinium enhancement in most of right ventricular wall (only sparing a small infero-septal area), interventricular septum and the epicardium of the left ventricular inferior wall. This patient underwent cardiac transplantation for severe right heart failure. (Images courtesy of MP Marra, MD, University of Padua, Italy)

more extensive substrate for VT using epicardium mapping than they do on RV endocardium.68 Due to the disappointing initial results endocardial ablation results,69 VT ablation as a line of therapy for patients with AC has been considered only in patients with end-stage AC, incessant VT, frequent implantable cardioverter defibrillator (ICD) interventions and intolerable antiarrhythmic drugs side-effects. However, more promising results have been recently published using more aggressive and sophisticated endocardial and epicardial substrate mapping and ablation techniques.68,70 Yet, those results, being exclusive to very highly experienced centers, may be difficult to reflect in general practice.

PHARMACOLOGICAL THERAPY Pharmacological treatment has been another challenging aspect in AC, given the small number of patient study populations and the near lack of randomized clinical trials. One of the largest series of pharmacologic therapy in AC is from Germany, first published in 1992 and updated in 2005 with 191 patients and 608 drug tests.71,72 Sotalol at a dosage of 320-480 mg/d was the most effective drug resulting in a 68% overall efficacy. Combinations of amiodarone and beta-blockers were also efficacious. Another large study was presented from the North American Registry in 2009.73 Of 108 patients in this registry, it was concluded that there was no clinically significant benefit in preventing malignant ventricular arrhythmias with beta-blockers. However there was a trend in the reduction in number of shocks in patients with implantable cardioverter defibrillator (ICD) and on a beta-blocker therapy. In opposition to the German registry, sotalol failed to show any clinical benefit, with worse outcomes associated with highest doses of sotalol. A small number (10 patients) were studied for amiodarone, and they showed 75% lower risk of any clinically relevant ventricular arrhythmias compared with all other patients. The mixed results from those two registries lead to the conclusion

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Figure 9. A: Bipolar endomyocardial electrovoltage mapping of a patient with arrhythmogenic cardiomyopathy.

Red color represents areas of a low voltage recordings , 0.5 mV indicating a dense scar tissue. B: Cardiac magnetic resonance of the same patient. Extensive fibrotic changes demonstrated by delayed gadolinium enhancement in most of the right ventricular free wall and extending into the interventricular septum (concordant to the electrovoltage mapping findings). (Images courtesy of MP Marra, MD, University of Padua, Italy)

that there is not sufficient evidence to adequately guide physicians considering pharmacological management of AC.74

IMPLANTABLE CARDIOVERTER DEFIBRILLATOR There is accumulating evidence that ICD provides life saving protection by effectively terminating ventricular arrhythmias in high-risk patients with AC, coining ICD therapy as the gold standard line of management for those patients. One of the most seminal mutli-center studies of ICD therapy in AC is the DARVIN I study. The study was published in 2003, with a study population of 132 AC patients, 80% of them received ICD implant because of a history of either cardiac arrest or sustained VT (secondary prevention). Over the study period of 39 ^ 25 months, 3 deaths occurred (only one SCD; one for infective endocarditis and one for heart failure), 48% of patients (64 out of 132) had at least one appropriate ICD intervention. Fifty-three of the 64 patients were receiving antiarrhythmic medication at the time of the first appropriate shock. Twelve percent of the patients received inappropriate ICD interventions and 16% had ICD-related complications (Figure 10).75 This was followed by the mutli-center DARVIN II study which focused on primary ICD prevention in high risk AC patients. The study included 106 patients with AC and no prior VF or VT who received an ICD because of one or more arrhythmic risk factors such as syncope, asymptomatic nonstained VT, familial SCD and inducibility of sustained VT by programmed ventricular stimulation in the electrophysiological laboratory. During a mean follow up of 4.8 years, no death occurred, and 24% of the patients received appropriate ICD interventions and 19% received inappropriate interventions.76 In a large single center study, Witcher et al., reported 60 AC patients who received ICD therapy and were followed up for a period of 80 ^ 43 months The majority of the cases received their ICD as a secondary prevention. With only 26% of event free follow up in the highest risk group, the study confirmed the improvement of long term prognosis of high risk AC patients who undergo ICD implantation.77

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Figure 10. DARVIN I Kaplan-Meier analysis of actual patient survival (upper line) compared with survival free of ventricular fibrillation/flutter (lower line) that in all likelihood would have been fatal in the absence of the ICD. The divergence between the lines reflects the estimated mortality reduction by ICD therapy of 24% at 3 years of follow up. Adapted from Corrado, D. et al. Implantable cardioverter-defibrillator therapy for prevention of sudden death in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia. Circulation 108, 3084-91 (2003).

SPORTS AND PRE-PARTICIPATION SCREENING The cornerstone in the management of AC as a cause of SCD relies on screening among high-risk population. Corrado et al., have shown that the risk of sudden death from AC has been estimated to be 5.4 times greater during competitive sports than during sedentary activity (Figure 11).13 This can be

Figure 11. Incidence and relative risk (RR) of sudden death (SD) for specific cardiovascular causes among athletes and non-athletes. ARVC: arrhythmogenic right ventricular cardiomyopathy; CAD: coronary artery disease; CCA: congenital coronary artery anomaly; MVP: mitral valve prolapse. Adapted from Corrado, D., Basso, C., Rizzoli, G., Schiavon, M. & Thiene, G. Does sports activity enhance the risk of sudden death in adolescents and young adults? J Am Coll Cardiol. 42, 1959-63 (2003).

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attributable to the fact that physical exercise acutely increases the RV afterload and causes cavity enlargement, which in turn may elicit ventricular arrhythmias by stretching the diseased RV myocardium. This theory has been confirmed by Kirchof et al., in an experimental study on heterozygous plakoglobin-deficient mice, when compared with wild-type controls, the mutant mice had increased RV volumes, reduced RV function, and more frequent and severe VT of RV origin. Endurance training accelerated the development of RV dysfunction and arrhythmias in the plakoglobin-deficient mice.78 For more than 30 years, a systemic pre-participation screening for athletes, based on 12-lead ECG, in addition to history and physical examination, has been in practice Italy. A time trend analysis of the incidence of SCD in athletes aged 12 to 35 years in the Veneto region in Italy between 1979 and 2004 has proved compelling evidence of the efficiency of this life saving screening strategy. The annual incidence of SCD in athletes decreased by 89%, from 3.6 per 100,000 during the prescreening period, to 0.4 per 100,000 in the late screening period.79 This is highly attributable to the efficiency of ECG in detecting HCM and AC as the most common causes of SCD amongst athletes. Moreover, during long-term follow-up, no deaths were recorded in the disqualified athletes with HCM, suggesting that restriction from competition may reduce the risk of SCD.80 CONCLUSION The evolution of the clinical, investigational, and basic sciences has changed much of our understanding on ARVC shifting it to the wider concept of AC. The scientific community is yet challenged with a long path of research to fully unveil the many faces of this potentially lethal condition.

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OPEN ACCESS

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Research article

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An electronic medical record-linked biorepository to identify novel biomarkers for atherosclerotic cardiovascular disease

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Zi Ye1,2, Fara S Kalloo1, Angela K. Dalenberg1, Iftikhar J Kullo1,*

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ABSTRACT

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1

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Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN, USA Division of Cardiovascular disease, Shanghai Huashan Hospital, Fudan University, Shanghai, China

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*Email: kullo.iftikhar@mayo.edu

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Background: Atherosclerotic vascular disease (AVD), a leading cause of morbidity and mortality, is increasing in prevalence in the developing world. We describe an approach to establish a biorepository linked to medical records with the eventual goal of facilitating discovery of biomarkers for AVD. Methods: The Vascular Disease Biorepository at Mayo Clinic was established to archive DNA, plasma, and serum from patients with suspected AVD. AVD phenotypes, relevant risk factors and comorbid conditions were ascertained by electronic medical record (EMR)-based electronic algorithms that included diagnosis and procedure codes, laboratory data and text searches to ascertain medication use.

Results: Up to December 2012, 8800 patients referred for vascular ultrasound examination and non-invasive lower extremity arterial evaluation were approached, of whom 5268 consented. The mean age of the initial 2182 patients recruited was 70.4 ^ 11.2 years, 62.6% were men and 97.6% were whites. The prevalences of AVD phenotypes were: carotid artery stenosis 48%, abdominal aortic aneurysm 21% and peripheral arterial disease 38%. Positive predictive values for electronic phenotyping algorithms were . 0.90 for cases (and . 0.95 for controls) for each AVD phenotype, using manual review of the EMR as the gold standard. The prevalences of risk factors and comorbidities were as follows: hypertension 78%, diabetes 29%, dyslipidemia 73%, smoking 70%, coronary heart disease 37%, heart failure 12%, cerebrovascular disease 20% and chronic kidney disease 19%.

Conclusions: Our study demonstrates the feasibility of establishing a biorepository of plasma, serum and DNA, with relatively rapid annotation of clinical variables using EMR-based algorithms.

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Keywords: atherosclerotic vascular disease, biorepository, electronic medical records, electronic phenotyping

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http://dx.doi.org/ 10.5339/gcsp.2013.10 Submitted: 12 September 2012 Accepted: 6 March 2013 q 2013 Ye, Kalloo, Dalenberg, Kullo, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

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Cite this article as: Ye Z, Kalloo FS, Dalenberg AK, Kullo IJ,. An electronic medical record-linked biorepository to identify novel biomarkers for atherosclerotic cardiovascular disease, Global Cardiology Science & Practice 2013:10 http://dx.doi.org/10.5339/gcsp.2013.10

Page 2 of 9 Ye et al. Global Cardiology Science and Practice 2013:10

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BACKGROUND Atherosclerotic vascular disease (AVD) is a leading cause of mortality and morbidity worldwide.1 Several circulating biomarkers and genetic variants have been reported to be associated with AVD in cohorts of European ancestry.2 As AVD becomes increasingly prevalent in developing countries, there is an urgent need to identify biomarkers for early identification, prognostication and new drug development in diverse ethnic groups. Although significant progress has been made in identifying novel risk factors of coronary heart disease, little is known about genetic susceptibility variants and circulating biomarkers for peripheral vascular diseases1 – a group of diverse diseases characterized by atherosclerotic lesions in carotid arteries, aorta and lower extremity arteries. As a step towards identifying novel genetic and circulating biomarkers, we describe our approach to create an electronic medical record (EMR)-linked vascular disease-specific biorepository of DNA, plasma and serum. The biorepository includes patients with carotid artery stenosis (CAS), abdominal aortic aneurysm (AAA), and peripheral arterial disease (PAD), with linkage of biospecimens to clinical characteristics.

The EMR archives billing information, laboratory and imaging results, medications, and clinical documentation, thereby serving as a resource for genotype-phenotype association studies. A key issue we attempted to address was the feasibility and accuracy of capturing relevant clinical data using EMRbased phenotyping algorithms. Such algorithms have the potential to cost-effectively and efficiently ascertain phenotypes and relevant clinical covariates for conducting genomic studies3 – 5, whereas traditional manual review of medical records to ascertain clinical covariates can be time-consuming and expensive.

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METHODS The study protocol was approved by the Institutional Review Board of the Mayo Clinic. Enrollment of patients and collection of biospecimens started in June 2009 and is still ongoing. The recruitment process is summarized in Figure 1 and the project infrastructure is depicted in Figure 2.

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Figure 1. Pattern of recruitment for the Vascular Disease Biorepository. EMR ¼ electronic medical record.

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Participant recruitment Consecutive adult patients with known or suspected CAS, AAA, or PAD, referred for non-invasive vascular ultrasound or lower extremity arterial evaluation, were approached for participation in the biorepository. Definitions of three AVD phenotypes for the study are listed in Table 1. All potential participants were checked against records of patients already enrolled or those who had refused research consent (Figure 1). The informed consent document (see supplement) conformed to the guidelines regarding Bioethics Resources and human subject research on National Institutes of Health

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Figure 2. Overview of the Vascular Disease Biorepository. RLIMS ¼ research laboratory information management systems; CAS ¼ carotid artery stenosis; AAA ¼ abdominal aortic aneurysm; PAD ¼ peripheral arterial disease.

Web (http://nih.gov/sigs/bioethics), and International Society of Biological and Environmental Biorepositories Web (http://www.isber.org).

The study coordinator described the objectives of the study, the risks and potential benefits of participation in the study, and the storage and future use of the samples. The consent form had separate check-off boxes seeking consent for biospecimens to be re-used or shared with collaborating investigators. Lack of immediate benefit for health, the potential to improve risk stratification of AVD, and the right to withdraw from the study any time after consent were specified. A questionnaire on sociodemographic information, cardiovascular health history, physical activity, lifestyle, past medical history and family history, was given to each participant at the time of consent. Once returned, the barcoded questionnaire was reviewed, scanned and added to the study database.

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Collection, aliquoting, and storage of peripheral blood samples Blood was collected in the fasting state whenever possible and the time from blood draw to storage was limited to , 1 h to minimize sample degradation; 52 ml of peripheral blood were drawn into the appropriate collection tubes, labeled with a Mayo-generated barcode ID number, and sent through a pneumatic tube system to the Biospecimens Accessioning and Processing (BAP) laboratory. Blood was

153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

Table 1. Criteria for ascertaining atherosclerotic vascular disease phenotypes.

Carotid artery stenosis (1) $ 40% stenosis in internal carotid artery/bulb (peak systolic velocity $ 150 cm/second) on either side evaluated with Doppler; OR 2) at least moderate atheromatous plaque in any of the following locations: common carotid artery, bulb, bifurcation or internal carotid artery of either side, or postoperative change of carotid endarterectomy or presence of stent in either side demonstrated by conventional, computed tomography or magnetic resonance angiography; OR 3) any procedure reports of carotid endarterectomy or stenting.

Abdominal aortic aneurysm (1) Distal, infrarenal or juxtarenal abdominal aortic anteroposterior diameter $ 3 cm, measured with ultrasound, conventional or computed tomography or magnetic resonance angiography, or evidence of abdominal aortic aneurysm repair on imaging; OR 2) any procedure reports of open or endovascular abdominal aortic aneurysm repair; OR 3) abdominal aortic aneurysm documented in physician’s note.

Peripheral artery disease (1) Rest or 1 min post exercise ankle-brachial index (ABI) # 0.9 or rest ABI $ 1.4 or lower extremity systolic BP $ 255 mm Hg in either leg; OR 2) at least moderate stenosis in lower extremity arteries in either side (distal to abdominal aortic bifurcation) on imaging; OR 3) postoperative change of lower extremity angioplasty, stenting, open vascular bypass or amputation on imaging or reports of these procedures for lower extremity arterial occlusive disease.

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centrifuged and EDTA plasma and serum were aliquoted into 0.5 ml tubes and stored in 2 808C freezers. DNA was extracted by Gentra AutoPure chemistries (Gentra systems Inc., Minneapolis, MN) from 5 ml of whole blood contained in EDTA and quantified by ultraviolet absorbance and quality control by 260/280 optical density ratio. In a subset of patients, lymphocytes were cryopreserved for future (see supplement).

174 175 176 177 178 179 180 181 182 183 184 185 186 187

Laboratory Information Management System An in-house software system, Research Laboratory Information Management System (RLIMS), was used to record and monitor sample processing. All tubes and plates that contained an individual’s samples were barcode-labeled by patient numbering program (PNP) and entered into the RLIMS. The program contains demographic information and an assigned study number to de-identify participants enrolled and track each one after recruitment. The unique number assigned to each subject is in no way related to his or her identity. The current PNP is web-based and contains a security layer and a logging mechanism for tracking by RLIMS. RLIMS allots unique IDs for all barcoded biospecimens including input (sample tubes) and output (DNA/plasma/white blood cell) tubes. Based on barcoding, RLIMS records the time biospecimens were received, the time of the DNA extraction and the quality of DNA. All pipetting was performed by robotic workstations that incorporate barcode scanners to track the transfer of the biological material from tube to tube, tube to plate, and plate to plate. Extensive integrity checks were made within the tracking system to reduce the risk for error.

188 189 190 191 192 193 194 195 196 197 198 199 200 201 202

Annotation of biospecimens with phenotype data Broadly, there are two types of data in the EMR: codified data that can be abstracted directly including billing codes, demographics, and laboratory data; and narrative data in free-text format that can be mined by text searches using natural language processing (NLP). Electronic phenotyping algorithms were used to obtain patient characteristics including AVD phenotypes, conventional risk factors, comorbidities, and medication use. A federated warehouse of patient data – the Mayo Clinic Life Sciences Trust, derived from EMR data sources throughout the Mayo Clinic, was used to obtain relevant demographic and clinical data. It accommodates most EMR contents for . 7 million patients, including highly annotated, full-text clinical notes, laboratory tests, diagnostic findings, demographics, and related clinical data. Since 1999, all medical records have been entered in this integrated EMR system.

Billing codes, including International Classification of Disease (ICD) diagnosis and procedure codes version 9-CM, and Current Procedural Terminology (CPT) codes version 4, were used to obtain diagnoses and procedure information from Mayo’s billing systems.

203 204 205 206 207 208 209

Demographics Birth date, gender, race/ethnicity, and current residency, were mined directly from the EMR. The categories of self-reported race were “American Indian/Alaskan Native,” “Asian/Pacific Islander,” “Black,” “choose not to disclose,” “Native Hawaiian,” “other,” “Unknown,” and “white.” Current residency information included city, state, and zip code where patient currently resides. The geographic distribution of the enrolled patients was ascertained by zip codes.

210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

Conventional risk factors for vascular disease Hypertension, diabetes, dyslipidemia, and smoking status were ascertained by electronic phenotyping algorithms as previously described.3 These algorithms were constructed based on laboratory test values, medications, and ICD-9-CM diagnosis codes. The time window for ICD-9-CM codes to ascertain relevant clinical covariates was any time before and up to 6 months after the enrollment, and for laboratory data, one year around enrollment. Plasma glucose, hemoglobin A1c, total and high-density lipoprotein cholesterol and triglyceride levels were extracted from the laboratory database. Resting systolic and diastolic blood pressure (BP) values were mined as structured observations from the vital signs section. Hypoglycemic agents or insulin, lipid-lowering and anti-hypertensive medications were ascertained by NLP from the current medications, admission medications and dismissal medications sections in clinical notes. Hypertension was defined as either systolic BP $ 140 mmHg or diastolic BP $ 90 mmHg at two serial measurements within 3 months closest to the enrollment, or a prior diagnosis of hypertension with use of antihypertensive medication. Diabetes was defined as fasting blood glucose $ 126 mg/dL, random glucose $ 200 mg/dL, hemoglobin A1c $ 6.5%, or a prior

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diagnosis with oral hypoglycemic or insulin therapy. Dyslipidemia was defined as total cholesterol $ 220 mg/dL, or high-density lipoprotein cholesterol # 40 mg/dL in men or # 45 mg/dL in women, triglycerides $ 200 mg/dL, or the use of lipid-lowing medications. Smoking status was ascertained by NLP as described previously6 and smokers were defined as either current or past smokers.

230 231 232 233 234 235 236 237 238 239 240 241 242

Comorbid conditions We used the following ICD-9-CM diagnosis and procedure codes to identify comorbid conditions: cerebrovascular disease: 433.xx – 434.xx (cerebral infarction), 435.x (cerebral ischemia), 436 – 437.x (vascular disorders of the entire brain); heart failure: 428.0, 428.1, 428.21 – 22, 428.4x and codes given as primary or secondary diagnosis; coronary heart disease: 410.xx (acute myocardial infarction), 412 (old myocardial infarction), 413.xx (angina), 414.0x and 414.2x (chronic ischemic heart disease), procedure codes 36.0x (percutaneous coronary intervention) and 36.1x (by-pass surgery); chronic kidney disease 585.x (chronic kidney disease stage I – IV), 586 (end stage renal disease), 588.x (renal failure). Fifty patients were randomly selected to validate algorithms for risk factors and comorbid conditions. Manual medical record review was used as gold standard to generate a positive predictive value (PPV) for each algorithm.

243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

AVD phenotypes We used ICD-9-CM and CPT-4 codes to ascertain the three AVD phenotypes of interest: CAS, AAA, and PAD (Table 2). The algorithms were developed to identify cases and controls for each phenotype. To identify AVD phenotype with high specificity, we required that the relevant diagnosis codes had to be present at least twice in the EMR. For controls, we required that the relevant diagnosis codes had to be absent in the EMR. PPV was calculated to assess the accuracy of each algorithm to ascertain cases and controls. Manual review of random samples was used to improve the algorithm (criteria listed in Table 1) and repeated to obtain a PPV . 90% for cases and controls. We reviewed 50 cases and 50 controls for each phenotype at each step of algorithm development and for final validation. Finalized algorithms were run in the entire dataset and a separate dataset of random samples from the Mayo Phase I eMERGE (electronic MEdical Records and GEnomics) cohort to test the performance of the phenotyping algorithms. The Mayo eMERGE study cohort consists of 1687 patients with PAD and 1725 controls recruited from non-invasive vascular laboratory and stress electrocardiography laboratory respectively, as previously described.3 Accuracy of algorithms to ascertain vascular intervention or surgeries was validated by manually reviewing a random set of 25 cases and 25 non-cases for each phenotype.

Table 2. Algorithms to ascertain atherosclerotic vascular disease cases and controls.

261 262 263 264 265 266

Carotid artery stenosis Abdominal aortic aneurysm Peripheral arterial disease

267 268 269 270 271 272 273

Vascular procedures Carotid stenting or endarterectomy Abdominal aortic aneurysm repair Lower extremity revascularization or surgery

ICD-9-CM codes to ascertain cases

ICD-9-CM codes to rule out controls

433.1, 433.10, 433.11 – Occlusion and stenosis of carotid artery 441.3-4, 441.6-7 – abdominal and thoracoabdominal aneurysm 440.21 –24 – atherosclerotic limb with claudication, rest pain, ulcer or gangrene

433.xx – occlusion and stenosis of precerebral arteries 441.xx – aortic aneurysm and dissection 440.20–24, 440.0, 440.4, 443.9 – atherosclerotic extremity, peripheral vascular disease

ICD-9-CM codes: 38.12 and 00.62; CPT-4: 35,301 and 0075 T Open vascular repair: ICD-9-CM codes: 38.44, 39.52, 38.34, 38.64, 38.4, 38.6; CPT-4 codes: 33,877; Endovascular repair: CPT-4 codes: 34,800-05: Open vascular bypass: ICD-9-CM codes: 38.08, 38.18, 38.48, 38.48, 39.25; Angioplasty with or without stenting: ICD-9-CM codes: 39.50, 39.90; CPT-4 codes: 73,725, 75,635, 75,716; Major amputation: ICD-9-CM codes: 84.13–84.17

274 275

CPT-4: current procedural terminology codes version 4; ICD-9-CM: international classification disease codes version 9-CM.

276 277 278 279 280

RESULTS From June, 2009 to December 2012, 8800 patients scheduled for vascular ultrasound or lower extremity arterial evaluation in the Gonda Vascular Center were approached, of whom 5268 consented.

Demographics and clinical characteristics for the initial 2182 participants are summarized in Table 3.

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Demographics and clinical characteristics Our study population (Table 3) was predominantly white (97.6%) and 62.7% were men, with mean age 70.43 ^ 11.21 years. All participants were U.S. residents, with 85% from the Upper Midwest. We manually reviewed the “patient-provided information summary” section in the EMR for 50 patients. No mismatches for sex, race and address information were noted between EMR mined data and manually reviewed data.

287 288 289

Table 3. Demographics and clinical characteristics.

290

Variables

n ¼ 2182

291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309

Age (years) Men Non-Hispanic white ethnicity Upper midwest residency* Atherosclerotic vascular disease phenotype Carotid artery disease Carotid endarterectomy/carotid stenting Abdominal aortic aneurysm Repair of abdominal aortic aneurysm Peripheral arterial disease Lower extremity revascularization/amputation Conventional risk factors Hypertension Diabetes Dyslipidemia Ever Smoking Comorbid conditions Coronary artery disease Coronary revascularization Heart failure Cerebrovascular disease Chronic kidney disease

70.4 ^ 11.2 1367 (62.7%) 2029 (97.6%) 1856 (84.9%) 1041 (48%) 521 (24%) 448 (21%) 190 (9%) 834 (38%) 224 (10%) 1712 (78%) 632 (29%) 1585 (73%) 1538 (70%) 841 (37%) 513 (24%) 255 (12%) 427 (20%) 418 (19%)

*Upper Midwest includes the following states: Minnesota, Iowa, Illinois, Wisconsin, Michigan, North and South Dakota.

310 311 312 313 314 315

The most prevalent risk factor was hypertension (78%), followed by dyslipidemia (73%). More than one third (37%) of the participants had coronary heart disease, one fifth (20%) had heart failure and chronic kidney disease (19%) respectively. The PPVs for algorithms were: hypertension 0.96, dyslipidemia 1.00, diabetes 0.98 and smoking 0.90, cerebrovascular disease 0.90, heart failure 0.88, coronary heart disease 0.90 and chronic kidney disease 1.00.

316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336

Vascular disease phenotypes The most common vascular disease in our biorepository was CAS (48%), followed by PAD (38%) and AAA (21%). History of carotid endarterectomy or carotid stenting was present in half of the patients with CAS, history of aneurysm repair was present in 48% of patients with AAA and history of lower extremity revascularization or amputation was present in 34% of patients with PAD. More than 40% of patients had atherosclerotic disease in two or more vascular beds. To get the final PPVs for vascular disease phenotypes, we manually reviewed patients detected by algorithms as cases and controls for each phenotype. The causes of false positives for the final algorithms were ascertained in 50 cases and 50 controls and listed in Table 4. For cases, false positives were due to: 1) codes for a specific phenotype given at the time of non-invasive testing or clinical evaluations even though results were normal subsequently; 2) mild disease not meeting the criteria we used in the present study. For controls, false positives were due to: 1) lack of specific codes for a subphenotype, such as poorly compressible arteries in the case of PAD; 2) codes assigned in error. The accuracy of the algorithms to ascertain history of carotid stenting or endarterectomy in patients with CAS or to ascertain history of aneurysm repair in patients with AAA was 100%. The accuracy of procedure codes to identify vascular interventions in patients with PAD was 98%, with 1 patient who underwent renal artery stenting procedure detected as PAD case and 1 patient with superior femoral artery stenting detected as PAD control by the algorithm. To test the specificity of the EMR-based algorithms, we validated these in random samples from the Mayo eMERGE phase I cohort, in which 49% of the patients have PAD. The

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337 338

Table 4. Accuracy of electronic phenotyping algorithms – comparison of EMR-based algorithms to manual medical record review in cases (n 5 50) and controls (n 5 50) in each dataset.

339

PPV (VDB dataset)

340

PPV (Validation dataset)

Causes of false positives

341 342 343 344 345 346 347 348 349 350 351 352

Carotid artery stenosis Cases 0.94 Controls 0.98 Abdominal aortic aneurysm Cases 0.96 Controls 1.0 Peripheral arterial disease Cases 0.92 Controls

0.96

0.90 0.98

mild atherosclerotic plaque or stenosis < 40%; abnormal carotid ultrasound with codes assigned for cerebrovascular disease, not for carotid artery stenosis

0.94 1.0

ectasia of abdominal aorta diameter < 30 mm

0.98

lower extremity aneurysm; diabetic neuropathy or non-ischemic ulcer only noncompressible artery in the lower extremity

0.98

353 354 355 356 357

PPVs of cases were lower for CAS and AAA, higher for PAD. The PPVs were similar for controls for each vascular disease (Table 4). The false positives for comorbid conditions mainly resulted from billing codes assigned at the time of non-invasive testing.

358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381

DISCUSSION AVD is the leading cause of death globally despite the development of effective therapies.7 Changes in lifestyle due to urbanization, industrialization, and longer life expectancy are some of the factors leading to increase in prevalence of cardiovascular disease in developing countries.8 Varying genetic susceptibility as well as novel circulating biomarkers may help explain some of the disparities in the prevalence of AVD globally. However, to date, most of the attention has focused on coronary heart disease in whites, whereas other AVD phenotypes and ethnic groups remain relatively understudied.

To reduce the global burden of AVD, there is a need to identify novel biomarkers for early detection and prognostication, especially in patients of non-European ancestry. We describe the creation of a biorepository of DNA, serum and plasma from patients with AVD encountered in clinical practice. The biorepository was annotated with demographic information, AVD phenotypes, conventional risk factors and comorbidities by using electronic phenotyping algorithms.

The need for biomarker studies of cardiovascular diseases has led to the establishment of biorepositories in several countries, predominantly in the developed world. The Generation Scotland project (n ¼ 15,000) enrolled participants from Scotland’s population to identify genetic variants accounting for variations in quantitative traits underlying heart disease, diabetes and mental disease.9 The UK Biobank (n ¼ 500,000) aims to investigate the association of common complex diseases including stroke or coronary heart disease with genetic and lifestyle factors by recruiting volunteers aged 40 – 69 years and following them through linked population-level health related medical records.10 deCode Genetics leverages Iceland’s genealogy data and medical records to investigate genetic and molecular causes of common diseases including myocardial infarction and aneurysmal disease11. Recently, disease-focused biorepositories have been initiated to study the association of genetic variants with atherosclerosis.12 – 14

382 383 384 385 386 387 388 389 390 391 392

Electronic phenotyping To maximize the value of a biorepository, collection of clinical information should not be limited to characteristics for the specific disease, but should include laboratory and imaging reports, treatments, medications, and past medical history as well.15 Abstracting clinical data from medical records by manual review can be time-consuming and costly. Electronic phenotyping has several advantages over the classic abstraction approach, including rapid and inexpensive generation of large case-control cohorts.16 An example of EMR-coupled biorepositories is the eMERGE (electronic Medical Records and Genomics) Network, an NHGRI-supported consortium of five institutions, including Mayo Clinic, to explore the potential of DNA repositories linked to EMR for genomic studies.17 Other examples include

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BioVU, the Vanderbilt DNA databank16 and the Marshfield Clinicâ&#x20AC;&#x2122;s Personalized Medicine Research Project (PMRP), a population-based DNA biobank.18 Accurate ascertainment of phenotypes depends on the approach to establish the diagnoses. Using ICD-9-CM codes alone to ascertain cardiovascular risk factors such as hypertension or diabetes from the EMR is sensitive but not accurate.19 Combining diagnosis codes, medications, laboratory data, and text searches using NLP may increase accuracy.16,20 We used a similar approach, including codified data such as billing codes and laboratory results, and narrative data in physician notes, to ascertain risk factors and increase accuracy of electronic algorithms. We validated the accuracy of algorithms in a separate dataset and found similar PPVs for cases and controls. We found high PPVs of electronic phenotyping algorithms based on manual review of the EMR; supporting the view that EMR-based phenotyping could be used instead of traditional manual abstraction. However, billing codes do not provide information on the location of disease in a particular vascular bed and may lead to a significant number of false positives. We have previously demonstrated that the use of text searches by NLP to ascertain PAD in radiology reports21 can provide information on the extent and location of atherosclerosis.

Ethical and psychosocial issues Advances in bioinformatics allow the merging of datasets from different centers, for data sharing, and re-analysis in the future. However, this raises ethical and psychosocial issues, such as whether the initial informed consent allows the use of biospecimens for secondary research and the potential aggregation of data into different databases, using and sharing existing databases, and best approaches to avoid participant identifiability. Additional ethical and psychosocial issues that are unique to a particular ethnic group/geographic location may also need to be addressed. To ensure that procedures conform to what has been established during the informed consent process, different approaches have been used as described above. Phenotypic information needs to be used and stored in a manner that protects patientsâ&#x20AC;&#x2122; confidentiality. For example, a redacted version of the data would be created for those who are eligible and wish to use it. The Mayo Institutional Review Board, a Biospecimen Trust Oversight Group and involvement of bioethicists in our study allow rapid adaptation to issues evoked by policy changes and scientific advancement.

Limitations Billing codes to ascertain relevant covariates and comorbidities are easily available at a relatively low cost, but systematic misclassification and exclusion of conditions or procedures not pertinent to reimbursement are potential limitations to their use.22 The availability of phenotypes in the EMR may be affected by whether a patient gets care at one or multiple medical institutions. The relatively high prevalences of vascular diseases and related risk factors may have inflated PPVs for our algorithms.

Using NLP to conduct more comprehensive and specific free-text search in radiology and procedure reports will increase precision and generalizability of the electronic phenotyping algorithms. Obtaining data for environmental factors such as physical activity or diet from the EMR is difficult, limiting the ability to study gene-environment interactions. EMRs are not in widespread use yet in developing countries. However, study questionnaires could serve as an alternative means of obtaining information on covariates needed to conduct biomarker and genetic studies.

436 437 438 439 440 441 442 443

CONCLUSION In summary, we describe methodology for establishing a biorepository of plasma, serum and DNA from patients with AVD and demonstrate the use of electronic phenotyping algorithms to annotate such a biorepository with relevant covariates. These methods may inform the establishment of similar biorepositories in different geographic regions of the world, facilitating the identification and validation of novel biomarkers of AVD in diverse ethnic groups.

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Authorsâ&#x20AC;&#x2122; contributions IJK conceived of the study and participated in its design and helped to draft the manuscript. ZY participated in the design of the study and drafted the manuscript. FSK participated in the study design.

All authors read and approved the final manuscript.

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449

Acknowledgement

450

This work was funded by a Marriott Award in Individualized Medicine to I.J.K.

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OPEN ACCESS

Images in cardiology

Further insights into the syndrome of prolapsing non-coronary aortic cusp and ventricular septal defect Akhlaque N Bhat1*, Ahmad Sallehuddin1, Mohammad Riyas1, Reyaz Ahmad Lone1, Pawel Tyrsarowski1, Suresh Kumar1, Jiju John1, Pradeep Bhaskar1, Syed Zin1, Magdi H Yacoub2 1

CCS Department, Section of Pediatric Cardiac Surgery, Hamad Hospital, Doha, Qatar 2 Director Qatar Cardiovascular Research Centre, Doha, Qatar *Email: abhat@hmc.org.qa

ABSTRACT Ventricular septal defect (VSD) with prolapse of the right coronary cusp and aortic regurgitation can be managed surgically with the anatomical correction technique. However when the VSD is located underneath the non coronary cusp surgical management differs due to anatomical constraints and secondary pathological changes seen in the non coronary cusp. It is therefore important that the location of the VSD and the morphology of prolapsing cusp be characterised preoperatively in order to plan appropriate surgical repair. We present a case study in which we discuss the salient differences in the surgical management of the prolapsing right and the prolapsing non coronary cusps.

Keywords: aortic regurgitation, ventricular septal defect, aortic cusp prolapse, sinus of valsalva

http://dx.doi.org/ 10.5339/gcsp.2013.11 Submitted: 1 February 2012 Accepted: 6 March 2013 q 2013 Bhat, Sallehuddin, Riyas, Lone, Tyrsarowski, Kumar, John, Bhaskar, Zin, Yacoub, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Bhat AN, Sallehuddin A, Riyas M, Lone RA, Tyrsarowski P, Kumar S, John J, Bhaskar P, Zin S, Yacoub MH. Further insights into the syndrome of prolapsing non-coronary aortic cusp and ventricular septal defect, Global Cardiology Science & Practice 2013:11 http://dx.doi.org/10.5339/gcsp.2013.11

Page 2 of 5 Bhat et al. Global Cardiology Science and Practice 2013:11

INTRODUCTION We have previously identified the lack of continuity between the ventricular septum and the aortic annulus as the salient pathological feature of the syndrome of prolapsing right coronary cusp, dilatation of the sinus of valsalva, and ventricular septal defect (VSD).1 In keeping with this salient pathological feature, we devised a simple technique of anatomic correction that addressed all the components of the pathology. This consists of using a transaortic approach, closing the VSD with interrupted pledgeted sutures that elevate the crest of the septum to the aortic media, thus restoring the position of the aortic annulus and plicate the excessive tissue of the unsupported sinus of valsalva with the same sutures.

Management of patients whose VSD is located under the non coronary sinus has generally been ignored in literature. Characterisation of the cusp prolapsing into the VSD is important to determine the type of correction because prolapse of the non-coronary cusp is not amenable to anatomical correction.

CASE STUDY We present the case of a male child in heart failure with NYHA class II symptoms, who weighed only 21 kilograms at 9 years of age. Echocardiography showed that he had a VSD located underneath the noncoronary aortic cusp with left-to-right shunt. He had significant prolapse of the non-coronary cusp, dilation of the corresponding sinus of valsalva, and severe aortic regurgitation. The VSD shunt was restricted by the prolapsed cusp. His left ventricle was dilated but had preserved function. His mitral, tricuspid and pulmonary valves were normal and his right ventricular outflow tract was widely patent.

At operation the heart was significantly enlarged. Aortic valve was tricuspid. Non-coronary aortic sinus was significantly dilated and its corresponding leaflet had extensive secondary changes in the form of thickening, and a significantly redundant free margin. The commissure between the noncoronary cusp and the right coronary cusp was displaced down into the non-coronary aortic sinus. The adjoining free edge of the right cusp was also moderately thickened. A ventricular septal defect, shaped like a transverse oval, was located underneath the non-coronary cusp.

Figure 1. Short axis view of the aortic valve showing the dilated non coronary cusp (NCC), the right coronary cusp (RCC) and the left coronary cusp (LCC).

Page 3 of 5 Bhat et al. Global Cardiology Science and Practice 2013:11

Figure 2. Operative photograph showing the redundant non coronary cusp (NCC). RA (right atrium)

We attempted aortic valve repair by performing commissuroplasty that resuspended the non-right coronary commissure at its proper level and eliminated part of the redundant free margin of the noncoronary cusp. We performed additional plication of the free margin to accurately match the length of the free margins of the non coronary cusp with the other two cusps. A reasonably good final coaptation was achieved. The VSD was closed with a goretex patch using continuous prolene suture, taking care to safeguard the atrioventricular node. On removing the aortic cross clamp the left ventricle immediately dilated indicating severe aortic regurgitation. Cardioplegia was immediately re-administered directly

Figure 3. Operative photograph showing the non coronary cusp (NCC) retracted with a forceps revealing the ventricular septal defect (VSD) underneath it. RA (right atrium)

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Figure 4. Cartoon showing discontinuity between the aortic media and the ventricular septal crest. (With permission J Thorac Cardiovasc Surg 1997;113:253â&#x20AC;&#x201C; 261.)

Figure 5. Cartoon depicting interrupted pledgeted sutures taken through the ventricular septal crest and into the right coronary cuspas placating sutures before finally being tied down in the right coronary sinus. (With permission J Thorac Cardiovasc Surg 1997;113:253â&#x20AC;&#x201C; 261.)

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Figure 6. Cartoon depicting the final repair with the sutures tied down causing obliteration of the ventricular septal defect and plication of the right coronary aortic sinus. (With permission J Thorac Cardiovasc Surg 1997;113:253â&#x20AC;&#x201C;261.)

into coronary ostia, and cardiac standstill was achieved. We replaced the aortic valve by performing a Ross procedure using the root replacement technique. Right ventricular outflow tract was reconstructed with a pulmonary homograft. The child made an uneventful recovery and was discharged home on the seventh post-operative day.

LESSONS LEARNT Preoperative characterisation of the prolapsing cusp is important to plan repair in patients with VSD, aortic valve prolapse and aortic regurgitation. In patients whose VSD is located underneath the non coronary sinus, the atrioventricular node is in close proximity to the non-coronary cusp and is therefore extremely vulnerable to damage if direct closure of the VSD is undertaken. Also, the non-coronary cusp is extensively involved by secondary pathological changes which diminish the chances of a successful repair. In our patient, patch closure of the VSD and failure to simultaneously plicate the non coronary sinus added to the redundancy of tissues of the non-coronary sinus. This resulted in failure to restore the normal position of the aortic annulus and caused severe aortic regurgitation.

Early diagnosis of this disease and clear delineation of aortic valve prolapse and aortic regurgitation can be easily achieved with transthoracic echocardiography. Close follow-up is important to diagnose the onset of aortic valve prolapse. As soon as the aortic valve shows signs of prolapse the patient should be referred for repair before the onset of significant aortic regurgitation. Late diagnosis in our patient contributed significantly to the severity of his aortic regurgitation.

Finally, a Ross procedure can easily be performed in this group of patients in spite of the presence of significant pathology in the region of the right ventricular outflow tract and pulmonary valve.

REFERENCE [1] Yacoub MH, Khan H, Stavri G, Shinebourne E, Radley-Smith R. Anatomic correction of the syndrome of prolapsing right coronary aortic cusp, dilatation of sinus of valsalva, and ventricular septal defect. J Thorac Cardiovasc Surg.

1997;113:253â&#x20AC;&#x201C; 261.

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Letter to the Editor

Letter by Elshazly Regarding Article “Primary Coronary Angioplasty for ST-Elevation Myocardial Infarction (STEMI) in Qatar: First Nationwide Program” Mohamed B Elshazly* Department of Medicine, Institution, Osler Residency Program, Johns Hopkins Hospital, Balimore, MD, USA *Email: melshaz1@jhmi.edu

http://dx.doi.org/ 10.5339/gcsp.2013.9 Submitted: 2 January 2013 Accepted: 31 March 2013 q 2013 Elshazly, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Dear Editor: In their article “Primary Coronary Angioplasty for ST-Elevation Myocardial Infarction (STEMI) in Qatar: First Nationwide Program”, Gehani et al. developed an impressive plan to implement primary percutaneous coronary intervention (PCI) for the first time in Qatar.1 As a graduate of Weill Cornell Medical College in Qatar, I have witnessed immense improvement in the Qatari healthcare system over the past few years. From building the new state of the art Heart Hospital to developing the first unified nationwide primary PCI program in the world, there is no doubt that Qatar has made an immense leap towards implementing world-class cardiovascular healthcare in the Middle East.

The authors are applauded for their immense efforts to develop such a complicated protocol from scratch. Strategies such as 12-lead ECG transmission by EMS, a single easily reachable dedicated phone line, near campus accommodations for the primary PCI team and a nationwide awareness program are all crucial to sustaining a door to balloon (DTB) time of ,90 min, which is the essence of primary PCI2,3. Given the importance of achieving DTB time ,90 min for the success of this program, it is important to outline some areas for improvement.

Accurate diagnosis of STEMI is the most important step of this program especially in the extremely busy ED and Primary Health Centers of Hamad General Hospital (HGH). Most patients with STEMI present to the ED and outpatient clinics, instead of calling EMS. Therefore, efficient standard protocols for the evaluation of patients presenting with chest pain should be developed. These protocols should outline specific time intervals from first contact with medical personnel to arrival at the catheterization lab such as the Mayo clinic protocol4 and should be printed and distributed to all healthcare providers in Qatar. An online training course about primary PCI should be offered to all medical personnel.

In the current high-tech era, an electronic medical record (EMR) system is imperative for faster diagnosis of equivocal cases of STEMI. For example, rapid access to baseline ECGs can help differentiate a new Left Bundle Branch Block from an old one. Therefore, developing an EMR for ECGs that is easily accessible to the on call interventional cardiologist is very crucial.

HGH is the biggest hospital in the country. It incorporates all specialties other than cardiology, which has relocated to the Heart Hospital. HGH patients who develop STEMI will require transfer to the Heart Hospital for primary PCI. This is a time consuming process that will waste 20– 30 min of transport.

Having a fully equipped catheterization lab at HGH as well as other large hospitals such as Al-Wakrah is an idea worth considering for maintaining a DTB ,90 min. Training specialized transport teams that understand the concept of “time is life” in STEMI is also crucial.

Cite this article as: Elshazly MB. Letter regarding article “Primary coronary angioplasty for ST-Elevation Myocardial Infarction in Qatar: First nationwide program”, Global Cardiology Science & Practice 2013:9 http://dx.doi.org/10.5339/gcsp.2013.9

Page 2 of 2 Elshazly. Global Cardiology Science and Practice 2013:9

Finally, it is important to recognize that timely feedback starting at day one will help significantly improve this program at a faster pace. 24 – 48 h audits and feedback on each case will help identify flaws that can be fixed reliably and quickly.

REFERENCES [1] Gehani A, Al Suwaidi J, Arafa S, Tamimi O, Alqahtani A, Al-Nabti A, Arabi A, Aboughazala T, Bonow RO, Yacoub M. Primary coronary angioplasty for ST-Elevation Myocardial Infarction in Qatar: first nationwide program. Global Cardiol Sci Pract. 2012;23, http://dx.doi.org/10.5339/gcsp.2012.23 [2] Authors/Task Force Members, Steg PG, James SK, Atar D, Badano LP, Blo¨mstrom-Lundqvist C, Borger MA, Di Mario C, Dickstein K, Ducrocq G, Fernandez-Aviles F, Gershlick AH, Giannuzzi P, Halvorsen S, Huber K, Juni P, Kastrati A, Knuuti J, Lenzen MJ, Mahaffey KW, Valgimigli M, van’t Hof A, Widimsky P, Zahger D. ESC guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: the task force on the management of ST-segment elevation acute myocardial infarction of the European Society of Cardiology (ESC). Eur Heart J. 2012;33(20):2569– 2619.

[3] O’Gara PT, Kushner FG, Ascheim DD, Casey DE Jr, Chung MK, de Lemos JA, Ettinger SM, Fang JC, Fesmire FM, Franklin BA, Granger CB, Krumholz CB, Linderbaum JA, Morrow DA, Newby LK, Ornato JP, Ou N, Radford MJ, Tamis-Holland JE, Tommaso JE, Tracy CM, Woo YJ, Zhao DX, CF/AHA Task Force. 2013 ACCF/AHA Guideline for the Management of ST-Elevation Myocardial Infarction: Executive Summary: A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2012.

[4] Nestler DM, Noheria A, Haro LH, Stead LG, Decker WW, Scanlan-Hanson LN, Lennon RJ, Lim CC, Holmes DR Jr, Rihal CS, Bell MR, Ting HH. Sustaining improvement in door-to-balloon time over 4 years: the Mayo Clinic ST-Elevation Myocardial Infarction Protocol. Circu Cardiovasc Qual Outcomes. 2009;2(5):508 –513.

OPEN ACCESS

Letter to the Editor

Response to the letter of Elshazly Abdurrazzak Gehani* Consultant Interventional Cardiologist, Director, Primary Coronary Angioplasty Program, Heart Hospital, Hamad Medical Corporation, Doha, Qatar *Email: aa.gehani@gmail.com

http://dx.doi.org/ 10.5339/gcsp.2013.12 Submitted: 30 March 2013 Accepted: 31 March 2013 q 2013 Gehani, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Dear Editor, Thank you for forwarding the letter of Dr. Mohamed Elshazly, in relation to our paper “Primary Coronary Angioplasty for ST-Elevation Myocardial Infarction (STEMI) in Qatar: First Nationwide Program”. A particular delight is that Dr. Mohamed is a previous student with us from Weill Cornell Medical College in Qatar. His knowledge of the health care system in Qatar was reflected in the good points he raised.

Dr. Mohamed touched on important issues that should guarantee a successful primary PCI program, especially in relation to two issues.

One is the awareness of all health care professionals of how to timely access and refer patients with STEMI, aiming at a door-to-balloon Time (DBT) of , 90 min. This was outlined in our paper and practical protocols similar to that of the Mayo Clinic1 are being prepared.

The second issue is the protocols and procedures to guarantee full compliance with the speed at every step during the diagnosis, referral and transfer of the patients to the Cardiac Catheterization Laboratory.

Another issue which Dr. Mohamed has also touched on, and which is of major interest to us, is the awareness of the patients themselves to the possible symptoms of “heart attack”. This is because in Qatar, as in many other countries, the patient delay in reporting the symptoms and seeking medical attention is one of the major causes of delay.

Furthermore, in Qatar as in many countries, patients tend to transport themselves to the hospital, rather than call the ambulance. The disadvantage of this practice is that they may land themselves in a unit without a primary PCI facility, or one with low awareness of the importance of PPCI and the time limits related to it.

Admittedly this out-of-hospital delay is vital, but is not reflected in the DBT, since the latter evaluates the time from moment the patient seeks medical advice to balloon dilatation of the infarct related artery in the cath lab. DBT is a measure of the in-hospital, or healthcare delay, rather than the prehospital delay, which can be quite significant.

It may sound odd, but some large registries have suggested that the adjusted in-hospital mortality does not increase significantly with increasing pre-hospital delay. However, there is some evidence that the results obtained by DBT are affected by the pre-hospital delay. An intriguing relationship between the effect of pre-hospital and in-hospital delay times on the outcome of primary-PCI patients was depicted in a study by Brodie et al.2, using data from the CADILLAC and HORIZONS – AMI trials. They revealed that the DBT made a larger impact on survival in patients who presented early after the appearance of symptoms (short pre-hospital delay) than who came late.

This makes sense since the infarction process does not start when the patient arrives to the hospital, but when, or even before the symptoms are appear. It is for this reason that we emphasized the importance of an awareness program for the public. However, Dr. Mohamed’s idea of an online training course for all health care professionals in Qatar is worth considering as part of our national awareness program for both the public and the health care professionals. The other note about timely feedback and regular audit to identify and fix flaws reliably and quickly is also a good addition that have been already considered.

Cite this article as: Gehani A. Response to the letter of Elshazly, Global Cardiology Science & Practice 2013:12 http://dx.doi.org/10.5339/gcsp.2013.12

Page 2 of 2 Gehani. Global Cardiology Science and Practice 2013:12

Acknowledgements We thank Dr. Mohamed for his valuable comments and for the editors for giving us the opportunity to respond.

REFERENCES [1] Nestler DM, Noheria A, Haro LH, Stead LG, Decker WW, Scanlan-Hanson LN, Lennon RJ, Lim CC, Holmes DR Jr, Rihal CS, Bell MR, Ting HH. Sustaining improvement in door-to-balloon time over 4 years. The mayo clinic ST-elevation myocardial infarction protocol. Circ Cardiovasc Qual Outcome. 2009;2(5):508 â&#x20AC;&#x201C;513.

[2] Brodie BR, Gersh BJ, Stuckey T, Witzenbichler B, Guagliumi G, Peruga JZ, Dudek D, Grines CL, Cox D, Parise H, Prasad A, Lansky AJ, Mehran R, Stone GW. When Is door-to-balloon time critical? Analysis from the HORIZONS-AMI (Harmonizing Outcomes with Revascularization and Stents in Acute Myocardial Infarction) and CADILLAC (Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications) Trials. J Am Coll Cardiol. 2010;56:407â&#x20AC;&#x201C;413.

TABLE OF CONTENTS: VOL 2013 (1) Editors’ page Robert B Bonow, Iacopo Olivitto, Magdi H Yacoub

Acute coronary syndrome in the Middle East: The importance of registries for quality assessment and plans for improvement Jassim Al Suwaidi

CADUCEUS, SCIPIO, ALCADIA: Cell therapy trials using cardiac‐derived cells for patients with post myocardial infarction LV dysfunction, still evolving Magdi H Yacoub, John Terrovitis

The zebrafish model system in cardiovascular research: A tiny fish with mighty prospects Robert B Bonow, Iacopo Olivitto, Magdi H Yacoub

Self‐assembled metal‐organic polyhedra (MOPs): Opportunities in biomedical applications Mohamed H Alkordi

Temperature management in cardiac surgery Hesham Saad, Mostafa Aladawy

Science and practice of arrhythmogenic cardiomyopathy: A paradigm shift Mohamed ElMaghawry, Federico Migliore, Nazar Mohammed, Despina Sanoudou, Mohammed Alhashemi

An electronic medical record-linked biorepository to identify novel biomarkers for atherosclerotic cardiovascular disease Zi Ye, Fara S Kalloo, Iftikhar J Kullo

Further insights into the syndrome of prolapsing non-coronary aortic cusp and ventricular septal defect Akhlaque N Bhat, Ahmad Sallehuddin, Mohammad Riyas, Reyaz Ahmad Lone, Pawel Tyrsarowski, Suresh Kumar, Jiju John, Pradeep Bhaskar, Syed Zin, Magdi H Yacoub

Letter regarding article "Primary coronary angioplasty for ST‐Elevation Myocardial Infarction in Qatar: First nationwide program" Mohamed Badreldin Elshazly

Response to the letter of Elshazly Abdurrazzak Gehani

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