STEM Today

Special Edition on Impaired Performance Due to Reduced Muscle Mass, Strength & Endurance

STEM TODAY February 2016, No. 5

Muscle Protein Turnover During Long Duration Space Flight Prolonged space flight足induced alterations in the structure and function of human skeletal muscle fibres Exercise Countermeasures Project (ECP) Mission

STEM TODAY February 2016 , No. 5

CONTENTS Special Edition on Risk of Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance Human Spaceflight Evidence Effects of Sex and Gender on Adaptation to Space: Musculoskeletal Health NASA's Muscle Risk revised MLDs Genetics of muscle strength and power IGF足1 colocalizes with muscle satellite cells following acute exercise in humans Muscle Atrophy Research and Exercise System ( MARES ) HUMAC NORM Exercise Countermeasures Project (ECP) Mission Blood flow足restricted exercise in space

Editorial Editor: Mr. Abhishek Kumar Sinha Advisor: Mr. Martin Cabaniss

Cover Page Good Morning From the International Space Station NASA astronaut Scott Kelly (@StationCDRKelly), currently on a year-long mission on the International Space Station, took this photograph of a sunrise and posted it to social media on Aug. 10, 2015. Kelly wrote, "GoodMorning to those in the western USA. Looks like there’s a lot going on down there. #YearInSpace" The space station and its crew orbit Earth from an altitude of 220 miles, traveling at a speed of approximately 17,500 miles per hour. Because the station completes each trip around the globe in about 92 minutes, the crew experiences 16 sunrises and sunsets each day.

Image Credit: NASA

Back Cover First Flower Grown in Space Station’s Veggie Facility On Jan. 16, 2016, Expedition 46 Commander Scott Kelly shared photographs of a blooming zinnia flower in the Veggie plant growth system aboard the International Space Station. Kelly wrote, "Yes, there are other life forms in space! #SpaceFlower #YearInSpace" This flowering crop experiment began on Nov. 16, 2015, when NASA astronaut Kjell Lindgren activated the Veggie system and its rooting "pillows" containing zinnia seeds. The challenging process of growing the zinnias provided an exceptional opportunity for scientists back on Earth to better understand how plants grow in microgravity, and for astronauts to practice doing what they’ll be tasked with on a deep space mission: autonomous gardening. In late December, Kelly found that the plants "weren’t looking too good," and told the ground team, "You know, I think if we’re going to Mars, and we were growing stuff, we would be responsible for deciding when the stuff needed water. Kind of like in my backyard, I look at it and say ’Oh, maybe I should water the grass today.’ I think this is how this should be handled."

The Veggie team on Earth created what was dubbed "The Zinnia Care Guide for the On-Orbit Gardener," and gave basic guidelines for care while putting judgment capabilities into the hands of the astronaut who had the plants right in front of him. Rather than pages and pages of detailed procedures that most science operations follow, the care guide was a one-page, streamlined resource to support Kelly as an autonomous gardener. Soon, the flowers were on the rebound, and on Jan. 12, pictures showed the first peeks of petals beginning to sprout on a few buds.

Image Credit: NASA. STEM Today , February 2016

Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance

Impaired Performance Due to Reduced Muscle Mass,Strength, and Endurance There is a growing research database that suggests that skeletal muscles, particularly postural muscles of the lower limb, undergo atrophy and structural and metabolic alterations during space flight. However, the relationships between in-flight exercise, muscle changes, and performance levels are not well understood.

Human Spaceflight Evidence Mercury and Gemini Programs The initial biomedical problem faced by Project Mercury (which ran from 1959-1963) was establishment of selection criteria for the first group of astronauts. Medical requirements for the Mercury astronauts were formulated by the NASA Life Sciences Committee, an advisory group of distinguished physicians and life scientists.

Final selection criteria included results of medical testing as well as the candidates’ technical expertise and experience. Aeromedical personnel and facilities of the Department of Defense (DoD) were summoned to perform the stress and psychological testing of astronaut candidates. The screening and testing procedures defined for the selection of Mercury astronauts served as the basis for subsequent selection of Gemini and Apollo astronauts when those programs were initiated. While the Mercury flights were largely demonstration flights, the longest Mercury mission lasting only approximately 34 hours, Project Mercury clearly demonstrated that humans could tolerate the spaceflight environment without major acute physiological effects, and some useful biomedical information was obtained, which included the following : • Pilot performance capability was unaltered by spaceflight.

• All measured physiological functions remained within acceptable normal limits.

• No signs of abnormal sensory or psychological responses were observed.

• The radiation dose received was considered insignificant from a medical perspective.

• Immediately after landing, an orthostatic rise in heart rate and drop in systemic blood pressure were noted, which persisted for 7 to 19 hours post-landing.

Because of the short mission durations of Project Mercury, there was little concern about loss of musculoskeletal function; thus, no exercise hardware or protocols were developed for use during flight. However, the selection criteria ensured that astronauts were in excellent physical condition before flight.

Biomedical information acquired during the Mercury flights provided a positive basis on which to proceed with the next step, the Gemini Program, which took place during the 20 months from March of 1965 to November of 1966. The major stated objective of the Gemini Program was to achieve a high level of operational confidence with human spaceflight. To prepare for a lunar landing mission, three major goals had to be realized, namely,[1] to accomplish rendezvous and docking of two space vehicles; [2] to perform extravehicular activities and to validate human life support systems and astronaut performance capabilities under such conditions; and [3] (germane to this report) to develop a better understanding of how humans tolerate extended periods of weightless flight exposure. Thus, Project Gemini provided a much better opportunity to study the effects of microgravity on humans. In the 14-day Gemini 7 flight, salient observations were undertaken to more carefully examine the physiological and psychological responses of astronauts as a result of exposure to spaceflight and the associated microgravity environment.

The Gemini Program resulted in approximately 2000 man-hours of weightless exposure of U.S. astronauts.

Additional observations included the presence of post-flight orthostatic intolerance that was still present for up to 50 hours after landing in some crewmembers, a decrease in red cell mass of 5-20% from preflight levels, and radiographic indications of bone demineralization in the calcaneus. No significant decrements in performance of mission objectives were noted, and no specific measurements of muscle strength or endurance were obtained that compared pre-flight, in-flight, and post-flight levels.

Apollo Program The Apollo (1968-1973) biomedical results were collected from 11 crewed missions that were completed within the five-year period of the Apollo Program, from pre-lunar flights (missions 7 through 10); the first lunar landing (mission 11), and five subsequent lunar exploratory flights (missions 12 through 17). Apollo 13 did not complete its intended lunar landing mission because of a pressure vessel explosion in the Service Module. Instead, it returned safely to Earth after attaining a partial lunar orbit.

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance Essential to the successful completion of the Apollo Program was the requirement for some crewmembers to undertake long and strenuous periods of extravehicular activity (EVA) on the lunar surface. Naturally, there was concern about the capability of crewmembers to accomplish the lunar surface excursions planned for some of the Apollo missions. Although reduced lunar gravity was expected to make some tasks less strenuous, reduced suit mobility coupled with a complex and ambitious timeline led to the prediction that metabolic activity would exceed resting levels for extended periods. Because the nature and magnitude of physiological dysfunction resulting from microgravity exposure had not yet been established (and is still not concisely defined), suitable physiological testing was completed within the constraints of the Apollo Program to determine whether crewmember physiological responses to exercise were altered as a consequence of spaceflight.

Initial planning for the Apollo Program included provisions for in-flight measurements of salient parameters of concern, including physiological responses to exercise. However, the fire in the Apollo 204 spacecraft (also known as Apollo 1), fatal to astronauts Grissom, White, and Chaffee, resulted in the initiation of changes in the program by NASA management that eliminated such prospects. Thus, investigators were left with only the possibility to conduct preflight and post-flight exercise response studies and to assume that these findings reflected alterations of cardiopulmonary and skeletal muscle function secondary to microgravity exposure. It was realized early on that within the context and constraints imposed by the realities of the Apollo missions, the inability to control certain experimental variables would present challenges to many biomedical investigations.

First, re-adaptation to Earth gravity begins immediately upon re-entry into the Earth’s gravitational field, which likely changes key physiological responses from their measurements during spaceflight. Second, crew recovery procedures introduced additional challenges to a well-controlled experiment design, as Apollo crewmembers spent variable amounts of time in an uncomfortably warm spacecraft bobbing in the ocean, and additionally, orbital mechanics constraints on re-entry times imposed crew recovery times that prevented the possibility of conducting pre- and post-flight testing within a similar circadian schedule. The impact of these uncontrollable conditions and that of other physical and psychological stresses could not be separated from responses attributable to microgravity exposure alone. Thus, data related to the physiological responses to exercise stress in Apollo astronauts must be interpreted within this overall context.

No standardized in-flight exercise program was planned for any of the Apollo flights; however, an exercise device (Figure 1) was provided on some missions. Crewmembers, when situated in the Command Module (CM), typically used the exerciser several times per day for periods of 15-30 min. The pre- and post-flight testing consisted of graded exercise stress tests conducted on a bicycle ergometer. Heart rate was used to determine stress levels , and the same heart rate levels were used for pre- and postflight testing. Although the exact duration of each stress level was adjusted slightly (1-2 minutes) for the later Apollo missions to obtain additional measurements, the graded stress protocol included exercise levels of 120, 140, and 160 beats per minute, corresponding to light, medium, and heavy work, respectively, for each individual. For the Apollo 9 and 10 missions, a stress level of 180 beats per minute was added. The entire test protocol was conducted 3 times within a 30-day period before lift-off. Post-flight tests were conducted on recovery (landing) day and once more at 24 to 36 hours after recovery.

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance During each test, workload, heart rate, blood pressure, and respiratory gas exchange (O2 consumption, CO2 production, and minute volume) measurements were made. For the Apollo 15 to 17 missions, cardiac output measurements were obtained by the single-breath technique . Arteriovenous oxygen differences were calculated from the measured oxygen consumption and cardiac output data.In brief, reduced work capacity and oxygen consumption of significant degree was noted in 67% (18 of 27) of the Apollo crewmembers tested on recovery. This decrement was transient, and 85% of those tested (23 of 27) returned to preflight baseline levels within 24- 36 hours. A significant decrement in cardiac stroke volume was associated with diminished exercise tolerance. It was not clear whether the exercise decrement had its onset during flight. If it did, the Apollo data did not reveal the precise in-flight time course because of lack of in-flight measurement capabilities. The astronauts’ performance on the lunar surface provided no reason to believe that any serious exercise tolerance decrement occurred during flight, except that related to lack of regular exercise and muscle disuse atrophy.

The studies completed during Apollo, although less than optimal, left no doubt that a decrement in exercise tolerance occurred in the period immediately after landing, although it is believed that such decrements were not present during surface EVA. It seems likely that multiple factors are responsible for the observed decrements. Lack of sufficient exercise and development of muscle disuse atrophy probably contributed. Catabolic tissue processes may have been accentuated by increased cortisol secretion as a consequence of mission stress and individual crewmember reaction to such stress. Additional factors associated with the return to Earth’s gravity may also be implicated. Thus, the observed diminished stroke volume (cardiac output) is certainly contributory and, in turn, is a reflection of diminished venous return and contracted effective circulating blood volume induced by spaceflight factors . Skeletal muscle atrophy is mentioned with respect to its possible contribution to exercise intolerance, and in some of the later Apollo flights, lower limb girth measurements were completed (data not published) that provided the first evidence for loss of muscle mass in the legs.

Skylab The Skylab Program (May 1973-November 1974) was from the onset intended to provide a life sciences laboratory in space. A significant number of experiments were conducted to provide physiological data from humans exposed to long-duration stays in a microgravity environment. A 56-day ground-based simulation of many of the Skylab experiments, conducted in an environmentally controlled, enclosed chamber, was termed the Skylab Medical Experiments Altitude Test (SMEAT) and represented the first mission. The three subsequent orbital missions were termed Skylab 2, 3, and 4. These three long-duration missions were 28, 56, and 84 days in duration, respectively. Collectively, the Skylab missions achieved a milestone in providing a vast array of human spaceflight biomedical information during missions of longer duration than any previous mission. With respect to the current issue of loss of muscle mass and function, two key studies were performed during the course of the three Skylab orbital missions. First, leg and arm volumes were calculated by measuring the girth (circumference) of

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance contiguous 3-centimeter arm and leg segments, with all the segments treated as a short tapered cylinder, and then summing the segment volumes to obtain the volume of each extremity.

The second study included the first muscle strength measurements by means of a dynamometer . In addition to measurements directly related to skeletal muscle strength and mass, indirect measurements were made that demonstrated that all Skylab crewmembers had a negative nitrogen balance indicative of skeletal muscle attrition. This was also observed 10 years later in short-duration Space Shuttle crewmembers.

Upper and lower limb volumes of the three crewmembers of Skylab 4 are shown in Figure 2. Fluid shifts contributed the largest changes to lower limb volumes, but loss of leg tissue mass is clearly evident, particularly in the Commander. As shown in the graphs, significant loss of leg volume occurs within the first few days of microgravity exposure, while changes in the upper limbs are less remarkable. Upon return to Earth, much of the loss of leg volume is corrected and there is often a short overcorrection or overshoot. Once this fluid shift resolves, the true loss of muscle mass remaining in the legs is revealed that more slowly returns to the baseline or preflight level (see Figure 2, leg during recovery on right side of graph for all three crewmembers).

In the Skylab 4 Commander, the loss in leg volume appears to be nearly 300 cc (Figure 2, topmost graph).

Because the complement of exercise equipment for this mission was the largest (consisting of a cycle ergometer, passive treadmill, and the "Mini gym," modified commercial devices that provided the capability for low-load resistive exercises), losses in muscle mass and strength were less than those in the previous two missions of shorter duration.

During the Skylab Program, exercises and exercise devices were added incrementally and the testing expanded with each mission. This produced a different exercise environment for each flight so that in reality there were three separate but related orbital experiments, each with N = 3. The results from each mission had a significant impact on the next . Pre-flight and post-flight evaluations of muscle strength were performed on the right arm and leg of each crewmember for all three Skylab orbital missions by means of a Cybex isokinetic dynamometer . The protocol completed on each crewmember included a thorough warm-up and 10 maximum-effort full flexions and extensions of the arm at the elbow and of the hip and knee at an angular rate of 45â—Ś /second. The isokinetic leg strength results from all three missions, as well as body weights and leg volumes, are presented in Figure 3.

On Skylab 2, only the bicycle ergometer was available for in-flight exercise, with testing performed 18 days before launch and 5 days after landing. While it was realized that these times were too temporally remote from the flight, this was the best that could be achieved due to schedule constraints. By the time day 5 of the mus-

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance cle testing was completed, some recovery in function had likely occurred; however, a marked decrement still remained. The decrement in leg extensor strength was nearly 25%; the arms suffered less but also exhibited marked losses (data not shown). The Commander’s arm extensors showed no loss, as he used these muscles in hand This illustrates a fundamental point in muscle conditioning: to maintain the strength of a muscle, it must be stressed to or near the level at which it will have to function. Leg extensor muscles important in standing and providing propulsive forces during walking are capable of generating forces of hundreds of pounds, while the arm extensor forces are measured in tens of pounds.

Forces developed while pedaling a bicycle ergometer are typically tens of pounds and are thus incapable of maintaining leg strength. The bicycle ergometer proved to be an excellent machine for aerobic exercise and cardiovascular conditioning, but it was not capable of developing either the type or level of forces needed to maintain strength for walking under 1 G . Immediately after Skylab 2, work was started on devices to provide adequate exercise to arms, trunk, and legs. A commercial device, termed "Mini Gym," was modified extensively and designated "MK-I." Only exercises that primarily benefited the arms and trunk were achievable with this device. While forces transmitted to the legs were greater than those from the cycle ergometer, they were still limited to an inadequate level, as this level could not exceed the maximum strength of the arms, which represents a fraction of leg strength. A second device, designated "MK-II," consisted of a pair of handles between which up to five extension springs could be attached, allowing development of maximum forces of 25 pounds per foot. These two devices were flown on Skylab 3, and in-flight nutrition support, exercise time, and food were increased. The crew performed many repetitions per day of their favorite maneuvers on the MK-I and, to a lesser extent, on the MK-II. Additionally, the average amount of work performed on the bicycle ergometer was more than doubled on Skylab 3, with all crewmembers participating actively.

It was perceived by Skylab life scientists that a device that allowed walking and running under forces equivalent to Earth gravity would provide more strenuous exercise . Immediately after completion of Skylab 2, work was begun on a treadmill for Skylab 4. As mission preparation progressed, the launch weight of Skylab 4 escalated so much that the final design of the treadmill was constrained by weight limitations. The final weight for the device was a mere 3.5 pounds. This passive device (Figure 4) consisted of a Teflon-coated aluminum walking surface attached to the Skylab iso-grid floor. Four rubber bungee cords provided an equivalent weight of approximately 80 kilograms (175 lbs) and were attached to a shoulder and waist harness worn by crewmembers during use.

By angling the bungee cords so that the user was pulled slightly forward, an equivalent to a slippery hill was created. High loads were placed on some leg muscles, especially in the calf, and fatigue was so rapid that the device could not be used for significant aerobic work because of the bungee/harness design. It was absolutely necessary to wear socks and no shoes to provide a low-friction interface with the Teflon surface.

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance On Skylab 4, the crew used the bicycle ergometer at essentially the same rate as that used on Skylab 3, as well as the MK-I and MK-II Mini Gym exercisers. In addition, they typically performed 10 minutes per day of walking, jumping, and jogging on the treadmill. Food intake had again been increased.

Upon their return to Earth and even before muscle testing, it was apparent that the Skylab 4 crewmembers were in very good physical condition. They were able to stand and walk for long periods without apparent difficulty on the day after landing (R+1), in contrast to the crewmembers from the earlier two missions. Results of strength testing confirmed a surprisingly small loss in leg strength even after nearly 3 months of microgravity exposure (Figure 3). In fact, knee extensor strength increased over the preflight level.

Space Shuttle A variety of investigations related to skeletal muscle function have been completed during the course of the Space Shuttle Program. One of the most comprehensive of these was a suite of investigations accomplished during the Extended Duration Orbiter Medical Project (EDOMP), which was carried out during 1989-1995 with missions of up to 16 days. Studies most relevant to the risk on which this report focuses include the following: DSO 475 - Direct assessment of muscle atrophy and biochemistry before and after short spaceflight; DSO 477 - Evaluating concentric and eccentric skeletal muscle contractions after spaceflight; DSO 606 - Assessing muscle size and lipid content with magnetic resonance imaging after spaceflight; DSO 617 - Evaluating functional muscle performance.

The collective specific aim of DSO 477 and DSO 617 was to evaluate functional changes in concentric and eccentric strength (peak torque) and endurance (fatigue index) of the trunk, arms, and legs of crewmembers before and after flight. LIDOr dynamometers located at the Johnson Space Center and at both the prime and contingency landing sites were used to evaluate concentric and eccentric contractions before and after flight.

Test subjects in this study exercised during flight for various durations, intensities, and numbers of days on the original Shuttle treadmill (Figure 5) (as opposed to the EDO treadmill, which flew on later Shuttle missions and was the basis for the ISS treadmill) as part of separate in-flight investigations. Exercise protocols included continuous and interval training, with prescriptions varying from 60% to 85% of preflight VO2-max as estimated from heart rate (HR).

Some subjects had difficulty in achieving or maintaining their target HR during flight. The speed of this passive treadmill was controlled at seven braking levels by a rapid-onset centrifugal brake (see Figure 5). A harness and bungee/tether system was used to simulate body weight by providing forces equivalent to an approximate 1-G body mass. Subjects on this non-motorized treadmill were required to walk and run at a positive percentage grade to overcome mechanical friction. Study participants were familiarized with the LIDOr test protocol and procedures approximately 30 days before launch (L-30), after which time six test sessions were conducted.

Three sessions were completed before launch (L-21, L-14, and L-8 days), and three were completed after landing (R+0, R+2, and R+7 to R+10 days). The muscle groups tested are shown in Table 1. Torque and work data were extracted from force-position curves. Peak torque, total work, and fatigue index measured in the three pre-flight test sessions were compared; when no differences were found between sessions, values from the three pre-flight sessions were averaged, and this average was used to compare pre-flight values with those on landing day and during the post-flight period.

Skeletal muscle strength was defined as the peak torque generated throughout a range of motion from 3 consecutive voluntary contractions for flexion and extension. Eccentric contractions are actions of the muscle whereby

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance force is generated while the muscle is lengthening, as opposed to concentric actions characterized by muscle shortening (contracting) while generating force. Skeletal muscle endurance was defined as the total work generated during 25 repetitions of concentric knee exercise, as determined from the area under the torque curve for a complete exercise set. Work was also compared between the first 8 and last 8 repetitions. Endurance parameters were measured during concentric knee flexion and extension activity only. On R+0, significant decreases in concentric and eccentric strength were shown in the back and abdomen compared with the pre-flight means (Table 1).

Concentric back extension and eccentric dorsiflexion remained significantly less than preflight values on R+7. Recovery (an increase in peak torque from R+0 to R+7) was demonstrated for the eccentric abdomen and the concentric and eccentric back extensors. However, the data depicted in Table 1 may be somewhat misleading because in some cases, there were significant differences in strength between crewmembers who exercised during flight versus those who did not.

For example, some crewmembers who exercised during flight actually gained isokinetically measured strength in the ankle extensor/flexor muscles (anterior versus posterior calf muscles, i.e., m. tibialis anterior versus the gastrocnemius/soleus complex) compared with crewmembers who did not exercise and who actually showed a decrease in isokinetically measured strength in these muscles (Figure 6).

With respect to endurance, the majority of the decrease in total quadriceps work occurred on R+0. This result likely reflects significant loss in the first third of the exercise bout (-11%). The declines in peak torque at the faster endurance test velocities are consistent with changes seen at the slower angular velocity used during the strength tests. Torque for the quadriceps at 75â—Ś /s was 15% less than preflight values but was 12% less than the pre-flight mean at 60â—Ś /s for the hamstrings. Endurance data showed little difference between pre-flight and R+7 test results, suggesting that crewmembers had returned to baseline by 1 week after landing.

Additionally, subjects who did exercise during flight compared with those who did not had significantly greater (p < 0.05) losses within 5 hours of landing in concentric strength of the back, concentric and eccentric strength

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance of the quadriceps (30◦ /sec), and eccentric strength of the hamstrings relative to the respective preflight values (data not shown here). Non-exercisers also had significantly less concentric strength of the quadriceps at 75◦ /s and lower total work extension, work first-third flexion, and work last-third extension immediately after landing than before flight. The conclusions reached by the investigators were that the data indicate that muscles are less able to maintain endurance and resist fatigue after spaceflight, as well as that exercise may prevent decrements in these aspects of endurance .

Conversely, crewmembers who exercised during flight had greater losses in trunk muscle strength measured at landing compared with the non-exercising group (Figure 7). However, preflight strength in trunk flexion and extension was substantially greater in the exercising group than in the non-exercising group. Apparently, treadmill exercise did not prevent decrements in trunk strength after 9-11 days of spaceflight, and the investigators offered the explanation that preservation of muscle function may be limited only to those muscles that are effectively used as part of the exercise regimen.

The specific aim of DSO 475, "Direct Assessment of Muscle Atrophy Before and After Short Spaceflight," was to define the morphological and biochemical effects of spaceflight on skeletal muscle fibers . To obtain myofiber biochemical and morphological data from Space Shuttle crewmembers, biopsies were conducted once before flight (L->21 days) and again on landing day (R+0). The subjects were eight crewmembers, three from a 5-day mission and five from an 11-day mission. Biopsies of the mid-portion of the m. vastus lateralis were obtained by means of a 6-mm biopsy needle with suction assistance. Muscle fiber crosssectional area (CSA), fiber distribution, and number of capillaries were determined for all crewmembers before and after flight.

The CSAs of slow-twitch (Type I) fibers in post-flight biopsies were 17% and 11% lower than those in preflight biopsies for 11- and 5-day flyers, respectively. Similarly, the CSAs of fast-twitch (Type II) fibers were 21% and 24% lower in post-flight compared with pre-flight biopsies for 11- and 5-day flyers. Due to the extremely small sample sizes, these numbers do not reflect significant differences but nevertheless provide evidence that space flight-induced muscle atrophy occurs at the cellular level. Interestingly, when samples were further analyzed for changes in Type II sub-types, significant CSA reductions were detected in Type IIA (-23%) and Type IIB (-36%) fibers from crewmembers involved in the 11-day mission. The relative proportions of the Type I and Type II fibers were different before and after the 11-day mission; the fiber distribution followed the same trend after the 5-day mission (increased Type II and decreased Type I fibers compared with pre-flight), but the sample size was too small to reach statistical significance. This shift is consistent with the observed reduction in the number of individual muscle fibers that expressed the Type I myosin heavy chain protein.

While no specific enzymatic activities involved in energy metabolism were found to be significantly different in muscle biopsy samples from returning crewmembers, the glycolytic/oxidative enzyme ratio of αglycerophosphate dehydrogenase/succinate dehydrogenase activity was found to be increased , suggesting a shift resulting in decreased oxidative and increased glycolytic capacity in muscle fibers. The implication of such a shift is the potential of reduced fatigue resistance of the muscle during work. The number of capillaries per fiber was significantly reduced after 11 days of spaceflight. However, because the mean fiber size was also reduced, the number of capillaries per unit of CSA of skeletal muscle tissue remained the same . Atrophy of both major myofiber types, with atrophy of Type II > Type I, is somewhat different from the more selective Type I myofiber atrophy observed in unloaded Sprague-Dawley and Wistar rat muscle , representing an uncommon case in which differences exist between responses of human and murine skeletal muscle. The purpose of DSO 606, "Quantifying Skeletal Muscle Size by Magnetic Resonance Imaging (MRI)," was to non-invasively quantify changes in size, water, and lipid composition in antigravity (leg) muscles after spaceflight. This experiment was the first attempt to measure limb volumes before and after flight since the less sophisticated methods of measuring limb girths during the Apollo and Skylab programs were used. The subjects included four Space Shuttle crewmembers from an 8-day mission. All subjects completed three pre-flight tests and two post-flight tests at R+1 and R+15/16. Testing involved obtaining a 1.5-Tesla MRI scan of the lower body. Multi-slice axial images of the leg were obtained to identify and locate various muscle groups. Muscle volumes for the calf, thigh, and lumbar regions were measured to assess the degree of skeletal muscle atrophy. Significant reductions were observed in the anterior calf muscles (-3.9%), the gastrocnemius/soleus muscles (-6.3%), hamstrings (-8.0%), and intrinsic back muscles (-10.3%). After two weeks of recovery, some residual atrophy still persisted. These whole muscle measures along with the cellular measurements clearly established that muscle atrophy begins rapidly in the unloaded environment of space and accounts, at least in part, for the observed losses in muscle strength.

The EDOMP provided significant knowledge on the effects of spaceflight on human physiology and, specifically, on alterations in skeletal muscle mass, strength, and function. Once again, losses of skeletal muscle mass, strength, and endurance were documented, despite the use of exercise countermeasures in some cases. However, some findings were encouraging, particularly indications that in-flight exercise does have a positive effect

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance in countering losses in muscle strength at least in the legs (see Table 1 and Figure 6), as predicted from the results of the 84-day Skylab 4 mission when multiple modes of exercise were used, including a unique "treadmill" device (see Figure 4). This unusual treadmill provided loads of sufficient magnitude to the legs in a manner approaching resistance exercise. However, the data provided by MRI volume studies indicate that not all crewmembers, despite utilization of various exercise countermeasures, escape the loss in muscle mass that has been documented during most of the history of U.S. human spaceflight since Project Mercury.

In addition to the EDOMP, the Life and Microgravity Spacelab (LMS) experiments represent another hallmark Space Shuttle Program initiative to better understand the physiological adaptations to spaceflight. LMS was conducted aboard STS-78 and involved four crewmember subjects who participated in each of the following muscle physiology studies during their 17-day mission.

Studies of muscle function and physiology Muscle atrophy was assessed during LMS by MRI using procedures similar to those used for STS-47 .

Post-flight muscle volumes were significantly reduced (7-12%) in back muscles, quadriceps, gastrocnemius, soleus, and gluteal muscles on landing day . By R+10, all changes in muscle volume had reverted to preflight levels. The observed reductions in gastrocnemius, soleus, and quadriceps muscles following the 17-day LMS mission were on average larger than those reported for the 8-day STS-47. The MRI results not only directly confirm that muscle atrophy is an early consequence of space flight, but they also suggest that muscle atrophy continues during longer exposures to microgravity. Whole muscle strength was measured in knee extensors and plantar flexors during LMS. The production of force by knee extensors was determined under isoinertial and isometric conditions. Pre-flight and post-flight measurements were obtained with an instrumented leg press device that uses inertial flywheels as the resistance mode. The device could also be locked in place at a 90-degree knee angle for the measurement of maximal isometric force. Consistent with the reported reduction in quadriceps CSA, knee extensor (leg press) strength was reduced post-flight (R+1). Maximal isometric force was reduced by 10.2%, whereas concentric and eccentric strength were reduced by 8.7% and 11.5%, respectively.

In separate experiments involving the same astronaut subjects, calf muscle performance was assessed before, during, and after STS-78 with a torque-velocity dynamometer (TVD) . The TVD was a mission-specific piece of hardware that measured ankle plantar flexion and dorsiflexion strength under isometric or isokinetic (fixed angular velocity) conditions. Angle-specific tests for isometric strength (80, 90, 100 degrees), isokinetic strength at speeds from 30-360 degrees/seconds, and isokinetic endurance were performed before, during and postflight.

In-flight tests were conducted on flight day (FD)2/3, FD8/9, and FD12/13. Post-flight tests were performed on R+2 and R+8. Muscle strength values were reported to be â&#x2030;&#x2C6;50% lower at the first two in-flight time points, but the charges were attributed to issues with the system that secured the TVD in place. The TVD was reported to be "lifting and floating" during testing. The issue was resolved prior to FD12/13 testing, at which time no differences in torque generation compared with pre-flight values were observed. Likewise, post-flight values were not significantly different than pre-flight values.

The authors of the investigation have suggested that the lack of change during 17 days of space flight may have been due to the nature in which the testing was conducted; that is, the in-flight testing may have served as an unexpected, yet effective, exercise countermeasure to protect the calf muscle from strength loss. The three

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance inflight calf muscle test sessions during STS-78 involved making ≈ 525 calf muscle contractions on the TVD , half of which were made at 80% to 100% of each individual’s maximal values. In contrast, the same LMS crew displayed significant deficits in both size and strength of the quadriceps , a muscle group that was not tested during flight. This result suggests that high-intensity muscle contractions, which are performed less than daily, may protect muscle strength during missions of up to 17 days.

Loss of skeletal muscle strength is a consequence not only of reduced muscle size, but also of decreased neural drive and myocellular damage. Studies were performed on the calf muscles (contralateral leg to that used in studies described above) before flight, during flight (four time points), and after flight to separate the causal effects of muscle atrophy from reduced neuromuscular recruitment to address this question. Surface electrodes were placed over the subjects’ gastrocnemius and soleus, and a percutaneous electrical muscle stimulator (PEMS) unit was used to directly cause forced whole-muscle contractions independent of any voluntary input provided by the crew member. No measureable losses in electrically evoked calf muscle performance were observed. However, post-flight (R+8) reductions in force production were observed. Given the lack of change during late in-flight testing (FD16), it was suggested that alterations are likely due to muscle damage due to gravitational reloading of the muscles during normal ambulation. This notion was supported by MRI analyses.

MRI transverse relaxation time (T2) of skeletal muscle is an indicator of increased tissue fluid volume and can be a marker of myocellular damage (inflammation/edema).

In these crewmembers, T2 values were elevated at R+2 and stayed elevated at R+10. Studies of muscle morphology and cellular function. Muscle biopsy samples were obtained from the 4 LMS crew members who participated in the whole-muscle size and function testing . Biopsies were obtained from the gastrocnemius and soleus muscles before flight and again within three hours of landing. Functional analyses of single muscle fibers provide the most direct evidence of space flight-induced changes in the function of the muscle mechanics without the influence of factors such as changes in neuromuscular recruitment patterns or differences in volitional effort. Using calcium-activated individual muscles, any observed alterations in mechanics can be attributed to alterations in the myofiber itself. Individual muscle fibers from the LMS crew were isolated and mounted between a force transducer and a servomotor for analyses. Space flight produced a small decrease (-6%) in type I single-fiber peak calciumactivated force production (Po) in samples from the gastrocnemius.

However, no difference was observed when these measurements were corrected for muscle fiber CSAs. No mean differences were found in Po or fiber CSA for fibers that either expressed type IIa myosin heavy chain (MHC) or co-expressed both type IIa and IIx MHC. While mean differences in fiber mechanics were not observed in subjects as a group, significant changes occurred within individual subjects when subject-by flight analyses were conducted (each subject had a cohort of fibers that were analyzed). In one subject, Po and CSA in Type IIa fibers were reduced by 19% and 12%, respectively. In another subject, Po was reduced by 23% in Type I fibers and 15% in Type IIa fibers, with reductions in fiber CSA of 7% for type I and 12% for type IIa. The investigators point out that the variability in space flight response seems to result, at least in part, from initial fiber size. Fibers with the greatest reduction in size and Po tended to come from the crew members who had larger pre-flight fibers.

In the soleus muscle, a calf muscle adjacent to the gastrocnemius but one that is more slow and oxidative in nature, 91% of muscle fibers expressed only type I MHC before flight . After space flight, the number of Type I fibers decreased to 79%. Space flight also resulted in a 21% decrease in mean Po. This decline in Ca2-activated peak force was paralleled by a 15% decrease in fiber CSA, which indicates that muscle atrophy accounted for most of the loss of function, although a 4% residual loss of Po remained when Po was normalized by individual fiber CSA. Skeletal muscle power is generally viewed as a functional measure of muscle performance because, like most physical tasks that require high levels of exertion, peak values actually occur at submaximal loads. The power of single fibers was measured in a manner similar to the Po measurements; however, instead of the measures being isometric, they were obtained with isotonic load clamps. No significant main effect of space flight was found on muscle power for single fibers from either the gastrocnemius or the soleus muscles. Despite some variability among crew members in the effect of space flight on Po in various muscle fiber types, the overall trend showed that increases in maximal shortening velocity (Vo), which are attributed to decreased thin filament density based on observations from electron microscopy , compensate for the loss of Po to maintain muscle power at the cellular level. Skeletal muscle is a highly metabolic tissue. As is true for muscle size, the intensity and volume of physical activity are also major determinants of the readily adaptable bioenergetic capacity and composition of the muscle. Portions of the biopsy specimens from the gastrocnemius and soleus were used to perform biochemical analyses of oxidative and glycolytic enzymes. Despite some evidence of a metabolic shift toward glycolysis-derived energy sources in biopsy samples after the 11-day STS-32 mission , no differences

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance were detected in citrate synthase, phosporylase, or β-hydroxyacyl-CoA dehydrogenase in samples after the 17day LMS mission . Accordingly, no post-flight changes were observed in muscle glycogen content. Therefore, while space-flight appears to promote a slow-to-fast shift in MHC, there does not appear to be a similar systemic metabolic shift.

Shuttle-Mir and NASA-Mir Programs During the seven NASA-Mir flights, seven U.S. astronauts trained and flew jointly with 12 Russian cosmonauts over a total period of 977 days (the average stay was 140 days) of spaceflight, which occurred during the period from March 1995 to June 1998. The major contribution of the joint U.S./Russian effort on the Mir space station relevant to the current risk topic was the first use of MRI to investigate volume changes in the skeletal muscles of astronauts and cosmonauts exposed to long-duration spaceflight. This began with the first joint mission, Mir-18, and continued until the final Mir-25 mission. The data indicated that loss of muscle volume, particularly in the legs and back, was greater than that in short-duration spaceflight but not as great as the data from short-duration flight may have predicted . A comparison between volume losses in the selected muscle groups in short-duration spaceflight on the Space Shuttle, long-duration (119 d) bed rest, and a (115 d) Shuttle-Mir mission demonstrates the relative time course of the losses (Figure 8).

There is a good correlation between long-duration bed rest and spaceflight of similar duration except that losses in the back muscles are much lower with bed rest. This result likely reflects the use of these muscles during bed rest to adjust body position and to reduce the potential for vascular compression and tissue injury.

During spaceflight, the back muscles are apparently less used because they do not have to support the upright body against Earthâ&#x20AC;&#x2122;s gravity and are not used with great force to make positional adjustments of the body as they are during the recumbency of bed rest.

International Space Station (ISS) The first ISS crew (Expedition 1) arrived in October 2000; since then, there have been 40 Increments. Two

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance major research study complements addressing the Risk of Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance were conducted during the early phase of ISS exercise countermeasures evaluation.

During these complements, subjects had access to the CEVIS cycle ergometer, the TVIS treadmill, and, importantly, the interim Resistive Exercise Device (iRED). iRED was an elastomer-based piece of resistance exercise hardware. This device was limited to a 300-pound maximum load. By comparison, the currently available ARED has a 600 pound load capacity. One investigation during the "iRED era" involved four ISS astronauts with mission durations of 161-194 days , and the other studied 10 astronauts and cosmonauts whose mission durations spanned a very similar 161-192 days in space . Each of these studies investigated changes in muscle size and strength, with one focusing on a larger array of muscle groups and the other performing a diverse set of whole muscle, cellular, and biochemical measures on the postural muscles of the calf.

Initial post-landing MRI data for both studies were obtained on a relatively similar timeline (5 Âą 1 and 4 Âą 1 days). Calf muscles were found to undergo greater decrements than thigh muscles (10-18% and 4-7% loss, respectively). Both studies reported the greatest loss in the soleus muscle (%) with less, but substantial, decrements in the gastrocnemius . Approximately half of the loss of muscle mass still existed up to two weeks following return to Earth . Although these MRI results highlighted a clear need for improved countermeasures hardware and/or strategies, they also demonstrate an incremental improvement in the countermeasures targeted to mitigating muscle loss compared with the more dramatic reductions observed during Shuttle-Mir missions . Muscle strength measurements in ISS crew members were not measured until approximately one week following landing. Nonetheless, strength losses accompanied muscle atrophy in both upper and lower leg muscles. Isokinetic strength measures in thigh knee extensor muscles revealed a 10% loss , whereas calf muscle strength was reduced by 24% , again demonstrating that the calf muscles are most susceptible to space-flightinduced decrements. The drop in torque production of the calf muscles was observable across the entire range of speeds used from 0-300 degrees/second . This reduction in calf muscle performance, initially measured one week post-landing, persisted until at least two weeks after return despite a partial restoration in muscle volume . Taken together, the results suggest that impairments in muscle strength are likely perturbed by muscle damage and/or soreness derived from gravitational reloading of the muscles.

Various structural and functional analyses were performed on muscle biopsy samples from the gastrocnemius and soleus muscles from nine ISS crewmembers. Mirroring what was observed at the whole-muscle level, individual muscle fiber analyses also revealed muscle atrophy at the cellular level . Crosssectional areas were determined in individual muscle fibers that were set at a standardized sarcomere length. The number of slow type I muscle fibers was reduced by 24% and 33% in the gastrocnemius and soleus muscles, respectively. The number of fast type II fibers (of all subtypes, excluding hybrids) was also reduced in the soleus muscle (29%) but was unchanged in the gastrocnemius. Measures of muscle fiber mechanics clearly demonstrated decrements of function at the cellular level . Peak calcium activated force, maximal shortening velocity, and peak power were all markedly reduced in post-flight samples taken from gastrocnemius and soleus muscles, with the most dramatic change being a 45% loss of power production in type I soleus muscles. This is in stark contrast to responses to short-term Space Shuttle flights, where increases in maximal shortening velocity were able to compensate for reduced force production to maintain peak power levels. Power was also reduced in type II fibers, with reductions to maximal shortening velocity and peak force being contributing factors for fibers from gastrocnemius and soleus muscles, respectively.

In both gastrocnemius and soleus muscles, a clear shift in the contractile machinery was observed with a slowerto-faster phenotype reported . This can be observed from MHC protein expression in the individual fibers that were analyzed for contractile properties. Both gastrocnemius and soleus muscles exhibited reductions in the amount of fibers expressing type I MHC. This corresponded to increases in the percentages of type IIa fibers and type I/IIa hybrid fibers from gastrocnemius muscle. A similar pattern occurred in the soleus muscle, although increases were primarily observed in the various hybrid fibers distributed in a manner such that significant changes were only detected in hybrid fibers grouped together.

Although limitations in the availability and accuracy of iRED loading data prevented investigators from making meaningful analyses of the relationships between resistance training loads and muscle adaptions during these ISS missions, a number of observations were made regarding treadmill running and alterations in the calf muscles . Treadmill use ranged from less than 50 minutes a week to greater than 300 minutes per week. Subjects who ran on the treadmill the most preserved muscle better than those who ran less. When total aerobic exercise (TVIS treadmill + CEVIS bicycle ergometer) was compared with changes in muscle volume, this correlation was lost. Data demonstrating that foot forces are much higher during treadmill running versus cycling aboard ISS support the argument that higher forces are vital to protecting against muscle atrophy during spaceflight.

Results for treadmill use were not restricted to in vivo whole-muscle observations. Subjects who used the TVIS treadmill more than 200 minutes per week generally fared better than those who ran less than 100 minutes per

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance week in terms of single fiber CSA, peak force, and power .

In addition to muscle mass and the function of the cellular contractile proteins, changes to the molecular mechanisms that control energy metabolism also have the potential to negatively affect human performance following exposure to long-duration space flight. Activities of a battery of oxidative and glycolytic enzymes were therefore measured in crewmembers before and after ISS missions . Overall, the observed spaceflight effects on metabolic enzymes in skeletal muscle were minimal. No changes in activities of citrate synthase, β-hydroxyacylCoA, lactate dehydrogenase, or phosphofructokinase were observed in calf muscles following 6 months aboard ISS. Rather, spaceflight and exercise countermeasures play a more limited role in select adaptions to metabolic enzymes in calf skeletal muscles. For example, the mitochondrial enzyme cytochrome oxidase was reduced in spaceflight by 35% in type I soleus muscle in all crewmembers studied. However, this result was entirely accounted for by the crewmembers in the low treadmill use group (less than 100 minutes/week), in which a 59% reduction occurred. Activity levels in the high treadmill use group were unchanged. In short, metabolic adaptations in skeletal muscle appear to be less sensitive to unloading compared with structural and functional changes related to morphology and contractility. Furthermore, countermeasure strategies that are insufficient to fully protect muscle from unloading-induced atrophy appear to be more effective in protecting against changes to the metabolic phenotype of the muscle.

These two major studies point to the need for high load intensity if prevention of muscle mass and strength is to be accomplished. In these early years, both hardware capabilities and reliability certainly contributed to this condition not being met. The iRED science requirement was to provide a load of up to an equivalent of 600 lb (273 kg); however, as mentioned above, the delivered hardware product provided only approximately half of that amount. Ground-based studies have shown that it does produce a positive training effect similar to that of equivalent free weights when used in a high-intensity program , but it will likely not provide sufficient loading in a zero-gravity environment to prevent loss of muscle and bone tissue, as determined from parabolic flight studies . For whole-body resistance exercises, such as squats, oneâ&#x20AC;&#x2122;s own body weight contributes a significant amount of load in a 1-G environment.

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance In the weightlessness of space, this contribution is lost. For this reason, load capacities for resistance exercise devices for use in space must be able to replace the body loads that are lost in the microgravity environment on top of the normal loads that one would use on the ground. Other problems in meeting load requirements were related to failures of the onboard exercise hardware with reduced utilization at other times, as well as use restrictions imposed due to transmission of forces into the structure of the space station itself. In fact, during the first eleven ISS Expeditions, there were only two short periods during Expeditions 3 and 4 when all three U.S. onboard exercise devices (CEVIS, TVIS, and iRED) were capable of being used under nominal conditions (Figure 9). The almost continuously suboptimal availability of exercise equipment likely has had a negative impact on maintenance of crew physical fitness during this time.

Since the time depicted in Figure 9, both the reliability and capability of the ISS exercise countermeasures hardware have continued to mature. The second-generation treadmill (T2) and the Advanced Resistive Exercise Device (ARED, Figure 10) were delivered to ISS in 2009 and 2010, respectively. The T2 allows for motor-driven running speeds up to 15 mph in addition to being able to be used in a passive resistance mode (the user rather than a motor drives the belt against resistance). ARED provides adjustable loads of 600 pounds provided by vacuum canisters that provide a constant force and inertial flywheels that simulate the inertial loads that would be experienced using free weights in 1-G. ARED allows for most multi-joint bar-based resistance exercises to be performed, including the squat, deadlift, heel raise, and bench press. Additionally, ARED can support cable pull exercises with loads up to 150 pounds. ARED was delivered to the ISS with expectations of improving muscle outcome measures due to the additional load capacity and the changes in exercise prescription that this improvement affords.

During ≈6-month ISS missions, iRED crewmembers lost 0.42±0.39 kg of total body lean mass while ARED users gained 0.77±0.30 kg, as determined by whole-body DXA scans (Table 2); a limitation of these measurements is that the mean post-landing time required to obtain these measurements was 13± 2 d and 8±1 d for iRED and ARED crewmembers, respectively. Regardless, a clear trend exists for the improved protection of muscle mass in more recent missions; this is likely due to a combination of enhanced resistance exercise loading (ARED) and improved caloric intake during flight , two key factors in skeletal muscle outcomes during unloading. ARED users lost more fat mass during flight, but because of an increase in muscle mass, ARED crewmembers had a smaller net decrease in total body mass compared with iRED crewmembers.

All United States Operating Systems (USOS; NASA, Japan Aerospace Exploration Agency, European Space

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance Agency, and Canadian Space Agency) crewmembers undergo specific medical requirement testing before and after their ISS missions. Part of this testing includes isokinetic muscle strength and endurance testing of the legs and trunk muscles. Post-flight testing occurs 5- 7 days after landing. In Figure 11, we present results as the percent change from pre-flight for isokinetic strength and endurance testing for crewmembers.

Results are divided into two groups: those who used iRED and those who used ARED during their flight. Isokinetic strength around the knee joint was measured at 60◦ /s. For iRED crewmembers, the mean decrements in knee extensor and knee flexor strength were -13.7% and -19.5%, respectively. ARED users still exhibited losses of knee extensor and flexor strength, but the values were improved at -6.9 and -11.1, respectively. Muscle endurance was measured in knee extensor and flexor muscles based on total work production during a 20 repetition effort at 180◦ /s. The average loss for iRED crewmembers was -10.7% in knee extensors and -8.9% in knee flexors. Mean values for loss of endurance in ARED users were lower than that in iRED users (-7.5% for both knee extension and flexion), but any improvements were more subtle than those for strength. Studies in bed rest analogs for long-duration spaceflight deconditioning have typically shown that calf muscle mass and strength are more difficult to protect than quadriceps muscle mass and strength . Here, authors show that calf muscle strength in ISS crewmembers using iRED was reduced 14.2% compared with pre-flight values. While this value is on par with knee extensor results (-13.7%), the improvement in ARED users’ calf muscle strength loss is more modest (-11.6% versus preflight) than that of ARED users for the knee extensors. Trunk extensor strength losses equaled -7.4% and -5.5% for iRED and ARED, respectively.

The current permissible outcome limit for muscle strength in returning crewmembers is at or above 80% of baseline values (NASA Space Flight Human System Standard Volume 1: Crew Health; NASA-STD-3001). Although ARED-era crewmembers have fared better than iRED-era crewmembers, on average, both groups have losses of less than the 20% standard. However, an examination of the individual data shows that many individuals have lost more than the targeted 20% threshold. It is also important to keep in mind that the medical requirement testing is conducted approximately one week after landing and therefore may not reflect a crewmember’s performance ability in the immediate post-landing time frame. While it would be ideal for all crewmembers to actually return with no loss of strength at all, it is important to note that this would not necessarily be reflective of crew ability to complete mission objectives. The Human Research Program aims to develop more performance-based strength standards that can better be used as benchmarks for mission success. Doing so will not only aid in designing better exercise countermeasure strategies, but will ultimately lead to greater assurance of crewmember safety.

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance While the delivery of ARED to the ISS already appears to be eliciting better strength maintenance, Human Research Program-funded research is beginning to examine how to better utilize ARED to not only improve strength and muscle mass outcomes, but also how to do so with a reduced weekly training volume. The Integrated Resistance and Aerobic Training Study (also known as SPRINT) is implementing a resistance training protocol based on greater intensity and reduced volume. Subjects are performing high load resistance training three days versus six days per week. In addition to pre- and post-flight muscle performance measures such as muscle mass, strength, power, and neuromuscular recruitment, in-flight measures of muscle mass will be tracked for the first time ever using ultrasound technology. A recent investigation examined the effects of iRED versus ARED use onboard the ISS on body composition. Eight iRED users and five ARED users were subjected to DXA analysis prior to flight and again anywhere from 5 to 45 days post-flight (mean 12Âą11 days post-flight). Total body mass was unchanged in both groups; however, lean body mass was increased in ARED users and fat mass was reduced. These data are consistent with the view that ARED use is better for musculoskeletal outcomes following ISS missions; however, the effect of space flight on postural skeletal muscles following space flight is difficult to assess via wholebody lean mass, as the target tissues do not likely represent a large enough portion of the total lean mass pool to detect changes with sufficient accuracy. Authors attempted to determine whether the changes in muscle strength shown in Figure 11 correlated with pre- to post-flight changes in lean body mass and found that changes in strength correlated poorly with changes in total body lean mass. This result may be due to the aforementioned delays in obtaining these measurements post-flight or to the generic nature of total body lean mass changes as opposed to the greater specificity of leg lean mass or, optimally, regional changes in the quadriceps and calf muscle. It appears that MRI and potentially ultrasound imaging technologies are required to adequately detect morphological changes associated with loss of muscle strength. Functional fitness test results for long-duration ISS crewmembers are presented in Table 3. Generally, iRED crewmembers experienced small to moderate decreases in performance of practical exercise tests such as pushups, pullups, bench press, and leg press. For all but one measure, ARED crewmembers fared better than their iRED counterparts, with most outcomes actually showing improvements after spaceflight.

Nutritional regulation of protein metabolism as it pertains to maintenance of muscle mass is a growing research topic with implications for aging populations and those undergoing unloading such as the ISS crew.

Numerous investigations have addressed the roles of protein and amino acid intake in bed rest analogs for long-duration spaceflight, whereas spaceflight data are much more limited. Aboard the ISS, protein intake has well-exceeded the U.S. Recommended Dietary Allowance (0.8 g/kg/d) both in the past (1.1 g/kg/d) and more recently (1.4 g/kg/d) . Total caloric intake has historically been a problem; Stein et al. reported significant decreases in body mass and protein synthesis after long-duration spaceflight on Mir. The reduction in protein synthesis was positively correlated with a decrease in energy intake during flight (r2 =0.86). These findings demonstrate the synergistic, deleterious effect of reduced energy intake on skeletal muscle metabolism and mass during mechanical unloading.

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D ID YOU KNOW ?

E LECTRICAL I MPEDANCE M YOGRAPHY (EIM)

Electrical Impedance Myography (EIM) is a non-invasive, painless clinical technique for the diagnosis and monitoring of a variety of neuromuscular diseases including amyotrophic lateral sclerosis and focal nerve injuries.

It involves the application of a low-intensity alternating current to a muscle group and the measurement of the consequent surface voltage patterns.

In contrast to conventional needle electromyography (EMG) and most standard neurophysiological techniques, EIM does not focus on measuring the inherent electrical activity of the tissues. Rather, similar to diagnostic ultrasound, measurements are made over a small area of interest, with energy being applied to the body and the resultant surface patterns analyzed. Unlike ultrasound, however, in which energy is in the form of sound waves and the main interest is image reconstruction, in the case of EIM, electrical current is used and the output is a set of quantitative parameters describing the state of the muscle, with presently little emphasis on imaging (though this remains possible). Although still in a relatively early stage of development, EIM may have important clinical uses in the future, both as an indicator of neuromuscular disease status and as a diagnostic tool.

All impedance methods rely upon the basic principle that if an alternating current is applied to a substance, energy will be dissipated as it travels through it, thus producing a measurable voltage. Moreover, the timing of the oscillations in the measured voltage will be out of phase with those of applied current. A variety of methods have been developed for the measurement of electrical impedance, such as the Wheatstone bridge, each with certain advantages and disadvantages depending on the frequency range of interest. Electrical impedance methods can be applied to virtually any substance or material, and thus have found widespread use in disciplines as disparate as medicine, metallurgy, geology, and forestry.

As suggested, several mechanisms are likely to account for changes in the impedance values in neuromuscular disease states. The simplest and most obvious explanation is that atrophy and loss of muscle fibers causes the basic "circuitry" of the muscle to change. With atrophy, resistance values will increase and reactance values will decrease. The elevation in resistance is partly due to the fact that the muscle itself is getting smaller, though intra- and extra-cellular fluid changes may also impact this.

Changes in muscle composition also will play a role, with localized edema of muscle or increased fatty infiltration or connective tissue also impacting the measured electrical impedance. Why and how these changes should affect the multifrequency spectrum, however, remains incompletely elucidated. Thus, although simple atrophy likely plays an important role in the changes in signature in disease, it is likely only one piece of the puzzle; for example, disuse states show a very different picture than atrophy due to myopathy or neurogenic change, with relatively reduced resistance and a better preservation of the frequency relationships.

A variety of questions about EIM remains unanswered and demand further study. These include: 1. the detailed mechanisms of how disease impacts the EIM signature and how it relates to standard EMG; 2. the effects of the skin-subcutaneous fat on the impedance measurements when "near-electrode" or rotational measurements are performed and how to account effectively for them; 3. the means of identifying individual muscles while using EIM; 4. the detailed effects of muscle excitation and contraction on the EIM signature.

Reference:

R

Rutkove SB. Electrical Impedance Myography: Background, Current State, and Future Directions. Muscle and nerve. 2009;40(6):936-946.

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Ogunnika OT, Scharfstein M, Cooper RC, Ma H, Dawson JL, Rutkove SB. A Handheld Electrical Impedance Myography probe for the assessment of neuromuscular disease. Conf Proc IEEE Eng Med Biol Soc. 2008; 2008: 3566-9.

Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance Effects of Sex and Gender on Adaptation to Space: Musculoskeletal Health Men and women differ in many aspects of the musculoskeletal system, with men generally having greater muscle and bone mass. Important questions for spaceflight application are whether the time course of loss with unloading is the same for men and women, whether the initial bone or muscle mass influences the rate of loss, whether that rate of loss is linear over an ≈3-year period (the most likely duration of initial exploration-class missions), and whether loss of bone and/or muscle over this period of time has secondary effects on other musculoskeletal tissues such as articular cartilage. If there are large sex differences in the time course of loss, this would be a compelling argument for sexspecific countermeasure development for exploration-class space missions. However, to the best of the authors’ knowledge, there are no published human studies that have directly assessed sex differences in either the time course of disuseinduced bone or muscle loss or the impact of starting values.

It is well established that the human musculoskeletal response to unloading is highly variable among individuals, with 10-fold differences in response among participants often observed. As an example, after 30 days of unilateral lower limb suspension, individual responses ranged from a 2.5% to a nearly 20% decline in plantarflexor cross-sectional area compared with before the suspension.1 Similarly, with actual spaceflight the loss of cancellous bone in the distal tibia after 6 months aboard Mir ranged from 2% to 24%; such changes range from a negligible loss to deficits equal to those observed after spinal cord injury.2 Understanding the factors that contribute to such large variability is an important step toward both selecting and protecting the first astronauts ˝ 3 year) exploration missions. The extent to which biological sex or sex-based horwho undertake very long (2U mones contribute to this variability is unknown. While this review is focused on sex differences in the response of the musculoskeletal system to the unloading of microgravity, it is important to remember that the overriding uncertainty about which factors contribute to individual differences is a significant issue.

The primary emphases of the literature review were to evaluate sex differences with respect to (1) the magnitude of response and time course of muscle/bone loss to unloading, (2) the influence of negative energy balance on muscle/bone loss, and (3) risk of joint injury and the impact on articular cartilage. This literature review evaluates sex differences in middle-aged, healthy adults and does not consider adolescent or early adult growth and development, menopause, osteoporosis, or old age. While these are all certainly important, there is very little, if any, literature related to spaceflight and these issues.

Time Course and Magnitude of Response: Muscle There is considerable individual variability with respect to loss of muscle size and function as a result of unloading. The precise extent to which sex differences contribute to this is unknown. There is limited evidence in the literature that sex differences related to muscle atrophy might exist. In the first 2 weeks of unloading, minimal sex differences are apparent in whole muscle atrophy (2%-4%) in side-by-side comparisons. If unloading extends beyond 2 weeks, women may experience greater reductions in whole muscle volume and fiber area, particularly in fast-type 2 fibers. Slow-type 1 fibers in both men and women exhibit preferential atrophy with unloading. There is limited evidence that women experience greater loss of strength in the first 30 days of bed rest, but this sex difference in rate of loss may be reversed with long duration unloading ( > 4 months). Women demonstrate greater impairment in neural activation of muscle after short-term unloading; future studies should determine if this leads to greater fatigue susceptibility in women in the first 2 weeks of unloading. There is one study suggesting that recovery of strength after unloading may be slower for women thanmen.

Taken together these data suggest that the time course of unloading-induced muscle loss may be sex specific.

There are also areas where sex differences appear quite unlikely. For both men and women, whole muscle and single muscle fiber atrophy does not fully account for the strength and power loss; the reduction in the force and cross-sectional area of type 1 fibers appears to be very similar in both genders. A significant gap in knowledge is whether sex differences in strength loss/neural activation translate to differences in functional performance (e.g., mission-related tasks).

Negative Energy Balance Some bed rest studies have restricted energy intake and allowed weight loss by design or allowed subjects to consume food at their discretion, so as to not coerce intake. The 60-day Women’s International Simulation for Space Exploration study was one of these studies, and as a result, these female subjects did lose body weight (lean tissue more than fat) during bed rest at a rate of 0.06 kg/day. In a similar 90-day study with male subjects conducted earlier at the same institution, men also lost weight at 0.04 kg/day (calculated from the published average weight loss). Due to the many differences in study design, it cannot be concluded with any certainty if this slight difference in rate of weight loss between men and women is of any significance. While "weightlessness" is a key aspect of space travel, an unexpected analog comes in the form of studies related to weight loss.

Though there is a fair amount of literature on weight loss and effects on bone similar to space-related research,

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance few studies have examined the effects of negative energy balance on bone with regard to gender, and those that have attempted are plagued bymany confounding factors (age, body size, diet- and/or exercise-induced weight loss, rate of weight loss, etc.), making drawing conclusions difficult.

Hence, there is a paucity of literature evaluating sex related differences relative to the effects of energy deficit on bone and muscle metabolism. Making comparisons across separate studies evaluating male and female responses is fraught with confounding factors. If one were to speculate, there do not appear to be major sex differences in the bone or muscle responses to energy deficit between men and women.

Joint Injury Sex-based differences have been identified in the incidence of osteoarthritis (OA), with OA of the knee, in particular, significantly more common in women. Sex-based risk factors explaining this include the loss of estrogenâ&#x20AC;&#x2122;s anabolic effect on cartilage after menopause, a higher incidence of predisposing knee injuries-such as anterior cruciate ligament tears-in women, and increased joint laxity. There is clear evidence from animal studies that regular mechanical loading is essential to cartilage health. In humans, 6 or more weeks of nonweight-bearing can produce changes in magnetic resonance imaging images of knee cartilage that resemble OA. However, sex-based differences in the response to joint unloading have not been elucidated.Because articular cartilage health is impacted by the quality of the underlying bone as well as the strength of muscles around the joint, assessment of the potential risk for articular cartilage injury imposed by unloading needs to include evaluation of all three tissues: bone, muscle, and cartilage. There is some evidence to suggest that osteopenia of subchondral bone underlying articular cartilage contributes to cartilage degeneration. Conversely, damaged cartilage releases receptor activator of nuclear factor kappa-B ligand (RANKL) and other inflammatory components, which can lead to the loss of adjacent bone. Since muscles serve to stabilize and dampen forces across joints, loss of muscle mass and strength after a prolonged unloading can contribute to joint injury risk and early degenerative joint changes, especially in the knee. However, sex-based differences in the relative impact of bone and muscle loss on joint health have not been defined. Specific interventions to increase loadbearing or strengthening activities in space will be indicated.They may also identify the need for progressive strengthening and joint loading upon arrival on a planetary surface after extended microgravity exposure, after return from space or after prolonged period of non-weight-bearing on Earth. Musculoskeletal injuries have been reported in-flight at a rate of 0.021 per flight day for men and 0.015 per flight day for women; hand injuries are the most common, with abrasions and small lacerations the most common manifestations. There are few data on the recovery of the musculoskeletal system following spaceflight and even less data on sex differences in recovery rates. Generally, international space station crew have substantial recovery ofmuscle strengthwithin a month following flight. The time course of recovery of bone mineral density has been evaluated but not specifically for sex differences. In general, half-lives for recovery of bone mineral density are â&#x2030;&#x2C6;150-200 days depending on site.

Muscle Risk revised MLDs

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance

Genetics of muscle strength and power Environmental and genetic factors influence muscle function, resulting in large variations in phenotype between individuals. Multiple genetic variants (polygenic in nature) are thought to influence exercise-related phenotypes, yet how the relevant polymorphisms combine to influence muscular strength in individuals and populations is unclear. In this analysis, 22 genetic polymorphisms were identified in the literature that have been associated with muscular strength and power phenotypes. Using typical genotype frequencies, the probability of any given individual possessing an "optimal" polygenic profile was calculated as 0.0003% for the world population. Future identification of additional polymorphisms associated with muscular strength phenotypes would most likely reduce that probability even further.

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IGF-1 colocalizes with muscle satellite cells following acute exercise in humans Muscle satellite cells (SC) are monopotent stem cells that represent between 2%-4% of all myonuclei. They are normally quiescent but become activated in response to appropriate cues and aid in muscle growth and repair .

The progression of a SC from quiescence through proliferation and into terminal differentiation is orchestrated by a network of transcription factors known as the myogenic regulatory factors (MRFs) ; however, many of the upstream cues driving this process remain unknown or poorly understood.

Insulin-like growth factor 1 (IGF-1) is a well-known regulator of muscle development. A role for IGF-1 in muscle growth is supported by reports demonstrating significant muscle hypertrophy in transgenic mice overexpressing IGF-1 . Additionally, work in our laboratory implicated local IGF-1 as a regulatory factor in SC activity in humans . Alternative spicing of IGF-1 results in 3 distinct splice variants IGF-1Ea, IGF-1Eb, and mechano growth factor (MGF). In vitro, C2C12 myoblasts exposed to MGF or IGF-1Ea proliferate and show impaired differentiation with MGF but not with exposure to IGF-1Ea . Authors work supports this concept, because they report a strong temporal relationship between IGF-1Ea and the mRNA abundance of a driver of differentiation MRF4 . In contrast, MGF expression was strongly correlated to Myf5 expression, a MRF known for its role in driving proliferation .

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance The purpose of the current study was to quantify the extent of IGF-1 colocalization with the SC post exercise in a fibre-type specific manner. Twenty-three healthy, untrained males (18-35 yrs) completed 16 weeks of progressive, whole-body resistance training. All participants were recreationally active and had not participated in a formal weight training program within the 12 months prior to commencement of the study.

Exercise training The subjects participated in 4 supervised resistance workouts per week, which consisted of 2 upper and 2 lower body sessions. The training program was designed to progress from 2 sets of each exercise at 70% of the subject’s 1 repetition maximum (RM), to 4 sets at 85% of 1 RMby the end of the 16 weeks. The last set of each exercise was performed until failure. At the end of each workout, all participants consumed a beverage that contained 30 g of whey protein to maximize gains in muscle . As described in Mitchell et al. (2013), supplementation was provided to maximize gains in muscle mass and strength; exercise training led to increases in strength and in the muscle fibre cross-sectional area of type I and II fibres.

Results Pax7 and IGF-1 colocalization IGF-1 colocalization with Pax7 was determined via immunofluorescent staining of muscle cross sections. Figure 1A shows a representative image of the IGF-1-Pax7 staining. Fibre-type specific immunofluorescent staining for IGF-1 and Pax7 demonstrated a significant increase in colocalization of IGF-1 and Pax7 by 72 h after a single bout of resistance exercise for type I (pre: 6 ± 3% SCs positive for IGF-1; post 72 h: 20 ±5% SCs positive for IGF-1; p < 0.05) and type II (pre: 8 ± 2% SCs positive for IGF-1; post 72 h: 26 ± 5% SCs positive for IGF-1; p < 0.05). Both type I and type II fibre associated satellite cells were similarly affected by the exercise (Fig. 1D and 1E). No difference in baseline colocalization was seen in either fibre type as a result of training (Fig. 1B and 1C).

IGF-1 mRNA No significant changes were observed after exercise in any IGF-1 splice variants (Fig. 2B). When comparing baseline mRNA before and after training, only IGF-1Ea showed a significant increase in gene expression following 16 weeks of progressive resistance exercise training (Fig. 2A) (p < 0.05).

Authors previously reported significant correlations between IGF-1 splice variants and MRF gene expression following an acute bout of muscle damaging exercise, inferring a role for IGF-1 in the regulation of SC. Furthermore, we identified, that SC were considered to be colocalized with IGF-1 following damaging exercise providing further support that IGF-1 plays a role in human SC activity . The present results extend on our initial findings by demonstrating for the first time in human muscle that the proportion of SC considered to be colocalized with IGF-1 increases after an acute bout of exercise.

Consistent with previous preliminary observations, authors observed a significant increase in IGF-1 colocalization with SC 72 h post exercise. It is interesting, however, that no fibre-type difference in the extent of IGF-1 colocalization with SC was observed. Both type I and type II associated SC colocalized with IGF-1 increased 72 h post exercise. Additionally, the extent of IGF-1 was considered to be colocalized with SC was not different before and after training. This suggests that the extent to which IGF-1 is considered to be colocalized with SC is a conserved response and is independent of training status and fibre type.

The demonstrated increase in IGF-1 associated with SC provides evidence of a role for IGF-1 in SC activity in vivo. Because MGF has been suggested to be involved in SC proliferation , it is conceivable that the increase in SC-specific IGF-1 contributes to the expansion of the SC pool following exercise. The later rise in IGF-1Ea may stimulate the progression of SC through the myogenic program towards differentiation.

Changes in whole-muscle IGF-1 mRNA expression following damaging exercise have been well described .

Surprisingly, no change in IGF-1 gene expression was observed in the acute time course following exercise. This could be attributed to the fact that the stimulus in the current study was of a lower intensity and volume than used in previous investigations. The specific intensity and volume used in this investigation was to reflect a more typical resistance exercise bout. Importantly, it is worth noting that we observed a significant increase in IGF-1Ea gene expression following training compared with pre-training baseline levels.

This is the first study to quantify SC-specific colocalization of IGF-1 and how it is impacted by acute exercise and exercise training. Although the exercise stimulus was not intense enough to detect a change in whole-muscle mRNA levels of IGF-1 splice variants acutely following exercise, it was enough to induce a significant increase in IGF-1 colocalization with SC. The increase in SC-specific IGF-1 colocalization suggests that local production of IGF-1 may be part of the normal post-exercise SC response and appears to be independent of training status and fibre type.

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D ID YOU KNOW ?

ACTN3 ACTININ ALPHA 3 ( GENE / PSEUDOGENE ) [H OMO SAPIENS ( HUMAN )]

This gene encodes a member of the alpha-actin binding protein gene family. The encoded protein is primarily expressed in skeletal muscle and functions as a structural component of sarcomeric Z line. This protein is involved in crosslinking actin containing thin filaments. An allelic polymorphism in this gene results in both coding and non-coding variants; the reference genome represents the coding allele. The non-functional allele of this gene is associated with elite athlete status.

An estimated 1.5 billion people worldwide are deficient in the skeletal muscle protein α-actinin-3 due to homozygosity for the common ACTN3 R577X polymorphism. α-Actinin-3 deficiency influences muscle performance in elite athletes and the general population. The sarcomeric α-actinins were originally characterised as scaffold proteins at the muscle Z-line.

In mouse and human skeletal muscle, the two sarcomeric α-actinin isoforms, α-actinin-2 and α-actinin-3 (encoded by the genes ACTN2 and ACTN3 respectively), form major components of the contractile apparatus at the Z-line where they cross-link and anchor actin filaments. The two isoforms are highly conserved (80% identical and 90% similar in humans) and are considered products of gene duplication . They share a common domain topology, consisting of a highly conserved N-terminal actin-binding domain, a central rod domain consisting of four spectrin-like repeats, and a C-terminal EF hand region . The expression of α-actinin-2 is ubiquitous in human muscle fibres, while α-actinin-3 is restricted to fast Type 2 fibres . This difference in expression pattern raises the possibility that α-actinin-3 has a specific function in fast muscle fibres that is independent from the function of α-actinin-2. α-Actinin-3 deficiency does not cause muscle disease, but has been shown to alter muscle function. The consequences of the ACTN3 R577X genotype in humans were first investigated in elite athletes. In an Australian cohort of 439 elite Caucasian athletes and 436 unrelated Caucasian controls, the frequency of the 577X al-

lele was significantly lower in both male (P<0.001) and female (P<0.01) sprint athletes while the frequency of 577XX genotype was higher in female endurance athletes (P<0.05) . The underrepresentation of ACTN3 577XX genotype in sprint and power athletes has been replicated in over 15 studies . A meta-analysis conducted on ten athlete cohorts also strongly confirmed this association . Two studies also report a higher frequency of the 577XX genotype in endurance athletes . Overall, the athlete studies suggest a strong association between ACTN3 genotype and athletic performance with α-actinin-3 deficiency being detrimental for power and sprint activities and potentially beneficial for endurance sports.

The ACTN3 R577X polymorphism has also been found to influence muscle performance in the general population. α-actinin-3 deficient women produced significantly less elbow flexor isometric strength than women homozygous for the ACTN3 577R allele who express α-actinin-3 and young ACTN3 577RR men had significantly higher knee extension strength compared to ACTN3 577XX men. Compared to their ACTN3 577RR and 577RX counterparts, adolescent boys deficient in α-actinin-3 took significantly longer to complete a 40m sprint while older males were slower to complete a 400m walk . These findings are consistent with the athlete data and suggest that α-actinin-3 deficiency has a detrimental effect on power performance in the general population.

The ACTN3 577X allele is estimated to be over one million years old , however, the genomic region surrounding the allele shows low levels of genetic variation and recombination suggestive of recent positive selection . The loss of α-actinin-3 appears to be beneficial in certain situations. Combined with evidence suggesting the ACTN3 577X allele may be beneficial for endurance and the varied frequency of the allele among different ethnic groups , the ACTN3 577X allele has been postulated to have conferred a selective advantage for modern humans as they moved out of Africa into a Eurasian environment.

The X-allele frequency is at its highest in places with reduced mean annual temperature and species diversity, and this increase occurs along a global latitudinal gradient . This has led us to postulate that the ACTN3 X-allele may have conferred a resistance to famine or enhanced cold tolerance . There are only two known examples in the human genome of a loss of function (LoF) variant resulting in a clear selection advantage (ACTN3 and CASP12 ). As such, there is significant interest in understanding how α-actinin-3 deficiency was advantageous during human evolution and why the absence of this protein alters human muscle function today.

Reference:

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ACTN3 actinin alpha 3 (gene/pseudogene) [ Homo sapiens (human) ] NCBI Website. (http://www.ncbi.nlm.nih.gov/gene/89)

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Kikuchi N, Miyamoto-Mikami E, Murakami H, Nakamura T, Min SK, Mizuno M, Naito H, Miyachi M, Nakazato K, Fuku N. ACTN3 R577X genotype and athletic performance in a large cohort of Japanese athletes. Eur J Sport Sci. 2015 Sep 1:1-8.

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Fiona X.Z. Lee, Peter J. Houweling, Kathryn N. North, Kate G.R. Quinlan, How does α-actinin-3 deficiency alter muscle function? Mechanistic insights into ACTN3, the ’gene for speed’, BBA - Molecular Cell Research (2016).

Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance

Muscle Atrophy Research and Exercise System (MARES)

Facility Description MARESâ&#x20AC;&#x2122; Main Box houses a powerful motor, battery, sensors, and its control electronics. The Microgravity Isolation Frame, to which the Main Box attaches, minimises disturbances to other payloads. The facility is completed by a Set of Human Adapters. These are pads and levers that adapt to different subject sizes and cover different movements. The Chair is a seat for the examined/exercising subject and is fixed to the Main Box by a pantograph arm. A Laptop computer provides the command and display interface to the subject. MARES is operated by dedicated experiment software. With a minimum effort, MARES can be tailored to a specific experiment: issuing automated instructions to the subject and setting the appropriate control algorithms for the motor.

MARES Instrumentation Torque and angular position/velocity measurements and training of the left and right extremities are supported on the following joint movements:

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance MARES ANGULAR MOVEMENTS • Ankle :Dorsi-plantar flexion • Knee :Flexion-extension • Hip :Flexion- extension • Trunk :Flexion- extension • Shoulder :Flexion- extension • Elbow :Flexion- extension • Wrist :Flexion- extension Radial-ulnar deviation Pronation-supination

MARES LINEAR MOVEMENTS • Arm press: One arm Both arms • Leg press: One leg Both legs

MARES supports experiments on seven different joints for a total of nine different angular movements (either left or right limb) and for two additional linear movements (upper and lower limb). A careful coordination of the many capabilities of MARES is needed in order to ensure that measurements are made in a consistent and repeatable manner. In MARES, this is done by translating the steps of an experiment into a set of computerbased instructions, the so-called MARES Experiment Procedures. Instructions are available that would allow the scientist to define within an Experiment Procedure the following functions: • To prompt (via text instructions and/or graphical messages) the test subject to perform tasks, i.e.: – How to set-up the limb adapters.

– How to restrain himself.

– To power-on an external device.

– To perform, for example, a Maximum Voluntary Contraction.

– To perform a series of flexions / extensions.

• To accept and respond to feedback from the test subject.

• To activate a MARES Profile.

• To activate data displays for assessment by the test subject.

• To perform real-time data processing, the results from which could be used to influence the execution of the Experiment.

• To programme and command external devices.

• To select data for storage and/or downlink.

MARES can store internally a large number of such Experiment Procedures, with the possibility for new Experiment Procedures to be uploaded from the ground.

MOTOR CONTROL ALGORITHMS Any desired limb movement can be described as a sequence of elementary steps. These elementary steps are referred to as Basic Motion Units (BMUs), such that during any BMU the relationship between position or velocity and torque/force is governed by a single, relatively simple, mathematical formula. These BMUs cover the three possible basic modes of muscle contraction known from physiology (isometric, isotonic, isokinetic), plus

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance eleven more BMUs that can be used in support of more sophisticated experimental requirements. MARES offers fourteen predefined BMUs as listed below: MARES BASIC MOTION UNITS - BMUS • isometric • isotonic (concentric and eccentric) • isokinetic (concentric and eccentric) • spring • friction • additional moment of inertia/mass • pseudogravitational • position control • velocity control • torque/force control • power control • physical elements • extended torque/force control • quick release

Complex combination of these modes are possible. The control algorithms for the motor are defined as so called ’Profiles’ consisting of a linked sequence of BMUs. GRAPHICAL EDITING OF A MARES PROFILE MARES provides a simple graphical software tool that allows the experimenter to develop these kinds of Profiles according to his/her specific scientific needs. In this software tool, each BMU is graphically represented and the user can ’drag and drop’ any of the fourteen BMUs into a sequence, thereby creating a Profile. In doing so, the user will be prompted to define appropriate end conditions for each BMU introduced into the sequence. This tool also supports looping and branching within the Profile.

The BMU end conditions are important because they define the transition between the BMUs. These transitions can be made dependent on: time, position, torque, subject interaction, external signals, and/or any other parameter. This level of programmability opens up a whole range of operational possibilities, for example, to simulate the changing friction of rowing (back and forth), to design a complex motor-control experiment, etc.

EXTERNAL INSTRUMENTATION MARES is designed to work in connection with other devices like electro-miographs, electro-cardiographs, stimulators, etc. The Percutaneous Electrical Muscle Stimulator (PEMS II), (see image above) is one of the possible external instruments, delivers electrical charge pulse stimulation to non-thoracic muscle groups of the test subject, thus eliciting contractile responses. PEMS stimulates muscle groups on the limbs (ankle, knee, thumb, elbow, and wrist) with electrical pulses applied via skin electrodes. The pulse duration and current amplitude are programmable to either 50 µs for up to 0.8 A or 250 µs up to 0.16 A. It provides either single pulses or fully programmable pulse trains, and can be triggered either manually or remotely, typically by MARES. A typical application is to study muscle strength independently of the subject’s voluntary neural drive.

ACCOMMODATION and TRANSPORT MARES was launched in the HRF MARES Rack. For operation, MARES occupies the centre aisle and is attached to the ISPR.

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OPERATIONAL CONCEPT MARES is a component of the Human Research Facility in the Columbus laboratory. MARES uses the EPM portable computer for interacting with the subject/operator. MARES is capable of acquiring data, controlling and providing power to external devices (EPM, PEMS, EMG amplifiers, etc.), and transfers real time data to the NASA Workstation or EPM for downlink. It is also capable of stand-alone data collection. When not in use, all of MARESâ&#x20AC;&#x2122; elements are stored inside the ISPR. The flight operations of MARES are managed by ESA via the User Support and Operations Centre (USOC) CADMOS in Toulouse.

UTILISATION SCENARIO MARES is mainly intended for research, but it can work as well as an exercising device. It is an ideal tool to do research on the efficacy of countermeasures. The MARES performances are ahead of any commercial research dynamometer.

SCHEDULE MARES was launched to the ISS on board STS-131/19A Shuttle Flight on 5 April 2010.

Results/More Information A Muscle Atrophy Research and Exercise System (MARES) unit was delivered to the International Space Station (ISS) in early 2010 which enables scientists to study strength changes in crewmembers during and not just after space flights. It is unknown whether space flight-induced strength losses occur in a linear fashion or whether they mirror those of aerobic capacity that occur mostly in the first few weeks of space flight. A strength change timeline featuring greater losses during the first days of flight would likely have significant operational implications. In flight strength testing capabilities will enhance countermeasures evaluation and facilitate improved prescription of crewmembersâ&#x20AC;&#x2122; in flight exercise programs over the duration of their flight.

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In advance of its deployment on the ISS, a ground-based study was performed to evaluate the test-retest reliability of MARES, and determine its agreement with a standard, commercially available isokinetic dynamometer (the "HUMAC NORM" Testing and Rehabilitation System) often used for pre- and postflight medical assessment testing. Ten subjects participated in this project and completed 2 testing sessions each using NORM and MARES (4 total testing sessions). During each session, peak torque values were measured during 2 isokinetic testing sets: 5 maximal, discrete repetitions of knee extension and knee flexion at 60◦ /sec and 21 maximal, continuous repetitions of knee extension and knee flexion at 180◦ /sec.

Both devices show good similarity and acceptable test-retest reliability. However, within device standard deviations were 1.3 to 4.3 times larger for MARES than NORM indicating somewhat lower reliability for MARES. Only one dependent measure, knee extension peak torque at 60◦ /sec, exhibited good agreement between the two machines. Overall, agreement between NORM and MARES was poor. The results of this relatively small "n" (n = number of subjects) investigation demonstrate that MARES is a reasonably reliable device that renders consistent measurements between two sessions. However, MARES does not produce values that are in consistent agreement with NORM. Thus, until further research suggests otherwise, it is not advisable to compare values obtained on one device to those obtained on the other. This may necessitate testing crewmembers on both MARES and the commercially available dynamometer already in use for pre- and postflight medical assessment testing (English 2010).

HUMAC NORM

The HUMAC NORM is your solution for measuring and improving human performance in the clinic, training room, and research laboratory. In one machine the HUMAC NORM offers 22 isolated-joint movement patterns, four resistance modes (isokinetic, isotonic, isometric, and passive) and numerous reports to meet the measurement and exercise needs of today’s clinicians and researchers.

Measurement

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance Only with testing can you determine baselines, set goals, and track change. The HUMAC NORM offers two primary measurement solutions.

• Isometric Testing: when dynamic movement is a concern, isometric testing is the answer. The HUMAC NORM will safely position the patient to each angle in the protocol. Protocol options include angles, hold-times, rest periods, repetitions, and sets.

• Isokinetic Testing: to determine maximum dynamic capability throughout the range-of-motion isokinetic testing is the solution. The HUMAC NORM offers concentric and eccentric resistance testing. Isokinetic curve results make it easy determine areas of pain or weakness and determine capability.

Exercise Exercise is performed to improve mobility, stability, strength, and control. The HUMAC NORM offers four modes of resistance and numerous feedback options to meet these goals.

• Passive Mode: develop the mobility that the patient requires, from straight pattern movements to complex PNF patterns.

• Isometric Mode: stabilize the joint to perform anglespecific strength training.

• Isokinetic Mode: continue to strengthen using proven methods to enhance return-to-function including concentric and eccentric loading and deceleration training.

• Isotonic Mode: complete the return-to-function training using our simulated mass isotonic mode.

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Exercise Countermeasures Project (ECP) Mission

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Blood flow-restricted exercise in space Prolonged exposure to microgravity results in chronic physiological adaptations including skeletal muscle atrophy, cardiovascular deconditioning, and bone demineralization. To attenuate the negative consequences of weightlessness during spaceflight missions, crewmembers perform moderate- to high-load resistance exercise

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance in conjunction with aerobic (cycle and treadmill) exercise. Recent evidence from ground-based studies suggests that low-load blood flow-restricted (BFR) resistance exercise training can increase skeletal muscle size, strength, and endurance when performed in a variety of ambulatory populations.

BFR exercise prescription and training BFR resistance training BFR resistance exercise training (also known as KAATSU when specific equipment is utilized ) combines low training intensity (approximately 20%50% 1RM) with an external pressure cuff applied to the exercising limb . BFR exercise protocols vary and are primarily influenced by cuff size, pressure, and the circumference (CIRC) of the limb being exercised ; however, most use three to five sets of exercise with 30- to 90-s rest periods . Maintaining the cuff pressure (CP) during exercise and the rest interval also appears to be an important variable in order to increase the metabolic demand in both type I and type II muscle fibers . As a result, the total number of repetitions performed in a training session can vary and is also determined by whether the first set of BFR exercise is to volitional fatigue or a predetermined number of repetitions.

Overall, BFR exercise training studies have ranged from 6 to 90 days in duration (Figure 1) and most show dramatic muscular adaptations. For example, Fujita et al. reported 6.7% and 3% increases in KE strength and size, respectively, after 6 days of twicedaily BFR knee extension (KEx). Likewise, Yasuda et al. demonstrated even larger improvements in 1RM SQT (14%) and quadriceps muscle size (7.8%) from 2 weeks of twice-daily BFR SQT exercise. Moreover, BFR SQT and leg curl exercise performed 6 days per week for 2 weeks resulted in approximately 17% and 22% increases in SQT and leg curl 1RM, respectively, and an 8.5% increase in thigh cross-sectional area (CSA) . Studies implementing less frequent BFR resistance exercise sessions over longer training durations also show substantial muscular adaptations. Clark et al. observed an 8% increase in KE strength after 4 weeks of BFR exercise (3 days per week), while Takarada et al. reported approximately 10% increases in KE strength and size over 8 weeks (2 days per week) of training.

When studies are statistically combined, mean effect sizes ((post-mean - pre-mean)/pre-standard deviation and adjusted for sample size bias) for muscle hypertrophy and strength for BFR resistance exercise are 0.39 and 0.58, respectively (compared to 0.01 and 0.000 for low-load training without cuff inflation) . An important difference between high-load and BFR training is that increased muscle strength corresponds with muscle hypertrophy within the first 4 weeks of BFR exercise training, which is in contrast with the nervous system adaptations that result in enhanced muscle strength over the same duration of high-load resistance exercise training.

BFR aerobic training Similar to BFR resistance training, the BFR aerobic exercise training studies couple low-intensity walking or cycling (approximately 20%-40% of maximal oxygen consumption, VO2 max) with an external pressure cuff of approximately 200 mmHg applied to the upper legs . Walk training studies showed that the metabolic cost of walking is approximately 3% higher and that heart rate (HR) is approximately 30 bpm higher during walking with BFR compared to normal walking at the same speed . The training adaptations of 3-weeks of twice-daily BFR walking training at 20% of VO2 max included an increase in leg press and leg curl 1RM of approximately 8% and an increase in upper leg CSA of approximately 6%. No changes in muscle size or strength were observed in the control group that performed the same walking protocol without BFR . Similarly, 3 weeks of three times-per-week BFR cycling for 15 min at 40% VO2 max resulted in increases in thigh CSA (3.4%), KEx

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance strength (7.7%), and VO2 max(6.4%). The control group cycled for 40 min at the same intensity and showed no change in muscle strength or aerobic fitness . Traditionally, aerobic exercise is prescribed at approximately 75% of VO2 max to elicit improvement in aerobic fitness. BFR aerobic exercise not only improves aerobic fitness at a low intensity, but also increases muscle strength and size, which are not usually observed following an aerobic training program.

Muscle metabolism, motor unit recruitment, and fiber activation Evidence suggests that type II muscle fibers are recruited at a low load during BFR exercise . A current hypothesis indicates that BFR resistance exercise causes type I muscle fibers to fatigue quickly due to low oxygen availability; hence, activation of type II muscle fibers and a greater reliance on anaerobic metabolism are required . These events result in an accumulation of muscle metabolites that stimulate the production of systemic or local growth factors that initiate muscle protein transcription and translation . A convincing evidence for type II muscle fiber activation was demonstrated by Krustrup et al. who showed that phosphocreatine concentrations in both slow and fast twitch fibers following BFR resistance exercise were reduced to an equivalent concentration as compared to high-intensity resistance exercise. However, fast twitch fiber recruitment evaluated by inorganic phosphate splitting occurred in only 31% of participants performing one set of BFR resistance exercise compared to 70% of subjects performing one set of high-load exercise . When multiple sets of BFR exercise were performed with continued BFR during the rest intervals, inorganic phosphate splitting was similar to multiple sets of high-load resistance exercise . Therefore, the overall effort and threshold of fatigue reached during a session may facilitate the acute training response . Further evidence for insufficient oxygen availability and anaerobic type II fiber activation exists in studies reporting enhanced muscle biopsy lactate , blood lactate (La) , and decreased pH following BFR exercise compared to exercise at the same load without external CP. Systemic hormonal response Anabolic and catabolic hormonal responses have been frequently evaluated following acute and chronic BFR resistance exercise (Table 1). It is hypothesized that accumulation of metabolic by-products and/or the hypoxiainduced stimulation of afferent nerve fibers results in an increase in secretion of the growth hormone (GH) and GH-releasing hormone . Takarada et al. reported that circulating GH concentrations following an acute bout of BFR exercise were 290 times greater than the baseline; however, Pierce et al. showed a lower but still physiologically significant ninefold increase in GH concentration using a similar protocol. A corresponding increase in the circulating insulin-like growth factor-1 (IGF1) has been observed during BFR KEx exercise (20% 1RM, four sets to exhaustion, 160-180 mmHg) and at 10-30 min postexercise . In contrast, the circulating IGF1 concentration was not increased up to 180 min following an acute bout of BFR KEx exercise (20% 1RM, 200 mmHg, four sets, 75 total repetitions, 30-s rest periods) .

It is also argued that the increase in IGF1 observed in some studies could be related to a hemoconcentration as a result of plasma volume (PV) changes following BFR resistance exercise . Following a training period, there was a report of a progressive increase in circulating IGF1 at rest following 2 weeks of twice-daily BFR SQTand leg curl exercise (20% 1RM, three sets, 15 repetitions/set, 30-s rest periods) . Therefore, the overall re-

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance lationship between BFR resistance exercise and the GH-IGF1 axis remains controversial. The influence of acute and chronic BFR exercise on other anabolic hormones such as testosterone (T) is also unclear . For example, neither Reeves et al. nor Fujita et al. observed changes in total or free following arm or leg BFR resistance exercise, respectively. In contrast, Madarame et al. reported a post-exercise elevation in total T following three sets of BFR KEx and flexion. Chronic BFR walking or resistance exercise has also failed to show resting changes in T. Worthy of note, studies do show that circulating cortisol (Cort) is elevated following BFR and high-load resistance exercise , which suggests a similar stress response.

However, circulating Cort concentration is primarily associated with catabolism and muscle protein breakdown.

Recently, the overall association between the acute systemic hormonal response to resistance exercise and muscle hypertrophy has been questioned. West et al. reported no additional rise in muscle protein synthesis or the phosphorylation of signaling proteins following resistance exercise during elevated systemic concentrations of T, GH, and IGF1 compared to low systemic concentrations of the same anabolic hormones. Hence, local factors may provide greater stimuli to induce BFR resistance exercise adaptations.

Gene expression and cell signaling An acute bout of high-load resistance exercise elicits anabolic and catabolic responses that are altered in a complex temporal manner to achieve muscle hypertrophy. Briefly, muscle protein synthesis and myogenic gene transcripts (e.g., myogenin) are upregulated within 2 h post-exercise and peak approximately 8 h post-exercise, whereas proteolytic ligases (muscle-specific RING finger protein-1 (MuRF-1), Atrogin-1) are upregulated 1-4 h after exercise and downregulated within 8 h of exercise termination . An approximate threefold increase in the phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1), a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and regulator of translation initiation and elongation, and a 46% increase in the fractional synthesis rate (a measure of muscle protein synthesis) have been previously shown following BFR resistance exercise . mRNA expression of genes that regulate satellite cell activity (mechano-growth factor, IGF1 receptor, myogenin, MyoD), cell size (myostatin), and protein turnover (MuRF1, mTOR, S6K1) were not different between BFR and low-load exercise up to 3 h post-exercise .

At 8 h post-BFR or low-load exercise, myogenic gene transcripts (IGF1, MyoD, and myogenin) were not different from the baseline; however, proteolytic transcripts (Atrogin-1, MuRF-1, and Forkhead box O3 (FOXO3A)) were downregulated twofold from baseline in the BFR group only . Following 8 weeks of BFR resistance training, myostatin gene expression was downregulated to a similar extent compared to high-load training , with a trend (p = 0.06) toward decreased activin IIb (myostatin receptor). There were also elevations in genes associated with myostatin function (growth and differentiation factor-associated serum protein-1) and signaling (SMAD family) . Overall, the molecular pathways that regulate BFR resistance exercise-induced muscle hypertrophy have not been extensively studied. Given that muscle hypertrophy results from a positive net muscle protein balance (synthesis > breakdown) across a training period, greater examination of molecular events is needed to better understand how BFR resistance exercise stimulates muscle growth.

BFR exercise limitations The application of BFR exercise appears to be limited to peripheral muscle groups; thus, core, back, and neck muscles cannot be specifically targeted using this methodology. Higher perceptual ratings of perceived exertion and pain during the rest intervals of sets have also been reported, which could limit the application of this training methodology . The pressure applied to the blood vessel during BFR exercise is likely the root cause of discomfort and is associated with cuff width and the layer of soft tissue situated between the cuff and the vessel . The cuff sizes most frequently used in research studies are either narrow (approximately 5 cm) or wide (approximately 13 cm).

From the data, it appears that lower cuff pressures (90-120 mmHg) are required to occlude venous blood flow when the wider cuffs are used compared to the narrow cuffs (pressures, 160-180 mmHg. However, the narrow cuffs and associated pressures have repeatedly been shown to cause improvements in muscle strength and size when combined with low-load resistance exercise . Exercise training adaptations have been observed with wider cuffs with the same or lower cuff pressures ; however, BFR exercise performed at supra-systolic blood pressure (SBP) with wider cuffs may restrict blood flow to a level that reduces exercise volume and increases discomfort compared to narrower cuffs . Therefore, cuff width is an important variable for determining BFR exercise prescription and may be a limiting factor if not taken into consideration with CP. Hemostasis The potential for blood coagulation and venous thrombus following blood pooling is the most frequently discussed (hypothesized) risk, and therefore, it has been comprehensively evaluated in multiple acute studies (Table 2). Most recently, Clark et al. observed an increase in tissue plasminogen activator (tPA), a fibrinolytic

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance protein that catalyzes the conversion of plasminogen to plasmin, immediately following a single bout of BFR exercise. This finding is consistent with those of Nakajima et al. who reported that an acute bout of BFR exercise increased tPA antigen without altering plasminogen activator inhibitor-1 (PAI-1) or D-dimer (D-d). Together, these results suggest that BFR exercise may acutely increase fibrinolytic activity, thus reducing the risk for blood coagulation.

Acute cardiovascular stress The acute cardiovascular responses to BFR exercise training are important to consider because this type of exercise could provide the greatest benefit for individuals with a variety of health risks that preclude them from performing traditional resistance exercise. Although most BFR literature focus on muscular effects, it is important to note that resistance and aerobic BFR exercises cause increases in HR and blood pressure that are greater than those observed with exercise performed at a similar intensity without BFR. The increase in HR is an important aspect of BFR exercise because it allows cardiac output (CO) to be maintained, despite a decrease in venous return due to the CP . Since most BFR studies are conducted in healthy subjects, it is important to evaluate the cardiovascular response to BFR in individuals presenting with cardiovascular disease risk factors in a controlled setting.

Muscle damage and reperfusion Maintaining CP during the between-set rest interval is one essential feature of the BFR exercise prescription. As a result, higher levels of muscle soreness and perceived exertion have been reported during and/or following BFR-restricted exercise compared to the same low intensity exercise performed without CP . Delayed onset muscle soreness is a common occurrence following eccentric muscle actions during high-load resistance exercise . In contrast, it appears that concentric muscle actions compared to eccentric muscle actions result in greater muscle soreness following BFR resistance exercise .

It is unclear why this is the case; however, eccentric-induced muscle soreness is typically associated with mechanical stress, which may be attenuated with BFR exercise given the very low training loads. Systemic physiological markers of BFR resistance exercise-induced muscle damage are conflicting. Although neither creatine kinase nor myoglobin were elevated following two BFR resistance exercise bouts , one case of rhabdomyolysis has been reported in the literature .

In addition to the potential for muscle damage with BFR training, there is a hypothetical risk for microvascular dysfunction as a consequence of the reperfusion that occurs when blood flow is restored after a period of restriction or ischemia . During reperfusion, there is an acute release of inflammatory molecules, clotting factors, and reactive oxidative species that impair microvascular function .

Further, nitric oxide bioavailability (a mediator of vasodilation) decreases when blood flow is restored, causing impaired arterial vasodilation and increased sheer stress. Repeated reperfusion injury can eventually cause a wound that influences endothelial function. Renzi et al. have shown a significant reduction in flow-mediated vasodilation 20 min after BFR walking exercise, suggesting the potential for endothelial dysfunction. Recent evidence also suggests increased sarcolemma permeability (evidenced by staining of tetranectin) following BFR exercise, which may be caused by cell damage from increased production of reactive oxygen species.

Furthermore, although not statistically significant, Goldfarb et al. showed that both protein carbonyls and glutathione ratios (systemic indicators of oxidative stress) were almost doubled following BFR resistance exercise in seven male subjects. Given that the time course of reactive oxygen species generation was limited to 15 min post-exercise, it is essential that future acute studies have a sufficiently high number of subjects and extended

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Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance time courses. Overall, BFR exercise training encompasses a variety of new variables (cuff width, CP, cuff inflation duration) for exercise prescription, and understanding these interactions in terms of safety is complex . To date, the most comprehensive data set on side effects from BFR exercise training was established using survey methodology. The most reported incidents from approximately 13,000 people participating in KAATSU training were as follows: bruising (13.1%), numbness (1.3%), cerebral anemia (0.3%), cold feeling (0.1%), pulmonary embolism (0.01%), rhabdomyolysis (0.01%), deterioration of ischemic heart disease (0.02%), and venous thrombus (0.06%).

BFR exercise in space-potential applications Crewmembers commonly experience losses in aerobic capacity and muscular strength following long-duration spaceflight. Crewmembers that perform daily moderateto high-intensity exercise throughout the mission duration generally return in considerably better condition than their counterparts that engage in little or lowintensity activity. Exercise with BFR may provide a means to perform resistance exercise with a low load or perform aerobic exercise at a slower walking speed or lower pedaling resistance in the event of ISS exercise hardware failure or in future exploration missions with less robust exercise hardware.

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