STEM Today

Special Edition on Long Duration Spaceflight

STEM TODAY April 2016, No.7

STEM TODAY April 2016 , No. 7

CONTENTS Muscle physiology Skeletal Muscle Structure and Function Human Spaceflight Evidence Muscle Volume, Strength, Endurance, and Exercise Loads During 6足Month Missions in Space Human skeletal muscle after 6 months aboard the International Space Station Microgravity足Induced Fiber Type Shift in Human Skeletal Muscle Effects of Sex and Gender on Adaptation to Space: Musculoskeletal Health Effects of prolonged space flight on human skeletal muscle enzyme and substrate profiles Simulated Microgravity Contributes to Autophagy Induction by Regulating AMP足 Activated Protein Kinase

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

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Cover Page Daybreak at Gale Crater This computer-generated images depicts part of Mars at the boundary between darkness and daylight, with an area including Gale Crater, beginning to catch morning light. Northward is to the left. Gale is the crater with a mound inside it near the center of the image. NASA selected Gale Crater as the landing site for Curiosity, the Mars Science Laboratory. The mission’s rover will be placed on the ground in a northern portion of Gale crater in August 2012. Gale Crater is 96 miles (154 kilometers) in diameter and holds a layered mountain rising about 3 miles (5 kilometers) above the crater floor. The intended landing site is at 4.5 degrees south latitude, 137.4 degrees east longitude. This view was created using three-dimensional information from the Mars Orbiter Laser Altimeter, which flew on NASA’s Mars Global Surveyor orbiter. The vertical dimension is not exaggerated. Color information is based on general Mars color characteristics Image Credit: NASA/JPL-Caltech Background Stormy Seas in Sagittarius Some of the most breathtaking views in the Universe are created by nebulae - hot, glowing clouds of gas. This new NASA/ESA Hubble Space Telescope image shows the center of the Lagoon Nebula, an object with a deceptively tranquil name, in the constellation of Sagittarius. The region is filled with intense winds from hot stars, churning funnels of gas, and energetic star formation, all embedded within an intricate haze of gas and pitch-dark dust.

Image Credit: NASA, ESA, J. Trauger (Jet Propulson Laboratory) Back Cover New Gravity Map Gives Best View Yet Inside Mars A map of Martian gravity looking down on the North Pole (center). White and red are areas of higher gravity; blue indicates areas of lower gravity. The map was derived using Doppler and range tracking data collected by NASA’s Deep Space Network from three NASA spacecraft in orbit around Mars: Mars Global Surveyor (MGS), Mars Odyssey (ODY), and the Mars Reconnaissance Orbiter (MRO). Like all planets, Mars is lumpy, which causes the gravitational pull felt by spacecraft in orbit around it to change. For example, the pull will be a bit stronger over a mountain, and slightly weaker over a canyon. Slight differences in Mars’ gravity changed the trajectory of the NASA spacecraft orbiting the planet, which altered the signal being sent from the spacecraft to the Deep Space Network. These small fluctuations in the orbital data were used to build a map of the Martian gravity field.

Image Credit: MIT/UMBC-CRESST/GSFC

STEM Today , April 2016

Special Edition on Long Duration Spaceflight Muscular System

After a few days of exposure to microgravity, muscle atrophy begins and the urinary excretion of nitrogen compounds increases. This atrophy is characterized by structural and functional alterations in the muscle tissue.

There is a decrease in muscle fiber size, with no apparent change in fiber number. Atrophy is considerably greater for postural muscles, i.e., those muscles that support activities such as walking, lifting objects, and standing on Earth, as compared to the non-postural muscles, which undergo only marginal changes. Astronauts lose 10-20% of their muscle mass on short missions. On long-duration flights, the muscle mass loss might rise to 50% in the absence of countermeasures. The visible reduction in the leg circumference has been used as an indicator of muscle atrophy. However, this reduction is also influenced by the shift of fluids from the lower to the upper body in microgravity.

The muscle loss is presumably caused by changes in the muscle metabolism, i.e., the process of building and breaking down muscle proteins. Experiments performed during long-duration missions on board Mir have revealed a decrease of about 15% in the rate of protein synthesis in humans.

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In addition to pure muscle loss, the fibers involved in muscle contractions change their contractile properties and are weakened. Significant decreases in strength of the trunk, knee and shoulder muscles have been found in as few as 6 days in microgravity. Extensor muscles are more affected than flexor muscles. Animal studies also revealed that muscle fiber regeneration is less successful in space. The associated continued excretion of nitrogen may also have deleterious hormonal and nutritional effects.

Spaceflight also results in increased susceptibility of skeletal muscle to contraction damage, which occurs in muscular atrophies on Earth-bound patients. These effects may compromise the ability of astronauts to perform some of their activities in orbit. Likewise, they may not be able to withstand the stress of 1-g upon return to Earth.

In fact, the muscle weakness, fatigue, faulty coordination, and delayed-onset muscle soreness that astronauts experience after spaceflight mimics the changes seen in bed rest patients and the elderly. Finally, it is important to bear in mind that muscle atrophy caused by weightlessness also participates in the postural instability and locomotion difficulties seen after spaceflight.

Muscle physiology There are several types of muscle tissue in the human body. The muscles that are the most affected by spaceflight are the skeletal muscles, which are those directly attached to the skeleton. Skeletal muscles are the largest tissues in the body, accounting for 40-45% of the total body weight. These muscles are attached to the bones by tendons. Their contraction allows for the movement of joints in everyday activities, like walking, lifting objects and standing. The anti-gravity muscles, also known as postural muscles, owe their importance and strength to the presence of gravity.

Skeletal Muscle Structure and Function Functional Anatomy of Normal Human Skeletal Muscle The human skeletal muscle system comprises about 220 specific muscles with various sizes, shapes, locations, and functions in the body. Some are relatively small (<3 cm in length) such as some hand and foot muscles (interossei, lumbricales) used for complex finger/toe movement control (grasping, playing on instruments) or some deep medial column back muscles (rotators) for local segmental spine rotations. Some are even smaller (<3 mm) and used for fine-tuning of discrete movements (Alkner and Tesch 2004) (e.g., the little stapedius controls movements of the last of three middle ear bones) in acoustic sensation and hearing.

Others are longer (>40 cm, sartorius, erector spinae), while others are broader and rather powerful and fleshy (quadriceps femoris, latissimus dorsi, adductors) for use in body stabilization (posture, gait) and mobilization (movements, performance). Another functional group is located at deep muscle layers adjacent to bones running closely over one or more joints (single vs. multi-joint muscles) to facilitate local joint movement and stabilization . In general, almost 60 % of the functional skeletal musculature in the healthy human body is used for body stabilization and postural control during stance and body motions in normal everyday life on Earth and is thus termed "antigravity" muscles (Fig.1).

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Special Edition on Long Duration Spaceflight

Most of the skeletal muscles are usually grouped in the body by anatomic and functional compartments classified according to their body location and function in the trunk or extremities as ventral/dorsal, medial/lateral, and extensor/flexor compartments. For example, the soleus and gastrocnemius belong to the superficial dorsal calf plantar flexors (triceps surae) which insert via the Achilles tendon to the calcaneus bone (Fig. 2.1). The adductor muscles (short, long, and greater adductor, gracilis) belong to the medial thigh compartment (thigh adductors) for hip stabilization and leg adduction.

Muscle compartments are located underneath the main body fascia (similar to a whole body stocking or cat suit) separated from the skin above (Schleip et al. 2012). The muscle compartments underneath the main body fascia particularly of the arms and legs resemble long spaces separated by fibrous connective tissue sheets known as fascia sheets or box fascia which usually contain about two or three individual muscles (e.g., the long and short fibularis of the lateral calf compartment) or two-/three-headed single muscles (e.g., biceps/triceps of ventral arm flexors/dorsal arm extensors). Each compartment is comparable to a box with functionally grouped (extensor/flexor/adductor) muscles that receive a common neurovascular supply (nerve and blood vessels) from deep main arteries or peripheral nerves and thus participate in the functional anatomy of human skeletal muscle (Blottner 2013). In most body regions, the fascia layers are part of the soft muscle tissue (Fig. 2) and may not be easily palpable, while others are more stiff (i.e., great thoracolumbar fascia) and well palpable on the back superficial lumbar region or at the lateral hip region (iliotibial tract) of the lower limb.

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Special Edition on Long Duration Spaceflight

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Sarcomere, Sarcoplasmic Reticulum (SR), and More A whole skeletal muscle is composed of larger and smaller bundles of skeletal myofibers (secondary and primary fascicles) mostly running throughout the length of a muscle and separated by perimysial connective tissue layers (Fig.3). The secondary fascicles (sarcous fibers) are usually visible with the eye.

In the microscope, a single muscle fiber is seen as a spindle-shaped elongated cell tube (about 20 cm in length,

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Special Edition on Long Duration Spaceflight thickness comparable to a spiders thread, approx. 100 Âľm) closely packed with bundles of thin myofibrils composed of much thinner microfilaments (thin actin, thick myosin filaments), with the calcium-dependent regulatory proteins (tropomyosin, troponin) responsible for actin-myosin interaction and the accessory proteins titin (spring-like protein) and nebulin (actomyosin stiffness) responsible for structural integrity, elasticity, and stiffness within the basic force-producing contractile component known as the sarcomere with its typical striated patterns (seen in light and electron microscopy) of A- and I-bands of overlapping actin-myosin microfilaments each anchored to the subtended Z-disks as the microstructural demarcations of a sarcomere (lat. sarcos = flesh) (Fig. 4).

Numerous sarcomeres (little power chambers) are arranged in series of about 500/mm of a muscle fiber so that the microscopic shortenings of individual sarcomeres add up to the visible muscle contractions and macroscopic changes in muscle length, for example, during body motions. Skeletal muscle fibers contain all the typical cell organelles found in any other nucleated cell type (mitochondria, Golgi apparatus, endoplasmic reticulum, vesicles), but, unlike most single-nucleated cell types of the body, they contain about a hundred of myonuclei for gene transcriptional control of subcellularly partitioned microdomains for normal cell metabolism and adaptation.

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Muscle fibers also contain a complex and specialized endoplasmic network of tubules known as the sarcoplasmic reticulum (SR) that express a set of calcium release and calcium uptake proteins (ryanodine receptor, SERCA) which actively transport calcium ions from this major calcium store to the sarcosol and vice versa (influx/ efflux) to enable normal fiber contraction during excitation.

Human Skeletal Muscle Contraction Types Under Normal Gravity Conditions on Earth (1G) Dynamic skeletal muscle is attached to relatively stable or stiff bone (point of origin) via more flexible osseotendinous junctions (muscle-tendon-bone) and further runs over joints to their points of insertion on another bone (usually distal to the same joint). The main function of skeletal muscle is contraction (shortening) which brings the two bones (lever arms) closer together to initiate active mode body motions during voluntary movements. This type of a "visible" muscle activity is usually considered by most people as a muscle contraction with force production.

In passive mode body motions, however, muscles are passively moved (i.e., they are shortened or slightly stretched) against gravitational load (i.e., passive arm or leg movements without voluntary forces) usually performed without oneâ&#x20AC;&#x2122;s own muscle force, however, with the help of the force of a therapist . The muscle in action is termed the agonist (e.g., biceps brachii during arm bending), while its counterpart is termed the antagonist (e.g., triceps brachii during arm bending). In this example, active and passive stretching of skeletal muscle is performed alternatingly between agonist and antagonist contractions during normal elbow joint movements.

The basic muscular contractions include: 1.Isotonic contraction (muscle shortens in length under constant work / tension against gravity, e.g., arm lifting a heavy book from table for reading, maximal force is larger than the gravitational load of an object); 2.Isometric contraction (muscle remains the same length under constant work/tension against gravity, e.g., holding a heavy book in front of the body or carrying a heavy bag, the muscle force precisely matches the gravitational load); 3.Auxotonic contraction (simultaneous change in length and force under constant work against gravity, e.g., heavy weight lifters, muscle force becomes higher during lifting motions).

In addition, stopped preloaded contractions (e.g., masticatory pressure following teeth occlusion) and afterload contractions (e.g., lifting an object with changed effective lever arms) are composite types of contractions including isotonic, isometric, and auxotonic contractions (Brenner and Maasen 2013).

In muscle exercise, muscular strength (force) is usually increased by resistance (resistive) training which is a combination between concentric (shortening contraction, force generated while shortening occurs sufficient to overcome gravitational load) and eccentric muscle loading (lengthening contraction, force generated while muscle elongates under tension such as during deceleration of an object or lowering an object gently, the force generated becomes more and more insufficient to overcome gravitational load). Eccentric muscle contraction is typically performed during sit-ups, squatting exercises, or arm press-ups (body weight load) or with additional loading using barbells, dumbbells, or elastic straps in normal fitness or extensive strength training protocols.

Compared to concentric loading, heavy eccentric loading (muscle building, strength training) has some considerable risk of muscle damage (Faulkner 2003).

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Special Edition on Long Duration Spaceflight 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.

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• 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.

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

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Special Edition on Long Duration Spaceflight 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.

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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 Earthâ&#x20AC;&#x2122;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.

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 2436 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 astronautsâ&#x20AC;&#x2122; 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

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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 Earthâ&#x20AC;&#x2122;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 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 ex-

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ercise 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&#x2014;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 muscle 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 Commanderâ&#x20AC;&#x2122;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

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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.

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.

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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竏知ax 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 nonmotorized treadmill were required to walk and run at a positive percentage grade to overcome mechanical friction. Study participants were familiarized with the LIDO r 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 preflight 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 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 75笳ｦ /s was 15% less than preflight values but was 12% less than

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the pre-flight mean at 60◦ /s for the hamstrings.

Endurance data showed little difference between preflight 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 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.

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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 21and 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 Îą- glycerophosphate 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 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

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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 torquevelocity 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. Postflight tests were performed on R+2 and R+8. Muscle strength values were reported to be ≈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 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 par-

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Special Edition on Long Duration Spaceflight ticipated 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.

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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 glycolysisderived energy sources in biopsy samples after the 11-day STS-32 mission , no differences were detected in citrate synthase, phosporylase, or β-hydroxyacyl-CoA dehydrogenase in samples after the 17- day 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).

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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 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.

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Special Edition on Long Duration Spaceflight 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 spaceflight induced decrements.

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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 . Cross sectional 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 postflight 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.

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Special Edition on Long Duration 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 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-hydroxyacyl- CoA, lactate dehydrogenase, or phosphofructokinase were observed in calf muscles following 6 months aboard ISS.

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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.

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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 highintensity 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, one’s own body weight contributes a significant amount of load in a 1-G environment.

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 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

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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

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Special Edition on Long Duration Spaceflight 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 crewmemberâ&#x20AC;&#x2122;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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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 12-11 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.

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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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Muscle Volume, Strength, Endurance, and Exercise Loads During 6-Month Missions in Space In this study , authors report changes in the volume of skeletal muscle and strength in four crewmembers following missions of 161 - 194 days to the ISS . Authors also provide information on typical loads from ISS exercise countermeasures, including iRED, the Treadmill with Vibration Isolation System (TVIS), and the Cycle Ergometer with Vibration Isolation System (CEVIS), that these crewmembers completed together with data from exercise logs and daily monitoring so that effects of a given " dose " of the exercise countermeasure on the musculoskeletal system can be evaluated.

Four healthy male astronauts (49.5 ± 4.7 yr, 179.3 ± 7.1 cm, 85.2 ± 10.4 kg) volunteered to participate in this study and completed long-duration missions aboard ISS (181 ± 15 d). The study protocol was approved by the Committee for the Protection of Human Subjects at NASA’s Johnson Space Center, Houston, TX, and by the Institutional Review Boards at the Pennsylvania State University, State College, PA, and the Cleveland Clinic, Cleveland, OH.

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Results Muscle Volume The largest volume losses occurred in the plantar flexors (soleus: -19 ± 7% and medial gastrocnemius: -10 ± 5%) and ankle dorsiflexors (anterior calf: -10 ± 3%), while smaller decrements were observed in the muscle groups of the thigh (knee extensors: -6 ± 3%, knee flexors: 7 ± 4%, and adductors: -4 ± 3%, Fig. 2 , Table I ). There was little or no loss (0 - 1%) in the muscles of the upper extremities for the two crewmembers for whom data was available.

Results from the pre- and postflight STIR images revealed the absence of edema in the three subjects for whom this sequence was available.

Muscle Strength Fig. 3 (mean ± SD) and Table I (individual crewmembers) show the postflight changes in the peak isokinetic and isometric strength from the five-repetition protocol.

Isokinetic strength decreased in both the knee flexor ( -24 ± 8%) and knee extensor (-10% ± 11%) groups.

Ankle plantarflexor strength decreased almost three times more ( - 22% ± 6%) than dorsiflexor strength ( 8% ± 16%). The mean hip extensor isokinetic torque showed a slight mean gain (2% ± 16%), while mean strength decreased in the hip flexors (-8% ± 17%). This large variation includes one subject who showed a gain in strength postflight. Isometric strength data showed considerably greater loss in the plantar flexors ( - 20% ± 16%) compared to dorsiflexors ( -4% ± 22%), but variability in the latter test was large. Both knee extensors and flexors lost isometric strength ( - 15% ± 13% and - 20% ± 17%, respectively). The hip extensors and flexors isometric strength decreased ( -15% ± 26% and - 28% ± 9%, respectively). Isometric elbow extensor and flexor strength measured in two subjects exhibited losses ( -11% ± 4% and -8% ± 13%, respectively), as did isokinetic strength ( - 8% ± 1% and -17% ± 3%).

Endurance Total work performed during the knee endurance test decreased pre- to postflight ( - 14 ± 4%). There was also a loss in total knee flexion work postflight ( - 14 ± 8%). Fig. 4 shows the peak torque and work done during each effort in the endurance test where the data points represent the percentage change from the eighth repetition.

The initial postflight decrements in both peak torque and work performed are apparent in all tests, but there is a lower postflight rate of fatigue in both muscle groups (knee extensor work preflight vs. postflight decline: 1.55% vs. - 1.14% per contraction and knee flexor work preflight vs. postflight: - 1.0% vs. - 0.62% per contraction). The rate of fatigue (slopes of the linear fit) of the torque data shows similar trends (knee extensor peak torque decline: - 1.68% vs. - 1.16% postflight per contraction and knee flexors peak torque decline: - 1.04% vs.

- 0.64% postflight per contraction).

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Special Edition on Long Duration Spaceflight

Foot Forces and Logs from Exercise Activities Fig. 5 shows typical in-shoe forces from one foot during iRED exercise recorded at the maximum resistance setting used during the days when measurements were made. Peak single leg in-shoe forces were approximately 0.65 x bodyweight (BW), 0.55 x BW, 0.37 x BW, 1.30 x BW, and 0.92 x BW for heel raises, squats, dead lifts, single leg heel raises, and single leg squats, respectively. The peak in-shoe forces during running and cycling on ISS were approximately 1.28 x BW and 0.10 x BW ( Fig. 6 ). No preflight data using iRED were available.

Although authors only collected between 4-8 days of foot force data, the exercise logs provided information on exercise patterns for the entire mission. Fig. 1 indicates that these crewmembers were highly reliable in carrying out their specified exercise prescriptions throughout the mission, but that they emphasized different preferred exercises. For example, Subject A accumulated more than twice as many CEVIS sessions as subject D.

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Subjects D and B accumulated 110 and 121 TVIS sessions, respectively (approximately one every other day), while Subjects A and C only used TVIS for 39 and 68 days , respectively. Most notably, Subject B performed almost three times more iRED exercise than other crewmembers.

The mean changes in isometric strength ranged from about - 2% for the hip abductors to about - 35% for the hip adductors. Isokinetic concentric changes ranged from + 2% (an increase) in hip extensor strength to 24% (loss) in the knee flexors. There was about a 2.3 times greater loss in postflight peak isokinetic torque in the knee flexors compared to the knee extensors.

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Special Edition on Long Duration Spaceflight

All subjects engaged in regular prescribed exercise and it is notable that losses in muscle volume and strength were in all cases less than the values previously reported for 16 - 28-week Mir missions. The decrements in endurance expressed per week of spaceflight were also less than those seen during the longer Mir missions of 129 - 145 day. This suggests that the "dose" of countermeasures in these four ISS crewmembers was higher as a result of more exercise repetitions, higher intensity, and/or greater adherence to exercise protocols. The protocols performed varied from one subject to another, depending on such factors as availability of the device, scheduling constraints due to mission-related activities, and individual preferences. Data from exercise logs indicate that approximately 55%, 46%, and 85% of exercise sessions included the use of the TVIS, the CEVIS, and the iRED, respectively, over the course of a mission.

The ankle dorsiflexor group is worthy of special discussion since the foot is often placed in a foot loop during work on the ISS. Under such circumstances, the ankle dorsiflexors are used to align and move the body into a new position against the resistance of inertial forces. While the volume losses in the ankle dorsiflexors were close to the mean loss of all lower extremity muscles, the isometric and isokinetic strength losses were well below the mean of all muscles tested (particularly the isometric strength loss of - 4%), suggesting that foot loop use could have provided an additional form of "resistance training." Variability was, however, high in this muscle as in all the strength results and this limits the conclusions that can be drawn.

Despite these regular exercise sessions, the "dose" of exercise was probably insufficient to preserve the musculoskeletal system, as pointed out by both Trappe et al. and Lee et al. The measured loads from all exercise countermeasures were considerably less than the loads measured from similar exercises on Earth. For example, during resistance exercise on the ISS the maximum single leg load during a squat was 0.6 x BW. This contrasts with similar exercise on Earth, where a load of 1.4 x BW on the shoulders adds to the weight of the body to generate a quasi-static force under 1 foot of (1.4 + 1.0)/2 = 1.2 x BW. The maximum peak in-shoe forces from a single-leg squat (0.92 x BW) is just slightly less than that while performing the same exercise on Earth with no added load. The measured foot loads do not agree with the "nominal" load settings for the exercises as specified in the exercise prescription, which apparently overestimated the load capacity of the iRED. Similarly, the in-shoe forces during walking and running were approximately 25% less than walking on Earth (0.89 vs.

1.18 BW) and 46% less than typical running (1.28 vs. 2.36 BW) as measured from the same subjects using the same instrumentation on Earth.

The forces during cycling were extremely small - 0.1 x BW and 50% less than what was experienced on Earth.

The reason for the different responses seen in walking and running is likely due to a combination of speed and externally applied load. It should be noted that a speed limitation of 6 mph was placed on the treadmill during most of the time period studied, but it is likely that all crewmembers ran faster than 6 mph (10-min miles) during their exercise on Earth, which was the basis for comparison. Also, the load applied to the harness worn by the crewmembers during exercise on the TVIS was typically generated by either a combination of bungee

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Special Edition on Long Duration Spaceflight cords and clips or a SLD. These observations provide some additional context regarding magnitude of muscular force during countermeasure exercise to the information on the number of contractions and duration of muscle activity reported by Trappe et al..

Human skeletal muscle after 6 months aboard the International Space Station The aim of the study was to document the exercise program used by crewmembers aboard the ISS and examine its effectiveness for preserving skeletal muscle size and function. The focus was on the calf muscles, since they have been shown to atrophy more than other leg and upper body muscles with unloading.

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Ten crewmembers participated in this study . For the analysis presented, one crewmember had incomplete data sets and was not included. The subject population consisted of American astronauts and Russian cosmonauts.

The subjects’ ( n=9 ) age, height, weight, and days in space were 45 ± 2 yr, 176±2 cm, 81 ± 3 kg, and 177 ± 4 days (range = 161-192 days), respectively. An overview of each crewmember’s exercise history in the weeks preceding their launch is shown in Table 1. Before volunteering to participate in this skeletal muscle research, all crewmembers were briefed on the project objectives and testing procedures by a member of the investigative team. Crewmembers were informed of the risks and benefits with the research and gave their written consent to protocols approved by the Human Subjects Institutional Review Boards at Ball State University, Marquette University, and the National Aeronautics and Space Administration (NASA; Johnson Space Center). This study was conducted in accordance with the Declaration of Helsinki.

Exercise in space During the 6 month, the crewmembers were on the ISS, they had access to a treadmill (treadmill with vibration isolation system), two bicycle ergometers (cycle ergometer with vibration isolation system and a Velosiped, i.e., Russian bicycle exercise device), and an interim resistive exercise device (iRED). The crewmembers also had access to bungee cords, which they could use to provide resistance-type exercise for various muscle groups. The treadmill device could be used in a passive (subject driven) or active (motorized) mode of operation, which was selected by the crewmember during each exercise session. Crewmembers used a subject-loading device to fix themselves to the treadmill, which provided varying levels of loading relative to body weight (typical load was ≈70% of body weight) during use. In this way, the crewmembers could complete running or walking exercise while partially loaded. The bicycle ergometers provided typical loading in 1-W increments up to 350 W and had clipless pedals for securing their feet. The iRED is an elastomer-based resistance exercise device consisting of two canisters capable of producing up to ≈ 68 kg of force per canister.

Additional bungee cords can also be attached to increase the load characteristics. A known limitation of the iRED is the inability to precisely set and quantify workloads. The operational guidelines prescribed that crewmembers exercise while in space with up to 2.5 h allocated per day for 6 of 7 days of the week. The 2.5-h period included time needed for hardware setup, stowage, and personal hygiene. The exercise prescription was not fixed or targeted to a specific level of performance for a given physiological system. The exercise program was structured to allow for personal preference from the crewmembers along with guidance from trainers and staff within NASA and the Russian Space Agency.

To track the exercise profile while in space, crewmembers kept logbooks of their physical activity. In addition, analog data from the devices (treadmill and cycle ergometer) were downloaded (when the downlink was

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Special Edition on Long Duration Spaceflight operational) at various times while on-orbit and accounted for ≈ 65% of the treadmill and cycle ergometer data.

Members of our investigative team personally interviewed each crewmember after their mission. The combination of these three elements (logbook, downloaded data, and personal interviews) comprised the database that enabled us to profile the exercise program conducted by each crewmember while in space.

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Results Exercise training profile A summary of the aerobic exercise performed while in space is shown in Table 2. There was a wide range of aerobic training performed by the crewmembers. Aerobic exercise (cycle + treadmill) approached ≈ 5 h/wk or ≈ 50 min/day. On average, subjects completed 138 ± 26 min/wk of cycle exercise that generally ranged between 100 and 150 W. While in orbit, crewmembers used the cycle ergometer ≈60% of mission days. Four of the crewmembers (subjects A, B, C, and G) used the cycle ergometer ≈ 81% (range=70 - 90%) or more of mission days, whereas four others (subjects D, F, H, and I) averaged ≈ 37% (range= 28 - 41%) of mission days, and one crewmember (subject E) did minimal cycling. For the treadmill exercise, subjects averaged 146 ± 34 min/wk on a level grade at a speed ranging from 2.1 to 5.5 miles/h. The treadmill appeared to be used less frequently compared with the cycle ergometer, accounting for <50% of total mission days. However, there was a wide range of treadmill use, with four crewmembers (subjects B, E, G, and H) having a high volume (>200 min/wk) of walking/running activities. The other five crewmembers (subjects A, C, D, F, and I) used the treadmill much less (≤85 min/wk).

A summary of the resistance exercise performed while in space is shown in Table 3. Generally, all crewmembers performed a suite of leg exercise routines consisting of squats, heel raises, and dead lifts. The program varied for each crewmember, with everyone performing resistance exercise at least 3 days/wk and several conducting resistance training 5 - 6 days/wk. During the resistance training sessions, crewmembers averaged 3 - 6 sets of 12-20 repetitions for each leg exercise. Authors were unable to get an accurate account of the time involved with the resistance exercise.

Based on the number of contractions and assuming a 2-s count for concentric and eccentric contractions (from video), authors estimate crewmembers spent more than 1 h/wk with the leg muscles under tension during resistance exercise. Based on the exercise database and available videos of crewmembers while exercising (cy-

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cling, running, and lifting), authors were able to estimate the number of muscle contractions performed while in space (Table 4). On average, crewmembers performed more than 435,000 muscle contractions per leg during scheduled exercise while in space. As noted earlier, the exercise program was highly variable with a low of ≈ 200,000 (subject C) and a high approaching 1 million (subject B) muscle contractions for each leg. Generally, crewmembers performed exercise 6 of 7 days/wk. Of the total time in orbit (≈ 4,248 h), the exercise program presented (minus setup, stowage, and hygiene) constituted ≈ 3.4% of the time. When sleep, workday schedule, and leisure time were considered, the estimate for exercise time increased to 7-10% of the available time for the crewmembers while in space.

Muscle volume A summary of calf muscle volume before and after space flight is shown in Table 5. The gastrocnemius (medial and lateral) and soleus muscle were smaller (P < 0.05) after 6 mo in space. Combined, the gastrocnemius and soleus atrophied (P < 0.05) -13±2% pre- to postflight. The soleus (-15±2%) atrophied more (P < 0.05) than the gastrocnemius (-10±2%) pre- to postflight (Fig. 1). One crewmember (subject E) had insignificant (-1%) atrophy after the flight. Two of the crewmembers (subjects A and F) lost more than 20% of their calf muscle mass. Of the remaining six crewmembers, five lost more than 10% calf muscle mass. At R+19 after landing, the gastrocnemius was still 5-6% atrophied, but this was not significant. Conversely, the soleus was still reduced (P < 0.05) compared with preflight, averaging -9 ±1% for all crewmembers. Although calf muscle volume was

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still reduced -8±2%, it represented a partial recovery from the more immediate (R+4) flight measurement.

Muscle performance A summary of each crewmember’s calf muscle performance for MVC at one angle (neutral position) and a slow (60◦ /s) and fast (180◦ /s) isokinetic speed are shown in Table 6. MVC was reduced (P < 0.05) -14±2% at R+7 and remained lower (-13±5%; P < 0.05) at R+13. All nine crewmembers had a decline in MVC (range=7 to -22%) with flight. At R+13, seven of the nine crewmembers were lower (range=-9 to -33%), with two crewmembers (subjects A and H) having a 5-10% increase compared with preflight. At the slow isokinetic speed (60◦ /s) there was a -20±3% loss (P < 0.05), which was sustained (-19±4; P < 0.05) at R+13. This pattern was also evident at the faster (180◦ /s) isokinetic speed with a 25±10% reduction at R+7 and R+13. For both the slow and fast speeds, eight of the nine subjects had a decrease in muscle performance. A force-velocity curve for all subjects from pre- to postflight (R+7) is shown in Fig. 2. On average, force-velocity characteristics were reduced -20 to -29% across the velocity spectrum (P < 0.05). Peak power was 134±11, 91±10, and 94±13 W preflight, R+7, and R+13, respectively. On average, peak power declined 32% with spaceflight (P < 0.05).

Muscle fiber type Authors isolated and analyzed the MHC profile on a total of 4,328 single muscle fibers from the gastrocnemius and soleus muscles before and after flight. The breakdown was 1,960 muscle fibers (1,109 preflight, 851 postflight) for the gastrocnemius and 2,368 muscle fibers (1,277 preflight, 1,091 postflight) for the soleus. The average MHC profile of the gastrocnemius and soleus muscles from the crewmembers before and after space flight is shown in Fig. 3. Individual data from the gastrocnemius and soleus of each crewmember are shown in Tables 7 and 8, respectively. One individual (subject B) had a small muscle biopsy sample and therefore was not

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Special Edition on Long Duration Spaceflight included in these analyses. The gastrocnemius had a 12% decrease (P < 0.05) in MHC I fibers and an increase (P < 0.05) in MHC I/IIa (+4%) hybrid fibers and MHC IIa fibers (+9%). Seven of the eight subjects had a decrease in MHC I fibers (range=-6 to -31%). There were minimal MHC IIx and MHC I/IIa/IIx fibers detected in the pre- and postflight muscle samples. The 4% increase in hybrid muscle fibers appears to be the result of the MHC I/IIa hybrid fiber type.

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On average, the soleus had a 17% decrease (P < 0.05) in MHC I fibers. The shift away from MHC I fibers was distributed among the other fiber types (MHC I/IIa, IIa, IIa/IIx), with a nonsignificant increase of 4-5% within each MHC phenotype. Combined, the soleus had a 12% increase (P < 0.05) in hybrid MHC isoforms. Three of the crewmembers (subjects D, E, and I) did not have any major alterations in fiber type of the soleus. Four of the crewmembers (subjects A, C, F, and G) had a decrease in soleus MHC I fibers that ranged from -20 to -44%.

The main finding from this study was that the exercise program did not completely protect the calf muscles. Authors observed a substantial decrease in calf muscle mass and performance along with a slow-to-fast fiber-type transition in the gastrocnemius and soleus muscles, which are all traits associated with unloading in humans.

These data suggest that changes to the exercise countermeasure program are required to more fully protect human skeletal muscle while crewmembers are in space for extended periods.

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The average amount of muscle mass lost (13%) with spaceflight was slightly less than with previous long-duration stays on the Russian Space Station Mir (-17%). The current ISS and previous Mir calf muscle volume loss is about one-half that of long-duration (60- to 120day) bed rest studies showing a ≈29% decrease among the control subjects without countermeasures . These data imply that the exercise in space is having a beneficial effect but is not complete, with the soleus being more difficult to protect than the gastrocnemius.

The data suggest that the crewmembers with larger calf muscles had a greater degree of atrophy with long-duration spaceflight. Second, the volume of treadmill exercise may have provided a level of protection for calf muscle mass. The three individuals (subjects B, E, and H) who performed >200 min/wk on the treadmill lost about onethird of the calf muscle volume (-43 ± 19 cm3 ) compared with crewmembers who used the treadmill <100 min/wk (-135 ± 16 cm3). As a percentage, this translated into a -7 ± 3% muscle loss for the high-volume treadmill users and -17 ± 2% for the low-volume treadmill users.

Interestingly, two of the high-volume treadmill users who lost the least amount of muscle mass (subjects B and E) also had the smallest calf muscles before flight. Third, when the treadmill is used in passive mode, more force is needed to drive the belt during walking/running activities and may help protect against calf muscle atrophy. Finally, inadequate caloric intake may have contributed to the muscle atrophy. These combined factors (initial muscle volume, amount of treadmill exercise, mode of treadmill use, and negative caloric balance) likely contributed to the varied muscle mass findings in this study.

Microgravity-Induced Fiber Type Shift in Human Skeletal Muscle Spaceflight induces quantitative and qualitative modifications to skeletal muscle by markedly decreasing size, strength, and endurance . Despite exercise countermeasures, muscle mass has been shown to decrease from -13% to -17% during long-duration spaceflight. Furthermore, long mission studies conducted aboard the ISS, Skylab, and Mir have shown significant decreases (-20 - 35%) in muscle performance.

Human Skeletal Muscle Fiber Type Classifications Muscle fibers are composed of functional units called sarcomeres. Within each sarcomere are the myofibrillar proteins myosin (the thick filament) and actin (the thin filament). The interaction of these 2 myofibrillar proteins allows muscles to contract (Fig. 1).Several classification techniques differentiate fibers based on different myosin structures (isoforms) or physiologic capabilities. The myosin molecule is composed of 6 polypeptides: 2 heavy chains and 4 light chains (2 regulatory and 2 alkali). A regulatory and an alkali light chain are associated with each of the heavy chains. The heavy chains contain the myosin heads that interact with actin and allow muscle to contract (Fig. 1). The myosin heavy chain in the head region also contains an adenosine triphosphate (ATP) binding site and serves as the enzyme (adenosinetriphosphatase [ATPase]) for hydrolyzing ATP into adenosine diphosphate (ADP) and inorganic phosphate (PI), which provides the energy necessary for muscle contraction. The thin filament is made of actin and 2 regulatory proteins, troponin and tropomyosin. When the muscle fiber receives a stimulus in the form of an action potential, Ca2+ is released from the sarcoplasmic reticulum. The calcium then binds to troponin and, through tropomyosin, exposes a myosin binding site on the actin molecule (Fig. 1). In the presence of ATP, the myosin head binds to actin and pulls the thin filament along the thick filament, allowing the sarcomere to shorten. As long as Ca2+ and ATP are present, the myosin heads will attach to the actin molecules, pull the actin, release, and reattach. This process is known as cross-bridge

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Special Edition on Long Duration Spaceflight cycling. The speed at which cross-bridge cycling can occur is limited predominantly by the rate that the ATPase of the myosin head can hydrolyze ATP.

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Muscle Fiber Typing Initially, whole muscles were classified as being fast or slow based on speeds of shortening.This division also corresponded to a morphological difference, with the fast muscles appearing white in some species, notably birds, and the slow muscles appearing red. The redness is the result of high amounts of myoglobin and a high capillary content. The greater myoglobin and capillary content in red muscles contributes to the greater oxidative capacity of red muscles compared with white muscles. Histological analysis shows that there is a correlation between myosin ATPase activity and the speed of muscle shortening. This histochemical analysis led to the original division of muscle fibers into type I (slow) and type II (fast).

Currently, muscle fibers are typed using 3 different methods: histochemical staining for myosin ATPase, myosin heavy chain isoform identification, and biochemical identification of metabolic enzymes.

Myosin ATPase Staining In humans, myosin ATPase hydrolysis rates for fast fibers are 2 to 3 times greater than those of slow fibers.

However, myosin ATPase histochemical staining, which is widely used for classifying muscle fibers, does not evaluate myosin ATPase hydrolysis rates. Fibers are separated based solely on staining intensities because of differences in pH sensitivity, not because of the relative hydrolysis rates of ATPases. Advances in the histochemical staining technique used to evaluate myosin ATPase have led to 7 recognized human muscle fiber types (Fig. 2).

Originally, fibers were identified as type I, IIA, or IIB. More recently, types IC, IIC, IIAC, and IIAB, which have intermediate myosin ATPase staining characteristics, have been identified. The slowest fiber, type IC, has staining characteristics more like those of type I fibers, whereas the fastest fiber, type IIAC, stains more like type IIA. Type IIAB fibers have intermediate staining characteristics between type IIA and IIB fibers. Because these delineations are based on qualitative analysis of stained fibers, it remains possible that more fiber types will be identified in the future. In summary, the 7 human muscle fiber types, as identified by myosin ATPase histochemical staining are (from slowest to fastest): types I, IC, IIC, IIAC, IIA, IIAB, and IIB (Fig. 2). These divisions are based on the intensity of staining at different pH levels, and, as such, any given fiber could be grouped differently by different researchers. Furthermore, not all studies use all 7 fiber types. Some researchers place all muscle fibers into just the original 3 fiber types.

Myosin Heavy Chain Identification Identification of different myosin heavy chain isoforms also allows for fiber type classification (Fig. 2). The different myosin ATPase-based fibers correspond to different myosin heavy chain isoforms. This is not surprising because the myosin heavy chains contain the site that serves as the ATPase. The fact that each muscle fiber can contain more than one myosin heavy chain isoform explains the existence of myosin ATPase fiber types other than the pure type I, type IIA, and type IIB fibers. Although the human genome contains at least 10 genes for myosin heavy chains, only 3 are expressed in adult human limbmuscles. Myosin heavy chain isoforms can be identified by immunohistochemical analysis using antimyosin antibodies or by sodium dodecyl sulfatepolyacrylamide gel electrophoretic (SDSPAGE) separation.

The 3 myosin isoforms that were originally identified were MHCI, MHCIIa, and MHCIIb, and they corresponded to the isoforms identified by myosin ATPase staining as types I, IIA, and IIB, respectively. Human mixed fibers almost always contain myosin heavy chain isoforms that are "neighbors" (ie, MHCI and MHCIIa or MHCIIa and MHCIIb). Consequently, the histochemical myosin ATPase type IC, IIC, and IIAC fibers coexpress the MHCI and MHCIIa genes to varying degrees, whereas the type IIAB fibers coexpress the MHCIIa and MHCIIb genes.

Because of its quantitative nature, identifying myosin heavy chain isoforms using single-fiber electrophoretic separation (SDS-PAGE technique) probably represents the best method for muscle fiber typing. Electrophoretic separation allows for the relative concentrations of different myosin heavy chain isoforms to be detected in a mixed fiber. One point regarding human myosin heavy chain isoforms and fiber type identification may prove confusing to someone trying to read research literature in this area.

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Special Edition on Long Duration Spaceflight In small mammals, a fourth myosin heavy chain isoform, MHCIIx or MHCIId, is present that has an intermediate contractile speed between the MHCIIa and MHCIIb isoform. Based on several types of evidence, extending to the level of DNA analysis, what was originally identified in humans as MHCIIb is actually homologous to MHCIIx/d of small mammals.

As a result, what has been called MHCIIb in humans is actually MHCIIx/d, and humans do not express the fastest myosin heavy chain isoform (MHCIIb).

Because the histochemical myosin ATPase fiber type nomenclature was developed using human muscle, type IIB fibers, which we now know correspond to the MHCIIx/d myosin heavy chain isoform, are not likely to be renamed type IIX. Consequently, depending on the author, histochemical myosin ATPase-based human type IIB fibers may be associated with either MHCIIb or MHCIIx/d isoforms. It is important to remember that in human limb muscles only 3 myosin heavy chain isoforms are present (from slowest to fastest): MHCI, MHCIIa, and MHCIIx/d (formerly erroneously identified as MHCIIb). Humans do not express the fastest myosin heavy chain isoform, MHCIIb. We will associate MHCIIx/d in humans with the histochemhistochemical myosin ATPase-based type IIB fiber in the remainder of this article.

Biochemical A third classification scheme that is often used to classify muscle fibers combines information on muscle fiber myosin ATPase histochemistry and qualitative histochemistry for certain enzymes that reflect the energy metabolism of the fiber (Fig. 2). Histochemical myosin ATPase fiber typing is used to classify muscle fibers as type I or type II, which are known to correspond to slow and fast muscle fibers, respectively. The enzymes that are analyzed reflect metabolic pathways that are either aerobic/oxidative or anaerobic/glycolytic. This classification technique leads to 3 fiber types: fast-twitch glycolytic (FG), fast-twitch oxidative (FOG), and slow-twitch oxidative (SO). Although a good correlation exists between type I and SO fibers, the correlations between type IIA and FOG and type IIB and FG fibers are more varied. Therefore, the type IIB fibers do not always rely primarily on anaerobic/glycolytic metabolism, nor do the type IIA fibers always rely primarily on aerobic/ oxidative metabolism. Although, in general, fibers at the type I end of the continuum depend on aerobic/ oxidative energy metabolism and fibers at the type IIB end of the continuum depend on anaerobic/glycolytic metabolism, the correlation is not strong enough for type IIB and FG or type IIA and FOG to be used interchangeably.

Microgravity-Induced Fiber Type Shift Spaceflight and bed rest induce decreased MHC I fiber proportion while increasing fast hybrid types (particularly MHC IIa/IIx fibers). This alteration in muscle cell phenotype negatively impacts performance and induces undesirable metabolic adaptations. While exercise has been postulated to minimize the negative effects of microgravity on human muscle, past spaceflight countermeasures have insufficiently prevented fiber type shifts in humans.

Research supporting a MHC fiber type shift during spaceflight in humans has been increasing since the mid1990s. After several ISS missions and long-term bed rest experiments in the last decade, enough data now exists to draw conclusions on the presence of spaceflight related fiber type shifts in humans. Figure 4 contains compiled data from laboratory and others, lending support to the microgravity-induced fiber type shift paradigm in humans. Each of the studies report changes in fiber type from pre to post-spaceflight (or bed rest) in men and women measured via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Unloading duration ranged from 11 to 177 days, with an average of â&#x2030;&#x2C6; 81 days. The studies investigated one of three lower limb muscles: the vastus lateralis (VL), soleus (Sol), or gastrocnemius (Gas). MHC I (slow) fiber composition decreased and total hybrid fiber proportion increased in all studies by an average of -13% and +14%, respectively. While unloading duration probably dictates the transition magnitude, trends were similar regardless of duration, unloading mode, or the muscle studied.

Long-duration spaceflight excercise countermeasures Past exercise regimens onboard the ISS were varied among crewmembers, but generally included moderate

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intensity aerobic (â&#x2030;&#x2C6;5 days/wk) and resistance exercise (3-6 days/wk). The guidelines prescribed exercise for up to 2.5 h/day for 6-7 days/wk (time included hardware setup, stowage, and personal hygiene) utilizing a running treadmill, cycle ergometer, and resistance exercise device. These previous exercise countermeasures failed to completely preserve skeletal muscle size and function, warranting modifications to longduration mission exercise prescription and/or hardware.

For decades, ground-based exercise physiology studies have shown chronic highintensity exercise promotes positive skeletal muscle adaptations (i.e. increases strength and endurance) and alters fiber type composition.

Figure 5 illustrates fiber type changes (maintained MHC I, increased MHC IIa, decreased MHC IIx) following high-intensity and sprint cycle training in men and women ranging from 42 to 105 days in duration. These studies measured fiber type by SDS-PAGE or histochemical staining (standard technique of the 1970s and â&#x20AC;&#x2122;80s).

Hybrid fibers were not reported in these investigations. MHC I fiber percentage varied but was generally maintained (+1%), while MHC IIa composition increased (+6%) and MHC IIx composition decreased (-5%) on average. As opposed to spaceflight and bed rest, the trend-line compiled from these high-intensity/sprint cycling studies demonstrates a fast to relatively slower fiber type shift. MHC I fibers significantly increased (+6%), MHC IIa fibers were maintained, and MHC IIb (IIx) fibers significantly decreased (- 6%) after 105 days of sprint cycling, suggesting lengthier training durations might induce increases in MHC I proportions as their transition may take longer to manifest.

Data from Figures 4 and 5 suggest mitigation of the microgravity-induced slow to fast shift is possible by employing high-intensity exercise during spaceflight. The idea of high-intensity exercise preventing a shift in MHC phenotype during long-duration unloading was recently shown with bed rest (60 day), which has served as a guide for moving the exercise countermeasure program forward.Past exercise countermeasures onboard the ISS have insufficiently prevented fiber type shifts in humans (as seen in Figure 4). Moving forward, two key changes to the exercise program for spaceflight have occurred. The first was placement of new hardware on the ISS that allows for greater loading and comfort for performing more robust exercise. Figure 6 shows images of these devices, which include the Advanced Resistance Exercise Device (ARED), Cycle Ergometer with Vibration Isolation and Stabilization System (CEVIS), and Combined Operational Load Bearing External Resistance Treadmill (COLBERT). Second, was the implementation of a new high-intensity, low volume resistance and aerobic exercise prescription for astronauts. The new regimen alternates days of high-intensity interval training with continuous aerobic exercise (opposed to predominately continuous aerobic exercise) and 3 days/wk of highintensity resistance training (opposed to 3-6 days/wk at lower intensity) .

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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 â&#x2030;&#x2C6;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 mus-

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Special Edition on Long Duration Spaceflight culoskeletal 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 authorsâ&#x20AC;&#x2122; 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. 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. Understanding the factors that contribute to such large variability is an important step toward both selecting and protecting the first astronauts who undertake very long (2-3 year) exploration missions. The extent to which biological sex or sex-based hormones 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.

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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 Womenâ&#x20AC;&#x2122;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, 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 re-

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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, sexbased 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 of muscle strength within 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.

Effects of prolonged space flight on human skeletal muscle enzyme and substrate profiles The purpose of this study was to determine the effects of a 6-month spaceflight on the ISS on selected anaerobic and aerobic enzymes, and the content of glycogen and lipids in slow and fast fibers of the soleus and gastrocnemius. The effects of countermeasure exercise were evaluated by relating pre- and postflight enzyme patterns to the extent of in-flight treadmill exercise.

The crew members participating in this study flew aboard the ISS from increments 5 to 11 (2002-2005). In the overall study group there were 10 crew members: 5 American astronauts and 5 Russian cosmonauts; however, for the metabolic studies described here there were nine subjects (5 astronauts and 4 cosmonauts). Due to small sample size and/or problems in shipment from Russia to the United States, the histochemical and biochemical assays were performed on biopsy tissue from six or seven and eight crew members, respectively.

RESULTS Authors purpose was to relate the atrophy of a given fiber type to specific enzyme and substrate changes. As reported previously , the degree of fiber atrophy varied greatly between crew members, and considerable variability was also found in muscle enzymes. The LT crew members, running <100 min/wk, showed more fiber atrophy than the HT group, running 200 min/wk or more . For the soleus muscle, the average decline in diameter for the type I and II fibers was 20 and 16%, respectively, but the differences between the LT and HT groups was substantial for the soleus type I (29% LT vs. 8% HT), and type II (27% LT vs. 3% HT) fibers . Subjects were identified with the letters A to I using the same letter scheme . Subjects B, E, G, and H are in the HT group, and Subjects A, C, D, F, and I are LT members. Authors subdivided the crew into those who ran 200 min/wk or more (high treadmill, HT) in-flight from those who ran <100 min/wk (low treadmill, LT).

Soleus Muscle Histochemistry: Enzyme Activity and Substrate Content Figures 1 and 2 show cross-sections of pre- and postflight soleus muscle fibers reacted for actomyosin ATPase activity to illustrate fiber types in LT Subject C (Fig. 1, A and B) and HT Subject E (Fig. 2, A and B). In the acid-preincubated ATPase sections, type I slow-twitch oxidative fibers are darkly stained. Type IIa fast-twitch oxidative glycolytic fibers are lightly reactive. Based on immunostaining for fast and slow myosins (data not shown), the moderately reactive fibers are hybrid fibers containing both fast and slow myosin (Fig. 2A). His-

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tologically, there were insufficient numbers of IIx fibers in the soleus and gastrocnemius muscle sections to quantify cytochrome oxidase (CO), lipid, or glycogen content in this fiber type, so the measurements in Tables 2, 3, and 4 were restricted to type I and IIa fibers. Postflight atrophy is evident in the fibers of LT Subject C (Fig. 1B), whereas atrophy did not occur in the HT Subject E (Fig. 2B).

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In the preflight samples of all subjects, the mean CO activity for soleus type IIa fibers was significantly less than for the slow type I fibers. Postflight, the all crew member mean for CO activity for the fast type IIa fiber was unchanged, while the activity of this enzyme was significantly depressed in the slow type I fiber (Table 2).

Despite no change in the composite mean (HT and LT groups combined), soleus type IIa fibers of the HT group had postflight values significantly higher than preflight, while the LT group mean was unaltered (Table 2). The CO activities for Subjects C and E are shown in serial section (Fig. 1, C and D, and Fig. 2, C and D, respectively).

The CO reaction product indicates an abundance of mitochondria in subsarcolemmal clusters and

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within myofibrils. The area percent of CO reaction product per muscle fiber area was reduced by 64% in the postflight soleus of Subject C (Fig. 1, C and D; Table 2). The average reduction was 59% for the four LT subjects (Table 2). For Subject E, the CO activity was not significantly altered postflight (Fig. 2, C and D; Table 2). The three HT subjects on average showed no significant change in CO activity, but large individual variations were present in soleus type I fiber, with Subject B highly increased, Subject E unchanged, and Subject H significantly down (Table 2).

Soleus fiber lipid content was quantified in Oil Red O stained sections (Fig. 1, E and F, and Fig. 2, E and F). The lipid-stained fibers are serial to the acid ATPase sections. The lipid content preflight was higher in type I fibers than IIa fibers (Table 3). Postflight in the LT group, there was loss of lipid in type I fibers, while the lipid content was preserved in the HT group (Table 3). Pooling All crew members, the soleus type I fiber lipid was unchanged postflight (Table 3). Representative of the LT group, Subject CĹ s soleus type I fibers exhibited decreased lipid postflight (Fig. 1, E and F). In contrast, HT Subject E had higher type I fiber lipid preflight than Subject C, and the lipid content was maintained postflight (Fig. 2, E and F; Table 3).

Gastrocnemius muscle histology Atrophy was pronounced in the gastrocnemius type I and II fibers in the LT group. Figure 3 illustrates fiber atrophy in alkaline-preincubation actomyosin reacted sections from Subject D (Fig. 3, A and B). This individual exhibits a shift from mostly type I fibers (lightly stained) to expression of darkly stained type II fibers postflight (Fig. 3B). Immunostaining with myosin isoform specific antibodies for type IIa and IIx revealed that a small proportion of type II fibers contained IIx myosin (data not shown). Serial sections revealed that CO decreased postflight in large part due to loss of peripheral subsarcolemmal staining (Fig. 3, C and D). CO levels were significantly lower in gastrocnemius type I fibers of the LT group postflight (Table 2). For the LT and HT groups, lipid concentration in gastrocnemius type I and IIa fibers tended higher postflight but not significantly (Table 4). However, a significant increase in lipid content was observed postflight for Subject D and Subject E for the type I fiber, and Subject H for the type IIa fiber (Fig. 3, E and F, Table 4).

Fiber glycogen Fiber glycogen was assessed histologically with PAS staining. Postflight soleus glycogen was significantly elevated for slow type I and fast type IIa fibers when all crew were compared quantitatively for PAS staining by optical density (Table 3). Representative micrographs illustrate LT Subject C soleus in which glycogen significantly increased postflight in both type I and IIa fibers (Fig. 4, A and B). For the gastrocnemius muscle, the preflight levels of glycogen in type I and IIa fibers are higher than that in soleus (Tables 3 and 4), and all crew quantitation reveals maintenance of PAS staining in the postflight fibers (Table 4). When the individual changes are examined, there are significant increases as shown in Fig. 4, C and D, for Subject A, and decreases in gastrocnemius glycogen staining that are not explained by HT or LT exercise (Table 4).

Biochemical Analysis of Enzyme Capacity Soleus muscle The effect of prolonged spaceflight on the glycolytic enzymes LDH and phosphofructokinase, and the mitochon-

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drial enzymes β-hydroxyacyl-CoA dehydrogenase (βOAC) and citrate synthase (CS), in soleus type I and type II fibers is shown in Tables 5 and 6, respectively. When all crew members were grouped together, there was no change in the soleus type I fiber glycolytic or mitochondrial enzyme activity (Table 5). However, two of the three HT (Subjects E and H) showed significant increases in both βOAC and CS in the soleus type I fibers (Table 5).

One LT, Subject I, also showed an increased mitochondrial enzyme capacity, but the magnitude of the increase (11% and 25%) was considerably less than that for the HT subjects where the increase ranged from 40% to 87%. Regarding the soleus type I fiber, Subject E (HT group) exhibited the largest increase in oxidative enzymes and was the only subject to show a significant decrease in glycolytic enzyme activity. In contrast, Subject C (LT group) showed a large decline in cytochrome oxidase and a significant increase in the glycolytic enzyme LDH (Table 5). Figure 5 shows a plot of CS vs. LDH for Subjects C and E for those fibers in which the activity of these two enzymes were determined. The plot demonstrates the rather wide range of enzyme activities that exist within a given fiber type, the postflight shift to higher aerobic (CS) and lower glycolytic (LDH) enzyme activities in HT Subject E, and the elevated LDH in LT Subject C (Fig. 5). Glycolytic and mitochondrial enzyme activities of fast type II fibers in the soleus were, with few exceptions, unaltered by the 6 month of microgravity.

The exception was LDH, where Subjects C and H showed a significant decrease and Subject D a significant increase postflight (Table 6).

Gastrocnemius muscle The glycolytic and mitochondrial enzyme activities of the gastrocnemius slow type I and fast type II fibers, and their changes with prolonged space flight, are shown in Tables 7 and 8. From a qualitative perspective, the microgravity effects in the slow type I fiber were similar to those observed for this fiber type in the soleus, in that no changes were observed in group means for any enzyme. Examination of the individuals revealed that subjects who showed a significant increase in CS activity (Subjects A, E, and G) showed a significant decrease in LDH activity, and Subject I with a postflight decline in CS showed increased LDH (Table 7).

Figure 6 illustrates this point by showing a plot for βOAC vs. LDH for gastrocnemius type I fibers for Subjects D and G preand postflight. Similar to the soleus, within a fiber type there is a wide range of activities for a given enzyme, and Subject G with a significant postflight increase in βOAC showed a significant decline in LDH activity. In comparison, Subject D showed a significant increase in LDH, which was accompanied by a small but nonsignificant decline in βOAC. The gastrocnemius type II fiber showed no group mean changes with microgravity for any of the enzymes studied (Table 8). However, similar to the type I fiber, when a significant increase in oxidative enzyme was observed, as for Subject H, a significant decline was observed for LDH (Table 8).

Comparing the LT and HT group means, one sees that there were no significant differences pre- vs. postflight, but βOAC tended up and LDH tended down in the HT group, with the opposite observed for the LT group (Table 8). The reciprocal change for the HT group, with the glycolytic enzyme decreasing and the oxidative enzymes

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increasing postflight, is shown in Fig. 7 for Subject H. Figure 7 also shows the postflight increase in LDH for LT Subject A.

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Despite in-flight aerobic and strength exercise-training, prolonged weightlessness caused considerable declines

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Special Edition on Long Duration Spaceflight in fiber mass, force, and power, with the greatest effects observed in slow type I fibers of the soleus . These changes were partially attenuated by treadmill running. A consistent observation is that crew members during weightlessness experience increased fatigue, and this is particularly true during extravehicular activities . This is in part explained by declines in peak aerobic capacity and muscle atrophy, so that loads represent a higher percentage of the crew memberâ&#x20AC;&#x2122;s peak aerobic capacity . The extent to which the latter could be attributed to declines in stroke volume and thus cardiac output vs. a reduced tissue oxidative capacity is unknown. With short-duration space flight, authors observed that VO2 elicited at a HR equal to 85% of the crew membersâ&#x20AC;&#x2122; preflight maximum HR showed a rapid and continued decline between day 2 (-6.2%) and day 13 (-11.3%), and recovered rapidly postflight . Others have shown that short-duration space flight reduces mitochondrial protein, but the decline is less than fiber atrophy, so the oxidative enzyme activity remained unaltered or slightly elevated when activity was expressed per gram of dry weight.

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Based on Oil Red O staining, soleus type I fiber lipid was significantly reduced in the LT but not the HT crew members. Preflight glycogen content was higher in the fast type IIa than the slow type I fibers, and this was true in the soleus and gastrocnemius. The highest content was observed in the fast fibers of the gastrocnemius.

Postflight, the glycogen content was significantly increased in the fast and slow fibers of the soleus, but no change was observed in the gastrocnemius. It is not possible to determine whether the increase was caused by weightlessness or the fact that the crew consumed carbohydrate-rich foods 12 h before return to earth.

In conclusion, the results of this study demonstrate that, despite prolonged weightlessness, crew members are able to maintain their muscle aerobic and glycolytic enzyme capacity and that, with adequate amounts of treadmill running, even are able to improve muscle oxidative capacity. The decline in type I fiber intracellular lipid in the antigravity soleus muscle was correlated with fiber atrophy, and was prevented by treadmill running that exceeded 200 min/wk. The increased fatigability reported to occur with weightlessness cannot be directly attributed to a loss of muscle substrate or enzyme capacity, but likely is in part due to fiber atrophy, such that extravehicular activities require greater fiber recruitment, both an increased number of fibers and activation frequency, both of which lead to an earlier onset of fatigue.

Simulated Microgravity Contributes to Autophagy Induction by Regulating AMP-Activated Protein Kinase A clinostat is considered a useful model system for simulating microgravity in biological experiments. A clinostat is equipped with two independent rotating axes to disperse the gravity vector uniformly to a whole steric angle.

Thus far, simulated microgravity using the clinostat has provided insight into biological processes and has been used to examine the biological changes.

Autophagy is an intracellular degradation and recycling pathway, conserved from yeast to human life forms.

While the ubiquitin-proteasomal degradation system targets most soluble short-lived proteins, autophagy targets long-lived proteins and organelles such as mitochondria. Autophagic degradation is mediated by the for-

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mation of autophagosomes, which are double-membrane vesicles that engulf the cytoplasmic organelles and macromolecules. The autophagosome is then fused with a lysosome to form an autolysosome, in which the targeted protein is degraded via lysosomal hydrolases at a low pH. The degradation products such as amino acids can then be recycled.

Autophagy is induced by various stimuli, including nutrient depletion, accumulation of damaged organelles, and infection of cytoplasmic pathogens. A recent study identified the cellular signaling pathways of autophagy and autophagy regulation by nutrient depletion stimulus. Nutrient depletion promotes autophagy by activating AMPdependent protein kinase (AMPK), a key energy sensor that responds to an increased AMP/ATP ratio.

AMPK is also activated by various cellular stresses, such as hypoxia and heat shock. AMPK activation results to the activation of FoxO3, which leads to an increase in the expression of Beclin, LC3-II, and Gabarap11. In addition, autophagy is negatively regulated by mammalian target of rapamycin (mTOR), which responds to the nutrient signal. Both AMPK activation and mTOR suppression activates the autophagy-initiating kinase Ulk1 (ATG1 in yeast).

Muscle atrophy is a decrease in muscle mass that is typically caused by a variety of diseases or disuse. A decrease in muscle mass is caused by loss of muscle protein. The proteasomes degrade myofibrillar proteins via the ubiquitinproteasome pathway, and autophagy is responsible for long-lived protein and organelle degradation. To sensitively examine autophagy induction due to microgravity, HEK293 cells with stable expression of GFP-LC3 (GFPLC3 cells), an autophagosomal marker, and used the clinostat to evaluate the effect of microgravity on autophagy induction. Incubation of GFP-LC3 cells in the clinostat resulted in the alteration of autophagosomal marker expression, which was accompanied by AMPK activation and mTOR suppression.

For sensitive autophagy monitoring, authors used the HEK293 cell lines that stably express GFP-LC3, an autophagosomal marker. Various autophagy signals stimulate the formation of cytoplasmic GFP-LC3 punctuates, which reflect autophagy induction. GFP-LC3 cells were set on the clinostat to simulate microgravity and then rotated for 24 or 72 h. GFP-LC3 cells exposed to microgravity for 72 h showed cytoplasmic GFPLC3 punctuates, which are reflective of the autophagosomes, while cells exposed to other conditions did not (Fig. 2A, B). For motion control, authors rotated GFP-LC3 cells using the laboratory platform rocker at 5 rpm for 72 h; however, we did not observe the cytoplasmic GFP-LC3 punctuates (Fig. 2C, D). These results indicate that simulated microgravity positively regulates autophagosome formation.

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To biochemically analyze autophagy induction under simulated microgravity condition, authors examined the expression patterns of LC3 and p62, which are widely used as markers of autophagosomes. LC3 is initially produced in an unprocessed form and then converted into LC3-I by Cterminal domain cleavage. LC3-I is then modified into PEconjugated LC3-II, the autophagosomal marker.GFP-LC3 cells placed in the clinostat for 24 or 72 h and untreated control cells were collected and the cell lysates were subjected to western blot analysis with anti-LC3 antibody. While other conditions did not show a significant change in the density of GFP-LC3-II bands, clinorotation for 72 h resulted in an elevation of GFP-LC3-II level (Fig. 3A).

Authors then examined the level of p62, another autophagy marker. Because p62 (also called SQSTM1) is degraded by autophagy and inhibition of autophagy upregulates p62, p62 has been implicated as a potential autophagic marker.Western blot analysis with anti-p62 antibody showed reduced levels of endogenous p62 after 72 h of clinorotation (Fig. 3A).

Western blot results were consistent with the microscopy data, and these results collectively indicate that simulated microgravity positively regulates autophagy induction. Inhibition of autophagic flux using bafilomycin A1 resulted in the elevated level of endogenous LC3-II, indicating that autophagosomes were not formed due to inhibition of lysosomal degradation (Fig. 3B). Since autophagy is induced by simulated microgravity conditions, we attempted to determine the cellular signaling pathway involved in autophagy induction. Recent studies showed that AMPK and mTOR regulate autophagy directly via phosphorylation of Ulk1 (yeast ATG1).

Authors examined phosphorylation at two sites on AMPK (T172 and S485) and mTOR. AMPK is activated by phosphorylation at threonine 172 (T172) within the a subunit activation loop and is suppressed by phosphorylation at serine 485 (S485). Clinorotation for 24 and 72 h increased the level of phosphorylation of AMPK at T172 when compared with the control (Fig. 3C). The phosphorylation of AMPK at S485 is reduced by exposure

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In addition, authors examined the activation of AMPK in the motion control; however, the level of phosphorylation of AMPK at T172 was not significantly changed by rocking for 72 h (Fig. 3D). These results suggest that simulated microgravity activates AMPK and suppresses mTOR, and these events are potentially involved in autophagy regulation.

AMPK inhibition interferes with microgravity-induced autophagy To examine the role of AMPK in autophagy induction, authors used AMPK siRNA to suppress AMPK expression.

GFPLC3 cells were transfected with control siRNA, AMPK siRNA No. 1, or AMPK siRNA No. 2, and the transfected cells were set in the clinostat to simulate microgravity and rotated for 72 h.

Authors examined the level of AMPK expression and AMPK expression was efficiently silenced by AMPK siRNA transfection (Fig. 4A). While the control siRNA cells exhibited autophagy induction by clinorotation, the AMPKdepleted cells did not (Fig. 4B, C). AMPK-depleted cells did not show autophagosome formation in the cytoplasm nor did they exhibit changes in the expression of autophagosome markers, GFP-LC3-II and p62 (Fig. 4B). These results indicate that AMPK is involved in microgravity induced autophagy induction.

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Simulated microgravity-induced autophagy in C2C12 myotube cells Authors examined whether simulated microgravity induced autophagy in C2C12 myotube cells. C2C12 myoblast cells were incubated in the differentiation medium to form the multinucleated myotube cells, and the cells were clinorotated for 72 h to simulate microgravity.

Multinucleated myotube cells exposed to microgravity showed the enlarged and distinctive LC3 spots in the cytoplasm, suggesting that the simulated microgravity induced autophagy in C2C12 myotube cells (Fig. 5A). To confirm the results, the C2C12 myotube cells exposed to microgravity were collected and analyzed by western blots. The level of LC3-II was elevated, suggesting that simulated microgravity induced autophagy in C2C12 myotube cells (Fig. 5B). In addition, clinorotation for 72 h increased the level of phosphorylation of AMPK at T172 when compared with the control (Fig. 5B).

In this study, a 3D-clinostat was used to simulate microgravity to examine autophagy induction. The clinostat is a useful apparatus to study gravitational biology . Previously, a clinostat was used to examine the inhibitory effect of simulated microgravity on cell differentiation, as well as its stimulatory role in cellular apoptosis . The GFP-LC3 recombinant protein has been widely used for monitoring autophagy microscopically due to its sensitivity, and the detection of GFP-LC3 is especially useful for in vivo studies . Because the transient expression of GFP-LC3 often results in artifacts due to overexpression, authors used GFP-LC3 stable cells (HEK293 cells that stably express GFP-LC3) to reduce background and artifacts . This clinostat and GFP-LC3 stable cell system seems to provide a reliable platform with which to examine the effect of microgravity on autophagy induction.

Clinorotation for both 24 and 72 h resulted in the phosphorylation of AMPK (T172) (Fig. 3A), suggesting that AMPK is involved in microgravity-induced autophagy. To prove the role of AMPK in microgravity-induced autophagy, cells were treated with AMPK siRNA to block its activity. AMPK-depleted cells did not exhibit autophagy induction. These results collectively suggest that simulated microgravity contributes to autophagy, possibly via AMPK mediation.

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The data suggest that microgravity contributes to autophagy induction, which is potentially linked to muscle atrophy. For this reason, it is possible that microgravity affects muscle atrophy, at least in part by regulating autophagy. Moreover, it is possible that autophagy inhibitors could serve as a potential solution to microgravityinduced atrophy.

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