A novel approach for establishing fitness standards for occupational task performance.

PURPOSE: To identify strength and performance thresholds below which task performance is impaired. METHODS: A new weighted suit system was used to manipulate strength-to-body-weight ratio during the performance of simulated space explorations tasks. Statistical models were used to evaluate various measures of muscle strength and performance on their ability to predict the probability that subjects could complete the tasks in an acceptable amount of time. Thresholds were defined as the point of greatest change in probability per change in the predictor variable. For each task, median time was used to define the boundary between "acceptable" and "unacceptable" completion times. RESULTS: Fitness thresholds for four space explorations tasks were identified using 23 physiological input variables. Area under receiver operator characteristic curves varied from a low of 0.68 to a high of 0.92. CONCLUSION: An experimental analog for altering strength-to-body weight combined with a probability-based statistical model for success was suitable for identifying thresholds for task performance below which tasks could either not be completed or time to completion was unacceptably high. These results provide data for strength recommendations for exploration mission ambulatory task performance. Furthermore, the approach can be used to identify thresholds for other areas where occupationally relevant tasks vary considerably.

NASA SPRINT exercise program efficacy for vastus lateralis and soleus skeletal muscle health during 70 days of simulated microgravity.

The efficacy of the NASA SPRINT exercise countermeasures program for quadriceps (vastus lateralis) and triceps surae (soleus) skeletal muscle health was investigated during 70 days of simulated microgravity. Individuals completed 6° head-down-tilt bedrest (BR, n = 9), bedrest with resistance and aerobic exercise (BRE, n = 9), or bedrest with resistance and aerobic exercise and low-dose testosterone (BRE + T, n = 8). All groups were periodically tested for muscle (n = 9 times) and aerobic (n = 4 times) power during bedrest. In BR, surprisingly, the typical bedrest-induced decrements in vastus lateralis myofiber size and power were either blunted (myosin heavy chain, MHC I) or eliminated (MHC IIa), along with no change (P > 0.05) in %MHC distribution and blunted quadriceps atrophy. In BRE, MHC I (vastus lateralis and soleus) and IIa (vastus lateralis) contractile performance was maintained (P > 0.05) or increased (P < 0.05). Vastus lateralis hybrid fiber percentage was reduced (P < 0.05) and energy metabolism enzymes and capillarization were generally maintained (P > 0.05), while not all of these positive responses were observed in the soleus. Exercise offsets 100% of quadriceps and approximately two-thirds of soleus whole muscle mass loss. Testosterone (BRE + T) did not provide any benefit over exercise alone for either muscle and for some myocellular parameters appeared detrimental. In summary, the periodic testing likely provided a partial exercise countermeasure for the quadriceps in the bedrest group, which is a novel finding given the extremely low exercise dose. The SPRINT exercise program appears to be viable for the quadriceps; however, refinement is needed to completely protect triceps surae myocellular and whole muscle health for astronauts on long-duration spaceflights.NEW & NOTEWORTHY This study provides unique exercise countermeasures development information for astronauts on long-duration spaceflights. The NASA SPRINT program was protective for quadriceps myocellular and whole muscle health, whereas the triceps surae (soleus) was only partially protected as has been shown with other programs. The bedrest control group data may provide beneficial information for overall exercise dose and targeting fast-twitch muscle fibers. Other unique approaches for the triceps surae are needed to supplement existing exercise programs.

Influence of muscle strength to weight ratio on functional task performance.

Existing models of muscle deconditioning such as bed rest are expensive and time-consuming. We propose a new model utilizing a weighted suit to manipulate muscle strength, power, or endurance relative to body weight. The aims of the study were to determine as to which muscle measures best predict functional task performance and to determine muscle performance thresholds below which task performance is impaired. Twenty subjects performed seven occupational astronaut tasks (supine and upright seat egress and walk, rise from fall, hatch opening, ladder climb, object carry, and construction board activity), while wearing a suit weighted with 0-120 % of body weight. Models of the relationship between muscle function/body weight and task completion time were developed using fractional polynomial regression and verified with pre- and post-flight astronaut performance data. Spline regression was used to identify muscle function thresholds for each task. Upright seat egress and walk was the most difficult task according to the spline regression analysis thresholds. Thresholds normalized to body weight were 17.8 N/kg for leg press isometric force, 17.6 W/kg for leg press power, 78.8 J/kg for leg press work, 5.9 N/kg isometric knee extension and 1.9 Nm/kg isokinetic knee extension torque. Leg press maximal isometric force/body weight was the most reliable measure for modeling performance of ambulatory tasks. Laboratory-based manipulation of relative strength has promise as an analog for spaceflight-induced loss of muscle function. Muscle performance values normalized to body weight can be used to predict occupational task performance and to establish relevant strength thresholds.

Sweat Production During Continuous and Interval Aerobic Exercise.

INTRODUCTION:Aerobic exercise within the habitable volume of small spacecraft needed for space exploration beyond low Earth orbit is expected to challenge the capacity of environmental control systems. Moisture control is a primary concern. Crewmembers will contribute moisture to the cabin environment in the form of sweat while exercising. The effects of continuous aerobic exercise for improving and maintaining aerobic capacity is well characterized. Likewise, evidence suggests that high intensity interval exercise for shorter durations is also effective in building and maintaining aerobic capacity.METHODS: On separate days, measures of sweat and respiratory responses were made for continuous (30 min of steady state exercise at ∼75% of aerobic capacity) and two interval (4 × 4 min, 8 × 30 s) exercise protocols.RESULTS: We observed that the 4-min and 30-s interval protocols produce 16% and 66% less metabolic water loss vs. the continuous exercise protocol, respectively. These responses were highly correlated with the amount of work performed (R² = 0.81) and the amount of energy expenditure (R² = 0.83) during exercise.DISCUSSION: These results suggest that interval exercise may be a useful alternative to continuous aerobic exercise when metabolic water production is an environmental concern. The results may inform the choices of aerobic exercise countermeasure protocols for use in deep space exploration.Ryder JW, Crowell JB, Song HJ, Ewert M. Sweat production during continuous and interval aerobic exercise. Aerosp Med Hum Perform. 2023; 94(8):623-628.

High intensity training during spaceflight: results from the NASA Sprint Study.

Historically, International Space Station (ISS) exercise countermeasures have not fully protected astronauts' musculoskeletal and cardiorespiratory fitness. Although these losses have been reduced on more recent missions, decreasing the time required to perform in-flight exercise would permit reallocation of that time to other tasks. To evaluate the effectiveness of a new training prescription, ISS crewmembers performed either the high intensity/lower volume integrated Sprint resistance (3 d wk-1) and aerobic (interval and continuous workouts, each 3 d wk-1 in alternating fashion) exercise program (n = 9: 8M/1F, 48 ± 7 y, 178 ± 5 cm, 77.7 ± 12.0 kg) or the standard ISS countermeasure consisting of daily resistance and aerobic exercise (n = 17: 14M/3F, 46 ± 6 y, 176 ± 6 cm, 80.6 ± 10.5 kg) during long-duration spaceflight. Bone mineral density (dual energy X-ray absorptiometry (DXA)), muscle strength (isokinetic dynamometry), muscle function (cone agility test), and cardiorespiratory fitness (VO2peak) were assessed pre- and postflight. Mixed-effects modeling was used to analyze dependent measures with alpha set at P < 0.05. After spaceflight, femoral neck bone mineral density (-1.7%), knee extensor peak torque (-5.8%), cone agility test time (+7.4%), and VO2peak (-6.1%) were decreased in both groups (simple main effects of time, all P < 0.05) with a few group × time interaction effects detected for which Sprint experienced either attenuated or no loss compared to control. Although physiologic outcomes were not appreciably different between the two exercise programs, to conserve time and optimally prepare crewmembers for the performance of physically demanding mission tasks, high intensity/lower volume training should be an indispensable component of spaceflight exercise countermeasure prescriptions.

Efficacy of Testosterone plus NASA Exercise Countermeasures during Head-Down Bed Rest.

INTRODUCTION: Prolonged confinement to head-down bed rest (HDBR) results in musculoskeletal losses similar to those observed during long-duration space flight. Exercise countermeasures by themselves have not completely prevented the deleterious losses in muscle mass or function in HDBR or space flight. PURPOSE: The objective was to investigate the safety and efficacy of intermittent, low-dose testosterone treatment in conjunction with NASA exercise (SPRINT) countermeasures during 70 d of 6° HDBR. METHODS: Healthy men (35 ± 8 yr) were randomized into one of three groups that remained inactive (CON) or performed exercise 6 d·wk in addition to receiving either placebo (PEX) or testosterone treatment (TEX, 100 mg·wk). Testosterone/placebo injections were administered once a week for 2 wk, followed by 2 wk off and so on, during HDBR. RESULTS: Total, leg, and trunk lean body mass (LBM) consistently decreased in CON, increased in TEX, and had little or no changes in PEX. Total, leg, and trunk fat mass consistently increased in CON and PEX and decreased in TEX. Leg strength decreased in CON, whereas PEX and TEX were protected against loss in strength. Changes in leg LBM correlated positively with changes in leg muscle strength. CONCLUSIONS: Addition of a testosterone countermeasure enhanced the preventative actions of exercise against body composition changes during long-term HDBR in healthy eugonadal men. This is the first report to demonstrate that cycled, low-dose testosterone treatment increases LBM under conditions of strict exercise control. These results are clinically relevant to the development of safe and effective therapies against muscle atrophy during long-term bed rest, aging, and disease where loss of muscle mass and strength is a risk. The potential space flight applications of such countermeasure combinations deserve further investigations.

Exercise Training Mitigates Multisystem Deconditioning during Bed Rest.

INTRODUCTION: This study investigated the safety and effectiveness of a new integrated aerobic and resistance exercise training prescription (SPRINT) using two different sets of exercise equipment: a suite of large International Space Station-like exercise equipment similar to what is found on the International Space Station and a single device with aerobic and resistance exercise capability in the spaceflight analog of bed rest (BR). METHODS: Subjects (n = 34) completed 70 d of 6° head down tilt BR: 9 were randomized to remain sedentary (CONT), 9 to exercise training using traditional equipment (EX), 8 to exercise using traditional equipment and low-dose testosterone supplementation (ExT), and 8 to exercise using a combined resistance and aerobic flywheel device. Peak aerobic capacity, ventilatory threshold, cardiac morphology and function (echocardiography), muscle mass (magnetic resonance imaging) and strength/power (isokinetic, leg press, and vertical jump), and bone health (bone mineral density, blood and urine bone markers) were assessed before and after BR. RESULTS: The SPRINT protocol mitigated BR-induced muscle and cardiac deconditioning regardless of the exercise device used. Molecular markers of bone did not change in the CONT or EX groups. Peak aerobic capacity was maintained from pre- to post-BR in all exercise groups similarly, whereas significant declines were observed in the CONT group (~10%). Significant interaction effects between the CONT group and all EX groups were observed for muscle performance including leg press total work, isokinetic upper and lower leg strength, vertical jump power, and maximal jump height as well as muscle size. CONCLUSIONS: This is the first trial to evaluate multisystem deconditioning and the role of an integrated exercise countermeasure. These findings have important implications for the design and implementation of exercise-based countermeasures on future long-duration spaceflight missions.

Myocellular Responses to Concurrent Flywheel Training during 70 Days of Bed Rest.

PURPOSE: This investigation evaluated myocellular responses to an integrated resistance and aerobic training program during 70 d of bed rest. METHODS: Training was 6 d·wk on a small-footprint gravity-independent flywheel resistance and aerobic device; 3 d of maximal flywheel supine quadriceps and calf exercises with continuous rowing separated by 4 to 6 h, and 3 d of interval rowing. Vastus lateralis (VL) and soleus (SOL) muscle biopsies were obtained from eight healthy males (age, 28 ± 4 yr; BMI, 25 ± 3 kg·m; V˙O2max, 42 ± 6 mL·kg·min) before and after 6° head-down tilt bed rest. Vastus lateralis and SOL myosin heavy chain (MHC) I and IIa single muscle fiber size and functional characteristics, as well as overall fiber type distribution, capillarization, and metabolic enzyme activities were evaluated. RESULTS: In the VL, MHC I size and power (absolute and normalized) were preserved. The MHC IIa fibers hypertrophied (+6%, P < 0.05) without a change in absolute power, so normalized power declined (-7%, P < 0.05). In the SOL, MHC I fibers atrophied (-9%) and absolute power declined (-17%) (P < 0.05), whereas normalized power was maintained. Size, absolute power, and normalized power were protected in the less-abundant MHC IIa fibers. Reduced MHC coexpressing hybrid fibers, generally indicative of an exercise training effect, was apparent in the VL, whereas fiber type was maintained in the SOL. Capillarization and metabolic enzymes were generally preserved or increased in VL and SOL. CONCLUSIONS: The integrated resistance and aerobic training protocol on a device maintains several key myocellular characteristics during prolonged unloading, but further refinement of the exercise approach to fully protect the SOL is warranted.

Physiological and Functional Alterations after Spaceflight and Bed Rest.

INTRODUCTION: Exposure to microgravity causes alterations in multiple physiological systems, potentially impacting the ability of astronauts to perform critical mission tasks. The goal of this study was to determine the effects of spaceflight on functional task performance and to identify the key physiological factors contributing to their deficits. METHODS: A test battery comprised of seven functional tests and 15 physiological measures was used to investigate the sensorimotor, cardiovascular, and neuromuscular adaptations to spaceflight. Astronauts were tested before and after 6-month spaceflights. Subjects were also tested before and after 70 d of 6° head-down bed rest, a spaceflight analog, to examine the role of axial body unloading on the spaceflight results. These subjects included control and exercise groups to examine the effects of exercise during bed rest. RESULTS: Spaceflight subjects showed the greatest decrement in performance during functional tasks that required the greatest demand for dynamic control of postural equilibrium which was paralleled by similar decrements in sensorimotor tests that assessed postural and dynamic gait control. Other changes included reduced lower limb muscle performance and increased HR to maintain blood pressure. Exercise performed during bed rest prevented detrimental change in neuromuscular and cardiovascular function; however, both bed rest groups experienced functional and balance deficits similar to spaceflight subjects. CONCLUSION: Bed rest data indicate that body support unloading experienced during spaceflight contributes to postflight postural control dysfunction. Further, the bed rest results in the exercise group of subjects confirm that resistance and aerobic exercises performed during spaceflight can play an integral role in maintaining neuromuscular and cardiovascular functions, which can help in reducing decrements in functional performance. These results indicate that a countermeasure to mitigate postflight postural control dysfunction is required to maintain functional performance.

Abnormalities of glucose homeostasis and the hypothalamic-pituitary-adrenal axis in mice lacking hexose-6-phosphate dehydrogenase.

Hexose-6-phosphate dehydrogenase (EC 1.1.1.47) catalyzes the conversion of glucose 6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating reduced nicotinamide adenine dinucleotide phosphate. Reduced nicotinamide adenine dinucleotide phosphate is a necessary cofactor for the reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of hexose-6-phosphate dehydrogenase lack 11beta-hydroxysteroid dehydrogenase type 1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose use are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately up-regulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin, as assessed by homeostatic model assessment values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the hypothalamic-pituitary-adrenal axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions, despite elevated circulating levels of this hormone.

Panoramic ultrasound: a novel and valid tool for monitoring change in muscle mass.

BACKGROUND: The strong link between reduced muscle mass and morbidity and mortality highlights the urgent need for simple techniques that can monitor change in skeletal muscle cross-sectional area (CSA). Our objective was to examine the validity of panoramic ultrasound to detect change in quadriceps and gastrocnemius size in comparison with magnetic resonance imaging (MRI) in subjects randomized to 70 days of bed rest (BR) with or without exercise. METHODS: Panoramic ultrasound and MRI images of the quadriceps and gastrocnemius muscles were acquired on the right leg of 27 subjects (26 male, 1 female; age: 34.6 ± 7.8 years; body mass: 77.5 ± 10.0 kg; body mass index: 24.2 ± 2.8 kg/m2 ; height: 179.1 ± 6.9 cm) before (BR-6), during (BR3, 7, 11, 15, 22, 29, 36, 53, 69), and after (BR+3, +6, +10) BR. Validity of panoramic ultrasound to detect change in muscle CSA was assessed by Bland-Altman plots, Lin's concordance correlation coefficient (CCC), sensitivity, specificity, positive predictive value, and negative predictive value. RESULTS: Six hundred ninety-eight panoramic ultrasound CSA and 698 MRI CSA measurements were assessed. Concordance between ultrasound and MRI was excellent in the quadriceps (CCC: 0.78; P < 0.0001), whereas there was poor concordance in the gastrocnemius (CCC: 0.37; P < 0.0006). Compared with MRI, panoramic ultrasound demonstrated high accuracy in detecting quadriceps atrophy and hypertrophy (sensitivity: 73.7%; specificity: 74.2%) and gastrocnemius atrophy (sensitivity: 83.1%) and low accuracy in detecting gastrocnemius hypertrophy (specificity: 33.0%). CONCLUSIONS: Panoramic ultrasound imaging is a valid tool for monitoring quadriceps muscle atrophy and hypertrophy and for detecting gastrocnemius atrophy.

Isoform-specific regulation of 5' AMP-activated protein kinase in skeletal muscle from obese Zucker (fa/fa) rats in response to contraction.

Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5' AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPKalpha1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPKalpha1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPKalpha2 activity more than fivefold, whereas insulin did not alter either AMPKalpha1 or -alpha2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPKalpha1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPKalpha2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.

Sildenafil increases muscle protein synthesis and reduces muscle fatigue.

Reductions in skeletal muscle function occur during the course of healthy aging as well as with bed rest or diverse diseases such as cancer, muscular dystrophy, and heart failure. However, there are no accepted pharmacologic therapies to improve impaired skeletal muscle function. Nitric oxide may influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Here we show that augmentation of nitric oxide-cyclic guanosine monophosphate signaling by short-term daily administration of the phosphodiesterase 5 inhibitor sildenafil increases protein synthesis, alters protein expression and nitrosylation, and reduces fatigue in human skeletal muscle. These findings suggest that phosphodiesterase 5 inhibitors represent viable pharmacologic interventions to improve muscle function.

Signaling processes for initiating smooth muscle contraction upon neural stimulation.

Relationships among biochemical signaling processes involved in Ca2+/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca(2+)-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca2+](i), MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca2+](i) and kinase activation were 55 +/- 8 and 65 +/- 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 +/- 3 ms. [Ca2+](i), kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca2+](i) and also declined more rapidly than [Ca2+](i) during relaxation. The apparent desensitization of MLCK to Ca2+ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca2+](i), MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca2+ signaling complex as an elementary mechanism initiating contraction.

Enhanced skeletal muscle contraction with myosin light chain phosphorylation by a calmodulin-sensing kinase.

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.

Neuregulins mediate calcium-induced glucose transport during muscle contraction.

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction.

Effects of calcineurin activation on insulin-, AICAR- and contraction-induced glucose transport in skeletal muscle.

Skeletal muscle is composed of fast- and slow-twitch fibres with distinctive physiological and metabolic properties. The calmodulin-activated serine/threonine protein phosphatase calcineurin activates fast- to slow-twitch skeletal muscle remodelling through the induction of the slow-twitch skeletal muscle fibre gene expression programme, thereby enhancing insulin-stimulated glucose uptake and offering protection against dietary-induced insulin resistance. Given the profound influence of skeletal muscle fibre type on insulin-mediated responses, we determined whether the fast- to slow-twitch fibre-type transformation leads to alterations in insulin-independent glucose uptake in transgenic mice expressing a constitutively active form of calcineurin (MCK-CnA* mice). We determined whether skeletal muscle remodelling by activated calcineurin alters glucose transport in response to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-beta-D-ribofuranoside (AICAR) or muscle contraction, two divergent insulin-independent activators of glucose transport. While insulin-stimulated glucose transport was increased 52%, the AICAR effect on glucose transport was 27% lower in MCK-CnA* mice versus wild-type mice (P < 0.05). In contrast, glucose transport was similar between genotypes after in vitro muscle contraction. Fibre-type transformation was associated with increased AMPKgamma1, decreased AMPKgamma3 and unchanged AMPKgamma2 protein expression between MCK-CnA* and wild-type mice (P < 0.05). The loss of AICAR-mediated glucose uptake is coupled to changes in the AMPK isoform expression, suggesting fibre-type dependence of the AICAR responses on glucose uptake. In conclusion, improvements in skeletal muscle glucose transport in response to calcineurin-induced muscle remodelling are limited to insulin action.

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction.

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways.

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.