Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology.

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants.

Eliminating Nox2 reactive oxygen species production protects dystrophic skeletal muscle from pathological calcium influx assessed in vivo by manganese-enhanced magnetic resonance imaging.

KEY POINTS: Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn2+ did not affect muscle function or the health of the animal, and Mn2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non-invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy. ABSTRACT: Duchenne muscular dystrophy (DMD) is an X-linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese-enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long-term effects on animal health or muscle function. We conclude that MEMRI is a viable, non-invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle.

Physical Training for Long-Duration Spaceflight.

INTRODUCTION: Physical training has been conducted on the International Space Station (ISS) for the past 10 yr as a countermeasure to physiological deconditioning during spaceflight. Each member space agency has developed its own approach to creating and implementing physical training protocols for their astronauts. We have divided physical training into three distinct phases (preflight, in-flight, and postflight) and provided a description of each phase with its constraints and limitations. We also discuss how each member agency (NASA, ESA, CSA, and JAXA) prescribed physical training for their crewmembers during the first 10 yr of ISS operations. It is important to understand the operational environment, the agency responsible for the physical training program, and the constraints and limitations associated with spaceflight to accurately design and implement exercise training or interpret the exercise data collected on ISS. As exploration missions move forward, resolving agency differences in physical training programs will become important to maximizing the effectiveness of exercise as a countermeasure and minimizing any mission impacts.

Isokinetic Strength Changes Following Long-Duration Spaceflight on the ISS.

INTRODUCTION: Long-duration spaceflight results in a loss of muscle strength that poses both operational and medical risks, particularly during emergency egress, upon return to Earth, and during future extraterrestrial exploration. Isokinetic testing of the knee, ankle, and trunk quantifies movement-specific strength changes following spaceflight and offers insight into the effectiveness of in-flight exercise countermeasures. METHODS: We retrospectively evaluated changes in isokinetic strength for 37 ISS crewmembers (Expeditions 1-25) following 163 ± 38 d (mean ± SD) of spaceflight. Gender, in-flight resistance exercise hardware, and preflight strength were examined as potential modifiers of spaceflight-induced strength changes. RESULTS: Mean isokinetic strength declined 8-17% following spaceflight. One month after return to Earth, strength had improved, but small deficits of 1-9% persisted. Spaceflight-induced strength losses were not different between men and women. Mean strength losses were as much as 7% less in crewmembers who flew after the Advanced Resistive Exercise Device (ARED) replaced the interim Resistive Exercise Device (iRED) as the primary in-flight resistance exercise hardware, although these differences were not statistically significant. Absolute and relative preflight strength were moderately correlated (r = -0.47 and -0.54, respectively) with postflight strength changes. DISCUSSION: In-flight resistance exercise did not prevent decreased isokinetic strength after long-duration spaceflight. However, continued utilization of ARED, a more robust resistance exercise device providing higher loads than iRED, may result in greater benefits as exercise prescriptions are optimized. With reconditioning upon return to Earth, strength is largely recovered within 30 d.

Early-phase musculoskeletal adaptations to different levels of eccentric resistance after 8 weeks of lower body training.

PURPOSE: Eccentric muscle actions are important to the development of muscle mass and strength and may affect bone mineral density (BMD). This study's purpose was to determine the relative effectiveness of five different eccentric:concentric load ratios to increase musculoskeletal parameters during early adaptations to resistance training. METHODS: Forty male subjects performed a supine leg press and calf press training program 3 days week(-1) for 8 weeks. Subjects were matched for pre-training leg press 1-repetition maximum strength (1-RM) and randomly assigned to one of five training groups. Concentric training load (% 1-RM) was constant across groups, but within groups, eccentric load was 0, 33, 66, 100, or 138% of concentric load. Muscle mass (dual energy X-ray absorptiometry; DXA), strength (1-RM), and BMD (DXA) were measured pre- and post-training. Markers of bone metabolism were assessed pre-, mid- and post-training. RESULTS: The increase in leg press 1-RM in the 138% group (20 ± 4%) was significantly greater (P < 0.05) than the 0% (8 ± 3%), 33% (8 ± 5%) and 66% (8 ± 4%) groups, but not the 100% group (13 ± 6 %; P = 0.15). All groups, except the 0% group, increased calf press 1-RM (P < 0.05). Leg lean mass and greater trochanter BMD were increased only in the 138% group (P < 0.05). CONCLUSIONS: Early-phase adaptations to eccentric overload training include increases in muscle mass and site-specific increases in BMD and muscle strength which are not present or are less with traditional and eccentric underload training. Eccentric overload provides a robust musculoskeletal stimulus that may benefit bedridden patients, individuals recovering from injury or illness, and astronauts during spaceflight.

Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease resulting from mutations in the dystrophin gene. Increased oxidative stress and altered Ca(2+) homeostasis are hallmarks of dystrophic muscle. While impaired autophagy has recently been implicated in the disease process, the mechanisms underlying the impairment have not been elucidated. Here we show that nicotinamide adenine dinucleotide phosphatase (Nox2)-induced oxidative stress impairs both autophagy and lysosome formation in mdx mice. Persistent activation of Src kinase leads to activation of the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduces oxidative stress and partially rescues the defective autophagy and lysosome biogenesis. Genetic downregulation of Nox2 activity in the mdx mouse decreases reactive oxygen species (ROS) production, abrogates defective autophagy and rescues histological abnormalities and contractile impairment. Our data highlight mechanisms underlying the pathogenesis of DMD and identify NADPH oxidase and Src kinase as potential therapeutic targets.

Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase.

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.

Musculoskeletal adaptations to training with the advanced resistive exercise device.

UNLABELLED: Resistance exercise has been used as a means to prevent the musculoskeletal losses associated with spaceflight. Therefore, the National Aeronautics and Space Administration designed the Advanced Resistive Exercise Device (ARED) to replace the initial device flown on the International Space Station. The ARED uses vacuum cylinders and inertial flywheels to simulate, in the absence of gravity, the constant mass and inertia, respectively, of free weight (FW) exercise. PURPOSE: To compare the musculoskeletal effects of resistance exercise training using the ARED with the effects of training with FW. METHODS: Previously untrained, ambulatory subjects exercised using one of two modalities: FW (6 men and 3 women) or ARED (8 men and 3 women). Subjects performed squat, heel raise, and dead lift exercises 3 d·wk(-1) for 16 wk. Squat, heel raise, and dead lift strength (one-repetition maximum; using FW and ARED), bone mineral density (via dual-energy x-ray absorptiometry), and vertical jump were assessed before, during, and after training. Muscle mass (via magnetic resonance imaging) and bone morphology (via quantitative computed tomography) were measured before and after training. Bone biomarkers and circulating hormones were measured before training and after 4, 8, and 16 wk. RESULTS: Muscle strength, muscle volume, vertical jump height, and lumbar spine bone mineral density (via dual-energy x-ray absorptiometry and quantitative computed tomography) significantly increased (P ≤ 0.05) in both groups. There were no significant differences between groups in any of the dependent variables at any time. CONCLUSIONS: After 16 wk of training, ARED exercise resulted in musculoskeletal effects that were not significantly different from the effects of training with FW. Because FW training mitigates bed rest-induced deconditioning, the ARED may be an effective countermeasure for spaceflight-induced deconditioning and should be validated during spaceflight.

Foot-ground reaction force during resistive exercise in parabolic flight.

INTRODUCTION: An interim resistance exercise device (iRED) was designed to provide resistive exercise as a countermeasure to spaceflight-induced loss of muscle strength and endurance as well as decreased bone mineral density. The purpose of this project was to compare foot-ground reaction force during iRED exercise in normal gravity (1 G) vs. microgravity (0 G) achieved during parabolic flight. METHODS: There were four subjects who performed three exercises (squat, heel raise, and deadlift) using the iRED during 1 G and 0 G at a moderate intensity (60% of maximum strength during deadlift exercise). Foot-ground reaction force was measured in the three orthogonal axes (x, y, z) using a force plate, and the magnitude of the resultant force vector was calculated (r = square root(x2 + y2 + z2)). Linear displacement (LD) was measured using a linear transducer. Peak force (Fpeak) and an index of total work (TWi) were calculated using a customized computer program. Paired t-tests were used to test if significant differences (p < or = 0.05) were observed between 1 G and 0 G exercise. RESULTS: Fpeak and TWi measured in the resultant axis were significantly less in 0 G for each of the exercises tested. During 0 G, Fpeak was 42-46% and TWi was 33-37% of that measured during 1 G. LD and average time to complete each repetition were not different from 1 G to 0 G. CONCLUSIONS: Crewmembers who perform resistive exercises during spaceflight that include the movement of a large portion of their body mass will require much greater external resistive force during 0 G than 1 G exercise to provide a sufficient stimulus to maintain muscle and bone mass.

Training with the International Space Station interim resistive exercise device.

UNLABELLED: A unique, interim elastomer-based resistive exercise device (iRED) is being used on the International Space Station. PURPOSE: This study characterized iRED training responses in a 1-g environment by: 1) determining whether 16 wk of high-intensity training with iRED produces increases in muscle strength and volume and bone mineral density (BMD), 2) comparing training responses with iRED to free weights, and 3) comparing iRED training responses at two training volumes. METHODS: Twenty-eight untrained men were assigned to four groups of seven subjects each: a no exercise control group (CON), an iRED group who trained with three sets/exercise (iRED3), a free-weight group (FW) who trained with three sets/exercise, and an iRED group who trained with six sets/exercise (iRED6). Training exercises included squat (SQ), heel raise (HR), and dead lift (DL) exercises, 3 d.wk(-1) for 16 wk. RESULTS: For CON, no changes occurred pre- to posttraining. For iRED3, increases (P< or =0.05) in one-repetition maximum (1-RM) strength (SQ 21 +/- 4%, HR 17 +/- 4%, DL 29 +/- 5%), leg lean mass (3.1 +/- 0.5%) by dual energy x-ray absorptiometry (DXA), and thigh (4.5 +/- 0.9%) and calf (5.9 +/- 0.7%) muscle volume (by magnetic resonance imaging) occurred after training with no changes in BMD (DXA). For FW, increases in 1-RM strength (SQ 22 +/- 5%, HR 24 +/- 3%, DL 41 +/- 7%), whole body (3.0 +/- 1.1%) and leg lean mass (5.4 +/- 1.2%), thigh (9.2 +/- 1.3%) and calf (4.2 +/- 1.0%) muscle volumes, and lumbar BMD (4.2 +/- 0.7%) occurred after training. For iRED6, all responses were similar to iRED3. CONCLUSION: High-intensity training with the iRED produced muscle responses similar to FW but was not effective in stimulating bone. Bed rest and spaceflight studies are needed to evaluate the effectiveness of the iRED to prevent microgravity deconditioning.