Myosin post-translational modifications and function in the presence of myopathy-linked truncating MYH2 mutations.

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1) MyHCs presence/post-translational modifications using LC/MS; 2) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3) myosin activation with an unloaded in vitro motility assay; and 4) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2) faster actomyosin kinetics; and 3) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.

Human skeletal muscle acetylcholine receptor gene expression in elderly males performing heavy resistance exercise.

Muscle fiber denervation is a major contributor to the decline in muscle mass and function during aging. Heavy resistance exercise is an effective tool for increasing muscle mass and strength, but whether it can rescue denervated muscle fibers remains unclear. Therefore, the purpose of this study was to investigate the potential of heavy resistance exercise to modify indices of denervation in healthy elderly individuals. Thirty-eight healthy elderly men (72 ± 5 yr) underwent 16 wk of heavy resistance exercise, whereas 20 healthy elderly men (72 ± 6 yr) served as nonexercising sedentary controls. Muscle biopsies were obtained pre and post training, and midway at 8 wk. Biopsies were analyzed by immunofluorescence for the prevalence of myofibers expressing embryonic myosin [embryonic myosin heavy chain (MyHCe)], neonatal myosin [neonatal myosin heavy chain (MyHCn)], nestin, and neural cell adhesion molecule (NCAM), and by RT-qPCR for gene expression levels of acetylcholine receptor (AChR) subunits, MyHCn, MyHCe, p16, and Ki67. In addition to increases in strength and type II fiber hypertrophy, heavy resistance exercise training led to a decrease in AChR α1 and ε subunit messenger RNA (mRNA; at 8 wk). Changes in gene expression levels of the α1 and ε AChR subunits with 8 wk of heavy resistance exercise supports the role of this type of exercise in targeting stability of the neuromuscular junction. The number of fibers positive for NCAM, nestin, and MyHCn was not affected, suggesting that a longer timeframe is needed for adaptations to manifest at the protein level.

Preserved stem cell content and innervation profile of elderly human skeletal muscle with lifelong recreational exercise.

Muscle fibre denervation and declining numbers of muscle stem (satellite) cells are defining characteristics of ageing skeletal muscle. The aim of this study was to investigate the potential for lifelong recreational exercise to offset muscle fibre denervation and compromised satellite cell content and function, both at rest and under challenged conditions. Sixteen elderly lifelong recreational exercisers (LLEX) were studied alongside groups of age-matched sedentary (SED) and young subjects. Lean body mass and maximal voluntary contraction were assessed, and a strength training bout was performed. From muscle biopsies, tissue and primary myogenic cell cultures were analysed by immunofluorescence and RT-qPCR to assess myofibre denervation and satellite cell quantity and function. LLEX demonstrated superior muscle function under challenged conditions. When compared with SED, the muscle of LLEX was found to contain a greater content of satellite cells associated with type II myofibres specifically, along with higher mRNA levels of the beta and gamma acetylcholine receptors (AChR). No difference was observed between LLEX and SED for the proportion of denervated fibres or satellite cell function, as assessed in vitro by myogenic cell differentiation and fusion index assays. When compared with inactive counterparts, the skeletal muscle of lifelong exercisers is characterised by greater fatigue resistance under challenged conditions in vivo, together with a more youthful tissue satellite cell and AChR profile. Our data suggest a little recreational level exercise goes a long way in protecting against the emergence of classic phenotypic traits associated with the aged muscle. KEY POINTS: The detrimental effects of ageing can be partially offset by lifelong self-organized recreational exercise, as evidence by preserved type II myofibre-associated satellite cells, a beneficial muscle innervation status and greater fatigue resistance under challenged conditions. Satellite cell function (in vitro), muscle fibre size and muscle fibre denervation determined by immunofluorescence were not affected by recreational exercise. Individuals that are recreationally active are far more abundant than master athletes, which sharply increases the translational perspective of the present study. Future studies should further investigate recreational activity in relation to muscle health, while also including female participants.

Muscle-nerve communication and the molecular assessment of human skeletal muscle denervation with aging.

Muscle fiber denervation is a major contributor to the decline in physical function observed with aging. Denervation can occur through breakdown of the neuromuscular junctions (NMJ) itself, affecting only that particular fiber, or through the death of a motor neuron, which can lead to a loss of all the muscle fibers in that motor unit. In this review, we discuss the muscle-nerve relationship, where signaling from both the motor neuron and the muscle fiber is required for maximal preservation of neuromuscular function in old age. Physical activity is likely to be the most important single factor that can contribute to this preservation. Furthermore, we propose that inactivity is not an innocent bystander, but plays an active role in denervation through the production of signals hostile to neuron survival. Investigating denervation in human muscle tissue samples is challenging due to the shared protein profile of regenerating and denervated muscle fibers. In this review, we provide a detailed overview of the key traits observed in immunohistochemical preparations of muscle biopsies from healthy, young, and elderly individuals. Overall, a combination of assessing tissue samples, circulating biomarkers, and electrophysiological assessments in humans will prove fruitful in the quest to gain more understanding of denervation of skeletal muscle. In addition, cell culture models represent a valuable tool in the search for key signaling factors exchanged between muscle and nerve, and which exercise has the capacity to alter.

Histology and Function of the Rectus Abdominis Muscle in Patients With Incisional Hernia.

BACKGROUND: The aim of the current study was to examine different features of the rectus abdominis muscle (RA) in patients with and without a midline incisional hernia to characterize the effects of a hernia on abdominal wall skeletal muscle. MATERIAL AND METHODS: RA tissue from patients undergoing surgical repair of a large midline incisional hernia (n = 18) was compared with that from an intact abdominal wall in patients undergoing colorectal resection for benign or low-grade malignant disease (n = 18). In addition, needle biopsies were obtained from the vastus lateralis muscle (VL) of all subjects. Outcome measures were muscle fiber type and size, preoperative truncal flexion strength and leg extension power measured in strength-measure equipment, and RA cross-sectional area measured by computed tomography. RESULTS: In both the RA and VL, the fiber cross-sectional area was greater in the patients with a hernia. The RA cross-sectional area correlated significantly with the truncal flexion strength (r = 0.44, P = 0.015). Patients in the hernia group had a significantly reduced ratio between truncal flexion strength and RA cross-sectional area compared with the control group (41.3 ± 11.5 N/cm2versus 51.2 ± 16.3 N/cm2, P = 0.034). CONCLUSIONS: Anatomical displacement of the RA and lack of medial insertion in the linea alba rather than dysfunction secondary to alteration of muscle fiber structure may contribute to impairment of abdominal wall function in patients with midline incisional hernias. The study was registered at http://www.clinicaltrials.gov/(NCT02011048).

Age and prior exercise in vivo determine the subsequent in vitro molecular profile of myoblasts and nonmyogenic cells derived from human skeletal muscle.

The decline in skeletal muscle regenerative capacity with age is partly attributed to muscle stem cell (satellite cell) dysfunction. Recent evidence has pointed to a strong interaction between myoblasts and fibroblasts, but the influence of age on this interaction is unknown. Additionally, while the native tissue environment is known to determine the properties of myogenic cells in vitro, how the aging process alters this cell memory has not been established at the molecular level. We recruited 12 young and 12 elderly women, who performed a single bout of heavy resistance exercise with the knee extensor muscles of one leg. Five days later, muscle biopsies were collected from both legs, and myogenic cells and nonmyogenic cells were isolated for in vitro experiments with mixed or separated cells and analyzed by immunostaining and RT-PCR. A lower myogenic fusion index was detected in the cells from the old versus young women, in association with differences in gene expression levels of key myogenic regulatory factors and senescence, which were further altered by performing exercise before tissue sampling. Coculture with nonmyogenic cells from the elderly led to a higher myogenic differentiation index compared with nonmyogenic cells from the young. These findings show that the in vitro phenotype and molecular profile of human skeletal muscle myoblasts and fibroblasts is determined by the age and exercise state of the original in vivo environment and help explain how exercise can enhance muscle stem cell function in old age.

Molecular indicators of denervation in aging human skeletal muscle.

INTRODUCTION: Muscle fiber denervation increases with age, yet studies at the tissue level are sparse due to the challenging nature of establishing the relative role of regeneration and denervation. METHODS: Muscle biopsies were obtained from the vastus lateralis of 70 healthy men (aged 72 ± 6 years; range, 65-94). Messenger RNA (mRNA) levels of acetylcholine receptors (AchR) were measured, and sections were stained for embryonic myosin, neonatal myosin (MHCn ), and neural cell adhesion molecule (NCAM). RESULTS: Embryonic myosin+ fibers were rare, while MHCn+ and NCAM+ fibers were observed in all samples. Age (range, 65-94 years) was negatively associated with AchRγ mRNA. DISCUSSION: Muscle from healthy older individuals expressed developmental myosins to varying degrees but more than has been previously reported for young individuals. Along with the AchR correlations, we propose that these findings support the presence of neuromuscular junction destabilization, denervation, and reinnervation in aging human skeletal muscle.

Effects of high-intensity physical training on muscle fiber characteristics in poststroke patients.

INTRODUCTION: Stroke is a leading cause of disability worldwide. High-intensity physical training can improve muscle strength and gait speed, but adaptive mechanisms at the muscle cellular level are largely unknown. METHODS: Outpatients with poststroke hemiparesis participated in a 3-month rehabilitation program combining high-intensity strength and body-weight supported treadmill-training. Biopsies sampled bilaterally from vastus lateralis muscles, before, after, and at 1-year follow-up after intervention, were analyzed for fiber size, type, and capillarization. RESULTS: At baseline, paretic lower limbs had smaller muscle fiber size and lower type I and IIA and higher type IIX percentages than nonparetic lower limbs. Paretic lower limbs had increased type IIA fibers after training. At follow-up, no difference between the lower limbs remained. CONCLUSIONS: Although high-intensity training appeared not to induce changes in fiber size or capillarization, increased type IIA fiber percentages may contribute to muscle power and endurance, which is crucial for functional capacity. Muscle Nerve 56: 954-962, 2017.

Matters of fiber size and myonuclear domain: Does size matter more than age?

INTRODUCTION: The relationship between fiber size and myonuclear content is poorly understood. METHODS: Biopsy cross-sections from young and old trained and untrained healthy individuals were analyzed for fiber area and myonuclei, and 2 fiber-size-dependent cluster analyses were performed. RESULTS: When comparing fibers of similar size, no effect of training or age was found for myonuclear domain. There was a linear relationship between fiber area and myonuclei per fiber (r = 0.99; P < 0.001) and a non-linear relationship between fiber area and domain (r = 0.97-0.99; P < 0.0001), with a markedly smaller domain in fibers <3,000 µm(2). A higher proportion of type II fibers <3,000 µm(2) was observed in the old subjects. CONCLUSIONS: These findings suggest that age-related reductions in myonuclear domain size could be explained by the greater proportion of small fibers. The data also highlight the usefulness of determining fiber-size-based clusters for gaining mechanistic insight into the relationship between skeletal muscle fiber size and myonuclear content.

Exercise performance and cardiovascular health variables in 70-year-old male soccer players compared to endurance-trained, strength-trained and untrained age-matched men.

The aim was to investigate performance variables and indicators of cardiovascular health profile in elderly soccer players (SP, n = 11) compared to endurance-trained (ET, n = 8), strength-trained (ST, n = 7) and untrained (UT, n = 7) age-matched men. The 33 men aged 65-85 years underwent a testing protocol including measurements of cycle performance, maximal oxygen uptake (VO2max) and body composition, and muscle fibre types and capillarisation were determined from m. vastus lateralis biopsy. In SP, time to exhaustion was longer (16.3 ± 2.0 min; P < 0.01) than in UT (+48%) and ST (+41%), but similar to ET (+1%). Fat percentage was lower (P < 0.05) in SP (-6.5% points) than UT but not ET and ST. Heart rate reserve was higher (P < 0.05) in SP (104 ± 16 bpm) than UT (+21 bpm) and ST (+24 bpm), but similar to ET (+2 bpm), whereas VO2max was not significantly different in SP (30.2 ± 4.9 ml O2 · min(-1) · kg(-1)) compared to UT (+14%) and ST (+9%), but lower (P < 0.05) than ET (-22%). The number of capillaries per fibre was higher (P < 0.05) in SP than UT (53%) and ST (42%) but similar to ET. SP had less type IIx fibres than UT (-12% points). In conclusion, the exercise performance and cardiovascular health profile are markedly better for lifelong trained SP than for age-matched UT controls. Incremental exercise capacity and muscle aerobic capacity of SP are also superior to lifelong ST athletes and comparable to endurance athletes.

Marked irregular myofiber shape is a hallmark of human skeletal muscle ageing and is reversed by heavy resistance training.

BACKGROUND: Age-related loss of strength is disproportionally greater than the loss of mass, suggesting maladaptations in the neuro-myo-tendinous system. Myofibers are often misshaped in aged and diseased muscle, but systematic analyses of large sample sets are lacking. Our aim was to investigate myofiber shape in relation to age, exercise, myofiber type, species and sex. METHODS: Vastus lateralis muscle biopsies (n = 265) from 197 males and females, covering an age span of 20-97 years, were examined. The gastrocnemius and soleus muscles of 11 + 22-month-old male C57BL/6 mice were also examined. Immunofluorescence and ATPase stainings of muscle cross-sections were used to measure myofiber cross-sectional area (CSA) and perimeter. From these, a shape factor index (SFI) was calculated in a fibre-type-specific manner (type I/II in humans; type I/IIa/IIx/IIb in mice), with higher values indicating increased deformity. Heavy resistance training (RT) was performed three times per week for 3-4 months by a subgroup (n = 59). Correlation analyses were performed comparing SFI and CSA with age, muscle mass, maximal voluntary contraction (MVC), rate of force development and specific force (MVC/muscle mass). RESULTS: In human muscle, SFI was positively correlated with age for both type I (R2  = 0.20) and II (R2  = 0.38) myofibers. When subjects were separated into age cohorts, SFI was lower for type I (4%, P < 0.001) and II (6%, P < 0.001) myofibers in young (20-36) compared with old (60-80) and higher for type I (5%, P < 0.05) and II (14%, P < 0.001) myofibers in the oldest old (>80) compared with old. The increased SFI in old muscle was observed in myofibers of all sizes. Within all three age cohorts, type II myofiber SFI was higher than that for type I myofiber (4-13%, P < 0.001), which was also the case in mice muscles (8-9%, P < 0.001). Across age cohorts, there was no difference between males and females in SFI for either type I (P = 0.496/0.734) or II (P = 0.176/0.585) myofibers. Multiple linear regression revealed that SFI, after adjusting for age and myofiber CSA, has independent explanatory power for 8/10 indices of muscle mass and function. RT reduced SFI of type II myofibers in both young and old (3-4%, P < 0.001). CONCLUSIONS: Here, we identify type I and II myofiber shape in humans as a hallmark of muscle ageing that independently predicts volumetric and functional assessments of muscle health. RT reverts the shape of type II myofibers, suggesting that a lack of myofiber recruitment might lead to myofiber deformity.

Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients.

Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.

Improved glucose tolerance after high-load strength training in patients undergoing dialysis.

BACKGROUND/AIMS: The aim of this controlled study was to investigate the effect of high-load strength training on glucose tolerance in patients undergoing dialysis. METHODS: 23 patients treated by dialysis underwent a 16-week control period followed by 16 weeks of strength training three times a week. Muscle fiber size, composition and capillary density were analyzed in biopsies obtained in the vastus lateralis muscle. Glucose tolerance and the insulin response were measured by a 2-hour oral glucose tolerance test. RESULTS: All outcome measures remained unchanged during the control period. After strength training the relative area of type 2X fibers was decreased. Muscle fiber size and capillary density remained unchanged. After the strength training, insulin concentrations were significantly lower in patients with impaired glucose tolerance or type 2 diabetes (n = 14) (fasting insulin from 68 ± 12 (46-96) to 54 ± 10 (37-77) pmol/l, p < 0.05, 2-hour insulin from 533 ± 104 (356-776) to 344 ± 68 (226-510) pmol/l, p < 0.05, total insulin area under the curve from 1,868 ± 334 (1,268-2,536) to 1,465 ± 222 (1,094-1,913), p < 0.05). Insulin concentrations were unchanged in patients with normal glucose tolerance (n = 9). CONCLUSION: The conducted strength training was associated with a significant improvement in glucose tolerance in patients with impaired glucose tolerance or type 2 diabetes undergoing dialysis. The effect was apparently not associated with muscle hypertrophy, whereas the muscle fiber type composition was changed.

Increased rate of force development and neuromuscular activity after high-load resistance training in patients undergoing dialysis.

AIM: The aim of this study was to investigate the effects of high-load resistance training on the rate of force development and neuromuscular function in patients undergoing dialysis. METHODS: Twenty-nine patients were tested before and after 16 weeks of resistance training. The rate of force development was tested using the Good Strength dynamometer chair. Muscle strength and neuromuscular function in the m. Vastus lateralis was estimated using electromyography in a one repetition maximum test during dynamic knee extension and during a 20 s isometric knee extension with 50% of the one repetition maximum load. Muscle biopsies from the m. Vastus lateralis were analysed for morphologic characteristics. RESULTS: One repetition maximum in knee extension increased by 46% (P<0.001) after the training programme. Rate of force development increased by 21-38% (P<0.05). The electromyography amplitude increased during 200-300 msec from 183 ± 36 µV to 315 ± 66 µV, (P<0.05), whilst electromyography frequency remained unchanged. The electromyography signals, during isometric contractions, remained unchanged. A higher rate of force development was found to be significantly associated with larger type 2 muscle fibres (r=0.647). CONCLUSION: Muscle strength in patients undergoing dialysis was increased after 16 weeks of resistance training in parallel with changed neuromuscular function and greater rate of force development, both of which have important clinical implications in terms of improved physical performance.

The effects of high-load strength training with protein- or nonprotein-containing nutritional supplementation in patients undergoing dialysis.

OBJECTIVE: The aim of this study was to investigate the effects of high-load strength training and protein intake in patients undergoing dialysis with a focus on muscle strength, physical performance, and muscle morphology. DESIGN: This was a randomized controlled study conducted in three dialysis centers. SUBJECTS: Subjects for the study included 29 patients undergoing dialysis. INTERVENTION: The participants went through a control period of 16 weeks before completing 16 weeks of strength training. Before the training period, the participants were randomly assigned to receive a protein or a nonprotein drink after every training session. MAIN OUTCOME MEASURE: Muscle strength and power were tested using the good strength equipment and the leg extensor power rig. Physical performance and function were assessed using a chair stand test and the Short Form 36 questionnaire. Muscle fiber type size and composition were analyzed in biopsies obtained from the m. vastus lateralis. RESULTS: All variables remained unchanged during the control period. After training, muscle strength and power, physical performance, and physical function increased significantly. Muscle fiber composition was changed by a relative decrease in type 2x muscle fiber number whereas muscle size at the fiber level was unchanged. There were no effects of combining the training with protein intake. CONCLUSIONS: High-load strength training is associated with improvements in muscle strength and power, physical performance, and quality of life. The effects were surprisingly not associated with muscle hypertrophy, and the results did not reveal any additional benefit of combining the training with protein intake. The positive results in muscle strength and physical performance have clinically relevant implications in the treatment of patients undergoing dialysis.

Extensive monitoring through multiple blood samples in professional soccer players.

The aim of this study was to make a comprehensive gathering of consecutive detailed blood samples from professional soccer players and to analyze different blood parameters in relation to seasonal changes in training and match exposure. Blood samples were collected 5 times during a 6-month period and analyzed for 37 variables in 27 professional soccer players from the best Danish league. Additionally, the players were tested for body composition, V[Combining Dot Above]O2max and physical performance by the Yo-Yo intermittent endurance submax test (IE2). Multiple variations in blood parameters occurred during the observation period, including a decrease in hemoglobin and an increase in hematocrit as the competitive season progressed. Iron and transferrin were stable, whereas ferritin showed a decrease at the end of the season. The immunoglobulin A (IgA) and IgM increased in the period with basal physical training and at the end of the season. Leucocytes decreased with increased physical training. Lymphocytes decreased at the end of the season. The V[Combining Dot Above]O2max decreased toward the end of the season, whereas no significant changes were observed in the IE2 test. The regular blood samples from elite soccer players reveal significant changes that may be related to changes in training pattern, match exposure, or length of the match season. Especially the end of the preparation season and at the end of the competitive season seem to be time points were the blood-derived values indicate that the players are under excessive physical strain and might be more subjected to a possible overreaching-overtraining conditions. We suggest that regular analyses of blood samples could be an important initiative to optimize training adaptation, training load, and game participation, but sampling has to be regular, and a database has to be built for each individual player.

Fusion of myofibre branches is a physiological feature of healthy human skeletal muscle regeneration.

BACKGROUND: The occurrence of hyperplasia, through myofibre splitting, remains a widely debated phenomenon. Structural alterations and fibre typing of skeletal muscle fibres, as seen during regeneration and in certain muscle diseases, can be challenging to interpret. Neuromuscular electrical stimulation can induce myofibre necrosis followed by changes in spatial and temporal cellular processes. Thirty days following electrical stimulation, remnants of regeneration can be seen in the myofibre and its basement membrane as the presence of small myofibres and encroachment of sarcolemma and basement membrane (suggestive of myofibre branching/splitting). The purpose of this study was to investigate myofibre branching and fibre type in a systematic manner in human skeletal muscle undergoing adult regenerative myogenesis. METHODS: Electrical stimulation was used to induce myofibre necrosis to the vastus lateralis muscle of one leg in 5 young healthy males. Muscle tissue samples were collected from the stimulated leg 30 days later and from the control leg for comparison. Biopsies were sectioned and stained for dystrophin and laminin to label the sarcolemma and basement membrane, respectively, as well as ATPase, and antibodies against types I and II myosin, and embryonic and neonatal myosin. Myofibre branches were followed through 22 serial Sects. (264 µm). Single fibres and tissue blocks were examined by confocal and electron microscopy, respectively. RESULTS: Regular branching of small myofibre segments was observed (median length 144 µm), most of which were observed to fuse further along the parent fibre. Central nuclei were frequently observed at the point of branching/fusion. The branch commonly presented with a more immature profile (nestin + , neonatal myosin + , disorganised myofilaments) than the parent myofibre, together suggesting fusion of the branch, rather than splitting. Of the 210 regenerating muscle fibres evaluated, 99.5% were type II fibres, indicating preferential damage to type II fibres with our protocol. Furthermore, these fibres demonstrated 7 different stages of "fibre-type" profiles. CONCLUSIONS: By studying the regenerating tissue 30 days later with a range of microscopy techniques, we find that so-called myofibre branching or splitting is more likely to be fusion of myotubes and is therefore explained by incomplete regeneration after a necrosis-inducing event.

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations.

AIM: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. METHODS: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. RESULTS: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the ß/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered-relaxed conformation. CONCLUSIONS: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

Physical activity impacts resting skeletal muscle myosin conformation and lowers its ATP consumption.

It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as the super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize ATP consumption and skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 5- to 10-fold reduction in ATP turnover compared with DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibers from young men of various physical activity levels (sedentary, moderately physically active, endurance-trained, and strength-trained athletes) and ran a loaded Mant-ATP chase protocol. We observed that in moderately physically active individuals, the amount of myosin molecules in the SRX state in type II muscle fibers was significantly greater than in age-matched sedentary individuals. In parallel, we did not find any difference in the proportions of SRX and DRX myosins in myofibers between highly endurance- and strength-trained athletes. We did however observe changes in their ATP turnover time. Altogether, these results indicate that physical activity level and training type can influence the resting skeletal muscle myosin dynamics. Our findings also emphasize that environmental stimuli such as exercise have the potential to rewire the molecular metabolism of human skeletal muscle through myosin.

Progressive resistance training and cancer testis (PROTRACT) - efficacy of resistance training on muscle function, morphology and inflammatory profile in testicular cancer patients undergoing chemotherapy: design of a randomized controlled trial.

BACKGROUND: Standard treatment for patients with disseminated germ cell tumors is combination chemotherapy with bleomycin, etoposide and cisplatin (BEP). This treatment is highly effective, but the majority of patients experience severe adverse effects during treatment and are at risk of developing considerable long-term morbidity, including second malignant neoplasms, cardiovascular disease, and pulmonary toxicity. One neglected side effect is the significant muscular fatigue mentioned by many patients with testicular cancer both during and after treatment. Very limited information exists concerning the patho-physiological effects of antineoplastic agents on skeletal muscle. The primary aim of this study is to investigate the effects of BEP-treatment on the skeletal musculature in testicular cancer patients, and to examine whether the expected treatment-induced muscular deterioration can be attenuated or even reversed by high intensity progressive resistance training (HIPRT). DESIGN/METHODS: The PROTRACT study is a randomized controlled trial in 30 testicular cancer patients undergoing three cycles of BEP chemotherapy. Participants will be randomized to either a 9-week HIPRT program (STR) initiated at the onset of treatment, or to standard care (UNT). 15 healthy matched control subjects (CON) will complete the same HIPRT program. All participants will take part in 3 assessment rounds (baseline, 9 wks, 21 wks) including muscle biopsies, maximum muscle strength tests, whole body DXA scan and blood samples. PRIMARY OUTCOME: mean fiber area and fiber type composition measured by histochemical analyses, satellite cells and levels of protein and mRNA expression of intracellular mediators of protein turnover. SECONDARY OUTCOMES: maximum muscle strength and muscle power measured by maximum voluntary contraction and leg-extensor-power tests, body composition assessed by DXA scan, and systemic inflammation analyzed by circulating inflammatory markers, lipid and glucose metabolism in blood samples. Health related Quality of Life (QoL) will be assessed by validated questionnaires (EORTC QLQ-C30, SF-36). DISCUSSION: This study investigates the muscular effects of antineoplastic agents in testicular cancer patients, and furthermore evaluates whether HIPRT has a positive influence on side effects related to chemotherapy. A more extensive knowledge of the interaction between cytotoxic-induced physiological impairment and exercise-induced improvement is imperative for the future development of optimal rehabilitation programs for cancer patients. TRIAL REGISTRATION: Current Controlled Trials ISRCTN32132990.