Elevated muscle mass accompanied by transcriptional and nuclear alterations several months following cessation of resistance-type training in rats.

Rodent studies investigating long-term effects following termination of hypertrophy-inducing loading have predominantly involved exposures such as synergist ablation and weighted wheel running or ladder climbing. This research yielded a spectrum of results regarding the extent of detraining in terms of muscle mass and myonuclei number. The studies were also limited in their lack of sensitive performance measures and indirect relatedness to resistance training. Our research group developed and validated a relevant rat model of resistance-type training that induces increased muscle mass and performance. The aim of the present study was to determine to what extent these features persist 3 months following the termination of this training. While performance returned to baseline, muscle mass remained elevated by 17% and a shift in distribution to larger muscle fibers persisted. A 16% greater total RNA and heightened mRNA levels of ribosomal protein S6 kinases implicated preserved transcriptional output and ribosomal content. Remodeling of muscle fiber nuclei was consistent with these findings - increased nuclear number and a distribution shift to a more circular nuclear shape. These findings indicate that muscle mass detrains at a slower rate than performance and implicates multiple forms of myonuclear remodeling in muscle memory.

Improved impedance to maladaptation and enhanced VCAM-1 upregulation with resistance-type training in the long-lived Snell dwarf (Pit1dw/dw) mouse.

Snell dwarf mice with the Pit1dw/dw mutation are deficient in growth hormone, prolactin, and thyroid stimulating hormone and exhibit >40% lifespan extension. This longevity is accompanied by compromised muscular performance. However, research regarding young (3-month-old) Snell dwarf mice demonstrate exceptional responsivity to resistance-type training especially in terms of a shifted fiber type distribution and increased protein levels of vascular cell adhesion molecule-1 (VCAM-1), a possible mediator of such remodeling. In the present study, we investigated whether this responsiveness persists at 12 months of age. Unlike 12-month-old control mice, age-matched Snell dwarf mice remained resistant to training-induced maladaptive decreases in performance and muscle mass. This was accompanied by retainment of the remodeling capacity in muscles of Snell dwarf mice to increase VCAM-1 protein levels and a shift in myosin heavy chain (MHC) isoform distribution with training. Even decreasing training frequency for control mice, an alteration which protected muscles from maladaptation at 12 months of age, did not result in the overt remodeling observed for Snell dwarf mice. The results demonstrate a distinct remodeling response to resistance-type exercise operative in the context of the Pit1dw/dw mutation of long-lived Snell dwarf mice.

Age-dependent stress response DNA demethylation and gene upregulation accompany nuclear and skeletal muscle remodeling following acute resistance-type exercise in rats.

Efficacy of high-intensity resistance exercise becomes progressively compromised with aging. Previously, to investigate this, we developed a rodent model of high-intensity training consisting of stretch-shortening contractions (SSCs) and determined that following one month of training, young rats exhibit a robust stress response and 20% performance increase, whereas old rats display a muted stress response and 30% performance decrease. Whether these age-specific responses occur early in training and constitute primary factors in adaptation/maladaptation was not addressed. The aim of the present study was to characterize performance, remodeling, and stress response transcriptional profile 6-120 h following acute SSC exposure. For young rats, the stress response pathway was highly regulated (≥20 differentially expressed genes at each time point) and was accompanied by robust DNA demethylation, tissue remodeling, and isometric torque recovery. For old rats, a muted transcriptional profile (13 and 2 differentially expressed genes at 6 and 120 h, respectively) coincided with deficiencies in demethylation, muscle remodeling, and torque recovery. These findings occurred in the context of heightened chronic levels of stress response gene expression with aging. This demonstrates that age-related constitutive elevations in stress response gene expression was accompanied by diminished SSC-induced responsiveness in epigenomic regulation and tissue remodeling.

Reduced frequency of resistance-type exercise training promotes adaptation of the aged skeletal muscle microenvironment.

The purpose of this study was to characterize the growth and remodeling molecular signaling response in aged skeletal muscle following 1 mo of "resistance-type exercise" training. Male Fischer 344 × Brown Norway hybrid rats aged 3 (young) and 30 mo (old) underwent stretch-shortening contraction (SSC) loading 2 or 3 days/wk; muscles were removed 72 h posttraining. Young rats SSC loaded 3 (Y3x) or 2 days/wk (Y2x) adapted via increased work performance. Old rats SSC loaded 3 days/wk (O3x) maladapted via decreased negative work; however, old rats SSC loaded 2 days/wk (O2x) adapted through improved negative and positive work. Y3x, Y2x, and O2x, but not O3x, displayed hypertrophy via larger fiber area and myonuclear domains. Y3x, Y2x, and O2x differentially expressed 19, 30, and 8 phosphatidylinositol 3-kinase-Akt genes, respectively, whereas O3x only expressed 2. Bioinformatics analysis revealed that rats in the adapting groups presented growth and remodeling processes (i.e., increased protein synthesis), whereas O3x demonstrated inflammatory signaling. In conclusion, reducing SSC-loading frequency in aged rodents positively influences the molecular signaling microenvironment, promoting muscle adaptation. NEW & NOTEWORTHY Decreasing resistance-type exercise training frequency in old rodents led to adaptation through enhancements in performance, fiber areas, and myonuclear domains. Modifying frequency influenced the molecular environment through improvements in phosphatidylinositol 3-kinase-Akt pathway-specific expression and bioinformatics indicating increased protein synthesis. Reducing training frequency may be appropriate in older individuals who respond unfavorably to higher frequencies (i.e., maladaptation); overall, modifying the parameters of the exercise prescription can affect the cellular environment, ultimately leading to adaptive or maladaptive outcomes.

VCAM-1 upregulation accompanies muscle remodeling following resistance-type exercise in Snell dwarf (Pit1dw/dw ) mice.

Snell dwarf mice (Pit1dw/dw ) exhibit deficiencies in growth hormone, prolactin, and thyroid stimulating hormone. Besides being an experimental model of hypopituitarism, these mice are long-lived (>40% lifespan extension) and utilized as a model of slowed/delayed aging. Whether this longevity is accompanied by a compromised quality of life in terms of muscular performance has not yet been characterized. In this study, we investigated nontrained and trained muscles 1 month following a general validated resistance-type exercise protocol in 3-month-old Snell dwarf mice and control littermates. Nontrained Snell dwarf gastrocnemius muscles exhibited a 1.3-fold greater muscle mass to body weight ratio than control values although muscle quality, maximum isometric torque normalized to muscle mass, and fatigue recovery were compromised. For control mice, training increased isometric torque (17%) without altering muscle mass. For Snell dwarf mice, isometric torque was unaltered by training despite decreased muscle mass that rendered muscle mass to body weight ratio comparable to control values. Muscle quality and fatigue recovery improved twofold and threefold, respectively, for Snell dwarf mice. This accompanied a fourfold increase in levels of vascular cell adhesion molecule-1 (VCAM-1), a mediator of progenitor cell recruitment, and muscle remodeling in the form of increased number of central nuclei, additional muscle fibers per unit area, and altered fiber type distribution. These results reveal a trade-off between muscle quality and longevity in the context of anterior pituitary hormone deficiency and that resistance-type training can diminish this trade-off by improving muscle quality concomitant with VCAM-1 upregulation and muscle remodeling.

High-intensity stretch-shortening contraction training modifies responsivity of skeletal muscle in old male rats.

Utilization of high-intensity resistance training to counter age-related sarcopenia is currently debated because of the potential for maladaptation when training design is inappropriate. Training design is problematic because the influence of various loading variables (e.g. contraction mode, repetition number, and training frequency) is still not well characterized at old age. To address this in a precisely controlled manner, we developed a rodent model of high-intensity training consisting of maximally-activated stretch-shortening contractions (SSCs), contractions typical during resistance training. With this model, we determined that at old age, high-repetition SSC training (80 SSCs: 8 sets of 10 repetitions) performed frequently (i.e. 3 days per week) for 4.5 weeks induced strength deficits with no muscle mass gain while decreasing frequency to 2 days per week promoted increases in muscle mass and muscle quality (i.e. performance normalized to muscle mass). This finding confirmed the popular notion that decreasing training frequency has a robust effect with age. Meanwhile, the influence of other loading variables remains contentious. The aim of the present study was to assess muscle adaptation following modulation of contraction mode and repetition number during high-intensity SSC training. Muscles of young (3 month old) and old (30 month old) male rats were exposed to 4.5 weeks of low-repetition static training of 4 (i.e. 4 sets of one repetition) isometric (ISO) contractions 3 days per week or a more moderate-repetition dynamic training of 40 SSCs (i.e. 4 sets of 10 repetitions) 3 days per week. For young rats, performance and muscle mass increased regardless of training protocol. For old rats, no muscle mass adaptation was observed for 4 ISO training while 40 SSC training induced muscle mass gain without improvement in muscle quality, an outcome distinct from modulating training frequency. Muscle mass gain for old rats was accompanied by decreased protein levels of tumor necrosis factor alpha, a mediator of age-related chronic inflammatory signaling, to young levels. These findings suggest that while dynamic high-intensity training with a moderate number of repetitions has a limited capacity for altering muscle quality, such training is a viable strategy for countering age-related inflammatory signaling and modifying muscle mass.

Inflammaging and the Age-Specific Responsiveness to Stretch-Shortening Contractions.

With aging, muscle injury from rapid, continuous stretch-shortening contractions (SSC) is prolonged, and maladaptation to moderate-velocity, intermittent SSC is more common. We hypothesize that high baseline levels of inflammatory signaling and oxidative stress may underlie these outcomes, whereas careful modulation of high-intensity SSC training design resets basal conditions and permits muscle adaptation to SSC.

Agonist muscle adaptation accompanied by antagonist muscle atrophy in the hindlimb of mice following stretch-shortening contraction training.

BACKGROUND: The vast majority of dynamometer-based animal models for investigation of the response to chronic muscle contraction exposure has been limited to analysis of isometric, lengthening, or shortening contractions in isolation. An exception to this has been the utilization of a rat model to study stretch-shortening contractions (SSCs), a sequence of consecutive isometric, lengthening, and shortening contractions common during daily activity and resistance-type exercise. However, the availability of diverse genetic strains of rats is limited. Therefore, the purpose of the present study was to develop a dynamometer-based SSC training protocol to induce increased muscle mass and performance in plantarflexor muscles of mice. METHODS: Young (3 months old) C57BL/6 mice were subjected to 1 month of plantarflexion SSC training. Hindlimb muscles were analyzed for muscle mass, quantitative morphology, myogenesis/myopathy relevant gene expression, and fiber type distribution. RESULTS: The main aim of the research was achieved when training induced a 2-fold increase in plantarflexion peak torque output and a 19% increase in muscle mass for the agonist plantaris (PLT) muscle. In establishing this model, several outcomes emerged which raised the value of the model past that of being a mere recapitulation of the rat model. An increase in the number of muscle fibers per transverse muscle section accounted for the PLT muscle mass gain while the antagonist tibialis anterior (TA) muscle atrophied by 30% with preferential atrophy of type IIb and IIx fibers. These alterations were accompanied by distinct gene expression profiles. CONCLUSIONS: The findings confirm the development of a stretch-shortening contraction training model for the PLT muscle of mice and demonstrate that increased cross-sectional fiber number can occur following high-intensity SSC training. Furthermore, the TA muscle atrophy provides direct evidence for the concept of muscle imbalance in phasic non-weight bearing muscles, a concept largely characterized based on clinical observation of patients. The susceptibility to this imbalance is demonstrated to be selective for the type IIb and IIx muscle fiber types. Overall, the study highlights the importance of considering muscle fiber number modulation and the effect of training on surrounding muscles in exercise comprised of SSCs.

Enhancement of Skeletal Muscle in Aged Rats Following High-Intensity Stretch-Shortening Contraction Training.

Exercise is the most accessible, efficacious, and multifactorial intervention to improve health and treat chronic disease. High-intensity resistance exercise, in particular, also maximizes skeletal muscle size and strength-outcomes crucial at advanced age. However, such training is capable of inducing muscle maladaptation when misapplied at old age. Therefore, characterization of parameters (e.g., mode and frequency) that foster adaptation is an active research area. To address this issue, we utilized a rodent model that allowed training at maximal intensity in terms of muscle activation and tested the hypothesis that muscles of old rats adapt to stretch-shortening contraction (SSC) training, provided the training frequency is sufficiently low. At termination of training, normalized muscle mass (i.e., muscle mass divided by tibia length) and muscle quality (isometric force divided by normalized muscle mass) were determined. For young rats, normalized muscle mass increased by ∼20% regardless of training frequency. No difference was observed for muscle quality values after 2 days versus 3 days per week training (0.65 ± 0.09 N/mg/mm vs. 0.59 ± 0.05 N/mg/mm, respectively). For old rats following 3 days per week training, normalized muscle mass was unaltered and muscle quality was 30% lower than young levels. Following 2 days per week training at old age, normalized muscle mass increased by 17% and muscle quality was restored to young levels. To investigate this enhanced response, oxidative stress was assessed by lipid peroxidation quantification. For young rats, lipid peroxidation levels were unaltered by training. With aging, baseline levels of lipid peroxidation increased by 1.5-fold. For old rats, only 2 days per week training decreased lipid peroxidation to levels indistinguishable from young values. These results imply that, appropriately scheduled high-intensity SSC training at old age is capable of restoring muscle to a younger phenotype in terms of lipid peroxidation levels and muscle quality.

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

Age-dependent Muscle Adaptation after Chronic Stretch-shortening Contractions in Rats.

Age-related differences in contraction-induced adaptation have been well characterized especially for young and old rodent models but much less so at intermediate ages. Therefore, additional research is warranted to determine to what extent alterations in adaptation are due to maturation versus aging per se. The purpose of our study was to evaluate muscles of Fisher 344XBrown Norway rats of various ages following one month of exposure to stretch-shortening contractions (SSCs). With exposure, muscles mass increased by ~10% for 27 and 30 month old rats vs. ~20% for 3 and 6 month old rats (P < 0.05). For 3 month old rats, maximum isometric force and dynamic peak force increased by 22 ± 8% and 27 ± 10%, respectively (P < 0.05). For 6 month old rats, these forces were unaltered by exposure and positive work capacity diminished by 27 ± 2% (P = 0.006). By 30 months of age, age-related deficits in maximum isometric force, peak force, negative work, and positive work were apparent and SSC exposure was ineffective at counteracting such deficits. Recovery from fatigue was also tested and exposure-induced improvements in fatigue recovery were indicated for 6 month old rats and to a lesser extent for 3 month old rats whereas no such effect was observed for older rats. Alterations in fatigue recovery were accompanied by evidence of substantial type IIb to IIx fiber type shifting. These results highlight the exceptional adaptive capacity for strength at a young age, the inclination for adaptation in fatigue recovery at early adulthood, and diminished adaptation for muscle performance in general beginning at late adulthood. Such findings motivate careful investigation to determine appropriate SSC exposures at all stages of life.

Desensitized morphological and cytokine response after stretch-shortening muscle contractions as a feature of aging in rats.

Recovery from contraction-induced injury is impaired with aging. At a young age, the secondary response several days following contraction-induced injury consists of edema, inflammatory cell infiltration, and segmental muscle fiber degeneration to aid in the clearance of damaged tissue and repair. This morphological response has not been wholly established at advanced age. Our aim was to characterize muscle fiber morphology 3 and 10 days following stretch-shortening contractions (SSCs) varying in repetition number (i.e. 0, 30, 80, and 150) for young and old rats. For muscles of young rats, muscle fiber degeneration was overt at 3 days exclusively after 80 or 150 SSCs and returned significantly closer to control values by 10 days. For muscles of old rats, no such responses were observed. Transcriptional microarray analysis at 3 days demonstrated that muscles of young rats differentially expressed up to 2144 genes while muscles of old rats differentially expressed 47 genes. Bioinformatic analysis indicated that cellular movement was a major biological process over-represented with genes that were significantly altered by SSCs especially for young rats. Protein levels in muscle for various cytokines and chemokines, key inflammatory factors for cell movement, increased 3- to 50-fold following high-repetition SSCs for young rats with no change for old rats. This age-related differential response was insightful given that for control (i.e. 0 SSCs) conditions, protein levels of circulatory cytokines/chemokines were increased with age. The results demonstrate ongoing systemic low-grade inflammatory signaling and subsequent desensitization of the cytokine/chemokine and morphological response to contraction-induced injury with aging - features which accompany age-related impairment in muscle recovery.

Volitional Weight-Lifting in Rats Promotes Adaptation via Performance and Muscle Morphology prior to Gains in Muscle Mass.

Investigation of volitional animal models of resistance training has been instrumental in our understanding of adaptive training. However, these studies have lacked reactive force measurements, a precise performance measure, and morphological analysis at a distinct phase of training - when initial strength gains precede muscle hypertrophy. Our aim was to expose rats to one month of training (70 or 700 g load) on a custom-designed weight-lifting apparatus for analysis of reactive forces and muscle morphology prior to muscle hypertrophy. Exclusively following 700 g load training, forces increased by 21% whereas muscle masses remained unaltered. For soleus (SOL) and tibialis anterior (TA) muscles, 700 g load training increased muscle fiber number per unit area by ∼20% and decreased muscle fiber area by ∼20%. Additionally, number of muscle fibers per section increased by 18% for SOL muscles. These results establish that distinct morphological alterations accompany early strength gains in a volitional animal model of load-dependent adaptive resistance training.

Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies.

Loss of mobility influences the quality of life for patients with neuromuscular diseases. Common measures of mobility and chronic muscle damage are the six-minute walk test and serum creatine kinase. Despite extensive pre-clinical studies of therapeutic approaches, characterization of these measures is incomplete. To address this, a six-minute ambulation assay, serum creatine kinase, and myoglobinuria were investigated for the mdx mouse, a dystrophinopathy mouse model commonly used in pre-clinical studies. mdx mice ambulated shorter distances than normal controls, a disparity accentuated after mild exercise. An asymmetric pathophysiology in mdx mice was unmasked with exercise, and peak measurements of serum creatine kinase and myoglobinuria were identified. Our data highlights the necessity to consider asymmetric pathology and timing of biomarkers when testing potential therapies for muscular dystrophy.

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice.

BACKGROUND: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with ß-dystroglycan. This link extends to the extracellular matrix by ß-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. METHODS: To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. RESULTS: The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. CONCLUSIONS: The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

Genetic ablation of complement C3 attenuates muscle pathology in dysferlin-deficient mice.

Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in dysferlinopathy and dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.

Characterization of skeletal muscle effects associated with daptomycin in rats.

Daptomycin is a lipopeptide antibiotic with strong bactericidal effects against Gram-positive bacteria and minor side effects on skeletal muscles. The type and magnitude of the early effect of daptomycin on skeletal muscles of rats was quantified by histopathology, examination of contractile properties, Evans Blue Dye uptake, and effect on the patch repair process. A single dose of daptomycin of up to 200 mg/kg had no effect on muscle fibers. A dose of 150 mg/kg of daptomycin, twice per day for 3 days, produced a small number of myofibers (

Adenosine A(3) receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury.

Effective therapy to reduce skeletal muscle injury associated with severe or eccentric exercise is needed. The purpose of this study was to determine whether adenosine receptor stimulation can mediate protection from eccentric exercise-induced muscle injury. Downhill treadmill exercise (-15 degrees ) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in beta-sarcoglycan knockout dystrophic mice, animals that show an exaggerated muscle damage with the stress of exercise. In the vehicle-treated WT animals, eccentric exercise increased serum creatine kinase (CK) greater than 3-fold to 358.9 +/- 62.7 U/l (SE). This increase was totally abolished by stimulation of the A(3) receptor. In the dystrophic beta-sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 +/- 5,558 U/l. A(3) receptor stimulation in these animals reduced the CK response by nearly 50%. In the dystrophic mice at rest, 10% of the fibers were found to be damaged, as indicated by Evans blue dye staining. While this percentage was doubled after exercise, A(3) receptor stimulation eliminated this increase. Neither the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (0.05 mg/kg) nor the A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (0.07 mg/kg) protected skeletal muscle from eccentric exercise injury in WT or dystrophic mice. The protective effect of adenosine A(3) receptor stimulation was absent in mice, in which genes for phospholipase C beta2/beta3 (PLCbeta2/beta3) and beta-sarcoglycan were deleted. The present study elucidates a new protective role of the A(3) receptor and PLCbeta2/beta3 and points to a potentially effective therapeutic strategy for eccentric exercise-induced skeletal muscle injury.

Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan.

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by alpha-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin alpha7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Large(myd) muscles, which have an intact dystrophin-glycoprotein complex and lack only the laminin globular domain-binding motif on alpha-dystroglycan. Compromised sarcolemmal integrity is directly shown in Large(myd) muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding alpha-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage.

Sarcolemma-localized nNOS is required to maintain activity after mild exercise.

Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.