Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue does not necessarily correlate with elevated central or peripheral fatigue in patients1, and some patients experience severe fatigue without any demonstrable somatic disease2. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown2. With no treatment available, this form of inactivity is a major determinant of disability3. 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 nNOS signaling in skeletal muscle is required to maintain activity after mild exercise. Of significance, 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 signaling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signaling from sarcolemma-localized nNOS, which reduces cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Notably, sarcolemmal nNOS was reduced 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 signaling.

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 ␣-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin ␣7 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 ␣-dystroglycan. Compromised sarcolemmal integrity is directly shown in Large myd muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding ␣-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. dystroglycanopathy ͉ glycosylation ͉ integrin ͉ membrane damage ͉ muscular dystrophy

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.

With aging, the skeletal muscles of humans sustain decreases of approximately 30% in mass and maximum force. Contraction-induced injury may contribute to these declines. When a 225 lengthening contraction protocol (LCP) was administered to small, non-weight-bearing muscles of mice, muscles of young/adult mice recovered completely, whereas those of old mice sustained permanent deficits of 20% in muscle mass and maximum force. Despite these observations, whether a large, frequently recruited, weight-bearing muscle sustains such permanent damage is not known. The hypothesis tested is that after a severe contraction-induced injury, large, weight-bearing muscles of old mice sustain permanent reductions in mass and force. The LCP was administered to plantar flexor muscles of adult and old, male C57BL/6 mice. At 3 days, 1 mo, and 2 mo after the LCP, maximum isometric forces were measured, anesthetized mice were euthanized, and muscles were removed and weighed. Two months after the LCP, the muscles of the adult mice regained control values of mass and force, whereas for muscles of old mice the mass decreased by 24% and the maximum force decreased by 32%. We conclude that a severe contraction-induced injury to large, weight-bearing muscles of old mice causes permanent deficits in mass and force.

BackgroundMutations 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 β-dystroglycan. This link extends to the extracellular matrix by β-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.MethodsTo 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.ResultsThe 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.ConclusionsThe 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.

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 contractioninduced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contractioninduced 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. muscular dystrophy | eccentric contraction | titin | skeletal muscle | dystroglycan

By the age of 80 yr, the skeletal muscles of men and women decrease in mass and maximum force by approximately 30%. Severe contraction-induced injury may contribute to these age-related declines. One to two months after a 225 lengthening contraction protocol (LCP), muscles of young/adult male mice recovered completely, whereas those of old male mice sustained deficits of approximately 15% in mass and approximately 25% in maximum force. Although gender-related differences in the early events of contraction-induced injury have been reported, the recovery phase of muscles in old female animals has not been investigated. The hypothesis tested was that 2 mo after a severe LCP to the plantar flexor muscle group, the magnitude of recovery of mass and force for old female mice is less than that for adult female mice. The LCP was administered to muscles of adult and old, female C57BL/6 mice. At 3 days, 1 mo, and 2 mo following the LCP, maximum isometric force was measured, and muscles were removed and weighed. Two months following the LCP, the muscles of adult female mice recovered mass and force. In contrast, for old female mice, even after 2 mo, muscle masses were decreased by 11% and maximum forces by 38%. We conclude that, as reported previously for old male mice, a severe contraction-induced injury to muscles of old female mice results in prolonged deficits in mass and force.

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.