Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease.

Mice lacking α-, ß1- and ß2-syntrophins exhibit diminished function and reduced dystrophin expression in both cardiac and skeletal muscle.

Syntrophins are a family of modular adaptor proteins that are part of the dystrophin protein complex, where they recruit and anchor a variety of signaling proteins. Previously we generated mice lacking α- and/or ß2-syntrophin but showed that in the absence of one isoform, other syntrophin isoforms can partially compensate. Therefore, in the current study, we generated mice that lacked α, ß1 and ß2-syntrophins [triple syntrophin knockout (tKO) mice] and assessed skeletal and cardiac muscle function. The tKO mice showed a profound reduction in voluntary wheel running activity at both 6 and 12 months of age. Function of the tibialis anterior was assessed in situ and we found that the specific force of tKO muscle was decreased by 20-25% compared with wild-type mice. This decrease was accompanied by a shift in fiber-type composition from fast 2B to more oxidative fast 2A fibers. Using echocardiography to measure cardiac function, it was revealed that tKO hearts had left ventricular cardiac dysfunction and were hypertrophic, with a thicker left ventricular posterior wall. Interestingly, we also found that membrane-localized dystrophin expression was lower in both skeletal and cardiac muscles of tKO mice. Since dystrophin mRNA levels were not different in tKO, this finding suggests that syntrophins may regulate dystrophin trafficking to, or stabilization at, the sarcolemma. These results show that the loss of all three major muscle syntrophins has a profound effect on exercise performance, and skeletal and cardiac muscle dysfunction contributes to this deficiency.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels.

Facilitation and inactivation of P/Q-type calcium (Ca(2+)) currents through the regulation of voltage-gated Ca(2+) (CaV) 2.1 channels by Ca(2+) sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca(2+) entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10-30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50-100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.

Sarcolemmal targeting of nNOSµ improves contractile function of mdx muscle.

Nitric oxide (NO) is a key regulator of skeletal muscle function and metabolism, including vasoregulation, mitochondrial function, glucose uptake, fatigue and excitation-contraction coupling. The main generator of NO in skeletal muscle is the muscle-specific form of neuronal nitric oxide synthase (nNOSµ) produced by the NOS1 gene. Skeletal muscle nNOSµ is predominantly localized at the sarcolemma by interaction with the dystrophin protein complex (DPC). In Duchenne muscular dystrophy (DMD), loss of dystrophin leads to the mislocalization of nNOSµ from the sarcolemma to the cytosol. This perturbation has been shown to impair contractile function and cause muscle fatigue in dystrophic (mdx) mice. Here, we investigated the effect of restoring sarcolemmal nNOSµ on muscle contractile function in mdx mice. To achieve this, we designed a modified form of nNOSµ (NOS-M) that is targeted to the sarcolemma by palmitoylation, even in the absence of the DPC. When expressed specifically in mdx skeletal muscle, NOS-M significantly attenuates force loss owing to damaging eccentric contractions and repetitive isometric contractions (fatigue), while also improving force recovery after fatigue. Expression of unmodified nNOSµ at similar levels does not lead to sarcolemmal association and fails to improve muscle function. Aside from the benefits of sarcolemmal-localized NO production, NOS-M also increased the surface membrane levels of utrophin and other DPC proteins, including ß-dystroglycan, α-syntrophin and α-dystrobrevin in mdx muscle. These results suggest that the expression of NOS-M in skeletal muscle may be therapeutically beneficial in DMD and other muscle diseases characterized by the loss of nNOSµ from the sarcolemma.

A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease with no effective treatment. DMD muscle pathogenesis is characterized by chronic inflammation, oxidative stress, and fibrosis. Statins, cholesterol-lowering drugs, inhibit these deleterious processes in ischemic diseases affecting skeletal muscle, and therefore have potential to improve DMD. However, statins have not been considered for DMD, or other muscular dystrophies, principally because skeletal-muscle-related symptoms are rare, but widely publicized, side effects of these drugs. Here we show positive effects of statins in dystrophic skeletal muscle. Simvastatin dramatically reduced damage and enhanced muscle function in dystrophic (mdx) mice. Long-term simvastatin treatment vastly improved overall muscle health in mdx mice, reducing plasma creatine kinase activity, an established measure of muscle damage, to near-normal levels. This reduction was accompanied by reduced inflammation, more oxidative muscle fibers, and improved strength of the weak diaphragm muscle. Shorter-term treatment protected against muscle fatigue and increased mdx hindlimb muscle force by 40%, a value comparable to current dystrophin gene-based therapies. Increased force correlated with reduced NADPH Oxidase 2 protein expression, the major source of oxidative stress in dystrophic muscle. Finally, in old mdx mice with severe muscle degeneration, simvastatin enhanced diaphragm force and halved fibrosis, a major cause of functional decline in DMD. These improvements were accompanied by autophagy activation, a recent therapeutic target for DMD, and less oxidative stress. Together, our findings highlight that simvastatin substantially improves the overall health and function of dystrophic skeletal muscles and may provide an unexpected, novel therapy for DMD and related neuromuscular diseases.

Validation of ultrasonography for non-invasive assessment of diaphragm function in muscular dystrophy.

KEY POINTS: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease that is commonly studied using the mdx mouse. The mdx diaphragm muscle closely mimics the pathophysiological changes in DMD muscles. mdx diaphragm force is commonly assessed ex vivo, precluding time course studies. Here we used ultrasonography to evaluate time-dependent changes in diaphragm function in vivo, by measuring diaphragm movement amplitude. In mdx mice, diaphragm amplitude decreased with age and values were much lower than for wild-type mice. Importantly, diaphragm amplitude strongly correlated with ex vivo specific force values. Micro-dystrophin administration increased mdx diaphragm amplitude by 26% after 4 weeks. Diaphragm amplitude correlated positively with ex vivo force values and negatively with diaphragm fibrosis, a major cause of DMD muscle weakness. These studies validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in mdx diaphragm function in vivo. This technique will be valuable for testing potential therapies for DMD. ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease caused by dystrophin mutations. The mdx mouse is a widely used animal model of DMD. The mdx diaphragm muscle most closely recapitulates key features of DMD muscles, including progressive fibrosis and considerable force loss. Diaphragm function in mdx mice is commonly evaluated by specific force measurements ex vivo. While useful, this method only measures force from a small muscle sample at one time point. Therefore, accurate assessment of diaphragm function in vivo would provide an important advance to study the time course of functional decline and treatment benefits. Here, we evaluated an ultrasonography technique for measuring time-dependent changes of diaphragm function in mdx mice. Diaphragm movement amplitude values for mdx mice were considerably lower than those for wild-type, decreased from 8 to 18 months of age, and correlated strongly with ex vivo specific force. We then investigated the time course of diaphragm amplitude changes following administration of an adeno-associated viral vector expressing Flag-micro-dystrophin (AAV-µDys) to young adult mdx mice. Diaphragm amplitude peaked 4 weeks after AAV-µDys administration, and was 26% greater than control mdx mice at this time. This value decreased slightly to 21% above mdx controls after 12 weeks of treatment. Importantly, diaphragm amplitude again correlated strongly with ex vivo specific force. Also, diaphragm amplitude and specific force negatively correlated with fibrosis levels in the muscle. Together, our results validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in dystrophic diaphragm function in vivo, and for evaluating potential therapies for DMD.

Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle.

Calcium and the damage pathways in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. Experiments on the mdx mouse, a model of DMD, have shown that mdx muscles are particularly susceptible to stretch-induced damage. In this review, we discuss evidence showing that a series of stretched contractions of mdx muscle fibres causes a prolonged increase in resting intracellular calcium concentration ([Ca2+]i). The rise in [Ca2+]i is caused by Ca2+ entry through a class of stretch-activated channels (SACNSC) for which one candidate gene is TRPC1. We review the evidence for activation of SACNSC in muscle by reactive oxygen species (ROS) and suggest that stretch-induced ROS production is part of the pathway that triggers increased channel activity. When the TRPC1 gene was transfected into C2 myoblasts, expression occurred throughout the cell. Only when the TRPC1 gene was coexpressed with caveolin-3 did the TRPC1 protein express in the membrane. When TRPC1 was expressed in the membrane, it could be activated by ROS to produce Ca2+ entry and this entry was inhibited by PP2, an inhibitor of src kinase. These results suggest that stretched contractions activate ROS production, which activates src kinase. Activity of this kinase causes opening of SACNSC and allows Ca2+ entry. This pathway appears to be a significant cause of muscle damage in DMD.

Stretch-induced membrane damage in muscle: comparison of wild-type and mdx mice.

One component of stretch-induced muscle damage is an increase in the permeability of the cell membrane. As a result soluble myoplasmic proteins leak out of the muscle into the plasma, extracellular proteins can enter the muscle, and extracellular ions, including calcium, are driven down their electrochemical gradient into the myoplasm. In Duchenne muscular dystrophy, caused by the absence of the cytoskeletal protein dystrophin, stretch-induced membrane damage is much more severe. The most popular theory to explain the occurrence of stretch-induced membrane damage is that stretched-contractions cause transient mechanically-induced defects in the membrane (tears or rips). Dystrophin, which is part of a mechanical link between the contractile machinery and the extracellular matrix, is thought to contribute to membrane strength so that in its absence mechanically-induced defects are worse. In our view the evidence that stretch-induced muscle damage causes increased membrane permeability is overwhelming but the evidence that the increased permeability is caused by mechanically-induced defects is weak. Instead we review the substantial evidence that the membrane permeability is a secondary consequence of the mechanical events in which elevated intracellular calcium and reactive oxygen species are important intermediaries.

Simvastatin provides long-term improvement of left ventricular function and prevents cardiac fibrosis in muscular dystrophy.

Duchenne muscular dystrophy (DMD), caused by absence of the protein dystrophin, is a common, degenerative muscle disease affecting 1:5000 males worldwide. With recent advances in respiratory care, cardiac dysfunction now accounts for 50% of mortality in DMD. Recently, we demonstrated that simvastatin substantially improved skeletal muscle health and function in mdx (DMD) mice. Given the known cardiovascular benefits ascribed to statins, the aim of this study was to evaluate the efficacy of simvastatin on cardiac function in mdx mice. Remarkably, in 12-month old mdx mice, simvastatin reversed diastolic dysfunction to normal after short-term treatment (8 weeks), as measured by echocardiography in animals anesthetized with isoflurane and administered dobutamine to maintain a physiological heart rate. This improvement in diastolic function was accompanied by increased phospholamban phosphorylation in simvastatin-treated mice. Echocardiography measurements during long-term treatment, from 6 months up to 18 months of age, showed that simvastatin significantly improved in vivo cardiac function compared to untreated mdx mice, and prevented fibrosis in these very old animals. Cardiac dysfunction in DMD is also characterized by decreased heart rate variability (HRV), which indicates autonomic function dysregulation. Therefore, we measured cardiac ECG and demonstrated that short-term simvastatin treatment significantly increased heart rate variability (HRV) in 14-month-old conscious mdx mice, which was reversed by atropine. This finding suggests that enhanced parasympathetic function is likely responsible for the improved HRV mediated by simvastatin. Together, these findings indicate that simvastatin markedly improves cardiac health and function in dystrophic mice, and therefore may provide a novel approach for treating cardiomyopathy in DMD.

N-Acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three-week-old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-kappaB, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of beta-dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients.

Streptomycin reduces stretch-induced membrane permeability in muscles from mdx mice.

It is well-known that muscles from mdx mice are more susceptible to membrane damage from eccentric contractions than wild-type muscles. The present study tested the hypothesis that the stretch-induced membrane permeability in dystrophic muscle is due to Ca(2+) entry through stretch-activated channels (SACs) and the subsequent activation of Ca(2+) -dependent degradative pathways. Eccentric contractions were carried out on muscles from mdx and wild-type mice, both on isolated muscles and on intact mice subjected to downhill running on a treadmill. In isolated muscles the SAC blockers, streptomycin and GsMTx4, improved force and significantly reduced the uptake of procion orange dye into fibres from mdx muscles, which increased progressively over 60 min after the eccentric contractions. In experiments on intact mdx mice, streptomycin also partially prevented the reduced force and the increased membrane permeability (Evans Blue Dye uptake). The results suggest that Ca(2+) entry through SACs activates Ca(2+) -dependent pathways, which are the main cause of the increased membrane permeability in mdx muscle.

Enhanced autophagy as a potential mechanism for the improved physiological function by simvastatin in muscular dystrophy.

Autophagy has recently emerged as an important cellular process for the maintenance of skeletal muscle health and function. Excessive autophagy can trigger muscle catabolism, leading to atrophy. In contrast, reduced autophagic flux is a characteristic of several muscle diseases, including Duchenne muscular dystrophy, the most common and severe inherited muscle disorder. Recent evidence demonstrates that enhanced reactive oxygen species (ROS) production by CYBB/NOX2 impairs autophagy in muscles from the dmd/mdx mouse, a genetic model of Duchenne muscular dystrophy. Statins decrease CYBB/NOX2 expression and activity and stimulate autophagy in skeletal muscle. Therefore, we treated dmd/mdx mice with simvastatin and showed decreased CYBB/NOX2-mediated oxidative stress and enhanced autophagy induction. This was accompanied by reduced muscle damage, inflammation and fibrosis, and increased muscle force production. Our data suggest that increased autophagy may be a potential mechanism by which simvastatin improves skeletal muscle health and function in muscular dystrophy.

Simvastatin offers new prospects for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common and severe inherited neuromuscular disorder. DMD is caused by mutations in the gene encoding the dystrophin protein in muscle fibers. Dystrophin was originally proposed to be a structural protein that protected the sarcolemma from stresses produced during contractions. However, more recently, experimental evidence has revealed a far more complicated picture, with the loss of dystrophin causing dysfunction of multiple muscle signaling pathways, which all contribute to the overall disease pathophysiology. Current gene-based approaches for DMD are conceptually appealing since they offer the potential to restore dystrophin to muscles, albeit a partially functional, truncated form of the protein. However, given the cost and technical challenges facing these genetic approaches, it is important to consider if relatively inexpensive, clinically used drugs may be repurposed for treating DMD. Here, we discuss our recent findings showing the potential of simvastatin as a novel therapy for DMD.

ToF-SIMS of tissues: "lessons learned" from mice and women.

The ability to image cells and tissues with chemical and molecular specificity could greatly expand our understanding of biological processes. The subcellular resolution mass spectral imaging capability of time of flight secondary ion mass spectrometry (ToF-SIMS) has the potential to acquire chemically detailed images. However, the complexities of biological systems combined with the sensitivity of ToF-SIMS require careful planning of experimental methods. Tissue sample preparation methods of formalin fixation followed by paraffin embedding (FFPE) and OCT embedding are compared. Results show that the FFPE can potentially be used as a tissue sample preparation protocol for ToF-SIMS analysis if a cluster ion pre-sputter is used prior to analysis and if nonlipid related tissue features are the features of interest. In contrast, embedding tissue in OCT minimizes contamination and maintains lipid signals. Various data acquisition methodologies and analysis options are discussed and compared using mouse breast and diaphragm muscle tissue. Methodologies for acquiring ToF-SIMS 2D images are highlighted along with applications of multivariate analysis to better identify specific features in a tissue sections when compared to H&E images of serial sections. Identification of tissue features is necessary for researchers to visualize a molecular map that correlates with specific biological features or functions. Finally, lessons learned from sample preparation, data acquisition, and data analysis methods developed using mouse models are applied to a preliminary analysis of human breast tumor tissue sections.

Pathways of Ca²âº entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle.

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 µM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and µ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.

Duchenne muscular dystrophy--what causes the increased membrane permeability in skeletal muscle?

Duchenne muscular dystrophy is a severe muscle wasting disease caused by a mutation in the gene for dystrophin--a cytoskeletal protein connecting the contractile machinery to a group of proteins in the cell membrane. At the end stage of the disease there is profound muscle weakness and atrophy. However, the early stage of the disease is characterised by increased membrane permeability which allows soluble enzymes such as creatine kinase to leak out of the cell and ions such as calcium to enter the cell. The most widely accepted theory to explain the increased membrane permeability is that the absence of dystrophin makes the membrane more fragile so that the stress of contraction causes membrane tears which provide the increase in membrane permeability. However other possibilities are that increases in intracellular calcium caused by altered regulation of channels activate enzymes, such as phospholipase A(2), which cause increased membrane permeability. Increases in reactive oxygen species (ROS) are also present in the early stages of the disease and may contribute both to membrane damage by peroxidation and to the channel opening. Understanding the earliest phases of the pathology are critical to therapies directed at minimizing the muscle damage.

Skeletal muscle NADPH oxidase is increased and triggers stretch-induced damage in the mdx mouse.

Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91(phox), p67(phox) and rac 1 were increased 2-3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91(phox) and p67(phox) were increased 3-4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67(phox),which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca(2+) rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca(2+) entry, a key mechanism for muscle damage and functional impairment.

TRPC1 binds to caveolin-3 and is regulated by Src kinase - role in Duchenne muscular dystrophy.

Transient receptor potential canonical 1 (TRPC1), a widely expressed calcium (Ca(2+))-permeable channel, is potentially involved in the pathogenesis of Duchenne muscular dystrophy (DMD). Ca(2+) influx through stretch-activated channels, possibly formed by TRPC1, induces muscle-cell damage in the mdx mouse, an animal model of DMD. In this study, we showed that TRPC1, caveolin-3 and Src-kinase protein levels are increased in mdx muscle compared with wild type. TRPC1 and caveolin-3 colocalised and co-immunoprecipitated. Direct binding of TRPC1-CFP to caveolin-3-YFP was confirmed in C2 myoblasts by fluorescence energy resonance transfer (FRET). Caveolin-3-YFP targeted TRPC1-CFP to the plasma membrane. Hydrogen peroxide, a reactive oxygen species (ROS), increased Src activity and enhanced Ca(2+) influx, but only in C2 myoblasts co-expressing TRPC1 and caveolin-3. In mdx muscle, Tiron, a ROS scavenger, and PP2, a Src inhibitor, reduced stretch-induced Ca(2+) entry and increased force recovery. Because ROS production is increased in mdx/DMD, these results suggest that a ROS-Src-TRPC1/caveolin-3 pathway contributes to the pathogenesis of mdx/DMD.

Muscle damage in mdx (dystrophic) mice: role of calcium and reactive oxygen species.

1. Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease caused by a genetic mutation that leads to the complete absence of the cytoskeletal protein dystrophin in muscle fibres. 2. The present review provides an overview of some of the physiological pathways that may contribute to muscle damage and degeneration in DMD, based primarily on experimental findings in the mdx mouse, an animal model of this disease. 3. A rise in intracellular calcium is widely thought to be an important initiating event in the dystrophic pathogenesis. The pathway(s) leading to increased intracellular calcium in dystrophin deficient muscle is uncertain, but recent work from our laboratory provides evidence that stretch-activated channels are an important source of the calcium influx. Other possible routes of calcium entry are also discussed. 4. The consequences of elevated cytosolic calcium may include activation of proteases, such as calpain, and increased production of reactive oxygen species (ROS), which can cause protein and membrane damage. 5. Another possible cause of damage in dystrophic muscle involves inflammatory pathways, such as those mediated by neutrophils, macrophages and associated cytokines. There is recent evidence that increased ROS may be important in both the activation of and the damage caused by this inflammatory pathway in mdx muscle.