Nicholas P. Whitehead

Effects of stretch-activated channel blockers on [Ca2+]i and muscle damage in the mdx mouse.

The mdx mouse lacks dystrophin and is a model of human Duchenne muscular dystrophy. Single mdx muscle fibres were isolated and subjected to a series of stretched (eccentric) contractions while measuring intracellular calcium concentration ([Ca2+]i) with fluo‐3 and confocal microscopy. Following the stretched contractions there was a slow rise in resting [Ca2+]i and after 30 min both the [Ca2+]i during a tetanus (tetanic [Ca2+]i) and the tetanic force were reduced. Two blockers of stretch‐activated channels, streptomycin and the spider venom toxin GsMTx4, prevented the rise of resting [Ca2+]i and partially prevented the decline of tetanic [Ca2+]i and force. Reducing extracellular calcium to zero also prevented the rise in resting [Ca2+]i and prevented some of the decline in tetanic [Ca2+]i and force. Patch‐clamping experiments identified a stretch‐activated channel in both wild‐type and mdx myotubes which was blocked by GsMTx4. These data suggest that blockers of stretch‐activated channels can ameliorate the force reduction following stretched contractions by reducing the influx of Ca2+ into the muscle. We therefore tested whether in intact mdx mice streptomycin, added to the drinking water, was capable of reducing muscle damage. mdx mice show a period of muscle damage from 20 to 40 days of life and fibres which regenerate from this damage display central nuclei. We measured the frequency of central nuclei in control mdx mice compared to streptomycin‐treated mdx mice and showed that the incidence of central nuclei was significantly reduced by streptomycin treatment. This result suggests that blockers of stretch‐activated channels may protect against muscle damage in the intact mdx mouse.

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.

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‐κB, 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 β‐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.

Mechanisms of stretch‐induced muscle damage in normal and dystrophic muscle: role of ionic changes

Muscle damage, characterized by prolonged weakness and delayed onset of stiffness and soreness, is common following contractions in which the muscles are stretched. Stretch‐induced damage of this sort is more pronounced in the muscular dystrophies and the profound muscle damage observed in these conditions may involve similar pathways. It has been known for many years that damaged muscles accumulate calcium and that elevating calcium in normal muscles simulates many aspects of muscle damage. The changes in intracellular calcium, sodium and pH following stretched contractions are reviewed and the various pathways which have been proposed to allow ion entry are discussed. One possibility is that TRPC1 (transient receptor potential, canonical), a protein which seems to form both a stretch‐activated channel and a store‐operated channel, is the main source of Ca2+ entry. The mechanisms by which the changes in intracellular ions contribute to reduced force production, to increased protein breakdown and to increased membrane permeability are considered. A hypothetical scheme for muscle damage which incorporates these ideas is presented.

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.

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 (Ca2+)-permeable channel, is potentially involved in the pathogenesis of Duchenne muscular dystrophy (DMD). Ca2+ 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 Ca2+ 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 Ca2+ 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.

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 gp91phox, p67phox 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, gp91phox and p67phox 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 p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ 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 Ca2+ entry, a key mechanism for muscle damage and functional impairment.

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. Copyright

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.

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.