Ella W. Yeung

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.

A prospective cohort study of hamstring injuries in competitive sprinters: preseason muscle imbalance as a possible risk factor

Background: Hamstring injuries are common in sprinters. Identifying preseason risk factors is essential to target injury-prone athletes and develop injury preventive measures. Objective: To investigate the incidence of hamstring muscle injury in sprinters over an athletic season and to explore the preseason predictor of this injury. Design: Prospective cohort study. Participants: 44 sprinters from the Hong Kong Sports Institute, the Hong Kong Amateur Athletic Association and intercollegiate athletic teams were recruited. Methods: Preseason assessment of hamstring flexibility, concentric and eccentric isokinetic peak torque and peak torque angle were obtained at the beginning of an athletic season. The athletes were followed over 12 months and were asked to report all injuries resulting from training and competition. Results: Eight athletes sustained hamstring injuries over the season. The injury rate was 0.87 per 1000 h of exposure. The incidence of injuries was higher at the beginning of the season, with 58.3% injuries occurring in the first 100 h of exposure. Cox regression analysis revealed that athletes with a decrease in the hamstring : quadriceps peak torque ratio of less than 0.60 at an angular velocity of 180°/s have a 17-fold increased risk of hamstring injury. Conclusion: Performing preseason hamstring : quadriceps peak torque ratio assessments may be useful to identify sprinters susceptible to hamstring injury.

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.

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.

A systematic review of interventions to prevent lower limb soft tissue running injuries

Objectives—To assess the available evidence for preventive strategies for lower limb soft tissue injuries caused by running. Methods—An electronic database search was conducted using The Cochrane Musculoskeletal Injuries Group Specialised Register, The Cochrane Controlled Trials Register, Medline, Embase, Sport Discus, Heracles, Atlantes, Biosis, Cinahl, Scisearch, Current Contents, Index To Theses and Dissertation Abstracts. Any randomised or quasi-randomised trials evaluating interventions to prevent running injuries to lower limb soft tissue were included. The eligibility of trials for inclusion and the quality of the trials were independently assessed by two reviewers. Results—Exposure to a high training load (duration, frequency, or running distance) increases the risk of injury, and thus modification of the training schedule can reduce the incidence of injury. The effectiveness of stretching exercises and of insoles in the prevention of lower extremity soft tissue injuries caused by running is not known. Wearing a knee brace with a patellar support ring may be effective in the prevention of anterior knee pain caused by running. Conclusions—This review provides evidence for the effectiveness of the modification of training schedules in reducing lower limb soft tissue running injuries. More studies are required to quantify the optimal training loads and to confirm that knee braces can prevent knee pain. It is important to note that the studies included in this review had few female participants therefore the results may not be generalisable.

Gadolinium reduces short‐term stretch‐induced muscle damage in isolated mdx mouse muscle fibres

Duchenne muscular dystrophy is a lethal muscle disease caused by absence of the protein dystrophin which is part of a glycoprotein complex located on the intracellular surface of the surface membrane. The precise function of dystrophin and the reason why its absence causes severe muscle damage are unclear. Stretch‐induced muscle damage is well recognised in normal muscle and is more severe in muscles from animals lacking dystrophin (mdx mice). It has been proposed that stretch‐induced damage underlies the progression of damage in muscular dystrophy. In the present study we confirm that single fibres from mdx muscle are more susceptible to stretch‐induced damage and show that there is an associated rise in intracellular sodium concentration ([Na+]i) which is greater than in wild‐type mice. We show that this rise in [Na+]i can be prevented by Gd3+, which is an established blocker of stretch‐activated channels. mdx fibres have a higher than normal resting [Na+]i and this is also reduced by Gd3+. If Gd3+ is applied over the period in which [Na+]i rises following stretched contraction, it prevents one component of the reduced force. The other component of reduced force is caused by inhomogeneity of sarcomeres and can be minimised by stretching the muscle to its new optimum length. These experiments show that part of the short‐term damage caused by stretch in mdx fibres can be prevented by blocking stretch‐activated channels.

Development of T-tubular vacuoles in eccentrically damaged mouse muscle fibres

Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force‐length relation and a steepening of the force‐frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T‐tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T‐tubules were clearly delineated. Sulforhodamine B diffused out of the T‐tubules with a half‐time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the T‐tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half‐time of 6.3 ± 2.4 min and diffused out of the T‐tubules with a half‐time of 39 ± 4 s. Vacuole production was eliminated by application of 1 mm ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T‐tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na+ and water exceeds the capacity of the T‐tubules and causes vacuole production. The damage to the T‐tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.