SR Ca2+ handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wt littermates (original) (raw)
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Experimental Physiology, 2010
In the dystrophinopathies, skeletal muscle fibres undergo cycles of degeneration and regeneration, with regenerated fibres displaying a branched morphology. This study tests the hypothesis that regenerated branched fibres are mechanically weakened by the presence of branches and are damaged by contractions which do not affect unbranched dystrophin-negative fibres. Experiments were carried out on single fast-twitch fibres and whole muscle from the dystrophin-negative mdx mouse. Fura-2 was ionophoresed into fibres to measure intracellular Ca 2+ concentration ([Ca 2+ ] i ). Single branched mdx fibres have abnormal Ca 2+ kinetics, with the [Ca 2+ ] i transient at the peak of the twitch depressed, are damaged by fatiguing activation, resulting in a breakdown of Ca 2+ homeostasis, and break at branch points when submaximally activated in skinned fibre experiments. When old intact isolated mdx muscles, with >90% branched fibres, are eccentrically activated with a moderate eccentric protocol there is a 40 ± 8% reduction in maximal force. Isolated single fibres from these muscles show areas of damage at fibre branch points. This same eccentric protocol causes no force loss in either littermate control muscles or mdx muscles with <10% branched fibres. I present a two-stage hypothesis for muscle damage in the dystrophinopathies, as follows: stage 1, the absence of dystrophin disrupts ion channel function, causing an activation of necrotizing Ca 2+ -activated proteases, which results in regenerated branched fibres; and stage 2, branched fibres are mechanically damaged during contraction. These results may have implications when considering therapies for boys with Duchenne muscular dystrophy. In particular, any therapy aimed at rescuing the defective gene will presumably have to do so before the number of branched fibres has increased to a level where the muscle is mechanically compromised.
Reciprocal amplification of ROS and Ca2+ signals in stressed mdx dystrophic skeletal muscle fibers
Pflügers Archiv - European Journal of Physiology, 2009
Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca 2+ spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca 2+ sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca 2+ responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca 2+ transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca 2+ spark activity, which was suppressed by ROSreducing agents and by inhibitors of NAD(P)H oxidase. These Ca 2+ signals resulted in mitochondrial Ca 2+ accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca 2+ signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.
Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains
Proceedings of the National Academy of Sciences, 2008
The spatiotemporal properties of the Ca 2+ -release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca 2+ indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca 2+ -release sites in both mouse strains. The Ca 2+ microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca 2+ microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca 2+ -dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were ...
The role of branched fibres in the pathogenesis of Duchenne muscular dystrophy
Experimental Physiology, 2011
Branched fibres are a well-documented phenomenon of regenerating skeletal muscle. They are found in the muscles of boys with Duchenne muscular dystrophy (DMD), a severe condition of progressive muscle wasting caused by an absence of the sarcolemmal protein dystrophin, and in the muscles of the mdx mouse, an animal model of DMD. However, only a handful of studies have investigated how the physiological properties of these morphologically deformed fibres differ from those of normal fibres. These studies have found an association between the extent of fibre branching in mdx muscles and the susceptibility of these muscles to damage from eccentric contractions. They have also found that branched mdx muscle fibres cannot sustain maximal contractions in buffered Ca 2+ solutions, that branch points are sites of increased mechanical stress and that myofibrillar stucture is greatly disturbed at branch points. These findings have important implications for understanding the function of dystrophin. It is commonly thought that the role of dystrophin is mechanical stabilization of the sarcolemma, as numerous studies have shown that eccentric contractions damage mdx muscle more than normal muscle. However, the finding that branched mdx fibres are mechanically weakened raises the question, is it the lack of dystrophin or is it the fibre branching that leads to the vulnerability of mdx muscle to contractile damage? The importance of this question to our understanding of the function of dystrophin warrants further research into the physiological properties of branched fibres and how they differ from morphologically normal fibres.
Biochemical Journal, 2004
Although the primary abnormality in dystrophin is the underlying cause for x-linked muscular dystrophy, abnormal Ca 2+ -handling following sarcolemmal micro-rupturing appears to be the pathophysiological mechanism leading to muscle weakness. In order to develop novel pharmacological strategies eliminating Ca 2+ -dependent proteolysis, it is crucial to determine the fate of Ca 2+ -handling proteins in dystrophin-deficient fibres. Here we show that a key luminal Ca 2+ -binding protein, sarcalumenin, is affected in mdx skeletal muscle fibres. One-and two dimensional immunoblotting revealed that the relative expression of the 160 kDa sarcoplasmic reticulum protein is approximately 70% lower in mdx fibres as compared to normal skeletal muscle. This drastic reduction in sarcalumenin was confirmed by immuno fluorescence microscopy. Patchy internal labelling of sarcalumenin in dystrophic fibres suggests an abnormal formation of sarcalumenin domains. Differential co-immuno precipitation experiments and chemical crosslinking demonstrated a tight linkage between sarcalumenin and the SERCA1 isoform of the sarcoplasmic reticulum Ca 2+ -ATPase. However, the relative expression of the fast Ca 2+ -pump was not decreased in dystrophic membrane preparations. This implies that the reduction in sarcalumenin and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca 2+ -buffering in the dystrophic sarcoplasmic reticulum. Impaired Ca 2+shuttling between the Ca 2+ -uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion binding capacity might indirectly amplify the Ca 2+ -leak channel induced elevation of cytosolic Ca 2+ -levels. This confirms the idea that abnormal Ca 2+ -cycling is involved in Ca 2+ -induced myonecrosis. Hence, manipulating disturbed Ca 2+ -handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy. reticulum glycoprotein of 53 kDa; Up395, utrophin of 395 kDa.
American journal of physiology. Cell physiology, 2018
A striking pathological feature of dystrophinopathies is the presence of morphologically abnormal branched skeletal muscle fibers. The deterioration of muscle contractile function in Duchenne muscular dystrophy is accompanied by both an increase in number and complexity of these branched fibers. We propose that when number and complexity of branched fibers reaches a critical threshold, or "tipping point," the branches in and of themselves are the site of contraction-induced rupture. In the present study, we use the dystrophic mdx mouse and littermate controls to study the prediseased dystrophic fast-twitch extensor digitorum longus (EDL) muscle at 2-3 wk, the peak myonecrotic phase at 6-9 wk, and finally, "old," at 58-112 wk. Using a combination of isolated muscle function contractile measurements coupled with single-fiber imaging and confocal microscope imaging of cleared whole muscles, we identified a distinct pathophysiology, acute fiber rupture at branch node...
The Journal of Physiology, 2004
The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca(2+) release is altered in mdx fibres. Action potential-evoked Ca(2+)-dependent fluorescence transients were recorded, using both low and high affinity Ca(2+) indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N-benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca(2+) transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca(2+) transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca(2+) release flux is responsible for the decrease in the peak of the Ca(2+) transient in mdx fibres. Since the myoplasmic Ca(2+) concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.
The Journal of Physiology, 2002
Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca2+ ions in fibre degeneration. Flexor digitorum brevis muscles of 3-month-old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (d0 to d5) and their survival was assessed. Cytosolic [Ca2+], passive Mn2+ influx (indicative of Ca2+ influx) and activity of mechanosensitive/voltage-independent Ca2+ channels were studied over the same period. Survival of normal fibres declined steadily from d0 to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca2+] 10-fold. In the d0-d5 period, both mdx and normal fibres showed transient increases of Mn2+ influx and activity of the Ca2+ channels; these peaked at d1 and disappeared by d3–d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca2+]i and Mn2+ influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca2+]i remained within the 25–85 nm limits while Mn2+ influx varied more than 10-fold. The median for Mn2+ influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca2+]i medians in fibres from normal and mdx mice. Addition of 25–75 nm of a Ca2+ ionophore (4-bromo-A23187) to the medium did not affect the level of cytosolic [Ca2+] in both types of fibres, while markedly increasing the rate of Mn2+ influx, as expected. Thus, Ca2+ homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn2+ influx and/or elevated [Ca2+]i above 100 nm. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.