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Versican is increased with inflammation and fibrosis, and is upregulated in Duchenne muscular dystrophy. In fibrotic diaphragm muscles from dystrophic mdx mice, genetic reduction of versican attenuated macrophage infiltration and improved contractile function. Versican is also implicated in myogenesis. Here, we investigated whether versican modulated mdx hindlimb muscle pathology, where inflammation and regeneration are increased but fibrosis is minimal. Immunohistochemistry and qRT-PCR were used to assess how fiber type and glucocorticoids (α-methylprednisolone) modify versican expression. To genetically reduce versican, female mdx and male versican haploinsufficient (hdf) mice were bred resulting in male mdx-hdf and mdx (control) pups. Versican expression, contractile function, and pathology were evaluated in hindlimb muscles. Versican immunoreactivity was greater in slow versus fast hindlimb muscles. Versican mRNA transcripts were reduced by α-methylprednisolone in soleus, but not in fast extensor digitorum longus, muscles. In juvenile (6-wk-old) mdx-hdf mice, versican expression was most robustly decreased in soleus muscles leading to improved force output and a modest reduction in fatiguability. These functional benefits were not accompanied by decreased inflammation. Muscle architecture, regeneration markers, and fiber type also did not differ between mdx-hdf mice and mdx littermates. Improvements in soleus contractile function were not retained in adult (20-wk-old) mdx-hdf mice. In conclusion, soleus muscles from juvenile mdx mice were most responsive to pharmacological or genetic approaches targeting versican; however, the benefits of versican reduction were limited due to low fibrosis. Preclinical matrix research in dystrophy should account for muscle phenotype (including age) and the interdependence between inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow‐ to fast‐twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow‐twitch soleus and fast‐twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre‐type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders. [ABSTRACT FROM AUTHOR]

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy. [ABSTRACT FROM AUTHOR]

Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1a) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function. [ABSTRACT FROM AUTHOR]

Store-operated [Ca.sup.2+] entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of [Ca.sup.2+] from the transverse tubule into the junctional cleft during periods of depleting [Ca.sup.2+] release. In dystrophic muscle fibers, SOCE may be a source of [Ca.sup.2+] overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during [Ca.sup.2+] release. To examine this, [Ca.sup.2+] in the tubular system and cytoplasm were simultaneously imaged during direct release of [Ca.sup.2+] from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic [Ca.sup.2+] transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR [Ca.sup.2+] concentrations ([[[Ca.sup.2+]].sub.SR]). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orail proteins in dystrophic muscle. While the mdx fibers can introduce more [Ca.sup.2+] into the fiber for an equivalent depletion of [[[Ca.sup.2+]].sub.SR] via SOCE, it remains unclear whether this is deleterious. skeletal muscle; sarcoplasmic reticulum; muscular dystrophy; STIM1; Orail; excitation-contraction coupling doi: 10.1152/ajpcell.00524.2009.

In skeletal muscle fibers, the coupling between excitation of the surface membrane and the release of [Ca.sup.2+] from the sarcoplasmic reticulum is irreversibly disrupted if cytoplasmic [Ca.sup.2+] concentration ([[Ca.sup.2+]]) is raised to micromolar levels for a prolonged period. This excitation-contraction (EC) uncoupling may contribute to muscle weakness after some types of exercise and in certain muscle diseases and has been linked to structural alteration of the triad junctions, but its molecular basis is unclear. Both [mu]-calpain, a ubiquitous [Ca.sup.2+]-activated protease, and muscle-specific calpain-3 become autolytically activated at micromolar [Ca.sup.2+] and have been suggested to be responsible for the uncoupling. This study used controlled [Ca.sup.2+] exposure in mechanically skinned fibers from extensor digitorum longus muscle to show that EC uncoupling still occurs in muscle fibers of calpain-3-deficient mice, with a [Ca.sup.2+] dependence indistinguishable from that in normal mice and rats. Western blotting of muscle fibers that had been partially EC uncoupled by exposure to an intermediate [Ca.sup.2+] level (~5 [micro]M [Ca.sup.2+] for 3 rain, no ATP) showed the presence of autolytic activation of a proportion of the [mu]-calpain present, but with little or no activation of calpain-3. Homogenates of normal and calpain-3-deficient muscles exposed to micromolar [Ca.sup.2+] displayed similar levels of diffusible proteolytic activity, as gauged by the rate of decline of passive force in stretched, skinned muscle fibers. Exogenously added [mu]-calpain, preactivated by elevated [[Ca.sup.2+]] and applied in the presence of 1 [micro]M [Ca.sup.2+], disrupted EC coupling in a manner similar to raised [[Ca.sup.2+]]. We conclude that calpain-3 is not responsible for [Ca.sup.2+]-induced disruption of EC coupling, but that [mu]-calpain is a plausible candidate. calpain-3; [mu]-calpain; skinned fiber; muscle fatigue; knockout mice

[micro]-calpain and calpain-3 are [Ca.sup.2+]+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that [micro]-calpain becomes proteolytically active in the presence of 2-200 [micro]M [Ca.sup.2+]. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular [Ca.sup.2+] concentration levels ([[[Ca.sup.2+]].sub.i]; -1 [micro]M) than the levels at which [micro]-calpain activation occurs. We have demonstrated the [Ca.sup.2+]-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [[Ca.sup.2+]] levels (0, 2.5, 10, and 25 [micro]M). Autolysis of calpain-3 was found to occur across a [[Ca.sup.2+]] range similar to that for [micro]-calpain, and both calpains displayed a seemingly higher [Ca.sup.2+] sensitivity in human than in rat muscle homogenates, with ~15% autolysis observed after 1-min exposure to 2.5 [micro]M [Ca.sup.2+] in human muscle and almost none after 1- to 2-min exposure to the same [[[Ca.sup.2+]].sub.i] level in rat muscle. During muscle activity, [[[Ca.sup.2+]].sub.i] may transiently peak in the range found to autolyze [micro]-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s 'all-out' cycling, n = 8; and 70% V[O.sub.2] peak until fatigue, n = 3) on the amount of autolyzed po-calpain or calpain-3 in human muscle. No significant autolysis of [micro]-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [[[Ca.sup.2+]].sub.i] that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo. [Ca.sup.2+]-dependent proteases; proteolysis

Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in [Ca.sup.2+] sensitivity (+0.061 [+ or -] 0.004 and +0.049 [+ or -] 0.009 pCa units, respectively) and maximum [Ca.sup.2+]-activated force (to 104 [+ or -] 1 and 105 [+ or -] 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in [Ca.sup.2+] sensitivity of the contractile apparatus, and maximum [Ca.sup.2+]-activated force was ~106 [+ or -] 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus. ergogenic aid; muscle contraction; fatigue