Store-operated calcium (Ca2+) entry (SOCE) in skeletal muscle is rapidly activated across the tubular system during direct activation of Ca2+ release. The tubular system is the invagination of the plasma membrane that forms junctions with the sarcoplasmic reticulum (SR) where STIM1, Orai1 and ryanodine receptors are found. The physiological activation of SOCE in muscle is not defined, thus clouding its physiological role. Here we show that the magnitude of a phasic tubular system Ca2+ influx is dependent on SR Ca2+ depletion magnitude, and define this as SOCE. Consistent with SOCE, the influx was resistant to nifedipine and BayK8644, and silenced by inhibition of SR Ca2+ release during excitation. The SOCE transient was shaped by action potential frequency and SR Ca2+ pump activity. Our results show that SOCE in skeletal muscle acts as an immediate counter-flux to Ca2+ loss across the tubular system during excitation-contraction coupling.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca sensor of Ca dynamics in the junctional space. The sensor was used to assess the basal Ca-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca handling under defined ionic conditions.

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Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca 2+ transients elicited by MH triggers are unknown. Here, we show that Ca 2+ release in the absence of Mg 2+ is unaffected by the presence of dantrolene but that dantrolene becomes increasingly effective as cytoplasmic-free [Mg 2+ ] (free [Mg 2+ ] cyto ) passes mM levels. Furthermore, we found in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced repetitive Ca 2+ waves in the presence of resting levels of free [Mg 2+ ] cyto (1 mM). However, an increase of free [Mg 2+ ] cyto to 1.5 mM could increase the period between Ca 2+ waves. These results reconcile previous contradictory reports in muscle fibers and isolated RyRs, where Mg 2+ is present or absent, respectively, and define the mechanism of action of dantrolene is to increase the Mg 2+ affinity of the RyR (or "stabilize" the resting state of the channel) and suggest that the accumulation of the metabolite Mg 2+ from MgATP hydrolysis is required to make dantrolene administration effective in arresting an MH episode. malignant hyperthermia | dantrolene | ryanodine receptor | magnesium | skeletal muscle fiber R yanodine receptors (RyRs) are essential regulators of cytoplasmic Ca 2+ in muscle, heart, and brain (1-3). Congenital or acquired mishandling of Ca 2+ by RyRs is associated with organ dysfunction, myopathy, and the increased risk of sudden death (1, 4-7). Consequently, the search for drugs to modulate RyR function is an area of intense research (8-10). An example of a successful drug controlling the Ca 2+ mishandling of the RyR is the muscle relaxant dantrolene (11). It is primarily used to treat malignant hyperthermia (MH), a life-threatening condition in which genetically predisposed individuals adversely react to the exposure of volatile anesthetics (5, 12). Since its approval, the drug has cut the mortality rate associated with MH from more than 80% to below 2% (11). Despite this success, the exact mechanism of how dantrolene antagonizes MH episodes and depresses overactive Ca 2+ release during MH episodes remains unknown.Susceptibility to MH most commonly arises from mutations in the RyR1 gene, the Ca 2+ release channel of skeletal muscle. Mg 2+ is present in the muscle at ∼1 mM and exerts an inhibitory action over the RyR1, which needs to be overcome for normal voltagecontrolled Ca 2+ release (13-16). RyR1 variants have a lowered affinity for Mg 2+ (13,17), making them more prone to opening and resulting in increased sensitivity to RyR agonist. The ensuing abnormal Ca 2+ release in RyR variants (18-24) during an MH event leads to excessive heat production as the muscle attempts to clear the...

Follistatin (FST) binds to myostatin (MSTN), a potent negative regulator of skeletal muscle growth. Inhibition of MSTN activity by FST treatment has shown to enhance muscle growth as well as ameliorate symptoms of muscular dystrophy in animal models, illustrating the potential of FST as an agent to enhance muscle growth in animal agriculture or to treat muscle wasting conditions or disease in humans. Therefore, we designed a study to produce biologically active recombinant chicken FST315 (chFST315) in an Escherichia coli host. Since FST contains multiple intramolecular disulfide bonds, we expressed chFST315 protein in either a system that utilizes a periplasmic expression strategy, or a genetically modified E. coli system (SHuffle strain) that is capable of disulfide bond formation in the cytoplasm. Periplasmic expression of chFST315 using the pMAL-p5x vector system, which was designed to express maltose-binding protein (MBP) fusion protein, failed to produce a soluble recombinant protein. However, cytoplasmic expression of chFST315 using pMAL-c5x vector in SHuffle E. coli strain resulted in a soluble expression of the recombinant protein (MBP-chFST315). Combination of heparin and amylose resin affinity chromatography yielded about 6 mg/L purified MBP-chFST315. The purified MBP-chFST315 showed binding affinity to MSTN and activin in a pull-down assay, as well as inhibited MSTN and activin activity in an in vitro reporter gene assay. In conclusion, results of the study demonstrate that for the first time a recombinant, biologically active FST molecule can be produced in a soluble form in E. coli. The ability to produce FST in a cost-effective system is expected to allow us to investigate the potentials of FST as an agent to improve skeletal muscle growth of meat producing animals via suppression of MSTN.