STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.

Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.

associated with an increase in cytoplasmic free Ca 2þ , which may activate cytoplasmic proteases or potentially enter the mitochondria to increase the load of calcium inside this organelle. A major gap in our understanding the issues associated with RyR variants is the effect on Ca 2þ-handling by the cellular organelles, which we could better address if we had information on the total calcium content of the SR and mitochondria under conditions of known RyR mutations and associated Ca 2þ leak. We determined the RyR Ca 2þ leak in extensor digitorum longus muscle fibres of wild-type (WT), heterozygous (HET) and homozygous (HOM) mice for the RyR1 mutant p.G2435R (Lopez et al 2018, BJA) using recently developed techniques (Cully et al 2018, PNAS). We observed an increasing leak with increasing number of mutated RyR alleles, from WT to HET to HOM mice. Next, we used a membrane-lysis method to release all the total compartmentalized calcium onto the contractile apparatus to use the ensuing force response to determine SR calcium content (Fryer & Stephenson, 1996. J.Physiol; Lamboley et al 2013, J.Physiol). We modified this technique to isolate the depletion of mitochondrial calcium to allow determination of total calcium within this compartment. Our results show a progressive decrease in the SR total calcium content with increasing RyR Ca 2þ leak. The mitochondrial total calcium content increased significantly from WT, to that in the HET, to that in the HOM mice.

deep transient store depletion which in turn switched on SOCE in a wild type cell. The peak of the Ca 2þ transient (triggered by the entry of the external Ca 2þ into the cytoplasm following the internal store depletion) was normalized to the peak of the SR Ca 2þ release transient (1.0650.11, n=17 vs 0.850.08, n=9). On isolated FDB fibers under voltage clamp, we found that the voltage dependence of the normalized fluorescence of the calcium transients in response to membrane depolarizations ranging between À60 and þ30 mV, with a 10 mV increments were well fitted with a Boltzmann distribution (V0.5: À23.2251.35 mV vs À24.1550.77 mV with respective k values of 6.1451.15 vs. 6.9350.65). Fatigue inducing protocols are being currently tested. Collectively, these data point toward altered Ca 2þ homeostasis in the compact mice. Understanding the mechanisms behind them may help developing new strategies in muscle atrophy, ageing and wasting diseases. To our knowledge, we are the first to employ Mstn(Cmpt-dl1Abc) mice and perform a comparative study to characterize their Ca 2þ homeostasis in light of SOCE.