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.

PICOT (PKC-interacting cousin of thioredoxin) was previously shown to inhibit the development of cardiac hypertrophy, concomitant with an increase in cardiomyocyte contractility. To explore the physiological function of PICOT in the hearts, we generated a PICOT-deficient mouse line by using a gene trap approach. PICOT −/− mice were embryonic lethal indicating that PICOT plays an essential role during embryogenesis, whereas PICOT +/− mice were viable with no apparent morphological defects. The PICOT protein levels were reduced by about 50% in the hearts of PICOT +/− mice. Significantly exacerbated cardiac hypertrophy was induced by pressure overload in PICOT +/− mice relative to that seen in wild type littermates. In line with this observation, calcineurin-NFAT signaling was greatly enhanced by pressure overload in the hearts of PICOT +/− mice. Cardiomyocytes from PICOT +/− mice exhibited significantly reduced contractility, which may be due in part to hypophosphorylation of phospholamban and reduced SERCA activity. These data indicate that the precise PICOT protein level significantly affects the process of cardiac hypertrophy and cardiomyocyte contractility. We suggest that PICOT plays as a critical negative regulator of cardiac hypertrophy and a positive inotropic regulator.

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The histidine-rich Ca2+-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high capacity Ca2+ binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca2+ uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca2+ transients, and maximal SR Ca2+ uptake rates in the heart. However, after-contractions were developed in 50% of HRC-KO cardiomyocytes, compared to 11% in WT mice under stress conditions of high frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca2+ spontaneous SR Ca2+ release and delayed after depolarizations (DADs) with ISO in HRC-KO, compared to WT cells. The frequency of Ca2+ sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca2+ load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca2+-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca2+-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the role(s) of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca2+-uptake through SERCA1a (more than 35%) at micromolar Ca2+ but not at nanomolar Ca2+, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.