RyR1-mediated Ca2+ Leak and Ca2+ Entry Determine Resting Intracellular Ca2+ in Skeletal Myotubes

Department of Anesthesiology Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2010; 285(18):13781-7. DOI: 10.1074/jbc.M110.107300
Source: PubMed


The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RyR1 and RyR3. We demonstrate here that expression of RyR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RyR1-expressing cells is not a result of active gating of the RyR1 channel but instead is accounted for by the RyR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RyR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.

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Available from: Jose M Eltit, Apr 05, 2014
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    • "These phenomena have been attributed to the conversion by these Ca 2? releasers of ryanodine-sensitive RyRs into leak channels, which are insensitive to ryanodine . In contrast, bastadins at low micromolar concentrations reverse this phenomenon and restore the sensitivity of RyRs to ryanodine (Pessah et al. 1997; Eltit et al. 2010; Altamirano et al. 2012). While excessive mobilization of Ca 2? from endoplasmic reticulum stores by thapsigargin and other Ca 2? releasers has been implicated in their neurotoxicity (Silverstein and Nelson 1992; Pessah et al. 2010; Stirling et al. 2014), contrasting reports have appeared envisaging neuroprotective potential residing in the up-regulation of RyRs and in the ensuing increased release of Ca 2? from the ER (Supnet et al. 2010).These discrepant effects prompted us to consider bastadin 12 and ryanodine as pharmacological tools to get a deeper insight into the role of intracellular Ca 2? release by TBBPA via RyRs in cytotoxicity, which to our knowledge has not been directly tested in CGC or neurons cultured from any other brain region. "
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    ABSTRACT: The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.
    Neurotoxicity Research 07/2015; 28(3). DOI:10.1007/s12640-015-9546-8 · 3.54 Impact Factor
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    • "The on-going recycling of calcium between SR and the cytoplasm mediated by this leak is overwhelmed by the massive release of SR that occurs during cell depolarisation or when cells are stimulated by caffeine. Interestingly, and as suggested previously [64] the extent of the leak appears to contribute to the basal cytoplasmic calcium concentration, since calcium concentration reduced when cells (in the resting state) were exposed to the RYR1 antagonist, dantrolene, thereby excluding excitation-coupled calcium entry [7], [39]. In horses, dantrolene has been used both to treat [38] and prevent RER [65], [66]. "
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    ABSTRACT: Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells' calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.
    PLoS ONE 08/2014; 9(8):e105971. DOI:10.1371/journal.pone.0105971 · 3.23 Impact Factor
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    • "The RyR1 channels, which release Ca 2+ in response to plasma membrane depolarization, contribute to the characteristic excitation-contraction coupling process of skeletal muscle cells (Protasi et al., 2002). A recent report proposes that RyR1 channels, by controlling passive Ca 2+ efflux from the SR to the cytoplasm, represent key factors in the management of resting muscle cytoplasmic Ca 2+ concentration (Eltit et al., 2010). "
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    ABSTRACT: Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased GLUT4myc translocation, an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)/NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine-receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation.This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3) activated Ca(2+) channels, enacted through activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,β phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3 receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.
    Journal of Cell Science 02/2014; 127(9). DOI:10.1242/jcs.138982 · 5.43 Impact Factor
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