Accelerated Activation of SOCE Current in Myotubes from Two Mouse Models of Anesthetic- and Heat-Induced Sudden Death

Abstract

Store-operated calcium entry (SOCE) channels play an important role in Ca2+ signaling. Recently, excessive SOCE was proposed to play a central role in the pathogenesis of malignant hyperthermia (MH), a pharmacogenic disorder of skeletal muscle. We tested this hypothesis by characterizing SOCE current (ISkCRAC) magnitude, voltage dependence, and rate of activation in myotubes derived from two mouse models of anesthetic- and heat-induced sudden death: 1) type 1 ryanodine receptor (RyR1) knock-in mice (Y524S/+) and 2) calsequestrin 1 and 2 double knock-out (dCasq-null) mice. ISkCRAC voltage dependence and magnitude at -80 mV were not significantly different in myotubes derived from wild type (WT), Y524S/+ and dCasq-null mice. However, the rate of ISkCRAC activation upon repetitive depolarization was significantly faster at room temperature in myotubes from Y524S/+ and dCasq-null mice. In addition, the maximum rate of ISkCRAC activation in dCasq-null myotubes was also faster than WT at more physiological temperatures (35-37°C). Azumolene (50 µM), a more water-soluble analog of dantrolene that is used to reverse MH crises, failed to alter ISkCRAC density or rate of activation. Together, these results indicate that while an increased rate of ISkCRAC activation is a common characteristic of myotubes derived from Y524S/+ and dCasq-null mice and that the protective effects of azumolene are not due to a direct inhibition of SOCE channels.

Antioxidants Protect Calsequestrin-1 Knockout Mice from Halothane- and Heat-induced Sudden Death

“…Finally, together with previous studies, 19–24,26 our results indicate that the increased susceptibility of some individuals to MHS (and EHS) may result from defects in proteins that interact and regulate RYR1 Ca 2+ release in skeletal muscle.…”

“…22,54 Even under resting conditions (follow orange labeling), SR Ca 2+ leak (step 1) would lead to an increased energy demand (required for continual SERCA-mediated SR Ca 2+ re-uptake) and enhanced oxidative stress (step 2) due to increased mitochondrial content 21 and activity, a scenario that predisposes CASQ1-null mice to lethal reactions under stress. 19 During exposure to heat or halothane, the sequence of events leading to lethal crises involve (follow purple labeling): a) excessive RYR1 Ca 2+ leak and increased oxidative and nitrosative stress (steps 1 and 2 19,31 ); b) RYR1 S-nitrosylation, resulting in a feed-forward mechanism to further increase RYR1 leak (step 3 26 ); c) SR Ca 2+ store depletion (step 4 22 ); d) activation of SOCE resulting in massive Ca 2+ entry (step 5 24 ); e) uncontrolled contractures and rhabdomyolysis (steps 6 and 7 19 ).…”

“…3B and D and 5 A-E) and SOCE activation. 24 Whether RYR1 S-nitrosylation 26 occurs in CASQ1-null mice and the degree to which NAC and Trolox treatment prevents RYR1 S-nitrosylation and/or uncontrolled Ca 2+ entry through SOCE channels requires further investigation.…”

“…Resting Ca 2+ leak and oxidative stress are slightly elevated (compared to WT) in mice lacking CASQ1 (shown as orange junctional Ca 2+ ions). During exposure to heat or halothane, SR Ca 2+ leak (shown as additional purple junctional Ca 2+ ions) and oxidative stress (steps 1 and 2) are further increased, driving a feed-forward mechanism (step 3 26 ) that ultimately leads to SR Ca 2+ store depletion (step 4 22,53 ), activation of SOCE (step 5 24 ), and uncontrolled Ca 2+ influx resulting in severe contractures and rhabdomyolysis (steps 6 and 7 19 ). The protective effects of NAC and Trolox shown in this study likely result from direct effects of the antioxidants to reduce oxidative stress, thus blocking the feed-forward mechanism (steps 3 to 5) leading to SOCE activation.…”

“…Specifically, muscle fibers from CASQ1-knockout (CASQ1-null) mice exhibit impairments in Ca 2+ handling consistent with their enhanced heat- and anesthetic-sensitivity: a) resting Ca 2+ levels are slightly increased at physiological temperature (37°C) due to enhanced SR Ca 2+ leak, 19 b) total SR Ca 2+ store content is greatly reduced, 21 and c) SR depletion during repetitive stimulation is accelerated. 22–24 However, a recent study failed to identify any mutations in the CASQ1 gene in MHS individuals within the North American population. 25 …”