Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Abstract

Dantrolene reduces the elevated myoplasmic Ca2؉ generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca 2؉ release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest storeoperated Ca 2؉ entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1. Malignant hyperthermia (MH)2 is a potentially fatal pharmacogenetic syndrome in which exposure to volatile anesthetics triggers uncontrolled elevation of myoplasmic Ca 2ϩ concentrations ([Ca 2ϩ ] i ), skeletal muscle hypercontracture, and hypermetabolism, resulting in a dramatic rise in body temperature (1, 2). Mutations in the type 1 ryanodine receptor (RyR1), the major Ca 2ϩ release channel in skeletal muscle, are linked to MH susceptibility in pigs in an autosomal recessive manner (3-5). In humans, MH is transmitted as an autosomal dominant trait with incomplete penetrance, and the more than 80 mutations in RyR1 that have been identified appear in only about 50% of affected families (6, 7). Muscle bundles from MH-susceptible patients are hypersensitive to RyR1 agonists, including caffeine, Ca 2ϩ , and halothane (8, 9), the latter a member of the class of volatile anesthetics that triggers MH. Thus, the loss of control of RyR1-mediated Ca 2ϩ release likely contributes to the elevation of [Ca 2ϩ ] i observed in MH patients. To date, the only effective treatment for MH is dantrolene sodium, a skeletal muscle relaxant, which suppresses the uncontrolled rise in myoplasmic Ca 2ϩ , presumably by targeting RyR1 and suppressing its Ca 2ϩ channel activity (10 -12). Azumolene sodium is a structurally similar, equipotent analog of dantrolene, with an ϳ30-fold greater water solubility (13,14).Whereas hyperactivity or leakiness of the RyR1 channel has been described as the primary physiological defect in MH-susceptible muscle, the molecular mechanism underlying the suppression of [ -sensitive fluorescence measurements of RyR1-dependent intracellular Ca 2ϩ transients and extracellular Ca 2ϩ entry via SOCE, we show that azumolene inhibits SOCE both in skeletal muscle fibers and in cultured cells expressing RyR1. Our results reveal two modes of SOCE activation. One mode is RyR1-dependent and can be inhibited by the action of azumolene; the other is RyR1-independent and is insensitive to azumolene. EXPERIMENTAL PROCEDURESCell Culture-C1148 cells, a Chinese hamster ovary (CHO) ce...

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca 2+ channel and the type 1 ryanodine receptor

“…Although increased SR Ca 2+ leak may represent a sensitizing mechanism, it is almost certain that multiple mechanisms are at work in MH susceptibility. In this regard, any mechanism that causes sustained, partial SR Ca 2+ store depletion would presumably result in a sustained increase in storeoperated Ca 2+ entry (34,35) and thus increased resting myoplasmic Ca 2+ . Simply raising myoplasmic Ca 2+ would be expected to increase the efficacy of other activators (e.g., caffeine and volatile anesthetics).…”

New factors contributing to dynamic calcium regulation in the skeletal muscle triad—a crowded place

“…Likewise, RyR1 is not essentially required for SOCE, as SOCE was still present in dyspedic myotubes (Cherednichenko et al 2004), although some reduction in Mn 2+ quench rates in dyspedic myotubes (Lyfenko and Dirksen 2008) and in myotubes from mice lacking RyR1/RyR3 or the synaptophysin-familyrelated protein mitsugumin-29 in the triad junction suggests a modulation of SOCE by the release channels (Pan et al 2002;Zhao et al 2006). Other myotube studies have established a link between mitochondrial depolarization and reduced SOCE (Vandebrouck et al 2006) or sarcolemmal TRPC1 channels anchored to the dystrophin-associated glycoprotein complex via α1-syntrophin with compromised SOCE in dystrophic myotubes where this molecular lattice would be disrupted (Vandebrouck et al 2007).…”

“…This has to be kept in mind, as most studies on SOCE detected by Mn 2+ quench did not keep divalent ion concentrations constant (Hopf et al 1996;Boittin et al 2006;Zhao et al 2006). A further artificial condition in the quest for the physiological relevance of SOCE is introduced by severe SR depletion using high-dose caffeine or SERCA pump blockers to chronically deplete the store in Ca 2+ -free external solution with subsequent recordings of force, intracellular Ca 2+ , or Ca 2+ entry (Zhao et al 2006;Boittin et al 2006;Kurebayashi and Ogawa 2001;Cherednichenko et al 2004).…”

“…SOCE in nonexcitable cells develops in tens of seconds after store depletion (Bennett et al 1998) and also takes similar times (∼100 ms) in chronically store-depleted intact adult muscle fibers (Gonzalez Narvaez and Castillo 2007) or <1 min following halothane-induced Ca 2+ waves in mechanically skinned adult rat muscle fibers (Duke and Steele 2006). Myotubes even showed SOCE dynamics in the ∼10 min range (Zhao et al 2006). Launikonis and Rios (2007), however, speculated that SOCE in adult skeletal muscle should be much faster given the highly organized structure of the tubules and the SR, being determined for fast signaling.…”

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

“…The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca 2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules 9,11,12 . …”