Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia

Abstract

Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca(2+) from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1(R163C) knock-in mice that increased passive SR Ca(2+) leak causes an enlarged basal influx of sarcolemmal Ca(2+) that results in chronically elevated myoplasmic free Ca(2+) concentration ([Ca(2+)]i) at rest. We discovered that Gd(+3) and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1(E190Q) in reducing both Ca(2+) entry and [Ca(2+)]i, implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca(2+)]i. Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na]i is chronically elevated in MH-RyR1(R163C) muscle cells. [Ca(2+)]i and [Na(+)]i are persistently elevated in vivo and further increased by halothane in MH-RyR1(R163C/WT) muscle. These increases are markedly attenuated by local perfusion of Gd(+3) or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca(2+) and Na(+) overload both at rest and during the MH crisis.-Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

“…Perturbation of any of these mechanisms can change the resting intracellular Ca 2ϩ regulation in muscle cells. The present results confirm not only that [Ca 2ϩ ] r is elevated in MHS compared with MHN muscle fibers (19,21,22), but also that [Na ϩ ] r is also higher in MHS than MHN (19 ] r is primarily regulated by the Na ϩ ,K ϩ -ATPase (2), the NCX, and the amiloride-sensitive Na ϩ /H ϩ exchanger (40). An elevated [Na ϩ ] r would shift the balance of fluxes through NCX to favor more Ca 2ϩ influx resulting in an elevation of [Ca 2ϩ ] r .…”

“…The fact that Na ϩ ,K ϩ -ATPase expression levels were similar in vastus lateralis from MHS patients who underwent a diagnostic contracture test for susceptibility to malignant hyperthermia (41) and that the values of the V m in MHN and MHS muscle are similar, suggests that observed dysfunction in [Na ϩ ] r would most probably be associated with an increase in Na ϩ influx rather than a decrease in efflux. In addition, the fact that elevation of [Na ϩ ] r in murine MHS muscle cells could be attenuated by local application of agents that block Orai1 and nonselective cation entry through transient receptor potential channels 3/6 (TRPC3/6) suggests that the elevation is mediated by an increase in influx of Na ϩ and Ca 2ϩ through these channels (19). This potential mechanism is supported by a number of electrophysiological studies that demonstrate that TRPCs allow permeation of Na ϩ as well as Ca 2ϩ (42,43 (44,45).…”

“…The institutional animal care committees at the Instituto Venezolano de Investigaciones Científicas, the Harvard Medical Area, and the University of California, Davis, approved all experimental protocols. Ca 2ϩ and Na ϩ Selective Microelectrodes-Double-barreled Ca 2ϩ selective and Na ϩ selective microelectrodes were prepared as described previously (19). The calcium ionophore II, ETH-129 (Fluka, Sigma), and the Na ϩ ionophore I, ETH-227 (Fluka, Sigma), were used to back-fill the Ca 2ϩ and Na ϩ -selective microelectrodes, respectively.…”

“…After making measurements of [Ca 2ϩ ] r and [Na ϩ ] r all microelectrodes were recalibrated, and if the two calibration curves did not agree within 3 mV, data from that microelectrode were discarded. The drugs used in the present study did not interfere with Ca 2ϩ microelectrode sensitivity in the relevant ranges studied (from pCa6 to pCa7 and from 1 to 30 mM Na ϩ ] r were measured in vivo in MHN and MHS mice as described previously (19). In brief, mice were anesthetized with 100 mg/kg of ketamine and 5 mg/kg of xylazine, intubated, and ventilated with air using a mouse ventilator (Harvard Minivent, M-845, Holliston, MA).…”

“…MH susceptibility is associated with a dysfunction of regulation of intracellular resting Ca 2ϩ concentration ([Ca 2ϩ ] r ) (19 -21) and is characterized by the occurrence of a robust increase in [Ca 2ϩ ] r in response to exposure to halothane (19,22,23). In more than 70% of humans with MH susceptibility (MHS) the trait is linked to one of Ͼ185 mutations within RYR1 located on ch19q13.1, the gene that encodes the type 1 skeletal isoform of the ryanodine receptor (RYR1), the primary Ca 2ϩ release channel in the sarcoplasmic reticulum (SR) (24).…”