Altered [Ca2+] Regulation in Cortical Neurons of Mice Susceptible to Malignant Hyperthermia: An In Vivo Study

Malignant hyperthermia (MH) is a life-threatening hereditary pharmacogenetic syndrome that can occur when susceptible subjects are exposed to depolarizing muscle relaxants or halogenated inhalational anesthetic.1 The underlying molecular mechanism of MH is not fully understood; however, a common characteristic of MH muscle is an increase in resting intracellular [Ca2+].2–4 This disorder has been linked to mis-sense mutations in the type 1 ryanodine receptor (RyR1) and the voltage-gated Ca2+ channel subunit alpha-1S of the dihydropyridine receptor, both essential proteins involved in skeletal muscle functions.1Although MH is traditionally recognized as a pharmacogenetic syndrome of skeletal muscle, recent findings suggest that MH affects organs beyond the skeletal muscle, such as the central nervous system.5 Based on the presence of RyR1 in neurons,6 one of the primary loci for MH susceptibility, we measured intracellular Ca2+ or Na+ concentrations in vivo in cortical neurons and gastrocnemius muscle of (1) wild-type C57BL/6J mice and (2) C57BL/6J knock-in mice heterozygous for RyR1 variant that results in the amino acid change p.R163C in the RyR1 protein (MH-R163C).7 Determinations were carried out in anesthetized mice (37ºC): (1) before isoflurane inhalation, (2) after 1.5% isoflurane inhalation, (3) after 2.5 mg/kg intravenous dantrolene administration using the tail vein, and (4) after dantrolene pretreatment and 1.5% isoflurane inhalation. The criteria for successful intracellular ions measurements were consistent with previous reports.3,8 The mice were continuously monitored with pulse oximetry and rectal temperature. The study was conducted following the National Institutes of Health (Bethesda, Maryland) Guide for the Care and Use of Laboratory Animals guidelines and approved by the Institutional Animal Care and Use Committee of the Mount Sinai Research Institute (New York, New York).Simultaneous measurements of intracellular [Ca2+] or [Na+] in neurons and muscle fibers of MH-R163C mice revealed a significantly elevated baseline. Intracellular Ca2 + was 2.4 and 2.9 times higher in MH-R163C neurons and muscles, respectively, than in the wild type (fig. 1). Similarly, intracellular Na+ at baseline was 1.7 and 2 times higher in cortical neurons and skeletal muscle cells from MH-R163C compared to wild-type mice (fig. 1). These findings reveal, for the first time, an aberrant intracellular Ca2+ and Na+ in neuronal cells, mirroring the previously observed dysregulation in MH-R163C skeletal muscles.3,4 Moreover, our research has brought to light a remarkable discovery: exposure to 1.5% isoflurane results in a notable elevation in intracellular Ca2+ levels, reaching 3.1 times higher in neurons and 3.5 times higher in MH-R163C muscle. Furthermore, intracellular Na+ levels also exhibit a significant increase of 1.4 times in neurons and 1.6 times in muscle compared to the wild type (fig. 1). These alterations were accompanied by muscle contraction, decreased transcutaneous oxygen saturation, and increased rectal temperature. Conversely, no effects were observed in the wild-type group. The precise mechanisms underlying these changes in Ca2+ and Na+ in MH-R163C cells are not yet fully understood. However, previous studies have proposed that increased ryanodine Ca2+ leakage and enhanced Ca2+ and Na+ influx mediated by transient receptor potential channels may be the underlying mechanisms responsible for increased intracellular cations levels.3,9Intravenous administration of dantrolene (2.5 mg/kg), a known agent that reduced intracellular [Ca2+] in muscle cells and neurons,7,10 resulted in a notable reduction in baseline Ca2+ and Na+ levels in the neurons and muscles of both genotypes (fig. 1) and prevented intracellular Ca2+ elevations induced by isoflurane in MH mice. This finding leads us to hypothesize that, similar to MH skeletal muscle, neuronal RyR1 is involved in the observed disruption of Ca2+ homeostasis.Transient receptor potential canonical channels represent a family of cation channel proteins expressed in many cell types.11 We have previously demonstrated the contributions of transient receptor potential canonical channels 3 and 6 to elevated intracellular [Ca2+] in MH muscle at rest and during an episode of MH.3,9 Brain homogenates prepared from MH-R163C mice exhibited a marked upregulation in the expression levels of transient receptor potential canonical channels 3 (2.3-fold) and 6 (1.7-fold) when compared to the wild type (fig. 2). This upregulation of proteins is similar to what we had reported in skeletal muscle.3,9Although the current understanding of the pathophysiology of MH has been largely centered on skeletal muscle dysfunction, this study demonstrated that neurons appear to also be involved. Specifically, neurons in the MH cortex exhibit abnormal intracellular calcium and sodium concentrations and respond with a significant elevation of both ions after exposure to isoflurane, a known trigger anesthetic for MH. Furthermore, they show an upregulation of transient receptor potential canonical channels 3 and 6. These findings challenge the traditional characterization of MH as solely a skeletal muscle pharmacogenetic disorder and underscoring the extramuscular manifestations of this syndrome.This work was supported by the Florida Heart Research Foundation (Miami, Florida).The authors declare no competing interests.