Newly Published
Perioperative Medicine  |   June 2020
Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia
Author Notes
  • From the Department of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis, Davis, California (J.R.L., P.D.A.); the Department of Research (J.R.L.) and the Division of Neonatology (A.U., J.A.), Mount Sinai Medical Center, Miami, Florida; and the Malignant Hyperthermia Investigation Unit, St. James’ University Hospital, Leeds, United Kingdom (V.K., P.H., X.L., P.D.A.).
  • Part of the work presented in this article has been presented as a poster at the 63rd Annual Meeting of the Biophysical Society in Baltimore, Maryland, March 2–6, 2019.
    Part of the work presented in this article has been presented as a poster at the 63rd Annual Meeting of the Biophysical Society in Baltimore, Maryland, March 2–6, 2019.×
  • J.R.L. and V.K. contributed equally to this article.
    J.R.L. and V.K. contributed equally to this article.×
  • Submitted for publication December 26, 2019. Accepted for publication April 28, 2020.
    Submitted for publication December 26, 2019. Accepted for publication April 28, 2020.×
  • Correspondence: Address correspondence to Dr. Allen: Malignant Hyperthermia Investigation Unit, St. James’ University Hospital, Leeds, United Kingdom. p.d.allen@leeds.ac.uk. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Patient Safety / Pharmacology
Perioperative Medicine   |   June 2020
Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia
Anesthesiology Newly Published on June 5, 2020. doi:https://doi.org/10.1097/ALN.0000000000003387
Anesthesiology Newly Published on June 5, 2020. doi:https://doi.org/10.1097/ALN.0000000000003387
Abstract

Background: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca2+ release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca2+ dyshomeostasis in a mouse model relevant to malignant hyperthermia.

Methods: This study examined the mechanisms responsible for Ca2+ dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots.

Results: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca2+ and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene–dose–dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx.

Conclusions: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia–susceptible muscle, both at rest and during the malignant hyperthermia crisis.

Editor’s Perspective:

What We Already Know about This Topic:

  • The type 1 ryanodine receptor gene (RYR1) encoding the skeletal muscle sarcoplasmic reticulum Ca2+ release channel is the primary locus for malignant hyperthermia

  • Muscle expressing malignant hyperthermia-RYR1 mutations has chronically elevated intracellular resting calcium and sodium concentrations that increase several-fold on exposure to halothane or isoflurane

What This Article Tells Us That Is New:

  • The hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia susceptible muscle was tested in a RYR1-p.G2435R knock-in murine model of malignant hyperthermia

  • Skeletal muscle of mice expressing RYR1-p.G2435R had a significantly enhanced, extracellular Ca2+-dependent response to TRPC3/6 channel activators

  • The TRPC3/6 channel activator response could be prevented by TRPC3/6 channel blockers

  • Local administration of TRPC channel blockers during an active malignant hyperthermia crisis demonstrated that most of the rise in intracellular resting calcium in skeletal muscle comes from the extracellular space and not sarcoplasmic reticulum stores