Molecular Modification of Transient Receptor Potential Canonical 6 Channels Modulates Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia

Abstract

Background: Pharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia.

Methods: This study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots.

Results: RYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death.

Conclusions: These data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia-triggering agents.