CACNA2D4, a regulatory subunit of voltage-gated calcium channels, involved in exertional heat strokeective of effort-cost decision-making

Exertional Heat Stroke (EHS) is a life-threatening disease defined by a severe hyperthermia, with a body temperature above 40°C, associated with a sudden neurological dysfunction, observed in healthy individuals exposed to intensive physical exercise. EHS shares many clinical and biological similarities with Malignant Hyperthermia (MH), a pharmaco-genetic disease triggered by volatile halogenated agents mostly associated with mutations in the RYR1 gene, and more rarely in the CACNA1S and STAC3 genes. Several evidence support the existence of inherited genetic mutations that predispose some patients to EHS, but to date, the genetic basis of EHS remains poorly documented. Mutations in RYR1 have been associated with EHS with a much lower prevalence than in MH, which suggests the involvement of other genes. We report here the genetic study of a cohort of soldiers presenting with an EHS episode in whom mutations in RYR1 were previously excluded. Whole Exome Sequencing analysis led to the identification of variants in the CACNA2D4 gene in four unrelated patients. CACNA2D4 encodes the α2δ-4 isoform of the α2δ class of regulatory subunit of voltage-gated channels of the Cav family. This work aimed to investigate the implication of CACNA2D4 in the onset of exertional heat stroke with functional characterization going from in vitro to in vivo studies.  We show here that a CACNA2D4 transcript is expressed in mouse skeletal muscle. In vitro studies demonstrate that the four genetic variants  of CACNA2D4 found in EHS patients modify the functionality of the protein altering its membrane localization. The p.(Ser299Arg) variant localized in the MIDAS domain of α2δ-4 impacts the direct interaction of the protein with the α1 subunit of DHPR in a reconstituted system, as well as its channel properties. The mouse model with a knock-in for the p.(Ser299Arg)  (KI-S306R) mutation is viable and fertile. When submitted to intense exercise protocol, a significant increase in blood CPK levels, reflecting enhanced rhabdomyolysis, was observed in KI-S306R animals compared to Wild Type littermates In addition, female KI-S306R mice experienced EHS-like crisis associated with a significant elevation in core body temperature compared to Wild Type. We observed also an alteration in muscular calcium homeostasis in KI-S306 mice myotubes. Altogether, our data point to the CACNA2D4 gene as probably involved in skeletal muscle function and as a major gene mutated in EHS.