Functional analysis of RYR1 variants linked to malignant hyperthermia

Abstract

Malignant hyperthermia manifests as a rapid and sustained rise in temperature in response to pharmacological triggering agents, e.g. inhalational anesthetics and the muscle relaxant suxamethonium. Other clinical signs include an increase in end-tidal CO₂, increased O₂ consumption, as well as tachycardia, and if untreated a malignant hyperthermia episode can result in death. The metabolic changes are caused by dysregulation of skeletal muscle Ca²⁺ homeostasis, resulting from a defective ryanodine receptor Ca²⁺ channel, which resides in the sarcoplasmic reticulum and controls the flux of Ca²⁺ ions from intracellular stores to the cytoplasm. Most genetic variants associated with susceptibility to malignant hyperthermia occur in the RYR1 gene encoding the ryanodine receptor type 1. While malignant hyperthermia susceptibility can be diagnosed by in vitro contracture testing of skeletal muscle biopsy tissue, it is advantageous to use DNA testing. Currently only 35 of over 400 potential variants in RYR1 have been classed as functionally causative of malignant hyperthermia and thus can be used for DNA diagnostic tests. Here we describe functional analysis of 2 RYR1 variants (c. 7042_7044delCAG, p.ΔGlu2348 and c.641C>T, p.Thr214Met) that occur in the same malignant hyperthermia susceptible family. The p.Glu2348 deletion, causes hypersensitivity to ryanodine receptor agonists using in vitro analysis of cloned human RYR1 cDNA expressed in HEK293T cells, while the Thr214Met substitution, does not appear to significantly alter sensitivity to agonist in the same system. We suggest that the c. 7042_7044delCAG, p.ΔGlu2348 RYR1 variant could be added to the list of diagnostic mutations for susceptibility to malignant hyperthermia.

Keywords: anesthesia; calcium channel; malignant hyperthermia; ryanodine receptor; skeletal muscle.

Figure 1.

Pedigree diagram pedigree diagram showing inheritance of RyR1 variants from each parent and MH status where known.

Figure 2.

Immunoblot showing expression of RyR1 constructs Immunoblot of HEK293T cells transiently transfected with RYR1 expression plasmids. Total protein (270 µg) extracts were separated on a 7.5% SDS-PAGE gel and transferred onto a PVDF membrane. RyR1, > 250 kDa, and α-tubulin, 50 kDa, were detected using anti-RyR1 (34C) and anti-α-tubulin antibodies respectively. Lane 1, pcDNA3.1+ vector only; lane 2, pcRyR1 wildtype; lane 3, pcRyR1 Gly248Arg; lane 4, pcRyR1 Arg2452Trp; lane 5, pcRyR1 Thr214Met; lane 6, pcRyR1 Thr214Met/ΔGlu2348; lane 7, pcRyR1 ΔGlu2348.

Figure 3.

Co-localization of RyR1 with the endoplasmic reticulum. Immunofluorescence of transiently transfected HEK293T cells with RyR1 cDNA. Variants are indicated by their amino acid change. Primary antibodies that specifically recognize RyR1 (34C) and PDI as well as fluorescently-labeled secondary antibodies FITC (green) and TRITC (red) were used to visualize RyR1 and PDI respectively, while nuclei were visualised by staining the cells with DAPI (blue). Cells were examined by confocal fluorescence microscopy at a magnification of 1260 X; the scale bar in each merged image represents a length of 20 microns.

Figure 4.

Ca⁺ release assays in HEK293T cells. Ca²⁺-release is illustrated in concentration-response curves for transiently transfected HEK293T cells. Ca²⁺ released was measured for 4-CmC concentrations between 0 and 1000 μM, values were normalized to Ca²⁺ released at 1000 μM 4-CmC and represented as mean ± SEM (n ≥ 8). Curves were plotted using OriginLab Origin 8 software.