T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and...

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca²⁺ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca²⁺ concentration ([Ca²⁺]_i), Ca²⁺ release from the store, store-operated Ca²⁺ entry (SOCE), and mitochondrial inner membrane potential (ΔΨ_m) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca²⁺]_i and ΔΨ_m dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca²⁺]_i, diminished responses to Ca²⁺ mobilization with thapsigargin, and decreased rate of [Ca²⁺]_i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca²⁺]_i and ability of thapsigargin to mobilize Ca²⁺ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨ_m in WT T cells, it produced ΔΨ_m hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca²⁺ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca²⁺ level to the amplitude of thapsigargin-induced Ca²⁺ transient and an integral of changes in ΔΨ_m in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca²⁺ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca²⁺ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.