1. T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function.
- Author
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Yang, Lukun, Dedkova, Elena N, Allen, Paul D, Jafri, M Saleet, and Fomina, Alla F
- Subjects
T-Lymphocytes ,Intracellular Space ,Mitochondria ,Animals ,Mice ,Malignant Hyperthermia ,Calcium ,Thapsigargin ,Lymphocyte Activation ,Cell Proliferation ,Calcium Signaling ,Genotype ,Mutant Proteins ,Membrane Potential ,Mitochondrial ,Machine Learning ,Dantrolene sodium ,Intracellular Ca(2+) ,Mitochondrial potential ,RYR1-p.R163C knock-in mice ,Ryanodine receptor ,T lymphocytes ,1.1 Normal biological development and functioning ,Underpinning research ,2.1 Biological and endogenous factors ,Aetiology ,Intracellular Ca2+ ,Biochemistry and Cell Biology ,Physiology ,Medical Physiology ,Biochemistry & Molecular Biology - Abstract
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 (ΔΨm) 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 ΔΨm 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 ΔΨm in WT T cells, it produced ΔΨm 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 ΔΨm 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.
- Published
- 2021