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.

兰尼碱受体 1 (RyR1)中的功能获得性RyR1 -p.R163C 突变使骨骼肌中的 Ca ²⁺信号传导和线粒体功能失调，并在触发条件下导致人和小鼠发生恶性高热。我们研究了来自杂合RyR1 -p.R163C 敲入突变小鼠（HET T 细胞）的 T 淋巴细胞在静息细胞溶质 Ca ²⁺浓度（[Ca ²⁺ ] _i）、Ca ²⁺从储存中释放、储存中是否表现出可测量的异常-操作的Ca ²⁺进入 (SOCE) 和线粒体内膜电位 (ΔΨ _m) 与来自野生型小鼠的 T 淋巴细胞 (WT T 细胞) 进行比较。我们探讨了这些变量是否可用于区分具有正常和改变的 RyR1 基因型的 T 细胞。

从脾脏和淋巴结中分离出 HET 和 WT T 细胞，并使用植物血凝素 P在体外激活。分别使用 Fura 2 和四甲基罗丹明甲酯荧光染料检查[Ca ²⁺ ] _i和 ΔΨ _{m动力学。}与 WT T 细胞相比，活化的 HET T 细胞表现出升高的静息 [Ca ²⁺ ] _i，减少了对毒胡萝卜素对 Ca ²⁺动员的反应，并且降低了响应 SOCE 的 [Ca ²⁺ ] _{i升高率。}^{用兰尼定或丹曲林钠预处理 HET T 细胞可减少静息 [Ca 2+} ] _i的差异以及毒胡萝卜素在 HET 和 WT T 细胞之间动员 Ca ^{2+的能力。}虽然 SOCE 引起WT T 细胞中ΔΨ _{​​m的消散，但它在 HET T 细胞中产生 ΔΨ m}超极化。当用作分类变量时，毒胡萝卜素诱导的 Ca ²⁺瞬变的幅度在预测RyR1 -p.R163C 突变的存在方面表现出最好的前景。通过机器学习分析确定的其他重要变量是静息细胞溶质 Ca ²⁺水平与毒胡萝卜素诱导的 Ca ²⁺瞬态幅度的比率以及响应 SOCE的 ΔΨ _{m变化的积分。}

我们的研究表明，RyR1 的功能获得性突变显着影响 T 淋巴细胞中的 Ca ²⁺信号传导和线粒体虚构，这表明这种突变可能导致其载体的免疫反应发生改变。我们的数据将导致遗传性骨骼肌疾病的RyR1 -p.R163C 突变与免疫 T 细胞中 Ca ²⁺信号传导和线粒体功能的失调联系起来，并为基于 T 细胞的遗传性体外诊断测定建立了原理证明RyR1 功能亢进。