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T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function
Cell Calcium ( IF 4.3 ) Pub Date : 2020-12-01 , DOI: 10.1016/j.ceca.2020.102325
Lukun Yang 1 , Elena N Dedkova 2 , Paul D Allen 3 , M Saleet Jafri 4 , Alla F Fomina 5
Affiliation  

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.



中文翻译:

来自恶性高热易感小鼠的 T 淋巴细胞在细胞内钙信号传导和线粒体功能中表现出异常

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。

更新日期:2020-12-09
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