瞬时受体电位经典型 6 通道的分子修饰调节与恶性高热相关的小鼠模型中的钙动态失衡。
Molecular Modification of Transient Receptor Potential Canonical 6 Channels Modulates Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia.
出版信息
Anesthesiology. 2021 Feb 1;134(2):234-247. doi: 10.1097/ALN.0000000000003635.
BACKGROUND
Pharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia.
METHODS
This study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots.
RESULTS
RYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death.
CONCLUSIONS
These data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia-triggering agents.
背景
药理学调节先前表明，瞬时受体电位经典型（TRPC）通道在恶性高热的发病机制中起重要作用。本研究通过测试在与恶性高热相关的小鼠模型中，通过遗传抑制 TRPC6 的功能是否可以部分改善肌肉阳离子动态平衡和对氟烷的反应的假设。
方法
本研究使用钙和钠离子选择性微电极和 Western blot 检测在 20 个 RYR1-p.R163C 和 20 个野生型小鼠以及相同数量的非表达对照中转基因过表达肌肉特异性非传导性显性负 TRPC6 通道的效果。
结果
与野生型肌肉相比，RYR1-p.R163C 小鼠肌肉中的细胞内钙和钠离子水平长期升高。非传导性 TRPC6 通道的转基因表达将细胞内钙从 331 ± 34 nM（平均值 ± SD）降低至 190 ± 27 nM（P < 0.0001），并将钠离子从 15 ± 1 mM 降低至 11 ± 1 mM（P < 0.0001）。其表达将 TRPC6 特异性激活剂贯叶连翘素在 RYR1-p.R163C 肌纤维中引起的细胞内 Ca2+增加从 52%（348 ± 37 nM 至 537 ± 70 nM）降低至 14%（185 ± 11 nM 至 210 ± 44 nM）。TRPC3 和 TRPC6 表达的 Western blot 分析显示，过表达其显性负转基因引起的 TRPC6 预期增加，以及 TRPC3 表达的代偿性增加。尽管肌肉特异性显性负 TRPC6 的表达能够调节氟烷暴露期间和延长的生命过程中的细胞内钙增加（35 ± 5 min 与 15 ± 3 min；P < 0.0001），但氟烷暴露 20 分钟后，钙开始缓慢，稳定增加，最终导致死亡。
结论
这些数据支持先前的发现，即 TRPC 通道在引起与 RYR1 变体相关的细胞内钙和钠离子动态平衡紊乱方面起重要作用，而这些变体对恶性高热具有致病性。然而，它们还表明，单独调节 TRPC 通道不足以防止暴露于挥发性麻醉剂恶性高热触发剂的致死作用。
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