Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation
Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation
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DOI：
10.1007/s00424-020-02507-w
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发表时间：
2021-01-04
影响因子：
4.5
通讯作者：
Inoue, Ryuji
中科院分区：
文献类型：
--
作者：
Hu, Yaopeng;Kaschitza, Daniela Ross;Inoue, Ryuji
TRPM 4是一种Ca 2+激活的非选择性阳离子通道，参与心血管生理和病理生理。基于细胞实验和数值模拟，本研究旨在探讨CaMK II介导的TRPM 4通道过度激活与心脏Ca 2+失调的潜在促心律失常作用。共聚焦免疫荧光显微镜，蛋白质印迹和邻近连接试验（PLA）在HL-1心房心肌细胞和/或TRPM 4表达TSA 201细胞表明，TRPM 4和CaMK II蛋白是密切定位。TRPM 4和CaMKII delta或基于FRET的传感器Camui在HEK 293细胞中的共表达表明TRPM 4通道激活的程度与CaMKII活性的程度相关，表明它们的功能相互作用。应用膜片钳技术的电流钳模式记录HL-1细胞动作电位（AP）的复极相，发现血管紧张素II可诱导早期后去极化（埃兹）。在用TRPM 4通道阻断剂9-菲咯或CaMKII抑制剂KN-62处理后，这种代谢变化消失。为了定量评估CaMK II如何调节TRPM 4通道的门控行为，采用离子霉素透化的细胞贴附记录来获得电压依赖性参数，例如稳态开放概率和在不同[Ca 2 +]（i）下激活/失活的时间常数。将这些动力学数据合并到修改的HL-1模型中的数值模拟表明，TRPM 4电流密度的> 3倍增加在复极晚期诱导埃兹，并且CaMKII抑制（通过KN-62）完全消除它们。这些结果共同表明，一种新的致瘤机制，涉及过度的CaMK II活性，导致TRPM 4过度激活的压力心脏。
TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKII delta or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+](i). Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.