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 CaMKIIδ 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.

TRPM4 是一种 Ca ²⁺激活的非选择性阳离子通道，参与心血管生理学和病理生理学。基于细胞实验和数值模拟，本研究旨在探索 CaMKII 介导的 TRPM4 通道过度激活与心脏Ca ²⁺失调相关的潜在致心律失常性。HL-1 心房心肌细胞和/或表达 TRPM4 的 TSA201 细胞中的共聚焦免疫荧光显微镜、蛋白质印迹和邻近结扎试验 (PLA) 表明 TRPM4 和 CaMKII 蛋白紧密定位。TRPM4 和 CaMKIIδ 的共表达或基于 FRET 的传感器Camui在 HEK293 细胞中显示 TRPM4 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。血管紧张素 II 处理大大增强了这两种蛋白质的表达和相互作用，这在通过膜片钳技术的电流钳模式从 HL-1 细胞记录的动作电位 (AP) 的复极阶段诱导了早期后去极化 (EAD)。在用 TRPM4 通道阻滞剂 9-phenanthrol 或 CaMKII 抑制剂 KN-62 治疗后，这种心律失常变化消失了。为了定量评估 CaMKII 如何调节 TRPM4 通道的门控行为，²⁺ ]_我。将这些动力学数据纳入修改后的 HL-1 模型的数值模拟表明，TRPM4 电流密度增加 3 倍以上会在复极后期诱导 EAD，并且 CaMKII 抑制（通过 KN-62）完全消除了它们。这些结果共同表明了一种新的致心律失常机制，涉及过度的 CaMKII 活性，导致应激心脏中的 TRPM4 过度激活。