Lipid regulation of Cardiac Excitation-Contraction coupling

项目摘要 本申请中概述的实验提出了一种新的范式，即急性血管紧张素II(AngII)刺激 PIP2水解触发心脏CaV1.2通道内化，提供了一种快速调节细胞的手段 兴奋性和调节健康中的EC偶联。相反，我们认为，血浆中的持续缺陷 慢性Angii时膜CaV1.2的表达和PIP2的缺失可触发适应不良的代偿 交感反应在短期内改善心脏功能，但最终导致进行性， 病理性心脏重构、肥厚，以及潜在的致心律失常的钙信号失控。我们 提供令人信服的初步数据表明，PIP2在急性血管紧张素Ⅱ/AT1R/GQ下游的水解 激活，导致心脏CaV1.2通道的内吞。我们可以想象这种内吞作用的发生 在生理浓度的血管紧张素Ⅱ(100 NM)灌流时，活体心室肌细胞的动态变化。 初步结果表明，通道插入和移除之间的平衡发生了变化，从而使血管紧张素Ⅱ刺激 PM通道的去除，导致PM CaV1.2丰度降低约30%。我们观察到一个惊人的相似之处 %-在其他三种单独的实验方法中减少，在电生理学研究中发现ICA减少， 超分辨率成像中通道簇面积和表达减少，地表PM CaV1.2的损失 生物素化作用。我们分离出PIP2作为这一反应的关键执行者，不同于PKC和 花生四烯酸的产生伴随着AT1R的刺激，实验绕过了受体和 取而代之的是利用雷帕霉素刺激的二聚化系统将4‘，5’磷酸酶招募到膜上，并 耗尽PIP2。我们的结果支持PIP2在Cav1.2通道运输和表达中的一种新的机制作用 它可以根据生理信号级联反应进行调节，以在急性发作期间调节EC偶联 调节血压。我们进一步提出在AngiI/AT1R信号转导过程中PIP2的慢性耗竭 与心力衰竭相关的原因：(I)PM CaV1.2持续不稳定和长期表达缺陷； (Ii)促进心脏功能的代偿性交感反应，涉及激活PKA和CaMKII， 与AT1R直接刺激的CaMKII联合作用，增强CaV1.2和RyR2的磷酸化， 产生增强的Po和舒张期漏，刺激CaN/NFAT和肥大基因表达；(Iii) 增加IP3的产生，也刺激CaN/NFAT和肥大基因的表达，以及(Iv)细胞骨架 心肌保护性PI(3，4，5)P3耗尽和ROS诱导的微管增强所致的不稳定性 扰乱通道传递并促进生物力学不稳定、T-小管和二联体丢失的灾难。 我们建议在这里描述的两个具体目标中严格测试这些想法。

Project Summary Experiments outlined in this application, suggest a novel paradigm, that acute angiotensin II (AngII)-stimulated PIP2 hydrolysis triggers cardiac CaV1.2 channel internalization, providing a means to rapidly tune cellular excitability and modulate EC-coupling in health. In contrast, we propose that sustained deficits in plasma membrane CaV1.2 expression, and PIP2 depletion during chronic AngII can trigger a maladaptive compensatory sympathetic response that improves cardiac function in the short-term but ultimately leads to progressive, pathological cardiac remodeling, hypertrophy, and potentially arrhythmogenic Ca2+ signaling dysregulation. We provide compelling preliminary data indicating that PIP2 hydrolysis, downstream of acute AngII/AT1R/Gq activation, leads to endocytosis of cardiac CaV1.2 channels. We can visualize this endocytosis occurring dynamically in live ventricular myocytes upon perfusion with physiological concentrations of AngII (100 nM). Initial results indicate a shift in the balance between channel insertion and removal, such that AngII-stimulated removal of PM channels, leads to an ~30 % reduction in PM CaV1.2 abundance. We observe a strikingly similar %-reduction in three other separate experimental approaches, finding decreased ICa in electrophysiology studies, reduced channel cluster area and expression in super-resolution imaging, and a loss of PM CaV1.2 in surface biotinylation. We isolate PIP2 as the critical executor of this response, distinct from the activation of PKC and arachidonic acid production that accompanies AT1R stimulation with experiments that bypass the receptors and instead utilize a rapamycin-stimulated dimerization system to recruit a 4’,5’ phosphatase to the membrane and deplete PIP2. Our results support a novel mechanistic role of PIP2 on Cav1.2 channel trafficking and expression which can be tuned in response to physiological signaling cascades to modulate EC-coupling during acute regulation of blood pressure. We further propose that chronic depletion of PIP2 during AngII/AT1R signaling associated with heart failure causes: (i) sustained destabilization of PM CaV1.2 and long-lived expression deficits; (ii) a compensatory sympathetic response to boost cardiac function involving activation of PKA and CaMKII that acts in combination with direct AT1R-stimulated CaMKII to enhance CaV1.2 and RyR2 phosphorylation, producing enhanced Po and diastolic leak that stimulates CaN/NFAT and hypertrophic gene expression; (iii) enhanced IP3 production that also stimulates CaN/NFAT and hypertrophic gene expression, and (iv) cytoskeletal instability as a result of depletion of cardioprotective PI(3,4,5)P3 and enhanced ROS-induced microtubule catastrophe that disrupts channel delivery and promotes biomechanical instability, t-tubule and loss of dyads. We propose to rigorously test these ideas in two specific aims described herein.