Lipid regulation of Cardiac Excitation-Contraction coupling

项目摘要 在本申请中概述的实验提出了一种新的范例，急性血管紧张素II（AngII）刺激 PIP 2水解触发心脏CaV1.2通道内化，提供了一种快速调节细胞凋亡的方法。 兴奋性和调节健康的EC耦合。相反，我们认为，血浆中的持续缺陷 膜CaV1.2表达和慢性AngII期间PIP 2耗竭可触发适应不良的代偿性 交感神经反应，在短期内改善心脏功能，但最终导致进行性， 病理性心脏重塑、肥大和潜在的促炎性Ca 2+信号传导失调。我们 提供了令人信服的初步数据，表明急性AngII/AT 1 R/Gq下游的PIP 2水解 激活，导致心脏CaV1.2通道的内吞作用。我们可以看到这种内吞作用 在用生理浓度的AngII（100 nM）灌注后，在活心室肌细胞中动态地表达。 最初的结果表明，在通道插入和移除之间的平衡发生了变化，使得AngII刺激的血管内皮生长因子表达增加。 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 在其他三种单独的实验方法中，发现伊卡降低，在电生理学研究中， 在超分辨率成像中减少的通道簇面积和表达，以及在表面中PM CaV1.2的损失 生物素化我们分离出PIP 2作为这种反应的关键执行者，与PKC的激活不同， 伴随AT 1 R刺激的花生四烯酸产生，实验中绕过受体， 而是利用雷帕霉素刺激的二聚化系统将4 ′，5 ′磷酸酶募集到膜上， 耗尽PIP 2。我们的结果支持PIP 2对Cav1.2通道运输和表达的一种新的机制作用 其可以响应于生理信号级联而被调节，以在急性炎症期间调节EC偶联。 调节血压。我们进一步提出血管紧张素Ⅱ/血管紧张素Ⅱ受体1受体信号转导过程中PIP 2的慢性消耗， 与心力衰竭原因相关：（i）PM CaV1.2的持续不稳定和长期表达缺陷; (ii)增强心脏功能的代偿性交感神经反应，涉及PKA和CaMKII的激活， 与直接AT 1 R刺激的CaMKII联合作用以增强CaV1.2和RyR 2磷酸化， 产生刺激CaN/NFAT和肥大基因表达的增强的Po和舒张渗漏;（iii） 增强的IP 3产生，也刺激CaN/NFAT和肥大基因表达，和（iv）细胞骨架 由于心脏保护性PI（3，4，5）P3耗竭和ROS诱导微管增强而导致的不稳定性 这是一种破坏通道递送并促进生物力学不稳定性、T-小管和二联体损失的灾难。 我们建议在本文所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.