Lipid regulation of Cardiac Excitation-Contraction coupling

心脏兴奋-收缩耦合的脂质调节

• 批准号: 10451117
• 负责人: Rose Ellen Dixon
• 金额: $ 60.23万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10451117
• 关键词: Actins; Acute; Address; Adult; Angiotensin II; Arachidonic Acids; Architecture; Area; Biochemistry; Biological Assay; Biomechanics; Biotinylation; Bypass; Calcium; Cardiac; Cardiac Output; Cell Death; Cell membrane; Cell surface; Cells; Chronic; Consensus; Coupled; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Dimerization; Electrophysiology (science); Electrostatics; Endocytosis; Equilibrium; Excision; F-Actin; Functional disorder; Gene Expression; Goals; Health; Heart failure; Hydrolysis; Hypertension; Hypertrophy; Image; Infusion procedures; Ion Channel; Knowledge; Ligation; Link; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Microtubules; Modeling; Mus; Muscle Cells; Myocardial; Nanostructures; Nocodazole; Optics; Output; PTEN gene; Pathologic; Perfusion; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Play; Production; Regulation; Resolution; Role; RyR2; Sarcolemma; Signal Pathway; Signal Transduction; Sirolimus; Stress; Structure; Surface; System; Techniques; Testing; Vasoconstrictor Agents; Ventricular; actin capping protein; blood pressure control; blood pressure regulation; calmodulin-dependent protein kinase II; cardioprotection; experimental study; heart function; imaging approach; improved; in silico; innovation; latrunculin A; novel; patch clamp; prevent; receptor; recruit; response; theories; tool; trafficking

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.

项目摘要 在此应用中概述的实验表明了一个新的范式，即急性血管紧张素II（ANGII）刺激 PIP2水解触发心脏CAV1.2通道内在化，提供了一种快速调整细胞的手段 兴奋性和调节健康的EC耦合。相比之下，我们建议持续的等离子体定义 慢性血管中的膜CAV1.2表达和PIP2部署可以触发适应不良的补偿性 同情反应在短期内改善心脏功能，但最终导致渐进性， 病理心脏重塑，肥大和潜在的心律不齐CA2+信号失调。我们 提供引人注目的初步数据，表明PIP2水解，急性Angii/AT1R/GQ的下游 激活，导致心脏CAV1.2通道的内吞作用。我们可以可视化这种内吞作用 灌注后，在活心肌细胞中动态动态，其物理浓度的ANGII（100 nm）。 初始结果表明通道插入和去除之间的平衡发生了变化，因此Angii刺激了 去除PM通道，导致PM CAV1.2抽象降低约30％。我们观察到非常相似的 ％ - 在其他三种单独的实验方法中还原，在电生理研究中发现ICA的改善， 降低了通道簇面积和超分辨率成像中的表达，表面损失了PM CAV1.2 生物素化。我们将PIP2分离为此响应的关键执行者，与PKC和PKC的激活不同。 用绕过接收器和 取而代 DEPLETE PIP2。我们的结果支持PIP2在CAV1.2通道运输和表达中的新机械作用 可以根据物理信号级联反应调节，以调节急性期间的EC偶联 血压调节。我们进一步建议，在Angii/AT1R信号传导期间，PIP2的慢性耗竭 与心力衰竭的原因相关：（i）PM CAV1.2的持续不稳定和长期表达定义； （ii）对增强心脏功能的补偿性交感反应，涉及PKA和CAMKII的激活 与直接AT1R刺激的CAMKII结合起作用，以增强CAV1.2和RYR2磷酸化， 产生增强的PO和舒张期泄漏，从而刺激CAN/NFAT和肥厚基因表达； （iii） 增强的IP3产生也可以刺激CAN/NFAT和肥厚基因表达，并且（iv）细胞骨架 由于心脏保护PI（3,4,5）P3的耗竭并增强了ROS诱导的微管，因此不稳定 破坏通道传递并促进生物力学不稳定性，T型管和二元组损失的灾难。 我们建议在此处描述的两个具体目标中严格测试这些想法。