Calcineurin compartmentation and regulation of pathological cardiac remodeling

钙调神经磷酸酶的划分和病理性心脏重塑的调节

• 批准号: 10594426
• 负责人: Michael Seth Kapiloff
• 金额: $ 52.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594426
• 关键词: Adrenergic Agents; Adult; Agonist; Allosteric Site; Angiotensin II; Attenuated; Binding; Ca(2+)-Transporting ATPase; Calcineurin; Calmodulin; Cardiac; Cardiac Myocytes; Cell membrane; Cell physiology; Cells; Clinical; Co-Immunoprecipitations; Complex; Contracts; Coronary artery; Coupling; Data; Dependovirus; Development; Diagnosis; Diglycerides; Distal; Elements; Enzymes; Exhibits; Family; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Gene Targeting; Genetic; Guanosine Triphosphate Phosphohydrolases; Heart; Heart Hypertrophy; Heart failure; Hypertrophy; Image; Immunosuppressive Agents; In Vitro; Ion Channel; Ischemia; Knockout Mice; Left; Ligation; Mediating; Membrane; Membrane Proteins; Modeling; Monomeric GTP-Binding Proteins; Mus; Muscle Cells; Myocardial Infarction; Myocardial Ischemia; N-terminal; NFAT Pathway; Operative Surgical Procedures; Pathologic; Phosphoric Monoester Hydrolases; Phosphoserine; Prevention; Protein Isoforms; Proteins; Public Health; Pump; RHO Effector Domain; RNA Interference; Rattus; Regimen; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporter; Role; Sarcomeres; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Source; Specific qualifier value; Specificity; Stains; Stress; Structure; Syndrome; TRIP10 gene; Testing; Therapeutic; Threonine; VIVIT peptide; Ventricular; Vesicle; beta-adrenergic receptor; calcineurin phosphatase; cdc42 GTP-Binding Protein; experimental study; functional independence; heart function; improved; inhibitor; insight; member; mortality; mutant; myocardial injury; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; polyproline; pressure; receptor; recruit; response; rho; sensor; theories; therapeutic target

The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN, PP2B) is a pleiotropic signaling enzyme important for the regulation of cardiac hypertrophy. While CaN inhibition will attenuate pathological cardiac remodeling, therapeutic targeting of CaN is problematic as clinically established CaN inhibitors are immunosuppressant and as CaN targeting can worsen myocardial injury due to ischemia/reperfusion. In theory, however, if CaN signaling pathways mediating pathological cardiac hypertrophy could be targeted in isolation, a new therapeutic paradigm could be established. Two isoforms of CaNA, α and β, are expressed as proteins equally in the heart, and yet genetic targeting of CaNAβ (PPP3cb) is sufficient to blunt pathological hypertrophy in mice. We recently discovered that CaNAβ2 is targeted through its unique N-terminal polyproline (PP) domain to a myocyte compartment organized by the scaffold protein Cdc4-Interacting Protein 4 (CIP4, TRIP10). Imaging using Forster Resonance Energy Transfer (FRET) reporters revealed that CIP4-bound CaNAβ2 is activated by G-protein coupled receptor signaling, including angiotensin II, α- and β-adrenergic receptors, but not by pacing that induces myocyte contraction. Notably, both CIP4 gene targeting and adeno-associated virus-mediated PP- domain anchoring disruption inhibited cardiac remodeling and improved cardiac function in response to pressure overload. Thus, CaNAβ PP-dependent anchoring constitutes a novel mechanism for specification of CaN function, providing an explanation for the specific role of CaNAβ in the cardiac myocyte, as well as for the important question why hypertrophic CaN signaling is not active in normal contracting myocytes. In this project, we will test the novel hypothesis that in the cardiac myocyte PP-domain anchoring and CIP4 compartmentation confer CaNAβ2 action selectively promoting pathological cardiac hypertrophy, thereby comprising a new therapeutic target for the prevention and/or treatment of heart failure. Specific Aim 1: Localization and function of the CIP4 signaling compartment. This Aim will define CIP4 localization in the myocyte, study the relevance of the various CIP4 domains for myocyte hypertrophy, and explore whether CIP4-associated CaNAβ2 acts locally or distally from CIP4 complexes to promote hypertrophy. Specific Aim 2: Regulation of Ca2+ in the CIP4-CaNAβ2 compartment. Functional independence of a Ca2+ signaling compartment requires mechanisms for both local influx of Ca2+ and for insulating the compartment against elevations in Ca2+ associated with other cellular functions. In this Aim, we will define the source of Ca2+ and how this compartment is insulated using live myocyte imaging. Specific Aim 3: PP-anchored CaNAβ and ischemic heart disease. This aim will test whether targeting of PP-anchored CaNAβ can attenuate the development of heart failure without worsening myocyte survival in ischemic heart disease, These Aims will elucidate how PP-domain-mediated anchoring CaNAβ confers activation within a novel Ca2+ compartment, providing both novel insights regarding specificity in phosphatase signal transduction and further evidence supporting a novel therapeutic approach for heart failure.

Ca 2 +/钙调素依赖性磷酸酶钙调神经磷酸酶（CaN，PP 2B）是一种多效性信号酶 对调节心脏肥大很重要。而抑制CaN则可减轻病理性心肌损伤 重塑，CaN的治疗靶向是有问题的，因为临床上建立的CaN抑制剂是 免疫抑制剂和CaN靶向可使由于缺血/再灌注引起的心肌损伤恶化。理论上， 然而，如果介导病理性心脏肥大的CaN信号通路可以单独靶向， 可以建立新的治疗模式。CaNA的两种亚型α和β以蛋白质形式表达， 在心脏中同样如此，但CaNAβ（PPP 3cb）的遗传靶向足以钝化病理性肥大 对小鼠我们最近发现，CaNAβ2通过其独特的N端聚脯氨酸（PP）结构域被靶向 到由支架蛋白Cdc 4-相互作用蛋白4（CIP 4，TRIP 10）组织的肌细胞区室。成像 使用Forster共振能量转移（FRET）报告显示，CIP 4结合的CaNAβ2被激活， G蛋白偶联受体信号传导，包括血管紧张素II、α和β肾上腺素能受体，但不通过起搏 引发肌细胞收缩值得注意的是，CIP 4基因靶向和腺相关病毒介导的PP- 结构域锚定破坏抑制心脏重塑并改善对压力的反应的心脏功能 超载。因此，CaNAβ PP依赖的锚定构成了一种新的CaN特化机制 功能，为CaNAβ在心肌细胞中的特定作用以及 为什么肥大的CaN信号在正常收缩的肌细胞中不活跃。在本项目中， 我们将测试新的假设，即在心肌细胞PP结构域锚定和CIP 4区室化中， 赋予CaNAβ2选择性促进病理性心脏肥大的作用，从而包含一种新的 预防和/或治疗心力衰竭的治疗靶点。具体目标1：本地化和 CIP 4信号室的功能。本研究的目的是明确CIP 4在心肌细胞中的定位，研究CIP 4在心肌细胞中的作用， 各种CIP 4结构域与肌细胞肥大的相关性，并探索CIP 4相关的CaNAβ2 从CIP 4复合物局部或远端起作用以促进肥大。具体目标2：调节细胞中的Ca 2 + CIP 4-CaNAβ2隔室。Ca 2+信号室的功能独立性需要机制 对于Ca 2+的局部流入和对于隔离隔室以对抗与其它离子相关的Ca 2+升高， 细胞功能。在这个目标中，我们将定义Ca 2+的来源，以及如何使用带电的绝缘层来绝缘这个隔室。 肌细胞成像具体目标3：PP锚定的CaNAβ与缺血性心脏病。这一目标将检验 靶向PP锚定的CaNAβ是否可以减轻心力衰竭的发展而不恶化 缺血性心脏病中心肌细胞的存活，这些目的将阐明PP结构域介导的锚定 CaNAβ在一个新的Ca 2+隔室中赋予激活作用，提供了关于CaNA β特异性的新见解， 磷酸酶信号转导和支持心力衰竭的新治疗方法的进一步证据。