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.

Ca2+/钙调蛋白依赖性磷酸酶钙调神经磷酸酶 (CaN, PP2B) 是一种多效性信号酶 对于心脏肥大的调节很重要。虽然 CaN 抑制会减弱病理性心脏病 CaN 的重塑和治疗靶向存在问题，因为临床上建立的 CaN 抑制剂 免疫抑制剂和 CaN 靶向药物可能会加重缺血/再灌注引起的心肌损伤。理论上， 然而，如果可以单独针对介导病理性心脏肥大的 CaN 信号通路， 可以建立新的治疗范式。 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 (PPP3cb) 的基因靶向足以减弱病理性肥大 在小鼠中。我们最近发现 CaNAβ2 通过其独特的 N 末端聚脯氨酸 (PP) 结构域进行靶向 到由支架蛋白 Cdc4 相互作用蛋白 4（CIP4、TRIP10）组织的肌细胞区室。影像学 使用福斯特共振能量转移 (FRET) 记者发现，CIP4 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 从而诱导心肌细胞收缩。值得注意的是，CIP4 基因靶向和腺相关病毒介导的 PP- 域锚定破坏抑制心脏重塑并改善心脏功能对压力的反应 超载。因此，CaNAβ PP 依赖性锚定构成了 CaN 规范的新机制 功能，解释了 CaNAβ 在心肌细胞中的具体作用，以及 一个重要的问题是，为什么肥大的 CaN 信号在正常收缩的肌细胞中不活跃。在这个项目中， 我们将测试心肌细胞中 PP 结构域锚定和 CIP4 区室化的新假设 赋予 CaNAβ2 选择性促进病理性心脏肥大的作用，从而包含一个新的 预防和/或治疗心力衰竭的治疗目标。具体目标 1：本地化和 CIP4信号室的功能。该目标将定义 CIP4 在肌细胞中的定位，研究 各个 CIP4 结构域与肌细胞肥大的相关性，并探讨 CIP4 相关的 CaNAβ2 是否 CIP4 复合物在局部或远端发挥作用，促进肥大。具体目标 2：Ca2+ 的调节 CIP4-CaNAβ2 隔室。 Ca2+信号传导室的功能独立性需要机制 用于 Ca2+ 的局部流入以及隔离隔室以防止与其他相关的 Ca2+ 升高 细胞功能。在此目标中，我们将定义 Ca2+ 的来源以及如何使用带电隔离该隔间。 肌细胞成像。具体目标 3：PP 锚定的 CaNAβ 与缺血性心脏病。这个目标将考验 靶向 PP 锚定的 CaNAβ 是否可以减轻心力衰竭的发展而不恶化 缺血性心脏病中的心肌细胞存活，这些目标将阐明 PP 结构域介导的锚定如何 CaNAβ 在新型 Ca2+ 区室中赋予激活作用，提供了有关特异性的新见解 磷酸酶信号转导和支持心力衰竭新治疗方法的进一步证据。