Calcineurin compartmentation and regulation of pathological cardiac remodeling

钙/钙调蛋白依赖性磷酸酶钙调神经磷酸酶(CaN，PP2B)是一种多效性信号转导酶 对心肌肥厚的调节具有重要意义。而抑制CaN则可减轻病理性心脏 CaN的重塑和治疗靶向是有问题的，因为临床上已经建立的CaN抑制剂是 免疫抑制剂和AS-CAN靶向可加重缺血/再灌注性心肌损伤。从理论上讲， 然而，如果介导病理性心肌肥厚的CaN信号通路能够被孤立地靶向，那么 可以建立新的治疗模式。CANA的两种异构体α和β以蛋白质的形式表达 在心脏中也是如此，然而CANAβ(Ppp3cb)的基因靶向足以钝化病理性肥厚 在老鼠身上。我们最近发现，CANAβ2是通过其独特的N末端多聚脯氨酸(PP)结构域被靶向的 到由支架蛋白CDC4相互作用蛋白4(CIP4，TRIP10)组织的肌细胞室。成象 利用福斯特共振能量转移(FRET)记者发现，CIP4结合的CanAβ2是由 G蛋白偶联受体信号转导，包括血管紧张素II、α和β肾上腺素能受体，但不是通过起搏 这会导致心肌细胞收缩。值得注意的是，CIP4基因打靶和腺相关病毒介导的PP- 结构域锚定中断抑制心脏重塑并改善心功能 超载。因此，CANAβPP依赖的锚定构成了一种用于规范CAN的新机制 功能，解释了CANAβ在心肌细胞中的具体作用，以及 重要的问题是为什么肥大的CaN信号在正常收缩的心肌细胞中不活跃。在这个项目中， 我们将测试这一新的假设，即在心肌细胞PP-结构域锚定和CIP4区隔 赋予CANAβ2选择性促进病理性心肌肥厚的作用，从而构成一种新的 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。这一目的将确定CIP4在心肌细胞中的定位，研究 不同的CIP4域与心肌细胞肥大的相关性，并探讨CIP4相关的CANAβ2 作用于局部或远端的CIP4复合体以促进肥大。具体目标2：钙离子在细胞内的调节 CIP4-CANAβ2隔室。钙离子信号隔间的功能独立性需要机制 用于局部钙离子的内流，以及用于隔离隔室以防止与其他 细胞功能。在这个目标中，我们将定义钙离子的来源，以及如何使用LIVE绝缘这个隔间 心肌细胞成像。具体目的3：PP锚定的CANAβ与缺血性心脏病。这一目标将考验 靶向PP锚定的CANAβ能否在不加重的情况下延缓心力衰竭的发展 心肌细胞在缺血性心脏病中的存活，这些目的将阐明PP结构域是如何介导锚定的 CANAβ在一个新的钙离子隔间内激活，提供了关于 磷酸酶信号转导和进一步的证据支持心力衰竭的新治疗方法。

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.