CaMKII and IP3-Mediated Signaling in Cardiac Myocytes

描述(由申请人提供)： 心肌细胞的钙调节是兴奋-收缩偶联(ECC)的核心，也参与肥大的核信号。钙调素依赖的蛋白激酶II(CaMKII)和肌醇(1，4，5)P3受体(InsP3R)是心肌细胞中两个重要且普遍存在的钙调节系统，它们参与改变ECC、心律失常和核信号转导。然而，令人惊讶的是，人们对这些影响是如何发生的知之甚少。在肥厚(Hyp)和心力衰竭(HF)中参与转录调控的钙依赖途径包括CaMKII和CaN，这些途径可能通过改变转录的关键因子(NFAT和HDAC)的核转位发挥作用。我们的总体目标是更好地了解CaMKII和InsP3R在心肌细胞中如何在急性钙信号(ECC和心律失常的发生)以及在肥厚和心衰的核信号(通过NFAT和HDAC)中发挥作用。计划开展四个高度协同的多学科项目。项目I(BERS)从3个方面对CaMKII的细胞方面进行了研究：1)CaMKII对ECC的急性作用；2)钙依赖的核信号转导途径；3)Hyp和HF中CaMKII信号的改变(涉及ECC、心律失常和HDAC激活)。项目II(布拉特)集中在3个目标(均为qHyp和HF)中IP3Rs的细胞方面，涉及：1)IP3R介导的急性ECC效应，2)InsP3R在心律失常发生中的作用和CaMKII如何调节lnsP3R功能，以及3)钙编码和IP3R参与NFAT信号转导到细胞核。项目III(Miqnery)专注于1)核InsP3Rs面临的方向及其与CaMKII(&CaM和CaN)的物理相互作用，2)依赖CaMKII的InsP3R的磷酸化和功能调节，3)InsP3R-CaMKII相互作用的调控，4)InsP3R亚型在心房和心室肌细胞中的表达和定位(Hyp&HF)，5)产生新的荧光[InsP3]感受器(FIRS)。项目IV(Brown)侧重于在体内和生化水平上对CaMKII和InsP3R的调控，涉及：1)缺乏心脏LnsP3R2或CaMKIIlambda(主要肌细胞异构体)的基因敲除小鼠的Hyp&HF的发育；2)胞浆CaMKII与核CaMKII的差异激活，以及荧光CaMK活性传感器(CaMKAR)的开发；3)CaMKII异构体的差异靶向磷酸化；以及4)心脏InsP3的形成和CaMKII的调节。三个科学核心将支持这些目标。核心B(肌细胞和心力衰竭兔)将从小鼠和兔(包括Hyp小鼠和心力衰竭兔)分离心肌细胞。核心C(荧光成像)将为荧光成像提供仪器和专业知识。核心D(遗传小鼠和腺病毒)将开发独特的小鼠模型(如lnsPaR2-和CaMKIIlambda-KO)和腺病毒载体用于心肌细胞研究。拟议的工作汇集了具有高度互补性的专业知识和观点的经验丰富的调查人员，以高度互动的多学科方法解决这些问题。这些结果将大大加深我们对CaMKII和InsP3R在正常、Hyp和HF心肌细胞ECC、心律失常发生和核信号转导过程中的作用的理解。

DESCRIPTION (provided by applicant): Calcium regulation in cardiac myocytes is central to excitation-contraction coupling (ECC) and is also involved in hypertrophic nuclear signaling. Two important and ubiquitous Ca regulatory systems, Ca-calmodulin dependent protein kinase II (CaMKII) and inositol (1,4,5)P3 receptors (InsP3R) are present in myocytes, and have been implicated in altering ECC, arrhythmogenesis and nuclear signaling. However, surprisingly littlie is known about how these effects occur. Ca-dependent pathways implicated in regulating transcription in hypertrophy (Hyp) and heart failure (HF) include CaMKII & calcineurin (CaN) and these may function via nuclear translocation of key factors (NFAT & HDAC) which alter transcription. Overall goals here are to understand better how CaMKII and InsP3R function in cardiac myocytes with respect to acute Ca signaling (ECC & arrhythmogenesis) and in nuclear signaling (via NFAT & HDAC) in hypertrophy & HF. Four highly synergistic multidisciplinary projects are planned. Project I (Bers) focuses on cellular aspects of CaMKII in 3 aims concerning: 1) acute CaMKII effects on ECC, 2) Ca-dependent nuclear signaling via a proposed lnsP3R-CaMKII-HDAC pathway, & 3) altered CaMKII signaling in Hyp & HF (regarding ECC, arrhythmias & HDAC activation). Project II (Blatter) focuses on cellular aspects of IP3Rs in 3 aims (all qHyp & HF) concerning: 1) acute IP3R-mediated effects on ECC, 2) the role of InsP3R in arrhythmogenesis and how CaMKII modulates lnsP3R function, and 3) Ca coding and IP3R involvement in NFAT signaling to the nucleus. Project III (Miqnery) focuses on molecular characterization of 1) the direction that nuclear InsP3Rs face and their physical interactions with CaMKII (& CaM & CaN), 2) CaMKII-dependent phosphorylation of InsP3R and modulation of function, 3) manipulation of InsP3R-CaMKII interaction, 4) InsP3R isoform expression & localization in atrial & ventricular myocytes (Hyp & HF), 5) generating novel fluorescent [InsP3] sensors (FIREs). Project IV (Brown) focuses on CaMKII and InsP3R regulation at in vivo and biochemical levels concerning: 1) development of Hyp & HF in knockout mice lacking cardiac lnsP3R2 or CaMKIIlambda (the dominant myocyte isoforms), 2) differential activation of cytosolic vs. nuclear CaMKII, plus development of a fluorescent CaMK activity sensor (CaMKAR), 3) differential target phosphorylation by CaMKII isoforms, & 4) cardiac InsP3 formation and regulation by CaMKII. Three scientific cores will support these aims. Core B (Myocytes & HF Rabbits) will isolate myocytes from mice and rabbits (including Hyp mice and HF rabbits). Core C (Fluorescence Imaging) will provide instrumentation and expertise for fluorescent imaging. Core D (Genetic Mouse & Adenovirus) will develop unique mouse models (e.g. lnsPaR2- & CaMKIIlambda-KO) and adenoviral vectors for myocyte studies. The proposed work integrates experienced investigators with highly complementary expertise and perspective to tackle these questions in a highly interactive multidisciplinary approach. The results will greatly increase our understanding of the roles of CaMKII and InsP3R in cardiac myocytes during ECC, arrhythmogenesis and nuclear signaling in normal, Hyp and HF cardiac myocytes.