CaMKII and IP3-Mediated Signaling in Cardiac Myocytes

描述（由申请人提供）： 心肌细胞中的钙调节是兴奋-收缩偶联（ECC）的核心，也参与肥大核信号传导。钙调素依赖性蛋白激酶II（CaMKII）和肌醇（1，4，5）P3受体（InsP 3R）是两种重要的钙调节系统，它们参与了细胞外钙通道（ECC）、细胞核信号转导和细胞内钙离子生成。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。参与调节肥大（Hyp）和心力衰竭（HF）中的转录的Ca依赖性途径包括CaMK II和钙调神经磷酸酶（CaN），并且这些可以通过改变转录的关键因子（NFAT和HDAC）的核转位来起作用。本文的总体目标是更好地了解CaMKII和InsP 3R如何在心肌细胞中就急性Ca信号传导（ECC和心肌生成）和核信号传导（通过NFAT和HDAC）在肥大和HF中起作用。计划开展四个高度协同的多学科项目。项目I（Bers）专注于CaMKII的细胞方面，涉及3个目标：1）急性CaMKII对ECC的影响，2）通过提出的InsP 3R-CaMKII-HDAC途径的Ca依赖性核信号传导，以及3）Hyp和HF中改变的CaMKII信号传导（关于ECC，心律失常和HDAC激活）。项目II（Blatter）在3个目标（所有qHyp和HF）中关注IP 3R的细胞方面，涉及：1）对ECC的急性IP 3R介导的作用，2）InsP 3R在胚胎发生中的作用以及CaMKII如何调节InsP 3R功能，以及3）Ca编码和IP 3R参与NFAT向细胞核的信号传导。项目III（Miqnery）侧重于1）核InsP 3R面临的方向及其与CaMKII的物理相互作用的分子表征（& CaM & CaN），2）InsP 3R的CaMK II依赖性磷酸化和功能调节，3）InsP 3R-CaMK II相互作用的操纵，4）InsP 3R同种型在心房和心室肌细胞中的表达和定位（Hyp & HF），5）产生新型荧光[InsP 3]传感器（FIRE）。项目IV（Brown）专注于CaMKII和InsP 3R在体内和生化水平的调节，涉及：1）在缺乏心脏InsP 3R 2或CaMK II λ的敲除小鼠中Hyp和HF的发展（优势肌细胞同种型），2）胞质相对于核CaMK II的差异活化，加上荧光CaMK活性传感器（CaMKAR）的开发，3）CaMK II同种型的差异靶磷酸化，（4）心肌InsP 3的形成和CaMK Ⅱ的调节。三个科学核心将支持这些目标。核心B（肌细胞和HF兔）将从小鼠和兔（包括Hyp小鼠和HF兔）中分离肌细胞。核心C（荧光成像）将提供荧光成像的仪器和专业知识。核心D（基因小鼠和腺病毒）将开发独特的小鼠模型（例如lnsPaR 2-和CaMKII基因敲除）和用于肌细胞研究的腺病毒载体。拟议的工作将经验丰富的调查人员与高度互补的专业知识和观点相结合，以高度互动的多学科方法解决这些问题。这些结果将大大增加我们对CaMK Ⅱ和InsP 3R在正常、Hyp和HF心肌细胞ECC、心肌细胞凋亡和核信号传导中的作用的理解。

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.