CaMKII and IP3-Mediated Signaling in Cardiac Myocytes

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(InsPsR)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通路包括CaMKII，它可能通过激活II型组蛋白去乙酰基酶(HDAC)的核输出来发挥作用，否则会抑制转录。我们的总体目标是更好地了解CaMKII在心肌细胞中如何在急性钙信号转导(ECC和心律失常发生)中发挥作用，以及InsPsR和CaMKII如何参与肥厚和心衰的核信号转导(通过HDAC)。主要的实验方法有钙指示剂和其他荧光探针的共聚焦荧光成像和电压钳技术。肌细胞 将从小鼠(包括CaMKIIS或lnsPaR2基因敲除(KO)小鼠和受到主动脉缩窄的小鼠，Hyp)和兔(包括我们特征良好的非缺血性心律失常致心力衰竭模型)分离。这个项目集中在3个目标中的CaMKII的细胞方面。目的1将从4个方面阐述CaMKII对ECC的急性影响：钙电流易化、舒张期和ECC期间SR钙释放的改变、持续酸中毒期间SR运输的频率依赖性加速和恢复。这些将在CaMKIIS-KO小鼠和CaMKII抑制肽专门针对SR的小鼠中进行评估。目的2通过局部InsPsR-CaM-CaMKIIHDAC通路验证钙依赖核信号的新假说，该通路可能优先对神经体液刺激(如内皮素-1)作出反应，而不是与ECC相关的钙瞬变。将使用局部测量[Ca]和HDAC-GFP和荧光CaM的移位，以及药理学和分子解剖(例如，CaMKIISc-和lnsP3R2-KO小鼠)。AIM 3将评估Hyp和HF中有关ECC、心律失常和HDAC激活的CaMKII信号的变化。CaM、CaMKII和InsPsR在血管内皮细胞病变中的表达及作用 Brown将通过上述HDAC途径在HF兔(来自Core B)和Hyp小鼠中评估ECC、心律失常的发生和核/肥大信号。当Mignery和Brown在项目中开发时，用于[Insps]和CaMKII活性的新型双GFP FRET传感器也将被纳入这些研究。这项拟议的工作将与其他三个项目密切结合，并将提供关于CaMKII和InsPaR在ECC、心律失常发生和正常、Hyp和HF心肌细胞的核信号转导中在心肌细胞中的作用的全面新信息。

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 (InsPsR) are present in myocytes, and have been implicated in altering ECC, arrhythmogenesis and nuclear signaling. However, surprisingly little is known about how these effects occur. Ca-dependent pathways implicated in regulating transcription in hypertrophy (Hyp) and heart failure (HF) include CaMKII, which may function via activation of nuclear export of type II histone deacetylases (HDAC) which otherwise repress transcription. Overall goals here are to understand better how CaMKII functions in cardiac myocytes with respect to acute Ca signaling (ECC & arrhythmogenesis) and how both InsPsR and CaMKII may be involved in nuclear signaling (via HDAC) in hypertrophy & HF. Main experimental methods include confocal fluorescence imaging (of Ca indicators & other fluorescent probes) and voltage clamp in isolated adult cardiac myocytes. Myocytes will be isolated from mice (including CaMKIIS or lnsPaR2 knockout (KO) mice and mice subjected to aortic banding, Hyp) and rabbits (including our well characterized non-ischemic arrhythmogenic HF model). This project focuses on cellular aspects of CaMKII in 3 aims. Aim 1 will address acute CaMKII effects on ECC, with respect to 4 properties (Ca current facilitation, altered SR Ca release (during diastole and ECC), frequency-dependent acceleration of relaxation & recovery of SR transport during sustained acidosis). These will be assessed in CaMKIIS-KO mice and those in which a CaMKII inhibitory peptide is targeted specifically to the SR. Aim 2 will test a novel hypothesis about Ca-dependent nuclear signaling via a proposed local InsPsR-CaM-CaMKIIHDAC pathway that may respond preferentially to neurohumoral stimuli (e.g. endothelin-1) rather than the Ca transients associated with ECC. Local measurements of [Ca] and translocation of HDAC-GFP and fluorescent CaM will be used, along with pharmacological and molecular dissection (e.g. CaMKIISc- and lnsP3R2-KO mice). Aim 3 will assess altered CaMKII signaling in Hyp & HF regarding ECC, arrhythmias & HDAC activation. The expression and function of CaM, CaMKII & InsPsR in the alteration of ECC, arrhythmogenesis and nuclear/ hypertrophic signaling via the above HDAC pathway will be assessed in both HF rabbits (from Core B) and Hyp mice from Project by Brown. The novel dual GFP FRET sensors for [InsPs] and CaMKII activity will also be incorporated into these studies as they are developed in Projects by Mignery and Brown. The proposed work will be integrated closely with the other three projects and will provide comprehensive new information regarding the roles of CaMKII and InsPaR in cardiac myocytes during ECC, arrhythmogenesis and nuclear signaling in normal, Hyp and HF cardiac myocytes.