CaMKII and IP3-Mediated Signaling in Cardiac Myocytes

心肌细胞中的钙调节是兴奋-收缩耦合（ECC）的核心，也参与肥厚性核信号传导。两个重要且普遍存在的钙调节系统，钙钙调素依赖性蛋白激酶II （CaMKII）和肌醇（1,4,5）P3受体（InsPsR）存在于肌细胞中，并参与改变ECC、心律失常发生和核信号传导。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。参与调节肥厚（Hyp）和心力衰竭（HF）转录的钙依赖途径包括CaMKII，它可能通过激活II型组蛋白去乙酰化酶（HDAC）的核输出而起作用，否则会抑制转录。这里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传导（ECC和心律失常）方面发挥作用，以及InsPsR和CaMKII如何参与肥大和心衰的核信号传导（通过HDAC）。主要实验方法有离体成人心肌细胞共聚焦荧光成像（钙指示剂及其他荧光探针）和电压钳法。细胞

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.