CaMKII and IP3-Mediated Signaling in Cardiac Myocytes

心肌细胞中 CaMKII 和 IP3 介导的信号传导

• 批准号: 7139941
• 负责人: Donald M Bers
• 金额: $ 35.64万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7139941
• 关键词: acidosis; arrhythmia; biological signal transduction; calcium flux; calcium indicator; calmodulin dependent protein kinase; cardiac myocytes; cell nucleus; cytoplasm; endothelin; enzyme activity; enzyme mechanism; fluorescence resonance energy transfer; genetically modified animals; heart contraction; heart enlargement; heart failure; inositol phosphates; laboratory mouse; laboratory rabbit; molecular /cellular imaging; neuromuscular function; pathologic process; phosphorylation; sarcoplasmic reticulum; second messengers

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.

心肌细胞中的钙调节是兴奋-收缩耦合 (ECC) 的核心，也参与肥大的核信号传导。肌细胞中存在两个重要且普遍存在的 Ca 调节系统：Ca 钙调蛋白依赖性蛋白激酶 II (CaMKII) 和肌醇 (1,4,5)P3 受体 (InsPsR)，并且与改变 ECC、心律失常发生和核信号传导有关。然而，令人惊讶的是，人们对这些影响如何发生知之甚少。参与调节肥大 (Hyp) 和心力衰竭 (HF) 转录的 Ca 依赖性途径包括 CaMKII，它可能通过激活 II 型组蛋白脱乙酰酶 (HDAC) 的核输出发挥作用，否则会抑制转录。这里的总体目标是更好地了解 CaMKII 在急性 Ca 信号传导（ECC 和心律失常发生）方面如何在心肌细胞中发挥作用，以及 InsPsR 和 CaMKII 如何参与肥厚和心力衰竭的核信号传导（通过 HDAC）。主要实验方法包括共焦荧光成像（Ca 指示剂和其他荧光探针）和离体成体心肌细胞的电压钳。肌细胞 将从小鼠（包括 CaMKIIS 或 lnsPaR2 敲除 (KO) 小鼠和接受主动脉束带术的小鼠，Hyp）和兔子（包括我们充分表征的非缺血性心律失常性 HF 模型）中分离出来。该项目重点关注 CaMKII 的细胞方面的 3 个目标。目标 1 将解决 CaMKII 对 ECC 的急性影响，涉及 4 个特性（Ca 电流促进、改变 SR Ca 释放（在舒张期和 ECC 期间）、频率依赖性的松弛加速和持续酸中毒期间 SR 运输的恢复）。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。目标 2 将通过拟议的局部 InsPsR-CaM-CaMKIIHDAC 通路测试关于 Ca 依赖性核信号传导的新假设，该通路可能优先响应神经体液刺激（例如内皮素-1），而不是与 ECC 相关的 Ca 瞬变。将使用 [Ca] 的局部测量以及 HDAC-GFP 和荧光 CaM 的易位，以及药理学和分子解剖（例如 CaMKIISc- 和 lnsP3R2-KO 小鼠）。目标 3 将评估 Hyp 和 HF 中有关 ECC、心律失常和 HDAC 激活的 CaMKII 信号变化。 CaM、CaMKII 和 InsPsR 在改变中的表达和功能 将在 HF 兔（来自 Core B）和来自 Brown 项目的 Hyp 小鼠中评估通过上述 HDAC 途径的 ECC、心律失常发生和核/肥大信号传导。用于 [InsPs] 和 CaMKII 活性的新型双 GFP FRET 传感器也将纳入这些研究，因为它们是在 Mignery 和 Brown 的项目中开发的。拟议的工作将与其他三个项目紧密结合，并将提供有关 CaMKII 和 InsPaR 在 ECC 期间心肌细胞中的作用、正常、Hyp 和 HF 心肌细胞中的心律失常和核信号传导的全面新信息。