Molecular Signaling in Cardiac Sarcomeres

我们的长期目标项目1是了解心肌肌节水平的信号如何协调能量供应和能量消耗。我们的目的是验证这一假说，即在肌瘤蛋白水平上的修饰在心力衰竭的进展过程中是信号汇聚的重要部位。我们的初步数据表明，这是一种复杂的偶联，包括促进由肌瘤功能改变诱导的信号通路，并通过肌瘤蛋白的瑞丙醇翻译后修饰来协调控制能量供应和能量消耗。这些实验包括对新发现的研究，包括：1)在有氧条件下，表达与家族性肥厚型心肌病(FHC)相关的突变型肌钙蛋白I(CTn1)的心脏中AMPK的差异激活，2)AMPK对cTn1的磷酸化；Iii)鉴定与PKCE激活和扩张型心肌病相关的新的cTn1磷酸化位点，iii)预测代谢驱动的鞘脂信号转导到肌节的数据，以及iv)cTn1分子内相互作用的证据。具体目的是：目的1.比较AMPK(AMP激活的蛋白激酶)作为协调正常心脏和心脏能量供应和能量消耗的信号机制的作用，肌节蛋白的表达导致钙敏感性和FHC增加。目的#2.确定表达PKCE并表现为扩张型心肌病的小鼠的心脏表型与肌节磷酸化之间的时间相关性，以及缺乏唯一N末端的非磷酸化突变体TnL的表达是否改变了表型。目标#3.确定功能 CTn1区域与自身以及与其他细丝蛋白位点相互作用的意义，在协调能量需求和供应方面具有潜在的重要意义，并被AMP激酶(AMPK)、蛋白激酶D(PKD)和PKCE修饰。达到AIMS的方法包括在原位跳动的心脏、分离的心肌细胞和皮肤纤维的水平上的研究，重点是动力学和肌丝钙敏感性和肌节亚蛋白质组的评估。该项目与其他三个项目的目标密切互动，并与之相辅相成。这三个核心都强烈支持这一项目。由拟议的实验产生的数据将 开辟了一条新的研究途径，将代谢信号与肌节的相互信号联系起来，并提供了在开发对心力衰竭重要的新诊断和治疗策略方面具有重要意义的分子机制。

Our long term objective Project 1 is to understand how signals at the level of the cardiac sarcomere serve to coordinate energy supply and energy consumption. Our aims test the hypothesis that modifications at the level of the sarcomeric proteins serve as significant sites of signal convergence in the progression to heart failure. Our preliminary data indicate a complex coupling, which involves promotion of signaling pathways induced by altered sarcomeric function and that coordinately control energy supply and energy consumption through reciprocol post-translational modifications of sarcomeric proteins. The experiments include investigation of novel findings including: i) differential activation of AMP activated kinase (AMPK) in aerobic conditions in hearts expressing mutant troponin I (cTnl) linked to familial hypertrophic cardiomyopathy (FHC), ii) phosphorylation of cTnl by AMPK; iii) identification of novel cTnl sites of phosphorylation associated with PKCe activation and dilated cardiomyopathy, iii) data predicting metabolically driven sphingolipid signaling to the sarcomeres, and iv) evidence for functionally significant cTnl intra-molecular interactions The specific aims are: Aim #1. To compare the role of AMPK (AMP activated protein kinase) as a signaling mechanism coordinating energy supply and energy consumption in normal hearts and hearts stressed by expression of sarcomeric proteins inducing increases in Ca-sensitivity and FHC. Aim #2. To determine the temporal association of the cardiac phenotype of mice expressing PKCe and demonstrating dilated cardiomyopathy with sarcomeric phosphorylation and whether the phenotype is altered by expression of a non-phosphorylatable mutant Tnl lacking the unique N-terminus. Aim #3. To determine the functional significance of interactions of regions of cTnl with itself and with other thin filament protein sites, potentially significant in coordinating energy demand and supply and modified by AMP kinase (AMPK), protein kinase D (PKD), and PKCe. Approaches to the aims includes studies at the level of the in situ beating heart, isolated myocytes, and skinned fibers with focus on dynamics and evaluation of myofilament Ca-sensitivity and the sarcomere sub-proteome. This project interacts closely with and complements the aims of the other three projects. All three cores strongly support this project. Data generated by the experiments proposed will open a new avenue of research linking metabolic signaling with reciprocal signaling to the sarcomeres, and provide molecular mechanisms of significance in the development of novel diagnostic and therapeutic strategies important in heart failure.