Molecular Signaling in Cardiac Sarcomeres

我们的长期目标项目1是了解心脏肌节水平的信号如何协调能量供应和能量消耗。我们的目的是验证一个假设，即在心肌蛋白水平上的修饰是心衰过程中信号收敛的重要位点。我们的初步数据表明了一种复杂的耦合，其中包括由改变的肌体功能诱导的信号通路的促进，并通过肌体蛋白的相互翻译后修饰协调控制能量供应和能量消耗。实验包括对新发现的调查，包括：i)在有氧条件下，与家族性肥厚性心肌病（FHC）相关的表达突变肌钙蛋白i （cTnl）的心脏中AMP活化激酶（AMPK）的差异激活，ii) AMPK磷酸化cTnl；iii)鉴定与PKCe激活和扩张型心肌病相关的新的cTnl磷酸化位点，iii)预测代谢驱动的鞘脂信号到肌节的数据，iv)功能显著的cTnl分子内相互作用的证据。比较AMPK （AMP活化蛋白激酶）在正常心脏和应激心脏中作为协调能量供应和能量消耗的信号机制的作用，通过表达肌合成蛋白诱导钙敏感性和FHC的增加。目标# 2。确定表达PKCe并表现为扩张型心肌病的小鼠心脏表型与肌肉磷酸化的时间相关性，以及是否通过缺乏独特n端的非磷酸化突变体Tnl的表达改变了表型。目标# 3。来确定函数

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.