Molecular Signaling in Cardiac Sarcomeres

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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.