Project Summary / Abstract The Hippo-signaling pathway (HSP), a kinase cascade, inhibits nuclear localization and transcriptional activity of YAP, a transcription co-factor. In previous work, deletion of the HSP component Sav in mouse and pig cardiomyocytes (CMs) with established ischemic cardiomyopathy and heart failure (HF), results in reversal of HF and improved heart function due to increased nuclear YAP and activation of YAP target genes. These groundbreaking findings indicate that uncovering methods to modulate YAP activity is valuable to treat ischemic cardiomyopathy and HF, which is the goal of this research program. Importantly, YAP is primarily regulated by post-translational modifications (PTMs), which control YAP nuclear/cytoplasmic subcellular localization dynamics. HSP kinases inhibit YAP nuclear localization by phosphorylating conserved serine residues in YAP. When HSP activity is low, YAP enters the nucleus and complexes with Tead family transcription factors to activate genes involved in progression through multiple cell cycle phases. In addition to HSP-mediated phosphorylation, the recent discovery that a Lysine acetylation-deacetylation cycle controls YAP nuclear- cytoplasmic localization provides an opportunity to develop novel therapies for cardiomyopathy, which will be investigated in this research program. Protein acetylation-deacetylation is very sensitive to the metabolic state of the local cellular environment. During myocardial infarction (MI), metabolism quickly shifts from mitochondrial oxidative phosphorylation to glycolysis due to oxygen deficiency in CMs. Consequently, Lysine acetylation, one of the main PTMs closely associated with metabolism, is also altered. The notion that HF-altered myocardial metabolism contributes to disease pathogenesis and that regulating it may serve innovative therapeutic purposes underscores the importance of identifying the metabolic characteristics of HF patients. It is therefore critical to clarify how YAP responds to post-MI environmental factors or metabolic changes. The proposed studies are based on preliminary data revealing that following MI, YAP acetylation is required for YAP/cytoskeleton interactions, wherein YAP binds the non-muscle actin filaments. These data reveal an MI-induced metabolic shift pathway wherein acetylation promotes YAP sequestration with the stabilized microtubule network in the cytoplasm, which is detrimental to cardiac regeneration. The aims of these proposed studies are to investigate phosphorylated and acetylated YAP in the contexts of HF and low HSP activity using advanced genomics, gene therapies, and mouse HF models. These studies will reveal how metabolic shifts after MI regulate YAP activity via Lysine acetylation in CMs and how YAP regulates the CM cytoskeleton. Using clinically approved compounds that target metabolic substrates/intermediates, Lysine acetyltransferases, and Lysine deacetylases, these studies will guide the development of new st...