Human heart failure, a leading cause of death worldwide, is a prominent example of a chronic disease that may result from poor cell renewal. The Hippo signaling pathway is an inhibitory kinase cascade that represses adult heart muscle cell (cardiomyocyte) proliferation and renewal after myocardial infarction in genetically modified mice. Here, we investigated an adeno-associated virus 9 (AAV9)–based gene therapy to locally knock down the Hippo pathway gene Salvador (Sav) in border zone cardiomyocytes in a pig model of ischemia/reperfusion-induced myocardial infarction. Two weeks after myocardial infarction, when pigs had left ventricular systolic dysfunction, we administered AAV9-Sav–short hairpin RNA (shRNA) or a control AAV9 viral vector carrying green fluorescent protein (GFP) directly into border zone cardiomyocytes via catheter-mediated subendocardial injection. Three months after injection, pig hearts treated with a high dose of AAV9-Sav-shRNA exhibited a 14.3% improvement in ejection fraction (a measure of left ventricular systolic function), evidence of cardiomyocyte division, and reduced scar sizes compared to pigs receiving AAV9-GFP. AAV9-Sav-shRNA–treated pig hearts also displayed increased capillary density and reduced cardiomyocyte ploidy. AAV9-Sav-shRNA gene therapy was well tolerated and did not induce mortality. In addition, liver and lung pathology revealed no tumor formation. Local delivery of AAV9-Sav-shRNA gene therapy to border zone cardiomyocytes in pig hearts after myocardial infarction resulted in tissue renewal and improved function and may have utility in treating heart failure.

Rationale: During neonatal heart regeneration, the fibrotic response, which is required to prevent cardiac rupture, resolves via poorly understood mechanisms. Deletion of the Hippo pathway gene Sav in adult cardiomyocytes increases Yap activity and promotes cardiac regeneration, partly by inducing fibrosis resolution. Deletion of Yap in neonatal cardiomyocytes leads to increased fibrosis and loss of neonatal heart regeneration, suggesting that Yap inhibits fibrosis by regulating intercellular signaling from cardiomyocytes to cardiac fibroblasts (CFs). Objective: We investigated the role of Wntless ( Wls ), which is a direct target gene of Yap, in communication between cardiomyocytes and CFs during neonatal heart regeneration. Methods and Results: We generated 2 mouse models to delete Wls specifically in cardiomyocytes ( Myh6-Cas9 combined with AAV9-Wls-g RNAs, and Myh6 ^cre-ERT2/+ ; Wls ^flox/flox ). Reanalysis of single-cell RNA sequencing data revealed that Wnt ligands are expressed in cardiomyocytes, whereas Wnt receptors are expressed in CFs, suggesting that Wnt signaling is directional from cardiomyocytes to CFs during neonatal heart regeneration. Wls deletion in neonatal cardiomyocytes disrupted Wnt signaling, revealed by reduced noncanonical Wnt signaling in non-cardiomyocytes. Four weeks after neonatal heart myocardial infarction, heart function was measured by echocardiography. Wls deletion in neonatal cardiomyocytes after myocardial infarction impairs neonatal heart regeneration, marked by decreased contractile function and increased fibrosis. Wls mutant hearts display CF activation, characterized by increased extracellular matrix secretion, inflammation, and CF proliferation. Conclusions: These data indicate that during neonatal heart regeneration, intercellular signaling from cardiomyocytes to CFs occurs via noncanonical Wnt signaling to rebuild cardiac architecture after myocardial infarction.

BACKGROUND: The Hippo pathway effector YAP (Yes-associated protein) plays an essential role in cardiomyocyte proliferation and heart regeneration. In response to physiological changes, YAP moves in and out of the nucleus. The pathophysiological mechanisms regulating YAP subcellular localization after myocardial infarction remain poorly defined. METHODS: We identified YAP acetylation at site K265 by in vitro acetylation followed by mass spectrometry analysis. We used adeno-associated virus to express YAP-containing mutations that either abolished acetylation (YAP-K265R) or mimicked acetylation (YAP-K265Q) and studied how acetylation regulates YAP subcellular localization in mouse hearts. We generated a cell line with YAP-K265R mutation and investigated the protein-protein interactors by YAP immunoprecipitation followed by mass spectrometry, then validated the YAP interaction in neonatal rat ventricular myocytes. We examined colocalization of YAP and TUBA4A (tubulin α 4A) by superresolution imaging. Furthermore, we developed YAP-K265R and αMHC-MerCreMer (MCM); Yap-loxP/K265R mutant mice to examine the pathophysiological role of YAP acetylation in cardiomyocytes during cardiac regeneration. RESULTS: We found that YAP is acetylated at K265 by CBP (CREB-binding protein)/P300 (E1A-binding protein P300) and is deacetylated by nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide/sirtuins axis in cardiomyocytes. After myocardial infarction, YAP acetylation is increased, which promotes YAP cytoplasmic localization. Compared with controls, mice that were genetically engineered to express a K265R mutation that prevents YAP K265 acetylation showed improved cardiac regenerative ability and increased YAP nuclear localization. Mechanistically, YAP acetylation facilitates its interaction with TUBA4A, a component of the microtubule network that sequesters acetylated YAP in the cytoplasm. After myocardial infarction, the microtubule network increased in cardiomyocytes, resulting in the accumulation of YAP in the cytoplasm. CONCLUSIONS: After myocardial infarction, decreased sirtuin activity enriches YAP acetylation at K265. The growing TUBA4A network sequesters acetylated YAP within the cytoplasm, which is detrimental to cardiac regeneration.