Heart failure due to cardiomyocyte loss after ischemic heart disease is the leading cause of death in the United States in large part because heart muscle regenerates poorly. The endogenous mechanisms preventing mammalian cardiomyocyte regeneration are poorly understood. Hippo signaling, an ancient organ size control pathway, is a kinase cascade that inhibits developing cardiomyocyte proliferation but it has not been studied postnatally or in fully mature adult cardiomyocytes. Here, we investigated Hippo signaling in adult cardiomyocyte renewal and regeneration. We found that unstressed Hippo-deficient adult mouse cardiomyocytes re-enter the cell cycle and undergo cytokinesis. Moreover, Hippo deficiency enhances cardiomyocyte regeneration with functional recovery after adult myocardial infarction as well as after postnatal day eight (P8) cardiac apex resection and P8 myocardial infarction. In damaged hearts, Hippo mutant cardiomyocytes also have elevated proliferation. Our findings reveal that Hippo signaling is an endogenous repressor of adult cardiomyocyte renewal and regeneration. Targeting the Hippo pathway in human disease might be beneficial for the treatment of heart disease.

Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure (HF) commonly results in mortality1. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration2, is upregulated in human HF. We show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischemic HF after myocardial infarction (MI) induced a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared to controls. Using TRAP (translating ribosomal affinity purification), we isolated cardiomyocyte specific translating mRNA. Hippo deficient cardiomyocytes had increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control (MQC) gene, Park2. Genetic studies indicated that Park2 was essential for heart repair suggesting a requirement for MQC in regenerating myocardium. Gene therapy with a virus encoding Salv shRNA improved heart function when delivered at the time of infarct or after ischemic HF post-MI was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

Summary Myocardial infarction results in compromised myocardial function with heart failure due to insufficient cardiomyocyte self-renewal1. Unlike lower vertebrates, mammalian hearts only have a transient neonatal renewal capacity2. Reactivating primitive reparative ability in the mature heart requires knowledge of the mechanisms promoting early heart repair. By testing an established Hippo-deficient heart regeneration model for renewal promoting factors, we found that Pitx2 expression was induced in injured, Hippo-deficient ventricles. Pitx2-deficient neonatal hearts failed to repair after apex resection while Pitx2-gain-of-function in adult cardiomyocytes conferred reparative ability after myocardial infarction. Genomic analyses indicated that Pitx2 activated genes encoding electron transport chain components and reactive oxygen species scavengers. A subset of Pitx2 target genes was cooperatively regulated with the Hippo effector, Yap. Furthermore, Nrf2, a regulator of antioxidant response3, directly regulated Pitx2 expression and subcellular localization. Pitx2 mutant myocardium had elevated reactive oxygen species levels while antioxidant supplementation suppressed the Pitx2-loss-of-function phenotype. These findings reveal a genetic pathway, activated by tissue damage that is essential for cardiac repair.

Liver X receptors (LXRK K and LXRL L) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol e¥ux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR) 3=3 mice. T-0901317 signi¢cantly reduced the atherosclerotic lesions in LDLR 3=3 mice without a¡ecting plasma total cholesterol levels. This anti-atherogenic e¡ect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR 3=3 mice as well as in mouse peritoneal macrophages. T-0901317 also signi¢cantly induced cholesterol e¥ux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.

Summary The regenerative capacity of the adult mammalian heart is limited because of the reduced ability of cardiomyocytes (CMs) to progress through mitosis1. The regenerative capacity of endogenous CMs exists at birth but is lost postnatally, with subsequent organ growth occurring through CM hypertrophy2,3. The Hippo pathway, a conserved kinase cascade, inhibits CM proliferation in the developing heart to control heart size and in the adult heart to prevent regeneration4,5. The dystrophin glycoprotein complex (DGC), a multicomponent transmembrane complex linking the actin cytoskeleton to extracellular matrix, is essential for CM homeostasis. DGC deficiency in humans results in muscular dystrophy, including lethal Duchenne muscular dystrophy (DMD). We found that the DGC component dystroglycan 1 (DAG1) directly binds to Hippo pathway effector Yap to inhibit CM proliferation. The Yap-DAG1 interaction was enhanced by Hippo-induced Yap phosphorylation, revealing a connection between Hippo pathway function and the DGC. After injury, Hippo-deficient postnatal hearts maintained organ size control by repairing the defect with correct dimensions, whereas postnatal hearts doubly deficient for Hippo and the DGC showed CM overproliferation at the injury site. In mature Mdx mouse hearts—a model of DMD—Hippo deficiency protected against overload-induced heart failure.