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Congenital heart disease is one of the leading causes of pediatric morbidity and mortality, thus highlighting the importance of deciphering the molecular mechanisms that control heart development. As the terminal transcriptional effectors of the Hippo-YAP pathway, YAP and TEAD1 form a transcriptional complex that regulates the target gene expression and depletes either of these two genes in cardiomyocytes, thus resulting in cardiac hypoplasia. Vestigial-like 4 (VGLL4) is a transcriptional co-factor that interacts with TEAD and suppresses the YAP/TEAD complex by competing against YAP for TEAD binding. To understand the VGLL4 function in the heart, we generated two VGLL4 loss-of-function mouse lines: a germline Vgll4 depletion allele and a cardiomyocyte-specific Vgll4 depletion allele. The whole-body deletion of Vgll4 caused defective embryo development and perinatal lethality. The analysis of the embryos at day 16.5 revealed that Vgll4 knockout embryos had reduced body size, malformed tricuspid valves, and normal myocardium. Few whole-body Vgll4 knockout pups could survive up to 10 days, and none of them showed body weight gain. In contrast to the whole-body Vgll4 knockout mutants, cardiomyocyte-specific Vgll4 knockout mice had no noticeable heart growth defects and had normal heart function. In summary, our data suggest that VGLL4 is required for embryo development but dispensable for myocardial growth.

In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PRC2 writing of H3K27me3 is mechanistically required for gene silencing. Here, we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (embryonic ectoderm development) inactivation in the postnatal heart (Eed CKO ) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in Eed CKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized Eed CKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring.

Binding of the transcriptional co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP's mitogenic potency declines postnatally. While investigating factors that limit YAP's postnatal mitogenic activity, we found that the CM-enriched TEAD1 binding protein VGLL4 inhibits CM proliferation by inhibiting TEAD1-YAP interaction and by targeting TEAD1 for degradation. Importantly, VGLL4 acetylation at lysine 225 negatively regulated its binding to TEAD1. This developmentally regulated acetylation event critically governs postnatal heart growth, since overexpression of an acetylation-refractory VGLL4 mutant enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates TEAD stability and YAP-TEAD activity. These insights may improve targeted modulation of TEAD-YAP activity in applications from cardiac regeneration to cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair., (© 2016. Published by The Company of Biologists Ltd.)

GATA4 and GATA6 are central cardiac transcriptional regulators. The postnatal, stage-specific function of the cardiac transcription factors GATA4 and GATA6 have not been evaluated. In part, this is because current Cre-loxP approaches to cardiac gene inactivation require time consuming and costly breeding of Cre-expressing and "floxed" mouse lines, often with limited control of the extent or timing of gene inactivation. We investigated the stage-specific functions of GATA4 and GATA6 in the postnatal heart by using adeno-associated virus serotype 9 to control the timing and extent of gene inactivation by Cre. Systemic delivery of recombinant, adeno-associated virus 9 (AAV9) expressing Cre from the cardiac specific Tnnt2 promoter was well tolerated and selectively and efficiently recombined floxed target genes in cardiomyocytes. AAV9:Tnnt2-Cre efficiently inactivated Gata4 and Gata6. Neonatal Gata4/6 inactivation caused severe, rapidly lethal systolic heart failure. In contrast, Gata4/6 inactivation in adult heart caused only mild systolic dysfunction but severe diastolic dysfunction. Reducing the dose of AAV9:Tnnt2-Cre generated mosaics in which scattered cardiomyocytes lacked Gata4/6. This mosaic knockout revealed that Gata4/6 are required cell autonomously for physiological cardiomyocyte growth. Our results define novel roles of GATA4 and GATA6 in the neonatal and adult heart. Furthermore, our data demonstrate that evaluation of gene function hinges on controlling the timing and extent of gene inactivation. AAV9:Tnnt2-Cre is a powerful tool for controlling these parameters.

The adult mammalian heart exhibits limited regenerative capacity after myocardial injury, a shortcoming that is responsible for the current lack of definitive treatments for heart failure. A search for approaches that might enhance adult heart regeneration has led to interest in the Hippo/Yap signaling pathway, a recently discovered signaling pathway that regulates cell proliferation and organ growth. Here we provide a brief overview of the Hippo/Yap pathway and its known roles in the developing and adult heart. We discuss the implications of Hippo/Yap signaling for regulation of cardiomyocyte death and regeneration., (Copyright © 2014. Published by Elsevier B.V.)

Heart failure is a growing epidemic caused by cardiomyocyte depletion. Current therapies prolong survival by protecting remaining cardiomyocytes but are unable to overcome the fundamental problem of regenerating lost cardiomyocytes. Several strategies for promoting heart regeneration have emerged from decades of intensive study. Although some of these strategies remain confined to basic research, others are beginning to be tested in humans. We review strategies for cardiac regeneration and summarize progress of related clinical trials., (Copyright © 2014, American Association for the Advancement of Science.)

Rationale: Cardiomyocytes in adult mammalian hearts are terminally differentiated cells that have exited from the cell cycle and lost most of their proliferative capacity. Death of mature cardiomyocytes in pathological cardiac conditions and the lack of regeneration capacity of adult hearts are primary causes of heart failure and mortality. However, how cardiomyocyte proliferation in postnatal and adult hearts becomes suppressed remains largely unknown. The miR-17-92 cluster was initially identified as a human oncogene that promotes cell proliferation. However, its role in the heart remains unknown., Objective: To test the hypothesis that miR-17-92 participates in the regulation of cardiomyocyte proliferation in postnatal and adult hearts., Methods and Results: We deleted miR-17-92 cluster from embryonic and postnatal mouse hearts and demonstrated that miR-17-92 is required for cardiomyocyte proliferation in the heart. Transgenic overexpression of miR-17-92 in cardiomyocytes is sufficient to induce cardiomyocyte proliferation in embryonic, postnatal, and adult hearts. Moreover, overexpression of miR-17-92 in adult cardiomyocytes protects the heart from myocardial infarction-induced injury. Similarly, we found that members of miR-17-92 cluster, miR-19 in particular, are required for and sufficient to induce cardiomyocyte proliferation in vitro. We identified phosphatase and tensin homolog, a tumor suppressor, as an miR-17-92 target to mediate the function of miR-17-92 in cardiomyocyte proliferation., Conclusions: Our studies therefore identify miR-17-92 as a critical regulator of cardiomyocyte proliferation, and suggest this cluster of microRNAs could become therapeutic targets for cardiac repair and heart regeneration.

Heart growth is tightly controlled so that the heart reaches a predetermined size. Fetal heart growth occurs through cardiomyocyte proliferation, whereas postnatal heart growth involves primarily physiological cardiomyocyte hypertrophy. The Hippo kinase cascade is an important regulator of organ growth. A major target of this kinase cascade is YAP1, a transcriptional coactivator that is inactivated by Hippo kinase activity. Here, we used both genetic gain and loss of Yap1 function to investigate its role in regulating proliferative and physiologic hypertrophic heart growth. Fetal Yap1 inactivation caused marked, lethal myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation of YAP1 stimulated cardiomyocyte proliferation. Enhanced proliferation was particularly dramatic in trabecular cardiomyocytes that normally exit from the cell cycle. Remarkably, YAP1 activation was sufficient to stimulate proliferation of postnatal cardiomyocytes, both in culture and in the intact heart. A dominant negative peptide that blocked YAP1 binding to TEAD transcription factors inhibited YAP1 proliferative activity, indicating that this activity requires YAP1-TEAD interaction. Although Yap1 was a critical regulator of cardiomyocyte proliferation, it did not influence physiological hypertrophic growth of cardiomyocytes, because postnatal Yap1 gain or loss of function did not significantly alter cardiomyocyte size. These studies demonstrate that Yap1 is a crucial regulator of cardiomyocyte proliferation, cardiac morphogenesis, and myocardial trabeculation. Activation of Yap1 in postnatal cardiomyocytes may be a useful strategy to stimulate cardiomyocyte expansion in therapeutic myocardial regeneration.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes. [ABSTRACT FROM AUTHOR]

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinβ, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinβ in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinβ mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinβ knock-out mice—indicating a functional and genetic interaction between Xinβ and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinβ modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases. Intercalated discs ensure mechanical and electrochemical coupling during contraction of the heart. Here, the authors show that loss of Xinβ results in cardiomyocyte proliferation defects and cardiomyopathy by influencing the Hippo-YAP signalling pathway, thus affecting cardiac development and function. [ABSTRACT FROM AUTHOR]

Introduction: Shortly after acute myocardial infarction, innate immure response is initiated to recruit leukocytes into the injured myocardium. Toll-like receptors (TLRs), which recognize the death associated patterns (DAMPs) are crucial components of the innate immune system. Hippo-YAP signaling pathway is required for cardiac repair, however, whether YAP regulates cardiac repair by orchestrating innate immune response is unknown. Hypothesis: Transient activation of YAP with modified RNA is sufficient to reduce cardiomyocyte necrosis and cardiac inflammation after ischemia reperfusion stress Methods: In this study, we transiently activated YAP with active YAP modified RNA (aYAP modRNA) in the myocardium in a mouse ischemia reperfusion model. In short term studies, hearts were collected for histology and inflammation analysis one or two days after IR. In long term studies, mouse heart function was measured at three different time points. At the end of the long term study, hearts were collected for scar size measurement and histology analysis. We used cultured cardiomyocytes (CM) to study the molecular mechanism of YAP regulation of innate immune. Results: Two days after IR, aYAP modRNA treated mice had more CM proliferation, less cardiomyocytes (CMs) necrosis, less neutrophils infiltration and lower serum troponin T level. One month after IR, Yap modRNA treated mice had better heart function, reduced scar size and hypertrophic remodeling. Whole genome scale gene expression analysis showed that TLR signaling pathway was regulated by YAP/TEAD1. Depletion of Yap in the mouse heart increased the expression of Tlr2 and Tlr4. YAP modRNA treatment reduced the expression of Tlr2 and Tlr4 in the myocardium. In vitro data showed that activation of YAP was sufficient to abolish TLR4 activation induced CM necrosis. Conclusions: Our findings show that aYAP modRNA is sufficient to boost cardiac repair by reducing CM necrosis and cardiac inflammatory response. Mechanically, YAP suppresses CM necrosis and cardiac inflammation by regulating the TLR4 signaling. [ABSTRACT FROM AUTHOR]