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 development | physiological hypertrophy

Normal heart development is orchestrated by a set of highly conserved transcription factors that includes GATA4, Nkx2-5, and Tbx5. Heterozygous mutation of each of these genes causes congenital heart disease in humans. In mouse models, haploinsufficiency for Nkx2-5 or Tbx5 resulted in an increased incidence of structural heart disease, confirming that normal heart development is sensitive to small changes in expression levels of Nkx2-5 and Tbx5. However, mice haploinsufficient for GATA4 have not been reported to have cardiac abnormalities. We generated two new GATA4 alleles, GATA4(H) and GATA4(flox). GATA4(flox/flox) embryos expressed 50% less GATA4 protein in the heart and survived normally. In contrast, GATA4(H/H) embryos expressed 70% less GATA4 protein in the heart and died between days 13.5 and 16.5 of gestation. These embryos had common atrioventricular canal (CAVC), double outlet right ventricle (DORV), hypoplastic ventricular myocardium, and normal coronary vasculature. Myocardial hypoplasia was associated with diminished cardiomyocyte proliferation. Hemodynamic measurements demonstrated that these embryos had normal systolic function, severe diastolic dysfunction, and atrioventricular regurgitation. Surprisingly, expression levels of the putative GATA4 target genes ANF, BNP, MEF2C, Nkx2-5, cyclin D2, and BMP4 were unchanged in mutant hearts, suggesting that GATA4 is not a dose-limiting regulator of the expression of these genes during later stages of embryonic cardiac development. These data demonstrate that multiple aspects of embryonic cardiac morphogenesis and function are exquisitely sensitive to small changes in GATA4 expression levels.

Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at embryonic day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P19CL6 prevented differentiation into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic factor gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming growth factor ␤-activated kinase 1. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.Nkx2.5 (also referred to as Csx) is a member of the NK2 class of homeoproteins (20,22). NK2 class homeoproteins are expressed in a tissue-specific manner, suggesting a role in tissue specification, differentiation, and patterning (13). Murine Nkx2.5 was identified as a homologue of the Drosophila tinman gene (5), which is required for subdivision of the dorsal mesoderm into the cardiac and visceral mesoderm (20,22). In mice, expression of Nkx2.5 begins as early as embryonic day (ED) 7.5 and continues through adulthood (17,20,22). Nkx2.5-null mice die before ED 11 with an arrest of cardiac development (23,36).Myocardin is expressed in cardiac myocytes and smooth muscle cells, and its expression begins in the cardiac crescent at ED 7.75 (42). Myocardin is a transcription cofactor and transactivates the serum response element (SRE) (also referred to as CArG box) dependent promoters by forming a ternary complex with serum response factor (SRF). Expression of a dominant-negative mutant of myocardin in Xenopus embryos prevents heart formation (42). However, regulation of myocardin gene expression is unknown.Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ␤ (TGF-␤) superfamily. TGF-␤-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase family and a mediator of TGF-␤ and BMP signal transduction (43). By using the teratocarcinoma P19 cells and their clonal derivative P19CL6 cells in an in vitro system, BMPs have been shown to be essential for cardiac myocyte differentiation (15, 28). BMP signaling regulates Nkx2.5 activity during cardiomyogenesis in P19 cells (15). In P19CL6 cells, TAK1, Nkx2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway (28). In addition, overexpression of ...

Abstract-Both genetic and epigenetic factors, such as abnormal hemodynamics, affect cardiac morphogenesis and the pathogenesis of congenital heart disease. Diastolic function is an important determinant of cardiac function, and tools for evaluating diastolic function in the embryo would be very valuable for assessment of cardiac performance. Using histological measurements of ventricular myoarchitecture, Doppler assessment of ventricular inflow velocities, and direct measurement of ventricular pressure, we investigated developmental changes of ventricular diastolic function in the mouse embryos from embryonic days 9.5 to 19.5. Regression analysis showed that peak velocity of A wave (an index of passive compliance) correlated with the area of trabecular myocardium in right ventricle (RV) (r 2 ϭ0.92, PϽ0.0001) and left ventricle (LV) (r 2 ϭ0.93, PϽ0.0001). Peak velocity of E wave (an index of active relaxation) exponentially correlated with the area of compact myocardium in RV (r 2 ϭ0.98, PϽ0.0001) and LV (r 2 ϭ0.97, PϽ0.0001). We used these techniques to analyze FOG-2 null embryos. FOG-2 null embryos had thin compact myocardium, higher EDP and E/A ratio, smaller ϪdP/dt, and diminished sucking pressure than wild-type littermates, indicating that decreased ventricular diastolic function might be the primary cause of embryonic lethality. In conclusion, during embryogenesis the development of compact myocardium tightly regulates the development of ventricular distensibility. Our study in normal mice forms the basis for future studies of embryonic cardiac function in genetically manipulated mice with abnormalities of the cardiovascular system.