Calvin T. Hang

Chromatin regulation by Brg1 underlies heart muscle development and disease

Cardiac hypertrophy and failure are characterized by transcriptional reprogramming of gene expression. Adult cardiomyocytes in mice primarily express α-myosin heavy chain (α-MHC, also known as Myh6), whereas embryonic cardiomyocytes express β-MHC (also known as Myh7). Cardiac stress triggers adult hearts to undergo hypertrophy and a shift from α-MHC to fetal β-MHC expression. Here we show that Brg1, a chromatin-remodelling protein, has a critical role in regulating cardiac growth, differentiation and gene expression. In embryos, Brg1 promotes myocyte proliferation by maintaining Bmp10 and suppressing p57kip2 expression. It preserves fetal cardiac differentiation by interacting with histone deacetylase (HDAC) and poly (ADP ribose) polymerase (PARP) to repress α-MHC and activate β-MHC. In adults, Brg1 (also known as Smarca4) is turned off in cardiomyocytes. It is reactivated by cardiac stresses and forms a complex with its embryonic partners, HDAC and PARP, to induce a pathological α-MHC to β-MHC shift. Preventing Brg1 re-expression decreases hypertrophy and reverses this MHC switch. BRG1 is activated in certain patients with hypertrophic cardiomyopathy, its level correlating with disease severity and MHC changes. Our studies show that Brg1 maintains cardiomyocytes in an embryonic state, and demonstrate an epigenetic mechanism by which three classes of chromatin-modifying factors—Brg1, HDAC and PARP—cooperate to control developmental and pathological gene expression.

Endocardial Brg1 Represses ADAMTS1 to Maintain the Microenvironment for Myocardial Morphogenesis

Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

Chromatin Remodeling in Cardiovascular Development and Physiology

Chromatin regulation provides an important means for controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various transcription factors in the fine regulation of gene expression, essential for all aspects of cardiovascular biology. Aberrant cardiac gene expression, triggered by a variety of pathological insults, can cause heart diseases in both animals and humans. The severity of cardiomyopathy and heart failure correlates strongly with abnormal cardiac gene expression. Therefore, controlling cardiac gene expression presents a promising approach to the treatment of human cardiomyopathy. This review focuses on the roles of ATP-dependent chromatin-remodeling factors and chromatin-modifying enzymes in the control of gene expression during cardiovascular development and disease.

Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development

Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21cip1, to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.

Spatial and temporal regulation of coronary vessel formation by calcineurin-NFAT signaling

Formation of the coronary vasculature requires reciprocal signaling between endothelial, epicardially derived smooth muscle and underlying myocardial cells. Our studies show that calcineurin-NFAT signaling functions in endothelial cells within specific time windows to regulate coronary vessel development. Mouse embryos exposed to cyclosporin A (CsA), which inhibits calcineurin phosphatase activity, failed to develop normal coronary vasculature. To determine the cellular site at which calcineurin functions for coronary angiogenesis, we deleted calcineurin in endothelial, epicardial and myocardial cells. Disruption of calcineurin-NFAT signaling in endothelial cells resulted in the failure of coronary angiogenesis, recapitulating the coronary phenotype observed in CsA-treated embryos. By contrast, deletion of calcineurin in either epicardial or myocardial cells had no effect on coronary vasculature during early embryogenesis. To define the temporal requirement for NFAT signaling, we treated developing embryos with CsA at overlapping windows from E9.5 to E12.5 and examined coronary development at E12.5. These experiments demonstrated that calcineurin-NFAT signaling functions between E10.5 and E11.5 to regulate coronary angiogenesis. Consistent with these in vivo observations, endothelial cells exposed to CsA within specific time windows in tissue culture were unable to form tubular structures and their cellular responses to VEGF-A were blunted. Thus, our studies demonstrate specific temporal and spatial requirements of NFAT signaling for coronary vessel angiogenesis. These requirements are distinct from the roles of NFAT signaling in the angiogenesis of peripheral somatic vessels, providing an example of the environmental influence of different vascular beds on the in vivo endothelial responses to angiogenic stimuli.

Inducible Cardiomyocyte-Specific Gene Disruption Directed by the Rat Tnnt2 Promoter in the Mouse

We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010.

Epigenetic response to environmental stress: Assembly of BRG1-G9a/GLP-DNMT3 repressive chromatin complex on Myh6 promoter in pathologically stressed hearts

Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin-marked by H3K9 and CpG methylation-on a key molecular motor gene (Myh6), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6, reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1-G9a/GLP-DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1-G9a-Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Use of Whole Embryo Culture for Studying Heart Development

Congenital heart defects occur in approximately 1% of newborns and are a major cause of morbidity and mortality in infants and children. Many adult cardiac diseases also have developmental basis, such as heart valve malformations, among others. Therefore, dissecting the developmental and molecular mechanisms underlying such defects in embryos is of great importance in prevention and developing therapeutics for heart diseases that manifest in infants or later in adults. Whole embryo culture is a valuable tool to study cardiac development in midgestation embryos, in which ventricular chambers are specified and expand, and the myocardium and endocardium interact to form various cardiac structures including heart valves and trabecular myocardium (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008). This technique is essentially growing a midgestation embryo ex utero in a test tube. One of the strengths of embryo culture is that it allows an investigator to easily manipulate or add drugs/chemicals directly to the embryos to test specific hypotheses in situations that are otherwise very difficult to perform for embryos in utero. For instance, embryo culture permits pharmacological rescue experiments to be performed in place of genetic rescue experiments which may require generation of specific mouse strains and crosses. Furthermore, because embryos are grown externally, drugs are directly acting on the cultured embryos rather than being degraded through maternal circulation or excluded from the embryos by the placenta. Drug dosage and kinetics are therefore easier to control with embryo culture. Conversely, drugs that compromise the placental function and are thus unusable for in utero experiments are applicable in cultured embryos since placental function is not required in whole embryo culture. The applications of whole embryo culture in the studies of molecular pathways involved in heart valve formation, myocardial growth, differentiation, and morphogenesis are demonstrated previously (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008; Nature 446: 62-67, 2010). Here we describe a method of embryo culture in a common laboratory setting without using special equipments.

Erratum: Chromatin regulation by Brg1 underlies heart muscle development and disease (Nature (2010) 466 (62-67))

This corrects the article DOI: 10.1038/nature09130