Role of CHF1 in Cardiovascular Development

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DESCRIPTION (the applicant's description verbatim): The development of cardiac myocytes from primordial mesoderm, the specification of atrial and ventricular cardiac myocytes, and the terminal differentiation of cardiac myocytes are critical events in vertebrate embryonic development. In addition, diseases of the myocardium, such as ischemic heart disease and heart failure, are the leading causes of death in western society. After infarction, cardiac myocytes are unable to regenerate, due to their postmitotic, terminally differentiated state. An understanding of the transcriptional programs that regulate cardiac myocyte ontogeny may lead to better therapies for cardiovascular disease. We have recently identified cardiovascular basic helix-loop-helix protein 1 (CHF1), a novel protein that is distantly related to the hairy family of transcriptional repressors. Members of the hairy family are known to play important roles in control of neuronal cell fate and timing of neuronal differentiation. CHF1 is expressed primarily in the developing ventricle, concurrently with MLC2v, the earliest known marker of ventricular myocytes. This expression persists until birth, and is downregulated as the cardiac myocytes terminally differentiate and withdraw from the cell cycle. We hypothesize that CHF1 plays an important role in cardiac myocyte differentiation, presumably by interacting with intracellular proteins. Our experimental aims are: Aim 1: Study the effect of CHF1 on cardiac phenotype determination in vitro. We hypothesize that CHF1 plays an important role in cardiac ventricular myocyte cell fate determination. We will perform gain of function and loss of function studies in mouse embryonic stem cells that can be made to differentiate into cardiac myocytes. We will compare the ability of wild type, heterozygous and homozygous CHF1 knockout cells to differentiate into cardiac myocytes in vitro. In addition, we will infect wild type ES cells with a recombinant adenovirus that overexpresses CHF1 and then measure the timing of differentiation into cardiac myocytes in vitro. Aim 2: Study the effect of CHF1 on ventricular myocyte phenotype in vivo. We hypothesize that CHF1 plays an important role in the development of the cardiovascular system. We will use the heterozygous knockout ES cells described in Aim 1 to generate knockout mice that lack CHF1. In addition, we will generate transgenic mice that express CHF1 under the control of the alpha-MHC promoter to assess the effect of persistent expression in the adult myocardium. Aim 3: Determine the intracellular interaction partners of CHF1 that mediate its effects. We hypothesize that CHF1 exerts an effect on ventricular phenotype by interaction with other cellular proteins. We will perform a yeast 2-hybrid screen with the full length, bHLH and repressor domains of CHF1 to identify potential heterodimerization partners and transcriptional corepressors present in a mouse embryonic library.