A rhythmic heart beat is coordinated by the pace making action potentials of the cardiac conduction system. Our retroviral cell lineage studies of the chick embryo have demonstrated conclusively that impulse-conducting cells differentiate from working myocytes and are induced by local signals derived from the endocardial and coronary vasculature. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic myocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its G protein-coupled receptors expressed by myocytes. Strikingly, the ability of myocytes to convert their phenotype in response to ET declines as embryos mature, concurrent with a gradual decrease in the expression of the receptor; adult myocytes respond to ET by becoming hypertrophic. In addition, we have found for the first time that myocytes undergoing Purkinje fiber differentiation express at least two factors similar to myoD/myogenin members of the bHLH transcription factor gene family. Finally, murine Purkinje fibers differentiate exclusively along the endocardium, suggesting that mammalian as well as avian species, exhibit endothelial-myocyte interactions during Purkinje fiber differentiation. These data lead to the central hypotheses that: a) signals from endothelial cells recruit embryonic myocytes to a conduction cell fate in both mammalian and avian species; b) this inductive responsiveness is controlled by the developmentally regulated expression of ET-receptors; and c) the conversion from myocyte to conducting fiber involves transcriptional regulation distinct from that of working myocytes. We will test these three specific hypotheses by: 1 ) testing the necessity of endothelial cell-derived signals in conduction cell development of the mouse heart; 2) determining the role of ET-receptors in the developmentally regulated potential of myocyte to Purkinje fiber conversion; and 3) creating an ectopic site of myoD/myogenin-like transcription factor expression in the developing heart and assaying Purkinje fiber gene expression in vivo to determine their requirement for conduction cell differentiation. The studies proposed here will identify the regulators of three critical mechanisms in establishing the Purkinje fiber network and build a foundation for rational therapeutics for conduction disorder in adults.
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