The cardiac conduction system consists of several subcomponents, including subendocardial and intramyocardial Purkinje fibers. It is unknown how these distinct conducting elements are induced, patterned, and integrated into an entire conduction system network. We have shown that: 1) conduction cells differentiate from myocytes during embryogenesis; 2) this conversion of contractile myocytes to a conduction cell fate is induced by the stretch/pressure-induced vessel-derived factor, endothelin (ET); and 3) ET-induced differentiation is triggered only when ET is proteolyticaly activated from its precursor by the ET-converting enzyme (ECE-1). We have recently found that ECE-1 is first expressed in the endocardium at the onset of heart tube beating, and a week later, its expression begins in coronary arteries. Importantly, conversion of subendocardial and periarterial myocytes into conduction cells precisely follows this sequence of ECE-1 expression. Furthermore, retroviral co-expression of exogenous ECE-1 with ET precursor in the embryonic heart is sufficient to ectopically convert myocytes to Purkinje fibers. Finally, we have shown that differentiation of murine Purkinje fibers, which occurs exclusively along the endocardium, is also induced and maintained by paracrine interactions with endocardial endothelial cells. ? ? We therefore hypothesize that: a) hemodynamics directs the cardiac ECE-1 expression that defines the Purkinje fiber differentiation site; b) subendocardial and periarterial Purkinje fibers differentiate independently and their in situ linkage establishes the entire Purkinje network; and c) conversion of myocytes to a conduction cell fate is a conserved process that can be used to engineer mammalian Purkinje fibers. ? ? We will test these hypotheses by: 1) both activating and inhibiting biomechanical transducers in the embryonic heart and assaying the resulting ECE-1 expression and Purkinje fiber differentiation; 2) determining the coupling mechanisms between subendocardial and periarterial conduction elements; and 3) testing the potential of murine bone marrow-derived stem cells to differentiate into Purkinje fibers. 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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