Induction and patterning of the Purkinje fibre network.

Impulse-conducting Purkinje cells differentiate from myocytes during embryogenesis. In the embryonic chicken heart, this conversion of contractile myocytes into conduction cells occurs subendocardially and periarterially. The unique sites of Purkinje fibre differentiation suggest that a shear stress-induced paracrine signal from the endocardium and arterial beds may induce adjacent myocytes to differentiate into conduction cells. Consistent with this model, Purkinje fibre marker genes can be induced in cultured embryonic myocytes by endothelin (ET), an endothelial cell-derived signalling peptide. This inductive response is, however, gradually lost from myocytes as embryos develop, and mature myocytes express only genes characteristic of hypertrophy in response to ET. In vivo, active ET is produced, through proteolytic processing, from its precursor by ET-converting enzyme 1 (ECE1) and triggers signalling by binding to its receptors, ETA and ETB. In the embryonic heart, the expression of these ET signalling components changes dynamically, defining the site and timing of Purkinje fibre differentiation within the ventricular myocardium during chick embryogenesis.

Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression.

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ET(A), ET(B) and ET(B2), in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ET(A) was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ET(B) was predominantly expressed in the atrium and the left ventricle. ET(B2) expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ET(A) and ET(B) increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ET(A) in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart.

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.