Abstract
Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.
Highlights
Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS)
To investigate whether cardiac fibroblasts trigger neighboring cardiomyocyte specialization into the conduction phenotype, we developed a monolayer co-culture consisting of a cardiomyocyte-enriched area, followed by a contact zone and a fibroblast-enriched area (Fig. 1)
Even though the existence of any influence mediated by cardiac fibroblast transmembrane proteins cannot be excluded, these results demonstrate that cardiac fibroblast-conditioned media is sufficient to induce the expression of VCS markers in cardiomyocytes in a paracrine fashion and exclude the possibility of cardiac fibroblasts being responsible for the increased expression of VCS-related genes in the mixed co-cultures rather than cardiomyocytes (Fig. 4)
Summary
Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). We provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro. Fibroblasts are one of the most abundant non-myocyte cells in the h eart[1] They are primarily known for their role in extracellular matrix homeostasis and remodeling during disease progression, but recent evidence suggests their influence in cardiomyocyte embryonic development and adult hypertrophy, as well as stabilization of cardiomyocyte contraction through heterocellular c oupling[2,3]. Transcription factors physically interact and are required for myocyte recruitment to differentiate into fast conducting fibers and PVCS postnatal maturation[10,12,19,22,23]
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