NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro

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.

Bioengineering Systems for Modulating Notch Signaling in Cardiovascular Development, Disease, and Regeneration

“…A study by Ribeiro da Silva et al cocultured neonatal mice CMs with a CFs-conditioned medium to understand the influence of CFs secretomes on CM differentiating into the ventricular conduction system cells (conduction phenotype). The results indicate that fibroblast secretomes are necessary for the activation of Notch1 to induce the conduction phenotype of the CMs [71]. Moreover, the promotion of valve formation through Notch activation is found to be modulated by the endocardial secretome.…”

Analysis of endogenous NOTCH1 fromPOFUT1 S162Lpatient fibroblasts reveals the importance of theO-fucose modification on EGF12 in human development

“…N1 signaling plays an important role in human fibroblasts during tissue repair and fibrosis. Activation of N1 signaling in fibroblasts can promote their proliferation and regulate the expression of several genes involved in fibroblast survival (Ribeiro da Silva, A., Neri, E.A., et al 2020). N1 also participates in fibroblast differentiation, guiding them to mature into specialized fibroblast subtypes (Wei, K., Korsunsky, I., et al 2020), or fibroblast-derived cells such as myofibroblasts, in response to appropriate environmental cues mediated by collagen secretion (Dees, C., Tomcik, M., et al 2011).…”

“…It was recently reported that loss of N1 in dermal fibroblasts leads to enhanced healing from diabetic wounds demonstrating the importance of N1 signaling in fibroblasts (Shao, H., Li, Y., et al 2020). Additionally, induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro is dependent on N1 signaling in cardiac fibroblasts (Ribeiro da Silva, A., Neri, E.A., et al 2020). Loss of N1 signaling in POFUT1 S162L patient cells could be due to a N1 trafficking defect or from loss of Notch-ligand interactions.…”

“…2020). Additionally, induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro is dependent on N1 signaling in cardiac fibroblasts (Ribeiro da Silva, A., Neri, E.A., et al 2020). Loss of N1 signaling in POFUT1 S162L patient cells could be due to a N1 trafficking defect or from loss of Notch-ligand interactions.…”

Multicellular 3D Models for the Study of Cardiac Fibrosis

“…As an example, inclusion of stromal cells can help tissue modelling protocols to reach a more advanced differentiation state and render them more performing [ 32 , 33 , 34 ], thus more relevant also for translational studies, such as drug testing. This effect derives, among others, from the beneficial paracrine effects of stromal cells towards cardiomyocytes [ 35 , 36 ], including the important contribution of stromal cells to electrical conductance through connexin-43 (CX43) expression [ 32 ] and to the specific differentiation of cardiomyocytes of the conduction systems [ 37 ]. Functional electrophysiological cues were precisely discovered thanks to multicellular modelling itself [ 38 ].…”