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Updated: Dec 7, 2025
Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells
Published on: February 12, 2021
NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction
Agatha Ribeiro da Silva1, Elida A Neri1, Lauro Thiago Turaça1
1Lab Genetics & Molec Cardiology, Instituto do Coracao (InCor) da Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), São Paulo, Brazil.
Insights
Cardiac fibroblasts promote specialized ventricular conduction system (VCS) cell development in cardiomyocytes. This occurs through NOTCH1 signaling, influencing gene expression and electrical properties, suggesting fibroblast-cardiomyocyte crosstalk is key for VCS formation.
Area of Science:
- Cardiovascular Biology
- Cellular Signaling
- Cardiac Electrophysiology
Background:
- Cardiac fibroblasts are abundant in the myocardium, particularly around the ventricular conduction system (VCS).
- Abnormalities in the VCS are linked to arrhythmias, but the cellular basis (cardiomyocyte-autonomous vs. cell-cell interactions) remains unclear.
- Fibroblast influence on cardiomyocyte specialization within the VCS microenvironment is hypothesized.
Purpose of the Study:
- To investigate the role of cardiac fibroblasts in cardiomyocyte specialization towards a VCS-like phenotype.
- To elucidate the molecular mechanisms, including signaling pathways, by which fibroblasts influence cardiomyocytes.
Main Methods:
- Development of 2D and 3D cardiac cell culture models using neonatal rat cells.
- Treatment of cardiomyocytes with fibroblast-conditioned media (fCM).
- Analysis of VCS marker expression (NAV1.5, CONTACTIN 2), electrical properties (calcium transient duration, action potential), and gene expression (Irx3, Scn5a, Connexin 40).
- Investigation of the role of NOTCH1 signaling in fibroblast-mediated effects.
Main Results:
- Cardiomyocytes co-cultured with fibroblasts exhibited a two-fold increase in VCS markers and prolonged calcium transient duration, resembling Purkinje cells.
- Fibroblast-conditioned media induced VCS-related gene expression and action potential prolongation.
- Fibroblast-conditioned media effects were spatially dependent in 3D organoids, with enhanced marker expression on the outer surface.
- NOTCH1 activation in both cell types was essential for up-regulating connexin 40, a VCS phenotype marker.
Conclusions:
- Cardiac fibroblasts significantly influence cardiomyocyte specialization into VCS-like cells in vitro.
- NOTCH1 signaling mediates the fibroblast-induced VCS phenotype in cardiomyocytes.
- Cell-cell crosstalk between fibroblasts and cardiomyocytes is crucial for VCS development and function.
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.
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