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.

心脏成纤维细胞遍布整个心肌，并且在心室传导系统 (VCS) 周围的微环境中富集。几种形式的心律失常与 VCS 异常有关，但目前尚不清楚 VCS 畸形是心肌细胞自主的还是与不同细胞类型之间的串扰有关。我们推断成纤维细胞影响 VCS 细胞中心肌细胞的特化。我们开发了新生大鼠心肌细胞的 2D 和 3D 培养模型，以评估心脏成纤维细胞对心肌细胞的影响。与心脏成纤维细胞相邻的心肌细胞显示 VCS 标记物（NAV1.5 和 CONTACTIN 2）的表达和钙瞬变持续时间增加两倍，呈现出浦肯野样特征。成纤维细胞条件培养基 (fCM) 足以激活 VCS 相关基因（ Irx3 、 Scn5a 、Connexin 40）并诱导动作电位延长，这是 Purkinge 表型的标志。 fCM 介导的反应似乎具有空间依赖性，因为用 fCM 处理的心肌细胞类器官主要在其外表面增加了连接蛋白 40 和 NAV1.5 的表达。最后，心肌细胞和成纤维细胞中的 NOTCH1 激活是连接蛋白 40 上调（VCS 表型的代表）所必需的。总而言之，我们提供的证据表明，心脏成纤维细胞在体外通过 NOTCH1 信号传导影响心肌细胞专门化为 VCS 样细胞。