The integrity and functionality of the heart depends on different, highly specialized cell types including cardiomyocytes (CMs), cardiac fibroblasts (FBs) and endothelial cells. Different studies published within the last years highlight the interactions of CMs with cardiac FBs and their importance in physiological and pathophysiological states. Cardiac FBs were shown to influence the maturation of CMs in mice during development. Furthermore, cardiac FBs are known to improve the maturation of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), which represent a promising model system to study human heart development. However, the molecular pathways of this bidirectional CM-FB crosstalk are far from being understood and it is unclear whether chamber-specific mechanisms are existing between specific cardiac FB subtypes and CMs.

In this project, we aim to characterize the influence of human atrial fibroblasts (HAF) and ventricular fibroblasts (HVF) on the development and functionality of iPSC-CMs. Initial studies revealed that co-culture of HAF or HVF with iPSC-CMs in 3D engineered heart muscles (EHMs) led to the development of HAF-EHMs and HVF-EHMs with functional characteristics of atrial and ventricular heart tissue, respectively. These include an increased spontaneous beating frequency and shorter contraction time in HAF-EHMs compared to HVF-EHMs. Although HAF-EHMs and HVF-EHMs showed a comparable inotropic response to the stimulation with isoprenaline, a reduction of the isoprenaline-induced increase in contractile force by carbachol was specifically observed for HAF-CMs, revealing the significant effect of the FB-subtype on drug-response of the chamber-specific EHMs. To elucidate the underlying mechanisms, we analyzed the secretome of HAF and HVF and examined the effect of FB-conditioned medium on gene expression and functionality of iPSC-CMs. Using this combined approach, we identified distinct gene expression patterns in iPSC-CMs in response to conditioned medium from HAF or HVF, such as activation of the retinoic acid (RA) pathway and interleukin-6 (IL-6) signaling. However, analysis of the contractile function revealed that treatment of iPSC-CMs with FB-conditioned medium did not fully recapitulate the changes observed in the EHMs, indicating cell-cell contact may play an important role. We then performed co-culture experiments of HAF and HVF with iPSC-CMs in a 2D monolayer format, allowing the direct interaction of CMs and FBs. The distinct influence of HAF and HVF on contractile function of iPSC-CMs could be recapitulated in 2D co-cultures. HAF-CM co-cultures showed an increased beating rate as well as shortened contraction time and beating duration, in comparison to HVF-CM. Furthermore, HAF-CM cultures had increased expression of HCN1 and NR2F2, which may contribute to the increased beating rate and development of atrial characteristics.

Overall, our data demonstrate the distinct influence of atria- and ventricle-derived cardiac FBs on the functionality and drug response of iPSC-CMs and therefore highlight the relevance of the FB-subtype to establish model systems fully recapitulating physiological function. In ongoing studies, we further aim to understand the underlying molecular pathways involved in CM-FB crosstalk in atria and ventricle.