Roles of Neuromedin-U and Endothelin-1 in human cardiac organoid differentiation

During recent years, many new culture methods have been established to generate cardiomyocytes, using signaling and patterning factors that play important roles in mammalian heart development. Here, we identify two additional pathways of interest using a cardiac differentiation roadmap that we have generated, involving the endothelin 1 receptor EDNRA gene, and the Neuromedin family signaling peptide gene NMU. Treatment with NMU had most effect in ventricular lineages where it normally is not abundantly expressed, leading to a significant shift from ventricular cardiomyocyte (vCM) differentiation to non-myocytes, as shown with reporters and cell type-specific markers, which was associated with an increased beating frequency. This was not observed in atrial cultures. Treating atrial cultures with endothelin 1 (ET-1, EDN1) increased the number of atrial cardiomyocytes (aCM), while reducing repolarization times, increasing the contraction frequencies and altering the patterns of action potential propagations across the tissue. In ventricular cultures, a reduction in ventricular cardiomyocyte (vCM) numbers was observed, suggesting a combinatorial effect of ET-1 and retinoic acid (RA) on cardiomyocyte differentiation. These results identify ET-1 and NMU as important signaling factors for cardiomyocyte differentiation and electrophysiology of cardiac organoids, with ET-1 enhancing aCM differentiation when added around the time of posteriorization of the second heart field. NMU added without RA reduces the number of vCMs in the culture while it changes the organoids to exhibit a more atrial-like beating frequency.