Single nuclei multiomics analysis of cardiogenic transcription factors in a new murine model of cardiac development.

Transcription factors such as Tbx5, Gata4, Mef2c and Pitx2 are required during cardiac development, and in adult cardiac homeostasis. We demonstrate that the gene dosage and modulation of these factors are mediated in vivo by the miR-200 family. These microRNAs (miRs) are expressed during early stages of heart development, and they regulate a highly complex gene regulatory network (GRN). To study the in vivo and in vitro function of specific miR-200 family members we inhibited individual members of the miR-200 family during embryonic development. Inhibition of a single miR-200 family member within the cluster caused defects in the left ventricle and cardiomyocyte maturation during development. Inhibition of the entire miR-200 family resulted in a ventral septal defect and embryonic lethality by embryonic day (E)16.5. In cardiomyocytes, the miR-200 family targets the transcripts of Tbx5, Gata4, Mef2c and Pitx2. Inhibition of this family increased expression of Tbx5, Gata4, Mef2c and Pitx2 in both the left ventricle and atria across multiple stages of cardiac development. Embryos with reduced levels of a single miR-200 family member were non-lethal and pups survived into adulthood. At postnatal day (P) 1 these pups had a reduced heart rate and increased ventricular wall thickness. Each miR-200 family has distinct heart phenotypes in cell specific differentiation and maturation. snRNA-sequencing revealed a new cardiomyocyte cell state, suggesting these cells were less differentiated due to inhibition of the miR-200 family. We have identified several new transcription factors regulated by miR-200 during heart development. The miR-200 family are critical regulators of early cardiac development through maintaining gene dosage of Tbx5, Gata4, Mef2c and Pitx2, which affects cardiomyocyte differentiation and maturation. The multiomics analyses of early heart development after miR-200 inhibition reveals a new cardiomyocyte less differentiated cell state and chromatin modifications due to the function of the miR-200 family.