Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post-chamber formation

There is scant information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Here we simultaneously tracked three lineage-specific markers (Nkx2.5, MLC2v, and ANF) and confirmed that CPCs with an Nkx2.5⁺MLC2v²ANF^- phenotype are present in the embryonic (E) day 11.5 mouse ventricular myocardium. We demonstrated that these CPCs could give rise to working cardiomyocytes and conduction system cells. Using a two-photon imaging analysis, we found that the majority of CPCs are not capable of developing Ca²⁺ transients in response to β-adrenergic receptor stimulation. In contrast, Nkx2.5 ⁺ cells expressing MLC2v but not ANF are capable of developing functional Ca²⁺ transients. We showed that Ca²⁺ transients could be invoked in Nkx2.5⁺MLC2v⁺ANF⁺ cells only upon inhibition of Gi, muscarinic receptors, or nitric oxide synthase (NOS) signaling pathways. Our data suggest that these inhibitory pathways may delay functional specification in a subset of developing ventricular cells.