Calcium channel blockade in embryonic cardiac progenitor cells disrupts normal cardiac cell differentiation

We suggest that characterization of processes involved in differentiation of the pluripotential cardiac precursor cells in their embryonic environment will permit identifying pathways important for induction of diverse stem cells toward the cardiac phenotype. Phenotypic characteristics of cardiac cells are their contractile and electrical properties. The objective of the present study was to define whether calcium (Ca(++)) has a regulatory role in the pluripotential precursor cell population during commitment into cardiomyocytes. We used the chick embryo model because of ease of staging the embryos and visibility of heart development. Using the Ca(++) indicator Fluo-3/acetoxymethyl and confocal microscopy, we demonstrated the existence of higher free Ca(++) levels in the cardiogenic precursor cells than in neighboring cell populations outside of the heart fields. Subsequently, gastrulation stage 4/5 chick embryos were set up in modified New cultures in the medium containing either the L-type Ca channel blocker, diltiazem, or the N-type Ca channel inhibitor, ω-conotoxin. The embryos were incubated for 22-24 h during which time the control embryos developed, beating looping hearts. At the end of incubation, exposure to the L-type channel blockade with diltiazem resulted in an inhibition of cardiomyogenesis in the most posterior, uncommitted, part of the heart fields. N-type channel blockade with ω-conotoxin was less intense. Cells in the most anterior cardiogenic regions that were already committed at time of exposure continued to differentiate. Thus, regulation and maintenance of normal cytosolic Ca levels are necessary for the early steps of cardiomyocyte specification and commitment leading to differentiation.