Abstract 338: Cardiac-type Calcium Release Mechanism in Cardiac Precursor Cells
Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca2+ release is a shared property of many cell types, Ca+ release through RyR channels is muscle specific.
Aims: To test if cardiac-type Ca2+ release mechanism is present in CPCs and whether it may develop during the CSs stage.
Methods: Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca2+ transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca2+ release from intracellular stores was tested by exposures to caffeine (CAF, 10 mM) or ATP (200 μM) respectively in Ca2+-free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis.
Results: In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %; p<0.05); ATP-induced response was already present in pre-CSs cells and showed only a small increase in post-CSs ones (94.3 % vs 86.1 % p<0.05). The ratio between post-CSs and pre-CSs responses was 6.2 for CAF and 1.1 for ATP. Both CAF and ATP responses were suppressed by the SERCA inhibitor CPA (50 μM), thus confirming intracellular stores as the Ca2+ source. ATP response only was suppressed by the IP3-R blocker 2APB (10 μM). Ryanodine (20 μM) prevented the response to CAF, but not to ATP. In post-CSs RyR2 protein levels were higher than in pre-CSs and similar to those of adult myocytes. Immunoistochemistry analysis of post-CSs cells results in a distribution of RyR2 expression consistent with immature neonatal cardiomyocytes previously described.
Conclusions: At variance with IP3-mediated signaling, RyR mediated Ca2+ release develops during maturation within the CSs environment, along with expression of the cardiac RyR isoform. Detection of caffeine-induced Ca2+ responses may be useful in identifying cardio-specific functional maturation in progenitor cell populations.