Abstract 1019: Intracellular Calcium Cycling Mediates Proliferation and Differentiation of Human Cardiac Stem Cells
Human cardiac stem cells (hCSCs) are self-renewing, clonogenic and have the ability to differentiate into myocytes, smooth muscle and endothelial cells in vitro and in vivo. Since Ca2+plays a crucial role in mechanotransduction and activation of signalling pathways in mature cardiac cells, intracellular Ca2+cycling was studied in hCSCs to determine the function of this cation in cell division and commitment to the myocyte lineage. For this purpose, hCSCs were exposed to conditions favouring proliferation and differentiation and affecting intracellular Ca2+ homeostasis. Moreover, hCSCs were loaded with Fluo-3 and intracellular Ca2+ levels were monitored by 2-photon microscopy. hCSCs presented spontaneous Ca2+ spikes mediated by Ca2+release from the endoplasmic reticulum (ER). ATP and histamine, which stimulate InsP3R-mediated ER Ca2+ release, increased the occurrence of spikes leading to oscillations in intracellular Ca2+. 2-APB, an antagonist of InsP3R, inhibited spike formation and oscillatory events. Ryanodine, which acts on the ryanodine receptors, did not alter intracellular Ca2+ and thapsigargin, a Ca2+ pump blocker, prevented spontaneous and induced ER Ca2+ release. Store operated capacitative Ca2+ entry was evoked by increasing extracellular Ca2+ after depletion of the ER. Ca2+ entry was blocked by lanthanum. Additionally, patch-clamp experiments indicated the absence of the voltage-activated L-type Ca2+ current in hCSCs. Exposure of hCSCs to IGF-1 triggered acutely Ca2+ spikes and increased chronically their occurrence. Over a period of 24 hours, IGF-1 resulted in more than 100% increase in the proliferation of hCSCs measured by BrdU labelling. Similarly, ATP enhanced proliferation of hCSC by ~60%. Importantly, incubation with 2-APB reduced by ~50% BrdU incorporation and abolished the effect of IGF-1 and ATP on both Ca2+ spikes and cell proliferation. In the presence of differentiating medium, the frequency of Ca2+ spikes in active hCSCs increased significantly. Additionally, enhanced Ca2+ cycling increased the number of hCSCs committed to the myocyte lineage, while attenuations in this phenomenon blunted hCSC differentiation. Thus, InsP3R-mediated Ca2+ spikes play an obligatory role in hCSC growth and differentiation.