Abstract 579: Calcium Handling and Mechanisms of Automaticity in Human Embryonic Stem Cell Derived Cardiomyocytes
Human embryonic stem cell derived cardiac myocyte (hES-CM) function is poorly understood. Under normal conditions mature cardiac muscle is quiescent but immature CMs, including hES-CMs display automaticity. In mature nodal cells store Ca contributes to automaticity. Therefore, we tested the hypothesis that cytosolic intracellular store Ca transients in hES-CMs contributes to their spontaneous excitability. Relatively immature hES-CMs were chosen for the study (<40 days differentiation). We used a combination of approaches including RT-PCR, immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp and laser scanning confocal Ca-imaging of cells co-labeled with di-8-Anepps to delineate surface membrane. hES-CMs reveal two distinct types of Ca transients AP driven global Ca transients (AP-Ca transients) and local Ca transients (LCTs). The LCTs originate at the cell periphery, associated with plasma membrane (PM), do not propagate across the width of the cell and are not interactive with electrical activity. The AP-Ca transients are rhythmic high amplitude events that originate at the PM and spread as a propagated wave to the cell center. Caffeine-releasable store Ca was demonstrated by focal, temporally-limited puffs of caffeine and by long-lasting caffeine depletion of internal store Ca. Spontaneous activity was inhibited by 20 mM caffeine (>20s continual application). Ryanodine caused a progressive decrease in the amplitude of Ca-release and AP rate. Interestingly, 10μM TTX abolishes all Ca transients (AP-Ca transients and LCTs) suggesting that elevated cytosolic Na contributes to the LCTs. High-speed 2D confocal laser scanning imaging showed that LCTs occur out of phase with AP-Ca transients, and separate LCTs within a given cell are often out of phase with one another. This argues against LCTs as a primary rhythm generator in early stage hES-CMs. However ryanodine and caffeine effects argue for internal store Ca requirement for automaticity. We conclude that the molecular mechanism of automaticity in early stage hES-CMs relies on the interplay between AP-Ca and membrane potential. This study provides the first evidence that developing hES-CMs have functional internal store Ca.