Abstract 945: Local Diastolic Ca2+ Release and Na+/Ca2+ Exchange Trigger Spontaneous Action Potentials in Developing Cardiac Cells: a Minor Role of If
Local Ca2+ releases (LCRs) critically contribute to adult cardiac pacemaker cell function via activation of Na+/Ca2+ exchanger (NCX) during late diastolic depolarization. While spontaneous LCRs are also present in developing cardiac cells, their role in the mechanisms of automaticity in these cells still remains unclear. It is believed that once hyperpolarization-activated If (so called “the pacemaker” current) becomes expressed, it provides major mechanism for spontaneous activity of the developing cardiac cells. Using perforated patch-clamp combined with confocal Ca2+ measurements, we studied the mechanisms of automaticity in two types of spontaneously beating developing cardiac cells: neonatal rat ventricular cardiomyocytes (NVCs) and late stage (>7+9 days) mouse embryonic stem cell-derived cardiocytes (ESCs), expressing both If and diastolic LCRs. Specific blockade of If by 7 μM UL-FS-49 (zatebradine) or by 2 mM Cs2+ reduced the beating rate of ESCs by ~19% and ~25%, respectively (n=5,8 cells). In contrast, occurrence of diastolic LCRs was essential for cell spontaneous beating. The cell automaticity was strongly suppressed or abolished in both ESCs and NVCs, when LCRs were inhibited by blocking SR Ca2+ pumping (0.1 μm thapsigargin, or 20 μm cyclopiazonic acid), or by disabling the Ca2+ release channel (20 μm ryanodine) (n=5– 8 cells/drug). Short-term NCX blockade with Li+ also abolished spontaneous action potentials (n=4) in both cell types. Under voltage clamp at −50 mV, excluding voltage-dependent interplay of activation of ion channels (including If), the oscillatory LCRs of both ESCs and NVCs caused rhythmic inward NCX currents with an amplitude of about 25 pA (n=4). We conclude that the mechanisms of automaticity of spontaneously beating developing cardiac cells are mainly linked to spontaneous diastolic LCRs. Similar to adult cardiac pacemaker cells, the diastolic LCRs trigger action potentials in these cells likely by activation of inward NCX current.