Mitochondrial Ca(2+) flux modulates spontaneous electrical activity in ventricular cardiomyocytes

INTRODUCTION: Ca(2+) release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na(+)-Ca(2+) exchanger (NCX). Mitochondria participate in Ca(2+) cycling. We studied the role of mitochondrial Ca(2+) flux in ventricular spontaneous electrical activity. METHODS: Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19–21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca(2+) transients were determined by fluorescent imaging. RESULTS: In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca(2+) channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca(2+) release from RYR2 or inositol trisphosphate receptors (IP(3)R), the pacemaker current (I(f)) and mitochondrial Ca(2+) uptake by the mitochondrial Ca(2+) uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca(2+) flux influenced spontaneous beating independently of the SR Ca(2+) release from RYR2, and the mitochondrial effect was dependent on IP(3)R SR Ca(2+) release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca(2+) flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels. CONCLUSIONS: Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca(2+) uptake plays a modulatory role. Mitochondrial Ca(2+) uptake through MCU is influenced by IP(3)R-dependent SR Ca(2+) release.