Mitochondrial Ca²⁺ flux modulates spontaneous electrical activity in ventricular cardiomyocytes

<div><p>Introduction</p><p>Ca²⁺ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na⁺-Ca²⁺ exchanger (NCX). Mitochondria participate in Ca²⁺ cycling. We studied the role of mitochondrial Ca²⁺ flux in ventricular spontaneous electrical activity.</p><p>Methods</p><p>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 Ca²⁺ transients were determined by fluorescent imaging.</p><p>Results</p><p>In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca²⁺ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca²⁺ release from RYR2 or inositol trisphosphate receptors (IP₃R), the pacemaker current (I_f) and mitochondrial Ca²⁺ uptake by the mitochondrial Ca²⁺ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca²⁺ flux influenced spontaneous beating independently of the SR Ca²⁺ release from RYR2, and the mitochondrial effect was dependent on IP₃R SR Ca²⁺ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca²⁺ 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.</p><p>Conclusions</p><p>Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca²⁺ uptake plays a modulatory role. Mitochondrial Ca²⁺ uptake through MCU is influenced by IP₃R-dependent SR Ca²⁺ release.</p>