Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes

• Myocytes derived from newborn rat heart and human induced pluripotent stem cells pace spontaneously.
• Ca2+-mediated pacing mechanisms are similar in both developing cell types.
• Pacing depends on activation of INCX and Ca2+-transients, but not on If and acute ICa block and Vm change.
• Regionally diverse mitochondrial Ca2+ signals may initiate and regulate pacing in both cell types.

The quintessential property of developing cardiomyocytes is their ability to beat spontaneously. The mechanisms underlying spontaneous beating in developing cardiomyocytes are thought to resemble those of adult heart, but have not been directly tested. Contributions of sarcoplasmic and mitochondrial Ca2+-signaling vs. If-channel in initiating spontaneous beating were tested in human induced Pluripotent Stem cell-derived cardiomyocytes (hiPS-CM) and rat Neonatal cardiomyocytes (rN-CM). Whole-cell and perforated-patch voltage-clamping and 2-D confocal imaging showed: (1) both cell types beat spontaneously (60–140/min, at 24 °C); (2) holding potentials between −70 and 0 mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at −50 mV activated cytosolic Ca2+-transients, accompanied by in-phase inward current oscillations that were suppressed by Na+-Ca2+-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca2+- and If-channels blockers; (4) spreading fluorescence images of cytosolic Ca2+-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50 nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca2+-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca2+-uptake and release signals activating during INCX oscillations; (7) If-channel was absent in rN-CM, but activated only negative to −80 mV in hiPS-CM; nevertheless blockers of If-channel failed to alter spontaneous pacing.

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