Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes.
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 Ca(2+)-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 0mV had no significant effects on spontaneous pacing, but suppressed action potential formation; (3) spontaneous pacing at -50mV activated cytosolic Ca(2+)-transients, accompanied by in-phase inward current oscillations that were suppressed by Na(+)-Ca(2+)-exchanger (NCX)- and ryanodine receptor (RyR2)-blockers, but not by Ca(2+)- and If-channels blockers; (4) spreading fluorescence images of cytosolic Ca(2+)-transients emanated repeatedly from preferred central cellular locations during spontaneous beating; (5) mitochondrial un-coupler, FCCP at non-depolarizing concentrations (∼50nM), reversibly suppressed spontaneous pacing; (6) genetically encoded mitochondrial Ca(2+)-biosensor (mitycam-E31Q) detected regionally diverse, and FCCP-sensitive mitochondrial Ca(2+)-uptake and release signals activating during INCX oscillations; (7) If-channel was absent in rN-CM, but activated only negative to -80mV in hiPS-CM; nevertheless blockers of If-channel failed to alter spontaneous pacing.
Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA.
Xiao-Hua
XH
Zhang
Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA.
Hua
H
Wei
Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
Tomo
T
Šarić
Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
Jürgen
J
Hescheler
Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA.
Lars
L
Cleemann
Cardiac Signaling Center of USC, MUSC, & Clemson University, Charleston, SC, USA. Electronic address: moradm@musc.edu.
Martin
M
Morad
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D006339:Heart Rate / Q000502:physiology*; D006801:Humans; D051379:Mice; D008929:Mitochondria, Heart / Q000502:physiology*; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology
D002121:Calcium Channel Blockers; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger
Calcium; Cardiomyocytes derived from human induced pluripotent stem cells; Electrophysiology; Genetically engineered fluorescent probes; Ion channels; Mitochondria; Pacing; Rat neonatal cardiomyocytes; Sarcoplasmic reticulum