Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes

Abstract

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 to 0mV had no significant effects on spontaneous pacing, but suppressed action potential formation; 3) Spontaneous pacing at −50mV 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 (~50nM), 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 −80mV in hiPS-CM; nevertheless blockers of If-channel failed to alter spontaneous pacing.

Calcium signaling in human stem cell-derived cardiomyocytes: Evidence from normal subjects and CPVT afflicted patients

“…In this respect, it appears to be a common finding that the rate of spontaneous beating is lower in CPVT1 mutant cell lines as compared to control lines, possibly because of the lower SR Ca 2+ content. This finding is consistent with the hypothesis that Ca 2+ signaling mechanism, rather than the activation of I f regulates spontaneous pacing activity in hiPSC-CMs [45]. …”

“…Caffeine concentrations of 3–5 mM triggered large global Ca 2+ transients that activated inward I NCX , of about 2.5 pA/pF (Fig. 2D) as compared to ~1.0 pA/pF in rat neonatal cardiomyocytes, confirming functionally the existence of large SR Ca 2+ stores in hiPSC-CM [45]. The larger density of I NCX activated on application of caffeine as compared to values of ~1.0 pA/pF in adult cardiomyocytes of rat, rabbit, mouse, and human hearts suggests either larger stores of Ca 2+ accessed by caffeine in hiPSC-CM or a greater density of NCX in hiPSC-derived myocytes.…”

“…(B) Images show baseline fluorescence (F 0 ) and color-coded regions of interest (ROI, with “n” indicating nuclei) and 4 differential fluorescence images (measured at the times indicated along the traces) showing repeatable mitochondrial Ca 2+ signals before FCCP application (1, 2, 3) and smaller, more localized responses after (4). Modified from [45]…”

Excitation–contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

“…RyRs in hiPSC-CMs exhibit rapid, large releases of Ca 2+ in response to the agonist caffeine (Fatima et al, 2011 ; Itzhaki et al, 2011 ; Lee et al, 2011 ; Kim et al, 2015 ; Zhang et al, 2015 ), and antagonism with ryanodine reduces whole cell Ca 2+ transient amplitude. SR Ca 2+ content, assessed by caffeine application, is similar to that found in adult rabbit cells (Hwang et al, 2015 ).…”

A spatiotemporal ventricular myocyte model incorporating mitochondrial calcium cycling

“…It has also been shown that mitochondrial depolarization and other metabolic stress affect the Ca 2+ cycling dynamics, including Ca 2+ spark (1,2), Ca 2+ alternans (3)(4)(5)(6)(7)(8), and spontaneous Ca 2+ release and waves (9,10). Mitochondrial depolarization and Ca 2+ cycling can also affect the action potential dynamics, such as promoting early afterdepolarizations (EADs) (11,12), and lower membrane excitability by opening of K ATP channels (13)(14)(15) to promote arrhythmias, or modulating the pacemaking activity of cardiomyocytes (16,17).…”