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Abstract
IntroductionCa2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes.Methods and resultsCa2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks.ConclusionsFor the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.
Highlights
Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes
The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism
The pluripotent status of Human induced pluripotent stem cell (hiPSC) was confirmed by their expression of pluripotent markers and by their pluripotent differentiation potential including embryoid body (EB) formation and cardiac differentiation in vitro and teratoma formation in vivo. hiPSCs were maintained on mouse embryonic fibroblasts (MEF) feeder in human embryonic stem cells (hESCs) medium (80% Knockout Dulbecco’s Modified Eagle Medium or DMEM, 20% Serum replacement, 1% non-essential amino acid, 1 mM Lglutamine, 0.1 mM beta-mercaptoethanol and 4 ng/mL bFGF)
Summary
Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. It is unclear if they share similar calcium handling property. RyR Ca2+ release channel is tightly linked to the gating of L-type Ca2+ channel and plays a key role in the intracellular Ca2+-handling in cardiac myocytes [4]. Such property of Ca2+ sparks may reflect the organizational maturity of RyRs in the cardiomyocytes [5]
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