Calcium Homeostasis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and ResultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9273-3) contains supplementary material, which is available to authorized users.

Mitochondrial disruption occurs downstream from β-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: Differential activation of stress and survival pathways

“…Increased basal calcium has been previously described in several models of cell differentiation, including cardiac cells (Fu et al, 2006), mouse embryonic stem cells (ES) (Metzger et al, 1994), human induced pluripotent stem cells (hiPSCs) (Lee et al, 2011) and rat neonatal cardiomyocytes (Gomez et al, 1994). β-receptor activation modulates L-type calcium channels and ryanodine receptors (RyR) (the major sarcoplasmic reticulum calcium release channels in cardiac and skeletal muscles), as well as sarcoplasmic reticulum (SR) Calcium- (Harris, 1977), although differentiated H9c2 myoblasts gradually decreased cAMP and AC activity (Pagano et al, 2004), which may serve as adaptation to increased cytosolic calcium during differentiation.…”

Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of <i>SCN5A</i>-D1275N-Related Cardiac Sodium Channelopathy

“…We also assessed the Ca 2+ transient properties associated with the contraction of CMs, such as Ca 2+ transient amplitude, maximal upstroke velocity (Vmax upstroke), Vmax upstroke time to peak, and Ca 2+ transient duration at 90% decay (CTD 90). 24 Isoproterenol administration slightly, but not significantly, shortened Vmax upstroke time and CTD90 in both cells. We found no significant differences in all measurements between the D1275N and Control hiPSC-CM before and after isoproterenol administration ( Figure S3C).…”

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

“…In addition, spontaneous Ca 2+ transients were reported in 20-day-old hESC-CMs derived from H7 and HES3 cells (Lee et al, 2011), and in 18-24-day-old HES2-derived CMs on electrical stimulation (Liu et al, 2007). Cardiomyocytes derived from HES2 cells have been reported to begin contracting spontaneously as early as day 8 of differentiation.…”

“…In the case of reporters such as Fluo-3 (Lee et al, 2011), Fluo-4 (Satin et al, 2008Apáti et al, 2013), and Fura-2 (Dolnikov et al, 2006;Liu et al, 2007), cells were loaded with the dye via incubation with the respective acetoxymethyl (AM) ester. However, the problems of using such dyes for measuring Ca 2+ signals inside cells with regards to the uneven distribution of the dye both within single cells via reporter compartmentalization and in beating clusters of cells, have been emphasized for both hESCs and hESC-CMs (Apáti et al, 2012(Apáti et al, , 2013.…”