Spontaneous Ca2+ Oscillations in Beating Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (HIPSC-CM) and Rat Neonatal Cardiomyocytes (RN-CM)

Abstract

Isolated 4-6 day old RN-CM and HIPSC-CM beat spontaneously under culture conditions and appear to express all the ionic channels associated with the cardiac myocyte phenotype. Both Cell types continued spontaneous beating and Ca2+ oscillations at −60mV holding potentials where Na+, K+, If, and Ca2 currents were not allowed to activate. Nevertheless, inward transients of NCX currents (INCX) occurred regularly (50-150 beats per minute at ∼25 oC) with spontaneous releases of Ca2+, using Cs+-based dialyzing solutions containing 0.1-0.2 mM concentrations of Fura 2 or EGTA. Caffeine-induced Ca2+-releases also activated large INCX in both RN-CM (1.7 pA/pF, n=13) and HIPSC-CM (3.9 pA/pF, n=9) that were 2-4 times larger than those of mature rat or human myocytes. The rate and magnitude of Ca2+-oscillations and INCX increased on adrenergic stimulation, and rapid increases of [Ca2+]o from 2-5 mM. Withdrawal of [Ca2+]o and application of NCX-blocker (KBR-7943) or tetracaine also rapidly and reversibly inhibited spontaneous Ca2+-oscillations. Xestospongine C, and 2-APB applications, to probe the role IP3-signaling failed to alter spontaneous beating, as did Ca2+-channel blocker (nifedipine) and NO-synthase inhibitor L-NAME. Surprisingly, application of low concentrations of mitochondrial uncouplers (5-50 nM FCCP or 100-200 μM DNP) suppressed the spontaneous Ca2+-oscillations rapidly and reversibly in both cell types and inhibited the rate of uptake of caffeine-induced release of Ca2+. Blockers of mitochondrial-uniporter, or mitochondrial NCX were ineffective in modulating the frequency of spontaneous beating. Our data suggests that mechanisms of spontaneous pacing are similar in HIPSC-CM and RN-CM, and are mediated by possible Ca2+cross-talk between NCX, RyR/SR, and mitochondria. It is as yet unclear whether the mitochondrial Ca2+ cycling primarily initiates or modulates the spontaneous Ca2+-oscillations and beating. (NIH HL16152 and HL 107600).