Na+/Ca2+Exchanger is a Determinant of Excitation–Contraction Coupling in Human Embryonic Stem Cell–Derived Ventricular Cardiomyocytes

Abstract

In adult cardiomyocytes (CMs), the Na⁺/Ca²⁺ exchanger (NCX) is a well-defined determinant of Ca²⁺ homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofibrillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2v-dsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While I_Ca-L, I_f, and I_Kr were robustly expressed, I_Ks and I_K1 were absent in dsRed+ ventricular hESC-CMs. Upon differentiation (7+40 to +90 days), the basal [Ca²⁺]_i, Ca²⁺ transient amplitude, maximum upstroke, and decay velocities significantly increased (P < 0.05). The I_Ca-L antagonizer nifedipine (1μM) decreased the Ca²⁺ transient amplitude (to ∼30%) and slowed the kinetics (by ∼2-fold), but Ca²⁺ transients could still be elicited even after complete I_Ca-L blockade, suggesting the presence of additional Ca²⁺ influx(es). Indeed, Ni²⁺-sensitive I_NCX could be recorded in 7+40− and +90-day dsRed+ hESC-CMs, and its densities increased from −1.2 ± 0.6 pA/pF at −120 mV and 3.6 ± 1.0 pA/pF at 60 mV by 6- and 2-folds, respectively. With higher [Ca²⁺]_i, 7+90-day ventricular hESC-CMs spontaneously but irregularly fired transients upon a single stimulus under an external Na⁺-free condition; however, without extracellular Na⁺, nifedipine could completely inhibit Ca²⁺ transients. We conclude that I_NCX is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation–contraction coupling.