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(+)/Ca(2+) exchanger (NCX) is a well-defined determinant of Ca(2+) 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 MLC2vdsRed 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 hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca(2+)](i), Ca(2+) transient amplitude, maximum upstroke, and decay velocities significantly increased (P < 0.05). The I(Ca-L) antagonizer nifedipine (1 microM) decreased the Ca(2+) transient amplitude (to approximately 30%) and slowed the kinetics (by approximately 2-fold), but Ca(2+) transients could still be elicited even after complete ICa-L blockade, suggesting the presence of additional Ca(2+) influx(es). Indeed, Ni(2+)-sensitive INCX 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 [Ca(2+)](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 Ca(2+) transients. We conclude that I(NCX) is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation-contraction coupling.

FIG. 1.

DsRed+ cells differentiated from stably LV-MLC2v-dsRed-transduced human embryonic stem cells (hESCs) displayed a ventricular phenotype. (A) Representative images of MLC2v-dsRed hESCs differentiating embryoid bodies (EBs) at different stages. Beating areas are circled by dash lines. Bars represent 50 μm. (B) A representative single isolated and FACS-sorted 7+90-day dsRed+ hESC-cardiomyocytes (CMs). Bar indicates 20 μm. (C) dsRed+ cells were stained positively for MLC2v (upper) but not MLC2a staining (lower). Bar indicates 20 μm. (D) Typical ventricular action potential (AP) recorded from a single dsRed+ hESC-CM. Atrial AP could only be observed in non-dsRed hESC-CMs. (E) ZD7288-sensitive pacemaker current (I_f), but no I_K1, were expressed in dsRed+ hESC-CMs. (F) E4031-sensitive I_Kr, but not I_Ks, were expressed in dsRed+ hESC-CMs. Color images available online at www.liebertonline.com/scd.

FIG. 2.

Ca²⁺ transients recorded from 7+40- to 7+90-day dsRed+ ventricular hESC-CMs. (A) Representative tracings of Ca²⁺ transients. (B–E) Comparison of the basal [Ca²⁺]_i (B), amplitude (C), maximum upstroke velocity (V_max-upstroke, D), and maximum decay velocity (V_max-decay, E) of Ca²⁺ transients in 40-day (n = 9) and 90-day (n = 10) dsRed+ ventricular hESC-CMs. ^*P < 0.05, ^**P < 0.01.

FIG. 3.

The inhibitory effect of nifedipine on Ca²⁺ transients during ventricular hESC-CM differentiation. (A) L-type Ca²⁺ current (I_Ca,l) in 7+40-day dsRed+ ventricular hESC-CMs and (B) the corresponding current–voltage (I-V) relationship. (C) Representative tracings of Ca²⁺ transients in 7+40- and 7+90-day dsRed+ cells before and after nifedipine treatment. Bar graphs of normalized amplitude (D) and maximum upstroke velocity (V_max-upstroke, E) of Ca²⁺ transients after nifedipine. ^**P < 0.01.

FIG. 4.

Developmental changes of NCX current (I_NCX) in dsRed+ ventricular hESC-CMs. (A) The transcriptional expression of Na⁺/Ca²⁺ exchanger (NCX) 1, NCX2, and NCX3 in hESCs, hESC-ventricular (V) CMs, fetal VCMs (FV), and adult VCMs (AV). (B) Representative tracings of Ca²⁺ transients in 7+40- and 7+90-day dsRed+ cells before and after Ni⁺ treatment. (C) Protocol and typical tracings of I_NCX, and (D) I/V curve of I_NCX in 7+40- and 7+90-day dsRed+ cells. ^*P < 0.05, ^**P < 0.01.

FIG. 5.

I_NCX contributes to E-C coupling of ventricular hESC-CMs. (A) Representative tracings of Ca²⁺ transients in 40-day and 90-day dsRed+ cells with and without external Na⁺. Normalized basal [Ca²⁺]_i (B), amplitude (C), and maximum decay velocity (V_max-decay, D) of Ca²⁺ transients in 7+40- and 7+90-day ventricular hESC-CMs after replacing with a Na⁺-free external solution. ^*P < 0.05; ^**P < 0.01. (E) Ca²⁺ transients were completely blocked in ventricular hESC-CMs treated with nifedipine and Na⁺-free solution.