IK1-enhanced human-induced pluripotent stem cell-derived cardiomyocytes: an improved cardiomyocyte model to investigate inherited arrhythmia syndromes

Abstract

Currently available induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) do not ideally model cellular mechanisms of human arrhythmic disease due to lack of a mature action potential (AP) phenotype. In this study, we create and characterize iPS-CMs with an electrically mature AP induced by potassium inward rectifier (IK1) enhancement. The advantages of IK1-enhanced iPS-CMs include the absence of spontaneous beating, stable resting membrane potentials at approximately -80 mV and capability for electrical pacing. Compared with unenhanced, IK1-enhanced iPS-CMs calcium transient amplitudes were larger (P < 0.05) with a typical staircase pattern. IK1-enhanced iPS-CMs demonstrated a twofold increase in cell size and membrane capacitance and increased DNA synthesis compared with control iPS-CMs (P < 0.05). Furthermore, IK1-enhanced iPS-CMs expressing the F97C-CAV3 long QT9 mutation compared with wild-type CAV3 demonstrated an increase in AP duration and late sodium current. IK1-enhanced iPS-CMs represent a more mature cardiomyocyte model to study arrhythmia mechanisms.

Keywords: LQTS; arrhythmia; iPS-CM; potassium ion channel.

Fig. 1.

Potassium inward rectifier (I_K1) from I_K1-enhanced induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). A: representative I_K1 recorded from iPS-CMs in baseline (dark grey), barium (500 μM, light grey), and barium-sensitive current (black). B: average current density from I_K1-enhanced iPS-CMs n = 7. Recordings were done using a ramp protocol shown in inset. C: sample I_K1 current recordings from iPS-CMs baseline (black), barium (dark grey), and barium-sensitive current (light grey). D: sample action potentials from 2 different iPS-CMs showing spontaneous activity and different maximum diastolic potentials. Results from the analysis of spontaneous activity are reported in Table 1. E: protocol used to record capacitance is shown at top and representative capacitance traces from iPS-CMs (black) and I_K1-enhanced iPS-CMs (grey) are shown at bottom.

Fig. 2.

Action potential (AP) characteristics from I_K1-enhanced ventricular-like iPS-CMs. Representative AP from ventricular-like (A) I_K1-enhanced iPS-CMs when paced at 0.5 Hz, 1 Hz, 2 Hz and 3 Hz. Scale bar is applicable to all APs. Resting membrane potentials from ventricular-like (B) I_K1-enhanced iPS-CMs at various pacing frequencies. Maximum upstroke velocity from ventricular-like (C) I_K1-enhanced iPS-CMs various pacing frequencies. Action potential duration at 10% (APD₁₀), 50% (APD₅₀), 70% (APD₇₀), and 90% (APD₉₀) at pacing frequencies of 0.5 Hz (back), 1 Hz (dark grey), 2 Hz (grey), and 3 Hz (white) calculated from ventricular-like (D) I_K1-enhanced iPS-CMs are shown. *Significant difference on comparison with respective APD at 0.5 Hz; #significant difference on comparison with respective APD at 1 Hz. Statistical significance was calculated by one-way ANOVA with Bonferroni correction. N = number of cells, n = number of AP. B and D report N within the bars.

Fig. 3.

Characterization of the calcium transients from I_K1-enhanced iPS-CMs and viral control-infected iPS-CMs. A: representative calcium recordings from viral infected control iPS-CMs (black) and I_K1-enhanced iPS-CMs (grey). Spontaneous activity was recorded from the groups shown at left, the same cells paced at 0.5 and 1 Hz are reported at middle and right, respectively. B: average data of the basal calcium concentration (left), the peak amplitude (middle), and the time constant of calcium reuptake (right) for viral control-infected (black) and I_K1-enhanced (grey) iPS-CMs. The peak amplitude and time constant were calculated at both the 0.5- and 1-Hz pacing frequency. *P < 0.01.

Fig. 4.

I_K1-enhancement changes iPS-CMs capacitance and size. A: capacitance of I_K1-enhanced iPS-CMs compared with uninfected and CAV3 WT/F97C-infected iPS-CMs. B: cell size measurement of I_K1-enhanced iPS-CMs compared with viral control-infected iPS-CMs. C: I_K1-enhanced iPS-CMs immunostained for α-actinin (green), MLC2V (red), and DAPI (blue). D: GFP-infected iPS-CMs immunostained for α-actinin (green), MLC2V (red), and DAPI (blue). Scale bar = 25 μm. *P < 0.01.

Fig. 5.

I_K1-enhanced iPS-CMs demonstrate increased DNA synthesis compared with viral control-infected iPS-CMs. A: representative photomicrographs of viral control-infected iPS-CMs (a, b, and c), and I_K1-enhanced iPS-CMs (d, e, and f) stained with DAPI (blue, a and d), 5-bromo-2-deoxyuridine (BrdU; red, b and e), and merged (c and f). Scale bar = 400 μM. B, top: the percentage of BrdU-positive cells are increased in I_K1-enhanced iPS-CMs compared with viral control-infected iPS-CMs (P = 0.0001). B, bottom: percentage of binucleated cells in increased in I_K1-enhanced iPS-CMs compared with viral control (*P = 0.004). C: representative photomicrographs of viral control-infected iPS-CMs (g, h, and i) and I_K1-enhanced iPS-CMs (j, k, and l) stained with BrdU (blue, g and j), Kir2.1 (red, h and k), and merged (i and j). Regions identified in i and l are zoom in i1, i2, and l1, l2, respectively, with the colors as identified before. Scale bar = 50 μm.

Fig. 6.

Long QT syndrome 9 (LQT9) causing F97C-CAV3 mutation increases I_Na-L. A: representative traces of I_Na in the presence of WT-CAV3 (top, left), F97C-CAV3 (bottom, left), I_K1-enhanced + WT-CAV3 (top, right), and I_K1-enhanced + F97C-CAV3 (bottom, right). B: average current-voltage (I–V) relationship for I_Na in iPS-CMs expressing WT-CAV3 (black, squares, n = 10) or F97C-CAV3 (red, circles, n = 10) or I_K1-enhanced + WT-CAV3 (black, diamonds, n = 13) and I_K1-enhanced + F97C-CAV3 (red, hexagons, n = 11). Inset: protocol used to record I_Na. C: representative traces of I_Na-L recording from iPS-CMs expressing WT-CAV3 (black) and F97C-CAV3 (red), I_K1-enhanced + WT-CAV3 (green), and I_K1-enhanced + F97C-CAV3 (blue). Inset: protocol used to measure the current. D: average data reporting ratio of I_Na-L to I_Na-P at −20 mV from iPS-CMs expressing WT-CAV3 (black, n = 9) and F97C-CAV3 (red, n = 9), I_K1-enhanced + WT-CAV3 (green, n = 10), and I_K1-enhanced + F97C-CAV3 (blue, n = 11). *Significant difference between the 2 groups.

Fig. 7.

Expression of F97C-CAV3 in I_K1-enhanced ventricular iPS-CMs prolongs APD and induces early afterdepolarizations (EADs). A, D, and G: representative AP from I_K1-enhanced iPS-CMs expressing WT-CAV3 paced at 0.5, 1, and 2 Hz, respectively. B and E: representative AP from I_K1-enhanced iPS-CMs expressing F97C-CAV3 paced at 0.5 and 1 Hz, respectively. C: APD₁₀, APD₅₀, APD₇₀, and APD₉₀% repolarization from peak at a pacing frequencies of 0.5 calculated from ventricular-like I_K1-enhanced iPS-CMs expressing WT-CAV3 (black) or F97C-CAV3 (grey). F: APD₁₀, APD₅₀, APD₇₀, and APD₉₀% repolarization from peak at a pacing frequencies of 1 Hz calculated from ventricular-like I_K1-enhanced iPS-CMs expressing WT-CAV3 (black) or F97C-CAV3 (grey). H: EADs generation in I_K1-enhanced iPS-CMs expressing F97C-CAV3 when paced at 0.33Hz. I: resting membrane potential reported at 3 pacing frequencies from iPS-CMs coexpressing Kir2.1 and WT/F97C-CAV3. *Significant difference between the 2 groups at the respective APD. N = number of cells, n = number of AP (scale bars in G applicable to all but H).