Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (I_k1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking I_k1-like currents with BaCl₂ increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with I_k1's role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.

Keywords: Ik1; Kir2.1; all-optical electrophysiology; cardiac electrophysiology; cell density; human iPSC-cardiomyocytes; maturity; optogenetics.

Fig. 1.

Experimental design using KCNJ2/Kir2.1 quantification and all-optical electrophysiology to probe the effects of I_k1-like current contributions in syncytia of human iPSC-CMs. A: 96-well plate format was used for functional tests. High (H), medium (M), and low (L) cell densities were achieved by plating: 50,000, 25,000, and 12,500 cells per well, respectively. Shown are example immunofluorescence images from the 3 density groups, using α-actinin (red) and nuclear DAPI labeling (blue). Scale bar, 20 μm. B: protein quantification for human adult heart tissue samples, rat heart, and human iPSC-CMs grown in 6-well plates was done using standard Western blot, while 96-well format small samples of hiPSC-CMs at 2 densities were processed using capillary-based Western blot (Wes by ProteinSimple); Cells-to-CT by Invitrogen was used for quantification of mRNA by qPCR. B was created with BioRender.com. C: functional measurements were done using all-optical electrophysiology. Example traces and image of a dual-labeled sample for voltage and calcium recordings are shown. Scale bar, 50 μm. D: pharmacological probing for I_k1 contributions by sequential application of increasing doses of BaCl₂. Functional (voltage and calcium) data were simultaneously collected 35 min after each dose treatment under spontaneous conditions (S) and under optical pacing. I_k1, inward rectifier potassium current; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocytes; KCNJ2, potassium inwardly rectifying channel subfamily J member 2.

Fig. 2.

Protein quantification of Kir2.1 in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), adult human ventricle, adult and neonatal rat ventricular tissue, and wild-type HeLa cells as a negative control. A: Western blot results for Kir2.1 protein expression and GAPDH as a loading control. Kir2.1 protein is detected at around 48 kDa. B: Western blot quantification based on normalized Kir2.1/GAPDH ratio (n = 4–9 per sample type, data pooled from 5 different runs). Tissue samples came from female and male adult human left ventricle as n = 1 biological replicate per sex, with n = 5 and 4 technical replicates, neonatal and adult rat hearts as n = 2, and 1 biological replicate with n = 8 and 4 technical replicates; iPSC-CM samples as n = 2 biological replicates with n = 9 technical replicates came from different cell cultures. Data were normalized by the values for the female human ventricle (which was included as a sample in all 5 runs) and shown as box-whisker plot.

Fig. 3.

Gene expression [KCNJ2 (potassium inwardly rectifying channel subfamily J member 2)] and protein expression (Kir2.1) are reduced in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured at lower plating densities for female and male hiPSC-CMs. Gene expression of KCNJ2 (A and C) and protein expression of Kir2.1 (B and D) were quantified in high-density (50,000 cells/well) and low- to medium-density (18,500 cells/well) of hiPSC-CM cultures in a 96-well format, using qPCR and Wes, respectively. Normalization was done by gene or protein levels of GAPDH in the same sample. Female hiPSC-CMs (the standard iCell2), orange, and male donor hiPSC-CMs (MyCell iPSC-CM-1X 01395 donor), gray, were used. qPCR data are presented as n = 3 biological replicates with n = 3 technical replicates per density and sex. WES data are presented as n = 3 biological replicates per density and sex. Male and female cell samples were done in separate runs. For WES controls, wild-type (WT) HeLa cells were used as a negative control and human left ventricle (LV) as a positive control for Kir2.1. Analysis of variance yielded significant differences in the qPCR data due to cell density and due to sex, whereas the protein data did not reach significance but followed a similar trend.

Fig. 4.

Unmasking the contributions of inward rectifier potassium current (I_k1)-like current to spontaneous activity across different cell culture densities using BaCl₂. A: spontaneous frequency (Hz) of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) decreases with the increase of cell culture density (*P < 0.05); 2-fold and 1.3 times higher frequency in low- and medium-density groups compared with high-density samples. B: pharmacological probing with different doses of BaCl₂ and changes in spontaneous frequency (%) from baseline. The syncytial high-density group shows a dose-dependent increase in the frequency of oscillations, as expected from I_k1 block (*P < 0.05). Effects of BaCl₂ without clear dose dependence in low-density samples and weaker effects in medium-density samples are potentially indicative of minimal I_k1. Data for this plot were from several locations within n = 6 multicellular samples (5 for the low-density group with several isolated regions), subjected to the full sequential drug treatment protocol (12 cases of density and drug dose), data are presented as box-whisker plot. Additional experiments with parallel (nonsequential) administration of BaCl₂ doses were done with a different batch of cells, yielding similar outcomes, as presented in Supplemental Fig. S3.

Fig. 5.

Irregular activity as function of cell density and suppression of inward rectifier potassium current (I_k1)-like current by BaCl₂. A: irregular activity is prominent in the low-density group at baseline (no BaCl₂) and is brought about in medium- and high-density groups by high doses of BaCl₂. B: example traces (spontaneous voltage and calcium) for regular and irregular activity. These results were from multiple records n = 11–29 per case (12 cases of density and drug dose); see Table of record numbers in the Supplemental Material.

Fig. 6.

Effects of BaCl₂ blocking of inward rectifier potassium current (I_k1)-like current on optically paced action potentials in syncytium human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and computer-simulated blocking of I_K1 in ventricular cells. A: experimentally obtained maximum upstroke velocity (a.u.) from voltage-sensitive fluorescence dye’s records (∆F/F) of optically paced action potentials, 0.7 Hz.B: action potential duration at 90% repolarization (APD₉₀) under different BaCl₂ doses. Upstroke velocity decreases as function of BaCl₂ dose, whereas APD₉₀ shows minor changes with BaCl₂ treatment. Data are presented as box-whisker plot (n = 4 to 9 high-density multicellular samples for each of the drug doses; multiple locations averaged). C: computational results for upstroke velocity in adult ventricular cardiomyocytes as a function of I_k1, analogous to A. D: APD₉₀ in adult ventricular cardiomyocytes as a function of I_k1, analogous to B. E: example traces of simulated action potentials paced at 0.7 Hz for different levels of I_k1. Red boxes in C and D outline the relevant low-I_k1 (<30%) regions of the plots as likely seen in the experimental hiPSC-CMs data in A and B, found to agree.

Fig. 7.

Sensitivity to BaCl₂ blocking of inward rectifier potassium current (I_k1)-like current as function of cell density. Dose response of experimentally obtained maximum upstroke velocity (a.u.) of optically paced action potentials when BaCl₂ is applied. Effects vary between the high (H)-, middle (M)-, and low (L)-density syncytium human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) cultures. Inset: changes of means in upstroke of BaCl₂ treated (0.5 mM) samples and control samples for the 3 cell densities, with the most pronounced seen in the high-density group. Data (n = 3–9 multicellular samples for each of the 12 cases of density and drug dose, multiple locations averaged) are shown as box-whisker plot.