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

Fig. 1.. Experimental design using KCNJ2/Kir2.1 quantification and all-optical electrophysiology to probe the effects of Ik1-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 Ik1 contributions by sequential application of increasing doses of BaCl2.…

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…

Fig. 4.. Unmasking the contributions of inward rectifier potassium current (Ik1)-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 Ik1 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 Ik1. 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…

Fig. 5.. Irregular activity as function of cell density and suppression of inward rectifier potassium current (Ik1)-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 (Ik1)-like current on optically paced action potentials in syncytium human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and computer-simulated blocking of IK1 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 Ik1, analogous to A. D: APD90 in adult ventricular cardiomyocytes as a function of Ik1, analogous to B. E: example traces of simulated action potentials paced at 0.7 Hz for different levels of Ik1. Red boxes in C and D outline the relevant low-Ik1 (<30%) regions of the plots as likely seen in the experimental hiPSC-CMs data in…

Fig. 7.. Sensitivity to BaCl2 blocking of inward rectifier potassium current (Ik1)-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.