A Multiwell Cardiac μGMEA Platform for Action Potential Recordings from Human iPSC-Derived Cardiomyocyte Constructs

Figure 1. Structural and Functional Maturation of hiPSC-CMs (A) Timeline of hiPSC cardiomyocyte production and maturation. At day 10 the cardiomyocytes were quality controlled by FACS analysis of TNNT2 and MLC2v and only cultures resulting in >90% cardiomyocytes were cryopreserved for future experiments. hiPSC-CMs were thawed and cultured until day 30 prior to plating on the μGMEA. Electrophysiology (EP) experiments were conducted on 2- to 15-day-old constructs. (B) Confocal images of α-actinin, Cx43, cTnI, and DAPI nuclear stain in hiPSC-CMs. The inlet shows the zoomed in area marked with an asterisk. Images were taken with 60× objective. Calibration bar represents 50 μm. n = 3 independent experiments. DIC, differential interference contrast. (C) FACS analysis of TNNT2 and MLC2v. n = 3 independent experiments. (D) Representative calcium transients (CaTs) at 0.5 Hz (top) and 1 Hz (bottom) using ratiometric Fura-2 dye and IonOptix system. (E) CaT amplitude. (F) Vmax upstroke (maximal departure velocity of the calcium release phase). (G) Vmax decay, measurement of the speed of CaT relaxation at 0.5 and 1 Hz. Data in (B)–(G) were from day 30 hiPSC-CMs. (C) Data expressed as mean ± SEM, n = 3…

Figure 2. Long-Term Electrophysiological Studies of Cardiac μGMEA Constructs (A) Average beating frequencies (n = 41 constructs). (B) Average FP peak-to-peak amplitude. Note a significant increase in the amplitude at day 10 marking electrophysiological maturation (n = 41 constructs). (C) Average RR interval (n = 341 electrodes). (D) Average FPD (n = 341 electrodes). Data expressed as mean ± SEM. Data obtained from three independent batches. See also Figures S2 and S3.

Figure 3. Electroporation-Mediated APs Using μGMEA (A) Representative pre-, during- and post-electroporation potential recordings from an electrode across three constructs. The scale bar represents 5 s and the y axis represents voltage in mV. AP and FP recordings were obtained at 10 kHz sampling rate. (B) APs traces obtained from the same cell site over days. Note that multiple electroporation did not affect AP signal quality. (C) Representative traces of APs recorded from independent constructs using patch clamp. Scale bars represent 500 ms. (D) TTX response on AP amplitude (n = 11 constructs across three independent batches). Data expressed as mean ± SEM. See also Figure S4.

Figure 4. APD Heterogeneity across Constructs (A) APD parameter histograms representing intra- and inter-constructs variability in a single batch. APs were examined according to APD30 and APD80 parameters (total of 1,162 waveforms). y axis represents percentage frequency. (B) The summation of all histograms per category with gaussian distribution fit from batches 1, 2, and 3. The inlet represents quantile-quantile (Q-Q) plots showing normality. See also Table S1.

Figure 5. Triangulation and Fractional Shortening (A) Triangulation (APD80 − APD30) and fractional shortening (APD80 − APD30/APD80) histograms using the data in Figure 4A. (B) The summation of all histograms per category as described in Figure 4B. See also Table S1.

Figure 6. Classification of AP Waveform Morphology (A) AP waveform distribution from 363 electrodes across 41 constructs based on APD50/APD90 and APD50 − APD90. APD difference and ratio histograms in the left and right insets represent the data in the scatterplot where the blue lines indicate the ranges containing 90% of the values (302 electrodes). (B and C) APD90 − APD50 and APD50/APD90 plots, respectively, to show the overall network composition of the constructs across the array. The box plots display the interquartile range (box), median (red bar), and whiskers represent the ±1.5 times the interquartile range (IQR). Outliers are labeled with a red plus (+). N = 302 electrodes. The solid and dotted line ranges represent ventricle and atrial AP phenotype of adult cardiomyocytes, respectively. Data obtained from three independent batches. See also Table S1.

Figure 7. μGMEA Construct Response to Pharmacological Modulation (A) NE response on FPs (top) and APs (bottom). Right panel, average t-estimates (milliseconds) of percentage APDs at different doses. N = 7 constructs. (B) Same as above for Iso. N = 8 constructs. (C) E4031 response on FP (top) and AP (bottom). Note the EADs elicited by 100 nM and above. Right panel, average t-estimates (milliseconds) of APD90 at different doses. N = 9 constructs. Data expressed as mean ± SEM. Data obtained from three independent batches. The scale bar represents 2 s.