Novel method for action potential measurements from intact cardiac monolayers with multiwell microelectrode array technology

Abstract

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

Figure 1

LEAP induction transforms field potentials to action potentials. (a) CytoView MEA 96-well plate with inset showing Bright Field 4x Magnification image of iCell CM² cultured in a single well atop eight planar microelectrodes. (b) Example signal transformation on a single planar electrode from before and after LEAP induction. (c) LEAP and FP waveforms (averaged across 5 beats) recorded from neighboring electrodes in the same well. Markers for depolarization (LEAP: rise time; FP: AMP) and repolarization (LEAP: APD at various stages of repolarization; FP: FPD) are indicated. Gray dashed line highlights that FPD occurs between APD50 and APD90.

Figure 2

LEAP is stable over long time courses unlike electroporation. Electroporation was applied across 42 wells on a Classic MEA 48-well plate cultured with iCell CM². Following electroporation decay, LEAP was applied to the same wells. (a) Representative electroporation signal showing rapid decay back to FP. Inset shows FP shape at two minutes post-induction. (b) Representative LEAP signal with stable amplitude. Inset shows stable LEAP shape persists at two minutes post-induction. (c) Percentage of wells with a stable AP signal at 0, 2, 5, 10, and 20 minutes post-induction. LEAP persisted for more than 20 minutes, whereas electroporation rapidly decayed.

Figure 3

LEAP does not disrupt cardiomyocyte function or behavior. (a) FPs were recorded before and 48 hours after LEAP induction on all electrodes of a Classic MEA 96-well plate with iCell CM². The FP was not affected by LEAP induction. (b) FPDc before and after LEAP were tightly correlated (R² = 0.84, p < 0.001). Each dot represents the FPDc before and 48 hours after LEAP induction for a well, with the unity line in black for comparison. (c) Here, LEAP induction was applied to half of the electrodes in half of the wells on a Classic MEA 96-well plate with iCell CM². FPs were recorded before and 60 minutes after LEAP induction. Bar plots represent the percent change (mean ± standard deviation across wells) in BP and FPDc, measured from the FP signal, from before to 60 minutes post-LEAP induction. Changes in BP and FPD did not differ between control (no LEAP) and LEAP wells (Mann Whitney U-Test, BP p = 0.097, FPDc p = 0.77).

Figure 4

Simultaneous LEAP and FP measurements establish translation between FP and AP signals. LEAP was induced on half of the microelectrodes in each well of a Classic MEA 48-well plate of iCell CM². (a) Representative well showing LEAP waveforms from eight electrodes (left) and FPs from eight electrodes (right). (b) Despite differences in signal amplitude, LEAP shapes were consistent across electrodes in a well. LEAP waveforms (averaged across 5 beats) from each of eight electrodes are shown in gray with the mean across electrodes overlaid in black. APD30, 50, and 90 are marked with gray dots on each electrode trace. (c) APD90 and 50 were correlated with FPD across all wells. Each dot represents the FPD and APD90 (black) or APD50 (gray) from a single well (n = 48 wells). A best fit linear regression is plotted for APD90 (black) and APD50 (gray) as a function of FPD (t-test for slope vs zero, p < 0.001). (d) Example FP and LEAP signals from the same well when dosed with DMSO (left) or E-4031 (middle, right). With vehicle control, depolarization and repolarization aligned between the FP and LEAP. With hERG block, both the FP and LEAP were prolonged and EADs developed. EADs were automatically detected on the LEAP signals (filled white triangles) and visually identified on the FP (open white triangles) for direct comparison of features.

Figure 5

LEAP signals reproduce expected responses to ion channel blockers. Cardiomyocytes (iCell CM²) plated on Classic MEA 48-well plates were dosed with vehicle control or one of four doses of Nifedipine (a), E-4031 (b), and Verapamil (c). For each compound, amplitude normalized LEAP waveforms (averaged across 5 beats) from representative wells are overlaid for DMSO (black) and the four increasing concentrations of each compound (dark gray, light gray, orange, and teal). Triangles indicate automatically detected EADs. Bar plots represent the mean ± standard deviation of APD30, 50, and 90 across replicate wells (n = 8 for DMSO, n = 5 for each concentration of Nifedipine and E-4031, n = 3 for each concentration of Verapamil). The highest dose of Verapamil stopped cardiomyocyte beating.

Figure 6

LEAP quantifies rise time and triangulation to reveal subtle changes in action potential morphology. (a) Coyne hiPSC-derived cardiomyocytes were plated on a Classic MEA 96-well plate and dosed with vehicle control or one of four doses of Astemizole (a,b,c). The rising phase of LEAP waveforms (averaged across 5 beats) from representative wells are overlaid for DMSO (black) and the four increasing concentrations of Astemizole (dark gray, light gray, teal, orange). Astemizole prolonged the rise time of the AP. (b) Bar plots represent the mean ± standard deviation of rise time across replicate wells (n = 13 for DMSO, n = 6 for each concentration of Astemizole). One of six wells became quiescent (Q) in response 100 nM of Astemizole. (c) Prolonged rise time corresponded to a reduction in FP AMP, with several wells becoming quiescent (Q) particularly at higher doses. (d) iCell CM²cardiomyocytes were dosed with vehicle control or one of four doses of Terodiline (d,e,f). LEAP waveforms (averaged across 5 beats) from representative wells are overlaid for DMSO (black) and three increasing concentrations of Terodiline (light gray, teal, orange). Terodiline shutdown beating in all replicates at the fourth and highest dose (Q, 10 µM). (e) Bar plots represent the mean ± standard deviation of the triangulation ratio across replicate wells (n = 10 for DMSO, n = 5 for each concentration of Terodiline). (f) AP triangulation corresponded to broadening and shrinking of the T-wave of the corresponding FP. FPs (averaged over 5 beats) at baseline (black) and dosed with 3 µM Terodiline (teal) are shown for a representative well.

Figure 7

LEAP morphology from hiPSC-derived and primary rodent cardiomyocytes. (a) LEAP repolarization timing is shown for four sources of hiPSC-derived cardiomyocytes. Repolarization is represented as the mean ± standard deviation of APD across wells for APD0 to APD90 (iCell CM2 n = 20 wells, Cor.4U n = 17 wells, Coyne n = 24 wells, Pluricyte n = 5 wells). (b) LEAP repolarization timing is shown for GFP-transfected (n = 17) and Tbx18-transfected (n = 8) primary neonatal rat ventricular cardiomyocytes. (c) Tbx18 transfection altered AP morphology by decreasing APD30 and APD90 relative to GFP-transfection (Mann Whitney U-Test, APD30 p < 0.001, APD90 p < 0.001). (d) GFP-transfected cardiomyocytes exhibited more regular beating than Tbx18 (GFP BP CoV = 8.47 ± 8.50, Tbx18 BP CoV = 39.39 ± 29.23, p < 0.001).

Figure 8

Optical and electrical pacing of LEAP signals can be used to control beat period and reveal rate-dependent effects. ChR2-transduced Cor.4U were paced using 5 ms blue light pulses at 1.5, 2, 2.5, and 3 Hz for 60 seconds each (a,b,d). Similarly, iCell CM² cardiomyocytes were paced using a dedicated stimulation electrode (50 µA, 200 µs) at 0.83, 1, 1.25, 1.5, and 2 Hz (c,e). Both experiments were carried out on CytoView MEA 24-well plates. (a) BP, FPD, and APD90 are shown from one example well across the four rates. (b) Optically paced Cor.4U LEAP waveforms (averaged across 5 beats) from a representative well are shown at each rate. (c) Electrically paced iCell CM² LEAP waveforms (averaged across 5 beats) from a representative well are shown at each rate. (d) When Cor.4U cardiomyocytes were dosed with 100 µM Sotalol, the AP was prolonged. Due to this prolongation, pacing at higher rates was not achieved. (e) Similarly, when iCell CM² cardiomyocytes were dosed with 30 µM Sotalol, the AP was prolonged with greatest prolongation occurring at slower rates. EADs were present at slow rates, but not at faster rates.