The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stemcell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor. 4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneouslymonitors 2 wavebands of emitted fluorescence, allowing ratiometricmeasurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though themean and variance differed significantly (APD(90): 229 +/- 15ms vs 427 +/- 49ms [mean +/- SD, P< 0.01]; average spontaneous cycle length: 0.99 +/- 0.02 s vs 1.47 +/- 0.35 s [mean +/- SD, P< 0.01], Cor. 4U vs iCell CMC, respectively). The 10-90% rise time of the AP (T-rise) was similar to 6ms and was normally distributed when expressed as 1/T-rise(2) in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide aminimally invasive, quantitative, and accuratemethod to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.