Abstract 17205: Contractility in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes is Highly Dependent on Stochastic Variability in Myofilament Development

Abstract

Introduction: Modeling heart diseases using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) enables direct study of cellular and molecular mechanisms. However, the variable phenotypes of individual cells has limited the reproducibility of contractile measurements. We hypothesized that variability in myofibrillar development in hiPSC-CMs influences the contractile kinetics and could be controlled for by visualizing and quantifying myofilaments in vitro . Methods and Results: Control hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and purified. hiPSC-CMs were replated at day ~25 onto 8.7 kPa polyacrylamide gels containing fluorescent microbeads and micropatterned with fibronectin in 7:1 aspect ratio rectangles (1750 um 2 ). Single iPSC-CMs grew into the rectangular shapes and exhibited spontaneous contractions by day 2 of replating. hiPSC-CMs were stained with live-cell actin dye and imaged on day 5-7 for fluorescent bead displacements and actin videos depicting myofilaments. Traction force analysis of bead displacements was performed to calculate whole-cell force vs. time curves at 33 msec temporal resolution. Myofilament area was measured by thresholding a binary mask on the actin live cell images. Myofilament number varied markedly among individual iPSC-CMs. iPSC-CMs with few myofilaments (myofilament area / cell area <30%) developed low force (<400 uN per cell) and were not included in subsequent analyses. Among cells with myofilament area >30%, the mean traction force was 1368±357 uN and the myofilament area demonstrated a linear correlation with maximum force (R 2 =0.19, runs test p=0.8) with slope of 0.8±0.2 uN/um 2 (p=0.002 vs zero slope). Maximum contraction velocity strongly correlated with maximum force (R 2 =0.31, runs test p=0.4) with slope of 3.9±0.9 sec -1 (p<0.0001 vs zero slope), as did maximum relaxation velocity (R 2 =0.27, runs test p=0.6, slope 4.1+1.0 sec -1 , p=0.0003 vs zero slope). Similar results were observed in two control cell lines and with two different purification methods. Conclusion: Variability in myofilament development is a critical determinant of contractile force and kinetics in individual iPSC-CMs that should be accounted for in iPSC-CM contractile experiments.