Two Small Molecule Inhibitors of Myosin Decrease Force and Increase Rates of Relaxation in Demembranated Rat Left Ventricular Tissue

Abstract

Cell-cell and cell-substrate interactions alter the contractile behaviour of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cells are often cultured as a mono-layer on rigid tissue culture plastic (TCP) or glass, in contrast to the flexible extracellular matrix of myocardium. We examine the contractile behaviour of hiPSC-CMs on a range of substrates including standard TCP to determine the influence on the time course and synchronicity of cell contraction. HiPSC-CM (Ncardia Cor.4U or CDI iCell 2 ) were seeded on day À2 on either fibronectin-coated TCP or fibronectin-coated recombinant collagen-like polypeptide-based hydrogel (Fujifilm, Tilburg NL) with an estimated stiffness of 10kPa. Spontaneous contractility was monitored using video recordings made on days 0, 1, 3, 5 and 7 (D0-7). To assess the contractile behaviour, video frames (100fps) were subdivided into a 30x30 grid, and each grid square analysed using the MUSCLEMOTION algorithm (Sala et al., 2018). The % of single peaked-transients and the 10 th -90 th percentile difference (IP90) of contraction duration at 50% amplitude (CD50-IP90) and contraction start time (TStart-IP90) values were analysed. Values are reported as means5SD, and groups were compared to each other. Contraction synchronicity (IP90 TStart) and duration (IP90 CD50) in cultures on TCP and hydrogel were not significantly different. However, the percentage of grid squares with single-peaked transients was less in the TCP group (80.150.7% on D1 decreasing to 60.953.9% on D7) than on hydrogel (98.650.1% on D1 decreasing to 84.4513.1% on D7). The time course associated with cultures on TCP was unphysiological and indicates that the substrate is not satisfactory for hiPSC-CMs. In contrast, a single-peaked twitch contraction observed routinely on the collagen hydrogel is consistent with the normal contraction-relaxation cycle in individual cardiomyocytes. These results underline the importance of a suitable substrate for hiPSC-CM culture.