Engineered Cardiac Tissue As a Novel in Vitro Human Myocardium Model for Studying Cardiac Stretch.

Heart disease is the largest contributor to death worldwide and research is limited by a lack of adequate human tissue in vitro models. To address this, we developed a protocol for generating 3D, atrial-like engineered cardiac tissue (ECT) comprised of cardiomyocytes (CM) and cardiac fibroblasts, derived from human-induced pluripotent stem cells (hiPSC). Control and atrial-like hiPSC-CM were produced using GiWi differentiation supplemented without and with 0.75 µM retinoic acid, respectively. ECTs were generated using hiPSC-cardiac fibroblasts and Day 30 hiPSC-CM combined in a fibrin matrix and molded via a FlexCell Tissue TrainTM system. ECTs were cultured for 30 days and evaluated for action potentials (AP) as well as mRNA and protein expression profiles. A subset of control ECTs were cyclically and incrementally stretched for 7 days up to 18% elongation. Stretch-induced changes were evaluated via transmission electron microscopy (TEM) and cell culture medium (LC-MS/MS). Atrial-like ECTs recapitulated various atrial phenotypes. They exhibit an atrial-like electrophysiology with a shorter AP duration and increased repolarization fraction (p<0.05). Atrial-like ECT show atrial-specific mRNA/protein expression, such as lower ventricular-like MYL2 and higher atrial-like MYL7 levels (p<0.05). They also demonstrate distinct contraction mechanics with faster kinetics (p<0.05) and decreased normalized contraction force, compared to control ECT. Cyclic stretch reduced cardiomyocyte membrane convolution index (estimating membrane tension) and caveolae membrane structure density (p<0.05). The latter was rescued via neutral sphingomyelinase inhibitor GW4869 (20 µM, p<0.05). LC-MS/MS of stretch-conditioned culture medium revealed decreases in relative sphingomyelins and phosphatidylcholines (p<0.05), ostensibly indicating membrane damage which was reversed by GW4869. Overall, our findings demonstrate retinoic acid-induced atrial-like phenotypes in hiPSC-CM are sustained in ECT form. We also introduce a novel ECT stretch protocol that recapitulates changes observed in animal models of heart pressure overload.