Engineered Single Human iPSC-Cardiomyocytes to Assay Contractile Defects Induced By Drugs and Disease States

Human induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hiPSC-CMs) and have the potential to model cardiac function. We developed, studied and validated a platform where hiPSC-CMs are engineered to present a more mature structural organization and are also functionally assayed. We specifically analyzed different parameters related to the contractile function of these engineered hiPSC-CMs and tested their ability to model the effects of drugs and disease states in cardiac function.

Introduction: The contractility of cardiomyocytes enables the beating of the heart. Sarcomeres are the contractile units of cardiomyocytes. In mature cardiomyocytes sarcomeres are distributed in series along myofibrils that are well aligned in the intracellular space. Within each sarcomere, the contractile interactions of myosins with actin filaments induce sarcomere shortening and cell beating. However, hiPSC-CMs lack the distribution and organization of sarcomeres and myofibrils that characterize mature cardiomyocytes. We engineered single hiPSC-CMs on polyacrylamide hydrogels with physiological stiffness to induce a mature cell shape and more mature organization of sarcomeres and myofibrils. We tested how the contractility of engineered hiPSC-CMs varied under disease conditions and due to drugs known to affect heart contractile activity. We also investigated the role of extracellular physical cues in driving the structural maturity in engineered hiPSC-CMs via a tension-driven mechanism.

 

Materials and Methods:To engineer single hiPSC-CMs, we seeded cells singularized from beating monolayers on polyacrylamide substrates of physiological stiffnesses with micropatterned rectangles composed of Matrigel. Rectangular micropatterns in association with a substrate stiffness of 10 kPa drove hiPSC-CMs to have a more mature shape and an aligned organization of sarcomeres and myofibrils. We dispersed fluorescent microbeads in the polyacrylamide substrate to calculate contractile and kinetic parameters of cell beating from traction force microscopy measurements. We also labeled actin in sarcomeres with LifeAct in live cells to measure the dynamics of sarcomere shortening and myofibril organization. We tested the role of tension in cell function by varying substrate stiffness and the length/width ratio of micropatterns. We analyzed variations in contractility and sarcomere activity induced by mutations that model heart disease, such as MYBPC3 gene knockout and mutations in GATA4 and MYH7 genes. Contractile variations induced by incubating cells in caffeine, isoproterenol and omecamtiv mercanbil were also studied.

 

Results and Discussion: We observed that cell shape and the stiffness of the substrate induced structural maturation of sarcomeres and myofibrils in engineered hiPSC-CMs via a tension mechanism. We detected different variations in the contractility of cells due to disease states and induced by isoproterenol and omecamtiv mercanbil.

 

Conclusions and Future Work: We validated a platform for systematic measuring the contractility of hiPSC-CMs and modeling cardiac activity in a dish to study the effects of heart disease and drugs in a dish. We also identified intracellular tension as a key cue in the structural maturation of these engineered cells. Altering intracellular and extracellular biological properties may further control tension in structurally maturing hiPSC-CMs.