Contractile Properties of Myofibrils from hiPSC-Derived Cardiomyocytes of Patients with Duchenne Muscular Dystrophy

Abstract

Duchenne Muscular Dystrophy (DMD) is a wasting disease of striated muscle resulting from membrane fragility. We modeled DMD cardiac disease using urine-derived cells from a patient, reprogrammed to induced pluripotent stem cells (hiPSCs) and differentiated into cardiomyocytes (DMD-hiPSC-CMs). DMD-hiPSC-CMs were dystrophin-deficient (exon 50 deletion) and manifested physiological consequences of the disease such as calcium-handling abnormalities. Here we report, for the first time, isolation and functional characterization of myofibrils from hiPSC-CMs to study DMD cardiomyopathy.At day 20 post-differentiation, DMD-hiPSC-CMs and control hiPSC-CMs (from a healthy volunteer) were replated onto fibronectin-coated nanopatterned coverslides and cultured until day 80. Both DMD- and controls-CMs exhibited more mature morphology with aligned myofibrils and clearly defined Z-bands, cell lengths of 100-150µm and widths of 40µm. For mechanical measurements, cells were harvested and skinned in a rigor solution containing Triton 1% for 5 minutes.Control hiPSC-CM myofibrils had mechanical and kinetic proporties more similar to human fetal skeletal (Racca, 2013) or cardiac myofibrils than human adult cardiac myofibrils. Preliminary data for DMD-hiPSC-CMs showed lower force development, prolonged duration of early, slow phase relaxation kinetics (tREL slow), but no differences in the other kinetic properties. Preliminary data from intact DMD-hiPSC-CMs paced at 1Hz showed no difference in the rate of cell shortening, but depressed magnitude and prolonged relaxation (t50). We previously reported (Xuan G, 2014) prolongation of Ca2+ transient decay.Together these data suggest DMD-hiPSC-CMs have slower relaxation due to both myofibril properties and Ca2+ sequestration, compared with control hiPSC-CMs, and both cell lines have myofibril mechanic and kinetic properties more similar to fetal than adult myofibrils.In conclusion, we demonstrated that isolated functional myofibrils from hiPSC-CMs can be obtained after growing on nano-patterned surfaces in culture to study cardiac diseases in vitro.