Description

Gaspard Pardon2,3, Alex Chang2,3, Beth Pruitt1,3, Helen M. Blau2,3

1Department of Bioengineering at University of California, Santa Barbara; 2Department of Microbiology & Immunology, Baxter Laboratory for Stem Cell Biology, Stanford; 3Cardiovascular Institute at Stanford

Duchenne Muscular Dystrophy (DMD) is an X-linked disease affecting ~1:3500 boys per year that culminates in heart failure in early adulthood. DMD results from >200 possible genetic mutations on dystrophin. The lack of dystrophin disrupts the anchoring of the cell sarcomere to the extracellular matrix (ECM), affecting cardiomyocyte contraction. With disease progression, tissue increases in stiffness due to fibrosis and changes in ECM composition in accordance with a dilated cardiomyopathy phenotype. We hypothesize that this entails a positive feedback loop involving multiple mechanosensing pathways. Here, we use a single-cell platform to model the fibrotic remodelling in DMD. We measure the force production of single human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on hydrogel substrates with a stiffness matching that of healthy or fibrotic tissue. Furthermore, we enhance the hiPSC-CMs structural maturity and standardize our measurements by patterning single iPSC-CMs in an elongated 1:7 aspect ratio using microcontact printing of ECM proteins. We compute the contractile strength as a function of bead displacement in the hydrogel substrate using Digital Image Correlation (DIC) and Fourier Transform Traction Cytometry (FTTC). We show that DMD hiPSC-CMs have a dramatically reduced ability to produce force on stiffer substrates compared to their isogenic controls. This loss of function correlates with an increase in reactive oxygen species (ROS) and mitochondrial dysfunction. The effect of stiffness in this difference in contractile function uncovers a potent role of mechanosignaling mediated by the dystroglycan complex. This platform will increase our understanding of the biophysics underlying cardiomyocyte mechanosensing.