Mechanobiology of Myosin Mutations and Myofibril Remodeling in iPSC-Cardiomyocytes

Abstract

Hypertrophic cardiomyopathies are the most prevalent form of heritable cardiovascular disease and are caused by a host of mutations that result in hypercontractility, though the largest number of known mutations are found in beta cardiac myosin heavy chain (βMYH). Myosin is a key protein responsible for muscle contraction and is arranged into a dynamic, hierarchical structure of sarcomeres and myofibrils within cardiomyocytes. We have previously measured differences in the kinetics of isolated myosin proteins with different hypertrophic cardiomyopathy mutations in βMYH, but it has been difficult to determine how these alterations manifest at the cellular level. Induced pluripotent stem cell-derived cardiomyocytes provide a powerful tool for studying specific features of human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization. We have developed a micropatterned hydrogel platform that promotes myofibril alignment, cardiomyocyte force generation, and other metrics of maturation. Using CRISPR-Cas9 gene-editing, we have created cell lines with specific βMYH mutations, including D239N and P710R. We used traction force microscopy in a preliminary study and measured increased forces in the mutant cells compared to isogenic controls, and we have observed larger cell size in one of the mutant lines in both patterned and unpatterned contexts. Infection with LifeAct adenovirus also allows for the visualization of actin structures including myofibrils and sarcomeres during cell spreading and the recovery of beating in cells recently transferred to patterned substrates. Immunostaining for βMYH revealed higher expression and more regular organization of sarcomeres in patterned cells, and we plan to use these techniques to quantify differences in myofibril and sarcomere organization in cells with βMYH mutations. This work provides valuable insight into the etiology of hypertrophic cardiomyopathy and the utility of induced pluripotent stem cell-derived cardiomyocytes as a model for human cardiac biology and disease.