Hypertrophic cardiomyopathy (HCM) is a hereditary disease in which the myocardium becomes hypertrophied, making it more difficult for the heart to pump blood. HCM is commonly caused by a mutation in the β-cardiac myosin II heavy chain. Myosin is a motor protein that facilitates muscle contraction by converting chemical energy from ATP hydrolysis into mechanical work and concomitantly moving along actin filaments. Optical tweezers have been used previously to analyze single myosin biophysical properties; however, myosin does not work as a single unit within the heart. Multiple myosin interacts to displace actin filaments and do not have the same properties as ensembles versus single molecules. We have engineered a more physiologically accurate optical trapping approach using a hierarchical cytoskeleton structure consisting of multiple myosin between two actin filaments that more closely models how myosin behaves within the heart. The model was verified using fluorescent microscopy, and we analyzed the biophysical properties of healthy myosin-actin complexes to lay the foundation for studying diseased models in the future. For the healthy myosin model, we have measured displacement profiles and force generation capacities using optical tweezers. This assay allows us to not only analyze myosin in a more physiologically relevant environment but also to study how multiple myosin interact within cardiac muscle cells
