Abstract 901: Muscle-specific Stress Fibers Give Rise to Sarcomeres in Cardiomyocytes

Abstract

The sarcomere is the contractile unit that drives muscle contraction. Despite its importance, little is understood about how a disordered acto-myosin distribution converts into an ordered contractile array during sarcomere assembly. Here, we take advantage of a sarcomere assembly assay we developed using human induced pluripotent stem cell derived cardiomycytes to image the formation of sarcomeres, using live-cell high resolution microscopy. Our data show that a population of muscle specific stress fibers (MSFs) are essential sarcomere precursors. Interestingly, MSFs are formed at the leading edge of cells, undergo retrograde flow, and transition into sarcomere-containing myofibrils on the dorsal surface of cardiomyocytes. This is in direct contradiction to a recent report claiming sarcomeres are formed from adhesions on the ventral surface of cardiomyocytes. We have been able to recapitulate this other group's published experiments and definitively show that sarcomeres are not forming from the bottom of the cells or streaming out of adhesions. Instead, our 3D microscopy data shows that this group was imaging sarcomeres which were already formed on the dorsal surface and were traveling to the ventral surface of the cells. After this important clarification, we used our assay to show that the transition of MSFs to sarcomere-containing myofibrils requires formin-mediated actin polymerization and the non-muscle myosin IIA and myosin IIB. We conclude that sarcomeres form by a "templating" mechanism similar to that originally postulated by Howard Holtzer >30 years ago. Furthermore, our data show short β cardiac myosin II filaments are themselves templated by "non-muscle" myosin II filaments. Subsequently, the short β cardiac myosin II filaments grow to form ~1.5 μm long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). Taken together, our data show that the differentiation of cardiomyocytes from stem cells is a powerful tool for dissecting the mechanisms controlling sarcomere assembly.