(117d) Elasticity Effects On the Origin of Heartbeat

Recent studies have shown that using stem-cell derived cardiomyocytes is a promising avenue for cardiovascular therapy. Current efforts are invested towards developing a culture system that increases the yield of cardiomyocytes from stem cell cultures. Spontaneous beating is a commonly used phenotype for determining the percent of cardiogenesis in a stem cell culture. However, at the single cell-level, even at early stages of differentiation, stem cell-derived cardiomyocytes have already differentiated into different sub-phenotypes: atrial, ventricular, and sinusnode-like, all with different beating behaviors. Designing a culture substrate that optimizes a certain long-term spontaneous beating behavior may drive cardiogenesis towards a single sub-phenotype. Use of an atrial-rich or sinusnode-like rich cardiomyocyte populations in clinical therapy may lead to poor electrical coupling of grafted cells and later arrhythmias in the heart. Therefore it is important to understand the effects substrate may have on cardiomyocyte sub-phenotype differentiation. This study focuses on the long-term effects of substrate elasticity on cardiomyocytes in culture. Early-stage and late-stage cardiomyocytes are isolated from avian embryos and cultured for several days on ligand-coated polyacrylamide gels (E = 11 kPa), which have previously been shown to prolong spontaneous beating in culture and stiff ligand-coated polyacrylamide gels (E = 34 kPa). Cardiomyocytes are classified into sub-phenotypes based on morphology, myofibril organization,and local calcium release. The populations of early-stage cardiomyocytes in long-term culture are compared to the populations of late-stage cardiomyocytes in short-term culture. Additionally, the beating behavior of early-stage cardiomyocytes plated on polyacrylamide gels attached to gridded coverslips are observed over several days to assess changes in sub-phenotype differentiation.