Abstract 240: Temporally-Stiffening Hydrogel Enhances Cardiomyocyte Maturation via Activation of Mechanosensitive Pathways
As cells migrate and differentiate throughout development, they secrete and assemble extracellular matrix, giving rise to time-dependent, tissue-specific stiffness, i.e. myocardium stiffens ~10-fold during maturation. When mimicked in vitro with a thiolated hyaluronic acid/poly(ethylene glycol) diacrylate hydrogel, myocardial matrix stiffening enhanced cardiac specific gene expression and myofibril organization in isolated embryonic cardiomyocytes vs. static hydrogels. Mechanical cues alone, even when time-dependent, were insufficient to induce cardiogenesis in mouse embryonic stem cell (mESCs), but a combination of precisely timed developmentally-relevant chemical cues (e.g. BMP4, Activin A, DKK-1) and dynamic matrix mechanical cues at the cardiac mesoderm induction stage enhanced maturation of mESC-derived cardiomyocytes vs. static hydrogels. On dynamic hydrogels, measurement of sarcomere length shows mESC-derived cardiomyocytes exhibited greater than 30% more mature myofibrils compared to cells plated on static, stiffer matrices. While active mechanotransduction aided maturation, the specific proteins responsible for responding to time-dependent stiffness remain unknown. In order to assess matrix-mediated mechanotransduction, we examined the expression and phosphorylation state of 890 protein kinases, as well as organization of mechanosensitive proteins, e.g. focal adhesion kinase and vinculin, of embryonic cardiomyocytes plated on matrices with either dynamic or static cardiac tissue-specific stiffness. Microarray analysis of protein kinases showed differential expression as a function of mechanics, confirmed by ratiometric western blotting. Several cardiogenic pathways exhibited time-dependent up-regulation on dynamic vs. static matrices, including PI3K/Akt and p38 MAPK, while GSK3β, a known inhibitor of cardiomyocyte maturation was down regulated. These data indicate that mechanically driven maturation is at least partially achieved via active mechanosensing at focal adhesions. Identifying mechanosensitive pathways that are active in cardiomyogenesis can lead to a better understanding of how stem cell differentiation and development are mediated by extracellular matrix properties.
- © 2013 by American Heart Association, Inc.