Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here we study isolated embryonic cardiomyocytes on a series of flexible substrates and show that matrices which mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1 Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells over-strain themselves, lack striated myofibrils and stop beating; on softer matrices, cells preserve contractile beating for days in culture but exhibit fewer striations and do very little work. Optimal matrix produces a strain match between cell and matrix and suggests dynamic differences in intracellular protein structures. A “Cysteine Shotgun” method of labeling the in situ proteome reveals differences in several cytoskeletal proteins, including vimentin, filamin, and myosin. Combined with recent results that show stem cell differentiation is also highly sensitive to matrix elasticity, the results here highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy.