Adult cardiomyocytes (CM) retain little capacity to regenerate, which motivates efforts to engineer heart tissues that can emulate the functional and mechanical properties of native myocardium. Although the effects of matrix stiffness on individual CM have been explored, less attention was devoted to studies at the monolayer and the tissue level. The purpose of this study was to characterize the influence of substrate mechanical stiffness on the heart cell phenotype and functional properties. Neonatal rat heart cells were seeded onto collagen-coated polyacrylamide (PA) substrates with Young's moduli of 3, 22, 50, and 144 kPa. Collagen-coated glass coverslips without PA represented surfaces with effectively "infinite" stiffness. The local elastic modulus of native neonatal rat heart tissue was measured to range from 4.0 to 11.4 kPa (mean value of 6.8 kPa) and for native adult rat heart tissue from 11.9 to 46.2 kPa (mean value of 25.6 kPa), motivating our choice of the above PA gel stiffness. Overall, by 120 h of cultivation, the lowest stiffness PA substrates (3 kPa) exhibited the lowest excitation threshold (ET; 3.5 +/- 0.3 V/cm), increased troponin I staining (52% positively stained area) but reduced cell density, force of contraction (0.18 +/- 0.1 mN/mm 2), and cell elongation (aspect ratio = 1.3-1.4). Higher stiffness (144 kPa) PA substrates exhibited reduced troponin I staining (30% positively stained area), increased fibroblast density (70% positively stained area), and poor electrical excitability. Intermediate stiffness PA substrates of stiffness comparable to the native adult rat myocardium (22-50 kPa) were found to be optimal for heart cell morphology and function, with superior elongation (aspect ratio > 4.3), reasonable ET (ranging from 3.95 +/- 0.8 to 4.4 +/- 0.7 V/cm), high contractile force development (ranging from 0.52 +/- 0.2 to 1.60 +/- 0.6 mN/mm(2)), and well-developed striations, all consistent with a differentiated phenotype. Biotechnol. Bioeng. 2010; 105: 1148-1160. (C) 2009 Wiley Periodicals, Inc.