Cardiovascular diseases are a major cause of mortality resulting in serious health concerns and deteriorating quality of life. The minimal regeneration capacity of the myocardium revamps the need for an alternative therapeutic strategy such as myocardial tissue engineering. Human heart generates in a mechanically active environment beginning from organogenesis and persistent throughout adulthood. Hence, mechanical loading may have great utility in the control of cardiomyocyte (CM) development for cardiac tissue engineering and in the study of molecular mechanisms of CM function and pathology. This dissertation is aimed at utilizing mechanical stretch loading to physiologically mimic these CM developmental conditions and guide murine P19 pluripotent embryonic stem cell differentiation to CM lineage. The beating dynamics and genomics development of contracting CMs were improved or at comparable levels for stretch loading alone case compared with those from known soluble factor induction with 5-Azacytidine, suggesting that stretch loading may serve as a potent trigger to induce functional CM development. In addition, potential regulatory role of molecular mechanosensors such as focal adhesion kinase and Rho-associated protein kinase were tested as mechanotransduction pathways. The next part of the dissertation was focused on evaluating the spontaneous contraction of CMs, as a measure of their functional health. A non-invasive, robust, and unbiased method is presented for novel spatial-temporal characterization of CM contraction. The method developed enables to adaptively select reference frame when applying digital image correlation (DIC) to observe CM contraction, overcoming the lack of static reference frame and resultant noise buildup in conventional DIC methods. Based on the physiological criteria of individual sarcomere displacement length of 0.14 µm as a contraction threshold, novel spatiotemporal parameters were defined to account for the homogeneity, propagation, and synchronicity of CM contraction. To reiterate the elasticity of mechanically active native myocardium for the myocardial regenerative medicine, the final part of the dissertation attempted to develop cardiac tissue patches on electrospun polyurethane nanofiber scaffolds. Relative to aligned nanofibers or flat control, more number of contractile CM tissues were obtained on randomly oriented polyurethane nanofiber substrates, which may thus be better suited for the myocardial tissue engineering application
