The inability of the myocardium to regenerate after injury plus the inadequate number of available hearts for transplantation have drawn attention to the creation of functional tissue constructs for implantation within the injured heart. In addition, there is an increasing interest in developing in vitro models to study heart physiology and pathology as well as to evaluate drug efficacy. Formation of these in vitro models and tissue constructs requires highly specific conditions to mimic the normal environment of cells in the body. Firstly, in this study, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes, and investigating their development in different mechanical microenvironments. Immunofluorescence microscopy and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as the sarcomere length, fiber alignment, and beating amplitude and frequency of the cardiomyocytes, are regulated by microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the aforementioned characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development. Secondly, a silicone substrate with tunable elasticity is characterized for biological studies. Uniaxial tensile testing and microindentation show that these substrates could cover the biological range of stiffness for normal and pathological conditions. Spectrophotometry demonstrates that the transmittance of these substrates is comparable to those of glass and Sylgard 184. Atomic force microscopy shows that the surface roughness of samples is lower than that of widely-used Sylgard 184. Contact angle measurements before and after exposure to air plasma indicate that these samples are compatible with plasma lithography patterning. Thirdly, a new technique for cell patterning is developed which utilizes selective plasma lithography to modify protein adhesion on the substrate. This approach is based on controlling the conformation of Pluronic F-127 layer adsorbed on the surface by modifying surface wettability. Contact angle measurements show that both PDMS and plastic petri dish are compatible with this technique. X-ray photoelectron spectroscopy and atomic force microscopy confirm the adsorption of PF-127 layers with controlled conformation. Fluorescent and bright-field microscopy demonstrate selective adhesion of proteins and attachment of cells merely on plasma-treated areas. Finally, micropillar arrays are employed to determine the effects of two proteins associated with regulation of thin filament length, i.e. Lmod2 and Tmod1, on contractile force generation at the cellular level. Our results demonstrate that the contractile force of single isolated Lmod2-KO cardiomyocytes decreases compared to the wildtype control. Transduction of Lmod2 in the knockout cardiomyocytes restores their contractile force to the level of their WT counterparts, verifying that the observed contractile dysfunction is specific to the loss of Lmod2. Our data demonstrate that overexpression of Tmod1 in cardiomyocytes decreases their contractile force compared to the WT cells and confirm the effects of Lmod2 knockout on contractile force generation.