Tissue engineering approaches are currently being investigated for the restoration of myocardial function in heart failure patients, most commonly by combining cells with a substrate to form myocardial-like constructs (MCs). The final properties of these constructs are dependant on the characteristics of both the substrate and the cells used for fabrication. To create a construct with the appropriate mechanical properties required for any future therapeutic, we tailored an acrylated polypropylene glycol triol (aPPGT) substrate to the elastic modulus of heart tissue and then investigated the fabrication of MCs. We first assessed the aPPGT substrate alone in vivo, both under normal conditions and in an infarct model in mice, and found that there was a mild foreign body response with good integration of the substrate into the epicardial surface in mice hearts. We next studied the fabrication and properties of MCs by culturing mouse embryonic cardiomyocytes on the aPPGT substrate. To achieve myocardial-like concentrically contractile constructs, cocultures with supportive stromal cells were found to be essential and both mouse heart-derived stromal cells or bone-derived mouse mesenchymal stromal progenitor cells (mMSCs) could be used. These different stromal cell types produced MCs with different properties. The average beating rate of the constructs formed from mouse heart-derived stromal cells was significantly higher those constructs formed using mMSCs. Conversely, the constructs formed using mMSCs had reduced fibrotic extracellular matrix secretion and increased hepatocyte growth factor expression. Both of these mMSC construct properties may enhance integration and therapeutic efficacy of the construct postimplantation on the surface of the infarcted heart. This study thus demonstrates the formation of MCs using mechanically tailored aPPGT substrate and also demonstrates the effects of different stromal cell populations have on the properties of the resultant MCs, both of which are critical for future applications of tissue engineering in heart failure patients.