Cardiac tissue engineering is targeting the repair of the heart by the design of a myocardial patch, needed in repair of congenital heart defects or acquired heart disease, often due to myocardial infarction. Both conditions affect a large patient population, facing shortage of available transplants and/or problems with existing surgical solutions. A natural scaffolding material is desirable and expected to outperform the synthetic material (Dacron) used in current surgical treatments (Dor procedure). In this study, an extracellular matrix (ECM) was examined as a patch material, prepared from urinary bladder after cell removal and with preservation of essential components, such as ECM proteins and growth factors. This natural scaffold, termed UBM, has intricate three-dimensional architecture, with distinctly different sides, as seen in scanning electron microscopy images. The luminal side is characterized with a smoother surface relief, while the abluminal side is comprised of fine mesh of nano- and microfibers. We asked the question whether the local microtopography alone can affect the growth and function of the cardiomyocytes - essential for proper surgical implantation of the matrix. UBM has been used successfully for tissue repair and restoration of various tissues such as the lower urinary track, esophagus, blood vessels etc. The biodegradability of the ECM facilitates tissue repair and restoration. UBM, located beneath epithelial cells of the bladder, is a distinct collection of proteins including laminin, collagen type IV and entactin. Cardiomyocytes, isolated from the hearts of newborn rats were used to form in vitro cardiac tissue on the two surfaces of the UBM. Fluorescence-based imaging technique (calcium-sensitive dye, Fluo-4, and a fast sensitive photodetector) was used to assess the electromechanical performance of the cardiomyocyte networks assembled on the UBM. Response of intracellular calcium concentration to electrical stimulation carries information about the ability of the replacement tissue to generate adequate contraction force. The kinetic parameters of the obtained calcium signals were analyzed using specialized software. Standard statistical tests were performed to estimate differences in the performance of the cardiac myocytes on the two sides of the matrix. The preliminary data (n=2 samples for the abluminal side and n=5 for the luminal side) demonstrated a trend for shorter calcium transients with faster recovery velocities for the cells on the luminal side. However, due to the small number of the tested samples no statistical significance in differences between the two sides was seen. We have acquired more data since, which are being analyzed. The study will be extended to structural characterization of the cells and their intimate interaction with the fibers of the matrix to elucidate the importance of the local nano- and microtopography. The results of this study will help in designing proper implantation surgical procedures (luminal vs. abluminal side) to optimize cardiomyocyte growth and function and speed up the healing process. Ultimately, using a natural ECM scaffold instead of the currently employed synthetic materials is expected to significantly reduce scar formation and to achieve better heart tissue regeneration. This work was supported by funding from the Whitaker Foundation (RG-02-0654) and the Simons Foundation.