Data Supplement

Expanded Materials and Methods:

Animal Protocol: Approximately 200 Sprague Dawley rats were used in the experiments for this study. The rats were euthanized via CO2 inhalation followed by cervical dislocation. Embryos were dissected out of the uterus and immediately decapitated. This animal protocol has been approved by the IACUC, through the University of South Carolina, reference number 1100.

Video Capture: Video micrographs of cardiac cultures were captured using a Nikon Diaphot inverted microscope that is equipped with an environmental chamber that was maintained at 37°C. Adobe Premier v6.0 software was used to import videos from a Panasonic digital GP-KR222 video camera mounted on the Nikon microscope. Total magnification is 255X. Whole tube images were taken with the Panasonic digital GP-KR222 video camera mounted on a Nikon SMZ1500 stereomicroscope. Images were taken at a magnification of .75X and also at no magnification.

Files in this Data Supplement:

• Cell-morphometrics - The table (A) and graph (B) shows the results of quantifying the cell area of cardiac myocytes grown on the five different substrates. E15 rat ventricular primary cultures were seeded on the ACol I, FN, Col I, PL, and Tube substrates. Cultures were stained with the sarcomeric myosin-specific monoclonal antibody MF-20 and TroPro III, a nuclear stain. A Z-series using confocal microscopy was used to determine the middle of the cell and subsequently, the image was captured. The area of the cell was determined by measuring the MF-20 positive portion of the myocytes plus the nucleus using programs found within the LaserSharp 2000 version 4.3 (Bio-Rad) and Photoshop 6.0 (Adobe) suits of software programs. A minimum of 15 cardiac myocytes was analyzed for each substrate. The cell area measurements were subjected to statistical analysis and shown in Table A and Graph B. Cardiac myocytes grown on the FN substrate were found to be significantly (P< 10^-9) larger than cells grown on all other substrates. Five representative fields of E15 rat ventricular primary cultures grown on each of the five different culturing substrates are also included in this supplement. The five examples of each culturing substrates follows the title of each substrate.
• Video - The movie is a collection of short segments of video images of tube cultures taken at different time points. Early time points (days 4, 5, and 6 at 255X) show the myocytes beating, but they are unsynchronized. Later time points (days14 and 21 at 255X) demonstrate myocytes forming cell-cell connections resulting in the synchronized beating of the cells. Videos taken at lower magnification show the entire tube contracting as a syncytium (days 11 mouse at .75X and 21 at no magnification).