WB F344 cells co-cultured with neonatal cardiac cells. A: Proliferating WB F344 cells that attached away from cardiac cells formed nests of undifferentiated WB F344 cells (black triangle). B and C: Differentiating WB F344-derived cardiomyocytes (blue cell) adjacent to neonatal cardiomyocytes. C: Binucleated, WB F344-derived cardiomyocyte. D: WB F344-derived cardiomyocyte expressing GFP. Original magnifications, ×40.

WB F344 myocytes express cardiac-specific protein. Undifferentiated WB F344 cells and WB F344-derived myocytes express β-galactosidase (blue cells in a–d). WB F344-derived cardiomyocytes co-express cardiac myosin, striated red fluorescence in a, 2 and 3 (13 days co-culture); cardiac-specific cTnI, red fluorescence in b (13 days co-culture); cardiac-specific cTnT, striated red fluorescence in c (7 days co-culture); connexin 43, green fluorescence in d (13 days co-culture). Select confocal microscopic stacks in a and b demonstrate that neonatal myocytes do not overlie or underlie the WB F344-derived myocytes. Expression of myosin and TnI is inherent to the WB F344-derived myocytes that co-express β-galactosidase. Undifferentiated WB F344 cells are predominant in the 13-day co-cultures (a and b). Arrow in a3 depicts the nucleus of a neonatal cardiomyocyte. Arrow in a7 depicts the body of the same neonatal cardiomyocyte in contact with the WB F344-derived myocyte. The distribution of connexin 43 throughout the WB F344-derived myocyte cell membranes (d) is consistent with the immaturity of the myocyte. The arrow at the top in d represents connexin 43 expression in an adjacent neonatal cardiac myocyte that does not express β-galactosidase. Original magnifications, ×40.

Top: Cellular coupling in co-cultures measured with FRAP. Neonatal myocyte (red) adjacent to another neonatal myocyte (A), WB F344-derived myocyte (green) adjacent to a neonatal myocyte (B), undifferentiated WB F344 cell adjacent to neonatal myocyte (C), and WB F344 nests (D) are shown. Confocal images of co-cultures (optical thickness, 3 μm) were obtained with a ×63/1.4 NA oil immersion objective (scale bar, 50 μm). Bottom: The rate of fluorescence recovery for the cell relations described at the top are provided here. Column A, neonatal cardiomyocyte adjacent to another neonatal cardiomyocyte; Column B, WB F344-derived cardiomyocyte adjacent to a neonatal cardiomyocyte; Column C, undifferentiated WB F344 cell adjacent to neonatal cardiomyocyte; and Column D, WB F344 cells within a nest of WB F344 cells.

Myofibrillogenesis of WB F344-derived myocytes. Nascent myofibrils in A and B and sarcoplasmic reticulum (black triangle) in a WB F344-derived myocyte. Electron dense crystalline deposits (arrows in A and B) are product of β-galactosidase reaction. Scale bars: 10 μm (A); 20 μm (B).

Double FISH on mouse neonatal cell/WB F344 cell co-cultures. The FISH was conducted after performing the β-gal reaction and the immunochemistry to demonstrate β-galactosidase and cardiac-specific protein expression in WB F344-derived cardiomyocytes. The mouse line 1 DNA and the rat-specific DNA probes were used simultaneously in the same probe mix on the same mouse/rat co-culture. A: The mouse L1 DNA probe hybridized with a mouse myocyte nuclear DNA. Punctate rhodamine-positive speckles in the nuclei in A2 (arrow) represent clusters of mouse L1 DNA. Some are artificially enhanced to differentiate their signal from the Texas Red striations in the sarcomeres. The nuclei of the same cell are shown with the 4,6-diamidino-2-phenylindole stain in A4. This mouse-derived myocyte does not express β-galactosidase (A3), nor does it hybridize with the rat DNA probe (A1). B: The rat-specific DNA probe hybridizes with the rat WB F344-derived myocyte nuclei. The two green fluorescent (fluorescein isothiocyanate-positive) dots in the nuclei of a WB F344-derived myocyte (B1, arrow) reflect high-affinity hybridization sites of the rat DNA probe with the Y-chromosome of the male WB F344-derived myocyte and with less affinity with alleles on chromosome 3 and/or 12.23 This WB F344-derived myocyte does not hybridize with mouse L1 DNA (B2). The nuclei of this WB F344-derived myocyte are shown with the 4,6-diamidino-2-phenylindole stain in B4. Unlike the mouse-derived myocyte, this WB F344-derived myocyte expresses β-galactosidase. Both the mouse neonatal myocyte and the WB F344-derived myocyte express cTnT (striations), probed with mAb 13-11. mAb 13-11 recognizes a cTnT epitope conserved across species and does not recognize skeletal muscle TnT. A Texas Red-labeled anti-mouse antibody was used as a secondary antibody. Using a long-pass filter, the Texas Red emission bled through the rhodamine range, and sarcomeric cTnT striations are evident in the myocytes (A2 and A4, B2 and B4). Only the WB F344-derived myocyte (B3) expresses β-galactosidase. Original magnifications, ×100.

Intracellular [Ca²⁺]_i transients. 1: Highlighted fiber optics overlayed on image of co-culture with neonatal cardiac myocyte (A) and WB F344-derived myocyte expressing GFP (B), intracellularly labeled with indo-1 (scale bar, 100 μm). 2: [Ca²⁺]_i transients recorded at sites A and B. Measured change in [Ca²⁺]_i recorded at sites A and B demonstrated conduction block when the pacing stimulus increased from 1 Hz to 2 Hz. Recorded signals were normalized for fiber optic A and B and offset for display.