Adult rat cardiac myocytes in culture: 'Second-floor' cells and coculture experiments.

OBJECTIVE: To compare adult rat cardiomyocytes in primary culture for up to 28 days with those in primary culture for 10 days plus up to 18 days in a coculture (CC) system. The phenomenon of 'second-floor' cells in primary cultures and the behaviour of CC myocytes were studied over varying times with regard to protein content and attachment to underlying cells. METHODS: Qualitative confocal microscopy and quantitation using the Imaris program (Bitplane, Switzerland) for the measurement of the fluorescence intensity in the confocal microscope were used. The protein content of actin, myosin, desmin, tubulin, titin, myomesin, cadherin and connexin was determined. Cell death was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method for apoptosis and using propidium iodide applied to unfixed cultures to detect necrosis. RESULTS: Compared with controls, second-floor cells contained only 14% of the actin and 4.9% of the tubulin at 10 days, whereas these proteins were well preserved in CC cells. All other proteins slowly declined in second-floor cells, whereas they were still present in normal amounts in CC cells. Cell death was evident in second-floor cells but absent in CC myocytes. Cellular attachment was still evident through original in vivo adherens junctions in second-floor cells but numerous newly developed cadherin- and connexin-containing junctions were visible in CC cells. It appears from the present study that second-floor cells are mummified dead cardiomyocytes, whereas CC myocytes survive and start to dedifferentiate. CONCLUSION: The absence of actin and tubulin, together with nuclear changes, are indicators of loss of cell viability despite preservation of the cells' rod shape and cross-striation, as observed in second-floor cells. In contrast, the establishment of a CC system of cardiomyocytes results in survival and organization of a three-dimensional cellular system, which may in the future be useful for tissue engineering attempts for replacement of lost tissue after myocardial infarction.