DIFFERENCES IN THE STRUCTURAL CHARACTERISTICS OF ADULT GUINEA-PIG ANDRAT CARDIOMYOCYTES DURING THEIR ADAPTATION AND MAINTENANCE IN LONG-TERM CULTURES - CONFOCAL MICROSCOPY STUDY

In this study, we used laser confocal scanning microscopy and immunofl uorescent markers to describe the establishment of long-term (1-5 week ) cultures of adult guinea pig cardiomyocytes and adult rat cardiomyoc ytes. Providing that the preparation of freshly isolated guinea pig ca rdiomyocytes consists mostly (> 80%) of rod-shaped, Ca2+-tolerant, and quiescent cells and these are plated under optimal conditions and den sity (10(5) cells/cm(2)), these myocytes have the following characteri stics: (1) they remain elongated with regular ultrastructural characte ristics and quiescent for 1 week; (2) within 10-14 days, they reestabl ish intercellular contacts and resume contractile activity, which beco mes synchronous all through the confluent layer; (3) their myofibrilla r striations remain regular all through the adaptation to culture cond itions without any sign of dedifferentiation or redifferentiation; (4) they form adherence junctions (as indicated by their immunoreactivity to an antibody against N-cadherin) over the entire cellular surface; (5) they appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate. In contrast, rat cardiomyo cytes cultured under very similar and optimal conditions exhibit very different characteristics during their adaptation in long-term culture s: (1) although 85-90% of freshly isolated cells are also rod-shaped a nd Ca2+ tolerant they exhibit slow spontaneous contractions; (2) they round up during the first few days, and during the first week they ded ifferentiate, losing their regular striated appearance; (3) they sprea d, becoming irregularly shaped, and, unlike the guinea pig cardiomyocy tes, they do not form confluent layers, no matter what the plating den sity is; (4) they atrophy at a very early stage in the cultures, so th at by the fourth week, they have lost most of their myofibrils; (5) th e initial rounding up is largely eliminated by exposure for 24 h to 1 mu M ryanodine or 20 mM butanedione monoxime, compounds that suppress the spontaneous contractions. In conclusion, our studies demonstrate t hat adult guinea pig cardiomyocytes adapt and survive in long-term (1- 5 week) cultures much better than do adult rat cardiomyocytes, indicat ing that the longterm cultures of adult guinea pig ventricular myocyte s provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial funct ion under the acute or chronic influence of various intrinsic and/or e xtrinsic factors. (C) 1997 Academic Press.