Differences in the structural characteristics of adult guinea pig and rat cardiomyocytes during their adaptation and maintenance in long-term cultures: confocal microscopy study.
In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term (1-5 week) cultures of adult guinea pig cardiomyocytes and adult rat cardiomyocytes. Providing that the preparation of freshly isolated guinea pig cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca(2+)-tolerant, and quiescent cells and these are plated under optimal conditions and density (10(5) cells/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for 1 week; (2) within 10-14 days, they reestablish intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layer; (3) their myofibrillar striations remain regular all through the adaptation to culture conditions 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 cardiomyocytes cultured under very similar and optimal conditions exhibit very different characteristics during their adaptation in long-term cultures: (1) although 85-90% of freshly isolated cells are also rod-shaped and Ca2+ tolerant they exhibit slow spontaneous contractions; (2) they round up during the first few days, and during the first week they dedifferentiate, losing their regular striated appearance; (3) they spread, becoming irregularly shaped, and, unlike the guinea pig cardiomyocytes, they do not form confluent layers, no matter what the plating density is; (4) they atrophy at a very early stage in the cultures, so that by the fourth week, they have lost most of their myofibrils; (5) the initial rounding up is largely eliminated by exposure for 24 h to 1 microM ryanodine or 20 mM butanedione monoxime, compounds that suppress the spontaneous contractions. In conclusion, our studies demonstrate that adult guinea pig cardiomyocytes adapt and survive in long-term (1-5 week) cultures much better than do adult rat cardiomyocytes, indicating that the long-term cultures of adult guinea pig ventricular myocytes provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under the acute or chronic influence of various intrinsic and/or extrinsic factors.
Horackova
M
M
Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada. Magda.Horackova@Dal.Ca
Byczko
Z
Z
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000032:analysis; D015415:Biomarkers; D015820:Cadherins / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D064211:Connectin; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006352:Heart Ventricles; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009124:Muscle Proteins / Q000032:analysis; D009206:Myocardium / Q000166:cytology* / Q000648:ultrastructure; D051381:Rats; D013045:Species Specificity
D015415:Biomarkers; D015820:Cadherins; D064211:Connectin; D009124:Muscle Proteins; D009320:Atrial Natriuretic Factor