Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle.
OBJECTIVE
Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts.


METHODS
Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting.


RESULTS
Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities.


CONCLUSIONS
These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.
Kwak
B R
BR
Department of Medical Physiology and Sports Medicine, Utrecht University, The Netherlands.
van Kempen
M J
MJ
Théveniau-Ruissy
M
M
Gros
D B
DB
Jongsma
H J
HJ
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D018031:Connexin 43 / Q000032:analysis*; D017630:Connexins / Q000032:analysis; D017629:Gap Junctions / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D000097003:Gap Junction alpha-5 Protein
D018031:Connexin 43; D017630:Connexins; D000097003:Gap Junction alpha-5 Protein