Connexin expression in cultured neonatal rat myocytes reflects the patternof the intact ventricle

Objective: Primary cultures of neonatal rat ventricular myocytes have becom e 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 hybr idization studies on developing rats, however, showed that Cx40 mRNA displa ys a dynamic and heterogeneous pattern of expression in the ventricular myo cardium around birth. The present studies were performed to examine the exp ression pattern of the Cx40 protein in neonatal rat heart, and to examine t he connexins present in cultures of ventricular myocytes obtained from thos e hearts. Methods: Cryosections were made of hearts of I-day-old Wistar rat s. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain >75, similar to 5 0%, and <25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 wer e detected by immunofluorescence and immunoblotting. Results: Immunohistoch emical stainings confirmed that gap junctions in the atrium and His-Purkinj e system were composed of at least Cx43 and Cx40. From the subendocardium t owards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wa ll. In ventricular myocytes cultured at high density (>75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolab eling which showed a homogeneous distribution pattern, Cx40 staining was he terogeneous, i.e. in some clusters of cells abundant labeling was present w hereas in others no Cx40 staining could be detected. The pattern of Cx43 im munoreactivity was not altered by the culture density. In contrast, in isol ated ventricular myocytes cultured at low density (<25% confluency) the rel ative number of cell-cell interfaces that were Cx40-immunopositive decrease d as compared to high density cultures (35 vs. 70%). Western blots did nor 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 v entricular myocardium with regard to their composition of gap junctions. Th is implicates that these cultures may serve as a good model for studying sh ort-term and long-term regulation of cardiac gap junction channel expressio n and function. (C) 1999 Elsevier Science BN. All rights reserved.