Question: 

Recently we have shown that a- and b-adrenoceptoragonists and angiotensin II cause an increase in connexin-43 (Cx43) expression in neonatal cardiomyocytes. The aim of our study was to evaluate the interaction between fibroblasts (FB) and cardiomyocytes (CM) in regard to Cx43 expression.

Methodology: 

We used three different types of cell cultures: Type 1: cell cultures with a 90min pre-plating step to enrich the CM fraction (95% CM and 5% FB). Type 2: cell cultures of the whole ventricle (without a pre-plating step) to obtain a co-culture of about 50% FB and 50% CM (distribution of the cells was analysed by FACS). Type 3: co-culture of FB and CM separated by a microporus membrane in a Transwell®-system. All cell cultures were exposed to either 0.1mM phenylephrine (Phe) or 0.1mM isoproterenol (Iso) or 0.1mM angiotensin II (AT) for 24 hours. Subsequently, Western blot analysis was carried out for Cx43-protein and real-time PCR analysis for Cx43-mRNA expression. To further evaluate signal transduction pathways the expression of phospho-ERK1/2 (extracellular signal-regulated kinase 1/2) was investigated by Western blot and the translocation of AP1 (activator protein 1) into the nucleus by EMSA (electromobility shift assay).

Results: 

Type 1-culture: Compared with non-stimulated controls we observed a significant increase in Cx43-protein expression in cardiomyocytes after treatment with Iso (174±15%), Phe (141±11%) or AT (150±9%) and an elevation of Cx43-mRNA levels (Iso:141± 4,37%, Phe: 143±12%, AT 164±12). Type 2-culture: AT led to a significant increase in Cx43-protein (183±12%) and -mRNA (142±5%) expression in co-cultures of FB and CM. In contrast the Iso and Phe Cx43-signals were suppressed. ERK 1/2 phosphorylation and AP1 translocation were significantly up-regulated after AT but not after Phe or Iso stimulation. Type 3-culture: In Transwell® cultured cells AT also led to a significant increase in Cx43-protein expression in FB (159±17%) and CM (123±7%) whereas adrenoceptor-stimulation did not affect Cx43-protein expression.

Conclusion: 

FB seem to impair Cx43-protein and -mRNA expression in cardiomyocytes after adrenoceptor stimulation. In contrast the AT signalling pathway appears to be preserved.