Signal transduction and transcriptional control of cardiac connexin43 up-regulation after α₁-adrenoceptor stimulation

Syncytial behavior of cardiac tissue is mainly controlled by the expression of cardiac gap junction proteins, and of these, connexin43 (Cx43) represents the predominant connexin in the working myocardium. Because the α₁-adrenoceptor is involved in many cardiac diseases, the following experiments were performed to clarify the pathway whereby α₁-adrenoceptor stimulation may control Cx43 expression. Cultured neonatal rat cardiomyocytes were stimulated with phenylephrine for 24 h, and Cx43 expression was investigated. Moreover, we investigated activation of p38 mitogenic-activated protein kinase (MAPK), p42/44-MAPK, and c-JUN NH ₂-terminal kinase (JNK) by phosphospecific enzyme-linked immunosorbent assay and nuclear translocation of the transcription factors c-fos and activator protein 1 (AP1). For verification of our results, a Cx43-promoter-enhanced green fluorescent protein (EGFP) construct using the complete promoter [2771 base pairs (bp)] or fragments (0-2421 bp) with EGFP under control of the Cx43 promoter was transfected into cardiomyocytes, and fluorescence intensity was investigated. Phenylephrine exposure caused approximately 2-fold up-regulation of Cx43 protein with an EC₅₀ of approximately 5 nM, which was significantly inhibited by bisindolylmaleimide I [protein kinase C (PKC) inhibitor], 4-(4-fluorophenyl)-2-(4- methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580; p38 inhibitor), or 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059; p42/44 inhibitor). Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44, and JNK. Moreover, we found translocation of c-fos and AP1 to the nucleus. Phenylephrine stimulation of Cx43-promoter EGFP-transfected cardiomyocytes significantly increased fluorescence, depending on the length of promoter fragments. A 91-bp fragment containing the first AP1 binding site produced approximately 50% of the fluorescence intensity of the complete promoter. Therefore, we conclude that α₁-adrenoceptor stimulation up-regulates cardiac Cx43 expression via a PKC p38- and p42/44 MAPK-regulated pathway, possibly involving AP1.