Opposing and synergistic effects of cyclic mechanical stretch and α- or β-adrenergic stimulation on the cardiac gap junction protein Cx43.

In the heart the most prominent cardiac gap junction protein is connexin43 (Cx43). Increased Cx43 expression has been identified in cardiac hypertrophy and may contribute to arrhythmias. Besides acute effects on gap junction channel function, chronic regulation of Cx43 expression can affect intercellular communication. Since both cyclic mechanical stretch (CMS) and catecholamines play an important role in cardiac physiology and pathophysiology, we wanted to elucidate whether a prolonged β- or α-adrenoceptor stimulation may modulate the effects of CMS on Cx43 expression. Neonatal rat cardiomyocytes were cultured on flexible 6-well plates. Thereafter, cells were kept static without any treatment or stimulated with 0.1μmol/L isoprenaline or phenylephrine for 24h without or with additional CMS (1Hz; 10% elongation). Isoprenaline and phenylephrine given alone significantly increased Cx43-protein and -mRNA level. Also CMS resulted in a significant Cx43-protein and -mRNA up-regulation. The combined treatment of the cells with either isoprenaline or phenylephrine and stretch also resulted in an up-regulation of Cx43-protein and -mRNA, which did not exceed those of stretch, isoprenaline or phenylephrine alone. However, while CMS reduced the Cx43-protein/mRNA ratio, adrenergic stimulation increased Cx43-protein/mRNA ratio. While isoprenaline and phenylephrine increased Cx43-phosphorylation, additional CMS significantly reduced P-Cx43/Cx43 ratio. For further investigation of the underlying signal transduction pathway, we examined the phosphorylated forms of ERK1/2, GSK3β and AKT and could demonstrate that these protein kinases are also significantly up-regulated following stretch or adrenoceptor stimulation. Again the combined treatment of cardiomyocytes with CMS and isoprenaline or phenylephrine had no additive effects. Thus, the combination of α- or β-adrenoceptor stimulation and CMS up-regulates Cx43 expression and leads to phosphorylation of ERK1/2 and AKT (=activation) and of GSK3β (=inactivation). There were no significant additive effects compared to CMS or adrenergic stimulation alone indicating a possible ceiling effect. However, CMS and adrenergic stimulation differentially affected Cx43-protein/mRNA ratio and Cx43-phosphorylation.