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It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.

5-hydroxytryptamine-4 (5-HT(4)) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT(4) receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT(4) receptors were present and real-time RT-PCR analysis revealed that 5-HT(4b) was the predominant isoform. Serotonin (1 microM) significantly reduced cAMP concentration unless a selective 5-HT(4) inhibitor (GR113808 or ML10375, both 1 microM) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT(4) inhibitor but strongly reduced when 5-HT(2A) and 5-HT(2B) receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT(4), 5-HT(2A) and 5-HT(2B) were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT(4) (mainly 5-HT(4b)), 5-HT(2A) and 5-HT(2B) receptors coexisted in auricular myocytes of newborn rat, that 5-HT(4) activation reduced cAMP concentration, I(Ca)(L) and intercellular coupling whereas 5-HT(2A) or 5-HT(2B) activation conversely enhanced GJIC., (Copyright 2009 Elsevier Inc. All rights reserved.)

Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up- or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The junctional channels that couple the cardiomyocytes are responsible for this propagation process. These channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Four Cxs - Cx30.2, -40, -43 and -45--have been demonstrated to be synthesized in the cardiomyocytes. In addition, each of these Cxs has a unique expression pattern in the myocardium. A fruitful approach of the role of these Cxs in the cardiac functions came with the development of transgenic mouse models. It has been shown that Cx43 was mainly involved in influx propagation in the ventricles and that inactivation in the cardiomyocytes of the gene of this Cx predisposed to development of cardiac abnormalities. Cx40 very significantly contributes to the propagation of electrical activity in the atria and the conduction system. Cx45 is essential to coordinate the synchronization of contractile activities of embryonic cardiomyocytes and for the normal progress of cardiogenesis. Finally, Cx30.2 contributes to the slowing of propagation of excitation in the atrioventricular node. These observations enable to better understand the relationships between alteration in Cx expression or gap junction remodelling and arrhythmias in the human heart.