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Chronic effects of endothelin 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells ¹

Institute of Pharmacology, Martin Luther University of Halle-Wittenberg, D-06097 Halle, Germany

²Correspondence: Klinik fuer Herzchirurgie, Universitaet Leipzig, Struempellstr. 39, University of Leipzig, D-04289 Leipzig, Germany. E-mail: stefan.dhein{at}medizin.uni-halle.de

SPECIFIC AIMS

Regulation of the gap junction-mediated intercellular communication by endothelin 1 (ET-1) and angiotensin II (AT-II) may influence heart function and its response to cardiac injury. This study examined the effects of ET-1 and AT-II on the major cardiac gap junction proteins connexin 43 (Cx43) and connexin 40 (Cx40) in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) and the role of MAPK signaling in the ET-1- and AT-II-induced responses.

PRINCIPAL FINDINGS

ET-1 and AT-II stimulate Cx43 but not Cx40 expression in cardiomyocytes
ET-1 or AT-II were added to the cell culture medium in concentrations of 10, 50, 100, 500, and 1000 nM for 24 h The changes observed in the Cx43 expression shown in Fig. 1 demonstrated that the total Cx43 content in the cardiomyocytes incubated with either peptide was enhanced. In addition, the extend of the protein phosphorylation increased in a dose-dependent manner, as can be clearly detected by the appearance of the slower migrating phosphorylated isoform of Cx43 in the blots. The concentration-response curves for ET-1 for Cx43 expression exhibited a pEC₅₀ (=negative logarithm of the concentration producing 50% of maximum effect) of 6.8 ± 0.1, whereas its pEC₅₀ for the Cx43 phosphorylation was 10-fold higher (pEC₅₀=7.9±0.1). In the case of AT-II, the concentration-response curves yielded a drug-effective concentration in the range of 10^-7 M for both Cx43 expression (pEC₅₀=7.24±0.08) and phosphorylation (pEC₅₀=7.03±0.04).

View larger version (23K): [in this window] [in a new window]   Figure 1. Western blot analysis of Cx43 in the neonatal rat ventricular cardiomyocytes treated for 24 h with either ET-1(A) or AT-II (B). Immunoblotting of the cell lysates with anti-Cx43 antibody demonstrates the effects of both peptides on expression and phosphorylation of the gap junction protein. The antibody recognized a single band migrating at 42 kDa in the untreated cells. After a 24 h incubation with different concentrations of either peptide, the second band representing the slower migrating phosphorylated isoform of Cx43 was also detected in the blots.

In contrast to Cx43, the level of Cx40 expression did not change in either ET-1- or AT-II-treated cardiomyocytes.

ET-1 and AT-II increased gap junction-mediated intercellular communication between the cultured cardiomyocytes
The mean junctional conductance between NRC under control conditions was 28 ± 4 nS (n=5). After 24 h treatment of cardiomyocyte cultures with 500 nM of either ET-1 or AT-II, a significant increase in electrical coupling up to 47 ± 3 nS (n=5) and 53 ± 3 nS (n=5), respectively, was detected between the cells (Fig. 2 ).

View larger version (21K): [in this window] [in a new window]   Figure 2. Effects of ET-1 and AT-II on intercellular communication between cardiomyocytes. A) Diagram of the macroscopic gap junctional conductance (Gj) between the cell pairs (n=5; P<0.05). B) Original registrations of gap junctional current between pairs of the neonatal rat ventricular cardiomyocytes under control conditions and in cultures treated with 500 nM of ET-1 or AT-II for 24 h.

ET-1 regulates Cx43 via ETA receptor subtype and AT-II-induced up-regulation of Cx43 is mediated by stimulation of AT₁ receptor
The 24 h treatment of cardiomyocytes with ET-1 performed in the presence of 100 nM of either ET_A receptor antagonist BQ123 or ET_B receptor antagonist BQ788 demonstrated that Cx43 up-regulation and phosphorylation could be diminished by the ET_A receptor antagonist BQ123, whereas the ET_B receptor antagonist BQ788 had only a weak effect on Cx43.

The increase in the AT-II-induced Cx43 expression was completely suppressed if cell cultures were incubated with AT-II in the presence of 100 nM of the AT₁ receptor antagonist losartan.

Involvement of MAP kinases in the regulation of Cx43
The cardiomyocyte cultures were incubated with either 500 nM of ET-1 or 100 mM of AT-II in the cell culture medium for 24 h with either no inhibitors or in the presence of 1) 25 µM of MEK inhibitor PD 98059; 2) 2 µM of p38 inhibitor SB 202190; or 3) 25 µM of a negative control for MAP kinase inhibition studies SB 202474. Control cultures were treated with the same set of the MAP kinase inhibitors to check their effects on the basal level of Cx43 expression.

Western blot analysis showed that all MAP kinase inhibitors were ineffective under control conditions. In the ET-1-treated cardiomyocyte cultures, Cx43 expression and phosphorylation returned to the control level if the MEK inhibitor was added to the cells, whereas the AT-II-induced increase in Cx43 expression could be prevented by inactivation of both MEK and p38 kinases.

CONCLUSIONS AND SIGNIFICANCE

The blocking or activation of gap junctions may influence heart function under normal and pathological conditions. For the first time, the role of the vasoactive peptides ET-1 and AT-II in functional regulation of cardiac gap junctions was investigated. ET-1 and AT-II both induced an increase in expression level and phosphorylation of Cx43 in a dose-dependent manner. The effective concentrations of ET-1 and AT-II experimentally determined in the cell culture of neonatal rat ventricular cardiomyocytes were between 10 and 100 nM. This concentration range could be comparable to that detected in the diseased heart tissue. The two- to threefold increase in the Cx43 expression level observed in the cardiomyocytes could result in changes in electrical coupling between the cells that may affect the heart tissue physiology. Indeed, the cardiomyocytes cultures treated with either peptide demonstrated a twofold increase in the electrical coupling between the cells. This first finding of a functional importance of both peptides for the electrical impulse propagation between the heart muscle cells may have clinical relevance and help improve therapy for heart disease.

The physiological effects of ET-1 in the heart are mediated by two subtypes of endothelin receptors: ET_A and ET_B. Two types of AT-II receptors (AT₁ and AT₂ receptors) are found in the rat heart. The ET-1 and AT-II receptor systems are both coupled via G_q/11 protein to the PLC/IP₃/DAG cascade as the major signal transduction pathway. The detailed mechanisms of the ET-1 and AT-II action on cardiac connexins are not known. Therefore, to define the pathways of the ET-1 and AT-II signaling leading to Cx43 up-regulation in the cardiomyocytes, we treated cells with either peptide in the presence of antagonists of the specific receptor subtypes and analyzed Cx43 expression. The ET-1-induced up-regulation of the Cx43 expression and phosphorylation could be significantly inhibited with 100 nM ET_A receptor antagonist BQ123. The ET_B receptor antagonist BQ788 had no effect on Cx43 in the ET-1-treated cell cultures. The effect of AT-II on Cx43 expression in NRC has been shown to be mediated through AT₁ receptors. We also observed that 100 nM of losartan added to the AT-II-treated cultures significantly inhibited the AT-II-induced increase in Cx43 expression and phosphorylation. Therefore, ET-1 regulates Cx43 via ET_A receptor and AT-II-induced up-regulation of Cx43 is mediated by stimulation of AT₁ receptor in cultured cardiomyocytes (Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized involvement of ET-1 and AT-II in the regulation of cardiac gap junctions via MAP kinase signaling pathways.

Evidence for interactions between the endothelin system and the renin–angiotensin system have recently been obtained in vitro as well as in vivo. Therefore, one might assume that the effect of AT-II on the Cx43 expression in our cell culture model might be partially mediated by the ET-1 produced by nonmyocytes in response to AT-II. However, an analysis of the effect of the ET_A receptor blocker BQ123 on Cx43 expression in the cardiomyocyte cultures treated with AT-II performed to prove this hypothesis showed that the paracrine production of ET-1 is not responsible for the stimulation of Cx43 in the AT-II-treated cardiomyocyte cultures.

One of the signaling pathways activated by both peptides in neonatal rat cardiomyocytes includes MAP kinases. The MAP kinases are well-known second messengers transducing the signals from different extracellular stimuli. However, the role of MAP kinases in the regulation of gap junctions in cardiac tissue is uncertain. Therefore, we studied the role of MAPK signaling in the ET-1- and AT-II-induced responses of cardiomyocytes. The important finding was the fact that ET-1 and AT-II can selectively modulate the specific expression of cardiac Cx43 through the signal transduction pathways, including ERK1/2 and p38. The present work demonstrates for the first time the involvement of ERK1/2 and p38 in the regulation of the cardiac connexin expression in cells treated with ET-1 or AT-II for 24 h. We showed that the ET-1-induced increase in the Cx43 content in the cardiomyocytes could be prevented by inhibition of MEK only, whereas the AT-II effects were blocked by both MEK and p38 inhibitors (Fig. 3) . The detailed mechanisms of this selective regulation could be a subject for future investigation.

Note that the level of Cx40 expression did not change in either ET-1- or AT-II-treated cardiomyocyte cultures. Thus, Cx43 but not Cx40 was the main target for the signal transduction induced by either peptide in the cultured ventricular cardiomyocytes. Therefore, the differential regulation of these two cardiac connexins was observed in the cells treated with either ET-1 or AT-II.

Taken together, our data indicate that the ET-1 and AT-II-induced stimulation of ERK and p38 signal pathways may play an important role in the molecular regulation of gap junctions under (patho)physiological conditions.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0381fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0381fje

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