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Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation¹

STEPHAN WENG, MELANI LAUVEN^*, THOMAS SCHAEFER, LIOUDMILA POLONTCHOUK, RAJIV GROVER^* and STEFAN DHEIN²

Clinic for Cardiac Surgery, University of Leipzig, Heart Center, 04289 Leipzig,
Institute of Pharmacology, University of Halle, 06097 Halle (Saale); and
^* Institute of Pharmacology, University of Cologne, 50931 Köln, Germany

²Correspondence: Clinic of Cardiac Surgery, Herzzentrum, University of Leipzig, Strümpellstr. 39, D-04289 Leipzig, Germany. E-mail: dhes{at}medizin.uni-leipzig.de

SPECIFIC AIMS

Antiarrhythmic peptides are small peptides that can improve intercellular communication and exert antiarrhythmic effects. The aim of the present study was to elucidate the underlying molecular mechanism of action. We examined effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH₂) on gap junction current and phosphorylation of the major cardiac gap junction protein connexin43 (Cx43) and the signal transduction of this effect in cardiomyocytes and transfected HeLa-Cx43 cells.

PRINCIPAL FINDINGS

AAP10 enhances gap junction conductance in cardiomyocytes
When applying transcellular voltages of -50 to + 50 mV (200 ms pulses) to pairs of adult guinea pig cardiomyocytes, a linear current-voltage relationship became obvious from which the gap junction conductance could be calculated via linear regression analysis. The initial gap junction conductance (g_j) was 35.4 ± 4 nS (n=18). There were no significant differences between the experimental series regarding the initial values of these parameters. During equilibration under control conditions gap, junction conductance g_j continuously decreased with time (control series: -0.292±0.130 nS/min (n=6; P<0.05) over 70 min. This time-dependent decline in g_j could be prevented by application of 50 nmol/L AAP10 (increase in g_j by+0.290±0.231 nS/min, n=6; Fig. 1 ; Fig. 2 ; P<0.05). The effect of AAP10 could be washed out within 20 min.

View larger version (13K): [in this window] [in a new window]   Figure 1. Original registration of gap junction currents in a pair of isolated guinea pig cardiomyocytes. One cell was held at -40 mV and the other was clamped to -90 to +10 mV, thereby establishing a transcellular potential ranging from -50 to 50 mV. The pulses were 200 ms in duration. The figure shows gap junction conductance before (filled circles) and after application of 50 nmol/L AAP10 (open circles). AAP10 was administered to the bath solution.

View larger version (35K): [in this window] [in a new window]   Figure 2. Rundown of gj (nS/min) in the absence (instead vehicle was applied, n=9) and presence of AAP10 (50 nM, n=6) with or without additional treatment with the PKC inhibitors bisindolylmaleimide I (BIM; 0.2 µM, n=6) or CGP54345 (CGP; 10 µM, n=6). In each series of experiments, there was a 30 min control period (CON), followed by a 30 min treatment period with 50 nM AAP10. While AAP10 was applied extracellularly into the bath solution, the PKC inhibitors were given intracellularly via the pipettes throughout the entire experiment. All values are means ± SE of n experiments. Significant differences vs. the vehicle control are indicated by an asterisk; significant differences vs. AAP10 alone are marked by a #P < 0.05.

The effect of AAP10 on g _j in cardiomyocytes depends on protein kinase C (PKC)
The effect of 50 nM AAP10 could be blocked if the PKC inhibitor bisindolylmaleimide I (0.2 µmol/L) was present in the pipette solution (-[0.295±0.05 nS/min; n=6; Fig. 2 ; P<0.05). Using the PKC subtype-specific inhibitor CGP 54345 (10 µM), the AAP10 effect (inhibition of the time-dependent decrease in g_j) could also be significantly antagonized by CGP 54345 (P<0.05). From an initial conductance of 55 ± 5 nS, g_j constantly decreased by -0.400 ± 0.180 nS/min (n=6) under control conditions. If 50 nM AAP10 were applied in the presence of CGP 54345 (in the pipette), we found a similar time-dependent decrease in g_j (-0.250±0.170 nS/min, n.s. vs. control) (Fig. 2) . Thus, PKC inhibition with CGP54345 antagonized the effect of AAP10 (P<0.05 vs. AAP10 alone).

AAP10 exerts similar electrophysiological effects on HeLa-Cx43 cells
In HeLa-Cx43 cells (kindly provided by Prof. Klaus Willecke, Institute of Genetics, University of Bonn, Germany), a similar effect of AAP10 became obvious: from an initial value of 11.4 ± 2.5 nS g_j decreased with time by -0.313 ± 0.111 nS/min. Similar to cardiomyocytes, this decrease in g_j was reversed by 50 nM AAP10 (+0.078±0.034 nS/min, P<0.05).

AAP10 activates PKC (ELISA assay)
AAP10 induced a 1.97 ± 0.21-fold increase in PKC activity in HeLa-Cx43 cells. This AAP10-induced stimulation of the enzyme could be prevented by addition of the specific PKC inhibitor CGP54345 (10 µM) (activity=0.98±0.05). Basal PKC activity in the cells treated with CGP54345 alone was slightly (but not significantly) reduced compared with control cultures.

AAP10 induces phosphorylation of Cx43
Incubation of the HeLa-Cx43 cells with ³²P resulted in radioactive labeling of Cx43. Under control conditions (in the absence of AAP10), the Cx43 band was significantly radioactively labeled (650±20 densitometric units in the autoradiography). Under the influence of 50 nM AAP10, incorporation of ³²P into Cx43 was significantly enhanced (1269±30 vs. 600±20 densitometric units). This AAP10-induced incorporation of ³²P into Cx43 was completely prevented by the PKC inhibitor BIM I (496±20 densitometric units).

In a second series of experiments, G-protein coupling was inhibited by 1 mM GDPßS (introduced using lipofectamine). In the presence of GDPßS, radioactive labeling of Cx43 was 1233 ± 30 densitometric units in the autoradiography in the absence of AAP10 and was unchanged in the presence of 50 nM AAP10 (1066±30 densitometric units).

AAP10 binds to a membrane protein: radioligand binding study
Incubation of cardiac membrane preparation with 10^-7 mol/L ¹⁴C-AAP10 for 2 min to 200 min at 4°C, 21°C, and 37°C revealed an equilibrium binding after an incubation period of 20 min at RT. Saturation binding assay with ¹⁴C-AAP10 as radioligand and the AAP10 derivative AAP11TT (VAGHypPY) as heterologous competitor for determination of unspecific binding resulted in a saturable specific binding of the radioligand. Using Scatchard analysis, we found a K_D of 0.41 nmol/L and a B_max of 47.2 pmol/mg (slope: 1.1±0.1; r²=0.92). If AAPnat (GPHypGAG) was used as heterologous competitor similar binding was found with a K_D of 0.88 nmol/L and a B_max of 2.2 pmol/mg (r²=0.88). Displacement binding studies were performed using ¹⁴C-AAP10 as radioligand and increasing concentrations of AAPnat (GPHypGAG) as the competitor, revealing a biphasic displacement of ¹⁴C-AAP10 binding by AAPnat with a high- and a low-affinity binding site (K_D.High=19 nmol/L; K_D.Low=23 µmol/L; r²=0.97).

A 200 kDa membrane protein binds to AAP10 in experiments using affinity chromatography and chemical cross-linking
Finally, we tried to isolate this binding protein using affinity chromatography and chemical cross-linking. After separation of rabbit cardiac membrane proteins on the affinity chromatography column (using AAP10-agarose), and fractionated elution (20 fractions of 1 mL), we found in 12% SDS-PAGE a 200 kDa protein band in fraction 5 (and to a lower extent in fractions 3, 4 and 6, 7). A second very weak band appeared at 84 kDa. For control, we incubated FITC-AAP10 with rabbit cardiac membrane proteins in the presence of the cross-linker disuccinimidylsuberate and separated the solution using SDS-PAGE. We found a single fluorescent band at 200 kDa. This could also be reproduced using ¹⁴C-AAP10 as ligand.

CONCLUSIONS AND SIGNIFICANCE

The results of our study demonstrate that 1) AAP10 increases g_j in adult cardiomyocytes and transfected HeLa-Cx43-cells, 2) AAP10 activates PKC, 3) AAP10 induces enhanced phosphorylation of connexin 43, 4) the effects of AAP10 on g_j, Cx43 phosphorylation and PKC activity were sensitive to treatment with PKC inhibitors, 5) PKC is involved, and 6) specific binding of antiarrhythmic peptides to a membrane protein exists.

Double cell voltage clamp experiments indicated a time-dependent rundown of g_j in good accordance with the observations of others. The common explanation for this g_j rundown is a dephosphorylation of the connexins, which might be due to inhibition of protein kinases, activation of phosphatases, or a loss of ATP. The latter usually can be overcome by addition of ATP to the pipette solution (as used in this study) in concentrations corresponding physiological intracellular level of 3–7.5 mM. Our results demonstrate that AAP10 treatment reverses this rundown and increases g_j. The results showed that the electrophysiological effect of AAP10 depended on PKC (inhibition by BIM and CGP54345). In support of this, PKC-ELISA showed that AAP10-induced activation of PKC was sensitive to CGP 54435. Since BIM I inhibits isoforms , ß, , and and CGP 54345 is specific for PKC, we conclude that AAP10 seems to act via PKC. Activation of PKC may lead to phosphorylation of Cx43. In our study, enhanced phosphorylation of Cx43 in the presence of AAP10 depended on PKC as became obvious from its antagonization by BIM I. Such a change in phosphorylation may then lead to the changes observed in gap junction conductance. Since PKC is activated in most cases via a membrane receptor, we performed radioligand binding, affinity chromatography, and chemical cross-linking studies, all of which showed that AAP10 binds to a membrane protein. We found a saturable binding with a K_D in the range of 1 nM, which fits well the EC₅₀ of 5 nM determined in previous functional experiments in hearts. The specificity was also demonstrated by displacement with derivatives of AAP10. However, since these peptides are closely related chemically and ¹⁴C has a low specific activity, the B_max determined in these studies might be too high and thus should be considered preliminary. However, no other substances are known to act in the same way as AAP10 or its derivatives. Thus, a further detailed determination of the receptor density should be carried out when other ligands for the receptor are available. These experiments support the idea of a membrane protein serving as receptor for the antiarrhythmic peptide AAP10. This idea is further corroborated by the finding of a 200 kDa membrane protein that binds to the agarose-N-AAP10 column (affinity chromatography) and the finding that labeled AAP10 (FITC-AAP10 and ¹⁴C-AAP10) could be covalently bound with the cross-linker to a 200 kDa protein. We cannot say at at this time that both proteins are identical and we were not able to analyze the amino acid sequence. The amount of protein was too small for sequencing. Thus, further upscaled experiments using higher amounts need to be done for a final identification. This 200 kDa protein may be found to represent a part of the membrane receptor in search.

The idea of a membrane receptor for antiarrhythmic peptides is supported by the finding that the AAP10-induced phosphorylation of Cx43 could be completely abolished by pretreatment of the cells with GDP-ß-S. Sensitivity to GDP-ß-S typically indicates G-protein-coupled signaling. Thus, the signaling cascade for AAP10 leading to PKC activation might involve a G-protein. Identification of the membrane protein binding AAP10 could be a subject for future investigation. As summarized in Fig. 3 , our data indicate that AAP10 binds to a membrane protein and, possibly via a G-protein cascade, activates PKC, which then phosphorylates Cx43 resulting in enhanced gap junction conductance. The pharmacological influence on gap junctions may open new horizons for therapeutic developments.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized molecular mechanism of action for the antiarrhythmic peptide AAP10.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-098fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0918fje.

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