[635] Serine 297 and Serine 306: Two New Phosphorylation Sites Involved in the Regulation of Connexin43 during Ischemia

Lene N Axelsen, MA, Shabaz Mohammed, PhD, Anne Louise Kjølbye, PhD, Morten S Nielsen, PhD, Niels-Henrik Holstein-Rathlou, MD, Professor, Ole N Jensen, Professor, Søren Andersen, Technisian, James K Hennan, PhD, Martin Stahlhut, PhD. Zealand Pharma, Glostrup, Denmark; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Department of Medical Physiology, Copenhagen Heart Arrhythmia Research Center (CHARC), the Panum Institute, University of Copenhagen, Copenhagen, Denmark; Wyeth Research, Philadelphia, PA

Introduction: Slow and heterogeneous conduction caused by uncoupling of gap junction (GJ) communication plays an important role in the genesis of ischemia-induced ventricular tachycardia. Previous studies suggest that dephosphorylation of serine 368 (S368) in the GJ protein, connexin43 (Cx43), is related to GJ uncoupling; however, a systematic analysis of phosphorylation status of all serine sites on Cx43 has never been performed. The aim of this study was to examine the effect of ischemia on site-specific Cx43 serine phosphorylation. In addition, we studied the changes in Cx43 serine phosphorylation following treatment with the antiarrhythmic peptide analogue, ZP123, shown to prevent ischemia-induced GJ uncoupling. Methods: Isolated perfused rat hearts were exposed to 0, 7, 15, 30 and 45 min of global ischemia (n=10/group). An additional group was pre-treated with 10 nM ZP123 and exposed to 30 min of global ischemia (n=10). Cx43 was purified using immuno-affinity chromatography. Characterization of Cx43 phosphorylation was performed using matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and liquid chromatography tandem MS. Results: MS analysis revealed that at least 13 different serine sites were phosphorylated in the carboxy-terminal domain of Cx43 during non-ischemic conditions (S255, S257, S262, S296, S297, S306, S325, S328, S365, S368, S369, S372 and S373). Three of these sites (S296, S297, and S306) have not been described previously. Within the first 7 min of ischemia, S306 was fully dephosphorylated whereas S330 was phosphorylated. Between 15 and 30 min of ischemia S297 and S368 were also fully dephosphorylated. During the same time period all un-treated hearts developed asystole. Treatment with ZP123 suppressed dephosphorylation of S297 and S368 and significantly increased time to ischemia-induced asystole (Cox analysis, p<0.001). Conclusion: Ischemia leads to dephosphorylation of S306 (0-7 min), S297 and S368 (15-30 min), and phosphorylation of S330 (0-7 min) in Cx43. The changes in phosphorylation status of S297 and S368 correlate with the onset of asystole and with the antiarrhythmic effect of ZP123 suggesting that these changes may be involved in functional gating of Cx43 during ischemia.

Session Info: Gap Junctions and Cellular Electrophysiology - Monday, November 14, 2005, 10:45 AM - 12:00 PM
Presentation Time: 10:45 AM
Room: Dallas Convention Center, D170/172