S262A mutation abolishes protective effects of connexin 43 against hypothermic preservation-induced injury in cardiomyocytes

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• 作者：He-jing Xu ; MD^a ; Wen-liang Chen ; MPhil^b ; Yang Wang ; MD^a ; Ming-zhi Zheng ; MD^c ; Ying-ying Chen ; PhD^b ; Yue-liang Shen ; MD^b ; ^{shenyueliang@zju.edu.cn}
• 关键词：heart ; cold preservation ; connexin 43 ; phosphorylation
• 刊名：The Journal of Heart and Lung Transplantation
• 出版年：2012
• 期刊代码：161_10532498
• 类别：med
• 出版时间：June, 2012
• 卷：31
• 期：6
• 页码：663-669
• 文件大小：1494 K

摘要

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Background

Alleviation of cold preservation-induced injury is a critical part of the heart transplantation process. In this study we investigate the protective effect of connexin 43 (Cx43) overexpression against hypothermic preservation-induced injury in cardiomyocytes.

Methods

Total RNA was prepared from H9c2 cells using TRIzol reagent to construct a recombinant vector pEGFP-c1-Cx43, which was then transformed into Escherichia coli DH5伪 competent cells. The S262A Cx43 containing a mutant site was obtained by RT-PCR. The protein expression of total Cx43 and p-S262 Cx43 were assessed by Western blot. Cell viability and LDH release in the culture medium was measured.

Results

Restrictive enzyme reaction assay and DNA sequencing confirmed that the recombinant vector pEGFP-c1-Cx43 and pEGFP-c1-S262A-Cx43 were constructed correctly. After H9c2 cells were hypothermically preserved in Celsior solution for 12 to 48 hours, the cell viability decreased and LDH release increased. Compared with empty vector cells, overexpression of Cx43 prevented the hypothermic preservation-induced decrease in cell viability and increase in LDH release, which was independent of the absence of gap junctions. S262A mutation prevented S262 phosphorylation of Cx43 and also abolished protection of Cx43 overexpression against cold preservation-induced cardiomyocyte injury.

Conclusions

In H9c2 cells hypothermically preserved for up to 48 hours, overexpression of Cx43 could protect against cold preservation-induced injury. Phosphorylation of Cx43 at S262 may play an essential role in the preservation of donor hearts.