title: S262A mutation abolishes protective effects of connexin 43 against hypothermic preservation-induced injury in cardiomyocytes.
abstract: 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 DH5α 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.
authors.affiliation: Department of Physiology, Wenzhou Medical College, Wenzhou, China.
authors.forename: He-jing
authors.initials: HJ
authors.lastname: Xu
authors.forename: Wen-liang
authors.initials: WL
authors.lastname: Chen
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Ming-zhi
authors.initials: MZ
authors.lastname: Zheng
authors.forename: Ying-ying
authors.initials: YY
authors.lastname: Chen
authors.forename: Yue-liang
authors.initials: YL
authors.lastname: Shen
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism*; D015925:Cryopreservation; D007035:Hypothermia / Q000150:complications*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D023421:Models, Animal; D009154:Mutation / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation; D051381:Rats; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics
chemical_list: D018031:Connexin 43; D007770:L-Lactate Dehydrogenase