Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state.
OBJECTIVE
The cardioprotective agent fibroblast growth factor 2 (FGF-2) was found previously to promote phosphorylation of connexin-43 (Cx43) at protein kinase C (PKC) sites such as serine (S) 262 at levels above those of non-stimulated hearts. We asked if other PKC-dependent cardioprotective treatments cause a similar effect, and if Cx43 phosphorylation at S262 mediates resistance to injury.


RESULTS
Isolated perfused adult rat hearts were subjected to the following treatments: ischaemic preconditioning (PC); diazoxide perfusion; FGF-2 pre-treatment followed by 30 min global ischaemia; 30 min global ischaemia followed by 60 min reperfusion in the presence or absence of FGF-2. Cx43 phosphorylation was assessed by western blotting with phospho-specific antibodies. Neonatal cardiomyocyte cultures were used to examine the effect of expressing Cx43 incapable of being phosphorylated at S262 due to an S to alanine (A) substitution on simulated ischaemia-induced cell death (TUNEL staining) and injury (lactic dehydrogenase release). Ischaemic PC, diazoxide, and FGF-2 pre-ischaemic or post-ischaemic treatments elicited a P Cx43 state, defined as above-physiological levels of phospho-S262-Cx43 and phospho-S368-Cx43. P Cx43 was sustained during global ischaemia and was accompanied by attenuation of ischaemia-induced Cx43 dephosphorylation and prevention of Cx43 lateralization. Post-ischaemic FGF-2 treatment also diminished dephosphorylated Cx43. Modest overexpression of S262A-Cx43, but not wild-type Cx43, exacerbated cardiomyocyte death and injury caused by simulated ischaemia in vitro. It also prevented the cytoprotective effects of FGF-2 or overexpressed PKCepsilon.


CONCLUSIONS
P Cx43 marks a state of enhanced resistance to ischaemic injury promoted by PKC-activating treatments such as FGF-2 administration or ischaemic PC. Cx43 phosphorylation at S262 likely mediates PKCepsilon-dependent cardioprotection.
Srisakuldee
Wattamon
W
Institute of Cardiovascular Sciences, St Boniface Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6.
Jeyaraman
Maya M
MM
Nickel
Barbara E
BE
Tanguy
Stéphane
S
Jiang
Zhi-Sheng
ZS
Kardami
Elissavet
E
D019943:Amino Acid Substitution; D000818:Animals; D018031:Connexin 43 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003981:Diazoxide / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D016297:Mutagenesis, Site-Directed; D017202:Myocardial Ischemia / Q000235:genetics / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018031:Connexin 43; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D051744:Protein Kinase C-epsilon; D003981:Diazoxide