TY - JOUR
T1 - Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation
AU - Yang, Changjun
AU - Talukder, M. A.Hassan
AU - Varadharaj, Saradhadevi
AU - Velayutham, Murugesan
AU - Zweier, Jay L.
N1 - Funding Information:
This work was supported by the National Institutes of Health grants HL38324, HL63744, and HL65608 to J.L.Z.
PY - 2013/1/1
Y1 - 2013/1/1
N2 - Aims The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC.Methods and resultsRat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOSSer1176, AktSer473, and PKAThr197 phosphorylation was increased following IPC. I/R decreased eNOSSer1176 phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOSSer1176 phosphorylation and quantitative Western blots showed ∼24% modification of eNOSSer1176 following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt Ser473 or PKAThr197 phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOSSer1176 phosphorylation as well as cardioprotection.ConclusionThus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.
AB - Aims The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC.Methods and resultsRat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOSSer1176, AktSer473, and PKAThr197 phosphorylation was increased following IPC. I/R decreased eNOSSer1176 phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOSSer1176 phosphorylation and quantitative Western blots showed ∼24% modification of eNOSSer1176 following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt Ser473 or PKAThr197 phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOSSer1176 phosphorylation as well as cardioprotection.ConclusionThus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.
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U2 - 10.1093/cvr/cvs287
DO - 10.1093/cvr/cvs287
M3 - Article
C2 - 22977010
AN - SCOPUS:84871758700
SN - 0008-6363
VL - 97
SP - 33
EP - 43
JO - Cardiovascular Research
JF - Cardiovascular Research
IS - 1
ER -