Temporal changes in myocardial salvage kinases during reperfusion following ischemia: studies involving the cardioprotective adipocytokine apelin.
BACKGROUND
Activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, which incorporates phosphatidylinositol-3-OH kinase (PI3K)-Akt/protein kinase B (PKB) and p44/42 mitogen-activated protein kinase (MAPK), underlies protection against ischemia-reperfusion (I/R) injury. The temporal nature of the activation of these RISK pathway components during reperfusion is, however, uncertain. We examined Akt and p44/42 phosphorylation in hearts subjected to ischemia and varying periods of reperfusion in the absence or presence of the putative cardioprotectant, apelin-13. Akt activity was also measured.


METHODS
Langendorff perfused C57Bl/6J mouse hearts were subjected to 35 min global ischemia followed by 0, 2.5, 5 or 10 min reperfusion with or without 1 microM apelin-13. Basal and apelin-induced phosphorylation of Akt (at both the threonine 308 and serine 473 phosphorylation sites) and p44/42 during the reperfusion phase was determined by Western blotting and Akt activity measured using an Enzyme-Linked ImmunoSorbent Assay (ELISA).


RESULTS
Basal phosphorylation of both Akt and p44/42 increased progressively with time of reperfusion. Apelin enhanced Akt and p44/42 phosphorylation at all reperfusion time points. Akt activity did not change under basal conditions but was increased by apelin at 5 min (NS) and 10 min (p<0.05) reperfusion.


CONCLUSIONS
We conclude that under basal conditions Akt and p44/42 phosphorylation increases with time of reperfusion but that this is not accompanied by increased kinase (Akt) activity. On application of a cardioprotectant, however, kinase phosphorylation and activity are enhanced suggesting that it is the combination of these two mechanisms that may underly the tissue preserving actions of such agents.
Smith
Christopher C T
CC
The Hatter Cardiovascular Institute, University College London Hospital and Medical School, 67 Chenies Mews, London, WC1E 6HX, UK.
Mocanu
Michaela M
MM
Bowen
Jonathan
J
Wynne
Abigail M
AM
Simpkin
James C
JC
Dixon
Richard A
RA
Cooper
Michael B
MB
Yellon
Derek M
DM
D000818:Animals; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017202:Myocardial Ischemia / Q000201:enzymology; D015428:Myocardial Reperfusion Injury / Q000201:enzymology* / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*
D036341:Intercellular Signaling Peptides and Proteins; C495199:apelin-13 peptide; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases