Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway.
OBJECTIVE
The cholesterol-lowering drugs statins could enhance the activities of endothelial nitric oxide synthase (eNOS) and protect myocardium during ischemia and reperfusion. The aim of this study was to examine whether protein kinase A (PKA) was involved in statin-mediated eNOS phosphorylation and cardioprotection.


METHODS
6-Month-old Chinese minipigs (20-30 kg) underwent a 1.5-h occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD). In the sham group, the LAD was encircled by a suture but not occluded. Hemodynamic and cardiac function was monitored using a polygraph. Plasma activity of creatine kinase and the tissue activities of PKA and NOS were measured spectrophotometrically. p-CREB, eNOS and p-eNOS levels were detected using Western blotting. Sizes of the area at risk, the area of no-reflow and the area of necrosis were measured morphologically.


RESULTS
Pretreatment of the animals with simvastatin (SIM, 2 mg/kg, po) before reperfusion significantly decreased the plasma activity of creatine kinase, an index of myocardial necrosis, and reduced the no-reflow size (from 50.4%±2.4% to 36.1%±2.1%, P<0.01) and the infarct size (from 79.0%±2.7% to 64.1%±4.5%, P<0.01). SIM significantly increased the activities of PKA and constitutive NOS, and increased Ser(133) p-CREB protein, Ser(1179) p-eNOS, and Ser(635) p-eNOS in ischemic myocardium. Intravenous infusion of the PKA inhibitor H-89 (1 μg·kg(-1)·min(-1)) partially abrogated the SIM-induced cardioprotection and eNOS phosphorylation. In contrast, intravenous infusion of the eNOS inhibitor L-NNA (10 mg·kg(-1)) completely abrogated the SIM-induced cardioprotection and eNOS phosphorylation during ischemia and reperfusion, but did not affect the activity of PKA.


CONCLUSIONS
Pretreatment with a single dose of SIM 2.5 h before reperfusion attenuates myocardial no-reflow and infarction through increasing eNOS phosphorylation at Ser(1179) and Ser(635) that was partially mediated via the PKA signaling pathway.
Department of Cardiology, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Xiang-dong
XD
Li
Yue-jin
YJ
Yang
Yong-jian
YJ
Geng
Jing-lin
JL
Zhao
Hai-tao
HT
Zhang
Yu-tong
YT
Cheng
Yi-ling
YL
Wu
D000818:Animals; D000924:Anticholesteremic Agents / Q000627:therapeutic use*; D003402:Creatine Kinase / Q000097:blood; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006321:Heart / Q000187:drug effects; D006439:Hemodynamics / Q000187:drug effects; D009203:Myocardial Infarction / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D009206:Myocardium / Q000201:enzymology / Q000473:pathology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D019821:Simvastatin / Q000627:therapeutic use*; D013552:Swine; D013556:Swine, Miniature
D000924:Anticholesteremic Agents; D017362:Cyclic AMP Response Element-Binding Protein; D019821:Simvastatin; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D003402:Creatine Kinase