title: Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium.
abstract: Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 +/- 3.8%; 5 microM fasudil, 18.2 +/- 3.1%; 0.3 microM Y-27632, 19.4 +/- 4.4%; 5 microM Y-27632, 9.2 +/- 2.9%). When 5 microM Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 +/- 2.6% vs. control 33.4 +/- 4.4%, P<0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 +/- 6.1%) and N(omega)-nitro-L-arginine methyl ester (30.4 +/- 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, suggesting that ROCK activation at reperfusion may be deleterious through suppression of the RISK pathway.
authors.affiliation: Royal Veterinary College, University of London, London, UK.
authors.forename: Shabaz A
authors.initials: SA
authors.lastname: Hamid
authors.forename: Hugo S
authors.initials: HS
authors.lastname: Bower
authors.forename: Gary F
authors.initials: GF
authors.lastname: Baxter
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002316:Cardiotonic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D003326:Coronary Circulation / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D009203:Myocardial Infarction / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D000077191:Wortmannin; D054460:rho-Associated Kinases; D000099149:Ezrin
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000577:Amides; D000730:Androstadienes; D002316:Cardiotonic Agents; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines; D000077191:Wortmannin; D054460:rho-Associated Kinases; D000099149:Ezrin; C108830:Y 27632; C071058:moesin; C071776:radixin; C496316:Nos3 protein, rat; C049347:fasudil