title: Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury.
abstract: In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P<0.05). Conversely, treatment with the mPTP opener Atractyloside (5×10(-6)M) 10min before hypoxic postconditioning abolished its protective effect (n=7 patients; P<0.05). We conclude that hypoxic postconditioning effectively salvages human skeletal muscle from hypoxia/reoxygenation injury by inhibition of mPTP opening and preservation of ATP synthesis during reoxygenation.
authors.affiliation: Research Institute, The Hospital for Sick Children, University of Toronto, Ontario, Canada.
authors.forename: Andreas
authors.initials: A
authors.lastname: Naparus
authors.forename: Homa
authors.initials: H
authors.lastname: Ashrafpour
authors.forename: Stefan O P
authors.initials: SO
authors.lastname: Hofer
authors.forename: Toni
authors.initials: T
authors.lastname: Zhong
authors.forename: Ning
authors.initials: N
authors.lastname: Huang
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Cahoon
authors.forename: Sandra E
authors.initials: SE
authors.lastname: McAllister
authors.forename: Peter C
authors.initials: PC
authors.lastname: Neligan
authors.forename: Trisha
authors.initials: T
authors.lastname: Roy
authors.forename: Joan E
authors.initials: JE
authors.lastname: Lipa
authors.forename: Cho Y
authors.initials: CY
authors.lastname: Pang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000368:Aged; D002470:Cell Survival; D016572:Cyclosporine / Q000494:pharmacology; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D066298:In Vitro Techniques; D057775:Ischemic Postconditioning; D008875:Middle Aged; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors; D000083162:Mitochondrial Permeability Transition Pore; D010100:Oxygen; D017568:Rectus Abdominis / Q000503:physiopathology*; D015427:Reperfusion Injury
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D016572:Cyclosporine; D000255:Adenosine Triphosphate; C090438:(melle-4)cyclosporin; D010100:Oxygen