title: Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.
abstract: Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase-2 (MMP-2). It has been shown that simultaneous inhibition of MMP-2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML-7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co-administration of nitric oxide synthase (NOS) inhibitor (1400W or L-NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia-reoxygenation (H-R)-induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP-2 by Doxy (25-100 μM), MLCK by ML-7 (0.5-5 μM) and NOS by L-NAME (25-100 μM) or 1400W (25-100 μM) protected myocyte contractility after H-R in a concentration-dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H-R at the level of highest single-drug concentration. The combination of subthreshold concentrations of NOS, MMP-2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP-2. The observed protection with addition of L-NAME or 1400W was better than previously reported combination of ML-7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.
authors.affiliation: Department of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Bil-Lula
authors.affiliation: Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Han-Bin
authors.initials: HB
authors.lastname: Lin
authors.affiliation: Department and Clinic of Cardiology, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Biały
authors.affiliation: Department of Emergency Medicine, Wroclaw Medical University, Wroclaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wawrzyńska
authors.affiliation: Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Lucas
authors.initials: L
authors.lastname: Diebel
authors.affiliation: Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Sawicka
authors.affiliation: Department of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland.|Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Mieczyslaw
authors.initials: M
authors.lastname: Woźniak
authors.affiliation: Department of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland.|Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Sawicki
mesh_terms: D000818:Animals; D001381:Azepines; D002316:Cardiotonic Agents / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007097:Imines / Q000494:pharmacology; D015151:Immunoblotting; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000473:pathology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009281:Naphthalenes; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010100:Oxygen; D017207:Rats, Sprague-Dawley
chemical_list: D001381:Azepines; D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D007097:Imines; C496401:N-((3-(aminomethyl)phenyl)methyl)ethanimidamide; D009281:Naphthalenes; C070571:ML 7; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009219:Myosin-Light-Chain Kinase; D020778:Matrix Metalloproteinase 2; D004318:Doxycycline; D010100:Oxygen; D019331:NG-Nitroarginine Methyl Ester
keywords: cardiomyocytes; contractile proteins; hypoxia-reoxygenation; matrix metalloproteinase-2; nitric oxide; phosphorylation; synergism