title: Inducible NO synthase is constitutively expressed in porcine myocardium and its level decreases along with tachycardia-induced heart failure.
abstract: BACKGROUND
  The adverse effects of oxidative stress and the presence of proinflammatory factors in the heart have been widely demonstrated mainly on rodent models. However, larger clinical trials focusing on inflammation or oxidative stress in heart failure (HF) have not been carried out. This may be due to differences in the anatomy and physiology of the cardiovascular system between small rodents and large mammals. Thus, we investigated myocardial inflammatory factors, such as inducible NO synthase (iNOS) and oxidative stress indices in female pigs with chronic tachycardia-induced cardiomyopathy.
  
  
  METHODS
  Homogenous female siblings of Large White breed swine (n=15) underwent continuous right ventricular (RV) pacing at 170bpm, whereas five sham-operated subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were euthanized at subsequent stages of the disease: mild, moderate and severe HF. Left ventricle (LV) sections were examined with electron microscopy. The relative expression of iNOS in LV was determined by quantitative PCR. The protein level of iNOS was determined by Western blotting and immunohistochemistry. The level of the S-nitrosylated (S-NO) protein in LV was determined after S-NO moieties were substituted by biotin, followed by a colorimetrical detection with streptavidin. Malondialdehyde (MDA), a marker of lipid peroxidation, was evaluated in the LV and serum using thiobarbituric acid. The aconitase activity (based on measurement of the concomitant formation of NADPH from NADP(+)), a marker of oxidative stress, was analyzed in mitochondrial and cytosolic LV fractions. The concentration of interleukin-1β (IL-1β) was measured in LV homogenates using enzyme-linked immunosorbent assay.
  
  
  RESULTS
  RV pacing resulted in an impairment of LV systolic function, LV dilatation and neurohormonal activation. The electron microscopy revealed abnormalities within the cardiomyocytes of failing hearts, i.e. swollen mitochondria and myofibril derangement. iNOS was expressed in the control LV myocardium. The development of HF was accompanied by a decrease in iNOS mRNA (P<.05), which was also reflected at a protein level, and a decrease in the protein S-nitrosylation (P<.05). Both iNOS mRNA and S-NO relative moiety levels were inversely related to the dilatation of the LV (P<.05). There was no difference in the concentration of MDA in the LV and serum. Similarly, no differences in the concentration of IL-1β LV were found between diseased and healthy animals. Aconitase activity was decreased only in the LV mitochondrial fraction of pigs with severe HF.
  
  
  CONCLUSIONS
  iNOS was shown to be constitutively expressed within porcine LV. Its level decreases during the progression of systolic nonischemic HF in the pig model. Thus, it can be assumed that an up-regulation of proinflammatory factors is not involved in porcine tachycardia-induced cardiomyopathy and that the impact of oxidative stress may be restricted to the mitochondria in this HF model.
authors.affiliation: Department of Internal and Diseases with Clinic for Horses, Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-366 Wroclaw, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Paslawska
authors.affiliation: Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland. Electronic address: liliana.kiczak@up.wroc.pl.
authors.forename: Liliana
authors.initials: L
authors.lastname: Kiczak
authors.affiliation: Department of Food Hygiene and Consumer Health Protection, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Bania
authors.affiliation: Department and Clinic of Internal and Occupational Diseases and Hypertension, Wrocław Medical University, 50-367 Wroclaw, Poland.
authors.forename: Robert
authors.initials: R
authors.lastname: Paslawski
authors.affiliation: Department of Internal and Diseases with Clinic for Horses, Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-366 Wroclaw, Poland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Janiszewski
authors.affiliation: Department of Histology and Embryology, Wroclaw Medical University, 50-367 Wroclaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Dzięgiel
authors.affiliation: Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Zacharski
authors.affiliation: Department of Internal and Diseases with Clinic for Horses, Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-366 Wroclaw, Poland.
authors.forename: Alicja
authors.initials: A
authors.lastname: Tomaszek
authors.affiliation: Department of Internal and Diseases with Clinic for Horses, Dogs and Cats, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-366 Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Michlik
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002304:Cardiac Pacing, Artificial; D004195:Disease Models, Animal; D018450:Disease Progression; D015536:Down-Regulation; D005260:Female; D006333:Heart Failure / Q000201:enzymology / Q000209:etiology* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D018836:Inflammation Mediators / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D034421:Sus scrofa; D017180:Tachycardia, Ventricular / Q000150:complications* / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D016277:Ventricular Function, Left; D016278:Ventricular Function, Right
chemical_list: D015415:Biomarkers; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D017392:Thiobarbituric Acid Reactive Substances; D009569:Nitric Oxide; D008315:Malondialdehyde; D052247:Nitric Oxide Synthase Type II; D000154:Aconitate Hydratase
keywords: Inducible NO synthase; Oxidative stress; Pigs; S-Nitrosylated proteins; Tachyarrhythmic experimental model of heart failure