| Co-Investigator(Kenkyū-buntansha) |
KANG Dongchong KYUSHU UNIVERSITY, FACULTY OF MEDICINE, ASSOCIATE PROFESSOR, 医学部・附属病院, 助教授 (80214716)
UTSUMI Hideo KYUSHU UNIVERSITY, FACULTY OF MEDICINE, PROFESSOR, 薬学研究院, 教授 (20101694)
KUBOTA Toru KYUSHU UNIVERSITY, FACULTY OF MEDICINE, ADJUNCT ASSISTANT PROFESSOR, 医学研究院, 助手 (40325444)
KASAI Hiroshi UNIVERSITY OF OCCUPATIONAL AND ENVIRONMENTAL HEALTH, PROFESSOR, 産業生態科学研究所, 教授 (40152615)
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| Research Abstract |
Experimental and clinical studies have identified an increased oxygen radical species (ROS) including superoxide anion (・O_2-), H_2O_2 and hydroxyl radical (・OH) as a characteristic of the failing heart. Oxidative stress might play an important role in the pathophysiology of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of ROS in the failing myocardium remain unknown.
1) ROS are increased in the failing hearts by electron spin resonance (ESR) spectroscopy
To measure the amount of ROS(・O_2-,H_2O_2 and ・OH), myocardial tissue homogenates obtained from dogs with heart failure(HF), induced by rapid ventricular pacing at 240 beats per min for 4weeks, were reacted with the nitroxide radical, 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO), as a spin probe and its spin signals were detected by ESR spectroscopy. The rate of ESR signal decay, proportional to ・OH level, was significantly increased in HF, which was inhibited
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by the addition of dimethylthiourea (・OH scavenger) into the reaction mixture. Increased ・OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H_2O_2 scavenger) and Tiron (・O_2- scavenger), indicating that ・OH was originated from H_2O_2 and ・O_2-. Further, ・O_2- produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H_2O_2 and ・OH seen in the failing myocardium.
There was a significant positive relation between myocardial ・OH level and left ventricular contractile dysfunction. The activities of myocardial antioxidant enzymes such as SOD, catalase, and glutathione peroxidase were not decreased in HF, indicating that the antioxidant capacity is preserved normal in the failing heart.
2) Mitochondrial electron transport complex I is a potential source of ・O_2-
To determine the subcellular source of ・O_2-, its generation was directly assessed in the subcellular fractions by ESR spectroscopy with spin trapping agent, 5,5 '-dimethyl- 1 -pyrroline-N-oxide (DMPO), in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. ・O_2-production in the submitochondrial fractions was increased 2.8-fold in HF, which was due to the functional block of electron transport at complex I. Enzymatic activity of complex I was decreased in HF, which could result in the functional uncoupling of the respiratory chain and the deleterious ・O_2- production in the failing mitochondria. ・O_2- production in cytosolic and microsomal fractions were comparable between control and HF.
3) ROS produce mitochondrial DNA(mtDNA) damage and dysfunction
ESR spectroscopy demonstrated that OH was increased in the non-infarcted myocardium from mice with myocardial infarction (MI) created by coronary artery ligation for 4 weeks. MtDNA copy number relative to nuclear gene (18S rRNA) by Southern blot analysis was preferentially decreased in MI, associated with a parallel decrease (30-50%) in mtDNA-encoded gene transcripts including complex I, III, and IV. Consistent with these changes, enzymatic activities are also decreased, but complex II, encoded only by nuclear DNA, was preserved normal. An intimate link among ROS, mtDNA damage, and defects in the mitochondrial electron transport function, which lead to further generation of ROS, might play an important role in the development and progression of LV failure.
4) ROS are involved in LV remodeling via metalloproteinase (MMP) activation
Chronic administration of dimethylthiourea, OH scavenger (DMTU; 50 mg/kg, ip), into MI animals attenuated the increase of OH. Further, DMTU treatment ameliorated LV dysfunction and structural alterations (LV dilatation as well as hypertrophy and fibrosis of non-infarcted myocardium) in MI without affecting infarct size. Myocardial MMP-2 activity, measured by gelatin zymography, was increased in MI, which was also attenuated by DMTU. Thus the attenuation of increased myocardial ROS and MMP activity by ROS scavenger may contribute to its beneficial effects on LV remodeling and failure.
In conclusion, mitochondrial electron transport complex I is the potential source of O2^- inHF. OH was produced as reactive products of O2^- and H_2O_2 in the failing myocardium. Oxygen radicals and their reactive products might be responsible for the contractile dysfunction and structural damage seen in the failing heart. Less
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