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Mechanisms of oxygen radical generation in the mitochondria from the failing hearts

衰竭心脏线粒体中氧自由基产生的机制

基本信息

批准号：
12670676
负责人：
TSUTSUI Hiroyuki
金额：
$ 2.05万
依托单位：
KYUSHU UNIVERSITY GRADUATE SCHOOL OF MEDICAL SCIENCES
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670676/
关键词：
Heart failure Remodeling Oxidative stress Free radicals Mitochondria 活性酸素種抗酸化薬

项目摘要

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) spectroscopyTo 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 … More 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 dysfunctionESR 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) activationChronic 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

实验和临床研究已经发现，超氧阴离子（·O_2-）、H_2O_2和羟基自由基（·OH）等氧自由基（ROS）的增加是心脏衰竭的一个特征。氧化应激可能在充血性心力衰竭（HF）的病理生理中起重要作用。然而，衰竭心肌中ROS生成增强的细胞来源和机制尚不清楚。1)利用电子自旋共振（ESR）光谱法测定衰竭心脏中ROS（·O_2-、H_2O_2和·OH）的含量。将心衰犬心肌组织匀浆与4-羟基-2,2,6,6，-四甲基哌啶- n -氧基（羟基- tempo）作为自旋探针，用ESR光谱法检测其自旋信号。在HF中，ESR信号衰减速率与·OH水平成正比，显著增加，并通过在反应混合物中加入二甲基硫脲（·OH清除剂）来抑制。衰竭心脏中增加的·OH在去铁胺（铁螯合剂）、过氧化氢酶（H_2O_2清除剂）和铁（·O_2-清除剂）存在下被同样程度地清除，表明·OH来源于H_2O_2和·O_2-。此外，在抗霉素A（线粒体复合体III抑制剂）存在下，正常心肌中产生的·O_2-可以复制衰竭心肌中H_2O_2和·OH的增加。心肌·OH水平与左室收缩功能障碍呈显著正相关。心肌中SOD、过氧化氢酶、谷胱甘肽过氧化物酶等抗氧化酶活性未见明显下降，说明衰竭心脏的抗氧化能力保持正常。2)线粒体电子传递复合物I是·O_2-的潜在来源，为了确定·O_2-的亚细胞来源，在NADH和琥珀酸作为NADH-泛醌氧化还原酶（复合物I）和琥珀酸-泛醌氧化还原酶（复合物II）的底物存在下，用自旋捕获剂5,5 '-二甲基- 1 -吡啶- n -氧化物（DMPO）的ESR光谱直接评估了其在亚细胞组分中的生成。在HF中，亚线粒体部分O_2的产生增加了2.8倍，这是由于复合物I上电子传递的功能阻断，复合物I的酶活性在HF中降低，这可能导致呼吸链的功能解偶联和失效线粒体中有害的O_2-产生。在对照组和hf之间，细胞质和微粒体中O_2-的产生相当。3)ROS产生线粒体DNA（mtDNA）损伤和功能障碍。esr光谱显示，冠状动脉结扎造成心肌梗死（MI）小鼠的非梗死心肌中OH增加。通过Southern blot分析，线粒体DNA相对于核基因（18S rRNA）的拷贝数在心肌梗死中优先减少，与线粒体DNA编码的基因转录物（包括复合体I、III和IV）的平行减少（30-50%）有关。与这些变化一致，酶活性也下降，但仅由核DNA编码的复合体II保持正常。ROS、mtDNA损伤和线粒体电子传递功能缺陷之间的密切联系，导致ROS的进一步产生，可能在左室衰竭的发生和发展中发挥重要作用。4)活性氧通过金属蛋白酶（MMP）激活参与左室重构。心肌梗死动物长期给药OH清除剂二甲基硫脲（DMTU; 50 mg/kg, ip）可降低OH的增加。此外，DMTU治疗改善了心肌梗死的左室功能障碍和结构改变（左室扩张以及非梗死心肌的肥大和纤维化），而不影响梗死面积。明胶酶谱法测定心肌MMP-2活性，心肌梗死后心肌MMP-2活性升高，DMTU降低心肌MMP-2活性。因此，ROS清除剂对增加的心肌ROS和MMP活性的衰减可能有助于其对左室重构和衰竭的有益作用。综上所述，线粒体电子传递复合体I是O2^- inHF的潜在来源。在衰竭心肌中，OH是O2^-和H_2O_2的反应产物。氧自由基及其反应产物可能是导致心力衰竭的收缩功能障碍和结构损伤的原因。少