Mitochondria are one of the enzymatic sources of reactive oxygen species (ROS) and could also be a major target for ROS-mediated damage. We hypothesized that ROS may induce mitochondrial DNA (mtDNA) damage, which leads to defects of mtDNA-encoded gene expression and respiratory chain complex enzymes and thus may contribute to the progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). In a murine model of MI and remodeling created by the left anterior descending coronary artery ligation for 4 weeks, the LV was dilated and contractility was diminished. Hydroxyl radicals, which originated from the superoxide anion, and lipid peroxide formation in the mitochondria were both increased in the noninfarcted LV from MI mice. The mtDNA copy number relative to the nuclear gene (18S rRNA) preferentially decreased by 44% in MI by a Southern blot analysis, associated with a parallel decrease (30% to 50% of sham) in the mtDNA-encoded gene transcripts, including the subunits of complex I (ND1, 2, 3, 4, 4L, and 5), complex III (cytochrome b), complex IV (cytochrome c oxidase), and rRNA (12S and 16S). Consistent with these molecular changes, the enzymatic activity of complexes I, III, and IV decreased in MI, whereas, in contrast, complex II and citrate synthase, encoded only by nuclear DNA, both remained at normal levels. An intimate link among ROS, mtDNA damage, and defects in the electron transport function, which may lead to an additional generation of ROS, might play an important role in the development and progression of LV remodeling and failure.

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (27413 versus 1369 nmol min 1 mg 1 protein, P0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium. (Circ Res. 1999;85:357-363.) Key Words: antioxidant free radical heart failure myocardial contraction reactive oxygen species C ongestive heart failure (HF) is an important cause of morbidity and mortality in patients with various heart diseases. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of left ventricular (LV) failure have not yet been fully elucidated. Reactive oxygen species (ROS) such as superoxide anions (O 2) and hydroxy radicals (OH) cause the oxidation of membrane phospholipids, proteins, and DNAs, and they have been implicated in a wide range of pathological conditions including ischemia-reperfusion injury, neurodegenerative diseases , and aging. Under physiological conditions, their toxic effects are prevented by such scavenging enzymes as super-oxide dismutase (SOD), glutathione peroxidase, and catalase as well as by other nonenzymatic antioxidants. However, when the production of ROS becomes excessive, oxidative stress might have a harmful effect on the functional integrity of biological tissue. Oxygen free radicals have been shown to cause contractile failure and structural damage in the myo-cardium. 1,2 However, their significance has been demonstrated to limited subsets of cardiac diseases, which include ischemic heart disease 3 and adriamycin-induced cardiac toxicity. 4 Recent investigations have suggested the generation of ROS to increase in chronic HF. Lipid peroxides and 8-iso-prostaglandin F 2 , which are the major biochemical consequences of RO...

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ACE angiotensin converting enzyme BAL bronchoalveolar lavage CRT cardiac resynchronization therapy CT computed tomography 18 F-FDG fluorine-18 fluorodeoxyglucose 67 Ga gallium-67 HRCT high resolution computed tomography ICD implantable cardioverter defibrillator MRI magnetic resonance imaging PET positron emission tomography sIL-2R soluble interleukin 2 recepter SPECT single photon emission computed tomography JCS GUIDELINES

Abstract-Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion ( ⅐ O 2 Ϫ ), H 2 O 2 , and hydroxy radical ( ⅐ OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-N-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to ⅐ OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea ( ⅐ OH scavenger) into the reaction mixture. Increased ⅐ OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator

Background-Tumor necrosis factor-␣ (TNF-␣) and angiotensin II (Ang II) are implicated in the development and further progression of heart failure, which might be, at least in part, mediated by the production of reactive oxygen species (ROS). However, the cause and consequences of this agonist-mediated ROS production in cardiac myocytes have not been well defined. Recently, we demonstrated that increased ROS production was associated with mitochondrial DNA (mtDNA) damage and dysfunction in failing hearts. We thus investigated whether the direct exposure of cardiac myocytes to TNF-␣ and Ang II in vitro could induce mtDNA damage via production of ROS. Methods and Results-TNF-␣ increased ROS production within cultured neonatal rat ventricular myocytes after 1 hour, as assessed by 2Ј,7Ј-dichlorofluorescin diacetate fluorescence microscopy. TNF-␣ also decreased mtDNA copy number by Southern blot analysis in association with complex III activity, which was prevented in the presence of the antioxidant ␣-tocopherol. A direct exposure of myocytes to H 2 O 2 caused a similar decrease in mtDNA copy number. In contrast, Ang II did not affect mtDNA copy number, despite the similar increase in ROS production. TNF-␣-mediated ROS production and a decrease in mtDNA copy number were inhibited by the sphingomyelinase inhibitor D609. Furthermore, N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analogue, increased myocyte ROS production, suggesting that TNF-␣-mediated ROS production and subsequent mtDNA damage were mediated by the sphingomyelin-ceramide signaling pathway. Conclusions-The intimate link between TNF-␣, ROS, and mtDNA damage might play an important role in myocardial remodeling and failure.

Abstract-Oxidative stress might play an important role in the progression of left ventricular (LV) remodeling and failure that occur after myocardial infarction (MI). We determined whether reactive oxygen species (ROS) are increased in the LV remodeling and failure in experimental MI with the use of electron spin resonance spectroscopy and whether the long-term administration of dimethylthiourea (DMTU), hydroxyl radical ( ⅐ OH) scavenger, could attenuate these changes. We studied 3 groups of mice: sham-operated (sham), MI, and MI animals that received DMTU (MIϩDMTU). Drugs were administered to the animals daily via intraperitoneal injection for 4 weeks. ⅐ OH was increased in the noninfarcted myocardium from MI animals, which was abolished in MIϩDMTU. Fractional shortening was depressed by 65%, LV chamber diameter was increased by 53%, and the thickness of noninfarcted myocardium was increased by 37% in MI. MIϩDMTU animals had significantly better LV contractile function and smaller increases in LV chamber size and hypertrophy than MI animals. Changes in myocyte cross-sectional area determined with LV mid-free wall specimens were concordant with the wall thickness data. Collagen volume fraction of the noninfarcted myocardium showed significant increases in the MI, which were also attenuated with DMTU. Myocardial matrix metalloproteinase-2 activity, measured with gelatin zymography, was increased with MI after 7 and 28 days, which was attenuated in MIϩDMTU. Thus, the attenuation of increased myocardial ROS and metalloproteinase activity with DMTU may contribute, at least in part, to its beneficial effects on LV remodeling and failure. Therapies designed to interfere with oxidative stress might be beneficial to prevent myocardial failure. (Circ Res. 2000;87:392-398.)Key Words: antioxidant Ⅲ radicals Ⅲ heart failure Ⅲ myocardial infarction Ⅲ remodeling M yocardial infarction (MI) frequently produces left ventricular (LV) dilatation and hypertrophy of the noninfarcted myocardium. 1 These changes in LV geometry, referred to as remodeling, contribute to the development of depressed cardiac performance. 2 Thus, surviving patients with MI are at an increased risk for occurrence of heart failure (HF), reinfarction, arrhythmia, and sudden cardiac death. 2 LV remodeling is caused by the side-to-side slippage of cardiac myocytes at the infarcted region and the hypertrophic response of the noninfarcted myocytes, resulting in a progressive increase in the chamber diameter. 3 The underlying mechanisms responsible for these processes have been attributed to hemodynamic stress as well as the activation of neurohumoral factors, including the renin-angiotensin system. However, the details of contributing factors in LV remodeling remain to be elucidated.Reactive oxygen species (ROS) can produce myocardial contractile dysfunction and structural damage. 4 There is growing evidence that ROS are increased in HF and may contribute to disease progression. 5,6 Hill and Singal 7 showed that antioxidant enzyme activities are decreased and th...

Background-Mitochondrial DNA (mtDNA) copy number is decreased not only in mtDNA-mutation diseases but also in a wide variety of acquired degenerative and ischemic diseases. Mitochondrial transcription factor A (TFAM) is essential for mtDNA transcription and replication. Myocardial mtDNA copy number and TFAM expression both decreased in cardiac failure. However, the functional significance of TFAM has not been established in this disease state. Methods and Results-We have now addressed this question by creating transgenic (Tg) mice that overexpress human TFAM gene and examined whether TFAM could protect the heart from mtDNA deficiencies and attenuate left ventricular (LV) remodeling and failure after myocardial infarction (MI) created by ligating the left coronary artery. TFAM overexpression could ameliorate the decrease in mtDNA copy number and mitochondrial complex enzyme activities in post-MI hearts. Survival rate during 4 weeks of MI was significantly higher in Tg-MI than in wild-type (WT) littermates (WT-MI), although infarct size was comparable. LV cavity dilatation and dysfunction were significantly attenuated in Tg-MI. LV end-diastolic pressure was increased in WT-MI, and it was also reduced in Tg-MI. Improvement of LV function in Tg-MI was accompanied by a decrease in myocyte hypertrophy, apoptosis, and interstitial fibrosis as well as oxidative stress in the noninfarcted LV. Conclusions-Overexpression