克山病区粮喂养豚鼠心肌的抗氧化能力、线粒体细胞色素氧化酶活性及园二色性的改变及其发病学意义

克山病区粮喂养豚鼠10周,可见其血浆、红细胞及心肌线粒体中的硒和谷胱甘肽过氧化物酶活性明显降低,红细胞膜过氧化氢酶活性降低,超氧化物歧化酶活性趋向增加,膜脂受氧自由基作用产生的共轭双烯、荧光色脂明显增加,表明存在膜的氧化损伤。实验组心肌线粒体中的心磷脂(CL)减少,细胞色素氧化酶(CCO)活性降低,园二色性(CD)异常,而补充青菜组随 CL 的增加,可见其 CCO 酶活性,CD 谱均恢复到接近正常水平,各实验组心肌线粒体、亚线粒体及纯化 CCO 的 CD 谱异常,峰值(208及224nm)明显低于对照,说明其线粒体 CCO 构象出现了异常。文中讨论了克山病发病的分子病理机制。     

微小RNA对缺血再灌注损伤心肌线粒体作用的研究进展

心肌缺血再灌注损伤是造成心肌结构损伤、功能障碍的一种病理生理过程,进一步发展会导致级联的多器官功能障碍。线粒体是一种结构功能复杂且对外界环境反应敏感的细胞器,其稳态的维持依赖于正常形态、功能及数量的相对稳定状态。线粒体质量与代谢异常和心血管疾病尤其是心肌缺血再灌注损伤的发生密切相关。微小RNA是近年来研究较多的在缺血再灌注损伤心肌线粒体保护中具有重要作用的调控因子。本文通过微小RNA对心肌缺血再灌注损伤时线粒体形态、功能、线粒体自噬和线粒体DNA几个方面的调控机制与相关前沿进展进行综述,为微小RNA参与缺血再灌注心肌线粒体损伤的后续研究提供一定的理论依据。     

Decrease in mitochondrial oxidative protein damage parameters in the streptozotocin-diabetic rat

BACKGROUND: Many authors have shown that hyperglycemia leads to an increase in oxidative protein damage in diabetes. The aim of this study was to reveal the susceptibility of mitochondria from liver, pancreas, kidney, and skeletal muscle of diabetic Sprague-Dawley rats, a model of type 1 diabetes, to oxidative protein damage. METHODS: Mitochondrial fractions were obtained by differential centrifugation. To show the effect of hyperglycemia in promoting oxidative protein damage, we determined mitochondrial protein carbonyl, total thiol, nitrotyrosine, advanced oxidation protein products, and lipid hydroperoxide levels. The levels of the studied markers, except nitrotyrosine, were determined by colorimetric methods. Nitrotyrosine levels were measured by ELISA. All values were compared with those of the controls by using the Mann-Whitney U-test. RESULTS: Nitrotyrosine and lipid hydroperoxide levels were decreased and other parameters were not changed in liver mitochondria of diabetic rats. Protein carbonyl, nitrotyrosine, advanced oxidation protein products, and lipid hydroperoxide levels were decreased and total thiol levels were not changed in pancreas mitochondria of diabetic rats. Only advanced oxidation protein products and lipid hydroperoxide levels were decreased in kidney mitochondria of diabetic rats. The levels of the same parameters were not significantly different in muscle mitochondria of diabetic rats. CONCLUSIONS: The decrease in mitochondrial oxidative protein damage in diabetes may correspond to either an increase in antioxidant defense mechanisms or a different adaptive response of the cells to the increased extramitochondrial oxidative stress in diabetes. The mitochondrial oxidative protein damage-lowering mechanisms in diabetes remain to be clarified.     

The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana

The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15_K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.Iron-sulfur cluster (ISC) containing proteins conduct essential metabolic processes in all organisms. In plants, autonomous pathways for ISC assembly are present in plastids and mitochondria, whereas ISC biosynthesis and incorporation in cytosolic and nuclear proteins relies on export of bound sulfide from mitochondria (1). Because ISCs are sensitive to superoxide and its reaction products formed by aerobic metabolism, increasing oxidation of the atmosphere led to evolution of sophisticated machineries mediating and controlling the assembly and the transfer of ISCs to acceptor proteins (2). The entire machinery consisting of more than 14 proteins in plastids and 19 proteins in mitochondria includes proteins providing sulfur and iron atoms, scaffold proteins for cluster assembly and transfer proteins that insert ISCs into recipient apoproteins (3). The fundamental role of ISC assembly for building the machineries that are at the center of maintaining life is emphasized by the fact that mutants affecting genes of the ISC assembly pathways are frequently embryo-lethal (3). Among the proteins considered as transfer proteins are representatives of the type II subset of glutaredoxins (GRX), which are characterized by their CGFS monothiol active site motif and their ability to bind glutathione-bridged ISCs (4).Although evidence exists for the function of GRXs in the assembly of Fe-S proteins in yeast cells and vertebrates (5, 6), their significance in planta is still unclear. Null mutants for plastidic monothiol GRXS14 and GRXS16 are viable, which may be explained by partially overlapping activities (3). Nevertheless, the ability of both proteins to complement the yeast Δgrx5 mutant that lacks the mitochondrial monothiol Grx5p strongly hints to involvement in maturation of Fe-S proteins (7). Similarly, it has been shown that the cytosolic and nuclear GRXS17, both from poplar and Arabidopsis, can also complement the Δgrx5 mutant (7, 8). However, Arabidopsis grxs17 null mutants had only a minor decrease in cytosolic Fe-S enzyme activities, whereas a severe developmental phenotype is visible under elevated temperature and extended daylight. These data indicate that GRXS17 is likely not required for de novo ISC assembly in the cytosol, although it could play a role in cluster repair. Rather, it may be that GRXS17 functions as an oxidoreductase, regulating the function of its partners as BolA2, NF-YC11, or other unidentified targets (8, 9).However, the involvement of GRXS15 in the maturation of Fe-S proteins in mitochondria remains elusive in plants because, among poplar monothiol GRXs, it is the only isoform failing to rescue most phenotypes of the yeast Δgrx5 mutant (7). Furthermore, the subcellular localization of GRXS15 is ambiguous. Independent targeting experiments have reported GFP-tagged poplar GRXS15 in mitochondria (7) and Arabidopsis GRXS15 in the plastid stroma (10) or dual-targeted plastidic mitochondrial in bifunctional fluorescence complementation (BiFC) experiments with BolA4 (9). In proteome studies, GRXS15 has been repeatedly found in the mitochondria of Arabidopsis (11) and potato (12), but also in the chloroplast proteome of maize (13). The only phenotype of grxs15-null mutants described thus far is sensitivity toward H₂O₂, which led to the suggestion that GRXS15 may be involved in the maintenance of growth and development under oxidative stress conditions (10).Here, we provide evidence that GRXS15 is an essential component of the mitochondrial ISC machinery that for a long time has been controversial because of the lack of suitable mutants and the failure to complement yeast cells lacking the orthologous gene. The results from in vitro ISC reconstitution and transfer assays and complementation of null mutants in yeast and Arabidopsis with mutated variants of GRXS15 demonstrate that GRXS15 coordinates an ISC and is likely essential for the delivery of ISC to apoproteins in mitochondria, serving in particular for the maturation of ISC into aconitase.     

心脏交感神经顿抑机制的初步研究

目的 :探讨缺血后大鼠心脏交感神经顿抑现象及其内在机制。方法 :运用电场刺激 (频率 6Hz,电压 5V ,脉宽 2ms,持续 1min)诱导离体灌注鼠心去甲肾上腺素 (NA)胞裂释放 ,并通过自身前后两次刺激比较(S2 ∶S1)以判断心脏交感神经末梢NA的释放状况。结果 :①大鼠心脏缺血 30min后恢复灌注其交感神经末梢NA的释放受到明显抑制 (neuronalstunning ,神经顿抑 ) ,并随复灌时间的延长 (第 1、5、30分钟 ) ,交感神经顿抑得到逐渐恢复 (S2 /S1:0 .2 3± 0 .0 31,0 .4 1± 0 .0 4 3和 0 .6 2± 0 .0 5 ,均P <0 .0 1) ;②腺苷受体阻滞剂 8 苯基茶碱使缺血后大鼠心脏交感神经末梢NA的释放得到恢复 [(12 8.30± 8.2 9)∶(12 6 .4 0± 8.0 9) pmol/L ,P >0 .0 5 ],而浕2 肾上腺素能受体阻滞剂育亨宾则对其无明显影响 [(5 4 .70± 10 .2 7)∶(12 7.5 0± 7.87)pmol/L ,P <0 .0 1];③腺苷受体激动剂 2 苯异丙基腺苷使非缺血大鼠心脏交感神经末梢NA的释放受到明显抑制 (6 1.70± 10 .6 5∶12 6 .30±14 .18,P <0 .0 1) ,而α2 肾上腺素能激动剂可乐定则对其无明显影响 [(130 .0 0± 9.90 )∶(130 .4± 10 .33)pmol/L ,P >0 .0 5 ]。结论 :心脏较长时间的缺血可引起交感神经顿抑 ,且内源性腺苷起重要作用。     

大剂量硝酸甘油加重大鼠离体心脏缺血/再灌注损伤

目的:研究大剂量硝酸甘油（NG）对心肌缺血再灌注（MI/R）损伤的影响。方法:SD大鼠30只,制备离体MI/R模型,并将其随机分为2组:NG组和对照组。再灌注开始时NG组给予硝酸甘油（15μg/h,溶剂为950ml/L乙醇,加药速度50μl/h）,对照组给予950ml/L乙醇（50μl/h）,观察心功能指标,检测冠脉流出液中的肌酸激酶（CK）和乳酸脱氢酶（LDH）含量,用伊文氏兰-红四氮唑（TTC）染色和TUNAL法观测再灌注心肌坏死和凋亡程度。结果:大剂量NG抑制了再灌注后心功能的恢复,引起CK、LDH释放增加,促进心肌细胞坏死和凋亡。结论:大剂量NG加重了大鼠离体MI/R损伤。     

心肌去乙酰化酶SIR33对缺血／再灌注小鼠心律失常的影响

目的探讨心肌线粒体去乙酰化酶SIRT3对急性缺血再灌注（I／R）所致心律失常的影响。方法：以24只SIRT3基因敲除型小鼠为实验对象,用24只野生型小鼠为对照,两种小鼠均随机各分为对照组、假手术组、I／R模型组及I／R＋烟酰胺腺嘌呤二核苷酸（NAD＋）治疗组,每组6只小鼠（rt=6）。采用冠脉左前降支结扎缺血30 min再灌注2 h建立在体大鼠急性心肌I／R模型,于术中监测心电指标。取心肌组织检测SIRT3、锰超氧化物歧化酶（MnSOD）和过氧化氢酶（Catalase）蛋白的表达和心肌内氧自由基（ROS）的水平。结果：与野生型对照小鼠相比,SIRT3基因敲除型小鼠心肌中SIRT3、MnSOD和Catalase蛋白表达的水平显著降低。心律失常评分的结果显示,SIRT3基因敲除型小鼠的假手术组即可观察到心律失常。SIR13基因敲除可导致小鼠心肌I／R所致心律失常显著加重（与野生型模型组相比,P〈0-05）。心肌I／R后,SIRT3基因敲除型小鼠心肌中ROS的增加程度明显高于野生型模型组小鼠（P〈0-05）。预先采用NAD＋治疗,可显著提高野生型I／R小鼠心肌SIRT3的活性（与野生型小鼠模型组相比,P〈0-05）,显著增加心肌MnSOD的活性,进而有效地抑制I／R小鼠心肌中ROS的水平,有效缓解I／R所致心律失常（与野生型小鼠模型组相比,均P〈0-05）。但是,SIR73基因敲除后,NAD＋治疗引起的上述心肌保护作用基本消失。结论：心肌中SIRT3表达的降低可能是加重心肌I／R过程中氧化应激损伤并促发心律失常的重要机制。SIRT3正常活性的维持有助于对抗心肌I／R损伤（MIRI）的发生。     

In situ mitochondrial function in volume overload- and pressure overload-induced cardiac hypertrophy in rats

Objectives: Little comparative information is available on mitochondrial function changes during experimentally-induced hypertrophy. Respiratory control mechanisms are not exactly the same in situ and in isolated mitochondria. This study assessed in situ mitochondrial function in two myocardial hypertrophy models.Methods: Cytochrome aa3 (Cytaa3) and myoglobin (Mb) absorption changes were monitored in isolated rat hearts using dual wavelength spectrophotometry (Cytaa3: 605–630 nm, Mb: 581–592 nm). Hypertrophy was induced by creation of an aortic stenosis or of an aorto-caval fistula. Optical monitoring was performed on diastolearrested perfused hearts using the sequence O₂ perfusion, N₂ perfusion during 4 min, and reoxygenation. The plateaus of the Cytaa3 and Mb curves were used to quantify oxidation-reduction and oxygenation levels. Respiratory kinetics were characterized by the slopes of transition phase curves.Results: Myoglobin oxygenation was comparable in the hypertrophied and control hearts. However, Cytaa3 oxidation-reduction levels in the hypertrophied hearts showed a shift towards greater reduction in comparison with the controls (controls: 0.580 ±0.008 DO605/DO630 nm, n=34; fistula: 0.530±0.023, n=23; stenosis: 0.522±0.016, n=20, p<0.001). The rate of Cytaa3 reduction and the rate of myoglobin deoxygenation were significantly accelerated (p<0.005) in the volume overload group (0.507±0.043, n=23), whereas the respiratory rate in the pressure overload group (0.389 ±0.034, n=20) was comparable to that in the control hearts (0.358±0.026 DO 605 nm/DO630 nm. min^–1, n=34).Conclusion: We found mitochonorial function alterations in both volume overload- and pressure overload-induced cardiac hypertrophy, despite adequate cytosol oxygenation. The patterns of these alterations differed: the redox state showed a shift of similar magnitude toward greater reduction in both models, but the respiratory rate was increased in the volume-overloaded hearts and unchanged in the pressure-overloaded hearts. The modification in the oxidation-reduction state suggested that overload hypertrophy may induced changes in the metabolism of the myocardium, which may, in turn, load to persistent modifications in mitochondrial function. The differences between the two models suggest that adaptation to hypertrophy-inducing events exists at the level of the mitochondrion.     

Oxidative phosphorylation function of two mitochondrial preparations from heart: effects of ischaemia and cytochrome C

Summary The effects ofmyocardial ischaemia on twomitochondrial preparations (polytron and nagarse) were evaluated, using three different substrates to localize the defect in the electron transport chain. Coronary artery ligation and normothermic ischaemic arrest (NICA) of the isolated working perfused rat heart were used as models of ischaemia. The results showed that, although the degree to which these two mitochondrial preparations were affected by ischaemia might differ, mitochondrial function was altered in a similar manner. After 20 min of NICA, the polytron mitochondria appeared to be more susceptible to injury than the nagarse fraction. However, coronary artery ligation for 55 min depressed the QO₂ (State 3) values of polytron and nagarse mitochondria to the same extent. Moderate ischaemia (coronary artery ligation for 55 min or NICA for 20 min) depressed the oxidative phosphorylation function of both mitochondrial populations with glutamate as substrate only, indicating that the NADH-coenzyme Q region of the electron transport chain is most sensitive to ischaemic injury. As the period and severity of ischaemia increased, the rest of the electron transport chain and the phosphorylating capacity of the mitochondria became depressed. In moderate ischaemia, addition of exogenous cytochrome C caused a complete return to normal QO₂ values of both mitochondrial preparations. However, in severe ischaemia (55 min NICA), although cytochrome C improved mitochondrial oxygen uptake, the values were still significantly lower than those of control hearts.

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Zusammenfassung Die Auswirkungen einer Myokardischämie auf zwei Mitochondrienpräparationen (Polytron und Nagarse) wurden unter Benutzung von drei verschiedenen Substraten zur Lokalisation des Defektes in der Elektronentransportkette untersucht. Als Ischämiemodell wurde die Unterbindung einer Koronararterie sowie normothermer ischämischer Stillstand des isolierten, perfundierten Rattenherzens benutzt. Die Ergebnisse zeigen, daß die mitochondriale Funktion in ähnlicher Weise verändert wird, obwohl das Ausmaß der ischämischen Beeinträchtigung bei beiden Mitochondrienpräraten unterschiedlich sein kann. Nach 20 Minuten ischämischen Herzstillstands schienen die Polytron-Mitochondrien gegen die Schädigung empfindlicher zu sein als die Nagarse-Fraktion. Jedoch verminderte eine Koronarunterbindung über 55 Minuten die QO₂-(Stadium-3-)Werte von Polytron- und Nagarse-Mitochondrien in gleichem Ausmaß. Mäßige Ischämie (Koronararterienunterbindung über 55 Minuten) verminderte die oxidative Phosphorylierung beider Mitochondrienpopulationen, wenn nur Glutamat als Substrat vorlag, was darauf hinweist, daß die NADH-Coenzym-Q-Region der Ionentransportkette besonders empfindlich gegen eine ischämische Schädigung ist. Wenn Dauer und Schweregrad der Ischämie anstiegen, wurde die restliche Elektronentransportkette und die Phosphorylierungskapazität der Mitochondrien herabgesetzt. Bei mäßiger Ischämie verursachte die Zufuhr von Cytochrom C die komplette Wiederherstellung normaler QO₂-Werte bei beiden Mitochondrienpräparationen. Dagegen waren die Werte bei schwerer Ischämie (55 Minuten ischämischer Stillstand) noch signifikant geringer als die bei normalen Herzen, obwohl Cytochrom C die mitochondriale Sauerstoffaufnahme verbesserte.

     

Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential

Postnatal maturation of the heart depends on the switch from glycolytic to oxidative metabolism and it is associated with decreasing tolerance to oxygen deprivation. Therefore, changes in composition and function of cardiac mitochondria during postnatal development require detailed characterization. Left-ventricular myocardium of prenatal, and 1-, 2-, 5-, 10-, 20-, 28-, 50-, 60-, and 90-d-old male Wistar rats was studied. The expression of uncoupling proteins (UCPs), adenine nucleotide translocase (ANT), and peroxisome proliferator-activated receptor alpha (PPARalpha) genes was characterized by northern blotting (UCP2), real-time quantitative RT-PCR (UCP2, UCP3, ANT1, ANT2, and PPARalpha), and by immunoblotting (UCP3). In isolated mitochondria, cytochromes a + a(3) were quantified by a spectrophotometry, and mitochondrial membrane potential (MMP) was measured using Rhodamine 123 (by spectrofluorimetry and flow cytometry). The specific content of cytochromes in mitochondria increased two-fold between birth and day 30, similarly, as the expression of ANT1 and PPARalpha genes. Postnatal activation in the expression of UCP2, UCP3, ANT1 and PPARalpha genes resulted in the expression maxima between days 20 and 30. The content/expression declined following day 20 (UCP2, UCP3, and PPARalpha) or 30 (cytochromes and ANT1), while expression of ANT2 declined continuously during the first month of life. In 1-d-old animals a single population of mitochondria with a relatively high MMP was observed; with increasing age, a second population of mitochondria with a significantly lower MMP appeared. The results support the view that mitochondrial energy conversion in heart changes during ontogeny and suggest the involvement of UCP3 and/or ANT1 in the control mechanism.     

Stochastic mitochondrial DNA changes: bioenergy decline in type I skeletal muscle fibres correlates with a decline in the amount of amplifiable full-length mtDNA

Extra-long PCR (XL-PCR) was used to assess the relative concentration of functional full-length mitochondrial DNA (mtDNA) in single type I human vastus lateralis muscle fibres of defined cytochrome c oxidase (COX)activity. Type I muscle fibres rely more on mitochondrial oxidative phosphorylation for their energy demands, compared to the other common fibre types (IIa, IIab and IIb) that principally depend on glycolysis for their energy requirements. A total of 60 single type I fibres were analyzed from 15 individuals (8males and 7 females) of various ages. COX positive muscle fibres were shown to contain amplifiable full-length mtDNA together with a small number of mtDNA rearrangements. By contrast, COX negative fibres did not contain detectable full-length mtDNA, but did contain aheterogeneous mixture of rearranged mtDNA species with the frequency and occurrence of each deletion varying considerably from fibre to fibre. These data lead us to the conclusion that the level of COX activity in type I muscle fibres is reflected by the amount of amplifiable full-length mtDNA. It is proposed that the amount of amplifiable full-length mtDNA constitutes the functional fraction of the total mtDNA. A comprehensive hypothesis that relates the dynamics of mtDNA turnover, mtDNA mutations, mtDNA damage and repair to the ageing process is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of iron overload in the isolated ischemic and reperfused rat heart

Summary It has been suggested that iron might play a pivotal role in the development of reperfusion-induced cellular injury through the activation of oxygen free radical producing reactions. The present study examined the effects of myocardial iron overload on cardiac vulnerability to ischemia and reperfusion. Moreover, the effect of the iron chelator deferoxamine in reversing ischemia-reperfusion injury was studied. Animals were treated with iron dextran solution (i.m. injection, 25 mg every third day during a 5 week period). The control group received the same treatment without iron. Isolated rat hearts were perfused at constant flow (11 ml/min) and subjected to a 15 minute period of global normothermic ischemia followed by reperfusion for 15 minutes. The effects of iron overload were investigated using functional and biochemical parameters, as well as ultrastructural characteristics of the ischemic-reperfused myocardium compared with placebo values. The results suggest that (a) a significant iron overload was obtained in plasma and hepatic and cardiac tissues (×2.5, ×16, and ×8, respectively) after chronic intramuscular administration of iron dextran (25 mg); (b) during normoxia, iron overload was associated with a slight reduction in cardiac function and an increase in lactate dehydrogenase (LDH) release (×1.5); (c) upon reperfusion, functional recovery was similar whether the heart had been subjected to iron overload or not. However, in the control group left ventricular end-diastolic pressure remained higher than in preischemic conditions, an effect that was not observed in the iron-overloaded group. Moreover, LDH release was markedly increased in the iron-loaded group (×4.2); (d) iron overload was associated with a significant worsening of the structural alterations observed during reperfusion, particularly at the mitochondrial and sarcomere level; (e) after 15 minutes of reperfusion, the activity of the anti-free-radical enzyme, glutathione peroxidase (GPX), was significantly reduced in ironoverloaded hearts, whereas catalase activity was increased; (e) the overall modifications observed in the presence of iron overload were prevented by deferoxamine. In conclusion, this study underlines the possible role of cardiac iron in the development of injury associated with ischemia and reperfusion, and the possible importance of the use of an iron-chelating agent in antiischemic therapy.     

Mitochondria and the aging heart

The average human life span has markedly increased in modern society largely attributed to advances in medical and therapeutic sciences that have successfully reduced important health risks. However, advanced age results in numerous alterations to cellular and subcellular components that can impact the overall health and function of an individual. Not surprisingly, advanced age is a major risk factor for the development of heart disease in which elderly populations observe increased morbidity and mortality. Even healthy individuals that appear to have normal heart function under resting conditions, actually have an increased susceptibility and vulnerability to stress. This is confounded by the impact that stress and disease can have over time to both the heart and vessels. Although, there is a rapidly growing body of literature investigating the effects of aging on the heart and how age-related alterations affect cardiac function, the biology of aging and underlying mechanisms remain unclear. In this review, we summarize effects of aging on the heart and discuss potential theories of cellular aging with special emphasis on mitochondrial dysfunction.     

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.     

Increased susceptibility to Ca2+‐induced permeability transition and to cytochrome c release in rat heart mitochondria with aging: effect of melatonin

Abstract: Aging is associated with a decline of cardiac function. The mitochondrial permeability transition (MPT) may be a factor in cardiac dysfunction associated with aging. We investigated the effect of aging and long‐term treatment with melatonin (approximately 10 mg/kg b.w./day for 2 months), a known natural antioxidant, on the susceptibility to Ca²⁺‐induced MPT opening and cytochrome c release in rat heart mitochondria. The mitochondrial content of normal and oxidized cardiolipin as a function of aging and melatonin treatment was also analyzed. Mitochondria from aged rats (24 month old) displayed an increased susceptibility to Ca²⁺‐induced MPT opening, associated with an elevated release of cytochrome c, when compared with young control animals (5 month old). Melatonin treatment counteracted both these processes. Aging was also associated with an oxidation/depletion of cardiolipin which could be counteracted as well by melatonin. It is proposed that the increased level of oxidized cardiolipin could be responsible, at least in part, for the increased susceptibility to Ca²⁺‐induced MPT opening and cytochrome c release in rat heart mitochondria with aging. Melatonin treatment counteracts both these processes, most likely, by preventing the oxidation/depletion of cardiolipin. Our results might have implications in the necrotic and apoptotic myocytes cell death in aged myocardium, particularly in ischemia/reperfusion injury.     

Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified

Nuclear mitochondrial pseudogenes (numts) are nonfunctional copies of mtDNA in the nucleus that have been found in major clades of eukaryotic organisms. They can be easily coamplified with orthologous mtDNA by using conserved universal primers; however, this is especially problematic for DNA barcoding, which attempts to characterize all living organisms by using a short fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Here, we study the effect of numts on DNA barcoding based on phylogenetic and barcoding analyses of numt and mtDNA sequences in two divergent lineages of arthropods: grasshoppers and crayfish. Single individuals from both organisms have numts of the COI gene, many of which are highly divergent from orthologous mtDNA sequences, and DNA barcoding analysis incorrectly overestimates the number of unique species based on the standard metric of 3% sequence divergence. Removal of numts based on a careful examination of sequence characteristics, including indels, in-frame stop codons, and nucleotide composition, drastically reduces the incorrect inferences of the number of unique species, but even such rigorous quality control measures fail to identify certain numts. We also show that the distribution of numts is lineage-specific and the presence of numts cannot be known a priori. Whereas DNA barcoding strives for rapid and inexpensive generation of molecular species tags, we demonstrate that the presence of COI numts makes this goal difficult to achieve when numts are prevalent and can introduce serious ambiguity into DNA barcoding.     

Effects of electrical pacing to the preischemic rate during rewarming after hypothermic ischemia in the rat heart

Summary The effects of electrical pacing during the early reperfusion following hypothermic global ischemia (60 min, at 25°C) was studied in the isolated working rat heart model. The hearts were divided into three groups. Hearts in Group I (n=8) were control without hypothermia, ischemia or pacing. Hearts in Group II (n=16) were paced with ventricular rate at 300 beats/min with 1 m Volt for 10 min during the Langendorff mode after an initial 5 min of reperfusion. Hearts in Group III (n=14) were not paced. The recovery of aortic flow (both absolute and percent) was significantly better in Group II than in Group III, but was significantly lower in both groups than in control. No significant differences were noted, however, in heart rate, aortic pressure or coronary flow between Group II and III. In contrast, the tissue concentration of adenosine triphosphate (ATP) in Groups II and III decreased significantly by the end of reperfusion relative to Group I, but no difference in ATP existed between Group II and III. Myocardial ATP concentrations did not correlate with percent recovery of aortic flow. The myocardial concentration of calcium in Groups II and III increased by the end of reperfusion as compared with Group I, but no difference in calcium existed between Group II and III. The myocardial concentration of calcium demonstrated a significant correlation with percent recovery of aortic flow (r=0.71, n=30,p<0.005). Our results indicate that an electrical pacing during early reperfusion in the myocardium improves functional recovery of aortic flow.