Tissue-specific reductions in mitochondrial efficiency and increased ROS release rates during ageing in zebra finches,Taeniopygia guttata

Mitochondrial dysfunction and oxidative damage have long been suggested as critically important mechanisms underlying the ageing process in animals. However, conflicting data exist on whether this involves increased production of mitochondrial reactive oxygen species (ROS) during ageing. We employed high‐resolution respirometry and fluorometry on flight muscle (pectoralis major) and liver mitochondria to simultaneously examine mitochondrial function and ROS (H₂O₂) release rates in young (3 months) and old (4 years) zebra finches (Taeniopygia guttata). Respiratory capacities for oxidative phosphorylation did not differ between the two age groups in either tissue. Respiratory control ratios (RCR) of liver mitochondria also did not differ between the age classes. However, RCR in muscle mitochondria was 55% lower in old relative to young birds, suggesting that muscle mitochondria in older individuals are less efficient. Interestingly, this observed reduction in muscle RCR was driven almost entirely by higher mitochondrial LEAK-state respiration. Maximum mitochondrial ROS release rates were found to be greater in both flight muscle (1.3-fold) and the liver (1.9-fold) of old birds. However, while maximum ROS (H₂O₂) release rates from mitochondria increased with age across both liver and muscle tissues, the liver demonstrated a proportionally greater age-related increase in ROS release than muscle. This difference in age-related increases in ROS release rates between muscle and liver tissues may be due to increased mitochondrial leakiness in the muscle, but not the liver, of older birds. This suggests that age-related changes in cellular function seem to occur in a tissue-specific manner in zebra finches, with flight muscle exhibiting signs of minimising age-related increase in ROS release, potentially to reduce damage to this crucial tissue in older individuals.

     

Stimulation of oxidative energy metabolism in liver mitochondria from old and young rats by treatment with dehydroepiandrosterone (DHEA). A comparative study

Effects of treatment with DHEA (0.2 or 1.0 mg/kg body weight for 7 days) on oxidative energy metabolism of rat liver mitochondria from old (18–24 month old) and young (8–10 weeks old) male albino rats belonging to Charles-Foster strain were examined. Treatment with 1.0 mg DHEA resulted in increased body weights of the young rats without change in the liver weight. In the old animals the liver weight increased progressively with increasing dose of DHEA without affecting body weight. The state 3 respiration rates in liver mitochondria from old animals were, in general, lower than those in the young rats. The state 3 and state 4 respiration rates increased following DHEA treatment in dose-dependent manner bringing them close to values for young animals or beyond that with the effect being more pronounced at 1.0 mg dose. Treatment with DHEA also stimulated state 3 and state 4 respiration rates in young rats in dose-dependent manner. Contents of cytochrome aa₃, b and c + c₁ increased significantly in old animals in dose-dependent manner. In the young rats the lower dose (0.2 mg) of DHEA was more effective in bringing about a maximum increase in the contents of the cytochromes; the effect declined at the higher dose (1.0 mg). DHEA treatment also stimulated the mitochondrial ATPase activity in the old as well as in the young rats. The dehydrogenases activities were considerably low in the old rats compared to the values for the young animals. Treatment with DHEA stimulated dehydrogenases activities in old rats in dose-dependent manner bringing them close to values for the young animals or beyond. Treatment with lower dose (0.2 mg) of DHEA maximally stimulated dehydrogenases activities in young animals.     

Aging decreases expression and activity of glutathione peroxidase-1 in human endothelial progenitor cells

The mechanisms underlying effects of aging on functions of pro-angiogenic endothelial progenitor cells (EPCs) are poorly understood. Previous studies demonstrated that human EPCs express high levels of antioxidant enzymes as compared to mature endothelial cells. Here, we hypothesized that aging impairs antioxidant capacity of EPCs. So called “early EPCs” derived from cultured blood mononuclear cells were obtained from healthy young (average = 24 years old) and old (average = 72 years old) subjects. In EPCs of old subjects, the levels of glutathione peroxidase-1 (GPX1) protein and enzymatic activity were significantly reduced. The serum selenium levels in young and old subjects were not significantly different. Increasing selenium concentration in the cell culture also did not affect the protein levels of GPX1, suggesting the reduced GPX1 in old subject's EPCs is selenium independent. Expressions of catalase, Mn-superoxide dismutase (MnSOD), and CuZnSOD were not affected by aging. EPCs of old subjects were more sensitive to oxidative stress induced by H₂O₂ as compared with EPCs of young subjects, suggesting that impairment of GPX1 during aging may contribute to low survival ability of EPCs in response to oxidative stress. The results indicate that GPX1 may represent a potential therapeutic target for enhancement of regenerative capacity of EPCs in old subjects.     

Role of cellular defense against hydrogen peroxide-induced inhibition of myocyte respiration

Hydrogen peroxide (H₂O₂) serves as a precursor for highly reactive oxygen intermediates. However, the respiratory function of myocytes is relatively resistant to exogenously administered H₂O₂. In this study, we examined whether or not the reduction of cellular defense increases the toxicity of H₂O₂. Rat heart myocytes were isolated by collagenase digestion. Respiratory rates of myocytes, suspended in a medium containing sucrose, 3-N-morpholino-propanesulfonic acid, EGTA and bovine serum albumin, were determined polarographically in the presence of pyruvate and malate with or without 2,4-dinitrophenol (DNP). Mitochondrial membrane potentials were measured by using [³H]triphenylmethylphosphonium⁺. Cellular defense was attenuated by i) inhibiting the catalase activity by 3-amino-1,2,4-triazole (AT), ii) reducing the glutathione concentration by diethyl maleate (DEM) or ethacrinic acid (EA), and iii) permeabilizing the sarcolemmal membrane by saponin. The dose-response relationship between H₂O₂ (0.1–5 mM) and mitochondrial membrane potential was not greatly affected by these experimental conditions. Myocyte respiration was inhibited by 5 mM H₂O₂, particularly that measured in the presence of DNP (48% of control). DEM treatment did not significantly affect the respiratory inhibition by H₂O₂, whereas the degree of inhibition was somewhat greater following EA or AT treatment. By contrast, the sensitivity of cellular respiration to H₂O₂ was potentiated approximately two orders of magnitude by the permeabilization of sarcolemmal membrane; thus, 100 M H₂O₂ inhibited both DNP-stimulated and unstimulated respiration to 17% and 35% of control, respectively. The results indicate that factors existing in the sarcolemma and/or in the cytosol, which become ineffective and/or are diluted, respectively, following permeabilization with saponin, are important cellular defense mechanisms in alleviating the toxic effect of exogenous H₂O₂ on the respiration of mitochondria in situ in myocytes.     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin

Aims/hypothesis We studied the impact of the reactive oxygen species hydrogen peroxide (H₂O₂) and antioxidative enzymes on the pathogenesis of diabetes induced by multiple low doses of streptozotocin (MLD-STZ).Methods We isolated the islets of C57BL/6 mice. For ex vivo analyses, mice had been injected with MLD-STZ. For in vitro analyses, islets were incubated with different concentrations of STZ, with either of the two moieties of STZ, methylnitrosourea and d-glucose, with H₂O₂ or with alloxan. Levels of H₂O₂ generation were measured by the scopoletin method. We assessed mRNA expression of Cu/Zn and Mn superoxide dismutase, catalase, and glutathione peroxidase (GPX) by semiquantitative polymerase chain reaction. GPX activity was measured spectrophotometrically. In vitro, beta cell function was assayed by measuring basal and d-glucose-stimulated release of immunoreactive insulin using an ELISA kit.Results Ex vivo, MLD-STZ significantly increased H₂O₂ generation in male but not in female mice. It also increased GPX activity and mRNA expression of catalase, Cu/Zn and Mn superoxide dismutase, and GPX in female but not in male mice. In vitro, STZ significantly stimulated H₂O₂ generation in islets of male mice only. In male islets, alloxan increased H₂O₂ generation at a highly toxic concentration, but d-glucose and methylnitrosourea did not. Both STZ and H₂O₂ dose-dependently inhibited the release of immunoreactive insulin after a d-glucose challenge.Conclusions/interpretation The results indicate that H₂O₂ participates in the pathogenesis of MLD-STZ diabetes in male C57BL/6 mice, which do not up-regulate antioxidative enzymes in islets. Conversely, female mice are protected, probably due to an increment of several enzymes with the potential to detoxify H₂O₂.Abbreviations GPX glutathione peroxidase - H₂O₂ hydrogen peroxide - HD-STZ high-dose streptozotocin - HRP horse radish peroxidase - IRI immunoreactive insulin - MLD-STZ multiple low doses of streptozotocin - NF-B nuclear factor kappa B - O₂^–· superoxide radical - ^·OH hydroxyl radicals - ROS reactive oxygen species - SOD superoxide dismutase N. T. E. Friesen and A. S. Büchau contributed equally to this work, it being part of their doctoral theses     

Hydrogen peroxide-dependent arteriolar dilation in contracting muscle of rats fed normal and high salt diets

Objective: High dietary salt intake decreases the arteriolar dilation associated with skeletal muscle contraction. Because hydrogen peroxide (H₂O₂) can be released from contracting muscle fibers, this study was designed to assess the possible contribution of H₂O₂ to skeletal muscle functional hyperemia and its sensitivity to dietary salt. Methods: The authors investigated the effect of catalase treatment on arteriolar dilation and hyperemia in contracting spinotrapezius muscle of rats fed a normal salt (0.45%, NS) or high salt (4%, HS) diet for 4 weeks. Catalase‐sensitive 2′,7′‐dichlorofluorescein (DCF) fluorescence was measured as an index of H₂O₂ formation, and the mechanism of arteriolar dilation to H₂O₂ was probed in each group using pharmacological inhibitors. Results: DCF fluorescence increased with muscle contraction, but not if catalase was present. Catalase also reduced arteriolar dilation and hyperemia during contraction in both dietary groups. Exogenous H₂O₂ dilated arterioles in both groups, with greater responses in HS rats. Guanylate cyclase inhibition did not affect arteriolar responses to H₂O₂ in either group, but K_Ca or K_ATP channel inhibition equally reduced these responses, and K_ATP channel inhibition equally reduced functional hyperemia in both groups. Conclusions: These results indicate that locally produced H₂O₂ contributes to arteriolar dilation and hyperemia in contracting skeletal muscle, and that the effect of H₂O₂ on arteriolar tone in this vascular bed is mediated largely through K⁺ channel activation. High dietary salt intake does not reduce the contribution of H₂O₂ to active hyperemia, or alter the mechanism through which H₂O₂ relaxes arteriolar smooth muscle.     

The essential role of FoxO6 phosphorylation in aging and calorie restriction

This study was aimed at characterizing the functional progression of the endothelial (ECs) and smooth muscle cells (SMCs) of the coronary microvasculature between youth and old age, as well as at determining the mechanisms of the observed changes on the basis of the glucose tolerance, mitochondrial energy metabolism, and oxidative stress. Male rats were divided into four age groups (3, 6, 11, and 17 months for the young (Y), young adult (YA), middle-aged (MA), and old (O) animals). The cardiac mechanical function, endothelial-dependent dilatation (EDD) and endothelial-independent dilatation (EID) of the coronary microvasculature were determined in a Langendorff preparation. The mitochondrial respiration and H₂O₂ production were evaluated and completed by ex vivo measurements of oxidative stress. EDD progressively decreased from youth to old age. The relaxation properties of the SMCs, although high in the Y rats, decreased drastically between youth and young adulthood and stabilized thereafter, paralleling the reduction of mitochondrial oxidative phosphorylation. The ECs dilatation activity, low at youth, was stimulated in YA animals and returned to their initial level at middle age. That parameter followed faithfully the progression of the amount of active cardiac endothelial nitric oxide synthase and whole body glucose intolerance. In conclusion, the progressive decrease in EDD occurring with aging is due to different functional behaviors of the ECs and SMCs, which appear to be associated with the systemic glucose intolerance and cardiac energy metabolism.     

Superoxide production by mitochondria of insulin-sensitive tissues: mechanistic differences and effect of early diabetes

Obesity and mild hyperglycemia are characteristic of early or “prediabetes.” The associated increase in fatty acid flux is posited to enhance substrate delivery to mitochondria, leading to enhanced superoxide production that results in mitochondrial dysfunction and progressive worsening of the hyperglycemic state. We quantified superoxide production by gastrocnemius muscle, heart, and liver mitochondria in a rodent model that mimics the pathophysiology of prediabetes by administering low-dose streptozotocin to rats fed high fat (HF). Superoxide was rigorously determined indirectly as H₂O₂ largely released from the matrix and by electron paramagnetic resonance spectroscopy that directly detects superoxide released externally. Both HF and low-dose streptozotocin mildly increased glycemia (P < .05 by 2-way analysis of variance). Matrix and external superoxide production by gastrocnemius mitochondria respiring on the complex II substrate succinate and matrix superoxide production by liver mitochondria respiring on the complex I substrates glutamate plus malate were significantly reduced by HF feeding but not affected by mild hyperglycemia. Superoxide production was not significantly altered by either treatment in heart mitochondria fueled by either complex I or II substrates. The functional status of the mitochondria was assayed as simultaneous respiration and membrane potential that were not affected by HF or mild hyperglycemia. Comparison of substrate and inhibitor effects on superoxide release implied marked differences in the redox mechanisms regulating mitochondrial superoxide production from liver mitochondria compared with muscle and heart. In summary, superoxide production from mitochondria of different insulin-sensitive tissues differs mechanistically. However, in any case, excess superoxide production as an intrinsic property of mitochondria of insulin-sensitive tissues does not result from conditions mimicking the pathophysiology of pre- or early diabetes.     

Prolonged protective effect of propranolol on hypoxic heart muscle

To assess whether the in vivo administration of propranolol protects heart muscle against the deleterious effects of hypoxia and to establish how long the protection persists after cessation of therapy, rabbits were given propranolol, 2.0 mg/kg subcutaneously twice daily for 5 to 6 days. This dose regimen was used to provide plasma levels comparable with those obtained clinically. The hearts were isolated and perfused under either aerobic (partial pressure of oxygen [PO₂]greater than 600 mm Hg) or hypoxic (PO₂ less than 6 mm Hg) conditions. Heart rate was kept constant. Coronary effluent was collected and assayed for creatine klnase (CK) activity and myoglobin content. Resting and peak systolic tension was monitored and, after 60 minutes of perfusion, the mitochondria were harvested and assayed for respiratory activity (atoms oxygen used/mg mitochondrial protein per min [QO₂], respiratory control index [RCI]and nmol adenosine dlphosphate used/n atoms oxygen consumed [$\text{ADP}\text{O}{}_2$ ratio]) and Ca⁺⁺-accumulatlng activity. The control rabbit hearts responded to the hypoxia-perfusion state with release of CK and myoglobin and an increase in resting tension. Mitochondrial QO₂ and RCI were significantly depressed. Mitochondrial Ca⁺⁺-accumulating activity was enhanced. The propranolol-treated rabbit hearts were protected in that they released CK and myoglobin more slowly and had a lower rate of rise of resting tension. Mitochondrial respiratory activity (QO₂ and RCI) was also better maintained, and the mitochondria accumulated Ca⁺⁺ at a relatively slow rate. This protective effect of propranolol pretreatment was not accompanied by a changed tissue level of adenosine triphosphate, creatine phosphate or glycogen.The protective effect of propranolol persisted for up to 72 hours after the last dose of propranolol, and hence was present when beta adrenoceptor blockade was no longer effective. These results support the hypothesis that there may be secondary consequences of beta blockade.     

Functional changes of the coronary microvasculature with aging regarding glucose tolerance,energy metabolism,and oxidative stress

This study was aimed at characterizing the functional progression of the endothelial (ECs) and smooth muscle cells (SMCs) of the coronary microvasculature between youth and old age, as well as at determining the mechanisms of the observed changes on the basis of the glucose tolerance, mitochondrial energy metabolism, and oxidative stress. Male rats were divided into four age groups (3, 6, 11, and 17 months for the young (Y), young adult (YA), middle-aged (MA), and old (O) animals). The cardiac mechanical function, endothelial-dependent dilatation (EDD) and endothelial-independent dilatation (EID) of the coronary microvasculature were determined in a Langendorff preparation. The mitochondrial respiration and H₂O₂ production were evaluated and completed by ex vivo measurements of oxidative stress. EDD progressively decreased from youth to old age. The relaxation properties of the SMCs, although high in the Y rats, decreased drastically between youth and young adulthood and stabilized thereafter, paralleling the reduction of mitochondrial oxidative phosphorylation. The ECs dilatation activity, low at youth, was stimulated in YA animals and returned to their initial level at middle age. That parameter followed faithfully the progression of the amount of active cardiac endothelial nitric oxide synthase and whole body glucose intolerance. In conclusion, the progressive decrease in EDD occurring with aging is due to different functional behaviors of the ECs and SMCs, which appear to be associated with the systemic glucose intolerance and cardiac energy metabolism.     

Oxidative stress and intracellular pH in the young and old erythrocytes of rat

The effects of oxidative stress (OS) on the rat erythrocytes (RBCs) that were fractionated on the percoll/BSA gradient into young and old cells were studied to find out if the altered Na ⁺ /H⁺ and Cl⁻/HCO₃ ⁻ antiporters and in turn the intracellular pH (pH_i) could act as one of the promoters of cell death. Old cells were more spherical with lesser surface area, more fragile osmotically and had lesser protein sulphydryl content than the young cells. OS was induced in RBCs by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). AAPH increased the superoxide dismutase (SOD) activity and MDA level and, the changes between the young and old. Interestingly, vitamin C was effective in reducing MDA in the old. Further, in the old a rapid Na⁺-dependent acidification in the presence of AAPH and a marginal acidosis in the presence of vitamin C were evident. Old RBCs exhibited higher acidosis and vitamin C was less effective in lowering the stress-induced acidosis compared to the young. Our studies suggest that increased acidosis followed by low intracellular pH could be one of the determinant factors for the disappearance of old RBCs from circulation, and perhaps of the young too under OS.     

The Effect of Hydrogen Peroxide in Human Internal Thoracic Arteries: Role of Potassium Channels,Nitric Oxide and Cyclooxygenase Products

Introduction We investigated both the effect and the role(s) of potassium channels, nitric oxide (NO) and cyclooxygenase (COX) products in the effect of hydrogen peroxide (H₂O₂) in human internal thoracic artery (ITA) rings. Materials and methods Samples of redundant ITA obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Results H₂O₂ (10⁻⁷–10⁻⁴ M) produced concentration-dependent relaxation responses in human ITA precontracted by phenylephrine. The relaxant responses to H₂O₂ did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human ITA rings with superoxide dismutase (50 U/ml) did not affect the relaxant responses to H₂O₂, while 1,000 U/ml catalase caused a significant decrease. Incubation of endothelium-intact or endothelium-denuded human ITA rings with voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) significantly inhibited the relaxant responses to H₂O₂. COX inhibitor indomethacin (10⁻⁵ M) also caused a significant inhibition. Incubation with ATP-dependent potassium channel blocker glibenclamide (10⁻⁶ M) or Ca²⁺-activated potassium channel blocker iberiotoxin (10⁻⁷ M) or NO synthase (NOS) blocker -nitro-l-arginine methyl ester (10⁻⁴ M) did not alter relaxant responses of ITA rings to H₂O₂. Conclusion The findings of the present study suggested that H₂O₂-induced relaxation responses in human ITA were neither dependant on the endothelium nor blocked by NOS inhibition but they rather seem to depend on the activation of voltage-dependent potassium channels and COX.     

Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle

Aims/hypothesis Insulin resistance and type 2 diabetes are associated with mitochondrial dysfunction. The aim of the present study was to test the hypothesis that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, as a result of a reduction in the mitochondrial content. Materials and methods The O₂ flux capacity of permeabilised muscle fibres from biopsies of the quadriceps in healthy subjects (n = 8; age 58 ± 2 years [mean±SEM]; BMI 28 ± 1 kg/m²; fasting plasma glucose 5.4 ± 0.2 mmol/l) and patients with type 2 diabetes (n = 11; age 62 ± 2 years; BMI 32 ± 2 kg/m²; fasting plasma glucose 9.0 ± 0.8 mmol/l) was measured by high-resolution respirometry. Results O₂ flux expressed per mg of muscle (fresh weight) during ADP-stimulated state 3 respiration was lower (p < 0.05) in patients with type 2 diabetes in the presence of complex I substrate (glutamate) (31 ± 2 vs 43 ± 3 pmol O₂ s⁻¹ mg⁻¹) and in response to glutamate + succinate (parallel electron input from complexes I and II) (63 ± 3 vs 85 ± 6 pmol s⁻¹ mg⁻¹). Further increases in O₂ flux capacity were observed in response to uncoupling by FCCP, but were again lower (p < 0.05) in type 2 diabetic patients than in healthy control subjects (86 ± 4 vs 109 ± 8 pmol s⁻¹ mg⁻¹). However, when O₂ flux was normalised for mitochondrial DNA content or citrate synthase activity, there were no differences in oxidative phosphorylation or electron transport capacity between patients with type 2 diabetes and healthy control subjects. Conclusions/interpretation Mitochondrial function is normal in type 2 diabetes. Blunting of coupled and uncoupled respiration in type 2 diabetic patients can be attributed to lower mitochondrial content.     

Hydrogen peroxide generation by mitochondria isolated from regionally ischemic and nonischemic dog myocardium

Summary We occluded the left anterior descending coronary artery of anesthetized, open-chest dogs, for 1 or 2 h. Some hearts were reperfused for 1 h after 1 h of ischemia. We isolated mitochondria from the central ischemic zone (CIZ) and a surrounding nonischemic zone (NIZ) of the left ventricle, and assayed H₂O₂ production using a horseradish peroxidase-dual wavelength spectrophotometric technique. Mitochondria, studied in the absence of exogenous respiratory chain inhibitors, generated H₂O₂ during State 4 respiration with succinate as the substrate. NIZ mitochondria in all groups produced ca. 1.5 nmols H₂O₂/min/mg protein (no significant differences between groups). The State 4 O₂ consumption rates of NIZ mitochondria from hearts subjected to 1 h ischemia plus reperfusion, or 2 h of ischemia (ca. 30 nmols/min/mg) were significantly higher than that of NIZ mitochondria of hearts subjected to only 1 h of ischemia (23 nmols/min/mg). Thus, the ratio between H₂O₂ produced and State 4 O₂ consumption fell from 6.5% to 5%. Mitochondria from all CIZ samples had State 4 O₂ consumption rates that were not different from corresponding NIZ values. However CIZ mitochondria of hearts subjected to 1 h ischemia without reperfusion produced less H₂O₂ (1.1±0.1 nmols/min/mg), and had a slightly reduced H₂O₂/O₂ ratio (4.4±0.7%), compared with their NIZ samples (1.5±0.1 nmols/min/mg; 5.3%). Reperfusion after 1 h of ischemia abolished these regional differences. The CIZ mitochondria from hearts subjected to 2 h ischemia produced only 0.75±0.22 nmols H₂O₂/min/mg (2.5% of State 4 O₂ consumption). These values were 50% of corresponding NIZ values, and were significantly less than for any other group or tissue region. If similar phenomena occur in conscious animals subjected to incomplete regional ischemia, especially of relatively brief duration or if accompanied by reduced intracellular defenses against oxidants such as H₂O₂, they suggest that mitochondria persist as H₂O₂ sources and so may contribute to the oxidant load and myocardial dysfunction.Supported by grants from the National Institutes of Health (HL-29499) and from the American Heart Association of Michigan     

Age-related differences in cigarette smoke extract-induced H2O2 production by lung endothelial cells

Cigarette smoke causes oxidative stress in the lung resulting in injury and disease. The purpose of this study was to determine if there were age-related differences in cigarette smoke extract (CSE)-induced production of reactive species in single and co-cultures of alveolar epithelial type I (AT I) cells and microvascular endothelial cells harvested from the lungs (MVECLs) of neonatal, young and old male Fischer 344 rats. Cultures of AT I cells and MVECLs grown separately (single culture) and together (co-culture) were exposed to CSE (1, 10, 50, 100%). Cultures were assayed for the production of intracellular reactive oxygen species (ROS), hydroxyl radical (OH), peroxynitrite (ONOO⁻), nitric oxide (NO) and extracellular hydrogen peroxide (H₂O₂). Single and co-cultures of AT I cells and MVECLs from all three ages produced minimal intracellular ROS in response to CSE. All ages of MVECLs produced H₂O₂ in response to CSE, but young MVECLs produced significantly less H₂O₂ compared to neonatal and old MVECLs. Interestingly, when grown as a co-culture with age-matched AT I cells, neonatal and old MVECLs demonstrated ~ 50% reduction in H₂O₂ production in response to CSE. However, H₂O₂ production in young MVECLs grown as a co-culture with young AT I cells did not change with CSE exposure. To begin investigating for a potential mechanism to explain the reduction in H₂O₂ production in the co-cultures, we evaluated single and co-cultures for extracellular total antioxidant capacity. We also performed gene expression profiling specific to oxidant and anti-oxidant pathways. The total antioxidant capacity of the AT I cell supernatant was ~ 5 times greater than that of the MVECLs, and when grown as a co-culture and exposed to CSE (≥ 10%), the total antioxidant capacity of the supernatant was reduced by ~ 50%. There were no age-related differences in total antioxidant capacity of the cell supernatants. Gene expression profiling found eight genes to be significantly up-regulated or down-regulated. This is the first study to describe age-related differences in MVECLs exposed to CSE.     

Protective effect of pretreatment with verapamil,nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium

To establish whether the prophylactic use of verapamil, nifedipine or propranolol protects heart muscle against the deleterious effects of global ischemia and reperfusion, rabbits were injected subcutaneously twice daily with 2.0 mg/kg of one of these drugs for 4 to 5 days. The hearts were then isolated, paced and either perfused aerobically, made totally ischemic for 90 minutes or made ischemic for 90 minutes and then reperfused. At the end of this time some of the hearts were assayed for adenosine triphosphate (ATP), creatine phosphate (CP) and calcium (Ca⁺⁺). Other hearts were homogenized, the mitochondria harvested and monitored for oxidative phosphorylating and ATP-generating capacity and Ca⁺⁺ content. The effect of Ca⁺⁺ on the ATP-generatlng capacity of cardiac mitochondria was also determined.Hearts that were made ischemic gained Ca⁺⁺. The endogenous stores of ATP and CP were depleted; the mitochondria accumulated Ca⁺⁺ and the oxidative phosphorylating activity (respiratory control index and oxygen quotient) was impaired. During reperfusion, tissue and mitochondrial Ca⁺⁺ was substantially increased, the capacity of the mitochondria to use O₂ for state III respiration was further impaired and the ATP-generating capacity reduced. Resting tension increased and there was only a small recovery of active tension generation.Hearts from the rabbits treated with propranolol, verapamil or nifedipine were protected against ischemia, and ischemia with reperfusion-induced decline in the ATP-generating and O₂-utillzing capacity of the mitochondria. There was also a less marked increase in tissue and mitochondrial Ca⁺⁺ and the systolic tension-generating capacity of the hearts was better maintained.     

Altered mitochondrial bioenergetics and ultrastructure in the skeletal muscle of young adults with type 1 diabetes

Aims/hypothesis

A comprehensive assessment of skeletal muscle ultrastructure and mitochondrial bioenergetics has not been undertaken in individuals with type 1 diabetes. This study aimed to systematically assess skeletal muscle mitochondrial phenotype in young adults with type 1 diabetes.

Methods

Physically active, young adults (men and women) with type 1 diabetes (HbA_1c 63.0?±?16.0 mmol/mol [7.9%?±?1.5%]) and without type 1 diabetes (control), matched for sex, age, BMI and level of physical activity, were recruited (n?=?12/group) to undergo vastus lateralis muscle microbiopsies. Mitochondrial respiration (high-resolution respirometry), site-specific mitochondrial H₂O₂ emission and Ca²⁺ retention capacity (CRC) (spectrofluorometry) were assessed using permeabilised myofibre bundles. Electron microscopy and tomography were used to quantify mitochondrial content and investigate muscle ultrastructure. Skeletal muscle microvasculature was assessed by immunofluorescence.

Results

Mitochondrial oxidative capacity was significantly lower in participants with type 1 diabetes vs the control group, specifically at Complex II of the electron transport chain, without differences in mitochondrial content between groups. Muscles of those with type 1 diabetes also exhibited increased mitochondrial H₂O₂ emission at Complex III and decreased CRC relative to control individuals. Electron tomography revealed an increase in the size and number of autophagic remnants in the muscles of participants with type 1 diabetes. Despite this, levels of the autophagic regulatory protein, phosphorylated AMP-activated protein kinase (p-AMPKα^Thr172), and its downstream targets, phosphorylated Unc-51 like autophagy activating kinase 1 (p-ULK1^Ser555) and p62, was similar between groups. In addition, no differences in muscle capillary density or platelet aggregation were observed between the groups.

Conclusions/interpretation

Alterations in mitochondrial ultrastructure and bioenergetics are evident within the skeletal muscle of active young adults with type 1 diabetes. It is yet to be elucidated whether more rigorous exercise may help to prevent skeletal muscle metabolic deficiencies in both active and inactive individuals with type 1 diabetes.

     

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H₂O₂) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H₂O₂ production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22^phox, SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.     

Reduced muscle oxidative capacity is independent of O2 availability in elderly people

Impaired O₂ transport to skeletal muscle potentially contributes to the decline in aerobic capacity with aging. Thus, we examined whether (1) skeletal muscle oxidative capacity decreases with age and (2) O₂ availability or mitochondrial capacity limits the maximal rate of mitochondrial ATP synthesis in vivo in sedentary elderly individuals. We used ³¹P-magnetic resonance spectroscopy (³¹P-MRS) to examine the PCr recovery kinetics in six young (26 ± 10 years) and six older (69 ± 3 years) sedentary subjects following 4 min of dynamic plantar flexion exercise under different fractions of inspired O₂ (FiO₂, normoxia 0.2; hyperoxia 1.0). End-exercise pH was not significantly different between old (7.04 ± 0.10) and young (7.05 ± 0.04) and was not affected by breathing hyperoxia (old 7.08 ± 0.08, P > 0.05 and young 7.05 ± 0.03). Likewise, end-exercise PCr was not significantly different between old (19 ± 4 mM) and young (24 ± 5 mM) and was not changed in hyperoxia. The PCr recovery time constant was significantly longer in the old (36 ± 9 s) compared to the young in normoxia (23 ± 8 s, P < 0.05) and was not significantly altered by breathing hyperoxia in both the old (35 ± 9 s) and young (29 ± 10 s) groups. Therefore, this study reveals that the muscle oxidative capacity of both sedentary young and old individuals is independent of O₂ availability and that the decline in oxidative capacity with age is most likely due to limited mitochondrial content and/or mitochondrial dysfunction and not O₂ availability.