Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Cardiac hypertrophy in copper-deficient rats is owing to increased mitochondria

Dietary copper depletion results in cardiac hypertrophy and ultrastructural alterations. The objective of this study was to determine the components that contribute to cardiac enlargement. Two groups (n = 4) of male, weaning, Sprague-Dawley rats were fed ad libitum with copper-adequate or copper-deficient diets for five weeks. Cross sectional transmission electron micrographs from both groups were evaluated using image analysis to quantify absolute area occupied by myocyte, mitochondria, myofibril, and other intracellular material. Copper-deficient rats had larger myocytes, increased area of mitochondria, and increased ratio of mitochondria :myofibril as well as mitochondria:myocyte. Copper deficiency did not change the absolute area occupied by myofibrils. These data suggested that increase in the absolute mitochondria area is the major contributory factor to the cardiac hypertrophy in copper deficiency. Under the conditions used, myofibril has minimal role toward contributing to the hypertrophic state. The pathology reported resembles human forms of genetic mitochondrial cardiomyopathies. The copper-deficient rat may be a useful model to investigate the underlying biochemical or molecular responses when peptides of enzymes are deleted.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

The effects of copper deficiency on the pyridinium crosslinks of mature collagen in the rat skeleton and cardiovascular system

The 3-hydroxypyridinium crosslinks of collagen were quantified in tissues of the skeleton and cardiovascular system of normal and copper-deficient rats. The copper-deficient rats used in this study displayed retarded growth, cardiac hypertrophy, anemia, and lowered liver copper concentrations. Quantification of the crosslinks by high performance liquid chromatography indicated that there were lower concentrations of collagen crosslinks in the hearts of copper-deficient animals, a finding that was manifest in both right and left ventricles. This was in contrast to the collagen of the aorta where no alteration in crosslink concentration was observed. The femoral diaphysis of copper-deficient rats also had lower amounts of collagen crosslinks than copper-supplemented animals, whereas crosslinking in the tibial diaphysis and articular cartilage was relatively unaffected by copper deficiency. These results are discussed with reference to the cardiac and skeletal abnormalities that occur in copper-deficient animals.     

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.     

Mechanism of inhibition of mitochondrial ATP synthase by 17β−Estradiol

17β-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling (“slipping”) of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and V_FCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and V_olig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the F_o component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F₁-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.     

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.     

Copper and signal transduction: platelets as a model to determine the role of copper in stimulus-response coupling.

Platelets from copper-deficient rats have been used as a model to investigate the role of copper in receptor-mediated cellular responses. Copper deficiency doubles the rate of dense granule secretion and increases myosin association with the platelet cytoskeleton following thrombin stimulation. Mechanisms underlying the effects of copper deficiency on thrombin-induced signals that elicit dense granule secretion involve suppression of protein kinase C activity and impairment of Ca2+ release from intracellular stores. Copper deficiency also reduces the cellular GTP content of platelets. This may limit receptor effector coupling through GTP-dependent regulatory proteins leading to protein kinase C activation and the release of Ca2+ from intracellular stores. The reduction in GTP content during copper deficiency results from its utilization to maintain cellular ATP levels in response to severely inhibited cytochrome c oxidase activity in platelet mitochondria. Thus, the role of copper in maintaining normal signal transduction may be indirectly related to its biological function in mitochondria.     

Influence of adenine nucleotides on oligomycin inhibition of energy-transducing reactions in intact rat-liver mitochondria.

The inhibitory effect of oligomycin was investigated in intact mitochondria through oxidative phosphorylation and uncoupler induced ATPase activity. Results show that oligomycin inhibition curves can be either sigmoidal or hyperbolic depending on experimental conditions and chiefly on the metabolic state of mitochondria with regard to the distribution of mitochondrial endogenous adenine-nucleotides. Active respiration and uncoupler-induced ATPse activity produce sigmoidal titration curves for a high initial ATP : ADP ratio and hyperbolic curves for a low ATP : ADP ratio. Time-dependent inhibitions are observed for the two reactions. The maximal inhibitory action for low concentrations of the inhibitor is delayed by the initial presence of ATP or the possibility of generating from inorganic phosphate before adding oligomycin. Results presented here show that the initial adenine-nucleotide distribution is important for oligomycin sensitivity of energy-linked reactions. Although a limited conformational change of the oligomycin-sensitivity to the inhibitor, it is more likely that a gross structural change of the inner membrane induced by adenine-nucleotides modifies membrane permeability to oligomycin.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets

Experiments were conducted to determine if copper deficiency enhances the rate of thrombin-induced dense granule secretion by modifying the major signal transduction pathways of rat platelets. Platelets were obtained from male, weanling Sprague-Dawley rats fed diets containing either deficient ( < 0.5 μg/g diet) or adequate (5.5 μg/g diet) copper for 5 weeks. Following stimulation with thrombin (0.1 U/mL), the rate of dense granule secretion as measured by ATP release was 160% higher in platelets from copper-deficient than from control rats. Inhibition of the rate of thrombin-induced ATP release by (6-aminohexyl)-1-naphthalene-sulfonamide, a calmodulin antagonist was independent of copper status. However, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the rate of ATP release only in platelets from copper-deficient rats. Aspirin had no effect on ATP release from platelets obtained from either copper-deficient or control rats. This suggests that copper deficiency alters the role of protein kinase C in regulating dense granule secretion. Analysis of autoradiographs showing [³²P]-labeled platelet proteins indicated that the phosphorylation of a 40 kDa protein, a known substrate for protein kinase C in platelets, was significantly less following thrombin stimulation in platelets from copper-deficient than from control rats. When protein kinase C was activated by phorbol 12-myristate 13-acetate prior to thrombin stimulation, ATP release was attenuated regardless of copper status. These findings suggest that protein kinase C can still function as a feedback inhibitor of platelet dense granule secretion in copper deficiency, but impaired activation of this enzyme following thrombin stimulation may prevent it from achieving full regulatory capacity.     

Energy-storage capacity of the mitochondrial proton-motive force

Resting state respiration of rat-liver mitochondria in the presence of oligomycin was rapidly blocked with cyanide and the dissipation of the membrane potential was followed with a tetraphenylphosphonium-sensitive electrode. From the rate of this dissipation and the electric capacitance of the mitochondrial membrane the energy stored in form of the membrane potential was calculated as about 7 microJ/mg protein. In the absence of oligomycin, dissipation of the membrane potential was slower, as it was partly compensated by proton ejection by mitochondrial ATPase hydrolyzing endogenous ATP. This allowed to calculate the total energy storage capacity of the proton-motive force. It amounted to the equivalence of 3.3 nmol ATP/mg protein or about 130 microJ/mg protein. The stoichiometry of proton-pumping ATPase utilizing endogenous ATP was estimated as three protons per molecule ATP.     

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O₂ consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.     

Cardiac levels of fibronectin,laminin, isomyosins,and cytochrome c oxidase of weanling rats are more vulnerable to copper deficiency than those of postweanling rats

The relative quantities of cardiac laminin, fibronectin, cytochrome c oxidase (CCO), and isomyosin types were studied by gel electrophoresis in male rats fed copper-deficient diets beginning either from the time of weaning for 5 weeks or from 5 weeks postweaning for 6 weeks with one group of copper-repleted rats. Increased levels of fibronectin and V(3) isomyosin but decreased levels of CCO subunit IV and laminin were found in weanling copper-depleted rats. In contrast, postweanling copper-depleted rats exhibited only increased levels of fibronectin and decreased levels of cardiac CCO subunit IV. Repletion of copper-deficient rats for 6 weeks was not sufficient to restore CCO subunit IV to the same level as controls. These results confirm that biochemical lesions in the basal laminae are a result of copper restriction. The decreased nuclear encoded subunits of CCO may help explain some of the mitochondrial pathology observed in dietary copper restriction. Increased V(3) isomyosin levels with low ATPase activity may help to conserve to a limited extent the ATP levels in copper-deficient cardiac tissue. These protein changes are consistent with the known morphological alterations of hearts from copper-restricted rats.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.