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.     

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.     

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.     

Decrease lysyl oxidase activity in hearts of copper-deficient bovines

BackgroundLysyl oxidase (LOX) is a metalloenzyme that requires Cu as a cofactor and it is responsible for the formation of collagen and elastin cross-linking. The objective of this work was to measure the LOX enzyme activity in the heart of bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet.MethodsEighteen myocardial samples were obtained from Cu-deficient (n = 9) and control (n = 9) Holstein bovines during two similar assays. The samples were frozen in liquid nitrogen and stored at −70 °C to measure enzymatic activity. A commercial kit was used, following producer instructions.ResultsThe results showed that LOX activity from the hearts of Cu-deficient bovines is 29 % lower than the ones of control bovines, being this difference statistically significant (p = 0.03).ConclusionTo our knowledge, this is the first report that determined LOX enzymatic activity in bovine heart of Cu-deficient animals. The microscopic alterations found in these animals in our previous work, could be explained by a diminished LOX activity. The results are in agreement with other authors, who found a relationship between LOX activity and dietary Cu intake. The information provided by this work could help to clarify the pathogenesis of cardiac lesions in cattle with dietary Cu deficiency.     

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.     

Functional aspects of oxidative phosphorylation and electron transport in cardiac mitochondria of copper-deficient rats

Although dietary copper deficiency causes physiological, morphological, and biochemical abnormalities in cardiac mitochondria, the relationship observed between abnormalities of mitochondrial structure and function have been inconsistent in previous studies. The purpose of the present study was to re-evaluate the respiration rates of cardiac mitochondria from copper-deficient rats and to use several drugs that uncouple and inhibit mitochondrial respiration in order to clarify the mechanisms of mitochondrial dysfunction found in several laboratories. Copper deficiency reduced state 4 and state 3 cardiac mitochondrial respiration rates with all substrates tested. However, neither the ratio of ADP/oxygen consumed nor the acceptor control index was affected by copper deficiency. Cardiac mitochondria of copper-deficient rats showed a resistance to respiratory blockade by oligomycin and an increased ability to hydrolyze ATP in the presence of oligomycin compared with mitochondria of copper-adequate rats. This suggests that copper deficiency affects the function of the cardiac mitochondrial ATP synthase.     

Cytochrome c oxidase, Cu,Zn-superoxide dismutase, and ceruloplasmin activities in copper-deficient bovines

The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 μmol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.     

Nonenzymatic isolation and culture of adult islets from atrophic pancreata of copper-deficient rats: A morphologic analysis

Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated, as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating effects on islet function. This work was supported by a research grant from the Diabetes Research and Education Foundation.     

Post-translational modifications of ATP synthase in the heart: biology and function

The ATP synthase complex is a critical enzyme in the energetic pathways of cells because it is the enzyme complex that produces the majority of cellular ATP. It has been shown to be involved in several cardiac phenotypes including heart failure and preconditioning, a cellular protective mechanism. Understanding the regulation of this enzyme is important in understanding the mechanisms behind these important phenomena. Recently there have been several post-translational modifications (PTM) reported for various subunits of this enzyme complex, opening up the possibility of differential regulation by these PTMs. Here we discuss the known PTMs in the heart and other mammalian tissues and their implication to function and regulation of the ATP synthase.     

Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice

Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.     

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits , and of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for ATP synthase, while the and subunits increased progressively up to 26 months.     

Copper deficiency halves serum dehydroepiandrosterone in rats

DHEA (dehydroepiandrosterone) is a hormone often taken as a dietary supplement to prevent the normal decline with age and in the hope of preventing heart attacks. Rats in two experiments were made deficient in copper by standard methods and criteria. Copper deficiency decreased DHEA in serum by approximately 50%. People who associate higher serum concentrations of DHEA with health probably should eat a diet adequate in copper.     

Copper chaperones,intracellular copper trafficking proteins. Function,structure, and mechanism of action

This review summarizes findings on a new family of small cytoplasmic proteins called copper chaperones. The copper chaperones bind and deliver copper ions to intracellular compartments and insert the copper into the active sites of specific partners, copper-dependent enzymes. Three types of copper chaperones have been found in eukaryotes. Their three-dimensional structures have been determined, intracellular target proteins identified, and mechanisms of action have been revealed. The Atx1 copper chaperone binds Cu(I) and interacts directly with the copper-binding domains of a P-type ATPase copper transporter, its physiological partner. The copper chaperone CCS delivers Cu(I) to Cu,Zn-superoxide dismutase 1. Cox17 and Cox11 proteins serve as copper chaperones for cytochrome c oxidase, a copper-dependent enzyme.     

The Profile of Cardiac Cytochrome c Oxidase (COX) Expression in an Accelerated Cardiac-Hypertrophy Model

Summary The contribution of the mitochondrial components, the main source of energy for the cardiac hypertrophic growth induced by pressure overload, is not well understood. In the present study, complete coarctation of abdominal aorta was used to induce the rapid development of cardiac hypertrophy in rats. One to two days after surgery, we observed significantly higher blood pressure and cardiac hypertrophy, which remained constantly high afterwards. We found an early increased level of cytochrome c oxidase (COX) mRNA determined by in-situ hybridization and dot blotting assays in the hypertrophied hearts, and a drop to the baseline 20 days after surgery. Similarly, mitochondrial COX protein level and enzyme activity increased and, however, dropped even lower than baseline 20 days following surgery. In addition, in natural hypertension-induced hypertrophic hearts in genetically hypertensive rats, the COX protein was significantly lower than in normotensive rats. Taken together, the lower efficiency of mitochondrial activity in the enlarged hearts of long-term complete coarcted rats or genetically hypertensive rats could be, at least partially, the cause of hypertensive cardiac disease. Additionally, the rapid complete coarctation-induced cardiac hypertrophy was accompanied by a disproportionate COX activity increase, which was suggested to maintain the cardiac energy-producing capacity in overloaded hearts.     

Cardiac cytochrome-c oxidase deficiency occurs during late postnatal development in progeny of copper-deficient rats

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.     

Dietary chromium deficiency effect on sperm count and fertility in rats

Male rats raised on a low chromium diet containing less than 100 ppb chromium had decreased sperm counts and decreased fertility at age 8 months compared to the Cr-supplemented controls. Decreased sperm cell production and fertility were not apparent at age 4 months. At age 7–8 months the frequency of conception was 25 percent or less and the sperm count of the low chromium males was approximately 50 percent of that of the Cr-supplemented rats.     

Buried water molecules in helical transmembrane proteins

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.     

Architecture of helix bundle membrane proteins: an analysis of cytochrome c oxidase from bovine mitochondria.

We have analyzed the structure of mitochondrial cytochrome c oxidase in terms of general characteristics thought to be important for describing the architecture of helix bundle membrane proteins. Many aspects of the structure are similar to what has previously been found for the photosynthetic reaction center and bacteriorhodopsin. Our results lead to a considerably more precise general picture of membrane protein architecture than has hitherto been possible to obtain.