Rodent brain and heart catecholamine levels are altered by different models of copper deficiency

Limiting dopamine beta-monooxygenase results in lower norepinephrine (NE) and higher dopamine (DA) concentrations in copper-deficient Cu- tissues compared to copper-adequate Cu+ tissues. Mice and rat offspring were compared to determine the effect of differences in dietary copper Cu deficiency started during gestation or lactation on catecholamine, NE and DA, content in brain and heart. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 12 and 27. For both rat and mouse Cu- tissue, NE and DA changes were greater at later ages. Though Cu restriction began earlier in rats than mice in the gestational model, brain NE reduction was more severe in Cu- mice than Cu- rats. Cardiac NE reduction was similar in Cu- rodents in the gestation models. In the lactation model, mouse catecholamines were altered more than rat catecholamines. Furthermore, following lactational Cu deficiency Cu- mice were anemic and exhibited cardiac hypertrophy, Cu- rats displayed neither phenotype. Within a species, changes were more severe and proportional to the length of Cu deprivation. Lactational Cu deficiency in mice had greater consequences than in rats.     

Copper deficient rat heart can compensate for doxorubicin-induced oxidant stress

The hypothesis that copper (Cu) alters drug metabolizing enzymes and functions as an antioxidant nutrient in doxorubicin cardiotoxicity was tested. Male Sprague-Dawley rats were fed Cu adequate (⁺Cu; 5 mg Cu/kg of diet), marginally Cu deficient (MCu; 1.2 mg Cu/kg of diet), or severely Cu deficient (⁻Cu; 0.5 mg Cu/kg of diet) diets for 6 wk. Doxorubicin (1, 2, or 4 mg/kg body wt) or saline were administered intraperitoneally 1 time/wk for 4 wk. Compared to control hearts, Cu, Zn superoxide dismutase activity was decreased by 9% in MCu rats and by 21–40% in⁻Cu rats. Glutathione peroxidase activity was elevated 5–15% in⁻Cu rats. Doxorubicin administration increased heart Cu, Zn superoxide dismutase activity in⁺Cu and⁻Cu rats 18 h after the last of 4 injections, but not 18 h after 1 injection. There was no synergism between doxorubicin and Cu deficiency on lipid peroxidation, plasma creatine phosphokinase, cardiac hypertrophy, electrocardiographic abnormalities, or morphological changes. Heart glutathione S-transferase activity was decreased by Cu deficiency, and like Cu, Zn superoxide dismutase activity, returned to normal in⁻Cu rats given doxorubicin. Thus, the Cu deficient rat heart may be able to compensate for doxorubicin-induced oxidant stress by increasing the activity of Cu,Zn superoxide dismutase and glutathione S-transferase.     

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.     

Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

The Cardiac Copper Chaperone Proteins Sco1 and CCS are Up-Regulated,but Cox 1 and Cox4 are Down-Regulated,by Copper Deficiency

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long–Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS–PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.     

Increased contractility of cardiomyocytes from copper-deficient rats is associated with upregulation of cardiac IGF-I receptor

Hearts from severely Cu-deficient rats show a variety of pathological defects, including hypertrophy and, in intact hearts, depression of contractile function. Paradoxically, isolated cardiomyocytes from these rats exhibit enhanced contractile properties. Because hypertrophy and enhanced contractility observed with other pathologies are associated with elevation of insulin-like growth factor-I (IGF)-I, this mechanism was examined for the case of dietary Cu deficiency. Male, weanling Sprague-Dawley rats were provided diets that were deficient (approximately 0.5 mg Cu/kg diet) or adequate (approximately 6 mg Cu/kg diet) in Cu for 5 wk. IGF-I was measured in serum and hearts by an ELISA method, cardiac IGF-I and IGF-II receptors and IGFBP-3 were measured by Western blotting analysis, and mRNAs for cardiac IGF-I and IGF-II were measured by RT-PCR. Contractility of isolated cardiomyocytes was assessed by a video-based edge-detection system. Cu deficiency depressed serum and heart IGF-I and heart IGFBP-3 protein levels and increased cardiac IGF-I receptor protein. Cardiac IGF-II protein and mRNA for cardiac IGF-I and IGF-II were unaffected by Cu deficiency. A Cu deficiency-induced increase in cardiomyocyte contractility, as indicated by increases in maximal velocities of shortening (-dL/dt) and relengthening (+dL/dt) and decrease in time to peak shortening (TPS), was confirmed. These changes were largely inhibited by use of H-1356, an IGF-I receptor blocker. We conclude that enhanced sensitivity to IGF-I, as indicated by an increase in IGF-I receptor protein, accounts for the increased contractility of Cu-deficient cardiomyocytes and may presage cardiac failure.     

Effect of coenzyme Q10 supplementation on cardiac hypertrophy of male rats consuming a high-fructose,low-copper diet

Administration of coenzyme Q₁₀ to humans and animals has a beneficial effect on a number of cardiac diseases. The purpose of the present study was to determine if coenzyme Q₁₀ treatment could ameliorate cardiac abnormalities associated with the carbohydrate × copper interaction in rats. Weanling male rats were provided with a copper-deficient diet (0.6 μg Cu/g) containing either 62.7% starch (S−Cu) or fructose (F−Cu) for 5 wk. Half of the rats provided with the F−Cu diet were given daily oral supplements of 300 mg coenzyme Q₁₀/kg body weight (F−Cu+Q). Heart hypertrophy, liver enlargement, or pancreatic atrophy were not affected by, nor was body growth or anemia improved by, supplementation with coenzyme Q₁₀ when compared to rats fed only the F−Cu diet. Hearts from rats fed the F−Cu diet had severe inflammation, degeneration, fibrosis, and giant mitochondria with abnormal cristae. Hearts from F−Cu+Q rats had similar mitochondrial changes as the F−Cu rat hearts but without any apparent degenerative changes. None of the F−Cu+Q rats, but 30% of the F−Cu rats, died during the study as a result of heart rupture. These observations show that whereas coenzyme Q₁₀ treatment did not prevent the cardiac hypertrophy of the carbohydrate × copper interaction, it did play a role in maintaining the integrity of the heart. This work was presented in part at the 2nd International Symposium on Metal Ions in Biology and Medicine, Loutraki, Greece, May 15–22, 1992 (Metal Ions (1992), J. Anastassopoulou, P. Collery, J.C. Etienne, T. Theophanides, eds., John Libbey Eurotext, Montrouge, France, pp. 402–407).     

Iron and zinc status in rats with diet-induced marginal deficiency of vitamin A and/or copper

The hypothesis was tested that there are interactions of marginal copper and vitamin A deficiency regarding iron and zinc status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered copper concentrations in plasma and tissues of rats and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Vitamin A restriction (0 vs 4000 IU vitamin A/kg diet) reduced plasma retinol concentrations and induced a fall of blood hemoglobin and hematocrit. Neither copper nor vitamin A restriction for up to 42 d affected feed intake and body wt gain. There were no interrelated effects of vitamin A and copper deficiency on iron status. Copper deficiency slightly depressed liver, spleen, and kidney zinc concentrations. Vitamin A deficiency lowered zinc concentrations in heart, but only when the diets were deficient in copper.     

Tissue-specific alterations in lipoprotein lipase activity in copper-deficient rats

Copper deficiency results in alterations in lipid metabolism that include elevations in serum cholesterol and triglycerides and a decrease in whole-body respiratory quotient. Copper-deficient animals are also leaner even though electron micrographs of the myocardium present increased lipid droplet accumulation. To address whether a compromised copper status impacts triglyceride deposition in a tissue-specific manner, the activity of lipoprotein lipase was measured in adipose tissue and cardiac and skeletal muscle. Weanling rats fed a copper-restricted diet (<1 ppm) for 6 wk demonstrated a greater than twofold increase in cardiac lipoprotein lipase activity concomitant with a significant reduction in adipose tissue lipoprotein lipase activity. Skeletal muscle lipoprotein lipase activity was not altered by the copper-deficient state. The results of this study suggest that copper deficiency may induce a tissue-specific alteration in lipoprotein lipase activity in rats, which may contribute to the notable deposition of lipid substance in myocardium and the concomitant general body leanness.     

Regression of copper-deficient heart hypertrophy: reduction in the size of hypertrophic cardiomyocytes

Dietary copper (Cu) deficiency causes cardiac hypertrophy and its transition to heart failure in a mouse model. Cu repletion results in rapid regression of cardiac hypertrophy and prevention of heart failure. The present study was undertaken to understand dynamic changes of cardiomyocytes in the hypertrophic heart during the regression. Dams of FVB mice were fed a Cu-deficient (CuD) diet (0.3 mg Cu/kg) starting on Day 3 post-delivery, and weanling pups were fed the same diet until Cu repletion (6.0 mg Cu/kg) in the diet at 31 days of age. Heart samples were obtained at the end of CuD feeding or at 3, 7, 14 or 28 days after Cu repletion. Cu deficiency resulted in increases in the size and reduction in the number of cardiomyocytes in the heart. Cu repletion led to regression in the size of hypertrophic cardiomyocytes and normalization of the total number of cardiomyocytes. Although a direct reduction in the cell size would be significantly responsible for the regression of heart hypertrophy, some hypertrophic cardiomyocytes upon Cu repletion reentered the cell cycle as determined by Ki-67 staining in the cardiomyocyte-specific α-sarcomeric actin-stained cells and underwent division as determined by a mitosis-specific marker, phospho-histone 3. Quantitative analysis indicated that the replication of hypertrophic cardiomyocytes made a contribution of about one-third to the total mitosis of the regenerated myocardium. This study suggests that a direct reduction in the size of some hypertrophic cardiomyocytes and a replication of other hypertrophic cardiomyocytes with reduced size make a significant contribution to the regression of CuD heart hypertrophy, leading to normalization of the size and the number of cardiomyocytes in the heart.     

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.     

Development of copper deficiency in neonatal mice

Dietary copper (Cu) deficiency was produced in Swiss albino mice to determine the temporal relationship between depletion of Cu and changes in the cardiovascular and nervous system. Dams were placed on a Cu-deficient diet 4 days after parturition. Half the dams were provided with deionized water and their offspring are referred to as Cu-deficient (-Cu). Half the dams were given cupric sulfate in their drinking water (20 microg Cu/mL) and their offspring are referred to as Cu-adequate (+Cu). At 6 weeks of age a sample of the -Cu mice were repleted with CuSO(4). Mice were sampled 1 day after birth and at weekly intervals for 7 weeks. Both +Cu and -Cu mice grew at the same rate: birth weight increased 16-fold at 6 weeks of age. Liver Cu more than doubled between 1 and 7 days of age. At 2 weeks of age -Cu mice were anemic (lower hematocrit and hemoglobin) and had lower liver Cu and plasma ceruloplasmin activity compared to +Cu mice. Liver Fe was not elevated in -Cu mice until 2 weeks after anemia developed. At weaning first signs of altered catecholamine metabolism included elevation of dopamine in both heart and spleen. Norepinephrine concentrations and content, in contrast, were not both lowered in -Cu mice until 5 weeks of age. Heart weight was first elevated in -Cu mice at 6 weeks of age and relative weight (mg/g body wt) at 4 weeks of age. Liver Cu concentration was lower in 1-week repleted mice than in +Cu mice. Anemia preceded the development of cardiac hypertrophy and altered catecholamine levels in -Cu mice.     

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

Effects of moderate copper deficiency on carbon tetrachloride-induced hepatotoxicity in rats.

Extreme copper deficiency has been shown to enhance CCl4-induced injury in rats. CCl4 hepatotoxicity was studied in rats with copper deficiency moderated by limiting deficiency periods to 5 or 6 weeks, using minimally adequate dietary zinc and including a marginal copper diet. Also, housing some rats in groups of six, rather than individually, was found to moderate the effects of low copper intake. Weanling male rats were fed copper at either 6, 2, or 0.2 mg/kg diet (adequate, marginal, deficient). Copper-zinc superoxide dismutase activity levels for singly and group-housed marginal rats were 80% and 93%, respectively, of adequate values. Values for deficient rats were 35% (singles) and 47% (group). In singly housed rats, a CCl4 dose of 400 microliters/kg intraperitoneally increased serum sorbitol dehydrogenase activities, indicators of cell membrane hepatotoxicity, in inverse proportion to dietary copper. A lower dose (100 microliters/kg) also produced smaller sorbitol dehydrogenase increases in adequate rats compared with deficients, but produced lowest increases in the marginals. The latter pattern also occurred in group-housed rats given the higher CCl4 dose, but the difference for adequate and marginal rats was not significant. The higher CCl4 dose, in singly housed rats, decreased liver glucose-6-phosphatase activities independently of copper intake. These activities are inversely proportional to microsomal lipid damage. In conclusion, moderate copper deficiency enhanced CCl4 hepatotoxicity, but the effect depended on injury criteria, CCl4 dose, and actual copper status as assessed by copper-zinc superoxide dismutase activities.     

Multiple mechanisms account for lower plasma iron in young copper deficient rats

Copper deficiency lowers brain copper and iron during development. The reduced iron content could be due to hypoferremia. Experiments were conducted to evaluate plasma iron and “ferroxidase” hypotheses by determining copper and iron status of Holtzman albino rats following gestational/lactational copper deficiency. Copper deficient (Cu−) dams on treatment for 5 weeks, two of gestation and three of lactation, had markedly lower copper content of milk and mammary tissue, and lower milk iron. Newborn pups from Cu− dams had lower copper and iron concentrations. Compared to Cu+ pups, Cu− pups, analyzed between postnatal age (P) 0 and P26, were smaller, anemic, had lower plasma iron, cardiac hypertrophy, and near zero ceruloplasmin activity. Liver copper in Cu+ pups increased then decreased during development and major reductions were evident in Cu− pups. Liver iron in Cu+ pups decreased with age while nursing but increased after eating solid food. Liver iron was lower in Cu− pups at P0 and P13 and normal at P20 and P26. Small intestinal copper decreased with age in Cu+ pups and was lower in Cu− pups. Intestinal iron levels in Cu− pups were higher than Cu+ pups postweaning in some experiments. Reduction in plasma iron in Cu− pups is likely due to a decreased “ferroxidase” function leading to lower placental iron transport, a lower milk iron diet, and partial block in iron uptake from intestine but is not due to failure to mobilize hepatic iron, in contrast to older rats eating diet with adequate iron.     

Increased type I collagen content and DNA binding activity of a single-stranded, cytosine-rich sequence in the high-salt buffer protein extract of the copper-deficient rat heart

Dietary copper (Cu) deficiency not only causes a hypertrophic cardiomyopathy but also increases cancer risk in rodent models. However, a possible alteration in gene expression has not been fully examined. The present study was undertaken to determine the effect of Cu deficiency on protein profiles in rat heart tissue. Male Sprague-Dawley rats were fed diets that were either a Cu-adequate diet (6.0 microg Cu/g diet, n = 6) or a Cu-deficient diet (0.3 microg Cu/g diet, n = 6) for 5 weeks. The high-salt buffer (HSB) protein extract from heart tissue of Cu-deficient, but not Cu-adequate rats showed a 132 kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This protein band stained pink with Coomassie Blue, suggesting the presence of collagens or other proline-rich proteins. Dot immunoblotting demonstrated that total type I collagen was increased by 110% in HSB protein extract from Cu-deficient, relative to Cu-adequate, rats. Liquid chromatography with mass spectrometry analysis indicated that the 132 kDa protein band contained a collagen alpha (I) chain precursor as well as a leucine-rich protein 130 (LRP130) in HSB protein extract from Cu-deficient but not Cu-adequate rats. A gel shift assay showed that HSB protein extract from Cu-deficient rats bound to a single-stranded cytosine-rich DNA with higher affinity than the extract of Cu-adequate rats, similar to reports of an increase in LRP130 single-stranded DNA binding activity in several types of tumor cells. Collectively, these results not only suggest an additional feature of altered collagen metabolism with Cu deficiency but also demonstrate for the first time an increase in single-stranded cytosine-rich DNA binding in Cu-deficient rat heart.     

Resveratrol improves survival, hemodynamics and energetics in a rat model of hypertension leading to heart failure

Heart failure (HF) is characterized by contractile dysfunction associated with altered energy metabolism. This study was aimed at determining whether resveratrol, a polyphenol known to activate energy metabolism, could be beneficial as a metabolic therapy of HF. Survival, ventricular and vascular function as well as cardiac and skeletal muscle energy metabolism were assessed in a hypertensive model of HF, the Dahl salt-sensitive rat fed with a high-salt diet (HS-NT). Resveratrol (18 mg/kg/day; HS-RSV) was given for 8 weeks after hypertension and cardiac hypertrophy were established (which occurred 3 weeks after salt addition). Resveratrol treatment improved survival (64% in HS-RSV versus 15% in HS-NT, p<0.001), and prevented the 25% reduction in body weight in HS-NT (P<0.001). Moreover, RSV counteracted the development of cardiac dysfunction (fractional shortening -34% in HS-NT) as evaluated by echocardiography, which occurred without regression of hypertension or hypertrophy. Moreover, aortic endothelial dysfunction present in HS-NT was prevented in resveratrol-treated rats. Resveratrol treatment tended to preserve mitochondrial mass and biogenesis and completely protected mitochondrial fatty acid oxidation and PPARα (peroxisome proliferator-activated receptor α) expression. We conclude that resveratrol treatment exerts beneficial protective effects on survival, endothelium-dependent smooth muscle relaxation and cardiac contractile and mitochondrial function, suggesting that resveratrol or metabolic activators could be a relevant therapy in hypertension-induced HF.