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It is known that mitochondrial transcription factor A (mtTFA) plays a pivotal role in coordinating the expression of proteins in the nuclear and mitochondrial genomes as it pertains to mitochondrial biogenesis. Hearts from copper-deficient rats have elevated mtTFA levels compared to copper-adequate rats. This study evaluated whether two proteins that control activation of mtTFA by binding to its promotor, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), are also upregulated prior to any upregulation of mtTFA. Long-Evans male rats were fed either copper-adequate or copper-deficient diets from weanling for periods of time up to 26 d. At d 26, mtTFA levels were elevated in the hearts from the copper-deficient rats, but not at earlier time points of 14, 18, and 22 d. However, NRF-1 and NRF-2 levels were increased at d 14 and 18, but not at the other two later time-points. These results revealed that the upregulation of mtTFA and mitochondrial biogenesis is preceded by upregulation of NRF-1 and NRF-2, which is consistent with the known molecular events controlling mitochondrial biogenesis in other systems.

The mechanism(s) by which impaired mitochondrial respiratory function and the accumulation of lipid droplets and mitochondria in hearts of copper-deficient rats occur remains unclear. It is not known whether specific components of the regulatory pathway involved in mitochondrial biogenesis, such as mitochondrial transcription factor A (mtTFA) and nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2), are activated in copper deficiency. Little is known about gene expression of enzymes involved in fatty acid oxidation (FAO) in hearts of copper-deficient rats. Male weanling rats were fed copper-adequate (CuA), copper-deficient (CuD) or pair-fed (CuP) diets for 5 wk. Mitochondria and lipid droplet volume densities from electron micrographs were greater and there was an elevation in the mtTFA protein level in hearts of copper-deficient rats. DNA binding activities of NRF-1 and NRF-2 did not differ among the groups. Northern blot analysis of cardiac tissue revealed that transcripts of F(1)F(0)-ATP synthase subunit c were greater, but mRNA levels of ATP synthase beta subunit and the FAO enzyme, medium-chain acyl-CoA dehydrogenase (MCAD), were lower in hearts of copper-deficient rats. Long-chain acyl-CoA dehydrogenase (LCAD) mRNA levels did not differ among treatment groups. These results suggest that certain components of the mitochondrial biogenesis program are activated in hearts of copper-deficient rats. F(1)F(0)-ATP synthase beta subunit and MCAD transcript levels remain low, which may contribute to impaired mitochondrial respiratory function, decreased fatty acid utilization and lipid droplet accumulation in hearts of copper-deficient rats.

This study investigated whether a high fat diet in tandem with a marginal copper (Cu) diet exerts deleterious effects on copper status, cardiac morphology, and electrophysiology compared to a low-fat marginal copper diet. Male weanling Long-Evans rats were fed diets containing either marginal copper (42.5 mumol/kg) or adequate copper (97.6 mumol/kg), and low fat (50.0 g/kg) or high fat (150.0 g/kg) diet for 12 wk in a 2 x 2 factorial design. To simulate the western diet, fat was composed of a 1:2 polyunsaturated:saturated fatty acids using a coconut and corn oil mixture. High dietary fat increased liver Cu concentration. Marginal copper diets decreased liver Cu,Zn-superoxide dismutase activity. Dietary copper and fat level had no effect on volume densities of mitochondria and myofibril. However, lower mitochondrial pathologic scores were observed in the rats consuming the high fat diets. Marginal copper high fat diet prolonged atrial electric depolarization (PR) and ventricular electric depolarization and repolarization (QT) intervals. This study provided direct evidence that a high fat diet can exert detrimental effects on cardiac ultrastructure and lead to alterations in electrocardiograms. The combination of marginal copper-high fat diet appears to alter cardiac electric conductivity. Longer term studies should provide information more relevant to clinical situations and morphologic changes.

Objective: Rats with a genetic tendency to develop hypertensive, hypertrophic cardiomyopathy were fed copper-deficient diets and their cardiac responses were investigated., Methods: Five male weanling rats of the Long-Evans and SHHF/Mcc-fa(cp) strains were randomly selected to receive diets containing either adequate quantities of copper (94.5 micromol Cu/kg diet) or reduced quantities of copper (<15.8 micromol Cu/kg diet) for 6 weeks, (n=5 within each group). Echocardiograms and electrocardiograms were recorded and analyzed at the end of the 6-week interval., Results: Electrocardiograms from copper deficient groups showed longer Q-T intervals and increased QRS amplitudes than controls. Both the copper deficient and control SHHF groups demonstrated significant QRS complex prolongation compared to Long-Evans rats. Echocardiography analysis showed significant increases in left ventricular area, free wall dimension, and myocardial cross-sectional areas in rats fed a copper deficient diet. The frequency of systolic cardiac murmurs increased in copper deficient rats and were related to the presence of valvular regurgitation as determined from echocardiography., Discussion: However, the data do not suggest that a copper-deficient diet fed to a strain of rats genetically susceptible to heart disease later in life, hastens or worsens the onset of cardiac disease. The genetic predisposition and copper-deficient states exert independent effects upon the heart.

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.

The cuproenzymes lysyl oxidase, cytochrome-c oxidase, and superoxide dismutase are key factors in understanding the cardiac hypertrophy and cardiomyopathy associated with dietary copper restriction. The role of copper in cardiac lipid and energy metabolism as a consequence of changes in some of these enzyme activities in comparison with what is known about normal cardiac substrate utilization is discussed here. While the decrease in the nuclear encoded subunits of cytochrome-c oxidase in hearts from copper-deficient rats is known, new evidence suggests that other factors, such as ATP synthase metabolism may be exerting an influence upon this observation. While this review focuses on newer knowledge about energy and fatty acid metabolism in copper deficiency, the extracellular matrix is considered as well. This complex interplay of extracellular and cellular events in copper restriction is outlined as a model for further studies of this unique model of concentric hypertrophy.

Hearts from rats fed a copper-deficient (Cu-) diet have decreased levels of nuclear-encoded peptides of cytochrome c oxidase (CCO). Studies were conducted to determine whether iron deficiency would lead to a similar finding, whether mRNA transcripts and the chaperonin heat shock proteins (HSP) 60 and 70 from hearts of Cu- rats were decreased as compared with copper-adequate controls and whether synthesis of mitochondrial and nuclear encoded peptides differed as affected by diet copper. In study 1, weanling rats were assigned to one of three groups (n = 6 in each group): (1) control copper and iron adequate fed rats; (2) Cu- rats and (3) iron-deficient (Fe-) rats. Western blotting of nonmyofibrillar cardiac proteins revealed that the nuclear encoded peptides of CCO from the Cu- rats were markedly decreased as compared with control and Fe- rats. Mitochondrial encoded subunits did not appear to differ by treatment groups. Iron-deficient rats had similar nuclear encoded peptide levels as those of controls. In study 2, mRNA transcripts from Cu- (n = 4) and control copper adequate (n = 4) rats did not appear to differ for subunits II and IV, which correspond to mitochondrial and nuclear encoded subunits, respectively. In study 3, levels of HSP 60 and 70 from hearts of Cu- rats (n = 3) did not differ from Cu+ rats (n = 3). In study 4, infusion of 3H-(4,5)-leucine into the hearts of Cu+ and Cu- rats suggested there was no difference in synthesis of the nuclear encoded peptides by copper status and some indication there was enhanced breakdown of the nuclear encoded peptides among the Cu- rats. As expected, more isotope was incorporated into the mitochondria of Cu- rats than Cu+ rats. These results demonstrate an independent effect of copper upon the apparent decrease in the nuclear encoded subunits of CCO, the effect of copper upon the CCO subunits is probably post-transcriptional and that translocation of the nuclear encoded subunits from the ribosomes to the mitochondria via the chaperonin proteins is not a primary defect in explaining these observations in hearts from Cu- rats and synthesis of the nuclear encoded subunits of CCO in not impaired in copper deficiency.

Pigs were made copper (Cu)-deficient to evaluate cardiac function and pathology, and electrocardiography. Fifteen-day-old pigs were fed a Cu-restricted diet over an 8 wk period and compared to Cu-adequate diet-fed pigs. Cardiac effects were examined concerning gross morphometry and ultrastructure, echocardiography, and electrocardiography, as well as serum cholesterol levels. The Cu-restricted diet-fed pigs exhibited a marked deceleration of growth and lower hematocrit, hemoglobin, and liver and serum Cu concentrations compared to the Cu-adequate diet-fed pigs. The Cu-restricted diet-fed pigs developed a significantly greater heart weight:body weight ratio, along with greater diastolic measures of ventricular wall and internal dimension relative to body weight. Electrocardiography in the Cu-restricted diet-fed pigs revealed one instance of electrical alternans and an intraventricular conduction disturbance and several instances of T-wave inversion. The Cu-restricted pigs also displayed a prolonged QT interval at the closure of study. Increased mitochondrial volume density and mitochondria:myofibril volume density ratio were observed in the Cu-restricted pig electron micrographs along with excessive lipid and glycogen inclusion and focal degradation of Z-lines, intercalated disk, and sarcomeres. Copper-restriction in young pigs results in cardiac pathology and electrical disturbances. These alterations are similar to those reported for young Cu-restricted rodents. Given then that many cardiac manifestations of developed Cu-deficiency appear conserved across specie lines, the potential for human disturbances in response to severe Cu-deficiency may be plausible.

Important aspects of copper nutrition were collected from all 10 editions of the Recommended Dietary Allowance along with the history of the paradigm concept according to Thomas Kuhn. Important anomalies in copper nutrition, such as the easy accessibility to diets containing considerably less copper than the estimated safe and adequate daily dietary intake are reviewed. Important experiments with animals are compared with copper depletion experiments with humans. Data support the belief that people respond to diets low in copper similarly to animals and that measurement of copper in blood plasma generally is useless in assessing nutritional status. If common diets low in copper are consumed regularly during pregnancy, maternal stores of copper will be depleted. Although there is some evidence of lower copper in heart and major blood vessels in elderly people, it is premature to suggest different intakes for adults of different ages. Although consideration of cardiovascular data in establishing an RDA for copper may seem to be a new paradigm, considerations of general health and well being have long contributed to establishing RDAs. Dietary copper can be increased by adhering to the advice symbolized by the food pyramid.

The objective of this study was to determine if a high fat diet having a 2:1 saturated-polyunsaturated fatty acid ratio exacerbates signs of copper deficiency. Male weanling Long-Evans rats were randomly placed into one of the following treatment groups: adequate copper low fat or deficient copper high fat. The levels of fat used were 31 or 12% of daily energy, and copper concentrations were 94.5 micromol/kg and <15.8 micromol/kg in the copper-adequate and copper-deficient diets, respectively. Cardiac hypertrophy as well as lower liver copper levels and superoxide dismutase activity were observed in both groups of copper-deficient rats. Irrespective of copper level, consumption of the high fat diet resulted in the thickening of the interventricular septum and left ventricular free wall. Electrocardiograms revealed that the copper-deficient high fat diet led to a significantly smaller QT interval compared with all other groups. Significantly greater S-wave voltage due to copper deficiency was observed. Significantly lower heart cytochrome c oxidase (CCO) activity was found in the copper-deficient groups with the copper deficient high fat group showing the lowest activity. Western blots of the cardiac non-myofibrillar fraction demonstrated lower amounts of CCO nuclear encoded peptides in the copper-deficient groups, with the least amount seen in the copper-deficient high fat treatment. These data suggest that a high level of dietary fat exacerbates some of the signs of copper deficiency.

To determine if chronic ingestion of a diet containing a marginally low level of Cu could cause deleterious alterations in cardiac ultrastructure, male offspring were nursed by dams fed a diet containing either 6.7 or 2.8 mg Cu/kg from midgestation through lactation before weaning to the same diet. Conventional measures of Cu status, including growth, relative heart weight, tissue concentrations of Cu, ceruloplasmin activity, and tissue activity of Cu,Zn-superoxide dismutase (SOD) were similar in both dietary treatment groups at 5.5 months of age. However, significant increases in the number and volume of lipid droplets and an increased incidence of pathological abnormalities in mitochondria and basal laminae were observed in sections of hearts from rats chronically fed the diet containing 2.8 mg/kg Cu. Reduction of the dietary level of Cu from 2.8 to 1.3 mg/kg from 4 to 5.5 months of age caused significant reductions in the concentration of Cu in serum and liver, but Cu content, Cu,Zn-SOD activity, pathological scores, and morphometric parameters in hearts were not modified by the greater restriction of dietary Cu in adult rats. This study suggests that abnormalities in cardiac ultrastructure occurred in rats chronically fed diets marginally low in Cu, despite minimal changes in conventional biochemical indicators of Cu status.

The relative amounts of cardiac proteins such as laminin, fibronectin, cytochrome c oxidase, and isomyosin types were studied by gel electrophoresis and Western blotting in control and copper-deficient Sprague-Dawley rats of both sexes fed their respective diets from weanling for 3 weeks. Isomyosin types appeared to shift from V1 to greater levels of V3 in copper deficient rats for both genders. Male copper deficient rats had increased cardiac levels of fibronectin, decreased laminin levels, cardiac hypertrophy and anemia. Both male and female rats fed copper-deficient diet had lower levels of cytochrome c oxidase (CCO) subunit IV, and low liver copper, and high heart-to-body weight ratios compared with their respective controls.

Comparative cardiac ultrastructure, morphometry, and electrocardiography after dietary copper and selenium restriction were examined. Male weanling Long-Evans rats were fed diets that were either adequate in both copper and selenium (Cu+/Se+) or restricted in either Cu (Cu-) or Se (Se-) for 8 wk. At wk 8, electrocardiograms (ECG) and dP/dts were obtained and heart tissue was utilized for electron microscopy. Upon examination, Cu- rats were anemic, exhibited a greater heart: body weight ratio, and developed concentric hypertrophy characterized by an enhanced thickening of the left and right ventricular free walls, and interventricular septum. ECG recordings from lead aVF in the Cu- group showed a greater R wave amplitude in comparison to the Cu+/Se+ group. Se- rats recorded a greater left ventricular +dP/dtmax than both the Cu+/Se+ and Cu- groups. Cardiac myofibril volume densities were decreased in both Cu- and Se- rats in comparison to the Cu+/Se+ rats. In addition Cu- rats showed a greater mitochondria:myofibril ratio. Sarcomere contractile protein disarray was present in both the Cu- and Se- groups. Se- myocytes also showed evidence of edema and mitochondrial fragmentation. The subcellular alterations suggest that similarities exist in the cardiac remodeling processes associated with copper and selenium restrictions.

This study determined if reported decreases in the delta subunit of ATP synthase and nuclear-encoded cytochrome c oxidase subunits in hearts of copper-deficient rats were secondary to the heart disease pathology or due to lack of the trace element. Male weanling Long-Evans rats were randomly divided into six groups: rats fed a copper-adequate or copper-deficient diet (with free access) with or without 5% dimethyl sulfoxide (DMSO) in the drinking water and rats pair-fed the copper-adequate or copper-deficient diet without DMSO treatment. After 4 wk, rats in the groups fed the copper-deficient diet had lower liver superoxide dismutase and heart cytochrome c oxidase activities compared with groups fed the copper-adequate diet. Administration of DMSO, an antioxidant, and energy restriction (pair-feeding) partially blocked cardiac hypertrophy in rats fed the copper-deficient diet. Greater mitochondrial volume density and mitochondrial:myofibrillar ratio and disrupted myofibrils and basal laminae were observed in the hearts from rats fed the copper-deficient diet and not treated with DMSO compared with hearts from groups fed the copper-adequate diet. The DMSO-treated rats fed the copper-deficient diet had hearts with intact structure but enlarged mitochondria compared with other groups fed the copper-deficient diet. The delta subunit of ATP synthase and the nuclear-encoded cytochrome c oxidase subunits IV and V were depressed in rats fed a copper-deficient diet regardless of antioxidant treatment and pair-feeding. These data suggest that the effects of copper deficiency upon ATP synthase and cytochrome c oxidase proteins are not due to the cardiac pathology.

To determine the dual effect of exercise training and copper depletion on myocardial function and ultrastructure, postweanling rats were either trained or sedentary while fed copper-adequate or copper-deficient diets for 8 wk. Rats developed characteristic myocardial subcellular degeneration and increased cardiac mitochondrial volume density when copper depleted, despite lack of overt cardiac hypertrophy, hypertension, or anemia. Training combined with copper depletion induced mild left ventricular hypertrophy. Basal laminae appeared fractionated in areas at capillary-myocyte interface, with focal pericapillary and interstitial collagen accumulation, whereas overt fibrosis was absent or minimal. Electrocardiograms revealed increased QRS wave and QT duration and notching of QRS complex with copper depletion, consistent with intraventricular conductance disturbances. The oxidative capacity of soleus muscle increased with training in copper-adequate rats, but was reduced with progressive copper depletion. These data suggest that copper depletion and training are synergistic in effecting focal accumulation of collagen, with deleterious effect on exercise capacity.

Dietary copper restriction in rats results in cardiomyopathy. In rats fed copper-restricted diets from weaning for 5 to 8 weeks, a concentric hypertrophy is apparent, whereas postweaning copper restriction does produce cardiomyopathy without apparent hypertrophy. Both sets of circumstances appear to affect the integrity of the basal laminae of cardiac myocytes and capillaries. In rats fed copper-restricted diets from weaning, decreases in cytochrome c oxidase are related not only to copper's role as a coenzyme, but also to a marked decrease in the nuclear encoded subunits of the enzyme complex. Decreased levels of the delta-subunit of ATP synthase have been observed. However, such aberrations in mitochondrial enzymes, as well as morphologic alterations, apparently do not affect cardiac levels of ATP. This review suggests mechanisms of cardiac adaptation and initiation factors leading to cardiac hypertrophy. We present a hypothetical working model explaining the events leading to cardiac failure in the copper-deficient rat heart based on the present body of knowledge, and compare the pathology with other models of cardiomyopathies.

Dietary copper deficiency induces alterations of connective tissue metabolism that are associated with lesions in cardiovascular and other organ systems. To determine the impact of copper deficiency on characteristics of collagen in porcine myocardium and cardiac valves, weaned pigs were fed diets with adequate or deficient levels of copper. Although dietary copper did not affect the concentration of collagen in either myocardium or bicuspid valves, the degree of collagen cross-linking, as assessed by the level of hydroxylysylpyridinoline, was lower in both tissues of copper-deficient pigs. Proportions of type III collagen were increased in the left ventricle and bicuspid valves of copper-deficient pigs. Copper deficiency induced extensive remodeling, however, of the collagen fraction of cardiac interstitium. Reduction in left ventricular collagen cross-linking may provide the stimulus for the development of cardiac hypertrophy, which characterizes severe copper deficiency, by increasing the compliance of the ventricular wall. The shift in the phenotypic profile of collagen that is associated with this cardiac hypertrophy indicates synthesis of new collagen, which could affect collagen cross-linking irrespective of copper status.

Cardiac hypertrophy and function were evaluated in rats fed diets containing deficient, marginal or adequate levels of copper. The fat concentration of the diets was either 10 g/100 g corn oil, 10 g/100 g coconut oil or 10 g/100 g coconut oil + 1 g/100 g added cholesterol. Left ventricular (LV) wall thickening of hearts in rats fed copper-deficient diets was characterized by greater (P < 0.05) LV free wall width, regardless of dietary fat type, and greater intraventricular septum width in the rats fed corn oil. Rats fed the copper-deficient diet with coconut oil + cholesterol had LV chamber volumes that were twofold larger than those of rats fed the copper-deficient diet with coconut oil or corn oil. Copper deficiency reduced LV chamber volume only in rats fed coconut oil + cholesterol. Cardiac LV end diastolic pressure in rats fed copper-deficient diets was twofold larger than in copper-adequate and copper-marginal groups fed corn oil or coconut oil. Hearts from rats fed the copper-deficient diet with corn oil compared with those from rats fed the copper-deficient diet with coconut oil + cholesterol had greater right ventricular (RV) and LV end diastolic pressures, LV pressures and LV and RV maximal rates of positive pressure development. Our data suggest that cardiac adaptations in rats fed copper-deficient diets are influenced by dietary fat type: 1) hearts of rats fed the copper-deficient diet with corn oil were concentrically hypertrophied, whereas cardiac contractility was maintained in the presence of high preload; 2) preload and contractility in hearts of coconut oil-fed rats was greater than cardiac response to cholesterol addition to the coconut oil diet; 3) hearts in copper-deficient rats fed coconut oil + cholesterol exhibited eccentric hypertrophy and ventricular dysfunction.

1. Male weanling Long-Evans rats were randomly assigned to either copper-adequate (6.0 mg Cu/kg feed) or copper-deficient (< 1.0 mg Cu/kg feed) diets for a 5 week period. 2. Cardiac ATP levels and energy charges did not differ between the two groups. Levels of NAD and phosphocreatine as well as the sum of pyridine nucleotides were greater in copper-deficient rats compared with copper-adequate rats using HPLC analysis. 3. Mitochondrial respiratory control ratios and oxygen consumption rates from copper-deficient rat hearts were depressed, although ADP:O ratios were similar to copper-adequate rat hearts.

Copper feeding studies in rats are generally initiated at weaning. This study examined whether a 6-week feeding of low or marginal Cu levels (0.2 or 2.5 ppm) to rats initially weighing 135 g produced deleterious effects. Controls were fed 8 ppm Cu. Liver Cu-Zn superoxide dismutase activities paralleled Cu intake. Plasma ceruloplasmin activities were very low for both low and marginal Cu consumption. Low but not marginal Cu intake caused a low body weight, high plasma cholesterol level, anemia, cardiac hypertrophy, and a high degree of hepatic plasma membrane injury 24 hours after CCl4 injection (150 microL/kg intraperitoneally [IP]). In summary, low and marginal Cu intakes produced low Cu enzyme activities, while low Cu intake produced pathological symptoms and poor resistance to an oxidative stress.

Cardiac ultrastructural and functional characteristics were determined in copper-depleted and copper-repleted rats. Male weanling rats were randomized into five groups that were fed either copper-adequate or copper-deficient diets. After 5 wk, one group fed each diet was studied to obtain baseline values. At this time, one copper-adequate postweanling group continued to receive the adequate diet as control, one deficient postweanling group was fed the adequate diet to evaluate the effect of copper repletion and one postweanling adequate group was fed the deficient diet to evaluate copper depletion in relatively older rats. These dietary treatments were continued for six additional weeks. Copper-depleted rats of both ages exhibited significant cardiac ultrastructural pathology and electrocardiogram abnormalities and the postweanling copper-depleted rats exhibited these abnormalities in the absence of hypertrophy and anemia. Increased mitochondrial volume density, disarranged cristae, and nonaligned myofibrils with disturbances at Z-bands were displayed. Additionally, all copper-depleted rats demonstrated fragmented basal laminae at capillary-myocyte interface. Increased QRS amplitude and notching and greater QT intervals were displayed. Copper-repleted rats exhibited some, but not total, reversal of these abnormalities. These results suggest that capillary-myocyte interface changes may play an important role in the developing pathology of copper depletion.

The temporal sequence of events leading to cardiac dysfunction during copper restriction in the Long-Evans rat was studied over a 6-week period. Weanling rats were fed either copper-adequate (6 mg Cu/kg diet, n = 25) or copper-restricted (less than 1 mg Cu/kg diet, n = 25) diets for varying periods of time for up to 6 weeks. Beginning at 2 weeks after weaning and weekly thereafter, five rats from each diet were evaluated for cardiac function, and sacrificed, and indicators of copper deficiency were determined on several tissues. Electrocardiograms began showing indications of cardiac disease at Week 3 in the copper-restricted rats, at which time cardiac hypertrophy and other signs of copper deficiency were apparent. Greater QT intervals and QRS amplitudes were observed in copper-restricted rats at various weeks. Peak + and - dP/dt maxs did not differ by diet copper treatment for any of the time intervals studied, nor was any notable difference in developed left ventricular pressure apparent. Hematocrit and liver copper levels were decreased in copper-restricted rat hearts at all weeks. These results suggest that the onset of cardiac dysfunction in copper deficiency is rapid, with both dysfunction and hypertrophy apparent within 3 weeks after copper restriction and when liver copper levels have declined.

The purpose of this study was to examine, by transmission electron microscopy (TEM), the nature of the protective effect of dimethyl sulfoxide (DMSO) on hearts of copper-deficient (CuD) rats. Male, weanling Sprague-Dawley rats were fed, in a two-way design, CuD (0.45 micrograms/g) or copper-sufficient (CuS, 5.4 micrograms/g) diets with or without 5% DMSO in their drinking water. After 28 d, CuD rats showed typical signs of copper deficiency, including reduced liver and heart Cu, enlarged hearts, and anemia. DMSO-treated, CuD rats had lower heart weights and higher hematocrits than CuD rats. DMSO enhanced organ Cu concentrations in CuS, but not in CuD rats. TEM of CuD hearts showed myofibrillar distortion and enlarged, vacuolated mitochondria with fragmented cristae; morphometric measurements indicated an enhanced mitochondrial/myofibrillar ratio (mito/myo), but an increase of both mitochondrial and myofibrillar mass relative to CuS hearts. Compared to CuD hearts, DMSO-treated CuD hearts showed better mitochondrial morphology and myofibrillar organization, as well as a greater mito/myo, but lower mitochondrial and myofibrillar masses. Its function as a hydroxyl radical scavenger indicates that DMSO could protect CuD hearts, in particular their mitochondria, against oxidative damage. However, because measurements of thiobarbituric acid reactive substances were not consistent with this theory, other metabolic mechanisms, direct and indirect, must be examined.

Two 6-wk double-blind studies evaluated the effects of supplements of 2 or 3 mg Cu/d on serum copper, ceruloplasmin, red-blood-cell super oxide dismutase (RBC-SOD), total serum cholesterol, and serum lipoprotein-cholesterol fractions in adult males. Study I had 6 supplemented and 8 placebo subjects, whereas study II had 7 and 6, respectively. Copper supplementation did not appear to affect serum copper levels, RBC-SOD, hematocrit, and ceruloplasmin levels when assayed by radial immunoassay diffusion. Supplementation with 2 mg Cu/d produced an increase in LDL cholesterol and the percentage of cholesterol as LDL at wk 4 compared to the placebo group, and a concomitant decline in VLDL-cholesterol levels and the percentage of cholesterol as VLDL. At wk 6, the percentage of cholesterol as LDL increased and that of cholesterol as VLDL decreased compared to baseline values in the supplemented group. Supplements of 3 mg Cu/d increased hemoglobin levels, ceruloplasmin activity, and serum total-cholesterol levels at wk 6 compared to placebos. Differences in cholesterol may be partly explained by variability in the placebo groups in both studies. Copper supplementation effects on cholesterol deserves further investigation.

The effect of copper deficiency on cardiac function and structure was studied in a strain of rats (SHHS/Mcc-cp) known to develop cardiac failure as adults. Restriction of dietary copper (less than or equal to 1 mg/kg vs. 6 mg/kg in adequate diets) at weaning in both sexes for a 6-wk period produced cardiac hypertrophy. Male rats developed more severe copper-deficiency symptoms than their female counterparts. In both sexes of copper-deficient rats, there was an increase in cardiac length, width, free ventricular wall thickness and septum thickness. Electrocardiographic tracings revealed greater QRS height among male copper-deficient rats. Heart rate also was substantially reduced in this group. The increased volume of myocardium occupied by mitochondria in the copper-deficient male rats might result in increased electrical resistance that would increase the QRS height; hypertrophy or anemia also could be contributory. Some male copper-deficient rats had prolongation of the QRS in a bundle branch block pattern. Maximal rates of rise and fall for left ventricular pressure were reduced in male copper-deficient rats. The gross histology indicated that this type of heart failure was more concentric than eccentric. The copper-deficient male rat may serve as a useful model for studying the concentric cardiac hypertrophy that occurs in humans.

Histological aspects of Cu-deficient hypertrophied rat hearts were evaluated to determine causal factors. Twenty-four male weanling rats were fed either Cu-adequate (8.0 mg/kg diet) or Cu-deficient (0.4 mg/kg diet) diets until 9 or 11 wk of age. Copper-deficient rats had increased mitochondrial:myofibrillar ratios compared with Cu-adequate rats. Mitochondria were vacuolated. Cristae seemed fragmented and matrix seemed translucent and they tended to distort the myofibrils. Glycogen granules and lipid droplets were more frequently observed in Cu-deficient rats. When the Cu-deficient rats were examined separately, the larger hearts in the older rats had smaller mitochondrial:myofibrillar ratios, but had larger myofibrillar volume densities. Valves from Cu-deficient rats had less connective tissue and seemed fragmented in areas. In 9-wk-old Cu-deficient rats, there was a negative correlation between heart: body weight ratios and bicuspid valve scores, whereas 11-wk-old rats demonstrated the same relationship for tricuspid valves with myofibril volume density. These results suggest that there are two components contributing to cardiac hypertrophy in copper deficient rats: 1) an enlarged mitochondrial area and 2) myofibrillar enlargement. Hematocrit values did not seem to be related to cardiac hypertrophy.

The effect of copper deficiency on liver, cardiac and adipose fatty acids was studied in the Long-Evans rat. Rats were fed diets adequate in copper (8.5 mg Cu/kg diet, group AC, n = 10) or with no added copper (0.4 mg Cu/kg diet, group NC, n = 9) or were pair-fed an adequate copper diet in amounts eaten by group NC rats (group PF, n = 10), from weaning until 8 wk thereafter. Group NC rats exhibited typical copper deficiency signs such as decreased body weight, hematocrit and liver copper levels but increased heart/body weight ratios. Adipose and cardiac triglycerides of group NC rats had greater 18:0 to 18:1n-9 ratios. All tissues in group NC rats had higher levels of longer-chain polyunsaturated fatty acids in triglycerides than those in AC or PF rats. Specifically, the following triglyceride fatty acids were in greater concentration in NC rats than in AC or PF rats: for liver, 18:2n-6, 22:5n-6, 22:5n-3 and 22:6n-3; for cardiac, 20:3n-6, 20:4n-6 and 22:5n-6. Liver phospholipids had lower levels of 20:4n-6 in NC rats than in AC or PF rats. These results suggest that copper deficiency results in the accumulation of both n-3 and n-6 longer-chain polyunsaturated fatty acids in triglycerides of various tissues.

Myofibrillar and nonmyofibrillar proteins from hearts of copper-adequate (n = 9) and copper-deficient (n = 10) rats were compared. Male weanling Long-Evans rats were fed copper-deficient or copper-adequate diets for 9 wk. Twelve additional rats were fed similar diets and cardiac tissue was evaluated by transmission electron microscopy. Ventricular myocytes were glycerinated and homogenized in 0.1 M KCl and 1.5% Triton X-100, and suspensions were centrifuged at 1100 x g. The supernatant was removed and designated Triton X-100-soluble non-myofibrillar protein, and the pellet was resuspended and recentrifuged several times to obtain myofibrillar protein. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted on both protein fractions. Densitometer scans of SDS-PAGE pherograms of myofibrillar protein revealed no significant difference between copper-adequate and copper-deficient groups. Similar analysis of nonmyofibrillar protein revealed a consistent decrease or diminished level of a 23-kDa polypeptide among copper-deficient rat hearts. These results may be consistent with the findings that demonstrated fragementation of mitochondrial cristae and an increased area occupied by mitochondria in copper-deficient rat hearts.

The association of copper and zinc intake with serum total cholesterol, HDL-cholesterol, triglycerides, and glucose concentrations were studied in young adults (N=59). Three-day diet records, hair, fasting blood, and overnight urine samples were collected from each subject. Higher hemoglobin concentrations, diet zinc intake, and serum copper:zinc ratios were associated with lower HDL-cholesterol concentrations. Higher hemoglobin and hair copper concentrations were associated with higher cholesterol concentrations. Higher serum copper concentrations were associated with higher triglyceride concentrations and with lower glucose concentrations. Greater hematocrits were associated with lower triglyceride concentrations. Hemoglobin levels had the strongest relationship with cholesterol and HDL-cholesterol. These results appeared inconsistent with the hypothesis on copper:zinc imbalance and reports from animal studies.

A variety of behaviors was assessed in Long-Evans male rats placed on either a low copper diet, a marginal copper diet, or an adequate copper diet at weaning. Rats in the low copper group had slightly, but significantly, enlarged hearts and gained less weight than rats fed diets containing higher copper levels. Treatment effects were not detected in measurements of muricide, open-field activity, water intake, shock sensitivity, and shock avoidance and memory.