Decreased passive stiffness of cardiac myocytes and cardiac tissue from copper-deficient rat hearts

Passive stiffness characteristics of isolated cardiac myocytes, papillary muscles, and aortic strips from male Holtzman rats fed a copper-deficient diet for approximately 5 wk were compared with those of rats fed a copper-adequate diet to determine whether alterations in these characteristics might accompany the well-documented cardiac hypertrophy and high incidence of ventricular rupture characteristic of copper deficiency. Stiffness of isolated cardiac myocytes was assessed from measurements of cellular dimensional changes to varied osmotic conditions. Stiffness of papillary muscles and aortic strips was determined from resting length-tension analyses and included steady-state characteristics, dynamic viscoelastic stiffness properties, and maximum tensile strength. The primary findings were that copper deficiency resulted in cardiac hypertrophy with increased cardiac myocyte size and fragility, decreased cardiac myocyte stiffness, and decreased papillary muscle passive stiffness, dynamic stiffness, and tensile strength and no alteration in aortic connective tissue passive stiffness or tensile strength. These findings suggest that a reduction of cardiac myocyte stiffness and increased cellular fragility could contribute to the reduced overall cardiac tissue stiffness and the high incidence of ventricular aneurysm observed in 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.     

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.     

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.     

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.     

Early ultrastructural changes in the myocardium following thyroxine-induced hypertrophy

The model of myocardial hypertrophy induced by thyroxine was studied with particular regard to the early ultrastructural changes in fractional volume of the mitochondria and myofibrils, and capillary distribution. Following injections of L-thyroxine (25 mg/kg IP) for 9 consecutive days, rats were sacrificed by vascular perfusion and cardiac tissue samples from the mid-wall zone of the left ventricle were processed routinely for electron microscopy. Heart weight/body weight ratios of thyroxine treated (T) rats showed a significant increase (P less than 0.001) over the ratios in control (C) rats. Likewise, the fractional volume of mitochondria (42%) was significantly increased (P less than 0.001) in the myocardium of T rats when compared with C rats (31%). However, the fractional volume of myofibrils was significantly decreased in the myocardium of T rats (P less than 0.001) and there was no significant difference between the hearts of T and C rats with respect to capillary luminal area/myocyte area. The mitochondria/myofibril ratio was increased in the hearts of T rats (0.82) over that found in control hearts (0.52). These results suggest that in the early stages of thyroxine-induced myocardial hypertrophy there is not an immediate increase in capillary area which may account for the ischemia and significant increase in mitochondrial volume which characterized myocardial hypertrophy in this model.     

Effect of nutritional copper deficiency on carrageenin edema in the rat

The effects of nutritional copper deficiency on carrageenin edema in the rat were investigated with emphasis on studying the correlation between the degree of copper deficiency and the degree of edema. Carrageenin paw edema in both copper-sufficient and copper-deficient groups of rats was compared after either 20, 40, or 60 d on respective diets. The degree of copper deficiency was quantitated by analyzing total copper concentrations in a number of tissues. Other copper dependent parameters were also determined. Results indicated that: (1) although copper sufficient rats showed relatively little change in the degree of edema, copper-deficient rats showed a steady and significant increase in edema from d 20 to 40 to 60; (2) paw edema in copper-deficient animals was highly and negatively correlated to the concentrations of copper in the liver; the correlation with liver Cu,Zn-superoxide dismutase activity, however, was inconsistent; (3) paw edema was not correlated either to copper concentration in tissues other than liver or to plasma ceruloplasmin activity; and (4) aggravation of carrageenin edema in copper-deficient animals seemed to be mediated via an as yet unknown secondary effect of copper deficiency.     

Submaximal,aerobic exercise training exacerbates the cardiomyopathy of postweanling Cu-depleted rats

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, where-as 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.     

Comparison of copper status in rats when dietary fructose is replaced by either cornstarch or glucose

The purpose of this study was to determine what levels of starch or glucose replacement for fructose in the copper-deficient diet (copper) can minimize the fructose-copper interaction. Experimental diets contained either 100% fructose as the carbohydrate source, or the fructose was partially replaced with 50% starch, 50% glucose, 75% starch, or 75% glucose. Diets were either copper adequate (7-8 ppm) or inadequate (less than 1 ppm). Male weanling rats were fed their respective diet for 5 weeks and then fasted overnight. After decapitation, blood was collected and liver and heart were removed. Plasma copper was significantly reduced and ceruloplasmin was not detected in all copper-deficient groups. Copper deficiency increased plasma cholesterol, as well as heart and liver weight in the glucose groups, but not in the starch groups. Those organ weights were heavier in glucose-copper than starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper rats. Erythrocyte copper-zinc-superoxide dismutase activity was greater in starch-copper than glucose-copper rats regardless of carbohydrate amount. Hepatic copper concentration of the group fed starch-copper was twice levels observed in glucose-copper. The 50% glucose rats had lower hepatic copper than the 75% glucose rats. Hepatic copper-zinc-superoxide dismutase activity showed patterns similar to hepatic copper. Cardiac copper was greater in starch-copper than glucose-copper rats. Cardiac copper-zinc-superoxide dismutase activity was equally reduced in all copper-deficient groups. The 50% starch-replaced diet was more effective in minimizing copper deficiency than the 75% glucose-replaced diet. This poorer improvement of copper deficiency by glucose than starch may partially be due to a more severe reduction of food intake in glucose than in starch diets.     

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.     

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.     

Inhibition of elevated hepatic glutathione abolishes copper deficiency cholesterolemia.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (MHG-CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by L-buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG-CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 micrograms Cu/g, copper-deficient (Cu-D) and copper-adequate (Cu-A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu-D and 10 animals in each of the Cu-D and Cu-A groups were randomly assigned to receive 10 mM BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu-D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu-D rats, but had no influence on plasma cholesterol of Cu-A rats. Hepatic GSH was increased 39 to 82% in Cu-D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG-CoA reductase activity was significantly increased 85 to 288% in Cu-D rats and BSO administration abolished this increase in activity in Cu-D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG-CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.     

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.     

Glucose-6-phosphatase Activity in Copper-Deficient Rats

Copper deficiency has been reported to cause glucose intolerance in rats by interfering with normal glucose utilization. Accordingly, copper deficiency was produced in rats to study its effects on glucose-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase activities of hepatic glucose-6-phosphatase (EC 3.1.3.9), a major enzyme involved in maintaining glucose homeostasis. When measured in homogenates treated with deoxycholate, total glucose-6-P phosphohydrolase was 23% lower and total carbamyl-P:glucose phosphotransferase was 17% lower in copper-deficient rats compared to controls. Latency, or that portion of total activity that is not manifest unless the intact membranous components are disrupted with deoxycholate also was lower in copper-deficient rats. Glucose-6-P phosphohydrolase was 5% latent in copper-deficient rats compared to 24% in controls and carbamyl-P : glucose phosphotransferase was 55% latent in copper-deficient rats compared to 65% in controls. The decrease in latency appears to compensate for the lower total enzyme activities in such a manner as to allow the net expression of these activities in the intact membranous components of the homogenate to remain unaltered by copper deficiency. It thus appears unlikely that copper deficiency affects glucose homeostasis in vivo by altering the net rate of glucose-6-P hydrolysis or synthesis by glucose-6-phosphatase. These observations are interpreted on the basis of a multicomponent glucose-6-phosphatase system in which the total enzyme activity expressed in intact membranous preparation is limited by substrate specific translocases that transport substrate to the membrane-bound catalytic unit. A decrease in latency can then be interpreted as a functional increase in translocase activity and may constitute a compensating mechanism for maintaining constant glucose homeostasis when glucose-6-phosphatase catalytic activity is depressed as it is in copper deficiency.     

Dietary carbohydrate influences tissue fatty acid and lipid composition in the copper-deficient rat

Fructose and copper have been shown independently to influence long chain fatty acid metabolism. Since fructose feeding exacerbates copper deficiency, their possible interaction with respect to tissue long chain fatty acid and lipid composition was studied. Weanling male Sprague-Dawley rats were given diets containing 0.6 or 6 mg/kg copper. The carbohydrate source (627 g/kg) was either fructose or corn starch. After 3 wk, fatty acid profiles and total lipids in heart and liver were analyzed. Copper-deficient rats fed fructose had more severe signs of copper deficiency than those fed starch, according to heart/body wt ratio, hematocrit, and liver copper content. The fatty acid composition of heart and liver triacylglycerol was significantly different between groups, but the changes did not correlate with the severity of copper deficiency. In heart, phosphatidylinositol and phosphatidylserine, arachidonic acid and docosapentaenoic acid (n-6) were increased 193 and 217%, respectively, p<0.05) in rats given the copper-deficient diet containing fructose. Changes in the long chain fatty acids in heart phospholipids may be related to the higher mortality commonly observed in rats fed a copper-deficient diet containing fructose.     

In vivo apoprotein catabolism of high density lipoproteins in copper-deficient, hypercholesterolemic rats

High density lipoprotein (HDL) apoprotein catabolism was examined in male Sprague-Dawley rats deficient in dietary copper. Twenty-four rats were randomly divided into two groups: copper-adequate (control, 5 mg of copper/kg diet) and copper-deficient (0.6 mg of copper/kg diet). After 5 weeks, animals were administered a tracer dose of iodinated HDL protein previously isolated from donor rats that were subjected to the same dietary treatments as the test animals. Copper-deficient rats exhibited a 54% increase in plasma volume and a 26% increase in HDL protein concentration above controls. Consequently, the intravascular pool of total HDL protein was increased 2-fold. The fractional catabolic rate of total HDL protein was similar between groups. However, because of the increased intravascular HDL pool in copper-deficient animals, the absolute catabolic rate was greater (640 +/- 49 micrograms/hr vs 316 +/- 12 micrograms/hr in controls). Tissue uptake of total HDL protein in copper-deficient rats tended to be greater in the kidneys, spleen, and testes compared with controls; the heart exhibited a significant 2.3-fold increase. In contrast, the catabolic rate of HDL protein in the liver and adrenal gland were not different between treatment groups. That an obligatory increase in HDL protein uptake was not observed in the liver and adrenal gland (organs which are sensitive to and can further metabolize cholesterol) suggests that these organs may be regulated, possibly contributing to the observed hypercholesterolemia in this model. These data imply that total HDL apoprotein catabolism is increased in response to the increased intravascular pool of HDL in copper-deficient rats.     

Effects of dietary carbohydrate intake on antioxidant enzyme activity and copper status in the copper-deficient streptozotocin (STZ) diabetic rat

The aim of this study was to investigate how dietary lactose, compared with sucrose, in association with copper deficiency influences the antioxidant and copper status in the diabetic rat. Two groups of male rats (n = 12) were fed copper-deficient diets containing either 300 g/kg of sucrose or 300 g/kg of lactose in a pair-feeding regime for 35 days. Six rats from each group were injected with streptozotocin to induce diabetes. After a further 16 days the animals were killed and the liver, heart, and kidney removed for the measurement of copper levels and the activities of antioxidant and related enzymes. Diabetes resulted in higher hepatic and renal copper levels compared with controls. The copper content of the heart and kidney in diabetic rats consuming sucrose was also significantly higher than in those consuming lactose. Catalase activity in the liver, heart, and kidney was significantly increased in diabetic rats compared with controls. Hepatic glutathione S-transferase and glucose-6-phosphate dehydrogenase and cardiac copper zinc superoxide dismutase activities were also higher in diabetes. Sucrose, compared with lactose feeding, resulted in higher cytochrome c oxidase and glutathione peroxidase activities in the kidney while glucose-6-phosphate dehydrogenase activity was lower. The combination of lactose feeding and diabetes resulted in significantly higher activities of cardiac managanese superoxide dismutase and catalase and renal manganese superoxide dismutase and glucose-6-phosphate dehydrogenase. These results suggest that sucrose consumption compared with lactose appears to be associated with increased organ copper content and in general decreased antioxidant enzyme activities in copper-deficient diabetic rats.     

Development of copper deficiency in rats fed fructose or starch: weekly measurements of copper indices in blood

Copper deficiency was induced in weanling rats fed diets whose sole source of carbohydrates was starch or fructose for 7 weeks. Conventional parameters of copper status, plasma copper concentrations, ceruloplasmin activity, and erythrocyte superoxide dismutase (SOD) activity were longitudinally monitored weekly to follow the development of the deficiency and to correlate these indices with the degree of severity of the deficiency. Although 30% of the rats fed a copper-deficient fructose diet died and no deaths occurred in rats fed the copper-deficient starch diet, plasma copper, ceruloplasmin, and SOD activities were reduced to a similar extent in all rats fed copper-deficient diets regardless of the type of dietary carbohydrate. Thus, none of the indices used accurately reflected the greater degree of deficiency or mortality in rats fed the fructose diet deficient in copper. The results of the present study underscore the need for more sensitive tests or alternative parameters to assess copper status in living animals.