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

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

Electrocardiographic activity and cardiac function in copper-restricted rats.

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.     

Myocardial cross-bridge kinetics in transition to failure in Dahl salt-sensitive rats

The role of altered cross-bridge kinetics during the transition from cardiac hypertrophy to failure is poorly defined. We examined this in Dahl salt-sensitive (DS) rats, which develop hypertrophy and failure when fed a high-salt diet (HS). DS rats fed a low-salt diet were controls. Serial echocardiography disclosed compensated hypertrophy at 6 wk of HS, followed by progressive dilatation and impaired function. Mechanical properties of skinned left ventricular papillary muscle strips were analyzed at 6 wk of HS and then during failure (12 wk HS) by applying small amplitude (0.125%) length perturbations over a range of calcium concentrations. No differences in isometric tension-calcium relations or cross-bridge cycling kinetics or mechanical function were found at 6 wk. In contrast, 12 wk HS strips exhibited increased calcium sensitivity of isometric tension, decreased frequency of minimal dynamic stiffness, and a decreased range of frequencies over which cross bridges produce work and power. Thus the transition from hypertrophy to heart failure in DS rats is characterized by major changes in cross-bridge cycling kinetics and mechanical performance.     

Silicon facilitation of copper utilization in the rat

An eight-week, 2 x 4 factorial rat experiment using two levels of dietary copper and four levels of dietary silicon was conducted to further delineate a previously observed silicon-copper interaction in which silicon appears to mimic copper in its effect on the composition of the aorta. Dietary copper concentrations were 1.4 (deficient) and 5.4 (adequate) mg/kg diet, and silicon concentrations were 5, 135, 270, and 540 mg/kg diet. Compared with the lowest level of silicon and copper, weight gains were 15.5% higher for rats fed 540 mg silicon/kg diet and 14.3% higher for those fed 5.4 mg copper/kg diet. The growth-promoting effects of silicon and copper were additive. Evidence that silicon elevated the copper status of copper-deficient rats includes an increase in packed-cell volume by 540 mg silicon/kg diet in the otherwise packed-cell volume-depressed, copper-deficient rats, accompanied by a trend toward higher hemoglobin values and lower relative heart weights. In the copper-adequate rats, evidence that 540 mg silicon/kg diet elevated their copper status includes a two-fold increase in the blood-plasma copper concentration, a three-fold increase in ceruloplasmin activity, and an increase in cardiac, renal, and hepatic copper concentrations. In addition, 540 mg silicon/kg diet resulted in higher aortic dry mass and aortic elastin content in both copper-deficient and copper-adequate rats. While dietary silicon concentrations of 135, 270, and 540 mg/kg diet were all effective in increasing aortic elastin in the copper-adequate rats, only 540 mg silicon/kg diet increased aortic elastin in the copper-deficient rats. These data indicate that some of the metabolic effects attributed to silicon may be manifested through a silicon-facilitated increase in copper utilization.     

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.     

Sexual differences in the expression of copper deficiency in rats

The present investigation was undertaken to establish whether the severity of copper deficiency in rats fed diets containing fructose is affected by the presence and type of endogenous sex hormones. Intact and castrated male rats and intact and ovariectomized females were fed from weaning a copper-deficient diet (0.6 ppm) containing 62% fructose for 8 weeks. Regardless of castration, male rats were anemic, exhibited heart hypertrophy, and died of the deficiency. However, castration ameliorated the anemia and delayed the mortality. In contrast, none of the females died of the deficiency. It is suggested that in addition to the sex of the animal, levels of testosterone in the male may also play a role in the severity of copper deficiency.     

The effect of vitamin E on lipid peroxidation in the copper-deficient rat

The present study was designed to determine whether the supplementation of vitamin E in the copper-deficient diet would ameliorate the severity of copper deficiency in fructose-fed rats. Lipid peroxidation was measured in the livers and hearts of rats fed a copper-deficient diet (0.6 microg Cu/g) containing 62% fructose with adequate vitamin E (0.1 g/kg diet) or supplemented with vitamin E (1.0 g/kg diet). Hepatic lipid peroxidation was significantly reduced by vitamin E supplementation compared with the unsupplemented adequate rats. In contrast, myocardial lipid peroxidation was unaffected by the level of vitamin E. Regardless of vitamin E supplementation, all copper-deficient rats exhibited severe signs of copper deficiency, and some of the vitamin E-supplemented rats died of this deficiency. These findings suggest that although vitamin E provided protection against peroxidation in the liver, it did not protect the animals against the severity of copper deficiency induced by fructose consumption.     

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.     

Interaction Between Dietary Carbohydrate and Copper Nutriture on Lipid Peroxidation in Rat Tissues

The effects of the interactions between dietary carbohydrates and copper deficiency on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and their roles in peroxidative pathways were investigated. Weanling rats were fed diets deficient in copper and containing either 62% starch, fructose, or glucose. Decreased activity of SOD was noted in all rats fed the copper-deficient diets regardless of the nature of dietary carbohydrate. However, the decreased activity was more pronouced in rats fed fructose. Feeding the fructose diets decreased the activity of GSH-Px by 25 and 50% in the copper-supplemented and copper-deficient rats, respectively, compared to enzyme activities in rats fed similar diets containing either starch or glucose. The decreased SOD and GSH-Px activities in rats fed the fructose diet deficient in copper were associated with increased tissue per-oxidation and decreased hepatic adenosine triphosphate (ATP). When the fructose in the diet of copper-deficient rats was replaced with either starch or glucose, tissue SOD and GSH-Px activities were increased and these increases in enzyme activity were associated with a tendency toward reduced mitochondrial peroxidation when compared to the corre-sponding values for rats fed fructose throughout the experiment Dietary fructose aggrevated the symptoms associated with copper deficiency, but starch or glucose ameliorated them. The protective effects were more pronounced with starch than with glucose.     

Fructose metabolizing enzymes in the rat liver and metabolic parameters: Interactions between dietary copper,type of carbohydrates,and gender

This study was conducted to determine the effects of nutrient interactions between dietary carbohydrates and copper levels on fructose-metabolizing hepatic enzymes in male and female rats. Male and female rats were fed diets for 5 weeks that were either adequate or deficient in copper that contained either starch or fructose. Rats of both sexes fed fructose as compared with those fed starch showed higher activity of hepatic fructose metabolizing enzymes. There were also significant differences in fructose metabolism of liver between the male and female rats. Female rats had lower hepatic ketohexokinase and triose kinase but higher triosephosphate isomerase activities compared with male rats. Male rats fed copper-deficient diets had lower aldolase B activity compared with those fed copper-adequate diets. Female rats fed copper-deficient diets had higher triosephosphate isomerase activity compared with rats fed copper-adequate diets. Our data suggest that gender differences in hepatic fructose metabolism may not be the primary reason for the severity of copper deficiency syndrome in male rats fed copper-deficient diet with fructose.     

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.     

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.     

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.     

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.     

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.     

Occurrence and distribution of atrial natriuretic peptide-containing cells in the left ventricle of hypertensive rats

In the ventricles of adult mammalian hearts, production of atrial natriuretic peptide (ANP) is negligible, restricted to the impulse-conducting cells, the papillary muscles, and a minority of subendocardial myocytes. ANP expression is reinduced in the ventricles of pressure-overloaded and failing hearts and is frequently used as a marker for myocyte hypertrophy. Using an immunohistochemical approach, we have characterized the size distribution of ANP-containing myocytes in the left ventricle of the spontaneously hypertensive rat (SHR) before and after chronic antihypertensive therapy and compared the results to age-matched normotensive Wistar rats (WR). Our findings show that in SHR the frequency of cells presenting ANP granularity is positively correlated with myocyte size (r=0.746, P<0.02). The highest proportion of ANP-positive myocytes (55-57%) was measured among cells of diameters 30-34 microm. In any corresponding cell size, the proportion of ANP-presenting myocytes was five- to tenfold higher in SHR than in the normotensive WR. We studied the effects of the antihypertensive drugs captopril, hydralazine, and nifedipine and found that, regardless of their effect on blood pressure or hypertrophy, all three eliminated ANP immunoproducts from the majority of the left ventricular myocytes and reduced the level of ANP mRNA, captopril being the most effective. The positive correlation between myocyte size and ANP expression was not maintained in the hearts of drug-treated SHR. Myocytes on the border of fibrotic areas or in regions of ANP presentation within the normal heart resisted the suppressive effect of the antihypertensive therapy, indicating that blood pressure or hypertrophy are not the sole correlates for ANP expression.     

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.     

Glycogen synthase kinase-3alpha reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3beta. Pressure overload increases expression of GSK-3alpha but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK.