Atrial thrombosis, abnormal electrocardiograms and sudden death in mice due to copper deficiency

Atherosclerosis, 54 (1985) 213-224 Elsevier Scientific Publishers Ireland,

213 Ltd.

ATH03572

Atria1 Thrombosis, Abnormal Electrocardiograms and Sudden Death in Mice Due to Copper Deficiency Leslie M. Klevay U.S.Department of Agriculture,Agricultural

Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202 (U.S.A.) (Received 14 May, 1984) (Revised, received 4 September. 1984) (Accepted 5 September, 1984)

Summary

Approximately 20 years ago a diet high in lard and sucrose was described that produced extensive cardiovascular damage in adult mice. Atria1 thrombosis, myocardial necrosis and sudden death were frequent. These experiments were repeated as closely as possible; the adverse effects were prevented by a drinking solution containing 10 pg copper/ml. Lack of copper also was associated with anemia, cardiac enlargement and abnormal electrocardiograms. Bradycardia, coupled beats, ectopic ventricular foci, premature atria1 beats and prolonged PR interval were found. Lack of copper had no effect on cholesterol in plasma. The results may be germane to ischemic heart disease and the thrombotic susceptibility of women who use oral contraceptives or are pregnant frequently, because copper metabolism is altered in these conditions. Key words: Atrial

thrombosis - Atherosclerosis - Copper - Electrocardiograms Frequent pregnancy - Ischemic heart disease - Oral contraceptives Sudden death

-

Correspondence and reprint requests to: Leslie M. Klevay, M.D., USDA, ARS, Human Nutrition Research Center, P.O. Box 7166, University Station, Grand Forks, ND 48202, U.S.A. Proprietary statement: Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. These data were presented in part at ,the 68th Annaal Meeting of the American Institute of Nutrition (Abstract No. 3268), St. Louis, MO, 1984.

0021-9150/85/$03.30

Q 1985 Elsevier Scientific

Publishers

Ireland,

Ltd.

214

Introduction The search for the cause(s) of atherosclerosis probably began in the 19th century. Dietary lipids began to receive greater emphasis compared to other factors when cholesterol was added to the diet of rabbits and atherosclerosis was produced in 1913 [1,2]. In subsequent decades lesions were produced in other species; diets were modified and frequently became bizarre. Selye [3] listed many experimental regimens that produce myriad cardiovascular lesions. In 1963, Ball et al. [4] described a diet with improved nutritional qualities that induced cardiovascular lesions in mice. The dietary improvements were decreased fat, increased carbohydrate and use of a vitamin supplement. This work led to a series of publications [5-lo] in which some of the characteristics of the diet were modified, the cardiovascular lesions were described more thoroughly and the susceptibility of various strains of mice was compared. Mice of the Swiss strain were found to be particularly susceptible, with high mortality due to atria1 thrombosis, coronary necrosis, coronary thrombosis, myocardial necrosis, ventricular calcification and with most deaths occurring between 6 to 12 weeks of dietary exposure. Coronary arteries showed ‘fragmentation of elastic membranes, hyalinization and deposition of fat (sudanophilia)’ [S]. Some of the lesions described above [4-lo] resemble both ischemic heart disease and copper deficiency in animals. Similarities between ischemic heart disease and copper deficiency have been noted [l l-131. The diets [4-lo] so ruinous of mice were made with a salt mixture [14] containing no added copper. This mixture, No. 2, from USP XIII, consists of 7 salts; salts of iodine, copper, manganese and zinc are not among them. As the grades of the chemicals are specified [14] as either USP or USP reagent, contamination with the elements omitted is not likely to supply adequate dietary amounts when the mixture is diluted 25-fold with other dietary components. The major components of the diet, sucrose and lard, are quite low in copper. It was decided to repeat the experiments of Ball et al. as nearly as possible to test the hypothesis that copper deficiency was the cause of the thrombosis and death. The 4 elements known to be absent from the USP salt mixture were provided. Materials and Methods The major dietary ingredients are shown in Table 1; components of the vitamin mixture are shown in Table 2. This diet was fed to 64 female, Swiss mice obtained from Taconic Farms (Germantown, NY) and matched into 4 equal groups with equal mean weight (34 g) the day after they arrived at the Center. No mouse weighed less than 26 g. These albino mice also are called Taconic-Swiss Webster or TS mice. Four mice were placed in each cage; conditions were similar to those described [15]. Trace element supplements were supplied as drinking solutions; concentrations were based on personal experience [16], that of Schroeder et al. [17] and consideration of the recommendations of the National Research Council [18]. Solutions (per ml) were made in demineralized water (Continental Water Systems, Minneapolis,

215

TABLE

1

DIET COMPOSITION Ingredients

g/kg

sucrose a Salt mixture b No. 2, USP XIII I-Cystine b

575 40.0 5.0 80.0 20.0 280

Vitamin Vitamin Lard d a b ’ d

free casein ’ mixture

American Crystal Sugar Co., Moorhead, MN. Teklad, ARS/Sprague-Dawley, Madison, WI. ICN Nutritional Biochemicals, Cleveland, OH. Armour and Co., Phoenix, AZ. Analysis of the diet revealed 0.4.0.6 and zinc, respectively.

and 4 pg/g

for copper,

manganese

MN): group A, no salts; group B, 10 pg copper; group C, 10 pg copper, 10 pg manganese, 0.5 pg iodine, 50 pg zinc; group D, 10 pg manganese, 0.5 pg iodine, 50 pg zinc. Reagent grade MnSO, - H,O, KI and Zn(C,H,O,), .2H,O were obtained from Fisher Scientific (Fair Lawn, NJ). Reagent grade CuSO, +5H,O was obtained from Mallinckrodt Chemical Works (St Louis, MO). TABLE

2

COMPOSITION

OF THE VITAMIN

MIXTURE

a

g p-Aminobenzoic acid b Ascorbic acid b i-Inositol ’ Niacinamide ’ a-Tocopheryl acetate (powder 250 IV/g) ’ Vitamin A concentrate (5OOCtOOIU/g) ’

2.75 25.00 2.75 2.50 2.75 2.50 mg

Biotin b Folic acid ’ Calcium pantothenate b.c Cyancobalamine b Menadione d Pyridoxine hydrochloride ’ Riboflavin ’ Thiamine hydrochloride ’ Vitamin D, concentrate (500000

II-l/g) d

11.00 50.0 1500 0.75 1250 500 500 500 125.0

a Diluted to 500 g with sucrose. Weighing error was kept to less than 1% by weighing shown. b Grand Island Biochemical Co., Grand Island, NY. ’ ICN Pharmaceuticals, Cleveland, OH. d Nutritional Biochemicals Corp., Cleveland, OH.

to the accuracy

216

Cholesterol in plasma was measured by fluorometry [19]. Electrocardiograms were recorded as described [20,21] under intraperitoneal anesthesia with a barbiturate. Copper was measured by atomic absorption spectrometry or plasma emission spectrometry after destruction of organic matter with nitric and sulfuric acids and hydrogen peroxide [22]. Statistical examination of the data was by analysis of variance followed by Scheffe contrasts when appropriate [23]. Dead mice were examined in search of gross cardiovascular pathology. Results

The mice consumed the diet and gained weight. To our surprise (and, it seems, to that of the supplier), some mice were pregnant. When pregnancy became obvious the mice were placed in individual cages and the dietary regimen was continued. After delivery a few pups were taken for analysis and dams were returned to their original cages. Twelve mice delivered litters, some as early as the 7th day of the experiment. Distribution of litters across groups was approximately equal. Pups from dams given no supplemental copper contained slightly less copper than those of dams given copper (10.2 vs 14.9 pg/dry g (P < 0.08)). Longevity of mice is shown in Table 3. Median longevity of mice given supplemental copper was more than twice as long while eating the diet as that of mice without a copper supplement. The time at which 3/4 of the mice with supplemental copper had died was almost 6 times that of mice without a copper supplement. The first mouse unsupplemented with copper died after 40 days, the first mouse supplemented with copper died 51 days later. By this time (the 91st day of the experiment), only 3 mice were alive in group A and in group D. On the 127th day of the experiment 2 mice receiving supplemental copper were dead and 2 mice without supplemental copper were alive. A few days later, when no mice in groups A and D were alive, 7 mice in group B and 6 mice in group C died within 2 days of anesthesia for the recording of electrocardiograms, decreasing the numbers of mice in these groups to 8 and 9, respectively. On the 164th day of the experiment, the 9th mouse in group B died and median longevity for the group was determined. No more deaths occurred for 18 weeks. The 9th mouse in group C died on the 362nd day of

TABLE

3

LONGEVITY

(days “)

Group

Median

75% dead

A

15 146 332 75

90 532 484 90

$ D

’ Longevity is given for the mice in this experiment b Mice receiving copper in the drinking solutions.

after weaning,

growth

to adult size and travel.

217

the experiment. Three fourths of the mice in group B and C were dead after 532 and 484 days, respectively. Three fourths of the mice without supplemental copper were dead in 90 days. Some of the hearts were so large at autopsy that it seemed that pulmonary function must have been diminished. Heart weights are shown in Table 4. Uneven numbers among groups are the result of a few dead mice being partially eaten by their associates and others being alive. The hearts of mice receiving no copper supplement were significantly larger than those of mice receiving supplemental copper. The cardiac enlargement seemed to be predominantly the result of large, distended atria which were filled with occlusive thromboses. The gross appearance of the hearts usually was similar to that pictured in the original experiments on this phenomenon [4,5,9]. Sometimes the two atria were as large as the rest of the heart. No hearts from mice receiving copper had a similar appearance. Eight mice with hemothorax, 3 with hemopericardium and 2 with chylothorax received neither supplemental copper nor anesthesia near the time of death. One mouse receiving supplemental copper was found to have a hemothorax shortly after anesthesia. Several mice had pleural effusions; effusions without anesthesia occurred only in mice without extra copper. No significant difference in the concentration of cholesterol (Table 4) in plasma was noted on the 40th day of the experiment. Measurement of cholesterol 3 weeks later, when death decreased the number of samples, produced similar results. The mean hematocrits of mice with and without supplemental copper were 54% and 28%, respectively, a highly significant difference (P < 0.001). Supplementation with iodine, manganese and zinc did not affect hematocrit or cholesterol. On the 56th day of the experiment the heart rates (Table 4) of mice receiving demineralized water were significantly slower than rates of the other mice. Thirty four days later, heart rates of both groups of mice without supplemental copper were

TABLE

4

CHARACTERISTICS OF MICE Data are means& SE. Numbers in parentheses are number Probability values are based on analysis of variance 1231. Group

Plasma cholesterol

Heart weight

(mg/dB

(g)

Aa B C Da

133 f 5.4(16) 130+6.8(16) 125 f 9.2(16) 147+6.3(15)

0.51 f 0.24 + 0.32 f 0.51*

P

0.16

0.0001

0.036(14) b 0.019(7) = 0.036(7) = 0.039(15) b

of mice

on which

data

were obtained.

Heart rate (beats/min)

(ms)

402_+31.1(12) b 493 f 17.5(12) 507+ 18.0(15) ’ 507k18.1(13)c

52+2.2(H)& 40+0.6(11)= 4Of 1.6(14) ’ 48+2.2(11) b

0.003

0.0001

PR interval

a Mice receiving no copper in the drinking solution. b,c Groups with different superscripts are significantly different; weight, P <: 0.03; rate, P-c 0.02; interval, P -z 0.009. Only the data on heart rate were changed appreciably by exclusion of mice that bore litters; overall probability was similar with group A being slower than group C (P -C0.01).

218

significantly slower (P < 0.0001). The PR intervals of mice receiving no copper supplement were longer than those of mice receiving supplemental copper. The prolonged PR interval indicates first degree heart block. This slowing and lengthening was noticed even among mice who had no obvious abnormalities of the electrocardiogram such as ectopic beats, escaped beats or higher degree heart block. The lengthening of the PR interval seems to precede the slowing of the heart rate. Some electrocardiograms are shown in Fig. 1. The first (a) illustrates a normal rate of 519 beats/mm and a typical contour. Pulse rates in (b) to (f) ranged from 66 to 223. An atria1 premature beat is shown (b). Later in the tracing (not shown) a premature beat occurred without a P wave; this absence indicates a nodal beat. Coupled beats (c) occurred; the 2nd beat of each pair had no P wave. The 2nd degree heart block (d) changed from the 4 : 1 (shown) to 3 : 1 and 2 : 1, but the ventricular rate remained more or less constant at 66 beats/n-tin. P waves, which on occasion had a different shape, dropped out to vary the block ratio. A sinus bradycardia is shown (e). A tracing was made during cardiac arrest (f) without

Fig. 1. Electrocardiograms. Tracings were made from original charts. The time (days) elapsed between the recording of the electrocardiogram and the beginning of exposure to the diets is shown. Scale: 0 50 ms by 0.44 mV. (a) mouse B,,; normal electrocardiogram. (b) mouse A,; atria1 premature beat. (c) mouse A,; coupled beats. (d) mouse A,; 4: 1 heart block. (e) mouse D6; sinus bradycardia. (f) mouse D,,; unanesthetized, moribund mouse with ventricular bradycardia and 2 ectopic foci. The shape of the ventricular complexes in (b), ( c) and (e) seems normal.

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anesthesia; 2 ventricular foci are illustrated. The R-R shown in Fig. 1 (a-c, e) seems to be normal for mice. None of the abnormal characteristics shown (b-f) were found in mice receiving supplemental copper. Discussion The decreased longevity of the mice without supplemental copper is similar to that found in rats deficient in copper [20,21]. The greater median longevity of mice receiving copper, manganese, iodine and zinc compared to those receiving only copper may be fortuitous as deaths associated with anesthesia decreased group size to the point where 2 deaths or 1 death would determine the median. This latter group lived 48 days more than the former before 75% were dead in support of this belief. Cardiac enlargement has been associated with copper deficiency since 1939 [24]. Its occurrence has been reviewed [20,25,26]. The atria1 thrombosis found in mice receiving no supplemental copper usually resembled that shown in the original experiments on this phenomenon [4,5,9]. However, in the first few mice to die, thrombosis was minimal and was manifested mainly as dark discoloration. In some mice that died later, the combined atria1 size exceeded that of the ventricles. These findings did not occur in mice receiving supplemental copper. Hemothorax and hemopericardium are common in rats [20,21] and mice [27] deficient in copper. To my knowledge chylothorax has not been reported in copper deficiency, although Hunt and Carlton [28] mentioned ‘whitish fluid’ in the peritoneal cavity of a deficient rabbit. Pleural effusion has been found in rats [21] and mice [27] deficient in copper. Hypercholesterolemia has been associated with copper deficiency frequently [29-341. In this experiment an inadequate intake of copper did not produce hypercholesterolemia. However, electrocardiographic abnormalities were found. Electrocardiographic abnormalities from copper deficiency have been found in the absence either of anemia [25,35] or anemia and hypercholesterolemia [35]. Thus although copper deficiency can produce abnormal electrocardiograms, anemia and hypercholesterolemia, these abnormalities do not always occur together. There is no explanation for this lack of effect of copper deficiency on plasma cholesterol. Some of these electrocardiographic abnormalities are similar to those that have been found among rats deficient in copper (Fig. Id, f) [20,21]. Prolongation of the PR interval (first degree heart block) and slower heart rate were not found in the first description of the effects of copper deficiency on electrocardiograms [20]. However, in later experiments a significant increase in PR interval was noted [36]; a 15%, but insignificant, decrease in heart rate also was noted. Hearts from rats deficient in copper beat more slowly in vitro than do hearts from well nourished animals [26]. The electrocardiographic abnormalities described here are predominantly of supraventricular origin. Those abnormalities found earlier [20,21] were predominantly of ventricular origin, although some were supraventricular [21]. The supraventricular nature of the PR prolongation, the slower heart rates, the atrial prema-

220

ture beats, the coupled beats, and the sinus bradycardia are consonant with the atria1 pathology. The abnormalities reported seem to be largely the result of inadequate copper intake. Results are consonant with other deficiency experiments; some new characteristics of deficiency were found. Supplementation with iodine, manganese and zinc did not prevent pathology. If these elements have an effect on the pathological processes, the effect is small. The hypothesis was tested successfully. The diet formulated by Ball et al. (4) was thought to be low in copper because the salt mixture contained no copper salt and because some of the pathological changes produced by the diet resembled those found in copper deficiency. Two components of the diet, ascorbic acid and sucrose, are present in amounts likely to impair the utilization of copper. Mice do not require vitamin C [18]. This diet contains 1 g of ascorbic acid/kg. This concentration is almost 7 times that which produced hypercholesterolemia in rats [37]. Evans [38] reviewed the evidence that ascorbic acid inhibits the intestinal absorption of copper. Thus diets high in ascorbic acid increase the dietary requirement for copper. High sucrose intakes produce higher copper requirements. Rats fed diets low in copper had l/3 the concentration of liver copper when fed sucrose in comparison to rats fed starch [39]. Ball et al. [4] emphasized the improved qualities of their diet in comparison to earlier diets with similar pathological effects. This diet was lower in fat, and higher in carbohydrate and vitamins. They did not know that sucrose and ascorbic acid may have contributed to the induction of pathology or that 10 /.tg of copper/ml of drinking solution would have overcome the adverse characteristics of the diet. This diet is 28% lard on a weight basis. Nearly half the energy is from fat. With adequate copper, this amount of fat had no effect on pathology. Although Ball et al. [5] noted a long history of induction of atherosclerosis in animals with diets high in fat, they found that increasing the lard to 40% (at the expense of sugar) did not increase the degree of thrombosis. Although allusion to human disease is lacking in the original reports on this phenomenon [4-lo], one should consider the possibility that the induction of thrombosis, sudden death and abnormal cardiac electrophysiology by copper deficiency may be germane to human illness. More than 20 articles contain analytical data showing that daily diets in the United States frequently contain less copper than the amount required (2 mg) to compensate for average daily urinary and fecal loss by adults [40]. Three of these reports are among the more accessible [41-431; the others have been cited in review [ll-13,441. Similarities between animals deficient in copper and people with ischemic heart disease have been noted [ll-131; the importance of copper metabolism in the origin of the disease is hypothesized [ll-13,16,45-491. Emphasis has been placed on abnormalities of lipid, glucose and uric acid metabolism and on altered cardiac electrophysiology. The thrombotic aspects of atherosclerosis and ischemic heart disease have received little comment [ll], although the thrombotic process is known to be important in atherogenesis and acute myocardial infarction [50-531. The

221

present results provide a link between copper metabolism and thrombosis and confirm earlier results with rats [54], mice [27] and another experiment on thrombosis [55] in dogs in which abnormal copper metabolism may have been a hidden variable (below). A relationship between copper and thrombosis probably has escaped general notice as no reference common to copper and coagulation (or several related terms) has been found [56,57]. In emphasizing the improved qualities of their diet, Ball et al. [4] mentioned that it contained no cholesterol or cholic acid. Cholesterol and cholic acid have been shown to increase the dietary copper requirement of rats [58]. Mahley et al. [55] fed dogs an atherogenic diet containing cholesterol and cholic acid that produced thrombosis of the coronary, ileofemoral, internal carotid and mesenteric arteries and the terminal aorta. This diet also produced atherosclerosis and myocardial infarction. Repeated pregnancies and use of oral contraceptives predispose to thromboembolism [59-621. Cerebral and coronary thrombosis, myocardial infraction and pulmonary embolism are the more serious manifestations of this phenomenon. Also a post- or peripartum cardiomyopathy of unknown etiology, consisting of cardiac enlargement, mural thrombi and degeneration and fibrosis of myocardial fibers, has been described [61]. Pregnancy and oral contraceptives alter copper metabolism. Krebs discovered that copper metabolism is altered during pregnancy with his demonstration of increased serum copper [63]. This phenomenon has been confirmed repeatedly; several references are cited in review [64]. Oral contraceptives induce hypercupremia; references are cited in review [65]. Estrogen probably is responsible for the hypercupremia in both these situations; this hypercupremia probably is associated with loss of copper from liver [16,45]. Estrogen can induce hyperceruloplasminemia as well as hypercupremia [38,65]. When Douglas et al. [9] fed the diet used in the present experiment to mice, they found that those ‘which were bred were more susceptible to diet-induced damage and had a shorter survival time than unbred mice’. Although the present experiment did not confirm this finding, perhaps the unanticipated pregnancies were too few. Thus the production of severe thrombosis in mice fed a diet with insufficient copper may be relevant to human disease. Thrombosis contributes to atherosclerosis and myocardial infarction and to complications of pregnancy and oral contraceptive use. Copper metabolism is altered in all of these conditions. Acknowledgement I wish to thank

Forrest

H. Nielsen,

Ph.D., for helpful

discussion.

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