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The effect of zinc on vitamin D3-induced cardiac necrosis
j Mol Cell Cardiol 17, 109-117 (1985)
T h e Effect o f Z i n c on V i t a m i n D 3 - i n d u c e d Cardiac Necrosis Monika Anna Wrzo~ek
Laboratory of Eleclron Microscopy and Department of Pathomorphology, Academic Medical School, 80-210 Gdahsk, Poland (Received 16August 1983, accepted in revisedform 20 April 1984) M. A. WRZO~EK.The Effect of Zinc on Vitamin Da-induced Cardiac Necrosis. Journal of MolecularCellular Cardiology(1985) 17, 109-117. Multifocal heart muscle necrotic lesions in rats were induced with high oral doses of vitamin D 3 (300,000 iu/rat in three daily doses 100000 iu each). The calcium content increased over 100 fold in the hearts of rats receiving vitamin D 3. Parenteral pre-treatment with zinc sulphate (50 or 200 rag/rat in ten daily doses) resulted either in a reduction or in the total prevention of myocardial lesions on macroscopic, light and electron microscopic examination. The effect of zinc was dose-dependent. The administration of various doses of zinc sulphate resulted in a gradual normalisation of heart calcium content. KEy WORDS: Vitamin D ; Necrosis, Experimental cardiac ; Zinc ; Calcium.
Introduction
8, 20]. V i t a m i n D - i n d u c e d m y o c a r d i a l injury
I s o p r o t e r e n o l - i n d u c e d heart muscle i n j u r y is one of the most widely studied a n d best understood forms of e x p e r i m e n t a l m y o c a r d i a l necrosis. I t is believed t h a t intracellular calcium o v e r l o a d plays a crucial role in the p a t h o g e n esis of this form of cardionecrosis [6, 8, 12, 13, 16, 18-20]. This view is s u p p o r t e d by the beneficial effect of administering calcium channel blockers which protect the m y o c a r d i u m by p r e v e n t i n g isoproterenol-induced inflow of calcium to the h e a r t muscle cells [10, 11, 16]. T h e r e have been recent reports of p r o t e c t i n g m y o c a r d i u m against isoproterenol cardiotoxicity b y p a r e n t e r a l a d m i n i s t r a t i o n of zinc, in b o t h h e a l t h y a n d S H R rats [4, 17]. T h e r e d u c t i o n of necrotic areas in the hearts of z i n c - p r e t r e a t e d animals was found to be associated with a lower h e a r t calcium content, as c o m p a r e d with rats receiving isoproterenol alone [4]. H i g h doses of v i t a m i n D are also k n o w n to p r o d u c e multifocal areas of necrosis in rat hearts [-1, 15, 23, 24]. A l t h o u g h there are some suggestions that a n o t h e r m e c h a n i s m takes p a r t in the pathogenesis of this form o f myoc a r d i a l fiber necrosis [23], the calcium ion is supposed to p l a y the most i m p o r t a n t role [7,
is associated with a d r a m a t i c increase in h e a r t tissue calcium content. R o e n t g e n o g r a p h i c microanalysis reveals calcium deposits both in the c a r d i a c ceils a n d in the interstitium of v i t a m i n D - d a m a g e d hearts [23]. This study investigates the inf/uence o f p a r enteral zinc a d m i n i s t r a t i o n on the f o r m a t i o n of necrotic lesions in the hearts of rats treated with high doses of v i t a m i n Da. Calcium, zinc a n d m a g n e s i u m contents in the h e a r t were measured. T h e relationships between tissue levels of these metals a n d the extent of v i t a m i n D s - i n d u c e d m o r p h o l o g i c a l changes in the m y o c a r d i u m were evaluated.
0022 2828/85/020109 + 09 $03.00/0
Materials and Methods Forty-five virgin female W i s t a r rats, each w e i g h i n g a b o u t 250 g, fed with a s t a n d a r d l a b o r a t o r y diet, were used in the experiment. T h e animals were divided into the following g r o u p s : (1) controls (five rats); (2) treated with v i t a m i n D3 (eight rats); (3) treated with low doses of zinc; 5 m g Z n S O 4 x 7 H 2 0 / 2 4 h (eight rats); (4) treated with low doses of zinc a n d v i t a m i n D3 (eight rats); (5) treated with high doses of zinc; 20 m g Z n S O 4 x 7 H 2 0 / 2 4 I~) 1985 Academic Press Inc. (London) Limited
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h (eight rats); (6) treated with high doses of zinc and vitamin D 3 (eight rats). Zinc treated rats received intraperitoneal injections of ZnSO4 x 7H20 (Polskie Odczynniki Chemiczne, Gliwice) for 10 days consecutively. The animals from groups 4 and 6 were given 100000 iu of vitamin Da/24 h orally during the last 3 days of zinc administration, as described previously by Wrzotkowa and Zydowo [23]. The rats from group 2 received the same dose of vitamin D 3 without any zinc pre-treatment. One day after completion of the vitamin D 3 administration the animals were killed by decapitation, their hearts were examined macroscopically and the extent of vitamin D3-induced myocardial lesion was determined according to the following scale : (A) heart with normal macroscopic appearance; (B) about three or four pin-head size whitish foci observed in the heart; (C) single foci dispersed in the entire heart muscle; (D) multiple but not confluent loci; (E) multiple foci partly confluent but macroscopically unchanged muscle prevailing; (F) confluent foci dominate over unchanged muscle. The cross section from the middle part of the ventricles was taken for histopathological examination. Each section was stained with hematoxylin and eosin and the van Kossa's method for calcium [9]. The extent of vitamin D3-induced myocardial alteration was determined microscopically, according to the following scale : 0 = no histological alterations detected; 1 = single minute foci of necrosis, calcification and small clusters of cellular infiltrate in the entire section; 2 = about 10 separate foci of necrosis, calcification or infiltrate in the section ; 3 = some of the foci coalesce, creating bigger, heterogenous ones; 4 = numerous minute and bigger foci present in each microscopic field at magnification of x 70; 5 = foci of necrosis, calcification and infiltrate dominate, with only minute fragments of apparently unchanged muscle. Samples were taken from macroscopically unchanged parts of the interventricular septum near the apex for examination in the electron microscope. Semi-thin sections of these samples were prepared and areas free of microscopically detectable alterations were chosen for the ultrathin sections. These were
examined in t h e J E M 7 A electron microscope after they had been processed and stained by a standard procedure. The remaining parts of the heart tissue were weighed, mineralised with nitric acid at 105~ and used for analysis of calcium, zinc and magnesium content. The measurements were performed with Beckman atomic absorption spectrophotometer, model 1272. All the data are presented as mean ___standard deviation (S.D.). They were statistically evaluated by the Student's l test. Results
The extent of vitamin D3-induced myocardial lesions both macroscopic and histopathological are presented in Table 1. Table 2 shows calcium, zinc and magnesium contents in the necrotic hearts compared to the values in the control group. Administration of high doses of vitamin D 3 resulted in the development of necrotic lesions of various extent in rat hearts. The morphological appearance of the hearts, both macroscopic and histopathological, as well as the ultrastructural alterations, have been described previously by Wrzo|kowa and Zydowo [23]. In the vitamin D3-treated rats light microscopic examination revealed multiple scattered foci of coagulative necrosis and/or myocytolysis in the entire heart muscle (Figure 1), but predominantly in the subendocardial and subepicardial layer. Some necrotic cells were basophilic in hematoxylineosin staining. The van Kossa's reaction revealed calcium deposits mainly in the necrotic basophilic cells. Necrotic foci were often accompanied by a mononuclear or polymorphonuclear infiltrate. Electron microscopic examination revealed various forms of alteration of cardiomyocytes. Figure 2 showed normal myocytes. Damaged cells were interspersed with well preserved ones. The changes varied from cells with slight separation of myofibrils (Figure 3) to cells filled with various organelles, myelin figures, lipid spheres and calcium deposits. The latter cells could hardly be recognized as myocytes. Common features were the proliferation of rough endoplasmic reticulum and polysomes as well as the appearance of various amounts
Vitamin
111
D 3 , Zinc and Cardiac Necrosis
T A B L E 1. T h e effect of zinc on the vitamin D3-induced lesions of rat myocardium Animals treated with 3 x 100000 iu vitamin D 3 (Group 2) D E C D E
10 x 5 m g Z n S O 4 + 3 x 100000 iu vitamin D3 (Group 4)
(3) (4) (3) (3) (4)
A A C D A
10 x 20 mg Z n S O 4 + 3 x 100000 iu vitamin D 3 (Group 6)
(i) (1) (2) (3) (0)
A A A A A
(0) (0) (0) (0) (0)
D (3)
C (2)
a (0)
B (2) E (3)
B (I) D (3)
A (0) A (0)
The letters represent macroscopic estimation of the extent of the lesion in the 5 degree scale (see methods section for details), accompanied (in parentheses) by the number representing the extent of microscopic changes in the same animal.
o f c e l l u l a r a n d e x t r a c e l l u l a r c o n c e n t r i c electron dense deposits of calcium. I t c a n b e seen i n T a b l e 2 t h a t t h e c a l c i u m c o n t e n t o f h e a r t tissue in rats r e c e i v i n g v i t a m i n D 3 a l o n e w a s a b o u t 100 t i m e s h i g h e r than that of the controls. The highest calcium c o n t e n t w a s o b s e r v e d in t h e h e a r t s o f i n d i v i d uals with the most pronounced morphological
a l t e r a t i o n s . A n a p p r o x i m a t e l y t w o fold rise o f c a r d i a c tissue m a g n e s i u m a n d zinc c o n t e n t w a s also o b s e r v e d i n t h e v i t a m i n D r t r e a t e d rats. H o w e v e r n o r e l a t i o n s h i p w a s f o u n d b e t w e e n t h e h e a r t tissue c o n t e n t o f e i t h e r z i n c or m a g n e s i u m a n d t h e e x t e n t o f t h e lesions i n a n i n d i v i d u a l rat. T h e a r e a s o f necrosis i n t h e h e a r t s o f r a t s
T A B L E 2 Calcium, zinc a n d magnesium contents in the h e a r t tissue of rats treated with vitamin D 3 and zinc sulphate Calcium
Zinc
Magnesium
(jumol/g wet tissue) Group 1 (control, n = 5) Group 2 (vitamin D3, n = 8) Group 3 (low dose of zinc, n = 8) Group 4 (low dose of zinc + vitamin D3, n = 8) Group 5 (high dose of zinc, n = 8) Group 6 (high dose of zinc + vitamin Ds, n = 8)
0.68 4- 0.175
0.49 __+0.076
10.33 + 0.833
76.16 ___61.2 b
0.88 _ 0.343 c
23.76 _ 10.33 b
0.95 4- 0.408
0.83 4- 0.245 c
19.63 4- 4.132
21.08 __- 16.2 b
1.37 4- 0.588"
35.12 4- 16.528 a
0.75 4- 0.068
0.69 4- 0.098 b
12.40 4- 1.033
1.09 4- 0.136 a
0.69 4- 0.098 I'
9.30 4- 1.033
The numbers represent the mean content of the metal in the heart +s.D. The differences between a particular group and the control group as calculated with the Student's t test are at the following probability levels: ap < 0.001g bp < 0.01 ; cp < 0.02.
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M.A. Wrzotek
FIGURE 1. Myocardium ofvltamin D 3 treated rat. Focus of coagulative necrosis with mononuclear infiltrate. Some of the necrotic cells are fragmented (white arrows), others have a homogenous cytoplasm (black arrows), a number of them are devoid of nuclei. Hematoxylin and eosin x 190. pre-treated with low doses of zinc were generally smaller than in animals treated with vitamin D 3 alone. Their histopathological apRearance was similar to that after administering vitamin D 3 alone, but granulocytic infiltrates were observed only occasionally. Foci of necrosis and calcification without an inflammatory reaction were more frequent in the hearts of rats pretreated with low doses of zinc. T h e alterations in the heart ultrastructure were not as prominent as in rats receiving only vitamin Ds, although their general appearance was the same. The proliferation of endoplasmic reticulum and polysomes was less pronounced. The lipid spheres and myelin figures as well as deposits of electron dense concentric structures were scarce. I n the hearts of rats which had been pre-treated with low doses of zinc before they received vitamin D a the calcium content was only about 30 times higher than in the controls. As in the case of rats treated with vitamin D3 alone, the calcium content was highest in the individuals with most pro-
nounced morphological changes. The magnesium and zinc content of heart tissue in the low dose zinc pre-treated rats was about three times higher than the control values. T h e hearts of rats receiving high doses of zinc and vitamin D 3 showed a normal macroscopic and histopathological structure. The content of calcium was still higher than the control, but represented only 1.4% of that in vitamin Ds-treated animals. The zinc content was about 40% higher, while the magnesium content did not differ from the control values. Ultrastructural examination of the myocardium of rats pretreated with high doses of zinc revealed generalised mitochondrial swelling (Fig. 4). O t h e r ultrastructural alterations, usually present after vitamin D s administration, did not occur in the hearts of rats pre-treated with high doses of zinc. These mitochondrial alterations were probably due to zinc treatment itself as they were also observed in rats receiving only high doses of zinc. No changes were detected in the rats treated only with low zinc doses.
Vitamin D 3 , Zinc and Cardiac Necrosis
113
F I G U R E 2. Cardiomyocyte of control rat with normal ultrastructural appearance, Lead citrate and uranyl acetate x 35 300.
114
M.A. Wrzolek
F I G U R E 3. Cardiomyocyte of vitamin D 3 treated rat. The contractile apparatus is well preserved but the myofibrils are separated from each other and from the sarcolemma (arrow) by a homogenous substance of low electron density (*). Mitochondria have normal appearance. Lead citrate and uranyl acetate x 17 800.
Vitamin
D 3 , Zinc and Cardiac Necrosis
1!5
F I G U R E 4. Cardiomyocyte of rat treated with high doses of zinc and vitamin D 3. Swollen mitochondria between well preserved myofibrils. Lead citrate and uranyl acetate x 27 900.
116
M . A . Wrzotek
Discussion
A protective effect of parenteral zinc administration in isoproterenol-induced myocardial necrosis in healthy and spontaneously hypertensive rats has been recently reported [4, 17]. The limited extent of morphological alterations was found to be associated with the prevention of calcium overload of cardiac cells [4]. In the present study severe necrotic changes in the rat heart were produced by the administration of high doses of vitamin D 3. The results of the presented experiment demon.strate the protective role of zinc in the development of vitamin D3-induced cardiac necrosis. This is documented both by morphological observation and the values of heart tissue calcium content. The areas of necrosis induced by vitamin D 3 were smaller in the hearts of the rats receiving low doses of zinc and completely absent in animals pretreated with high doses thereof. The prevention of necrotic lesions in hearts by zinc pre-treatment was accompanied by a gradual normalisation of the heart tissue calcium content, which in the hearts of rats treated with vitamin D3 alone exceeded the normal level more than a hundredfold. The calcium content in the hearts of animals pre-treated with high doses of zinc was only slightly increased. After the administration of low doses of zinc and vitamin D 3 the mean calcium content was higher than the control, but still much lower than in the hearts of rats receiving vitamin D 3 alone. These results show an inter-dependence between the heart calcium content and the extent of cardiac necrosis. This is further supported by the observation that the heart calcium content was highest in the individual rats with the most dramatic necrotic changes. These data confirm the assumption about the crucial role of calcium in the pathogenesis of vitamin D r i n d u c e d myocardial lesions. Most probably zinc exerts its beneficial role in vitamin Dyinduced cardiac necrosis by preventing the calcium overload of cardio-
myocytes. However the mechanism of this action is unknown. Zinc has a stabilizing effect on various biomembranes [3] and it was postulated that it is the zinc-induced sarcolemmal stabilization that underlies its protective action on the heart muscle injured by high doses of isoproterenol ['4, 17]. It is possible that the stabilizing effect of zinc on the sarcolemma could be a result of an interaction of this divalent ion with the calcium channel. Nevertheless one cannot exclude the possibility that the action of zinc has quite a different mechanism. Its protective effect in pyrrolizidine alkaloid-induced hepatotoxicity in rats has been connected with a marked increase of metallothionein content in the liver [14]. Metallothioneins are cytoplasmatic metalloproteins of low molecular weight which are known to play an important role in the regulation of metabolism and toxicity of metals [2]. The studies of Chvapil indicate that zinc exerts a stabilizing effect also on the lysosomal membranes [5]. Although nothing is known about the role of these structures in vitamin D3-induced myocardial necrosis, lysosomal activation is supposed to play an important role in other forms of heart injury [21, 22]. One cannot exclude the possibility that the interaction of zinc with lysosomal membranes may be responsible, at least in part, for its protective effect on the vitamin D3-induced heart damage. The protective action of zinc in vitamin D3-induced cardiotoxicity in rats is evident from the observations of animals, as well as morphological and chemical studies. It is not clear why there is no obvious relation between the tissue content of zinc and calcium (the latter being related to the extent of tissue damage). The experiment was performed within the complex system of a complete animal organism and not in an isolated system. Therefore the possibility can not be excluded that the effects observed are the result of zinc action through some intermediate factors.
References
B*JuSZ,E. ConditioningFactorsfor CardiacNecroses.Basel: Karger (1963). CHEmAN, M. G., GOYER,R. A. Metallothioneins and their role in the metabolism and toxicity of metals (Minireview). LifeSei 23, 1-10 (1978).
Vitamin
3 4 5 6
7
8 9 10 11 12
13 14 15 16
17 18
19 20 21 22 23 24
D 3 , Zinc and Cardiac
Necrosis
I 17
CHVAPIL,M. Effect of zinc on cells and biomembranes. Med Clin North Am 60, 799-812 (1976). CrlVAI'IL, M., OwEn, J. A. Effect of zinc on acute and chronic isoproterenol induced heart injury, j Mol Cell Cardiol 9, 151-159 (1977). CaVAPXL,M., RYANJ. N., ZVKOSKIC. F. The effect of zinc and other metals on the stability oflysosomes. Proc Soc Exp Biol Med 140, 642-646 (1972). FEDELE~OV~.,M., ZlEGELH6FFErt, A., LUKNXROVXO., KOSTOLANSK) g. Prevention by K+,Mg2+-aspartate of isoproterenol-induced metabolic changes in the myocardium. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 59-73. Baltimore: University Park Press (1975). FLECKENSTEIN,A.,JA~KE,J., D6RINa, H.J., LEDER, O. The key role of Ca in the production ofnoncoronarogenic myocardial necroses. In Pathophysiology and Morphology of Myocardial Cell Alterations: Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 21-32. Baltimore: University Park Press (1975). FLECK~NSTEIN, A., JANKE, J., D6mNO, H. J., LEDER, O. Myocardial fibroneerosis due to intracellular Ca overload--a new principle in cardiac pathopysiology. In Myocardial Biology : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 4, N. S. Dhalla, Ed., pp. 563-580. Baltimore: University Park Press (1974). GANTER,P.,JoLLES, G. : In Histochimie Normale etPatologique, vol. 2, pp. 927-1027. Paris: Guthier-Villars (1970). GODFRAIND,T., KrIovRI, G., STURBOIS, X. The action of flunarizine and lidoflazine on isoprenaline induced cardiac lesions. Arch Int Pharmacodyn Ther 244, 330-332 (1980). GODFRAIND, T., STURBOIS, X. Inhibition by cinnarizine of heart ionic changes induced by isoprenaline. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 127-134. Baltimore: University Park Press (1975). JANKE,J., FLECKENSTEIN,A., HEIN, B., LEDER, O., 8IGEL,H. Pre'~ention of myocardial Ca overload and necrotization by Mg and K salts or acidosis. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 33-42, Baltimore: University Park Press (1975). KATZ,A., REUTER, H. Cellular calcium and cardiac cell death. AmJ Cardiol '14, 188-190 (1979). MIRANDA,C. L., HENDERSON,M. C., REED, R. L., SCHMITZ,J. A., BUI-ILER,D. R. Protective action of zinc against pyrrolizidine alkaloid-induced hepatotoxicity in rats. J Toxic Environ Health 9, 359-366 (1982). SELYE,H. Morphologische Studie tiber Veriinderungen nach Verfutterung von bestrahlten Ergosterin (Vigantol) bei der weissen Ratte. Krankheitforschung 7, 290 (1929). SmEL, H., JANKE, J., FLECKENSTEIN, A. Restriction of isoproterenoMnduced myocardial Ca uptake and necrotization by a new Ca-antagonistic compound (ethyl-4-(3,4,5-trimethoxycinnamoyl)piperazinyl acetate (vascoril). In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 121-126. Baltimore : University Park Press (1975). SINGAL,P. K., DHILLON,K. S., BEAMISH,R. E., DHALLA,N. S. Protective effect of zinc against catecholamineinduced myocardial changes. Electrocardiographic and ultrastructural studies. Lab Invest 44, 426-433 (1981). SLEZ.~.K,J.,TRIBULOV~.,N. Morphological changes after combined administration ofisoproterenol and K+,Mg 2+aspartate as a physiological Ca 2 + antagonist. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 75-84. Baltimore: University Park Press (1975). TAKENAKA,F. Effects ofisoproterenol and some other drugs on high energy phosphate metabolism in rat myocardium. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 151-158. Baltimore : University Park Press (1975). URm~NEK,E., VA~K~r,J., BEDNA~IK,B., PRASLI~KA,M., POSPI~IL,M. Electrolyte changes in myocardial injury. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A Fleckenstein, G. Rona, Eds, pp. 43-58. Baltimore: University Park Press (1975). W~LMAN,E., COLBECg,J. F., SELW'ZN, A. P., Fox, K. M., ORR, I. Plasma lysosomal enzyme activity in acute myocardial infarction and the effects of drugs. In Advances in Myocardiology, Volume 2, M. Tajuddin, B. Bhatia, H. H. Siddiqui, G. Rona, Eds, pp. 359-369. Baltimore: University Park Press (1980). WILDENTnAL,K., DECKER, R. S. The role of lysosomes in the heart. In Advances in Myocardiology, Volume 2, M. Tajuddin, B. Bhatia, H. H. Siddiqui, G. Rona, Eds, pp. 349-358. Baltimore: University Park Press (1980). WRZOe~OWA,T., ~'zDowo, M. Ultrastructural studies on the vitamin D induced heart lesions in the rat. J Mol Cell Cardiol 12, 1117-1133 (1980). ~YDOWO,M., WALENTYNOWmZ,O., WRZOe~OWA,T. AMP deaminase from the rat heart injured by high doses of vitamin D. Acta Biochim Po127, 233-240 (1980).
T h e Effect o f Z i n c on V i t a m i n D 3 - i n d u c e d Cardiac Necrosis Monika Anna Wrzo~ek
Laboratory of Eleclron Microscopy and Department of Pathomorphology, Academic Medical School, 80-210 Gdahsk, Poland (Received 16August 1983, accepted in revisedform 20 April 1984) M. A. WRZO~EK.The Effect of Zinc on Vitamin Da-induced Cardiac Necrosis. Journal of MolecularCellular Cardiology(1985) 17, 109-117. Multifocal heart muscle necrotic lesions in rats were induced with high oral doses of vitamin D 3 (300,000 iu/rat in three daily doses 100000 iu each). The calcium content increased over 100 fold in the hearts of rats receiving vitamin D 3. Parenteral pre-treatment with zinc sulphate (50 or 200 rag/rat in ten daily doses) resulted either in a reduction or in the total prevention of myocardial lesions on macroscopic, light and electron microscopic examination. The effect of zinc was dose-dependent. The administration of various doses of zinc sulphate resulted in a gradual normalisation of heart calcium content. KEy WORDS: Vitamin D ; Necrosis, Experimental cardiac ; Zinc ; Calcium.
Introduction
8, 20]. V i t a m i n D - i n d u c e d m y o c a r d i a l injury
I s o p r o t e r e n o l - i n d u c e d heart muscle i n j u r y is one of the most widely studied a n d best understood forms of e x p e r i m e n t a l m y o c a r d i a l necrosis. I t is believed t h a t intracellular calcium o v e r l o a d plays a crucial role in the p a t h o g e n esis of this form of cardionecrosis [6, 8, 12, 13, 16, 18-20]. This view is s u p p o r t e d by the beneficial effect of administering calcium channel blockers which protect the m y o c a r d i u m by p r e v e n t i n g isoproterenol-induced inflow of calcium to the h e a r t muscle cells [10, 11, 16]. T h e r e have been recent reports of p r o t e c t i n g m y o c a r d i u m against isoproterenol cardiotoxicity b y p a r e n t e r a l a d m i n i s t r a t i o n of zinc, in b o t h h e a l t h y a n d S H R rats [4, 17]. T h e r e d u c t i o n of necrotic areas in the hearts of z i n c - p r e t r e a t e d animals was found to be associated with a lower h e a r t calcium content, as c o m p a r e d with rats receiving isoproterenol alone [4]. H i g h doses of v i t a m i n D are also k n o w n to p r o d u c e multifocal areas of necrosis in rat hearts [-1, 15, 23, 24]. A l t h o u g h there are some suggestions that a n o t h e r m e c h a n i s m takes p a r t in the pathogenesis of this form o f myoc a r d i a l fiber necrosis [23], the calcium ion is supposed to p l a y the most i m p o r t a n t role [7,
is associated with a d r a m a t i c increase in h e a r t tissue calcium content. R o e n t g e n o g r a p h i c microanalysis reveals calcium deposits both in the c a r d i a c ceils a n d in the interstitium of v i t a m i n D - d a m a g e d hearts [23]. This study investigates the inf/uence o f p a r enteral zinc a d m i n i s t r a t i o n on the f o r m a t i o n of necrotic lesions in the hearts of rats treated with high doses of v i t a m i n Da. Calcium, zinc a n d m a g n e s i u m contents in the h e a r t were measured. T h e relationships between tissue levels of these metals a n d the extent of v i t a m i n D s - i n d u c e d m o r p h o l o g i c a l changes in the m y o c a r d i u m were evaluated.
0022 2828/85/020109 + 09 $03.00/0
Materials and Methods Forty-five virgin female W i s t a r rats, each w e i g h i n g a b o u t 250 g, fed with a s t a n d a r d l a b o r a t o r y diet, were used in the experiment. T h e animals were divided into the following g r o u p s : (1) controls (five rats); (2) treated with v i t a m i n D3 (eight rats); (3) treated with low doses of zinc; 5 m g Z n S O 4 x 7 H 2 0 / 2 4 h (eight rats); (4) treated with low doses of zinc a n d v i t a m i n D3 (eight rats); (5) treated with high doses of zinc; 20 m g Z n S O 4 x 7 H 2 0 / 2 4 I~) 1985 Academic Press Inc. (London) Limited
110
M.A. Wrzdek
h (eight rats); (6) treated with high doses of zinc and vitamin D 3 (eight rats). Zinc treated rats received intraperitoneal injections of ZnSO4 x 7H20 (Polskie Odczynniki Chemiczne, Gliwice) for 10 days consecutively. The animals from groups 4 and 6 were given 100000 iu of vitamin Da/24 h orally during the last 3 days of zinc administration, as described previously by Wrzotkowa and Zydowo [23]. The rats from group 2 received the same dose of vitamin D 3 without any zinc pre-treatment. One day after completion of the vitamin D 3 administration the animals were killed by decapitation, their hearts were examined macroscopically and the extent of vitamin D3-induced myocardial lesion was determined according to the following scale : (A) heart with normal macroscopic appearance; (B) about three or four pin-head size whitish foci observed in the heart; (C) single foci dispersed in the entire heart muscle; (D) multiple but not confluent loci; (E) multiple foci partly confluent but macroscopically unchanged muscle prevailing; (F) confluent foci dominate over unchanged muscle. The cross section from the middle part of the ventricles was taken for histopathological examination. Each section was stained with hematoxylin and eosin and the van Kossa's method for calcium [9]. The extent of vitamin D3-induced myocardial alteration was determined microscopically, according to the following scale : 0 = no histological alterations detected; 1 = single minute foci of necrosis, calcification and small clusters of cellular infiltrate in the entire section; 2 = about 10 separate foci of necrosis, calcification or infiltrate in the section ; 3 = some of the foci coalesce, creating bigger, heterogenous ones; 4 = numerous minute and bigger foci present in each microscopic field at magnification of x 70; 5 = foci of necrosis, calcification and infiltrate dominate, with only minute fragments of apparently unchanged muscle. Samples were taken from macroscopically unchanged parts of the interventricular septum near the apex for examination in the electron microscope. Semi-thin sections of these samples were prepared and areas free of microscopically detectable alterations were chosen for the ultrathin sections. These were
examined in t h e J E M 7 A electron microscope after they had been processed and stained by a standard procedure. The remaining parts of the heart tissue were weighed, mineralised with nitric acid at 105~ and used for analysis of calcium, zinc and magnesium content. The measurements were performed with Beckman atomic absorption spectrophotometer, model 1272. All the data are presented as mean ___standard deviation (S.D.). They were statistically evaluated by the Student's l test. Results
The extent of vitamin D3-induced myocardial lesions both macroscopic and histopathological are presented in Table 1. Table 2 shows calcium, zinc and magnesium contents in the necrotic hearts compared to the values in the control group. Administration of high doses of vitamin D 3 resulted in the development of necrotic lesions of various extent in rat hearts. The morphological appearance of the hearts, both macroscopic and histopathological, as well as the ultrastructural alterations, have been described previously by Wrzo|kowa and Zydowo [23]. In the vitamin D3-treated rats light microscopic examination revealed multiple scattered foci of coagulative necrosis and/or myocytolysis in the entire heart muscle (Figure 1), but predominantly in the subendocardial and subepicardial layer. Some necrotic cells were basophilic in hematoxylineosin staining. The van Kossa's reaction revealed calcium deposits mainly in the necrotic basophilic cells. Necrotic foci were often accompanied by a mononuclear or polymorphonuclear infiltrate. Electron microscopic examination revealed various forms of alteration of cardiomyocytes. Figure 2 showed normal myocytes. Damaged cells were interspersed with well preserved ones. The changes varied from cells with slight separation of myofibrils (Figure 3) to cells filled with various organelles, myelin figures, lipid spheres and calcium deposits. The latter cells could hardly be recognized as myocytes. Common features were the proliferation of rough endoplasmic reticulum and polysomes as well as the appearance of various amounts
Vitamin
111
D 3 , Zinc and Cardiac Necrosis
T A B L E 1. T h e effect of zinc on the vitamin D3-induced lesions of rat myocardium Animals treated with 3 x 100000 iu vitamin D 3 (Group 2) D E C D E
10 x 5 m g Z n S O 4 + 3 x 100000 iu vitamin D3 (Group 4)
(3) (4) (3) (3) (4)
A A C D A
10 x 20 mg Z n S O 4 + 3 x 100000 iu vitamin D 3 (Group 6)
(i) (1) (2) (3) (0)
A A A A A
(0) (0) (0) (0) (0)
D (3)
C (2)
a (0)
B (2) E (3)
B (I) D (3)
A (0) A (0)
The letters represent macroscopic estimation of the extent of the lesion in the 5 degree scale (see methods section for details), accompanied (in parentheses) by the number representing the extent of microscopic changes in the same animal.
o f c e l l u l a r a n d e x t r a c e l l u l a r c o n c e n t r i c electron dense deposits of calcium. I t c a n b e seen i n T a b l e 2 t h a t t h e c a l c i u m c o n t e n t o f h e a r t tissue in rats r e c e i v i n g v i t a m i n D 3 a l o n e w a s a b o u t 100 t i m e s h i g h e r than that of the controls. The highest calcium c o n t e n t w a s o b s e r v e d in t h e h e a r t s o f i n d i v i d uals with the most pronounced morphological
a l t e r a t i o n s . A n a p p r o x i m a t e l y t w o fold rise o f c a r d i a c tissue m a g n e s i u m a n d zinc c o n t e n t w a s also o b s e r v e d i n t h e v i t a m i n D r t r e a t e d rats. H o w e v e r n o r e l a t i o n s h i p w a s f o u n d b e t w e e n t h e h e a r t tissue c o n t e n t o f e i t h e r z i n c or m a g n e s i u m a n d t h e e x t e n t o f t h e lesions i n a n i n d i v i d u a l rat. T h e a r e a s o f necrosis i n t h e h e a r t s o f r a t s
T A B L E 2 Calcium, zinc a n d magnesium contents in the h e a r t tissue of rats treated with vitamin D 3 and zinc sulphate Calcium
Zinc
Magnesium
(jumol/g wet tissue) Group 1 (control, n = 5) Group 2 (vitamin D3, n = 8) Group 3 (low dose of zinc, n = 8) Group 4 (low dose of zinc + vitamin D3, n = 8) Group 5 (high dose of zinc, n = 8) Group 6 (high dose of zinc + vitamin Ds, n = 8)
0.68 4- 0.175
0.49 __+0.076
10.33 + 0.833
76.16 ___61.2 b
0.88 _ 0.343 c
23.76 _ 10.33 b
0.95 4- 0.408
0.83 4- 0.245 c
19.63 4- 4.132
21.08 __- 16.2 b
1.37 4- 0.588"
35.12 4- 16.528 a
0.75 4- 0.068
0.69 4- 0.098 b
12.40 4- 1.033
1.09 4- 0.136 a
0.69 4- 0.098 I'
9.30 4- 1.033
The numbers represent the mean content of the metal in the heart +s.D. The differences between a particular group and the control group as calculated with the Student's t test are at the following probability levels: ap < 0.001g bp < 0.01 ; cp < 0.02.
112
M.A. Wrzotek
FIGURE 1. Myocardium ofvltamin D 3 treated rat. Focus of coagulative necrosis with mononuclear infiltrate. Some of the necrotic cells are fragmented (white arrows), others have a homogenous cytoplasm (black arrows), a number of them are devoid of nuclei. Hematoxylin and eosin x 190. pre-treated with low doses of zinc were generally smaller than in animals treated with vitamin D 3 alone. Their histopathological apRearance was similar to that after administering vitamin D 3 alone, but granulocytic infiltrates were observed only occasionally. Foci of necrosis and calcification without an inflammatory reaction were more frequent in the hearts of rats pretreated with low doses of zinc. T h e alterations in the heart ultrastructure were not as prominent as in rats receiving only vitamin Ds, although their general appearance was the same. The proliferation of endoplasmic reticulum and polysomes was less pronounced. The lipid spheres and myelin figures as well as deposits of electron dense concentric structures were scarce. I n the hearts of rats which had been pre-treated with low doses of zinc before they received vitamin D a the calcium content was only about 30 times higher than in the controls. As in the case of rats treated with vitamin D3 alone, the calcium content was highest in the individuals with most pro-
nounced morphological changes. The magnesium and zinc content of heart tissue in the low dose zinc pre-treated rats was about three times higher than the control values. T h e hearts of rats receiving high doses of zinc and vitamin D 3 showed a normal macroscopic and histopathological structure. The content of calcium was still higher than the control, but represented only 1.4% of that in vitamin Ds-treated animals. The zinc content was about 40% higher, while the magnesium content did not differ from the control values. Ultrastructural examination of the myocardium of rats pretreated with high doses of zinc revealed generalised mitochondrial swelling (Fig. 4). O t h e r ultrastructural alterations, usually present after vitamin D s administration, did not occur in the hearts of rats pre-treated with high doses of zinc. These mitochondrial alterations were probably due to zinc treatment itself as they were also observed in rats receiving only high doses of zinc. No changes were detected in the rats treated only with low zinc doses.
Vitamin D 3 , Zinc and Cardiac Necrosis
113
F I G U R E 2. Cardiomyocyte of control rat with normal ultrastructural appearance, Lead citrate and uranyl acetate x 35 300.
114
M.A. Wrzolek
F I G U R E 3. Cardiomyocyte of vitamin D 3 treated rat. The contractile apparatus is well preserved but the myofibrils are separated from each other and from the sarcolemma (arrow) by a homogenous substance of low electron density (*). Mitochondria have normal appearance. Lead citrate and uranyl acetate x 17 800.
Vitamin
D 3 , Zinc and Cardiac Necrosis
1!5
F I G U R E 4. Cardiomyocyte of rat treated with high doses of zinc and vitamin D 3. Swollen mitochondria between well preserved myofibrils. Lead citrate and uranyl acetate x 27 900.
116
M . A . Wrzotek
Discussion
A protective effect of parenteral zinc administration in isoproterenol-induced myocardial necrosis in healthy and spontaneously hypertensive rats has been recently reported [4, 17]. The limited extent of morphological alterations was found to be associated with the prevention of calcium overload of cardiac cells [4]. In the present study severe necrotic changes in the rat heart were produced by the administration of high doses of vitamin D 3. The results of the presented experiment demon.strate the protective role of zinc in the development of vitamin D3-induced cardiac necrosis. This is documented both by morphological observation and the values of heart tissue calcium content. The areas of necrosis induced by vitamin D 3 were smaller in the hearts of the rats receiving low doses of zinc and completely absent in animals pretreated with high doses thereof. The prevention of necrotic lesions in hearts by zinc pre-treatment was accompanied by a gradual normalisation of the heart tissue calcium content, which in the hearts of rats treated with vitamin D3 alone exceeded the normal level more than a hundredfold. The calcium content in the hearts of animals pre-treated with high doses of zinc was only slightly increased. After the administration of low doses of zinc and vitamin D 3 the mean calcium content was higher than the control, but still much lower than in the hearts of rats receiving vitamin D 3 alone. These results show an inter-dependence between the heart calcium content and the extent of cardiac necrosis. This is further supported by the observation that the heart calcium content was highest in the individual rats with the most dramatic necrotic changes. These data confirm the assumption about the crucial role of calcium in the pathogenesis of vitamin D r i n d u c e d myocardial lesions. Most probably zinc exerts its beneficial role in vitamin Dyinduced cardiac necrosis by preventing the calcium overload of cardio-
myocytes. However the mechanism of this action is unknown. Zinc has a stabilizing effect on various biomembranes [3] and it was postulated that it is the zinc-induced sarcolemmal stabilization that underlies its protective action on the heart muscle injured by high doses of isoproterenol ['4, 17]. It is possible that the stabilizing effect of zinc on the sarcolemma could be a result of an interaction of this divalent ion with the calcium channel. Nevertheless one cannot exclude the possibility that the action of zinc has quite a different mechanism. Its protective effect in pyrrolizidine alkaloid-induced hepatotoxicity in rats has been connected with a marked increase of metallothionein content in the liver [14]. Metallothioneins are cytoplasmatic metalloproteins of low molecular weight which are known to play an important role in the regulation of metabolism and toxicity of metals [2]. The studies of Chvapil indicate that zinc exerts a stabilizing effect also on the lysosomal membranes [5]. Although nothing is known about the role of these structures in vitamin D3-induced myocardial necrosis, lysosomal activation is supposed to play an important role in other forms of heart injury [21, 22]. One cannot exclude the possibility that the interaction of zinc with lysosomal membranes may be responsible, at least in part, for its protective effect on the vitamin D3-induced heart damage. The protective action of zinc in vitamin D3-induced cardiotoxicity in rats is evident from the observations of animals, as well as morphological and chemical studies. It is not clear why there is no obvious relation between the tissue content of zinc and calcium (the latter being related to the extent of tissue damage). The experiment was performed within the complex system of a complete animal organism and not in an isolated system. Therefore the possibility can not be excluded that the effects observed are the result of zinc action through some intermediate factors.
References
B*JuSZ,E. ConditioningFactorsfor CardiacNecroses.Basel: Karger (1963). CHEmAN, M. G., GOYER,R. A. Metallothioneins and their role in the metabolism and toxicity of metals (Minireview). LifeSei 23, 1-10 (1978).
Vitamin
3 4 5 6
7
8 9 10 11 12
13 14 15 16
17 18
19 20 21 22 23 24
D 3 , Zinc and Cardiac
Necrosis
I 17
CHVAPIL,M. Effect of zinc on cells and biomembranes. Med Clin North Am 60, 799-812 (1976). CrlVAI'IL, M., OwEn, J. A. Effect of zinc on acute and chronic isoproterenol induced heart injury, j Mol Cell Cardiol 9, 151-159 (1977). CaVAPXL,M., RYANJ. N., ZVKOSKIC. F. The effect of zinc and other metals on the stability oflysosomes. Proc Soc Exp Biol Med 140, 642-646 (1972). FEDELE~OV~.,M., ZlEGELH6FFErt, A., LUKNXROVXO., KOSTOLANSK) g. Prevention by K+,Mg2+-aspartate of isoproterenol-induced metabolic changes in the myocardium. In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 59-73. Baltimore: University Park Press (1975). FLECKENSTEIN,A.,JA~KE,J., D6RINa, H.J., LEDER, O. The key role of Ca in the production ofnoncoronarogenic myocardial necroses. In Pathophysiology and Morphology of Myocardial Cell Alterations: Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 21-32. Baltimore: University Park Press (1975). FLECK~NSTEIN, A., JANKE, J., D6mNO, H. J., LEDER, O. Myocardial fibroneerosis due to intracellular Ca overload--a new principle in cardiac pathopysiology. In Myocardial Biology : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 4, N. S. Dhalla, Ed., pp. 563-580. Baltimore: University Park Press (1974). GANTER,P.,JoLLES, G. : In Histochimie Normale etPatologique, vol. 2, pp. 927-1027. Paris: Guthier-Villars (1970). GODFRAIND,T., KrIovRI, G., STURBOIS, X. The action of flunarizine and lidoflazine on isoprenaline induced cardiac lesions. Arch Int Pharmacodyn Ther 244, 330-332 (1980). GODFRAIND, T., STURBOIS, X. Inhibition by cinnarizine of heart ionic changes induced by isoprenaline. 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Morphologische Studie tiber Veriinderungen nach Verfutterung von bestrahlten Ergosterin (Vigantol) bei der weissen Ratte. Krankheitforschung 7, 290 (1929). SmEL, H., JANKE, J., FLECKENSTEIN, A. Restriction of isoproterenoMnduced myocardial Ca uptake and necrotization by a new Ca-antagonistic compound (ethyl-4-(3,4,5-trimethoxycinnamoyl)piperazinyl acetate (vascoril). In Pathophysiology and Morphology of Myocardial Cell Alterations : Recent Advances in Studies on Cardiac Structure and Metabolism, Volume 6, A. Fleckenstein, G. Rona, Eds, pp. 121-126. Baltimore : University Park Press (1975). SINGAL,P. K., DHILLON,K. S., BEAMISH,R. E., DHALLA,N. S. Protective effect of zinc against catecholamineinduced myocardial changes. Electrocardiographic and ultrastructural studies. Lab Invest 44, 426-433 (1981). SLEZ.~.K,J.,TRIBULOV~.,N. Morphological changes after combined administration ofisoproterenol and K+,Mg 2+aspartate as a physiological Ca 2 + antagonist. 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Plasma lysosomal enzyme activity in acute myocardial infarction and the effects of drugs. In Advances in Myocardiology, Volume 2, M. Tajuddin, B. Bhatia, H. H. Siddiqui, G. Rona, Eds, pp. 359-369. Baltimore: University Park Press (1980). WILDENTnAL,K., DECKER, R. S. The role of lysosomes in the heart. In Advances in Myocardiology, Volume 2, M. Tajuddin, B. Bhatia, H. H. Siddiqui, G. Rona, Eds, pp. 349-358. Baltimore: University Park Press (1980). WRZOe~OWA,T., ~'zDowo, M. Ultrastructural studies on the vitamin D induced heart lesions in the rat. J Mol Cell Cardiol 12, 1117-1133 (1980). ~YDOWO,M., WALENTYNOWmZ,O., WRZOe~OWA,T. AMP deaminase from the rat heart injured by high doses of vitamin D. Acta Biochim Po127, 233-240 (1980).