Summary. The effect of administration of carnitine on the severity of myocardial infarction in rats induced by isoproterenol was studied by following ...
Aust. J. Exp. Biol. Med. Sci., 64 (Pt. 1) 79-87 (1986)
©EFFECT OF ADMINISTRATION OF CARNITINE ON THE SEVERITY OF MYOCARDIAL INFARCTION INDUCED BY ISOPROTERENOL IN RATS by SALEENA MATHEW, P. V. G. MENON AND P. A. KURUP (From the Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum 695581, Kerala, India.) (Accepted for publication August 19, 1985.) Summary. The effect of administration of carnitine on the severity of myocardial infarction in rats induced by isoproterenol was studied by following histopathological and biochemical parameters. Carnitine afforded partial protection against myocardial infarction. Serum aspartate amino transferase (GOT) and creative phospho kinase (CPK) values, serum, heart and aortic lipids, serum protein-bound hexose and sialic acid and glyco-saminoglycans in the heart were lower in the carnitine-treated rats. Histopathological examination showed very little necrosis in the carnitine-treated rats when compared to the extensive necrosis in the untreated controls.
INTRODUCTION Many substances have been studied for their possible protective action against the induction of myocardial infarction by isoproterenol and these include hypocholesterolemic agents (clofibrate) (Wexler and Greenberg, 1978), IJ-adrenergic blocking agents (propranolol) (Reimer, 1976), antiarrhythmic agents (allopurinol) (Wexler and McMurtry, 1981), permeability-promoting agents (hyaluronidase) (Maroko et al., 1975) and cortico-steroids (Fox, Hoffstein and Weissmann, 1976). Any substance which can ward off an attack of myocardial infarction or accelerate the recovery phase from an attack is bound to have considerable clinical application. Isoproterenol, a B-agonist, stimulates the B-receptors in the heart resulting in an increased rate and force of cardiac contraction and enhanced cadiac output. It also acts on the B-receptors in the vascular bed, the activation of which causes vasodilatation, thus lowering the mean arterial blood pressure. Acute coronary insufficiency results when the balance between oxygen need and blood supply to the myocardium is disturbed. The immediate result is hypoxemia of the myocardium. Apart from deficiency of oxygen, the heart also suffers from deficiency of carnitine during infarction (Whitmer et al., 1978). The role of carnitine is of special significance in cardiac metabolism since the heart preferentially oxidises fatty acids for its energy production. Carnitine is required to transport the fatty acids for Abbreviations used in this paper: CPK, Creatine phospho kinase; GOT, Aspartate amino transferase; GAG, Glycosaminoglycans; ANT, Adenine nucleotide translocase.
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SALEENA MATHEW, P. V. G. MENON AND P. A. KURUP
their oxidation into the mitochondria. It was therefore considered desirable to find out whether the heart can oxidise fatty acids and produce energy more efficiently with the limited oxygen available if carnitine is made available. The effect of carnitine in isoproterenol-induced myocardial infarction was therefore studied in rats. Rats were pretreated with carnitine before administration of isoproterenol. Its administration was also continued during the phase of recovery. The parameters studied included the various factors known to be affected in myocardial infarction, viz., changes in serum enzyme profile, serum and tissue lipids (Saleena, Menon and Kurup, 1981), glycosaminoglycans in the heart (Saleena, Menon and Kurup, 1982b), serum glycoproteins (Saleena, Menon and Kurup, 1982a) and histopathology of the myocardium (Wexler and McMurty, 1981). The results are discussed in this paper. MATERIALS AND METHODS Female virgin albino rats (Sprague-Dawley strain, body weight 150-170 g) were used for these experiments. The rats were grouped as follows: 1. Control group 2. Carnitine-treated group. Carnitine in physiological saline was administered daily by the intramuscular route at a dose of 10 mg/100 g body weight per day. The control group received intramuscular injection of physiological saline. At the end of 10 days the animals were treated as follows: 1. Normal control group. 2. Normal untreated rats given isoproterenol. 3. Carnitine-treated rats given isoproterenol. 4. Normal rats treated with carnitine. Isoproterenol was given at a dose of 40 mg/100 g body weight (= 10" 'M) in two injections 24 h apart as previously described (Saleena et al., 1981, 1982). Rats of group 3 and 4 continued to receive carnitine as mentioned above. Rats of group 3 were given isoproterenol 3 h after administration of carnitine. The isoproterenol-treated rats of group 2 exhibited signs of shock, tachycardia, dyspnea, rapid respiration, anuria and prostration. Rats of group 3 showed these symptoms to a significantly lower extent. The surviving animals of group 3 continued to receive carnitine till 7 Vi days following the first injection of isoproterenol. Eight rats in each group were killed after overnight starvation at the end of \Vz, AVi and IVi days (except the control rats of group 1 and 4 which were killed after \Vi days). The tissues were removed to ice-cold containers for various estimations. Serum CPK and GOT, cholesterol and triglycerides in the serum, heart and aorta and protein-bound hexose and siaiic acid in the serum were estimated as previously described (Saleena et al., 1982). For the determination of total glycosaminoglycans (Scott, 1960), the papain digest of the acetone dry powder of the tissue was treated with sodium hydroxide to a concentration of 0-5 N. After keeping overnight, it was neutralized with hydrochloric acid, deproteinised with trichloroacetic acid and the supernatant after centrifugation dialysed against water till free of trichloroacetic acid. Potassium acetate was added to a final concentration of 1% followed by 4-5 volumes of ethanol. After keeping overnighi at 0°, the precipitate was collected by centrifugation and dissolved in 0-01 N NaOH. Total glycosaminoglycans were quantitated by estimating uronic acid by the modified carbazole method of Bitter and Muir (1962). For histopathological examination, the heart was fixed in ]O*Vo buffered formalin and sections of the entire heart were stained with haemotoxylin and eosin. Statistical analysis was carried out using Student's't' test (Bennet and Franklin, 1967).
RESULTS Survival rates of animals in different groups Four experiments using 20 rats in each group in each experiment were carried out to determine the survival rate. The survival rate (in %) was 90 ± 4-0
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CARNITINE AND MYOCARDIAL INFARCTION
in tbe case of rats receiving carnitine and given isoproterenol, wbile it was 70 ±5-0 in tbe case of rats treated witb isoproterenol alone. Tbus, tbe mortality rate was significantly lower in rats treated witb carnitine + isoproterenol wben compared to tbose treated witb isoproterenol alone. Serum GOT and CPK values Tbe results are given in Table 1. Rats treated witb isoproterenol sbowed a significant increase in serum GOT and CPK values at tbe peak period (1 Vi days after tbe 1st injection of isoproterenol) wben compared witb normal rats. Tbese values were still bigber at AVi and IVi days except in tbe case of CPK at IVi days. Rats pretreated witb carnitine and given isoproterenol sbowed lower levels of botb enzymes in tbe serum at tbe peak period of infarction wben compared to animals receiving isoproterenol alone. However, after AV2 and IV2 days tbere were no significant differences between tbe carnitine-treated group and isoproterenol controls. Compared to normal rats, tbe values were still bigber in tbe carnitine group, except in tbe case of CPK at IVi days wbicb was lower. Carnitine alone did not significantly affect tbe level of tbese serum enzymes. TABLE 1 Activity of serum GOT and CPK at the period of peak infarction and stages of recovery
Group 1. Normal control 2. Normal + Isoproterenol 3. Carnitine + Isoproterenol 4. Carnitine control
After first injection (days) l'/2 4'/2 7'/2 l'/2 4'/2 IVi —
GOT (n moles of pvruvate/min/litre at
il")
144-8 ±3-16 275-2 ±6-81* 212-4 ±5-20* 180-6 ±4-30* 248-7 ±6-38*TJ 208-1 ±5-8O*t 170-8 ±3-90* 139-3 ±7-98
CPK (n moles of Creatine/min/litre at 37°) 256-8 ± 5-45 628-8 ± 17-60* 347-5 ± 8-20* 216-4 ± 4-8* 560-8 ± 12-90*tt 316-8 ± 9-20*t 190-0 ± 4-8*t 246-3 ± 11-2*
Group 2 to 4 have been compared with group 1. Group 2 has been compared with group 3. Group 4 has been compared with group 3. * p < 0-01. t p between 0-01 and 0-05. t p < 0-01. (Values given are the mean from eight rats in each group ± SEM.)
Concentration of cholesterol and triglycerides in the serum heart and aorta Results are given in Table 2. Rats treated witb isoproterenol sbowed bigber serum cbolesterol and triglycerides at tbe peak period wben compared to normal rats. Triglycerides were also bigber at AVi and 7'/2 days, wbile cbolesterol was lower. Rats treated witb carnitine and isoproterenol sbowed lower serum cbolesterol only at tbe peak period wben compared to tbose treated witb isoproterenol alone. Tbere was no significant difference in serum triglycerides at tbe peak period or during recovery. Compared to normal rats, serum
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cholesterol was higher in the carnitine group only at the peak period, but serum triglycerides were higher during the period of recovery also. Carnitine alone did not significantly affect the concentration of serum cholesterol and triglyceride. Cholesterol and triglycerides in the heart were significantly higher in the isoproterenol group at the peak period when compared with normal rats. At 4'/2 and IV2 days cholesterol was not significantly different, but triglycerides were lower. Rats treated with carnitine and given isoproterenol showed lower triglycerides but not cholesterol at the peak period when compared to those treated with isoproterenol alone, while there was no significant alteration in cholesterol and triglycerides at AV2 and IV2 days. Compared to normal rats, there was no significant alteration of cholesterol in the heart in the carnitinetreated group at any time, while triglycerides showed a decrease. Aortic cholesterol and triglyceride were significantly higher in the isoproterenol group at the peak period and at AVi and IVi days when compared to isoproterenoltreated control rats. Carnitine-treated rats showed significantly lower levels of cholesterol and triglycerides at the period of peak infarction and at AVi and 7 Vi days when compared to isoproterenol-treated control rats. Compared to normal rats, aortic cholesterol was not significantly different at any time, while aortic triglycerides were higher at AVi andlVi days. Carnitine alone did not affect heart cholesterol but decreased aortic cholesterol. Cardiac triglycerides were not significantly affected, but aortic triglycerides showed a decrease in the rats treated with carnitine alone. Concentration of protein-bound hexose and sialic acid in the serum and total glycosaminoglycans (GAG) in the heart The results are given in Table 3. Rats treated with isoproterenol showed higher values for protein-bound hexose and sialic acid in the serum at the peak period when compared to normal rats. The values were not different at AVi and IVi days. Rats treated with carnitine + isoproterenol showed significantly lower values of protein-bound hexose and sialic acid in the serum at the peak period when compared to those treated with isoproterenol alone. However, at AVi and 7 V2 days there was no significant alteration. Compared to normal rats, the carnitine group showed no significant alteration in the serum protein-bound hexose and sialic acid at any time, except in the case of sialic acid at 7 Vi days. Rats treated with carnitine alone did not significantly alter these serum values. Rats of the isoproterenol group showed significantly higher values for total GAG in the heart at the period of peak infarction and during recovery when compared to normal rats. Lower concentration of total GAG in the heart was observed at the peak period in the rats treated with carnitine when compared to isoproterenol-treated controls, but there was no significant alteration at AVi and IV2 days. When compared to normal rats, the values were higher in the carnitine-treated group. Rats treated with carnitine alone did not significantly affect the total GAG in the heart.
84
SALEENA MATHEW, P. V. G. MENON AND P. A. KURUP
TABLE 3 Changes in serum glycoproteins and total glycosaminoglycans In the heart at different stages of infarction. Serum Group I. Normal control 2. Normal + Isoproierenol 3. Carnitine + Isoproterenol 4. Carnitine control
After first injection (days)
Heart
Protein-bound hexose (
Sialic acid
mg/100 ml
)
136-±5-50
20-2 ±0-52
IVi
188-4 +8-62* 150-6 ±5-15 146-8 ± 3-56 134-8 ±7-201 150-6 ±6-80 154-3 ±5-02t
28-92 ±0-84* 21-60 ±0-94 21-9 ±1-24 19-8 ±0-52t 20-6 ±0-74 25-2 ±0-82*t
—
130-9 ±9-45
19-77 ±0-87
\Vi
AVi IVt V/i
4'/2
Total GAG mg/lOOg tissue
86-4 ±3-9 230-4 189-8 170-6 U8-3 200-6 182-3
±7-28* ±4-66* ±7-28* +5-58*tt ±5-26*§ ±3-9*J
76-42 ± 4-43
Group 2 to 4 have been compared with group 1. Group 3 has been compared with group 2. Group 3 has been compared with 3. * p < 0-01. t p < 0-01. X p < 0-01. (Values given are the mean from 8 rats in each group ± SEM.)
Histopathological changes Histopathologicai changes were studied using the heart tissue from the experiments carried out on survival rats. Five hearts in each group were examined and sections of the entire heart were examined. Rats treated with isoproterenol alone showed extensive confluent necrosis of the myocardium at 1 Vi days. Interfascicular spaces of oedema were observed with degeneration of the muscle fibres resulting from sarcolysis and nuclear fragmentation. Confluent lesions were observed more in the apex and papillary muscles with focal lesions in the other areas of the ventricle (Plate IB). Rats treated with carnitine + isoproterenol exhibited very little evidence of myocardial necrosis on histopathological examination (Plate lC). The sections were found to be almost normal with intact muscle fibres. Small subendocardial focal areas of degeneration were noticed which were almost insignificant when compared to isoproterenol-treated controls (Plate ID). Rats treated with carnitine alone did not show any change on histopathological examination when compared to untreated normal heart. DISCUSSION The effect of carnitine on the induction of myocardial infarction in rats by isoproterenol was studied by following histopathological changes and certain biochemical parameters which are altered in myocardial infarction. Activity of serum CPK and GOT, serum, heart and aortic cholesterol and triglycerides, concentration of protein-bound hexose and sialic acid in the serum and concentration of total GAG in the heart are all increased on induction of myocardial infarction by isoproterenol. The results now obtained indicate that
CARNITINE AND MYOCARDIAL INFARCTION
85
Fig 1 Section of heart from the apical region stained with hematoxylin-eosin—Magn. x 450. lA. Normal. IB. Normal + Isoproterenol. lC. Carnitine + Isoprotereno!. ID. Carnitine + Isoproterenol (showing a small sub-endo cardial focal area of degeneration).
administration of carnitine decreases the value of these biochemical parameters in most cases to a varying extent, indicating that carnitine affords partial protective action against the induction of myocardial infarction by isoproterenol in rats. The lower values for serum CPK and GOT in rats pretreated with carnitine indicate that the extent of necrosis produced in the heart is lower. This is strongly supported by the higher survival-rate in the carnitine-treated rats and by the histopathological changes observed. Rats treated with carnitine exhibited very little evidence of necrosis in the myocardium in contrast to the extensive necrotic changes observed in the isoproterenol controls. The changes in the serum cholesterol, protein-bound hexose and sialic acid at the period of peak infarction were also minimal in rats treated with carnitine. Similar changes were noticed with respect to the concentration of cholesterol and triglyceride in the heart and the concentration of total GAG in the heart. All these clearly indicate at least partial protective action of carnitine against induction of myocardial infarction by isoproterenol. Carnitine itself did not affect these biochemical parameters significantly, except in the case of cholesterol and triglyceride in the aorta. However, the various parameters studied in the serum and heart were not significantly different at AVi and IVi days (during the early recovery phase) in the carnitine-treated rats when compared to the untreated isoproterenol control rats, except in the case of the aorta. One reason probably is that, in
86
SALEENA MATHEW, P. V. G. MENON AND P. A. KURUP
many cases, these values came down at 4/2 and IVi days in the isoproterenoltreated rats when compared to the values at the peak period. Lower concentration of cholesterol and triglycerides was observed in the aorta during the early stages of recovery in the carnitine-treated rats. The action of carnitine may probably be due to its role in the transport of fatty acids into the mitochondria for their oxidation. This is evident from the fact that the concentration of triglycerides in the heart in rats treated with carnitine + isoproterenol is significantly lower than that in the rats treated with isoproterenol alone. The role of carnitine is of special significance in cardiac metabolism, since the heart preferentially oxidises fatty acids for its energy production. Depletion of myocardial stores of carnitine has been reported in ischemic heart (Whitmer et al., 1978) and it has been suggested that this deficiency of carnitine may limit mitochrondrial oxidation of long chain fatty acids in the myocardium. Administration of carnitine may provide sufficient carnitine to the heart for the transport of fatty acids to the mitochrondria for their oxidation. In this connection, triglyceride lipase activity has been found to increase in the heart in rats administered isoproterenol (Saleena et al, 1981). Carnitine administration may help to transport the fatty acids liberated by the action of this enzyme and promote their mitochondrial oxidation. It has also been suggested that carnitine may be involved in the regulation of adenine nucleotide translocase (ANT), which is involved in the transport of ADP across the inner mitochondrial membrane in the exchange of ATP. Carnitine reverses the inhibition of ANT activity by long chain acyl CoA esters, and this may play a role in the control of mitochondrial respiration and creatine phosphate formation.
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