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Lipolysis Is an Important Determinant of Isoproterenol-Induced Myocardial Necrosis
Lipolysis Is an Important Determinant of Isoproterenol-Induced Myocardial Necrosis Pamarthi Mohan, PhD and Sherman Bloom, MD Department of Pathology, The University of Mississippi Medical Center, Jackson, Mississippi
11 The cardiotoxic effect of isoproterenol (ISO) is associated with, and possibly due to, calcium overload. Prior work suggests that calcium entry into cardiac myocytes after ISO administration occurs in two phases: an early rapid phase, followed by a slow phase beginning about 1 hour after ISO injection, leading to a peak myocardial calcium level after about 4 hours. We have tested the relationship of these phases to myocardial necrosis (MN) by determining the time after ISO administration at which the commitment to MN occurs. This was done by administration of propranolol at various times before and after ISO. In addition, since ISO induces lipolysis, and lipids can be toxic, experiments were conducted to determine if adrenergically-activated lipolysis could play a significant role in ISO-MN. We found that propranolol protected the myocardium equally well when administered anytime within 2 hours of ISO injection, but had no effect when given 4 hours after ISO. This showed that metabolic events taking place more than two hours after ISO injection are required for ISO-MN. As expected from prior work, there was a small and consistent amount of propranolol-resistant ISO-MN. Lipolysis, assessed by measuring serum glycerol levels, increased to tenfold above base line at one hour after ISO administration and returned to near basal levels at 4 hours. Potentiation of lipolysis by intravenous injections of phospholipase A2 (PLA2) or lipoprotein lipase (LPL) to rats treated with ISO substantially augmented MN. Propranolol completely blocked the increase in necrosis produced by PLA2 when given with ISO. Lipases induced only minimal necrosis in the absence of ISO. Administration of adenosine (an anti-lipolytic agent), oxfenicine (an inhibitor of mitochondrial palmitoyl carnitine transferase), or vitamin C (an anti-oxidant) resulted in a 55–60% reduction in MN. These results suggest that critical necrosis-determining events occur between 2 and 4 hours after ISO administration and imply a relationship between ISO-induced lipolysis, calcium influx, and ISO-MN. We hypothesize that importance of lipolysis as a determinant of ISO-MN is related to the generation of free fatty acids, their oxidized/metabolic products, or direct damage to plasma membrane. Cardiovasc Pathol 1999;8:255–261 © 1999 by Elsevier Science Inc.
ISO-MN has been recognized for at least four decades, but the mechanism by which it occurs remains uncertain. This is because ISO produces a number of biochemical or electrophysiological alterations which precede the histopathologic changes in the heart (1,2). It is believed that necrosis is related to altered myocardial energy generation, which may be related to calcium overload (3–5). Calcium influx after
Manuscript received March 10, 1999; revised May 24, 1999; accepted June 3, 1999. Address for reprints: Dr. S. Bloom, Department of Pathology, The University of Mississippi Medical Center, Jackson, MS 39206. Tel: 601-9841540; fax: 601-984-1531;
ISO (10 mg/kg body weight) in rats shows two phases: a rapid process occurring immediately after ISO administration and a later slow influx where intracellular calcium reaches maximum level by 4 hours. Since the initial phase of calcium influx is associated with histopathologic alterations of the myocardium (mitochondrial swelling and Z-disk thickening associated with hypercontraction), it was believed to be the key event in ISO-MN. Although morphological changes are observed within minutes of ISO injection, the time required for ISO to produce cell death is unknown. Furthermore, inhibition of calcium influx reduced—but did not completely block—ISO-MN, suggesting that factors other than calcium may also be involved.
Cardiovascular Pathology Vol. 8, No. 5, September/October 1999:255–261 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Among the proposed mechanisms for ISO-MN are myocardial hypoperfusion (6), glycogen depletion (7,8), electrolyte imbalance (9,10), lipid accumulation (11), and free radical damage (12–14). We have proposed that lipids mobilized by ISO may contribute to ISO-MN based on the following considerations: (i) ISO is a potent stimulator of lipolysis (15,16); (ii) calcium is necessary for lipolysis (17); (iii) lipids can be toxic substances (18–21); (iv) obese rats are much more vulnerable than lean counterparts (22,23); (v) ISO-induced lipolysis is much greater in obese than in lean rats (Mohan & Bloom, unpublished data); (vi) lipid droplets accumulate in the myocardium of ISO-injected rats (11). It is well known that during lipolysis most of the mobilized body fat is oxidatively metabolized by myocardium and skeletal muscle. However, with extensive lipid hydrolysis, free fatty acids formed can accumulate to toxic levels and impair metabolism (19,20). Toxicity of free fatty acids can be due to a direct effect on plasma membranes or through actions of their oxidized derivatives on cellular contents (18–21). Thus, it is reasonable to assume that ISOinduced lipid mobilization may play a role in ISO-MN. The aim of this study is twofold: to determine the time at which commitment to ISO-MN occurs, and to test the hypothesis that lipolysis plays an important role in the pathogenesis of ISO-MN.
Materials and Methods Animals were purchased from Harlan Sprague-Dawley Inc. and housed at the Laboratory Animal Facility of The University of Mississippi Medical Center. They were fed Purina lab chow and maintained at 218C with 12-hr light/ dark cycles. Isoproterenol, propranolol, phospholipase A2, lipoprotein lipase, and other chemicals were purchased from Sigma Chemical Company.
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expressed as the percentage of apical myocardium affected by the inflammatory reaction associated with ISO-MN.
Determination of Plasma Glycerol as a Measure of Lipolysis Male SD rats 7–8 weeks old (225–250 g body weight) were injected with ISO (10 mg/kg body weight), and at 0, 15, 30, 60, 120, or 240, minutes they were anesthetized with halothane and heart blood collected in heparinized tubes. Plasma was separated by centrifugation and stored frozen at 2708C until used. Glycerol was determined in deproteinized plasma by a coupled enzyme assay (25). For deproteinization, 200 ml plasma was mixed with an equal volume of water and protein precipitated with 30 ml of 60% perchloric acid. The resulting supernatant was neutralized with approximately 30 ml of 7.5 M KOH. It was then centrifuged, and the resulting supernatant was used for glycerol determination.
Infusion of Adenosine, Oxfenicine, and Vitamin C Adenosine is an anti-lipolytic agent (26) and oxfenicine is an inhibitor of carnitine palmitoyltransferase and myocardial fatty acid uptake (27). Vitamin C is an anti-oxidant and protects against free radicals in aqueous, as well as in hydrophobic, environment by regenerating vitamin E, another anti-oxidant (28). Male SD rats 7–8 weeks old (225–250 g body weight) were divided into four groups. Saline (0.25 ml), or saline containing either adenosine (7 mg/kg body weight), oxfenicine (100 mg/kg body weight), or vitamin C (40 mg/kg body weight) was injected into the dorsal penile vein under halothane anesthesia. Immediately thereafter, each rat was injected ip with ISO (10 mg/kg body weight). Forty-eight hours later, ISO-MN was determined as described above.
Time to ISO-MN To determine the time at which commitment to ISO-MN occurs, we measured the time between ISO administration and the loss of the propranolol protective effect. Male SD rats 7–8 weeks old (225–250 g body weight) were divided into groups and ISO (10 mg/kg body weight) was injected ip. Propranolol (20 mg/kg body weight) was administered either not at all, by ip injection 1 hour before ISO, together with ISO, or between 0.12 and 24 hours after ISO. The control group received saline instead of propranolol. Fortyeight hours after ISO administration, rats were anesthetized with halothane and their coronary arteries perfused with buffered formaldehyde. Sections of myocardium were then processed for histopathology. The apical myocardium was embedded in paraffin and five micron-thick H&E stained sections were prepared. ISO-MN was quantified by the point counting method of Zavier et al. (24). To avoid bias, slide identification was concealed before scoring. Data is
Infusion of Lipases The aim of this experiment was to test the effect of elevated levels of free fatty acids on ISO-MN. Since it was impractical to infuse large amounts of FFAs into rats, we resorted to the radical procedure of lipase infusion. PLA2 and LPL suspensions (250 units/ml) were freshly prepared in sterile saline and passed through 0.2 micron filters. Male SD rats 7–8 week old (225–250 grams body weight) were divided into six groups and treated with either ISO alone, 50 units PLA2 alone, 50 units PLA2 1 ISO, 50 units LPL alone, 50 units LPL 1 ISO, or 50 units PLA2 1 50 units LPL 1 ISO. Lipases were injected into the dorsal penile vein under halothane anesthesia. ISO (10 mg/kg body weight) was injected ip immediately after lipase injections. Forty-eight hours later, ISO-MN was determined as describe above.
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Statistics Data was analyzed by Students’ t test (29).
Results The effect of propranolol administered at various time intervals after ISO injection on MN is shown in Figure 1. This experiment was designed to determine the time at which ISO-MN occurred. This was achieved by blocking the adrenergic action of ISO at different time intervals with propranolol, a beta blocker. The results show that propranolol administered 1 hour before, or at 0.0, 0.12, 0.5, 1.0, and 2.0 hours after, ISO injection resulted in 64–75% reduction in MN compared to the control group. However, propranolol injected at 4, 8, 12, and 24 hours after ISO resulted in ISO-MN that was no different than that of the control group. This experiment shows that, subsequent to ISO injection, the myocardium is not severely affected for at least 2 hours. However between 2 and 4 hours after ISO injection, critical events occur that lead to established ISO-MN.
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The fact that propranolol did not prevent all of the ISO-MN is consistent with earlier studies (3) and implies that a component of ISO-MN can be characterized as propranolol-sensitive while another component is propranolol-resistant. The time course of ISO-induced lipolysis, as determined by measuring the plasma glycerol level, is shown in Figure 2. Plasma glycerol levels increased from baseline values (animals injected with saline) to a peak level by 1 hour, and declined thereafter, returning close to baseline levels after 4 hours. This shows that lipids were mobilized from adipose tissue to a maximum extent at about 1 hour after ISO, and that glycerol was cleared from the blood within 4 hours. This experiment also shows the overlap of lipolysis with ISO-MN induction. In other words, the disappearance of lipids from the blood and their uptake (myocardial) by 4 hours occurs within the time frame of the development of ISO-MN, suggesting that lipid accumulation by myocytes could be a critical component of ISO-MN. Since the above experiments suggested that lipid mobilization (ISO-induced lipolysis) occurred prior to irreversibility of propranolol-sensitive ISO-MN, it is possible that such
Figure 1. Effect of propranolol on ISO-MN in rats. Each bar represents a mean 6 standard error of mean (n 5 4–6 rats). Propranolol (10 mg/kg body weight) was administered at the times indicated after ISO injection (10 mg/kg body weight). The time point 21 represents the group of rats given propranolol one hour prior to ISO injection. (Asterisks indicate statistical significance of the group mean value at p , 0.05 compared to the control.)
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Figure 2. Serum glycerol levels as a function of time after ISO (10 mg/kg body weight) injection. Each point represents a mean 6 standard error of mean (n 5 3 rats). The zero time point represents saline injected rats.
lipolysis plays a role in development of propranolol-sensitive necrosis. This possibility was tested by determining the effects of inhibitors of lipid mobilization, fatty acid utilization, and peroxidation on ISO-MN. The inhibitors tested were, respectively, adenosine, oxfenicine, and vitamin C. The results of these experiments are shown in Figure 3. These agents reduced the amount of ISO-MN from the mean control level of 13.6% to 6.1%, 6.2%, and 5.0% respectively (p , 0.05 for each). The effect of lipase infusion is shown in Figure 4. Administration of ISO alone (control group) resulted in 10.4% MN, and PLA2 infusion alone caused 3.7% necrosis, but rats infused with PLA2 and injected with ISO showed 27.5% necrosis. LPL infusion generated 4.9% necrosis and, in combination with ISO, produced 15% necrosis, but rats injected with ISO and a mixture of PLA2 1 LPL (50 units each) showed 34.2% necrosis.
Discussion In this study we show for the first time that propranolol administered at two hours after ISO exerts a level of protection similar to propranolol administered together with ISO. It appears that ISO-MN development occurs in two forms: propranolol-resistant, and propranolol-sensitive. It is not clear at this time what biochemical factors are associated
with propranolol-resistant ISO-MN. Although the early phase of calcium overload (3) has been considered a primary factor in the initiation of events leading to ISO-MN, it is inconclusive, since propranolol administration significantly reduced ISO-MN but was not able to completely eliminate it. Earlier studies by Bloom & Davis (3) showed that high doses of propranolol (40 mg/kg body weight) blocked calcium overload by 100% but could not prevent ISO-MN completely, suggesting that factors other than calcium influx contribute to ISO-MN. Since lipids have the potential to exert toxic effects on the myocardium, and lipolysis due to the adrenergic action of ISO is sensitive to propranolol, this was considered a potential factor in ISO-MN. The heart derives a significant portion of its fatty acid substrates as free fatty acids derived by lipolysis from adipose tissue. Although lipid availability is important for the heart, excess levels of fatty acids in myocytes can be deleterious (18,30). Figure 2 implies that lipids mobilized from the adipose depot reach their highest level in blood one hour after ISO administration and are cleared by 4 hours. This means that although myocardial lipid uptake may begin early, accumulation to peak levels occurs anywhere between two to four hours after ISO injection. The fact that significant protection was afforded by vitamin C, together with earlier studies which showed that myocardial lipid peroxidation increased after ISO injection (31,32), and vitamin
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Figure 3. Effect of saline, adenosine, oxfenicine, and vitamin C on ISO-MN. Each bar represents a mean 6 standard error of mean (n 5 6–8 rats). Dosages: ISO 5 10 mg/kg body weight; adenosine 5 7 mg/kg body weight; oxfenicine 5 100 mg/kg body weight; Vitamin C 5 40 mg/kg body weight. All infusions except ISO were via dorsal penile vein under halothane anesthesia. (* indicates statistical significance of the group mean value at p , 0.05 compared to the control.)
E pretreatment protected the myocardium against ISO induced injury (5,14) suggests that lipid accumulation and peroxidation in the myocardium may be the key events that determine ISO-MN. Further support for this conclusion comes from reports showing that the heart has less anti-oxidant protection than the liver, lung, or kidneys (33,34), and therefore may provide conditions conducive to lipid peroxidation. It is possible that lipid peroxides and the spontaneous oxidation products of ISO (35) by their action on the sarcolemma cause leakiness and contribute to a second phase of calcium accumulation (3,5). This presumption is further supported by studies in cultured cardiomyocytes in which inhibition of fatty acid accumulation by phospholipase inhibitors protected the cells from calcium overload and morphological damage (36). Furthermore, the protective effect of propranolol, apart from blocking calcium influx, can also be due to its anti-oxidant activity (37,38) or other effects such as inhibition of myocardial fatty acid uptake, increased glucose uptake, reduced mechanical function of the heart (39,40), and altered myocardial utilization of fuel from fatty acids to carbohydrates (41). One of the aims of this study was to determine if increased blood lipid levels affect ISO-MN. This was achieved by infusion of PLA2 and LPL into rats. PLA2 acts on phos-
pholipids, and LPL on triglycerides of lipoproteins, with the release of free fatty acids. These fatty acids combine with those mobilized from adipose tissue by the adrenergic action of ISO to further increase total blood lipids. Lipase injections along with ISO did lead to increased ISO-MN, particularly in rats injected with a mixture of PLA2 and LPL, suggesting that increased plasma lipid levels and a higher metabolic activity of the heart due to adrenergic action of ISO may be optimal conditions for maximal MN. Since it is plausible that lipases can also cause injury by attacking the myocyte cell membranes, lipases were injected into rats in the absence of ISO. Lipases induced minimal MN. Whether this damage was due to elevated lipids or lipase action on myocyte cell membrane is not clear at this time. Studies by Steigen et al. (42) showed that exogenous lipases attacked energy-depleted cardiomyocytes in culture and had no effect on normal cells. Therefore, we conclude that both ISO and the blood lipids make the myocytes vulnerable to the actions of lipases. In conclusion, the results of this study show that critical biochemical events leading to induction ISO-MN do not occur until 2 hours after ISO injection, and this timeframe suggests a critical role for lipolysis. By increasing serum-free fatty acids with lipases, or inhibiting lipolysis with adeno-
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Figure 4. Effect of ISO, phospholipase A2 (PLA2), lipoprotein lipase (LPL) on ISO-MN. Each bar represents a mean 6 standard error of mean (n 5 6–8 rats). Dosages: ISO 5 10 mg/kg body weight; PLA2 5 50 units/rat; LPL 5 50 units/rat. Lipases infused into rats via dorsal penile vein under halothane anesthesia. (Asterisk indicates statistical significance of the group mean value at p , 0.05 compared to the control.)
sine, or inhibiting fatty acid oxidation and uptake by oxfenicine, or preventing lipid peroxidation by vitamin C, we show here that lipids play a major role in ISO-MN. In these respects, the model of ISO-MN appears to be similar to myocardial injury induced by ischemia/reperfusion (43).
References 1. Balaz T, Bloom S. Cardiotoxicity of adrenergic bronchodilator and vasodilating antihypertensive drugs. In: Van Stee, ed. Cardiovascular Toxicology. New York: Raven Press, 1982;199–220. 2. Rockhold RW. Cardiotoxicity of naturally occurring catecholamines. In: Baskin SI, ed. Principles of Cardiac Toxicology. Boca Raton, FL: CRC Press, 1991;347–417. 3. Bloom S, Davis DL. Calcium as mediator of isoproterenol-induced myocardial necrosis. Am J Pathol 1972;69:459–470. 4. Bloom S, Cancilla P. Myocytolysis and mitochondrial calcification after low doses of isoproterenol. Am J Pathol 1969;54:373–391. 5. Dhalla KS, Rupp H, Beamish RE, Dhalla NS. Mechanisms of alterations in cardiac membrane calcium transport due to excess catecholamines. Cardiovascular Drugs and Therapy 1996;10:231–238. 6. Rona G. Catecholamine Cardiotoxicity. J Mol Cell Cardiol 1985; 17:291–306. 7. Mraz M, Kren V, Krsiakova D, Vrana A, Hynie S. The role of myocardial glycogen content for the development of isoprenaline-induced myocardial lesions in different inbred strains of rats. Basic Res Cardiol 1995;90:467–474.
8. Scheuer, J, Stezoski WAS. Protective role of increased myocardial glycogen stores in cardiac anoxia in the rat. Circ Res 1970;27:835– 849. 9. Brembilla-Perrot B, Chaise TDL, Le VD, Beurrier D. Effect of isoproterenol on serum and potassium and magnesium. Eur Heart J 1993;14:677–681. 10. Singal PK, Dhillon KS, Beamish RE, Dhalla NS. Protective effect of zinc against catecholamine-induced myocardial changes. Lab Invest 1981;44:426–433. 11. Ferrans VJ, Hibbs RG, Walsh JJ, Burch GE. Histochemical and electron microscopical studies on the cardiac necrosis produced by sympathomimetic agents. Ann NY Acad Sci 1969;156:309–332. 12. Joseph X, Balaz T. Protection against isoproterenol-induced myocardial necrosis in rat by 6-mercaptopurine and -thioguanine or by irradiation. Res Commun Chem Pathol Pharmacol 1989;63:385–394. 13. Persoon-Rothert M, Van Der Valk-Kokshoorn EJ, Egas-Kenniphaas JM, Mauve I, Van Der Laarse A. Isoproterenol-induced cytotoxicity in neonatal rat heart cell cultures is mediated by free radical formation. J Mol Cell Cardiol 1989;21:1285–1291. 14. Singal PK, Dhillon KS, Beamish RE, Dhalla NS. Role of free radical in catecholamine-induced cardiomyopathy. Can J Pharmacol 1981;60:1390–1397. 15. Garcia-Sainz JA, Fain JN. Regulation of adipose tissue metabolism by catecholamines: roles of alpha-1, alpha-2 and beta-adrenoceptors. Trends Pharmacol Sci 1982;3:201–203. 16. Palmer WK, Kane TA. Hormone-stimulated lipolysis in cardiac myocytes. Biochem J 1983;216:241–243. 17. Schwartz DD, Williams JL, Malik KU. Contribution of calcium to isoproterenol stimulated lipolysis in the isolated perfused rabbit heart. Am J Physiol 1993;28:E439–E445.
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18. Brownsey RW, Brundt RV. The effect of adrenalin-induced endogenous lipolysis upon the mechanical and metabolic performance of ischemically perfused rat hearts. Clin Sci Mol Med 1977;53:513–521. 19. Buja ML. Lipid abnormalities in myocardial cell injury. Trends Cardiovasc Med 1991;1:40–45. 20. Dhalla NS, Elimban V, Rupp H. Paradoxical role of lipid metabolism in heart function and dysfunction. Mol Cell Biochem 1992;116:3–9. 21. Shug, AL, Shrago E. A proposed mechanism for fatty acid effects on energy metabolism of the heart. J Lab Clin Med 1973;81:214–218. 22. Balaz T, Sahashrabudhe MR, Grice HC. The influence of excess body fat on the cardiotoxicity of isoproterenol in rats. Toxicol Appl Pharmacol 1962;4:613–620. 23. Mohan PF and Bloom S. Food restriction protects and obesity increases isoproterenol induced myocardial necrosis. Cardiovasc Pathol 1997;6:287–291. 24. Zavier J, Bloom S, Pledger G, Balazs T. Determinants of resistance to the cardiotoxicity of isoproterenol in rats. Toxicol Appl Pharmacol 1983;69:199–205. 25. Eggstein M, Kuhlmann E. Triglycerides and glycerol determination after alkaline hydrolysis. In: Bergmeyer HU, ed. Methods in Enzymatic Analysis, Vol. 4. New York: Academic Press. 1974;1825–1835. 26. Carey GB, Sidmore KA. Exercise attenuates the anti-lipolytic effects of adenosine in adipocytes isolated from miniature swine. Int J Obesity & Related Metabol Disorder 1994;18:2155–2160. 27. Schwartz GG, Greyson C, Wisneski JA, Garcia J. Inhibition of fatty acid metabolism alters myocardial high-energy phosphates in vivo. Am J Physiol 1994;36:H224–H231. 28. Beyer RE. The role of ascorbate in antioxidant protection of biomembranes: Interaction with vitamin E and Coenzyme Q.J. Bioenergetics and Biomembranes 1994;26:349–358. 29. Glantz SA. The special case of two groups: The t-test. In: Primer of Biostatistics. New York: McGraw-Hill, Inc., 1992;67–109. 30. Vik-Mo H, Mjos OD. Influence of free fatty acids on myocardial oxygen consumption and ischemic injury. Am J Cardiol 1981;48:361–365. 31. Higuchi Y. Changes of lipid peroxides and a-tocopherol in rats with experimental induced myocardial necrosis. Acta Med Okayama 1982;36:113–124.
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32. Namikawa K, Okazaki Y, Nishida S, Kadota E, Tomura T, Hashimoto S. Studies on early stage changes of peroxide lipid in isoproterenolinduced myocardial injury. Yakugaku Zasshi 1992;112:557–562. 33. Cao G, Giovanoni M, Prior RL. Anti-oxidant capacity of different tissues of young and old rats. PSEBM 1996;211:359–365. 34. Krame JH, Mak IT, Weglicki WB. Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. Circ Res 1984;55:120–124. 35. Yates JC, Beamish RE, Dhalla NS. Ventricular dysfunction and necrosis produced by adrenochrome metabolite of epinephrine: Relation to pathogenesis of catecholamine cardiomyopathy. Am Heart J 1981; 102:210–221. 36. Jones RL, Miller JC, Hagler HK, Chien KR, Willerson JT, Buja LM. Association between inhibition of arachidonic acid release and prevention of calcium loading during ATP depletion in cultured rat cardiac myocytes. Am J Pathol 1989;135:541–546. 37. Freedman, AM, Kramer JH, Mak T, Cassidy MM, Weglicki WB. Propranolol preserves ultrastructure in adult cardiocytes exposed to anoxia/reoxygenation: A morphometric analysis. Free Radical Biol Med 1991;11:197–206. 38. Jenkins RR, Singore DCM, Sauer P, Skelly C. The effect of beta blocking drugs on lipid peroxidation in rat heart in vitro. Lipids 1992;27:539–542. 39. Marchetti G, Merlo L, Noseda V. Myocardial uptake of free fatty acids and carbohydrates after beta adrenergic blockade. Am J Cardiol 1968;22:370–374. 40. Opie LH, Thomas M. Propranolol and experimental myocardial infarction: Substrate effects. Postgraduate Medical Journal 1976;(Suppl 4):124–133. 41. Mueller HS, Ayres SM, Religa A, Evans RG. Propranolol in the treatment of acute myocardial infarction: Effect on myocardial oxygenation and hemodynamics. Circulation 1974;49:1078–1087. 42. Steigen TK, Myrmel T, Forsdahl K, Larsen TS. Myocardial cell vulnerability to exogenous phospholipase attack. Mol Cell Biochem 1992;116:47–52. 43. Katz AM, Messineo FC. Lipid-Membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 1981;48:1–16.
11 The cardiotoxic effect of isoproterenol (ISO) is associated with, and possibly due to, calcium overload. Prior work suggests that calcium entry into cardiac myocytes after ISO administration occurs in two phases: an early rapid phase, followed by a slow phase beginning about 1 hour after ISO injection, leading to a peak myocardial calcium level after about 4 hours. We have tested the relationship of these phases to myocardial necrosis (MN) by determining the time after ISO administration at which the commitment to MN occurs. This was done by administration of propranolol at various times before and after ISO. In addition, since ISO induces lipolysis, and lipids can be toxic, experiments were conducted to determine if adrenergically-activated lipolysis could play a significant role in ISO-MN. We found that propranolol protected the myocardium equally well when administered anytime within 2 hours of ISO injection, but had no effect when given 4 hours after ISO. This showed that metabolic events taking place more than two hours after ISO injection are required for ISO-MN. As expected from prior work, there was a small and consistent amount of propranolol-resistant ISO-MN. Lipolysis, assessed by measuring serum glycerol levels, increased to tenfold above base line at one hour after ISO administration and returned to near basal levels at 4 hours. Potentiation of lipolysis by intravenous injections of phospholipase A2 (PLA2) or lipoprotein lipase (LPL) to rats treated with ISO substantially augmented MN. Propranolol completely blocked the increase in necrosis produced by PLA2 when given with ISO. Lipases induced only minimal necrosis in the absence of ISO. Administration of adenosine (an anti-lipolytic agent), oxfenicine (an inhibitor of mitochondrial palmitoyl carnitine transferase), or vitamin C (an anti-oxidant) resulted in a 55–60% reduction in MN. These results suggest that critical necrosis-determining events occur between 2 and 4 hours after ISO administration and imply a relationship between ISO-induced lipolysis, calcium influx, and ISO-MN. We hypothesize that importance of lipolysis as a determinant of ISO-MN is related to the generation of free fatty acids, their oxidized/metabolic products, or direct damage to plasma membrane. Cardiovasc Pathol 1999;8:255–261 © 1999 by Elsevier Science Inc.
ISO-MN has been recognized for at least four decades, but the mechanism by which it occurs remains uncertain. This is because ISO produces a number of biochemical or electrophysiological alterations which precede the histopathologic changes in the heart (1,2). It is believed that necrosis is related to altered myocardial energy generation, which may be related to calcium overload (3–5). Calcium influx after
Manuscript received March 10, 1999; revised May 24, 1999; accepted June 3, 1999. Address for reprints: Dr. S. Bloom, Department of Pathology, The University of Mississippi Medical Center, Jackson, MS 39206. Tel: 601-9841540; fax: 601-984-1531;
ISO (10 mg/kg body weight) in rats shows two phases: a rapid process occurring immediately after ISO administration and a later slow influx where intracellular calcium reaches maximum level by 4 hours. Since the initial phase of calcium influx is associated with histopathologic alterations of the myocardium (mitochondrial swelling and Z-disk thickening associated with hypercontraction), it was believed to be the key event in ISO-MN. Although morphological changes are observed within minutes of ISO injection, the time required for ISO to produce cell death is unknown. Furthermore, inhibition of calcium influx reduced—but did not completely block—ISO-MN, suggesting that factors other than calcium may also be involved.
Cardiovascular Pathology Vol. 8, No. 5, September/October 1999:255–261 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1054-8807/99/$–see front matter PII S1054-8807(99)00017-4
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Among the proposed mechanisms for ISO-MN are myocardial hypoperfusion (6), glycogen depletion (7,8), electrolyte imbalance (9,10), lipid accumulation (11), and free radical damage (12–14). We have proposed that lipids mobilized by ISO may contribute to ISO-MN based on the following considerations: (i) ISO is a potent stimulator of lipolysis (15,16); (ii) calcium is necessary for lipolysis (17); (iii) lipids can be toxic substances (18–21); (iv) obese rats are much more vulnerable than lean counterparts (22,23); (v) ISO-induced lipolysis is much greater in obese than in lean rats (Mohan & Bloom, unpublished data); (vi) lipid droplets accumulate in the myocardium of ISO-injected rats (11). It is well known that during lipolysis most of the mobilized body fat is oxidatively metabolized by myocardium and skeletal muscle. However, with extensive lipid hydrolysis, free fatty acids formed can accumulate to toxic levels and impair metabolism (19,20). Toxicity of free fatty acids can be due to a direct effect on plasma membranes or through actions of their oxidized derivatives on cellular contents (18–21). Thus, it is reasonable to assume that ISOinduced lipid mobilization may play a role in ISO-MN. The aim of this study is twofold: to determine the time at which commitment to ISO-MN occurs, and to test the hypothesis that lipolysis plays an important role in the pathogenesis of ISO-MN.
Materials and Methods Animals were purchased from Harlan Sprague-Dawley Inc. and housed at the Laboratory Animal Facility of The University of Mississippi Medical Center. They were fed Purina lab chow and maintained at 218C with 12-hr light/ dark cycles. Isoproterenol, propranolol, phospholipase A2, lipoprotein lipase, and other chemicals were purchased from Sigma Chemical Company.
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expressed as the percentage of apical myocardium affected by the inflammatory reaction associated with ISO-MN.
Determination of Plasma Glycerol as a Measure of Lipolysis Male SD rats 7–8 weeks old (225–250 g body weight) were injected with ISO (10 mg/kg body weight), and at 0, 15, 30, 60, 120, or 240, minutes they were anesthetized with halothane and heart blood collected in heparinized tubes. Plasma was separated by centrifugation and stored frozen at 2708C until used. Glycerol was determined in deproteinized plasma by a coupled enzyme assay (25). For deproteinization, 200 ml plasma was mixed with an equal volume of water and protein precipitated with 30 ml of 60% perchloric acid. The resulting supernatant was neutralized with approximately 30 ml of 7.5 M KOH. It was then centrifuged, and the resulting supernatant was used for glycerol determination.
Infusion of Adenosine, Oxfenicine, and Vitamin C Adenosine is an anti-lipolytic agent (26) and oxfenicine is an inhibitor of carnitine palmitoyltransferase and myocardial fatty acid uptake (27). Vitamin C is an anti-oxidant and protects against free radicals in aqueous, as well as in hydrophobic, environment by regenerating vitamin E, another anti-oxidant (28). Male SD rats 7–8 weeks old (225–250 g body weight) were divided into four groups. Saline (0.25 ml), or saline containing either adenosine (7 mg/kg body weight), oxfenicine (100 mg/kg body weight), or vitamin C (40 mg/kg body weight) was injected into the dorsal penile vein under halothane anesthesia. Immediately thereafter, each rat was injected ip with ISO (10 mg/kg body weight). Forty-eight hours later, ISO-MN was determined as described above.
Time to ISO-MN To determine the time at which commitment to ISO-MN occurs, we measured the time between ISO administration and the loss of the propranolol protective effect. Male SD rats 7–8 weeks old (225–250 g body weight) were divided into groups and ISO (10 mg/kg body weight) was injected ip. Propranolol (20 mg/kg body weight) was administered either not at all, by ip injection 1 hour before ISO, together with ISO, or between 0.12 and 24 hours after ISO. The control group received saline instead of propranolol. Fortyeight hours after ISO administration, rats were anesthetized with halothane and their coronary arteries perfused with buffered formaldehyde. Sections of myocardium were then processed for histopathology. The apical myocardium was embedded in paraffin and five micron-thick H&E stained sections were prepared. ISO-MN was quantified by the point counting method of Zavier et al. (24). To avoid bias, slide identification was concealed before scoring. Data is
Infusion of Lipases The aim of this experiment was to test the effect of elevated levels of free fatty acids on ISO-MN. Since it was impractical to infuse large amounts of FFAs into rats, we resorted to the radical procedure of lipase infusion. PLA2 and LPL suspensions (250 units/ml) were freshly prepared in sterile saline and passed through 0.2 micron filters. Male SD rats 7–8 week old (225–250 grams body weight) were divided into six groups and treated with either ISO alone, 50 units PLA2 alone, 50 units PLA2 1 ISO, 50 units LPL alone, 50 units LPL 1 ISO, or 50 units PLA2 1 50 units LPL 1 ISO. Lipases were injected into the dorsal penile vein under halothane anesthesia. ISO (10 mg/kg body weight) was injected ip immediately after lipase injections. Forty-eight hours later, ISO-MN was determined as describe above.
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Statistics Data was analyzed by Students’ t test (29).
Results The effect of propranolol administered at various time intervals after ISO injection on MN is shown in Figure 1. This experiment was designed to determine the time at which ISO-MN occurred. This was achieved by blocking the adrenergic action of ISO at different time intervals with propranolol, a beta blocker. The results show that propranolol administered 1 hour before, or at 0.0, 0.12, 0.5, 1.0, and 2.0 hours after, ISO injection resulted in 64–75% reduction in MN compared to the control group. However, propranolol injected at 4, 8, 12, and 24 hours after ISO resulted in ISO-MN that was no different than that of the control group. This experiment shows that, subsequent to ISO injection, the myocardium is not severely affected for at least 2 hours. However between 2 and 4 hours after ISO injection, critical events occur that lead to established ISO-MN.
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The fact that propranolol did not prevent all of the ISO-MN is consistent with earlier studies (3) and implies that a component of ISO-MN can be characterized as propranolol-sensitive while another component is propranolol-resistant. The time course of ISO-induced lipolysis, as determined by measuring the plasma glycerol level, is shown in Figure 2. Plasma glycerol levels increased from baseline values (animals injected with saline) to a peak level by 1 hour, and declined thereafter, returning close to baseline levels after 4 hours. This shows that lipids were mobilized from adipose tissue to a maximum extent at about 1 hour after ISO, and that glycerol was cleared from the blood within 4 hours. This experiment also shows the overlap of lipolysis with ISO-MN induction. In other words, the disappearance of lipids from the blood and their uptake (myocardial) by 4 hours occurs within the time frame of the development of ISO-MN, suggesting that lipid accumulation by myocytes could be a critical component of ISO-MN. Since the above experiments suggested that lipid mobilization (ISO-induced lipolysis) occurred prior to irreversibility of propranolol-sensitive ISO-MN, it is possible that such
Figure 1. Effect of propranolol on ISO-MN in rats. Each bar represents a mean 6 standard error of mean (n 5 4–6 rats). Propranolol (10 mg/kg body weight) was administered at the times indicated after ISO injection (10 mg/kg body weight). The time point 21 represents the group of rats given propranolol one hour prior to ISO injection. (Asterisks indicate statistical significance of the group mean value at p , 0.05 compared to the control.)
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Figure 2. Serum glycerol levels as a function of time after ISO (10 mg/kg body weight) injection. Each point represents a mean 6 standard error of mean (n 5 3 rats). The zero time point represents saline injected rats.
lipolysis plays a role in development of propranolol-sensitive necrosis. This possibility was tested by determining the effects of inhibitors of lipid mobilization, fatty acid utilization, and peroxidation on ISO-MN. The inhibitors tested were, respectively, adenosine, oxfenicine, and vitamin C. The results of these experiments are shown in Figure 3. These agents reduced the amount of ISO-MN from the mean control level of 13.6% to 6.1%, 6.2%, and 5.0% respectively (p , 0.05 for each). The effect of lipase infusion is shown in Figure 4. Administration of ISO alone (control group) resulted in 10.4% MN, and PLA2 infusion alone caused 3.7% necrosis, but rats infused with PLA2 and injected with ISO showed 27.5% necrosis. LPL infusion generated 4.9% necrosis and, in combination with ISO, produced 15% necrosis, but rats injected with ISO and a mixture of PLA2 1 LPL (50 units each) showed 34.2% necrosis.
Discussion In this study we show for the first time that propranolol administered at two hours after ISO exerts a level of protection similar to propranolol administered together with ISO. It appears that ISO-MN development occurs in two forms: propranolol-resistant, and propranolol-sensitive. It is not clear at this time what biochemical factors are associated
with propranolol-resistant ISO-MN. Although the early phase of calcium overload (3) has been considered a primary factor in the initiation of events leading to ISO-MN, it is inconclusive, since propranolol administration significantly reduced ISO-MN but was not able to completely eliminate it. Earlier studies by Bloom & Davis (3) showed that high doses of propranolol (40 mg/kg body weight) blocked calcium overload by 100% but could not prevent ISO-MN completely, suggesting that factors other than calcium influx contribute to ISO-MN. Since lipids have the potential to exert toxic effects on the myocardium, and lipolysis due to the adrenergic action of ISO is sensitive to propranolol, this was considered a potential factor in ISO-MN. The heart derives a significant portion of its fatty acid substrates as free fatty acids derived by lipolysis from adipose tissue. Although lipid availability is important for the heart, excess levels of fatty acids in myocytes can be deleterious (18,30). Figure 2 implies that lipids mobilized from the adipose depot reach their highest level in blood one hour after ISO administration and are cleared by 4 hours. This means that although myocardial lipid uptake may begin early, accumulation to peak levels occurs anywhere between two to four hours after ISO injection. The fact that significant protection was afforded by vitamin C, together with earlier studies which showed that myocardial lipid peroxidation increased after ISO injection (31,32), and vitamin
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Figure 3. Effect of saline, adenosine, oxfenicine, and vitamin C on ISO-MN. Each bar represents a mean 6 standard error of mean (n 5 6–8 rats). Dosages: ISO 5 10 mg/kg body weight; adenosine 5 7 mg/kg body weight; oxfenicine 5 100 mg/kg body weight; Vitamin C 5 40 mg/kg body weight. All infusions except ISO were via dorsal penile vein under halothane anesthesia. (* indicates statistical significance of the group mean value at p , 0.05 compared to the control.)
E pretreatment protected the myocardium against ISO induced injury (5,14) suggests that lipid accumulation and peroxidation in the myocardium may be the key events that determine ISO-MN. Further support for this conclusion comes from reports showing that the heart has less anti-oxidant protection than the liver, lung, or kidneys (33,34), and therefore may provide conditions conducive to lipid peroxidation. It is possible that lipid peroxides and the spontaneous oxidation products of ISO (35) by their action on the sarcolemma cause leakiness and contribute to a second phase of calcium accumulation (3,5). This presumption is further supported by studies in cultured cardiomyocytes in which inhibition of fatty acid accumulation by phospholipase inhibitors protected the cells from calcium overload and morphological damage (36). Furthermore, the protective effect of propranolol, apart from blocking calcium influx, can also be due to its anti-oxidant activity (37,38) or other effects such as inhibition of myocardial fatty acid uptake, increased glucose uptake, reduced mechanical function of the heart (39,40), and altered myocardial utilization of fuel from fatty acids to carbohydrates (41). One of the aims of this study was to determine if increased blood lipid levels affect ISO-MN. This was achieved by infusion of PLA2 and LPL into rats. PLA2 acts on phos-
pholipids, and LPL on triglycerides of lipoproteins, with the release of free fatty acids. These fatty acids combine with those mobilized from adipose tissue by the adrenergic action of ISO to further increase total blood lipids. Lipase injections along with ISO did lead to increased ISO-MN, particularly in rats injected with a mixture of PLA2 and LPL, suggesting that increased plasma lipid levels and a higher metabolic activity of the heart due to adrenergic action of ISO may be optimal conditions for maximal MN. Since it is plausible that lipases can also cause injury by attacking the myocyte cell membranes, lipases were injected into rats in the absence of ISO. Lipases induced minimal MN. Whether this damage was due to elevated lipids or lipase action on myocyte cell membrane is not clear at this time. Studies by Steigen et al. (42) showed that exogenous lipases attacked energy-depleted cardiomyocytes in culture and had no effect on normal cells. Therefore, we conclude that both ISO and the blood lipids make the myocytes vulnerable to the actions of lipases. In conclusion, the results of this study show that critical biochemical events leading to induction ISO-MN do not occur until 2 hours after ISO injection, and this timeframe suggests a critical role for lipolysis. By increasing serum-free fatty acids with lipases, or inhibiting lipolysis with adeno-
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Figure 4. Effect of ISO, phospholipase A2 (PLA2), lipoprotein lipase (LPL) on ISO-MN. Each bar represents a mean 6 standard error of mean (n 5 6–8 rats). Dosages: ISO 5 10 mg/kg body weight; PLA2 5 50 units/rat; LPL 5 50 units/rat. Lipases infused into rats via dorsal penile vein under halothane anesthesia. (Asterisk indicates statistical significance of the group mean value at p , 0.05 compared to the control.)
sine, or inhibiting fatty acid oxidation and uptake by oxfenicine, or preventing lipid peroxidation by vitamin C, we show here that lipids play a major role in ISO-MN. In these respects, the model of ISO-MN appears to be similar to myocardial injury induced by ischemia/reperfusion (43).
References 1. Balaz T, Bloom S. Cardiotoxicity of adrenergic bronchodilator and vasodilating antihypertensive drugs. In: Van Stee, ed. Cardiovascular Toxicology. New York: Raven Press, 1982;199–220. 2. Rockhold RW. Cardiotoxicity of naturally occurring catecholamines. In: Baskin SI, ed. Principles of Cardiac Toxicology. Boca Raton, FL: CRC Press, 1991;347–417. 3. Bloom S, Davis DL. Calcium as mediator of isoproterenol-induced myocardial necrosis. Am J Pathol 1972;69:459–470. 4. Bloom S, Cancilla P. Myocytolysis and mitochondrial calcification after low doses of isoproterenol. Am J Pathol 1969;54:373–391. 5. Dhalla KS, Rupp H, Beamish RE, Dhalla NS. Mechanisms of alterations in cardiac membrane calcium transport due to excess catecholamines. Cardiovascular Drugs and Therapy 1996;10:231–238. 6. Rona G. Catecholamine Cardiotoxicity. J Mol Cell Cardiol 1985; 17:291–306. 7. Mraz M, Kren V, Krsiakova D, Vrana A, Hynie S. The role of myocardial glycogen content for the development of isoprenaline-induced myocardial lesions in different inbred strains of rats. Basic Res Cardiol 1995;90:467–474.
8. Scheuer, J, Stezoski WAS. Protective role of increased myocardial glycogen stores in cardiac anoxia in the rat. Circ Res 1970;27:835– 849. 9. Brembilla-Perrot B, Chaise TDL, Le VD, Beurrier D. Effect of isoproterenol on serum and potassium and magnesium. Eur Heart J 1993;14:677–681. 10. Singal PK, Dhillon KS, Beamish RE, Dhalla NS. Protective effect of zinc against catecholamine-induced myocardial changes. Lab Invest 1981;44:426–433. 11. Ferrans VJ, Hibbs RG, Walsh JJ, Burch GE. Histochemical and electron microscopical studies on the cardiac necrosis produced by sympathomimetic agents. Ann NY Acad Sci 1969;156:309–332. 12. Joseph X, Balaz T. Protection against isoproterenol-induced myocardial necrosis in rat by 6-mercaptopurine and -thioguanine or by irradiation. Res Commun Chem Pathol Pharmacol 1989;63:385–394. 13. Persoon-Rothert M, Van Der Valk-Kokshoorn EJ, Egas-Kenniphaas JM, Mauve I, Van Der Laarse A. Isoproterenol-induced cytotoxicity in neonatal rat heart cell cultures is mediated by free radical formation. J Mol Cell Cardiol 1989;21:1285–1291. 14. Singal PK, Dhillon KS, Beamish RE, Dhalla NS. Role of free radical in catecholamine-induced cardiomyopathy. Can J Pharmacol 1981;60:1390–1397. 15. Garcia-Sainz JA, Fain JN. Regulation of adipose tissue metabolism by catecholamines: roles of alpha-1, alpha-2 and beta-adrenoceptors. Trends Pharmacol Sci 1982;3:201–203. 16. Palmer WK, Kane TA. Hormone-stimulated lipolysis in cardiac myocytes. Biochem J 1983;216:241–243. 17. Schwartz DD, Williams JL, Malik KU. Contribution of calcium to isoproterenol stimulated lipolysis in the isolated perfused rabbit heart. Am J Physiol 1993;28:E439–E445.
Cardiovasc Pathol Vol. 8, No. 5 September/October 1999:255–261
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18. Brownsey RW, Brundt RV. The effect of adrenalin-induced endogenous lipolysis upon the mechanical and metabolic performance of ischemically perfused rat hearts. Clin Sci Mol Med 1977;53:513–521. 19. Buja ML. Lipid abnormalities in myocardial cell injury. Trends Cardiovasc Med 1991;1:40–45. 20. Dhalla NS, Elimban V, Rupp H. Paradoxical role of lipid metabolism in heart function and dysfunction. Mol Cell Biochem 1992;116:3–9. 21. Shug, AL, Shrago E. A proposed mechanism for fatty acid effects on energy metabolism of the heart. J Lab Clin Med 1973;81:214–218. 22. Balaz T, Sahashrabudhe MR, Grice HC. The influence of excess body fat on the cardiotoxicity of isoproterenol in rats. Toxicol Appl Pharmacol 1962;4:613–620. 23. Mohan PF and Bloom S. Food restriction protects and obesity increases isoproterenol induced myocardial necrosis. Cardiovasc Pathol 1997;6:287–291. 24. Zavier J, Bloom S, Pledger G, Balazs T. Determinants of resistance to the cardiotoxicity of isoproterenol in rats. Toxicol Appl Pharmacol 1983;69:199–205. 25. Eggstein M, Kuhlmann E. Triglycerides and glycerol determination after alkaline hydrolysis. In: Bergmeyer HU, ed. Methods in Enzymatic Analysis, Vol. 4. New York: Academic Press. 1974;1825–1835. 26. Carey GB, Sidmore KA. Exercise attenuates the anti-lipolytic effects of adenosine in adipocytes isolated from miniature swine. Int J Obesity & Related Metabol Disorder 1994;18:2155–2160. 27. Schwartz GG, Greyson C, Wisneski JA, Garcia J. Inhibition of fatty acid metabolism alters myocardial high-energy phosphates in vivo. Am J Physiol 1994;36:H224–H231. 28. Beyer RE. The role of ascorbate in antioxidant protection of biomembranes: Interaction with vitamin E and Coenzyme Q.J. Bioenergetics and Biomembranes 1994;26:349–358. 29. Glantz SA. The special case of two groups: The t-test. In: Primer of Biostatistics. New York: McGraw-Hill, Inc., 1992;67–109. 30. Vik-Mo H, Mjos OD. Influence of free fatty acids on myocardial oxygen consumption and ischemic injury. Am J Cardiol 1981;48:361–365. 31. Higuchi Y. Changes of lipid peroxides and a-tocopherol in rats with experimental induced myocardial necrosis. Acta Med Okayama 1982;36:113–124.
261
32. Namikawa K, Okazaki Y, Nishida S, Kadota E, Tomura T, Hashimoto S. Studies on early stage changes of peroxide lipid in isoproterenolinduced myocardial injury. Yakugaku Zasshi 1992;112:557–562. 33. Cao G, Giovanoni M, Prior RL. Anti-oxidant capacity of different tissues of young and old rats. PSEBM 1996;211:359–365. 34. Krame JH, Mak IT, Weglicki WB. Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. Circ Res 1984;55:120–124. 35. Yates JC, Beamish RE, Dhalla NS. Ventricular dysfunction and necrosis produced by adrenochrome metabolite of epinephrine: Relation to pathogenesis of catecholamine cardiomyopathy. Am Heart J 1981; 102:210–221. 36. Jones RL, Miller JC, Hagler HK, Chien KR, Willerson JT, Buja LM. Association between inhibition of arachidonic acid release and prevention of calcium loading during ATP depletion in cultured rat cardiac myocytes. Am J Pathol 1989;135:541–546. 37. Freedman, AM, Kramer JH, Mak T, Cassidy MM, Weglicki WB. Propranolol preserves ultrastructure in adult cardiocytes exposed to anoxia/reoxygenation: A morphometric analysis. Free Radical Biol Med 1991;11:197–206. 38. Jenkins RR, Singore DCM, Sauer P, Skelly C. The effect of beta blocking drugs on lipid peroxidation in rat heart in vitro. Lipids 1992;27:539–542. 39. Marchetti G, Merlo L, Noseda V. Myocardial uptake of free fatty acids and carbohydrates after beta adrenergic blockade. Am J Cardiol 1968;22:370–374. 40. Opie LH, Thomas M. Propranolol and experimental myocardial infarction: Substrate effects. Postgraduate Medical Journal 1976;(Suppl 4):124–133. 41. Mueller HS, Ayres SM, Religa A, Evans RG. Propranolol in the treatment of acute myocardial infarction: Effect on myocardial oxygenation and hemodynamics. Circulation 1974;49:1078–1087. 42. Steigen TK, Myrmel T, Forsdahl K, Larsen TS. Myocardial cell vulnerability to exogenous phospholipase attack. Mol Cell Biochem 1992;116:47–52. 43. Katz AM, Messineo FC. Lipid-Membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 1981;48:1–16.