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Attenuation of Isoproterenol-mediated Myocardial Injury in Rat by an Inhibitor of Polyamine Synthesis
Attenuation of Isoproterenol-mediated Myocardial Injury in Rat by an Inhibitor of Polyamine Synthesis Ulka R. Tipnis, PhD, Gui Ying He, Suzhen Li, Gerald Campbell, and Paul J. Boor Department of Pathology, The University of Texas, Medical Branch, Galveston, TX
ⴙⴙ Objective: Ornithine decarboxylase (ODC) is an initial rate-limiting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine) that play a role in cell growth and differentiation. Recent studies have shown that spermidine and spermine cause injury to a variety of cells including myocytes in vitro. In this investigation, we used ␣-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis to test the hypothesis that polyamines contribute to myocardial injury in rat. Methods: Male Sprague Dawley rats were treated with (i) saline (0.2 ml/day, s.c.), (ii) isoproterenol (ISO) (5 mg/kg/day for 8 days, s.c.) to produce necrotizing myocardial injury, or with (iii) DFMO ⫹ ISO. DFMO was started 2 days before the initiation of ISO and both ISO and DFMO were continued until the end of the experimental period. Myocardial injury was assessed by determining the increased release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) into the plasma, and by morphometric analysis of the lesion area in heart sections stained with Gomori trichrome. Results: ISO induced the release of CPK and LDH by 6 hr and 24 hr, respectively, and produced subendocardial necrosis, which was both acute and resolving following 8 days of ISO. DFMO treatment inhibited ISO-induced increases in (i) ODC activity and putrescine and spermidine levels in heart, (ii) CPK and LDH activity in plasma, and (iii) the area of subendocardial lesions. Conclusions: These observations suggest that polyamines are one of the intracellular factors that contribute to ISO-mediated cardiac injury in the rat. Cardiovasc Pathol 2000;9:273–280 © 2000 by Elsevier Science Inc.
Introduction
Manuscript received January 17, 2000; revised April 28, 2000; accepted May 8, 2000. Address for correspondence: Paul J. Boor, MD, Department of Pathology, Keiller Building, The University of Texas, Medical Branch, Galveston, TX 77555-0609, USA. Tel: (409) 772-2813; Fax: (409) 7471763; E-mail: [email protected].
end-diastolic pressure, left ventricular wall thickness, and increased myocardial deposition of fibronectin and laminin. Some of the mechanisms proposed to explain the mechanisms of ISO-induced damage to cardiac myocytes include hypoxia due to myocardial hyperactivity and coronary hypotension (14), calcium overload (15), depletion of energy reserves (16), and excessive production of free radicals resulting from oxidative metabolism of catecholamines (17). Besides these mechanisms, other intracellular factors and processes that may contribute to or exacerbate ISO-mediation of myocyte injury remain to be defined. In previous studies, we have shown that the administration of ISO produces a significant increase in ornithine decarboxylase (ODC) activity and polyamine levels in the heart (5, 8). The objective of the experiments in the current study was to determine if ISO-induced increases in ODC activity and polyamine levels contribute to myocardial lesions. Ornithine decarboxylase (ODC) is an initial rate-lim-
Cardiovascular Pathology Vol. 9, No. 5, September/October 2000:273–280 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Myocardial injury commonly occurs in hypertension (1– 3) or following ischemia and reperfusion (4). Isoproterenol (ISO), a -adrenergic agonist, is a well-known inducer of myocardial hypertrophy (5,6) and its supramaximal dosages produce acute myocardial necrosis and interstitial fibrosis (7–12). The histopathological lesions produced by ISO in rat resemble myocardial infarcts seen in atherosclerotic animals (7,13). Grimm and coworkers (1998) have shown that supramaximal dosages of ISO (80 mg/kg) lead to heart failure that is characterized by increased end diastolic volume,
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iting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine), cellular aliphatic cations ubiquitous in eukaryotes. It catalyzes the decarboxylation of ornithine, resulting in the formation of putrescine. The addition of propylamine groups to putrescine by spermidine and spermine synthatases leads to the formation of spermidine and spermine (19). Before degradation, spermidine and spermine are acetylated and the acetylated derivatives of polyamines are degraded by polyamine oxidases (PAO). During this degradative reaction, hydrogen peroxide, ammonia, and aminoaldehydes are produced (19–21). The polyamines, putrescine, spermidine, and spermine, are generally implicated in cellular growth and differentiation (19,20). The experiments in vitro have shown that polyamines contribute to programmed cell death (22) and cell injury (23,24). Increased activity of ODC has been linked to c-Mycinduced apoptosis in an interleukin-3-dependent murine myeloid cell line (22). In heat-shock stimulated Chinese hamster cells, excessive degradation of polyamines, production of H2O2, and increased oxidative stress have been implicated in polyamine-mediation of toxicity (25). Other studies have shown that spermine causes toxicity to BHK-21/C13 cells by its direct action and not through imposition of oxidative stress (22). Our recent studies in vitro have shown that the oxidation of polyamines by both extracellular and intracellular amine oxidases has a role in myocyte injury (26). To date, however, no in vivo studies have been reported on the role of polyamines in myocardial injury. To determine the role of ISO-stimulated increases in ODC activity and polyamines in myocardial injury and fibrosis, we used ␣-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis (27,28). Myocardial injury in this study was assessed by the determination of activities of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) in plasma and by the morphometric analysis of fibrosis in sections of the rat hearts.
Methods Animals All animal care and experimental protocols were in compliance with the USDA Animal Welfare Act and the NIH guide for the Care and Use of the Laboratory Animals (NIH Publication #85-23, 1985).
Materials Male Sprague Dawley rats (150–200 gm) were purchased from Harlan, Inc. (Indianapolis). ⫹/⫺ Isoproterenol, creatine phosphokinase assay kits, ornithine, and other chemicals were also purchased from Sigma Chemicals, Inc. (St. Louis). [3H]putrescine and [14C]-ornithine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). DFMO (a specific and
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irreversible inhibitor of ODC) was generously provided by Merrel Hoechst Marion Roussel, Inc., Research Institute.
Experimental Protocols Rats were divided into the following three treatment groups: (1) Control: saline only (0.2/ml/day, i.p). (2) ISO: ISO (5 mg/kg/day, s.c.). (3) DFMO⫹ISO: Both DFMO and ISO were given throughout the experiment. DFMO was given in drinking water (2%) as well as by intraperitoneal injections (200 mg/kg/day, i.p.). To ensure inhibition of ODC activity by DFMO, the treatment with DFMO was initiated two days before ISO administration and was continued throughout the experimental period. Saline or ISO was given for 8 days. Due to the limited amount of DFMO available to us, the effect of DFMO on each of the following parameters was noted at a single time point when maximum change was noted after ISO. The systolic blood pressure of conscious rats (n⫽4) from different groups was measured by tail cuff method with a sphygmomanometer (IITC, model 31). For each rat, three measurements of blood pressure were taken before and after the administration of ISO. At the end of the experiment, the rats were anesthetized with pentobarbital (50 mg/kg) at 6, 24, 48 hr, or 8 days after ISO treatment. Approximately 1 ml of blood was collected in heparinized syringes from the abdominal aorta and transferred to tubes. The blood samples were immediately centrifuged and plasma samples were assayed for CPK and LDH activity. The chest cavity was opened to remove the heart for determination of ODC activity or for histological examination.
Ornithine Decarboxylase Assay The ODC activity of heart was determined 6 hr after ISO administration as described previously (5,18). Approximately 100 mg of tissue was homogenized in three volumes of icecold 50 mM Tris, pH 7.2 containing dithiothreitol (2 mM), EDTA (0.1 mM), sodium fluoride (5.0 mM), pyridoxal phosphate (60 mol), Nonidet P40 (0.1%) and phenylmethylsulfonylfluoride (2mM). After centrifugation (10,000 Xg), the supernatant was assayed at 37⬚C for 30 minutes in the presence of 14C-ornithine (final concentration 0.5 mM). The reaction was terminated with 1M citric acid and released CO2 was absorbed on to filter paper presoaked with 1 N NaOH. The radioactivity on the filter paper was measured by scintillation spectrophotometry. Proteins in the supernatants were estimated by the bicinchoninic acid (BCA) method (Pierce). The enzyme activity was expressed as mean ⫾ SD of pmols of 14CO2 released/30 min/mg protein from three rats.
Creatine Phosphokinase Activity CPK activity was measured according to the instructions provided in a commercially available assay kit (Sigma
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chemicals) used in these experiments. Briefly, plasma samples at 1:2 dilution were incubated with two hundred microliters of ADP-Glutathione (0.1 mmol) for 30 minutes at 37⬚C. The reaction was stopped by the addition of 0.2 ml of p-hydroxymercuribenzoate (0.05mol/l) followed by addition of 1 ml of alpha-napthol, 1 ml of diacetyl solution and 7 ml of water. The contents were mixed and incubated at 37⬚C for color development. After centrifugation, the absorbance of the supernatants was measured at 520 nm. Creatine standards ranging from 0 to 160 Sigma units/ml were assayed to produce a calibration curve for determination of the values of experimental samples. Sigma units were then converted to international units and the enzyme activity in plasma based upon five rats was expressed as international units/ml.
Lactate Dehydrogenase Activity
samples. With this procedure, putrescine, spermidine, and spermine were separated with retention times of 28, 33, and 36 minutes, respectively.
Histology For studying the histological changes in heart, six control or six ISO-treated and four ISO⫹DFMO treated rats were sacrificed after 8 days of ISO administration. Two to four horizontal sections of hearts from the apex to the atrioventricular groove were fixed overnight in 10% formalin in 0.1 M phosphate buffer, pH 7.4. The tissues were processed for paraffin sectioning and 4-micron sections were stained with hematoxylin and eosin for histopathologic examination or with Gomori’s trichrome to identify the connective tissue.
Determination of Fibrosis in Rat Heart
LDH activity was measured by the consumption of pyruvate and reduced diphosphopyridine nucleotide (DPNH) as described previously (29). Essentially, the reaction mixture contained 1.45 ml of phosphate-pyruvate solution (0.05 M of potassium dihydrogen phosphate; 3.1 ⫻ 10⫺4 M of sodium pyruvate), 0.025 ml of reduced diphosphopyridine nucleotide (DPNH, 8 ⫻ 10⫺3 M) and 50 l of plasma sample. The enzyme activity was measured by the rate of decrease in optical density at 340 m due to the oxidation of DPNH. The absorbance was measured at every 30 seconds for 3 minutes. The enzyme activity based upon 5 rats was expressed as units/ml. One unit of enzyme activity according to Wroblewski and LaDue (30) is the change in optical density of DPNH at 340 m by 0.001 in one minute.
Determination of Polyamines Approximately 100 mg of heart homogenized in 10% trichloroacetic acid was centrifuged at 1000 Xg. The supernatants were dansylated and extracted with toluene as described previously (26). Dansylated derivatives of polyamines were separated on a C-18 reverse phase column using a methanol gradient (51–100%) at a flow rate of 1 ml/min. The polyamines were detected and quantitated by fluorescence detector (excitation wavelength 340 nm; emission wavelength, 515 nm). The standards for putrescine, spermidine, and spermine were dansylated and extracted at the same time as
Four-micron paraffin sections stained with Gomori trichrome were used for morphometric analysis. The 4 ⫻ objective of a Nikon Optiphot II microscope equipped with a CCD color video camera was used to display the image on a high-resolution video monitor. After digitization of the image, the percent area of the heart replaced by fibrosis was measured using Optimas software (version 4.10) running on a IBM-PC compatible computer. Four sections from each rat were analyzed to provide sufficient data for determination of differences in fibrosis between the treatment groups.
Statistical Analysis Statistical Analysis was performed using SigmaStat software program (Version 2.0, Jandel Scientific, San Rafel, CA). Comparison between groups was assessed by one way analysis of variance (ANOVA) followed by Tukey’s post-test. The results were expressed as mean and S.D. of 4–6 rats. Statistical significance was accepted at the level of P ⬍ 0.05.
Results As reported in earlier studies (7), ISO induced ODC activity and increased the levels of polyamines in rat hearts. DFMO produced greater than 90% inhibition of ODC activity and blocked increases in the levels putrescine and spermidine levels in rat heart stimulated by ISO (Table 1 and 2).
Table 1. Effect of DFMO Pretreatment on ISO Stimulation of ODC Activity, Heart Rate and Blood Pressure Group Control ISO ISO⫹DFMO
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ODC Activity Pmols/30min/mg Protein
Heart Rate
Blood Pressure mm Hg
236.0 ⫾ 3.0 2510.0 ⫾ 249.0 * 233.0 ⫾ 63.0 ♣
410.0 ⫾ 17.0 620.0 ⫾ 17.0 * 600.0 ⫾ 2.0 **
123 ⫾ 7.0 115 ⫾ 12.0 115 ⫾ 11.0
The data represent mean ⫾ SD (n ⫽ 3). ODC activity - control versus ISO *p ⬍ 0.05; ISO versus DFMO ♣p ⬍ 0.05. Heart rate - control versus ISO or ISO ⫹ DFMO **p ⬍ 0.05. ISO, Isoproternol; DFMO, difluoromethylornithine.
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Table 2. Effect of DFMO on polyamine levels in rat heart Group Control ISO ISO ⫹ DFMO
Putrescine
Spermidine nmol/heart
Spermine
17.0 ⫾ 2.0 54.0 ⫾ 4.0* 23.0 ⫾ 2.0♣
42.0 ⫾ 11.0 75.0 ⫾ 13.0* 54.0 ⫾ 2.0♣
55.0 ⫾ 14.0 110.0 ⫾ 20.0* 106.0 ⫾ 11.0
The data based upon 3 rats represents the mean 6 SD. *p ⬍ 0.05 indicates difference in putrescine, spermidine and spermine levels between control and ISO groups. ♣ p ⬍ 0.05 represents the difference in putrescine and spermidine levels in ISO versus ISO⫹DFMO Group.
ISO increased the heart rate, but we did not observe hypotension as reported previously by investigators who measured blood pressure by arterial cannulation (31,32). The failure to observe hypotension may be due to the fact that the tail-cuff does not monitor blood pressure on a continuous basis, and the ISO effect on blood pressure is transient and occurs within seconds. The dose of ISO used in these experiments (5 mg/kg) was lower than ISO doses used in other studies (33,34). As such, the mortality rate in our experiments was only 4–6% as compared to the 55–75% mortality rate in studies conducted using 85–170 mg/kg ISO.
Changes in Creatine Kinase Activity in Plasma Creatine phosphokinase activity in plasma samples was measured at 6, 24, and 48 hr after ISO. The CPK activity increased significantly at 6 hr and returned to a basal level by 24 hr (data not shown). Figure 1 shows CPK activity 6 hr after ISO administration. In controls, CPK activity in the plasma ranged from 235 to 748 units/ml. Six hours after ISO administration, CPK activity in plasma increased significantly by ninefold. Compared to the ISO group of rats, CPK activity in the plasma of the ISO ⫹ DFMO group of rats was reduced by 50% (p ⬍ 0.05, ISO versus ISO ⫹ DFMO) (Figure 1).
Figure 1. Effect of DFMO on ISO-mediated release of CPK into plasma. ISO (5 mg/kg/day for 8 days, s.c.), DFMO (2% in drinking water ⫹ 200 mg/kg, i.p.). CPK was measured 6 hr after ISO. Control versus ISO, *p ⬍ 0.05. ISO versus ISO ⫹ DFMO. **p ⬍ 0.05. The data represents mean ⫾ SD of rats (n ⫽ 5).
Changes in LDH Activity in Plasma Figure 2 shows the effect of DFMO on plasma LDH activity at 24 hr after ISO. LDH activity in saline-treated rats ranged from 300 to 952 units/ml. Isoproterenol induced a 5.7 fold increase in plasma LDH activity, which was reduced by approximately 75% in the ISO ⫹ DFMO group.
Fibrosis in Rat Heart The sections stained with Gomori trichrome were examined to study the distribution of collagen and the development of fibrosis in different experimental groups. The heart sections from control rats showed a homogeneous myocardium without fibrosis (Figure 3A). ISO produced extensive subendocardial fibrosis in the rat heart (Figure 3B). At high magnification, this area showed replacement by collagen (Figure 3C) with predominance of nonmyocytes, few residual myocytes, and resolving myocardial necrosis (Figure 3D). DFMO treatment caused a significant reduction in the extent of the subendocardial lesions in the ISO-treated rats (Figures 3E and 3F). The results of morphometric analysis, shown in Figure 4, revealed that, compared to saline-treated rats, the area of fibrosis in ISO-treated rats was increased by twentyfold. In rats receiving ISO ⫹ DFMO, the area of fibrosis increased by only eightfold compared to the saline treated rats, and it was reduced by 2.5-fold compared to rats treated with ISO alone. These observations on fibrosis corroborated the results of CPK and LDH assays, showing the protective effect of DFMO on ISO-mediated myocardial damage to rat heart.
Discussion The levels of catecholamines increase in circulation after stress (7,35,36) and in sympathetic nerve terminals follow-
Figure 2. Effect of DFMO on ISO-mediated release of lactate dehydrogenase at 24 hr after ISO (5 mg/kg). DFMO treatment was the same as stated in methods or Figure 1. The data based upon represents mean ⫾ SD of rats (n ⫽ 4). *p ⬍ 0.05 ISO versus control. **p ⬍ 0.05, ISO versus ISO ⫹ DFMO.
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Figure 3. DFMO attenuates ISO-induced necrosis and fibrosis in rat heart. The rats were sacrificed at 8 days. The hearts were fixed and processed for paraffin sectioning and staining as described in methods. (A) Low power histologic cross section of control rat heart shows homogeneous normal myocardium of left and right ventricles. (B) Section of heart from rat given ISO shows extensive subendocardial myocardial necrosis and fibrosis evident at this low power as pale areas. (C,D) Higher power views of heart from rat given ISO show extensive fibrosis (C, ⫻117) and resolving myocardial necrosis (D, ⫻375). (E,F) Heart section of rat treated with ISO ⫹ DFMO shows only small area of myocardial necrosis and fibrosis (E, arrow) which is confined to the immediate subendocardial area (F). Gomori trichrome stain; (A, B, and E, ⫻8; C and F, ⫻117; D, ⫻375).
ing myocardial ischemia (37), respectively. Excessive levels of circulating catecholamines have been shown to cause myocardial hypertrophy, myocyte damage, and cardiomyopathy (9). ISO acts through -adrenergic receptors, stimulating calcium influx (15), and augmenting the consumption of oxygen and depletion of ATP (16). Its main action is on
the heart, where it produces infarctlike lesions. Isoproterenol-induced myocardial necrosis is associated with dosedependent increases in left ventricular end diastolic pressure, left ventricular volume, and wall stress (33). Isoroterenol given alone has been shown not to cause damage to other organs, although it can potentiate hepatotoxicity by methy-
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Figure 4. Morphometric analysis showing the DFMO attenuation of lesion area in rat hearts exposed to ISO. The data mean ⫾ SD is based upon 4 – 6 rats/group. ISO versus control, *p ⬍ 0.05., ISO versus ISO ⫹ DFMO. **p ⬍ 0.05.
phenidate hydrochloride, a CNS stimulant drug (38). Thus, observed changes in CPK and LDH levels observed in this study would reflect the release of enzymes resulting from ISO-induced myocardial damage. The determinations of CPK and LDH are both useful parameters for assessing myocardial damage (39,40). However, the time when those changes occur may differ due to differences in ISO itself, the dose of ISO administered or the age of the animals. In these experiments, ISO-mediation of myocardial injury was evidenced by increases in CPK and LDH activities in plasma and subendocardial fibrosis in rat hearts. We observed that CPK was released at an earlier phase of ISO damage (6 hr), while the release of LDH occurred at 24–48 hr after ISO administration. Clinical studies also show that CPK is released during an earlier phase of myocardial infarction than LDH, which is released after the infarct has occurred (40). The precise mechanisms by which isoproterenol causes myocardial damage is not fully elucidated, however, the proposed mechanisms include production of ischemia (14), calcium overloading (41), production of free radicals (17), and lipid peroxidation (42–44). In the present investigation, we examined whether inhibition of ISO-induced ODC activity attenuated the myocardial damage in rat heart. Difluoromethylornithine, a specific and irreversible inhibitor of ODC activity (27,28), blocks the synthesis of putrescine and results in depletion of spermidine (18). In humans, DFMO has been used in clinical trials for chemoprevention of cancer. In these studies, the side effects of DFMO in humans include hearing loss (45), diarrhea, and thrombocytopenia (46,47). Studies in rats have shown that chronic administration by gavage at doses greater than 800 mg/kg for 52 weeks produces a significant reduction in organ to body weight ratios of brain, adrenal glands, and liver. DFMO between 800–1000 mg/kg produced necrotizing dermatitis of the skin, coagulation necrosis of the liver, and inflammation of the duodenal mucosa,
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while DFMO dose below 800 mg/kg for 52 days did not produce toxic effects (48). Thus, DFMO given for a total of 8 days in drinking water (2%) and by intraperitoneal administration (200 mg/kg) is unlikely to cause the type of cellular damage noted in those investigations (48). The results of this study show that DFMO inhibition of ISO-stimulated increases in ODC activity and levels of polyamines in rat heart is associated with decreased release of CPK and LDH into plasma and attenuation of fibrosis. These observations suggest that DFMO confers protection against ISO-mediated myocardial damage. Polyamines are generally implicated in cellular proliferation and differentiation (20). Earlier studies by Koenig and colleagues have shown that increases in polyamines in isolated perfused rat heart trigger calcium transport that is associated with loss of contractility, release of myoglobin, and cytosolic enzymes (49). ODC activity is also increased in brain areas that have undergone infarction due to ischemia (50,51). Evidence based on studies using DFMO in cats suggests that polyamines play a role in mediation of neuronal injury, post-traumatic vasogenic edema, and blood brain barrier defects after ischemic injury (52). Spermidine and spermine are oxidatively deaminated by bovine serum amine oxidases to produce aminoaldehydes, ammonia and H2O2 (53). These products of polyamine degradation have been suggested to play a role in the mediation of cytotoxicity (54). For example, ammonia, which is a product of polyamine degradation, has been shown to reduce lymphocyte viability (55), and inhibition of polyamine oxidase has been shown to attenuate the toxicity mediated by spermine to BHK-21/C13 cells (22). Our recent in vitro studies show that both intracellular and extracellular amine oxidases play a role in mediation of polyamine toxicity to cardiac myocytes (25). Our preliminary studies indicate that inhibition of polyamine oxidase by MDL 72.527, a specific inhibitor of polyamine oxidase, attenuates the release of ISO-induced CPK and LDH into plasma. Studies are currently in progress to determine whether any of these mechanisms, or the calcium influx triggered by polyamines, are involved in ISO-mediated cardiac injury. Understanding the role of polyamines in fibrosis is important because this pathological change is a common occurrence in humans with ischemic heart disease and symptomatic heart failure (56). The nonmyocyte population of the heart is made up of fibroblasts, endothelial cells, and smooth muscle cells. These cells produce proteins of the intercellular matrix and cytokines, including transforming growth factor 1 (TGF 1) (57). Using mRNA probes, Eghbali and coworkers (58) have shown that fibroblasts are the source of collagen type I and III. TGF plays an important role in proliferation and phenotypic modulation of fibroblasts (59). Studies in intestinal epithelial cells indicate that polyamines are essential for the expression of TGF- (60). Further studies are needed to determine the specific mechanisms by which polyamines contribute to myocyte injury
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and fibrogenesis in the rat heart and whether polyamines regulate the cardiac fibrosis by regulating the expression of cytokines and phenotypic modulation of nonmyocytes. The authors thank Tom Bedernak for photography and Merrel Hoechst Marion Roussel, Inc., Research Institute for providing DFMO. This research was supported by American Heart Association, National (# 96010090) awarded to Ulka R. Tipnis.
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in ornithine decarboxylase activity and polyamine levels in regions of the rat heart. J Cardiovasc Res XXIII, 1989;23:611–619. 19. Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol 1982;12:C212–C221. 20. Janne J, Alhonen L, Leinonen P. Polyamines: from molecular biology to Clinical applications. Annal Med 1991;23:241–259. 21. Seiler N, Atanassov CL. The natural polyamines and the immune system. In: Jucker E, ed. Progress in Drug Research by von Heraugegeben. Basel, Boston, Berlin: Berkhauser verlag, 1994;43:87–141. 22. Packham G, Cleveland J. Ornithine decarboxylase is a mediator of c-Mycinduced apoptosis. Mol Cell Biology 1994;14:5741–5747.
References 1. Weber KT, Sun Y, Tyagi SC, Cleutjens JP, MJ. Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. Mol. Cell. Cardiol. 1994;26:279–292. 2. Weber KT, Sun Y, Campbell SE, Slight SH, Ganjam VK, Griffing GT, Swinfard RW, Diaz-Arias AA. Chronic mineralocorticoid excess and cardiovascular remodeling. Steroids 1995;60:125–132. 3. Villareal FJ, Mckenna DA, Omens JH, Dillmann WH. Myocardial remodeling in hypertensive Ren-2 transgenic rats. Hypertension 1995;25:98–104. 4. Jordan JE, Zhao Z-Q, Vinten-Johansen J. The role of neutrophils in myocardial reperfusion-injury. Cardiovas Res 1999;43:860–878. 5. Tipnis UR, Chopra A, Campbell G, Boor P. Role of polyamines in atrial natriuretic peptide expression in isoproterenol-induced cardiac hypertrophy in rat. Biomedical Lett 1995;52:81–86. 6. Boluyt MO, Long X, Eschenhagen T, Mende U, Schmitz W, Crow MT, Lakatta EG. Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart. Am J Physiol 1995;269:H638–H647. 7. Rona G, Chappel CI, Balazas T, Gaundry R. An infarct like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. Arch Pathol 1959;67:443–445. 8. Brilla CG, Maisch B, Rupp H, Funck R, Zhou G, Weber KT. Pharmacological modulation of cardiac fibroblast function. Herz 1995;20: 127–134. 9. Rona G. Catecholamine cardiotoxicity. J Mol Cell Cardiol 1985;17: 291–306. 10. Todd GL, Baroldi G, Pieper GM, Clayton FC, Eliot RS. Experimental catecholamine-induced myocardial necrosis I. Morphology, quantification and regional distribution of acute contraction band lesions. J Mol Cell Cardiol 1985;17:317–338. 11. Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, Weber K. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circulation Res 1989;64:1041–1050. 12. Benjamin IJ, Jalil JE, Tan LB, Cho K, Weber KT, Clark W. Isoproterenol induced myocardial fibrosis in relation to myocyte necrosis. Circ Res 1989;65:657–670. 13. Wexler BC, Kittinger GW. Myocardial necrosis in rats: serum enzymes, adrenal steroid and histopathological alterations. Circ Research 1963;13:159–171. 14. Yeager JC, Iams SG. The hemodynamics of isoproterenol-induced cardiac failure in rats. Circ Shock 1981;8:151–163. 15. Bloom S, Davis DL. Calcium as a mediator of isoproterenol-induced myocardial necrosis. Am J Pathol 1972;69:459–470. 16. Fleckenstein A, Janke J, Doring HJ, Leder, O. Myocardial fiber necrosis due to intracellular calcium overload. A new principle in cardiac pathophysiology. In: Dhalla NS, ed. Recent Advances in Cardiac Structure and Metabolism, Vol. 4. Baltimore, MD: University Park Press, 1974;563–580. 17. Singal PK, Beamish RE, Dhalla NS. Potential oxidative pathways of catecholamines in the formation of lipid peroxides and genesis of heart disease. Adv Exp Med Biol 1983;161:391–440. 18. Tipnis UR, Frasier-Scott K, Skiera C. Isoproterenol-induced changes
23. Brunton VG, Grant MH, Wallace HM. Mechanisms of spermine toxicity in baby-hamster kidney (BHK) cells. The role of amine oxidases and oxidative stress. Biochem J 1991;280:193–198. 24. Gaugas JM, Dewey DL. Hog kidney diamine oxidase conversion of biogenic diamines to inhibitors of cell proliferation. Jour Pathol 1981;134:243–252. 25. Harari PM, Fuller DJM, Gerner EW. Heat shock stimulates polyamine oxidation by two distinct mechanisms in mammalian cell cultures. Int J Rad Oncology Biol Phys 1989;16:451–457. 26. Tipnis UR, He GY. Mechanism of polyamine toxicity in cultured cardiac myocytes. Toxicology in Vitro 1998;12:233–240. 27. Metcalf BW, Bey P, Danzin C, Jung MJ, Casara P, Vevert JP. Catalytic irreversible inhibition of ornithine decarboxylase (E.C.1.1.17) by substrate of product analogue. J Am Chem Soc 1978;100:2551–2553. 28. Danzin C, Mamont P. Polymins inhibition in vivo and in organ growth and repair. In: McCann PP, Pegg AE, Sjoerdsma A, eds. Inhibition of polyamine metabolism. Biological significance and basis for new therapies. New York: Academic Press, 1987;141–164. 29. Bergemeyer H-U, Bernt E, Hess B. Lactate dehydrogenase. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis. New York: Weinheim/Bergstr Academic Press, 1965;736–741. 30. Wroblewski F, LaDue JS. Lactate dehydrogenase activity in the blood. Proc Soc Exp Biol Med 1955;90:210–213. 31. Boor P J. Studies of hypotension and cardiac flow in isoproterenolinduced myocardial necrosis. Exp Mol Pathol 1980;34:110–121. 32. Viano DC. Cardiovascular injury from blunt thoracic impact of epinephrine and isoproterenol injected rabbits. Aviat Space and Environmen Med 1983;54:988–993. 33. Teerlink JR, Pfeffer JM, Pfeffer MA. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res 1994;75:105–113. 34. Grimm D, Elsner D, Schunkert H, Pfeiffer M, Griese D, Bruckschlegel G, Muders F, Riegger GAG, Kromer EP. Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system. Cardiovasc Res 1998;37:91–100. 35. Pignatelli D, Magalhaes MM, Magalhaes MC. Direct effect of stress on adrenocortical function. Hormone and Metabolic Research 1998; 30:464–474. 36. Marino F, Sockler JM, Fry JM. Thermoregulatory, metabolic and sympathoadrenal responses to brief exposure to cold. Scand Jour Clin Lab Invest 1998;58:537–545. 37. Schomig A, Dart AM, Dietz R, Mayer E, Kubler W. Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A. Locally mediated release. Circ Res 1984;55:689–701. 38. Roberts SM, Harbison RD, Roth L, James RC. Methylphenidateinduced hepatotoxicity in mice and its potentiation by -adrenergic agonist drugs. Life Sci 1994;55:269–281. 39. Bhargawa AS, Preus M, Gunzel P. Effect of Iloprost on serum creatine kinase and lactate dehydrogenase isoenzymes after isoprenalineinduced cardiac damage in rats. Arzneim-Forsch/Drug Res 1990;40: 248–252. 40. Mair J, Puschendorf B, Michel G. Clinical significance of cardiac contractile proteins for the diagnosis of myocardial injury. In: Spiegel HE, ed. Advances in Clinical Chemistry, Vol. 31. New York: Academic Press, 1994.
280
TIPNIS ET AL. MYOCARDIAL INJURY ATTENUATION
41. Nirdlinger EL, Bramante PO. Subcellular myocardial ionic shifts and mitochondrial alterations in the course of isoproterenol-induced cardiomyopathy of the rat. J Mol Cell Cardiol 1974;6:49–60. 42. Norhona-Dutra AA, Steen-Dutra EM, Woolf N. Epinephrine-induced cytotoxicity in rat plasma. Its effects on isolated cardiac myocytes. Lab Invest 1988;59:817–823. 43. Persoon-Rothert M, Valk-Kokshoorn VD, Egaskenniphas JM, Mauve I, Laarse, VD. Isoproterenol-induced cytotoxicity in neonatal heart cell cultures is mediated by free-radical formation. J Mol Cell Cardiol 1989;21:1285–1291. 44. Kondo T, Ogawa Y, Sugiyama S, et al. Mechanisms of isoproterenolinduced myocyte damage. Cardiovas Res 1987;21:248–254. 45. Croghan MK, Aickin MG, Meyskens FL. Dose-related alpha-difluoromethylornithine ototoxicity. Amer Jour Clin Oncol 1991;14:331– 335. 46. Ajani JA, Ota DM, Grossie VB, Levin B, Nishioka K. Alterations in polyamine metabolism during continuous intravenous infusion of alpha-difluoromethylornithine showing correlation of thrombocytopenia with alpha-difluoromethylornithine plasma levels. Cancer Res 1989;49: 5761–5765. 47. Tietze KJ, Gaska JA, Cosgrove EM. Thrombocytopenia and vomiting due to difluoromethylornithine. Drug Intell Clin Pharm 1987;21:627– 630. 48. Crowell JA, Goldenthal EI, Keloff GJ, Malone WF, Boone CW. Chronic toxicity studies of the potential cancer preventive 2-(difluoromethyl)-dl-ornithine. Fundam Appl Toxicol 1994;22:341–354. 49. Koenig H, Goldstone AD, Trout JJ, Lu CY. Polyamines mediate uncontrolled calcium entry and cell damage in rat heart in the calcium paradox. Jour Clin Invest 1987;80:1322–1331. 50. Dempsey RJ, Roy MW, Meyer K, Tai HH, Olson JW. Polyamines and prostaglandin markers in focal cerebral ischemia. Neurosurgery 1985;17:635–640.
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51. Dempsey RJ, Maley M, Cowen D, Olson JW. Ornithine decarboxylase activity and immunohistochemical localization in postischemic brain. J Cereb Blood Flow Metab 1988;8:843–847. 52. Schmitz MP, Combs D, Dempsey RJ. Difluoromethylornithine decreases postischemic brain edema and blood brain barrier breakdown. Neurosurgery 1993;33:882–888. 53. Mondovi B, Riccio P, Agostineli E. The biological functions of amine oxidases and their reaction products: an overview. Adv Exp Med Biol 1989;250:147–161. 54. Kawase M, Samejima K, Okada, M. Mode of action of 3-substituted propylamine cytotoxicity in culture cells. Biochem Pharmacol 1982;31:2883–2988. 55. Flescher E, Fossum D, Talal N. Polyamine-production of lymphocytotoxic levels of ammonia by human peripheral blood monocytes. Immunology Letters. 1991;28:85–90. 56. Beltrami CA, Finato N, Rocco M, Fergulio GA, Puricelli C, Cigola E, Quaini F, Sonnenblick EH, Olivetti G, Aneversara P. Structural basis of end-stage failure in ischemic cardiomyopathy in humans. Circulation 1994;89:151–163. 57. Eghbali M, Czaja MJ, Zedel M, Weiner FR, Zern MA, Seifter S, Blumefeld OO. Collagen mRNA isolated from young and adult rats. J Mol Cell Cardiol 1988;20:267–276. 58. Eghbali M. Cellular origin and distribution of transforming growth factor-1 in the normal rat myocardium. Cell Tissue Res 1989;256: 553–558. 59. Sigel AV, Centrella M, Eghabali-Webb M. Regulation of proliferative response of cardiac fibroblasts by transforming growth factor-1. J Mol Cell Cardiol 1996;28:1921–1929. 60. Wang JY, Viar MJ, Li J, Shi HJ, McCormack SA, Johnson LR. Polyamines are necessary for normal expression of the transforming growth factor-beta gene during cell migration. Am J Physiol 1997; 272:G713–G720.
ⴙⴙ Objective: Ornithine decarboxylase (ODC) is an initial rate-limiting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine) that play a role in cell growth and differentiation. Recent studies have shown that spermidine and spermine cause injury to a variety of cells including myocytes in vitro. In this investigation, we used ␣-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis to test the hypothesis that polyamines contribute to myocardial injury in rat. Methods: Male Sprague Dawley rats were treated with (i) saline (0.2 ml/day, s.c.), (ii) isoproterenol (ISO) (5 mg/kg/day for 8 days, s.c.) to produce necrotizing myocardial injury, or with (iii) DFMO ⫹ ISO. DFMO was started 2 days before the initiation of ISO and both ISO and DFMO were continued until the end of the experimental period. Myocardial injury was assessed by determining the increased release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) into the plasma, and by morphometric analysis of the lesion area in heart sections stained with Gomori trichrome. Results: ISO induced the release of CPK and LDH by 6 hr and 24 hr, respectively, and produced subendocardial necrosis, which was both acute and resolving following 8 days of ISO. DFMO treatment inhibited ISO-induced increases in (i) ODC activity and putrescine and spermidine levels in heart, (ii) CPK and LDH activity in plasma, and (iii) the area of subendocardial lesions. Conclusions: These observations suggest that polyamines are one of the intracellular factors that contribute to ISO-mediated cardiac injury in the rat. Cardiovasc Pathol 2000;9:273–280 © 2000 by Elsevier Science Inc.
Introduction
Manuscript received January 17, 2000; revised April 28, 2000; accepted May 8, 2000. Address for correspondence: Paul J. Boor, MD, Department of Pathology, Keiller Building, The University of Texas, Medical Branch, Galveston, TX 77555-0609, USA. Tel: (409) 772-2813; Fax: (409) 7471763; E-mail: [email protected].
end-diastolic pressure, left ventricular wall thickness, and increased myocardial deposition of fibronectin and laminin. Some of the mechanisms proposed to explain the mechanisms of ISO-induced damage to cardiac myocytes include hypoxia due to myocardial hyperactivity and coronary hypotension (14), calcium overload (15), depletion of energy reserves (16), and excessive production of free radicals resulting from oxidative metabolism of catecholamines (17). Besides these mechanisms, other intracellular factors and processes that may contribute to or exacerbate ISO-mediation of myocyte injury remain to be defined. In previous studies, we have shown that the administration of ISO produces a significant increase in ornithine decarboxylase (ODC) activity and polyamine levels in the heart (5, 8). The objective of the experiments in the current study was to determine if ISO-induced increases in ODC activity and polyamine levels contribute to myocardial lesions. Ornithine decarboxylase (ODC) is an initial rate-lim-
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Myocardial injury commonly occurs in hypertension (1– 3) or following ischemia and reperfusion (4). Isoproterenol (ISO), a -adrenergic agonist, is a well-known inducer of myocardial hypertrophy (5,6) and its supramaximal dosages produce acute myocardial necrosis and interstitial fibrosis (7–12). The histopathological lesions produced by ISO in rat resemble myocardial infarcts seen in atherosclerotic animals (7,13). Grimm and coworkers (1998) have shown that supramaximal dosages of ISO (80 mg/kg) lead to heart failure that is characterized by increased end diastolic volume,
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iting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine), cellular aliphatic cations ubiquitous in eukaryotes. It catalyzes the decarboxylation of ornithine, resulting in the formation of putrescine. The addition of propylamine groups to putrescine by spermidine and spermine synthatases leads to the formation of spermidine and spermine (19). Before degradation, spermidine and spermine are acetylated and the acetylated derivatives of polyamines are degraded by polyamine oxidases (PAO). During this degradative reaction, hydrogen peroxide, ammonia, and aminoaldehydes are produced (19–21). The polyamines, putrescine, spermidine, and spermine, are generally implicated in cellular growth and differentiation (19,20). The experiments in vitro have shown that polyamines contribute to programmed cell death (22) and cell injury (23,24). Increased activity of ODC has been linked to c-Mycinduced apoptosis in an interleukin-3-dependent murine myeloid cell line (22). In heat-shock stimulated Chinese hamster cells, excessive degradation of polyamines, production of H2O2, and increased oxidative stress have been implicated in polyamine-mediation of toxicity (25). Other studies have shown that spermine causes toxicity to BHK-21/C13 cells by its direct action and not through imposition of oxidative stress (22). Our recent studies in vitro have shown that the oxidation of polyamines by both extracellular and intracellular amine oxidases has a role in myocyte injury (26). To date, however, no in vivo studies have been reported on the role of polyamines in myocardial injury. To determine the role of ISO-stimulated increases in ODC activity and polyamines in myocardial injury and fibrosis, we used ␣-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis (27,28). Myocardial injury in this study was assessed by the determination of activities of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) in plasma and by the morphometric analysis of fibrosis in sections of the rat hearts.
Methods Animals All animal care and experimental protocols were in compliance with the USDA Animal Welfare Act and the NIH guide for the Care and Use of the Laboratory Animals (NIH Publication #85-23, 1985).
Materials Male Sprague Dawley rats (150–200 gm) were purchased from Harlan, Inc. (Indianapolis). ⫹/⫺ Isoproterenol, creatine phosphokinase assay kits, ornithine, and other chemicals were also purchased from Sigma Chemicals, Inc. (St. Louis). [3H]putrescine and [14C]-ornithine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). DFMO (a specific and
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irreversible inhibitor of ODC) was generously provided by Merrel Hoechst Marion Roussel, Inc., Research Institute.
Experimental Protocols Rats were divided into the following three treatment groups: (1) Control: saline only (0.2/ml/day, i.p). (2) ISO: ISO (5 mg/kg/day, s.c.). (3) DFMO⫹ISO: Both DFMO and ISO were given throughout the experiment. DFMO was given in drinking water (2%) as well as by intraperitoneal injections (200 mg/kg/day, i.p.). To ensure inhibition of ODC activity by DFMO, the treatment with DFMO was initiated two days before ISO administration and was continued throughout the experimental period. Saline or ISO was given for 8 days. Due to the limited amount of DFMO available to us, the effect of DFMO on each of the following parameters was noted at a single time point when maximum change was noted after ISO. The systolic blood pressure of conscious rats (n⫽4) from different groups was measured by tail cuff method with a sphygmomanometer (IITC, model 31). For each rat, three measurements of blood pressure were taken before and after the administration of ISO. At the end of the experiment, the rats were anesthetized with pentobarbital (50 mg/kg) at 6, 24, 48 hr, or 8 days after ISO treatment. Approximately 1 ml of blood was collected in heparinized syringes from the abdominal aorta and transferred to tubes. The blood samples were immediately centrifuged and plasma samples were assayed for CPK and LDH activity. The chest cavity was opened to remove the heart for determination of ODC activity or for histological examination.
Ornithine Decarboxylase Assay The ODC activity of heart was determined 6 hr after ISO administration as described previously (5,18). Approximately 100 mg of tissue was homogenized in three volumes of icecold 50 mM Tris, pH 7.2 containing dithiothreitol (2 mM), EDTA (0.1 mM), sodium fluoride (5.0 mM), pyridoxal phosphate (60 mol), Nonidet P40 (0.1%) and phenylmethylsulfonylfluoride (2mM). After centrifugation (10,000 Xg), the supernatant was assayed at 37⬚C for 30 minutes in the presence of 14C-ornithine (final concentration 0.5 mM). The reaction was terminated with 1M citric acid and released CO2 was absorbed on to filter paper presoaked with 1 N NaOH. The radioactivity on the filter paper was measured by scintillation spectrophotometry. Proteins in the supernatants were estimated by the bicinchoninic acid (BCA) method (Pierce). The enzyme activity was expressed as mean ⫾ SD of pmols of 14CO2 released/30 min/mg protein from three rats.
Creatine Phosphokinase Activity CPK activity was measured according to the instructions provided in a commercially available assay kit (Sigma
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chemicals) used in these experiments. Briefly, plasma samples at 1:2 dilution were incubated with two hundred microliters of ADP-Glutathione (0.1 mmol) for 30 minutes at 37⬚C. The reaction was stopped by the addition of 0.2 ml of p-hydroxymercuribenzoate (0.05mol/l) followed by addition of 1 ml of alpha-napthol, 1 ml of diacetyl solution and 7 ml of water. The contents were mixed and incubated at 37⬚C for color development. After centrifugation, the absorbance of the supernatants was measured at 520 nm. Creatine standards ranging from 0 to 160 Sigma units/ml were assayed to produce a calibration curve for determination of the values of experimental samples. Sigma units were then converted to international units and the enzyme activity in plasma based upon five rats was expressed as international units/ml.
Lactate Dehydrogenase Activity
samples. With this procedure, putrescine, spermidine, and spermine were separated with retention times of 28, 33, and 36 minutes, respectively.
Histology For studying the histological changes in heart, six control or six ISO-treated and four ISO⫹DFMO treated rats were sacrificed after 8 days of ISO administration. Two to four horizontal sections of hearts from the apex to the atrioventricular groove were fixed overnight in 10% formalin in 0.1 M phosphate buffer, pH 7.4. The tissues were processed for paraffin sectioning and 4-micron sections were stained with hematoxylin and eosin for histopathologic examination or with Gomori’s trichrome to identify the connective tissue.
Determination of Fibrosis in Rat Heart
LDH activity was measured by the consumption of pyruvate and reduced diphosphopyridine nucleotide (DPNH) as described previously (29). Essentially, the reaction mixture contained 1.45 ml of phosphate-pyruvate solution (0.05 M of potassium dihydrogen phosphate; 3.1 ⫻ 10⫺4 M of sodium pyruvate), 0.025 ml of reduced diphosphopyridine nucleotide (DPNH, 8 ⫻ 10⫺3 M) and 50 l of plasma sample. The enzyme activity was measured by the rate of decrease in optical density at 340 m due to the oxidation of DPNH. The absorbance was measured at every 30 seconds for 3 minutes. The enzyme activity based upon 5 rats was expressed as units/ml. One unit of enzyme activity according to Wroblewski and LaDue (30) is the change in optical density of DPNH at 340 m by 0.001 in one minute.
Determination of Polyamines Approximately 100 mg of heart homogenized in 10% trichloroacetic acid was centrifuged at 1000 Xg. The supernatants were dansylated and extracted with toluene as described previously (26). Dansylated derivatives of polyamines were separated on a C-18 reverse phase column using a methanol gradient (51–100%) at a flow rate of 1 ml/min. The polyamines were detected and quantitated by fluorescence detector (excitation wavelength 340 nm; emission wavelength, 515 nm). The standards for putrescine, spermidine, and spermine were dansylated and extracted at the same time as
Four-micron paraffin sections stained with Gomori trichrome were used for morphometric analysis. The 4 ⫻ objective of a Nikon Optiphot II microscope equipped with a CCD color video camera was used to display the image on a high-resolution video monitor. After digitization of the image, the percent area of the heart replaced by fibrosis was measured using Optimas software (version 4.10) running on a IBM-PC compatible computer. Four sections from each rat were analyzed to provide sufficient data for determination of differences in fibrosis between the treatment groups.
Statistical Analysis Statistical Analysis was performed using SigmaStat software program (Version 2.0, Jandel Scientific, San Rafel, CA). Comparison between groups was assessed by one way analysis of variance (ANOVA) followed by Tukey’s post-test. The results were expressed as mean and S.D. of 4–6 rats. Statistical significance was accepted at the level of P ⬍ 0.05.
Results As reported in earlier studies (7), ISO induced ODC activity and increased the levels of polyamines in rat hearts. DFMO produced greater than 90% inhibition of ODC activity and blocked increases in the levels putrescine and spermidine levels in rat heart stimulated by ISO (Table 1 and 2).
Table 1. Effect of DFMO Pretreatment on ISO Stimulation of ODC Activity, Heart Rate and Blood Pressure Group Control ISO ISO⫹DFMO
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ODC Activity Pmols/30min/mg Protein
Heart Rate
Blood Pressure mm Hg
236.0 ⫾ 3.0 2510.0 ⫾ 249.0 * 233.0 ⫾ 63.0 ♣
410.0 ⫾ 17.0 620.0 ⫾ 17.0 * 600.0 ⫾ 2.0 **
123 ⫾ 7.0 115 ⫾ 12.0 115 ⫾ 11.0
The data represent mean ⫾ SD (n ⫽ 3). ODC activity - control versus ISO *p ⬍ 0.05; ISO versus DFMO ♣p ⬍ 0.05. Heart rate - control versus ISO or ISO ⫹ DFMO **p ⬍ 0.05. ISO, Isoproternol; DFMO, difluoromethylornithine.
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Table 2. Effect of DFMO on polyamine levels in rat heart Group Control ISO ISO ⫹ DFMO
Putrescine
Spermidine nmol/heart
Spermine
17.0 ⫾ 2.0 54.0 ⫾ 4.0* 23.0 ⫾ 2.0♣
42.0 ⫾ 11.0 75.0 ⫾ 13.0* 54.0 ⫾ 2.0♣
55.0 ⫾ 14.0 110.0 ⫾ 20.0* 106.0 ⫾ 11.0
The data based upon 3 rats represents the mean 6 SD. *p ⬍ 0.05 indicates difference in putrescine, spermidine and spermine levels between control and ISO groups. ♣ p ⬍ 0.05 represents the difference in putrescine and spermidine levels in ISO versus ISO⫹DFMO Group.
ISO increased the heart rate, but we did not observe hypotension as reported previously by investigators who measured blood pressure by arterial cannulation (31,32). The failure to observe hypotension may be due to the fact that the tail-cuff does not monitor blood pressure on a continuous basis, and the ISO effect on blood pressure is transient and occurs within seconds. The dose of ISO used in these experiments (5 mg/kg) was lower than ISO doses used in other studies (33,34). As such, the mortality rate in our experiments was only 4–6% as compared to the 55–75% mortality rate in studies conducted using 85–170 mg/kg ISO.
Changes in Creatine Kinase Activity in Plasma Creatine phosphokinase activity in plasma samples was measured at 6, 24, and 48 hr after ISO. The CPK activity increased significantly at 6 hr and returned to a basal level by 24 hr (data not shown). Figure 1 shows CPK activity 6 hr after ISO administration. In controls, CPK activity in the plasma ranged from 235 to 748 units/ml. Six hours after ISO administration, CPK activity in plasma increased significantly by ninefold. Compared to the ISO group of rats, CPK activity in the plasma of the ISO ⫹ DFMO group of rats was reduced by 50% (p ⬍ 0.05, ISO versus ISO ⫹ DFMO) (Figure 1).
Figure 1. Effect of DFMO on ISO-mediated release of CPK into plasma. ISO (5 mg/kg/day for 8 days, s.c.), DFMO (2% in drinking water ⫹ 200 mg/kg, i.p.). CPK was measured 6 hr after ISO. Control versus ISO, *p ⬍ 0.05. ISO versus ISO ⫹ DFMO. **p ⬍ 0.05. The data represents mean ⫾ SD of rats (n ⫽ 5).
Changes in LDH Activity in Plasma Figure 2 shows the effect of DFMO on plasma LDH activity at 24 hr after ISO. LDH activity in saline-treated rats ranged from 300 to 952 units/ml. Isoproterenol induced a 5.7 fold increase in plasma LDH activity, which was reduced by approximately 75% in the ISO ⫹ DFMO group.
Fibrosis in Rat Heart The sections stained with Gomori trichrome were examined to study the distribution of collagen and the development of fibrosis in different experimental groups. The heart sections from control rats showed a homogeneous myocardium without fibrosis (Figure 3A). ISO produced extensive subendocardial fibrosis in the rat heart (Figure 3B). At high magnification, this area showed replacement by collagen (Figure 3C) with predominance of nonmyocytes, few residual myocytes, and resolving myocardial necrosis (Figure 3D). DFMO treatment caused a significant reduction in the extent of the subendocardial lesions in the ISO-treated rats (Figures 3E and 3F). The results of morphometric analysis, shown in Figure 4, revealed that, compared to saline-treated rats, the area of fibrosis in ISO-treated rats was increased by twentyfold. In rats receiving ISO ⫹ DFMO, the area of fibrosis increased by only eightfold compared to the saline treated rats, and it was reduced by 2.5-fold compared to rats treated with ISO alone. These observations on fibrosis corroborated the results of CPK and LDH assays, showing the protective effect of DFMO on ISO-mediated myocardial damage to rat heart.
Discussion The levels of catecholamines increase in circulation after stress (7,35,36) and in sympathetic nerve terminals follow-
Figure 2. Effect of DFMO on ISO-mediated release of lactate dehydrogenase at 24 hr after ISO (5 mg/kg). DFMO treatment was the same as stated in methods or Figure 1. The data based upon represents mean ⫾ SD of rats (n ⫽ 4). *p ⬍ 0.05 ISO versus control. **p ⬍ 0.05, ISO versus ISO ⫹ DFMO.
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Figure 3. DFMO attenuates ISO-induced necrosis and fibrosis in rat heart. The rats were sacrificed at 8 days. The hearts were fixed and processed for paraffin sectioning and staining as described in methods. (A) Low power histologic cross section of control rat heart shows homogeneous normal myocardium of left and right ventricles. (B) Section of heart from rat given ISO shows extensive subendocardial myocardial necrosis and fibrosis evident at this low power as pale areas. (C,D) Higher power views of heart from rat given ISO show extensive fibrosis (C, ⫻117) and resolving myocardial necrosis (D, ⫻375). (E,F) Heart section of rat treated with ISO ⫹ DFMO shows only small area of myocardial necrosis and fibrosis (E, arrow) which is confined to the immediate subendocardial area (F). Gomori trichrome stain; (A, B, and E, ⫻8; C and F, ⫻117; D, ⫻375).
ing myocardial ischemia (37), respectively. Excessive levels of circulating catecholamines have been shown to cause myocardial hypertrophy, myocyte damage, and cardiomyopathy (9). ISO acts through -adrenergic receptors, stimulating calcium influx (15), and augmenting the consumption of oxygen and depletion of ATP (16). Its main action is on
the heart, where it produces infarctlike lesions. Isoproterenol-induced myocardial necrosis is associated with dosedependent increases in left ventricular end diastolic pressure, left ventricular volume, and wall stress (33). Isoroterenol given alone has been shown not to cause damage to other organs, although it can potentiate hepatotoxicity by methy-
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Figure 4. Morphometric analysis showing the DFMO attenuation of lesion area in rat hearts exposed to ISO. The data mean ⫾ SD is based upon 4 – 6 rats/group. ISO versus control, *p ⬍ 0.05., ISO versus ISO ⫹ DFMO. **p ⬍ 0.05.
phenidate hydrochloride, a CNS stimulant drug (38). Thus, observed changes in CPK and LDH levels observed in this study would reflect the release of enzymes resulting from ISO-induced myocardial damage. The determinations of CPK and LDH are both useful parameters for assessing myocardial damage (39,40). However, the time when those changes occur may differ due to differences in ISO itself, the dose of ISO administered or the age of the animals. In these experiments, ISO-mediation of myocardial injury was evidenced by increases in CPK and LDH activities in plasma and subendocardial fibrosis in rat hearts. We observed that CPK was released at an earlier phase of ISO damage (6 hr), while the release of LDH occurred at 24–48 hr after ISO administration. Clinical studies also show that CPK is released during an earlier phase of myocardial infarction than LDH, which is released after the infarct has occurred (40). The precise mechanisms by which isoproterenol causes myocardial damage is not fully elucidated, however, the proposed mechanisms include production of ischemia (14), calcium overloading (41), production of free radicals (17), and lipid peroxidation (42–44). In the present investigation, we examined whether inhibition of ISO-induced ODC activity attenuated the myocardial damage in rat heart. Difluoromethylornithine, a specific and irreversible inhibitor of ODC activity (27,28), blocks the synthesis of putrescine and results in depletion of spermidine (18). In humans, DFMO has been used in clinical trials for chemoprevention of cancer. In these studies, the side effects of DFMO in humans include hearing loss (45), diarrhea, and thrombocytopenia (46,47). Studies in rats have shown that chronic administration by gavage at doses greater than 800 mg/kg for 52 weeks produces a significant reduction in organ to body weight ratios of brain, adrenal glands, and liver. DFMO between 800–1000 mg/kg produced necrotizing dermatitis of the skin, coagulation necrosis of the liver, and inflammation of the duodenal mucosa,
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while DFMO dose below 800 mg/kg for 52 days did not produce toxic effects (48). Thus, DFMO given for a total of 8 days in drinking water (2%) and by intraperitoneal administration (200 mg/kg) is unlikely to cause the type of cellular damage noted in those investigations (48). The results of this study show that DFMO inhibition of ISO-stimulated increases in ODC activity and levels of polyamines in rat heart is associated with decreased release of CPK and LDH into plasma and attenuation of fibrosis. These observations suggest that DFMO confers protection against ISO-mediated myocardial damage. Polyamines are generally implicated in cellular proliferation and differentiation (20). Earlier studies by Koenig and colleagues have shown that increases in polyamines in isolated perfused rat heart trigger calcium transport that is associated with loss of contractility, release of myoglobin, and cytosolic enzymes (49). ODC activity is also increased in brain areas that have undergone infarction due to ischemia (50,51). Evidence based on studies using DFMO in cats suggests that polyamines play a role in mediation of neuronal injury, post-traumatic vasogenic edema, and blood brain barrier defects after ischemic injury (52). Spermidine and spermine are oxidatively deaminated by bovine serum amine oxidases to produce aminoaldehydes, ammonia and H2O2 (53). These products of polyamine degradation have been suggested to play a role in the mediation of cytotoxicity (54). For example, ammonia, which is a product of polyamine degradation, has been shown to reduce lymphocyte viability (55), and inhibition of polyamine oxidase has been shown to attenuate the toxicity mediated by spermine to BHK-21/C13 cells (22). Our recent in vitro studies show that both intracellular and extracellular amine oxidases play a role in mediation of polyamine toxicity to cardiac myocytes (25). Our preliminary studies indicate that inhibition of polyamine oxidase by MDL 72.527, a specific inhibitor of polyamine oxidase, attenuates the release of ISO-induced CPK and LDH into plasma. Studies are currently in progress to determine whether any of these mechanisms, or the calcium influx triggered by polyamines, are involved in ISO-mediated cardiac injury. Understanding the role of polyamines in fibrosis is important because this pathological change is a common occurrence in humans with ischemic heart disease and symptomatic heart failure (56). The nonmyocyte population of the heart is made up of fibroblasts, endothelial cells, and smooth muscle cells. These cells produce proteins of the intercellular matrix and cytokines, including transforming growth factor 1 (TGF 1) (57). Using mRNA probes, Eghbali and coworkers (58) have shown that fibroblasts are the source of collagen type I and III. TGF plays an important role in proliferation and phenotypic modulation of fibroblasts (59). Studies in intestinal epithelial cells indicate that polyamines are essential for the expression of TGF- (60). Further studies are needed to determine the specific mechanisms by which polyamines contribute to myocyte injury
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and fibrogenesis in the rat heart and whether polyamines regulate the cardiac fibrosis by regulating the expression of cytokines and phenotypic modulation of nonmyocytes. The authors thank Tom Bedernak for photography and Merrel Hoechst Marion Roussel, Inc., Research Institute for providing DFMO. This research was supported by American Heart Association, National (# 96010090) awarded to Ulka R. Tipnis.
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in ornithine decarboxylase activity and polyamine levels in regions of the rat heart. J Cardiovasc Res XXIII, 1989;23:611–619. 19. Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol 1982;12:C212–C221. 20. Janne J, Alhonen L, Leinonen P. Polyamines: from molecular biology to Clinical applications. Annal Med 1991;23:241–259. 21. Seiler N, Atanassov CL. The natural polyamines and the immune system. In: Jucker E, ed. Progress in Drug Research by von Heraugegeben. Basel, Boston, Berlin: Berkhauser verlag, 1994;43:87–141. 22. Packham G, Cleveland J. Ornithine decarboxylase is a mediator of c-Mycinduced apoptosis. Mol Cell Biology 1994;14:5741–5747.
References 1. Weber KT, Sun Y, Tyagi SC, Cleutjens JP, MJ. Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. Mol. Cell. Cardiol. 1994;26:279–292. 2. Weber KT, Sun Y, Campbell SE, Slight SH, Ganjam VK, Griffing GT, Swinfard RW, Diaz-Arias AA. Chronic mineralocorticoid excess and cardiovascular remodeling. Steroids 1995;60:125–132. 3. Villareal FJ, Mckenna DA, Omens JH, Dillmann WH. Myocardial remodeling in hypertensive Ren-2 transgenic rats. Hypertension 1995;25:98–104. 4. Jordan JE, Zhao Z-Q, Vinten-Johansen J. The role of neutrophils in myocardial reperfusion-injury. Cardiovas Res 1999;43:860–878. 5. Tipnis UR, Chopra A, Campbell G, Boor P. Role of polyamines in atrial natriuretic peptide expression in isoproterenol-induced cardiac hypertrophy in rat. Biomedical Lett 1995;52:81–86. 6. Boluyt MO, Long X, Eschenhagen T, Mende U, Schmitz W, Crow MT, Lakatta EG. Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart. Am J Physiol 1995;269:H638–H647. 7. Rona G, Chappel CI, Balazas T, Gaundry R. An infarct like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. Arch Pathol 1959;67:443–445. 8. Brilla CG, Maisch B, Rupp H, Funck R, Zhou G, Weber KT. Pharmacological modulation of cardiac fibroblast function. Herz 1995;20: 127–134. 9. Rona G. Catecholamine cardiotoxicity. J Mol Cell Cardiol 1985;17: 291–306. 10. Todd GL, Baroldi G, Pieper GM, Clayton FC, Eliot RS. Experimental catecholamine-induced myocardial necrosis I. Morphology, quantification and regional distribution of acute contraction band lesions. J Mol Cell Cardiol 1985;17:317–338. 11. Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, Weber K. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circulation Res 1989;64:1041–1050. 12. Benjamin IJ, Jalil JE, Tan LB, Cho K, Weber KT, Clark W. Isoproterenol induced myocardial fibrosis in relation to myocyte necrosis. Circ Res 1989;65:657–670. 13. Wexler BC, Kittinger GW. Myocardial necrosis in rats: serum enzymes, adrenal steroid and histopathological alterations. Circ Research 1963;13:159–171. 14. Yeager JC, Iams SG. The hemodynamics of isoproterenol-induced cardiac failure in rats. Circ Shock 1981;8:151–163. 15. Bloom S, Davis DL. Calcium as a mediator of isoproterenol-induced myocardial necrosis. Am J Pathol 1972;69:459–470. 16. Fleckenstein A, Janke J, Doring HJ, Leder, O. Myocardial fiber necrosis due to intracellular calcium overload. A new principle in cardiac pathophysiology. In: Dhalla NS, ed. Recent Advances in Cardiac Structure and Metabolism, Vol. 4. Baltimore, MD: University Park Press, 1974;563–580. 17. Singal PK, Beamish RE, Dhalla NS. Potential oxidative pathways of catecholamines in the formation of lipid peroxides and genesis of heart disease. Adv Exp Med Biol 1983;161:391–440. 18. Tipnis UR, Frasier-Scott K, Skiera C. Isoproterenol-induced changes
23. Brunton VG, Grant MH, Wallace HM. Mechanisms of spermine toxicity in baby-hamster kidney (BHK) cells. The role of amine oxidases and oxidative stress. Biochem J 1991;280:193–198. 24. Gaugas JM, Dewey DL. Hog kidney diamine oxidase conversion of biogenic diamines to inhibitors of cell proliferation. Jour Pathol 1981;134:243–252. 25. Harari PM, Fuller DJM, Gerner EW. Heat shock stimulates polyamine oxidation by two distinct mechanisms in mammalian cell cultures. Int J Rad Oncology Biol Phys 1989;16:451–457. 26. Tipnis UR, He GY. Mechanism of polyamine toxicity in cultured cardiac myocytes. Toxicology in Vitro 1998;12:233–240. 27. Metcalf BW, Bey P, Danzin C, Jung MJ, Casara P, Vevert JP. Catalytic irreversible inhibition of ornithine decarboxylase (E.C.1.1.17) by substrate of product analogue. J Am Chem Soc 1978;100:2551–2553. 28. Danzin C, Mamont P. Polymins inhibition in vivo and in organ growth and repair. In: McCann PP, Pegg AE, Sjoerdsma A, eds. Inhibition of polyamine metabolism. Biological significance and basis for new therapies. New York: Academic Press, 1987;141–164. 29. Bergemeyer H-U, Bernt E, Hess B. Lactate dehydrogenase. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis. New York: Weinheim/Bergstr Academic Press, 1965;736–741. 30. Wroblewski F, LaDue JS. Lactate dehydrogenase activity in the blood. Proc Soc Exp Biol Med 1955;90:210–213. 31. Boor P J. Studies of hypotension and cardiac flow in isoproterenolinduced myocardial necrosis. Exp Mol Pathol 1980;34:110–121. 32. Viano DC. Cardiovascular injury from blunt thoracic impact of epinephrine and isoproterenol injected rabbits. Aviat Space and Environmen Med 1983;54:988–993. 33. Teerlink JR, Pfeffer JM, Pfeffer MA. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res 1994;75:105–113. 34. Grimm D, Elsner D, Schunkert H, Pfeiffer M, Griese D, Bruckschlegel G, Muders F, Riegger GAG, Kromer EP. Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system. Cardiovasc Res 1998;37:91–100. 35. Pignatelli D, Magalhaes MM, Magalhaes MC. Direct effect of stress on adrenocortical function. Hormone and Metabolic Research 1998; 30:464–474. 36. Marino F, Sockler JM, Fry JM. Thermoregulatory, metabolic and sympathoadrenal responses to brief exposure to cold. Scand Jour Clin Lab Invest 1998;58:537–545. 37. Schomig A, Dart AM, Dietz R, Mayer E, Kubler W. Release of endogenous catecholamines in the ischemic myocardium of the rat. Part A. Locally mediated release. Circ Res 1984;55:689–701. 38. Roberts SM, Harbison RD, Roth L, James RC. Methylphenidateinduced hepatotoxicity in mice and its potentiation by -adrenergic agonist drugs. Life Sci 1994;55:269–281. 39. Bhargawa AS, Preus M, Gunzel P. Effect of Iloprost on serum creatine kinase and lactate dehydrogenase isoenzymes after isoprenalineinduced cardiac damage in rats. Arzneim-Forsch/Drug Res 1990;40: 248–252. 40. Mair J, Puschendorf B, Michel G. Clinical significance of cardiac contractile proteins for the diagnosis of myocardial injury. In: Spiegel HE, ed. Advances in Clinical Chemistry, Vol. 31. New York: Academic Press, 1994.
280
TIPNIS ET AL. MYOCARDIAL INJURY ATTENUATION
41. Nirdlinger EL, Bramante PO. Subcellular myocardial ionic shifts and mitochondrial alterations in the course of isoproterenol-induced cardiomyopathy of the rat. J Mol Cell Cardiol 1974;6:49–60. 42. Norhona-Dutra AA, Steen-Dutra EM, Woolf N. Epinephrine-induced cytotoxicity in rat plasma. Its effects on isolated cardiac myocytes. Lab Invest 1988;59:817–823. 43. Persoon-Rothert M, Valk-Kokshoorn VD, Egaskenniphas JM, Mauve I, Laarse, VD. Isoproterenol-induced cytotoxicity in neonatal heart cell cultures is mediated by free-radical formation. J Mol Cell Cardiol 1989;21:1285–1291. 44. Kondo T, Ogawa Y, Sugiyama S, et al. Mechanisms of isoproterenolinduced myocyte damage. Cardiovas Res 1987;21:248–254. 45. Croghan MK, Aickin MG, Meyskens FL. Dose-related alpha-difluoromethylornithine ototoxicity. Amer Jour Clin Oncol 1991;14:331– 335. 46. Ajani JA, Ota DM, Grossie VB, Levin B, Nishioka K. Alterations in polyamine metabolism during continuous intravenous infusion of alpha-difluoromethylornithine showing correlation of thrombocytopenia with alpha-difluoromethylornithine plasma levels. Cancer Res 1989;49: 5761–5765. 47. Tietze KJ, Gaska JA, Cosgrove EM. Thrombocytopenia and vomiting due to difluoromethylornithine. Drug Intell Clin Pharm 1987;21:627– 630. 48. Crowell JA, Goldenthal EI, Keloff GJ, Malone WF, Boone CW. Chronic toxicity studies of the potential cancer preventive 2-(difluoromethyl)-dl-ornithine. Fundam Appl Toxicol 1994;22:341–354. 49. Koenig H, Goldstone AD, Trout JJ, Lu CY. Polyamines mediate uncontrolled calcium entry and cell damage in rat heart in the calcium paradox. Jour Clin Invest 1987;80:1322–1331. 50. Dempsey RJ, Roy MW, Meyer K, Tai HH, Olson JW. Polyamines and prostaglandin markers in focal cerebral ischemia. Neurosurgery 1985;17:635–640.
Cardiovasc Pathol Vol. 9, No. 5 September/October 2000:273–280
51. Dempsey RJ, Maley M, Cowen D, Olson JW. Ornithine decarboxylase activity and immunohistochemical localization in postischemic brain. J Cereb Blood Flow Metab 1988;8:843–847. 52. Schmitz MP, Combs D, Dempsey RJ. Difluoromethylornithine decreases postischemic brain edema and blood brain barrier breakdown. Neurosurgery 1993;33:882–888. 53. Mondovi B, Riccio P, Agostineli E. The biological functions of amine oxidases and their reaction products: an overview. Adv Exp Med Biol 1989;250:147–161. 54. Kawase M, Samejima K, Okada, M. Mode of action of 3-substituted propylamine cytotoxicity in culture cells. Biochem Pharmacol 1982;31:2883–2988. 55. Flescher E, Fossum D, Talal N. Polyamine-production of lymphocytotoxic levels of ammonia by human peripheral blood monocytes. Immunology Letters. 1991;28:85–90. 56. Beltrami CA, Finato N, Rocco M, Fergulio GA, Puricelli C, Cigola E, Quaini F, Sonnenblick EH, Olivetti G, Aneversara P. Structural basis of end-stage failure in ischemic cardiomyopathy in humans. Circulation 1994;89:151–163. 57. Eghbali M, Czaja MJ, Zedel M, Weiner FR, Zern MA, Seifter S, Blumefeld OO. Collagen mRNA isolated from young and adult rats. J Mol Cell Cardiol 1988;20:267–276. 58. Eghbali M. Cellular origin and distribution of transforming growth factor-1 in the normal rat myocardium. Cell Tissue Res 1989;256: 553–558. 59. Sigel AV, Centrella M, Eghabali-Webb M. Regulation of proliferative response of cardiac fibroblasts by transforming growth factor-1. J Mol Cell Cardiol 1996;28:1921–1929. 60. Wang JY, Viar MJ, Li J, Shi HJ, McCormack SA, Johnson LR. Polyamines are necessary for normal expression of the transforming growth factor-beta gene during cell migration. Am J Physiol 1997; 272:G713–G720.