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Circulation Research. 1997;80:11-20

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(Circulation Research. 1997;80:11-20.)
© 1997 American Heart Association, Inc.

Articles

Expression of Inducible Nitric Oxide Synthase in Rat Experimental Autoimmune Myocarditis With Special Reference to Changes in Cardiac Hemodynamics

Satoru Hirono, M. Omedul Islam, Mikio Nakazawa, Yutaka Yoshida, Makoto Kodama, Akira Shibata, Tohru Izumi, Shoichi Imai

the Department of Pharmacology (S.H., M.O.I., M.N., Y.Y., S.I.) and the First Department of Internal Medicine (M.K., A.S.), Niigata (Japan) University School of Medicine, and the Department of Internal Medicine (T.I.), Kitasato University School of Medicine, Sagamihara, Japan.

Correspondence to Shoichi Imai, MD, Department of Pharmacology, Niigata University School of Medicine, Asahimachi 1-757, Niigata 951, Japan. E-mail shimai@med.niigata-u.ac.jp


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Excessive NO produced by an inducible NO synthase (iNOS) has been implicated in many types of immune-associated disorders of the cardiovascular system, but it remains to be determined whether NO plays a role in myocarditis. Thus, the significance of iNOS expression in the development of experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, was investigated. Lewis rats were immunized with cardiac myosin and were killed 7, 14, 21, 28, and 49 days after immunization. The development of severe myocarditis was observed on days 14, 21, and 28 in association with significant deterioration of hemodynamics determined by cardiac catheterization, which peaked on day 21. In parallel with histological severity of myocarditis and deterioration of cardiac performance, iNOS activity in the heart measured by [14C]L-citrulline formation was markedly increased on days 14, 21, and 28. The expression of iNOS was confirmed by immunoblotting and was localized to the infiltrating inflammatory cells found in the vicinity of necrotic myocytes by immunohistochemical analysis. Aminoguanidine, a selective inhibitor of iNOS, significantly decreased the iNOS activity (1.04±0.37 compared with 29.1±8.62 pmol·min-1·mg protein-1 in untreated myosin-immunized rats, P<.01) and effectively attenuated histopathological changes of EAM on day 21. Hemodynamic parameters were also improved from 64±3 to 89±3 mm Hg for mean blood pressure, from 80±2 to 113±4 mm Hg for left ventricular systolic pressure, from 7.8±0.3 to 3.2±0.3 mm Hg for left ventricular end-diastolic pressure, from 2867±137 to 4180±102 mm Hg/s for +dP/dt, and from 2717±132 to 4180±184 mm Hg/s for -dP/dt (P<.01). The values after aminoguanidine treatment were not significantly different from the control values. These results suggest an important role for NO in mediating pathophysiological changes in myocarditis of autoimmune origin.


Key Words: myocarditis • cardiomyopathy • nitric oxide synthase • hemodynamics • aminoguanidine


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
In recent years, NO has been implicated in the pathogenesis of a wide variety of immunologically mediated disorders.1 2 3 4 5 6 7 8 9 A Ca2+-independent inducible form of NO synthase (iNOS), which is not constitutively present, becomes expressed in macrophages and many other cell types via immunological stimuli and produces a large amount of NO for an extended period.10 Nonspecific cytotoxicity of excessive NO produced by this enzyme is considered to be exerted even in host cells10 11 12 and to lead to immune-associated tissue injury. Pathological release of NO may also be the cause of diminished myocardial contractility during certain circumstances in which significant activity of iNOS is expressed, such as septic shock,13 14 cardiac allograft rejection,4 5 and some form of idiopathic dilated cardiomyopathy.2 3 However, the precise role played by NO in myocarditis has not yet been investigated.

Human myocarditis varies greatly in origin and in clinical manifestation.15 16 However, the disease is generally a self-limiting process; acute congestive heart failure may develop during the active phase, but cardiac function improves significantly in most cases as myocarditis resolves histologically.15 16 17 Nevertheless, myocardial damage remains to a significant degree in some patients who survive the diseases and may lead to myocardial fibrosis with postmyocarditic cardiac dysfunction,18 19 indicating that myocyte injury during the active phase of myocarditis can range from reversible modulation of myocyte contractile function to cell death.20 21 It is possible that the excessive production of NO by iNOS is responsible for the development of myocardial organic lesions as well as for functional changes in myocardial contractility during the active phase of the disease.

To assess the possible importance of NO produced by iNOS in the pathophysiology of human myocarditis, an attempt was made in the present study to demonstrate the expression and the localization of biologically active iNOS in the myocardium of Lewis rats with EAM produced by immunization with cardiac myosin.22 23 Extensive myocardial necrosis associated with the appearance of multinucleated giant cells and fatal congestive heart failure are the characteristic features of EAM, suggesting a close relation of this model to a fulminant form of human myocarditis, such as giant cell myocarditis.22 23 24 25 26 This model of myocarditis has also been demonstrated to develop into postmyocarditic dilated cardiomyopathy in the chronic phase, of which the histopathological findings closely resemble those of some form of human idiopathic dilated cardiomyopathy.27

In the present study, iNOS activity was found to be expressed in inflammatory cells infiltrating into the myocardium during the active phase of EAM, in association not only with the histopathological severity of myocarditis but also with the deterioration of cardiac function. Furthermore, a selective inhibitor of iNOS, AG,28 29 effectively suppressed both histological and hemodynamic manifestations of EAM.


*    Materials and Methods
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 *Materials and Methods
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Animals
Male Lewis rats were purchased from Charles River Japan Inc (Atsugi, Japan) and maintained under the specific pathogen-free conditions at the Facilities for Comparative Medicine and Animal Experimentation, Niigata (Japan) University School of Medicine.

Antigen
Purified cardiac myosin from the ventricular muscle of pig hearts was prepared according to a procedure previously described15 and used as an antigen.

Immunization
The antigen was dissolved at a concentration of 20 mg/mL in PBS containing 0.3 mol/L KCl and mixed with an equal volume of CFA containing 11 mg/mL of Mycobacterium tuberculosis (Difco Laboratories). Rats (11 to 13 weeks old) were injected into their footpads with 0.2 mL SC of antigen-adjuvant emulsion (n=110). Another group of age-matched rats (control group) was injected with 0.1 mL of saline mixed with an equal volume of CFA (n=30).

AG Treatment
Thirty myosin-immunized rats were randomly assigned to treatment with a selective inhibitor of iNOS, AG hemisulfate29 (Sigma Chemical Co), at two different stages of the disease.30 31 Intraperitoneal injection of AG (600 mg/kg per day) was conducted beginning with the day of immunization (day 0) to day 10 (n=15) or from days 11 to 21 (n=15).

Instrumentation
Six myosin-immunized and 5 control rats were randomly chosen and anesthetized with 1.0% to 1.5% halothane on days 7, 14, 21, 28, and 49 for instrumentation. Rats treated with AG from days 0 to 10 (n=9) and from days 11 to 21 (n=10) were anesthetized on day 21 in the same manner. For determination of the arterial blood pressure and the central venous pressure, polyethylene catheters (PE-50) connected to pressure transducers (Statham P50) were introduced into the right femoral artery and the vena cava superior through the right jugular vein. A Mikro-Tip pressure transducer (Millar Instruments) was introduced into the left ventricle through the left carotid artery for determination of the left ventricular pressure and its first derivative by means of a differentiator (NEC San-ei Instruments). Heart rate was calculated from the electrocardiogram (standard limb lead II).

Hemodynamic Study
As indexes of hemodynamics, heart rate, central venous pressure, mean arterial pressure, left ventricular systolic pressure, LVEDP, and both peak positive and negative dP/dt were used. Anesthesia was maintained with 0.5% halothane in O2, and the indexes were recorded simultaneously during spontaneous ventilation after an equilibration period of a minimum of 20 minutes.

Histopathology
After measurement of hemodynamic parameters, all rats were killed by KCl injection. At thoracotomy, macroscopic findings and the amount of pericardial effusion were recorded. Macroscopic findings were scored according to the following grades22 23 : 0, normal; 1, focal discolored area present; and 2, multiple or diffuse discolored areas present on the cardiac surface. The amount of pericardial effusion was scored as follows: 0, normal; 1, a small pericardial effusion <1.0 mL; 2, a moderate pericardial effusion <3.0 mL; and 3, a massive pericardial effusion >3.0 mL. Hearts were then excised above the origin of the great vessels and weighed immediately to calculate the heart weight–to–body weight ratio. The removed hearts were fixed in 10% formalin, then embedded in paraffin, and sliced at several levels to make transverse sections for histopathological study. Microscopic findings were expressed in terms of infiltration score and fibrosis score. The extent of inflammatory cell infiltration and myocyte necrosis was estimated using hematoxylin-eosin staining, and the extent of fibrosis was estimated using Azan-Mallory staining. The size of the lesion was graded22 23 as follows: 0, normal; 1, few small lesion not exceeding 0.25 mm2 present; 2, multiple small or few moderately sized lesions not exceeding 6.25 mm2 present; and 3, multiple moderately sized lesions or larger lesions exceeding 6.25 mm2 present. Microscopic findings were examined by three of the authors; two of them had no knowledge of the protocol.

Sample Preparation for NO Synthase Assay
Myosin-immunized rats were randomly selected and killed under ether anesthesia on days 7, 14, 21, 28, and 49 (n=5, each day). Control rats (n=5) and myosin-immunized rats treated with AG from days 0 to 10 (n=5) and from days 11 to 21 (n=5) were killed on day 21 in the same manner. The hearts were removed and cut into 2- to 3-mm-thick transverse slices. The left ventricular myocardium excised from the slices was washed thoroughly with ice-cold Krebs-Henseleit buffer, freeze-clamped in liquid nitrogen, and stored at -80°C. Some slices of the hearts from myosin-immunized and control rats were snap-frozen in Tissue-Tek OCT compound (Miles Inc) for immunohistochemical analysis.

Determination of NO Synthase Activity
NO formation was determined by measuring the production of radiolabeled [14C]L-citrulline from [14C]L-arginine using a method essentially the same as that described by Knowles et al.32 The freeze-clamped segments were homogenized and centrifuged at 100 000g for 30 minutes. The cytosolic fractions of the left ventricular myocardium were incubated in duplicates with 300 µmol/L [14C]L-arginine (radioactivity, 0.05 Ci/mmol; Du Pont/NEN Research Products) and 1 mmol/L EGTA in the presence or absence of 1 mmol/L NG-methyl-L-arginine (Molecular Probes, Inc) to determine the level of Ca2+-independent NO synthase activity. A portion of the cytosolic fraction was separately stored for immunoblotting analysis and for protein assay. Protein concentration was determined by a modified Lowry method with bovine serum albumin (Sigma) used as a standard.33

Immunoblotting Analysis
The cytosolic fractions from the homogenized left ventricular myocardium (50 µg protein/20 µL per lane) were separated on 6% SDS–slab polyacrylamide gel and then electrophoretically transferred to Clear Blot membrane-P (Atto). Nonspecific binding was blocked by incubation in PBS-T and 5% defatted milk powder for 2 hours at room temperature. The membrane was then incubated overnight at 4°C in PBS-T containing 5% defatted milk powder and 2 µg/mL rabbit polyclonal antibody specific to iNOS (Upstate Biotechnology Inc). The selectivity of this antibody to iNOS was confirmed by Western immunoblot and immunoprecipitation and determined on activated RAW macrophages (Upstate Biotechnology Inc). A horseradish peroxidase–conjugated anti-rabbit IgG (Amersham) at a dilution of 1:1000 was used as a secondary antibody, and an immunologically cross-reacting band was visualized with the enhanced chemiluminescence assay kit (Amersham).34

Immunohistochemistry
Frozen sections (4 µm) of the hearts were fixed in acetone, washed in PBS, and incubated with 5% normal goat serum (IBL) for 20 minutes at room temperature. The tissue sections were then incubated in PBS containing 5 µg/mL of the rabbit anti-iNOS antibody (Upstate Biotechnology Inc) for 1 hour, followed by sequential incubations with goat anti-rabbit IgG (IBL) diluted to 1:200 for 30 minutes and peroxidase–rabbit anti-peroxidase antibody complex (IBL) diluted to 1:100 for 30 minutes.35 The reaction products were visualized using 0.5% diaminobenzidine and 0.03% hydrogen peroxide. Sections were counterstained with hematoxylin.

Statistical Analysis
Data were presented as mean±SE. Statistical assessment of the significance among groups was made by one-way ANOVA followed by Tukey's method. Student's t test was used for the comparison between two groups. Differences were considered significant at P<.05.


*    Results
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 up arrowMaterials and Methods
 *Results
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Clinical Course of Myosin-Immunized Rats
All 70 untreated myosin-immunized rats became ill and immobile after the second week following immunization. Fifteen of the rats died from days 20 to 40, 11 died during the active phase, and 4 died during the healing phase of disease. All these rats showed macroscopic evidence of severe myocarditis and congestive heart failure at autopsy and thus were judged to have died of heart failure. Fig 1Down shows a schema detailing the clinical course and death of the animals during the experimental period. No control rats died spontaneously throughout the period of experiments.



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  		Figure 1. Clinical course and experimental protocols. 1, Diagrammatic representation of the histopathological changes and the clinical course of EAM. Rats that died of heart failure during the experimental period were plotted on the chart as closed circles (untreated myosin-immunized rats) and an open circle (myosin-immunized rat treated with AG from days 0 to 10). No control rats or myosin-immunized rats treated with AG from days 11 to 21 died spontaneously. 2 and 3, Experimental protocol for hemodynamic study and histopathological analysis (2) and iNOS assay (3). Rats were randomly injected either with antigen-adjuvant emulsion (n=110) or with saline-adjuvant emulsion (n=30). Thirty of 110 myosin-immunized rats were assigned to treatment with AG either from days 0 to 10 or from days 11 to 21. Rats from each group were killed according to the protocol.

Hemodynamic Study
The hemodynamic parameters of myosin-immunized and control rats determined by cardiac catheterization on days 7, 14, 21, 28, and 49 are summarized in Table 1Down. Central venous pressure was significantly higher in myosin-immunized rats than in control rats on day 21. Mean arterial pressure, left ventricular systolic pressure, and both peak positive and negative dP/dt were significantly lower in myosin-immunized rats compared with control rats on days 14, 21, and 28, and LVEDP was significantly higher on days 14, 21, 28, and 49. Changes peaked on day 21, indicating that deterioration of hemodynamics in EAM proceeded until day 21. Except for LVEDP, the hemodynamic parameters recovered to the control level on day 49.


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  		Table 1. Time Course of Changes in Hemodynamic Parameters of Rats After Immunization

Macroscopic Findings
The extent of discolored areas and the amount of pericardial effusion are summarized in Table 2Down. The hearts of all control rats and myosin-immunized rats on day 7 had a normal appearance. On days 14, 21, 28, and 49, the hearts of myosin-immunized rats were markedly enlarged, with grayish-colored areas on the surface. On day 49, all six myosin-immunized rats had dilated ventricular chambers and thinner ventricular walls compared with control rats, and two of them showed aneurysmal changes of the right ventricle. Pericardial effusion was observed in all 6 myosin-immunized rats on day 21, in 5 of 6 rats on days 14 and 28, and in 3 of 6 rats on day 49. The heart weight–to–body weight ratios (percentage) of myosin-immunized rats were as follows: 0.283±0.011 on day 7, 0.505±0.052 on day 14, 0.565±0.014 on day 21, 0.496±0.045 on day 28, and 0.371±0.046 on day 49; thus, these ratios were significantly higher compared with control rats on days 14, 21, 28, and 49.


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  		Table 2. Time Course of Changes in Histopathology in Rats After Immunization

Microscopic Findings
Microscopic findings were summarized in Table 2Up. There were no microscopic abnormalities in the hearts of the control rats (Fig 2CDown) and the myosin-immunized rats on day 7. By day 14, a large number of mononuclear cells infiltrated into the myocardium of myosin-immunized rats, forming clusters at the sites of necrotic myocytes. Myocardial necrosis and inflammatory cell infiltrations were more extensive on day 21 (Fig 2ADown), and interstitial edema became evident. Thereafter, active inflammation subsided gradually, and fibrosis became prominent on day 28. Multinucleated giant cells were frequently observed on days 21 and 28. Most of infiltrated cells disappeared on day 49, and the inflammatory lesions were replaced by fibrosis (Fig 2BDown); focal accumulations of mononuclear cells were rarely detected.





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  		Figure 2. Histological sections of the hearts of myosin-immunized and control rats. A, On day 21, myosin-immunized rats had severe myocarditis characterized by extensive myocardial necrosis with inflammatory cell infiltrations and interstitial edema. Multinucleated giant cells were frequently observed (hematoxylin-eosin staining, bar=100 µm). B, Myocardial fiber loss and replacement fibrosis were observed on day 49 (Azan-Mallory staining, bar=100 µm). C, A representative histological section from a control rat on day 21 is shown. There was no histological evidence of myocarditis (hematoxylin-eosin staining, bar=100 µm).

NO Synthase Activity in Myocardium
Fig 3Down (left) shows the time course of changes in activities of Ca2+-independent NO synthase, ie, iNOS, in cytosolic fractions obtained from the left ventricular myocardium of myosin-immunized rats. In control rats, iNOS activity was very small or even absent (0.43±0.15 pmol·min-1·mg protein-1). A marked increase in iNOS activity was observed beginning at day 14 in myosin-immunized rats, with a peak on day 21 (29.1±8.62 pmol·min-1·mg protein-1). The activity returned gradually to control levels by day 49. Activity of iNOS was associated with histological changes and the extent of inflammatory cell infiltration.



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  		Figure 3. Time course of changes in enzyme activity and corresponding protein expression of iNOS in the hearts of rats after immunization. Left, Each column represents mean±SE of iNOS activity in 100 000-g supernatants (cytosolic fractions) of the homogenized left ventricular myocardium, as measured by production of radiolabeled [14C]L-citrulline from [14C]L-arginine (*P<.05, **P<.01). Right, The cytosolic fractions from the myocardium of a control rat on day 21 (lane 1) and myosin-immunized rats on days 7, 14, 21, 28, and 49 (lanes 2, 3, 4, 5, and 6, respectively) were adjusted to a protein content of 50 µg per lane and separated on a 6% SDS–polyacrylamide gel. The gel was processed for immunoblotting analysis using an antibody specific to iNOS.

Immunoblotting Analysis
On immunoblotting with an anti-iNOS antibody, a band with a molecular mass of {approx}130 kD (corresponding to the estimated molecular weight of iNOS protein) was clearly detected in hearts of myosin-immunized rats on days 14, 21, and 28 (Fig 3Up, right; lanes 3, 4, and 5, respectively). The band was not detected in hearts of myosin-immunized rats on days 7 and 49 (lanes 2 and 6, respectively). Nor was it detected in hearts of control rats (lane 1). The expression level of iNOS protein in myocardium appeared to correlate with its enzyme activity.

Immunohistochemical Analysis
Immunostaining for iNOS was negative in the hearts of control rats and myosin-immunized rats on day 7. On day 14, iNOS was demonstrated exclusively in large mononuclear cells infiltrating into the myocardium of myosin-immunized rats. On days 21 and 28, infiltrations of inflammatory cells became more extensive; however, cells expressing iNOS were found only at sites of active inflammation in contact with necrotic myocytes (Fig 4Down). Focal accumulations of mononuclear cells were still sparsely observed on day 49; however, they were not stained for iNOS. No iNOS was detected in cardiac endothelial cells, vascular smooth muscle cells, or myocytes throughout the experimental period.



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  		Figure 4. Immunohistochemistry of iNOS in the hearts of myosin-immunized rats on day 21. A, Extensive inflammatory cell infiltration and myocyte necrosis were observed. Among these inflammatory cells, the invading mononuclear cells around the border of active inflammatory sites were stained for iNOS (counterstained with hematoxylin, bar=120 µm). B, Infiltrating large mononuclear cells in the vicinity of necrotic myocytes were stained intensely. Myocytes themselves were not stained for iNOS (bar=30 µm).

Effects of AG on the Clinical Course of Myosin-Immunized Rats
All 15 myosin-immunized rats treated with AG from days 0 to 10 had ruffled fur and crouched immobile in cages after the second week; one of them died on day 20, and its heart showed severe myocarditis at autopsy. No rats treated with AG from days 11 to 21 became ill and died; rats were killed for study on day 21.

Effects of AG on Hemodynamic Parameters of Myosin-Immunized Rats
Hemodynamic parameters were significantly improved in rats treated with AG from days 11 to 21 compared with untreated myosin-immunized rats on the same day of illness; also, they did not differ significantly from the parameters in control rats (Table 3Down). However, there were no detectable differences in hemodynamic parameters between untreated myosin-immunized rats and myosin-immunized rats treated with AG from days 0 to 10 (Table 3Down).


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  		Table 3. Effects of AG on Hemodynamic Parameters of Myosin-Immunized Rats on Day 21

Effects of AG on Histopathology in Myosin-Immunized Rats
The hearts of 7 of 10 myosin-immunized rats treated with AG from days 11 to 21 were microscopically normal on day 21, and the remaining 3 rats showed only a focal discolored area on its surface. Inflammatory cell infiltrations and myocardial necrosis were clearly reduced (Table 4Down); only small clusters of inflammatory cells were observed among myocytes, some of which were necrotic (Fig 5BDown). In contrast, AG given from days 0 to 10 failed to ameliorate macroscopic and microscopic severity of myocarditis on day 21 (Table 4Down and Fig 5ADown). The heart weight–to–body weight ratio of myosin-immunized rats treated with AG from days 11 to 21 was significantly lower compared with that of rats treated from days 0 to 10 (0.314±0.023% versus 0.493±0.038%, P<.01) and untreated myosin-immunized rats (P<.01).


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  		Table 4. Effects of AG on Histopathology in Myosin-Immunized Rats on Day 21




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  		Figure 5. Histological sections of the hearts of myosin-immunized rats treated with AG. A, The hearts of rats treated with AG from days 0 to 10 showed severe myocarditis on day 21. B, The histopathology of myocarditis was attenuated by AG when given from days 11 to 21. There was a marked reduction in cellular infiltrations, myocardial necrosis, and interstitial edema (hematoxylin-eosin staining, bar=100 µm).

Effect of AG on iNOS Activity in Myocardium
The iNOS activity in the myocardium of rats treated with AG from days 0 to 10 markedly increased on day 21 (21.4±5.02 pmol·min-1·mg protein-1) and did not differ significantly from that of untreated myosin-immunized rats on the same day of illness. In contrast, AG given from days 11 to 21 significantly decreased iNOS activity (1.04±0.37 pmol·min-1·mg protein-1) compared with that of untreated myosin-immunized rats and myosin-immunized rats treated with AG from days 0 to 10 (P<.01).


*    Discussion
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In the present study, severe myocarditis characterized by extensive myocardial necrosis with inflammatory cell infiltrations and interstitial edema developed consistently in myosin-immunized rats on and after day 14 and persisted over 2 weeks. Diffuse loss of myocardial muscle and replacement fibrosis were observed on day 49. Some rats died of congestive heart failure during the period. These results are in agreement with those of our previous study.27

Cardiac catheterization conducted during the active phase of myocarditis disclosed marked reductions in mean arterial pressure, left ventricular systolic pressure, and peak positive and negative dP/dt and a rise in LVEDP. However, hemodynamic parameters recovered to the control level as histological signs of myocarditis resolved, except for LVEDP, which remained high throughout the entire experimental period. Thus, cardiac dysfunction during the active phase of EAM was associated with histological severity of myocarditis and found to be reversible to a certain degree. Myocardial fibrosis was found in association with a persistent increase in LVEDP observed during the chronic phase of EAM. The course of events inclusive of the persistent rise of LVEDP appears to be compatible with that of severe forms of human myocarditis: most patients recover with a considerable improvement of cardiac function; nevertheless, various degrees of functional impairments remain in some patients as sequelae.15 16 17 18 19 The persistent derangement of cardiac function may be due to the presence of irreversibly damaged tissues.

Because recent studies have implicated NO in many types of immune-associated disorders of the cardiovascular system,1 2 3 4 5 13 14 it was attempted in the present study to clarify the participation of NO in the production of autoimmune myocarditis and in the accompanying mechanical dysfunction. The activity of iNOS in the left ventricular myocardium as measured by [14C]L-citrulline formation was found to be markedly increased during the active phase of EAM, and immunoblotting with an anti-iNOS antibody revealed an abundant expression of iNOS protein in the myocardium. Among numerous inflammatory cells, such as lymphocytes, macrophages, and neutrophils infiltrating the active inflammatory site of the heart, only large mononuclear cells found in the vicinity of necrotic myocytes were stained with an anti-iNOS antibody, suggesting the important role played by iNOS in the production of myocardial lesions.

It is known that NO produced by iNOS in activated macrophages in response to immunological stimuli exerts direct cytostatic and cytotoxic actions on target cells,10 11 actions potentially beneficial for host defense. The biochemical basis for the cytostasis and cytotoxicity induced by NO is most likely its capacity to combine with iron-containing enzymes involved in both the respiratory cycle and pathway for the synthesis of DNA.10 36 37 Although NO itself is cytotoxic, NO can also react with other free radicals to generate molecules, such as peroxynitrite, which enhance its cytotoxicity.38 However, NO when produced in an excessive amount and for an extended period of time can also be cytotoxic for host cells10 11 12 and may bring about an immune-associated tissue injury. It is thought to play a part in the pathogenesis of a wide variety of immunologically mediated disorders.4 5 6 7 8 9 10 12 Pathological release of NO produced by iNOS may also be responsible for the deterioration of cardiac function that occurs in association with immunologically mediated disorders of the cardiovascular system, such as septic shock,13 14 cardiac allograft rejection,4 5 and myocarditis. Although this hypothesis is supported by in vitro observations,13 14 39 the precise role played by iNOS in mediating in vivo hemodynamic changes during the active phase of myocarditis has not been investigated. In the present study, not only the severity and extent of myocarditis (as revealed histologically) but also deterioration of hemodynamics seem to be associated with the increased expression of iNOS in the myocardium during the active phase of EAM, with a peak change on day 21.

To confirm whether excessive NO produced by iNOS plays an important role in mediating EAM, we then investigated the effects of a selective inhibitor of iNOS, AG, on the development of the disease. In our preliminary study, AG given from days 0 to 21 successfully prevented the development of EAM on day 21 (n=5).40 Thus, the present study was designed to extend these initial observations and to elucidate the contribution of iNOS in mediating EAM at different stages of the disease. EAM has been shown to be a T cell–mediated autoimmune disease41 ; the initial immune response after antigen challenge probably involves the activation and proliferation of cardiac myosin-reactive myocarditogenic T-cell clones. Recruitment of myocarditogenic T cells to the target organ is the next stage, followed by effector-target interaction, which may involve macrophages, polymorphonuclear leukocytes, and various inflammatory mediators.31 It has been demonstrated that inflammatory cell infiltration with myocyte necrosis was observed histologically on day 1342 and thus could be regarded as the onset of myocarditis. There is no evidence of myocarditis on day 10; however, day 10 is crucial for the initiation of effector-target interaction in EAM, because a few OX6-positive mononuclear cells gathered in the perivascular space of the small vessels were first observed immunohistologically.42 These cells were major histocompatibility complex class II antigen–expressing dendritic cells that would be the primary target for myosin-reactive T cells.42 Therefore, in the present study, AG was systemically administered to myosin-immunized rats either from days 0 to 10 and from days 11 to 2130 31 to elucidate the contribution of iNOS in two different stages of immune response of EAM. AG given from days 11 to 21 successfully decreased iNOS activity and ameliorated both histopathological and functional changes in the hearts of rats with EAM on day 21; by contrast, AG given from days 0 to 10 was without apparent effect on the subsequent course of the development of myocarditis. These observations may suggest that NO produced by iNOS plays an important role in the effector stage of EAM but has less effect on the initial immune responses induced by antigen challenge. Recently, it has been shown that treatment with AG significantly reduced inflammatory demyelinations in spinal cords of mice with EAE.8 Since antigen-specific proliferation and EAE transfer were not affected by treatment with AG, it was concluded that AG did not act by affecting EAE-inducing T-cell clones but was effective at the effector stage of EAE.8 Although it has not been confirmed whether the same could be applied to EAM, these results may support our observations.

The decreased inflammatory cell infiltration was the intriguing finding with AG treatment from days 11 to 21, indicating that iNOS inhibition also had suppressed the underlying immune processes that would occur in this model on and after day 11 and not just the manifestations of excessive NO production due to iNOS induction in these cells. There is increasing evidence that NO plays a complex role in the modulation of inflammatory response.12 Several in vitro studies have demonstrated that NO synthase inhibitors could attenuate leukocyte-directed chemotaxis.43 44 45 More recently, it was reported that myocardial and systemic vascular endothelial barrier dysfunction during early cardiac allograft rejection was mediated by NO,46 which may enhance inflammatory response through the migration of inflammatory cells into the target organ and edema formation. These results may partly explain the reduced inflammatory cell infiltration with AG treatment observed in the present study and suggest a possible role for NO in recruitment of these cells to the target organ.

AG has been demonstrated to be a potent selective inhibitor of iNOS,28 29 but its selectivity for iNOS appears to be rather limited in certain experimental systems.47 A high concentration of AG is also known to produce a dose-dependent inhibition of endothelial constitutive NO synthase. Thus, the possibility that other isoforms of NO synthase were affected by AG cannot be ruled out. However, as its limited pressor effects in anesthetized rats48 demonstrate, the compound can be taken to be a selective inhibitor of iNOS at the dose used in the present study (600 mg/kg per day). AG has other well-characterized effects in addition to its inhibition of NO synthase, including inhibition of diamine oxidase49 and aldose reductase,50 inhibition of polyamine catabolism,51 and oxidative modification of low-density lipoprotein.52 It is also known to inhibit advanced glycosylation end-product formation in diabetes.53 None of these effects has a known role in the pathogenesis of EAM; thus, it would appear unlikely that these effects played a role in the prevention of the disease.

It is well established that physiological actions of NO are mediated through the activation of soluble guanylate cyclase and the consequent elevation of the intracellular cGMP level.10 Recent studies suggest that NO can modulate myocardial contractility through an increase in myocardial cGMP.13 It is reported that cGMP produces inhibition of Ca2+ influx into myocytes54 55 or attenuation of the positive inotropic effect of ß-adrenergic stimulation.56 However, it has not yet been determined whether the elevation of cGMP produced by NO can induce negative inotropic effects on the myocardium.57 58

Cytotoxicity of NO released from activated macrophages is thought to be mediated in part by its high affinity for iron-containing enzymes, such as aconitase in the citric acid cycle and complex I and II in the mitochondrial respiratory chain,10 11 36 37 which would result in an inhibition of aerobic energy metabolism and a reduction of ATP production in target cells. This metabolic inhibition by NO appears to be reversible, since enzymatic activity of aconitase could be restored by reconstitution of the iron-sulfur cluster.44 Accordingly, it is possible that excessive and prolonged production of NO by iNOS in infiltrating inflammatory cells modulates the mitochondrial respiration in surrounding viable myocytes, thus exerting reversible cardiac dysfunction.

NO is reported to play a part in edema formation at the site of inflammation by increasing local blood flow and vascular permeability59 60 and promoting leukocyte infiltration. It is hypothesized that myocardial interstitial edema is responsible, at least in part, for the decreased chamber compliance during acute rejection following cardiac transplantation.20 61 Conceivably, overproduction of NO in the myocardium may affect the diastolic function of the heart with myocarditis through the formation of interstitial edema.

Treatment of human myocarditis has been essentially palliative and leaves much to be desired.15 16 17 Although the results of the present study may provide a new treatment option for the disease, due caution seems to be necessary in applying this new regimen to humans. Needless to say, NO formation in macrophages and many other cell types is a primary defense mechanism against microbial organisms, including parasites, bacteria, and viruses.10 NO synthase inhibition in the early phase of viral myocarditis may alter the adequate immune response to invading cardiotropic virus, thus preventing the elimination of virus from the cardiac tissue. Furthermore, continuous release of NO by vascular endothelial cells plays an important role in the regulation of vascular tone and platelet aggregation as well as in the regulation of leukocyte adhesion to endothelium,10 62 an essential step in the development of acute inflammation. A successful regimen should inhibit the pathological production of NO during active myocarditis but spare the physiologic roles of NO produced by the vascular endothelium.

In summary, we have demonstrated that the induction of biologically active iNOS in inflammatory cells infiltrating the myocardium may play a critical role in the pathophysiology of EAM, a model of human myocarditis. Selective inhibition of iNOS may be useful in the treatment of human myocarditis of autoimmune etiology.


*    Selected Abbreviations and Acronyms
 
AG = aminoguanidine
CFA = complete Freund's adjuvant
EAE = experimental autoimmune encephalomyelitis
EAM = experimental autoimmune myocarditis
iNOS = inducible NO synthase
LVEDP = left ventricular end-diastolic pressure
PBS-T = PBS containing 0.1% Tween 20


*    Acknowledgments
 
We wish to thank Dr Tadashi Yamamoto, Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, for immunohistochemical analyses. We also thank Kan Yoshida for technical assistance.


*    Footnotes
 
Correspondence to Satoru Hirono, MD, First Department of Internal Medicine, Niigata University School of Medicine, Asahimachi 1-757, Niigata 951, Japan.

Received April 1, 1996; accepted October 4, 1996.


*    References
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