Low-Dose Nω-Nitro-l-Arginine Methyl Ester Treatment Improves Survival Rate and Decreases Myocardial Injury in a Murine Model of Viral Myocarditis Induced by Coxsackievirus B3
Abstract Recent reports demonstrated the expression of inducible-type NO synthase in the heart of viral myocarditis. Since NO has multiple biological actions, a substantial amount of NO produced in the diseased heart may act either as a cytotoxic or as a cytoprotective molecule in the process of myocarditis. In the present study, we examined the effect of inhibition of NO synthesis on the mortality and the extent of myocardial injury in a murine model of coxsackievirus B3–induced myocarditis. We fed the infected mice drinking water containing a relatively low concentration (0.37 mmol/L) of Nω-nitro-l-arginine methyl ester (L-NAME) for 14 days after virus inoculation. This dose of L-NAME did not change virus titers in the heart. However, L-NAME–fed mice showed a significant reduction in mortality compared with those fed normal drinking water (nontreated mice). On the contrary, mice given a higher concentration of L-NAME (3.7 mmol/L) exhibited increased mortality. In addition, mice fed a low concentration of L-NAME showed reductions in the severity of heart failure and in the area of myocardial necrosis. Although systemic blood pressure was reduced in nontreated mice, in mice fed a low concentration of L-NAME, it was maintained at a level similar to that in uninfected control mice. L-NAME–treated mice also exhibited a reduction in the degree of inflammatory cell infiltration associated with decreased production of tissue prostaglandin E2 levels in the heart compared with nontreated mice. Therefore, NO is likely to be involved in the pathogenic mechanisms of myocardial injury and resultant cardiac dysfunction in a murine model of coxsackievirus B3–induced viral myocarditis.
Nitric oxide is a molecule with multiple biological actions and is known to be important in a variety of physiological and pathological processes.1 2 3 Besides acting as a vasodilatory substance and a neurotransmitter, NO participates in the immune and inflammatory systems. NO is synthesized by NOS from l-arginine in a variety of cell types. The biochemical and molecular studies identified three types of isoforms of NOS: constitutive endothelial, neuronal, and inducible.4 5 6 7 8 Recently, we have reported that iNOS mRNA and protein are expressed in the heart in the murine model of viral myocarditis induced by CVB3.9
In viral myocarditis, the myocytes are first damaged by the direct cytolytic effect of the virus after viral infection.10 Then, the cell-mediated immunological responses play a predominant role in the inflammatory process and subsequent myocardial damage. Natural killer cells and cytotoxic T cells are suggested to be involved in the mechanisms.11 12 However, biochemical mechanisms of cell-mediated immune responses are still not well understood. In addition to its action as a vasodilator to maintain tissue perfusion, NO has been reported to exert an inhibitory effect on viral replication. NO is also shown to act as a cytotoxic molecule, modulate cardiac contractility, and influence cardiac function.13 14 15 16 Because of these multiple actions, NO produced in the inflammatory tissue may play either a protective or a detrimental role in the pathogenesis of viral myocarditis. A recent report showed that inhibition of NOS enhanced viral replication and resulted in an increase in the mortality in the murine model of viral myocarditis.17 On the other hand, another report suggested the cytotoxic effect of NO in the murine model of viral myocarditis.18 In a model of autoimmune myocarditis in rats, NO contributed to the progression of myocardial damage.19 20
The present study was undertaken to evaluate the effect of long-term supplementation of a NOS inhibitor after virus infection on the extent of myocardial injury and on the mortality rate in a murine model of viral myocarditis induced by CVB3. For this purpose, we fed mice a relatively low concentration of the NOS inhibitor L-NAME in their drinking water to yield low-dose L-NAME treatment (0.37 mmol/L). The results of the present study demonstrated that low-dose L-NAME treatment improved the mortality and decreased myocardial injury, whereas high-dose L-NAME (3.7 mmol/L) increased mortality; the study also suggested that NO acts as an important modulating factor of myocardial inflammation and injury in viral myocarditis.
Materials and Methods
The method used was similar to that described in our previous report.9 CVB3 (Nancy strain), a gift from Dr Hara (National Institute of Hygienic Science, Tokyo, Japan), was grown in Vero cells in MEM containing fetal bovine serum. The virus preparation had a titer of 1×107 pfu/mL and was stored at −130°C until use.
Animals and Virus Inoculation
Three-week-old C3H/He male mice (Nippon Clea Co, Osaka, Japan) were inoculated intraperitoneally with either 1×105 or 5×106 pfu CVB3 in 0.5 mL PBS. For examining mortality rate after virus inoculation, we used 5×106 pfu, which was revealed to produce death in 50% of the animals in the preliminary study. For examinations of histopathological changes in the heart, systemic blood pressure, virus titers, and PGE2 levels, we inoculated mice with 1×105 pfu CVB3, which resulted in low mortality rate (≈5%). The day of inoculation was defined as day 0 in the following study.
Effect of L-NAME on Mortality After Viral Inoculation
We examined the effect of inhibiting NO production during the process of viral myocarditis on mortality. After receiving 5×106 pfu CVB3, mice were randomly classified into two groups, a nontreated group and a group treated with L-NAME. Feeding of L-NAME (0.37 mmol/L) was started on the day of virus inoculation and continued until the animals were killed on day 14. The first group of mice was fed untreated (without L-NAME) drinking water (nontreated group, n=42). In the other group, L-NAME was dissolved in the drinking water at a concentration of 100 mg/L (0.37 mmol/L), yielding a daily intake of ≈5 to 10 mg/kg of L-NAME in each mouse (low-dose L-NAME–fed group, n=43). In these two groups of animals, mortality on 14 days after virus inoculation (5×106 pfu) was compared. In addition, we examined the effect of a higher concentration of L-NAME on the mortality after virus inoculation. For this purpose, we fed a relatively high concentration of L-NAME (3.7 mmol/L) to another group of infected (5×106 pfu CVB3) mice (high-dose L-NAME–fed group, n=32).
Effects of L-NAME on Histopathological Changes
In another set of experiments, we studied the effects of NO synthesis inhibition on the extent of myocardial damage in viral myocarditis. As in the mortality protocol, either low-dose (0.37 mmol/L, n=50) or high-dose (3.7 mmol/L, n=90) L-NAME feeding was started on the day of virus inoculation (1×105 pfu) and was continued until the animals died or were killed for study. Nontreated infected mice (n=50) were also used for the examination. In addition, another group of mice (n=8) was given drinking water containing D-NAME (0.37 mmol/L) after virus inoculation. For comparison of body and heart weights and cardiac pathological changes, saline injection instead of virus inoculation was performed in 6 uninfected control mice that received distilled water, in 10 uninfected mice fed low-dose L-NAME (0.37 mmol/L), and in 10 uninfected mice fed high-dose L-NAME (3.7 mmol/L).
After the mice were killed, body weights were determined, the hearts were removed, and heart weights were determined. The hearts were fixed in 10% formalin and embedded in paraffin, and two 5-μm-thick slices were taken at the papillary muscle level. The myocardial sections were stained with hematoxylin and eosin and Masson’s trichrome. Using these sections, two independent researchers operating in a blind manner examined the extent of inflammatory cell infiltration and the area of myocardial necrosis. Because the lesions of necrosis, inflammatory cell infiltration, and fibrosis overlapped each other and clear differentiation of each lesion was difficult, we measured the total area, including necrosis, inflammatory cell infiltration, and fibrosis, and defined it as the area of necrosis. For the determination of total myocardial area and the area of necrosis, we took pictures of each section, magnified its original size by ×40 using a computerized image analyzer, and then determined the margin of the area to trace. After obtaining each area by planimetry, percent necrotic area was calculated by dividing the sum of area of necrosis by the whole myocardial area of each section. The determination of the margin was sometimes difficult, but the interobserver difference in the measured area was <10%. The mean of two sections from one heart was used for statistical analysis.
We also semiquantitatively evaluated the degree of inflammatory cell infiltration in each section by using the pathological grade as described previously.21 Pathological grade was determined as follows: grade 1 (mild), one or two small foci in the whole myocardial area; grade 2 (slight), several small foci; grade 3 (moderate), multiple small foci or several large foci; grade 4 (severe), multiple large foci; and grade 5 (very severe), diffuse infiltration, necrosis, or calcification. If the grading score for each specimen differed between the two examiners, the third examiner decided the grading score. In each group, subsets of mice were killed on day 3 for examination of early pathological changes, on day 7 for pathological grading of cellular infiltration, and on day 14 for measurement of percent necrotic area and the pathological grade. D-NAME–fed and uninfected control mice were killed on day 14. In addition, histological examinations were also performed in some of the deceased mice used in “mortality protocol.”
Effect of L-NAME Treatment on Systemic Blood Pressure During Myocarditis
We measured systemic blood pressure in both control and infected conscious mice (n=5 for each group). Mice were anesthetized with pentobarbital sodium (80 mg/kg). With the mouse supine, a 1-cm inguinal incision was made under a microscope, and the right femoral artery was isolated from surrounding structures. The polyethylene catheter (single lumen SP8, Natsume Japan) was then inserted into the artery. The catheter was advanced to a 0.5-cm depth and secured in place. The catheter was then flushed with heparinized saline (100 U/mL saline), sealed with cyanoacrylate glue, tunneled to the back of the mouse, and tucked into a subcutaneous pocket on the back. The incision was sutured with 6-0 surgical silk. The mice were then allowed to recover in a standard rodent cage with food and water available ad libitum for 24 hours. After removal from the subcutaneous pocket, the arterial catheter was connected to a pressure transducer (MLT1050, AD Instruments Japan). Blood pressure was analyzed with Mac Lab/4s (AD Instruments Japan). Baseline mean blood pressure measurement was started 24 hours after surgery and continued for a total of 3 hours when the mice were noted to be quietly resting and not sleeping or engaging in grooming or feeding activities. The average value of blood pressure obtained during this period was used for the assessment.22 23
Virus Titer in the Heart
For the infectivity assay, hearts of the infected mice (1×105 pfu) were removed aseptically on days 2, 3, 6, 10, and 14, and virus titers in the heart were measured in the nontreated and the low-dose L-NAME (0.37 mmol/L)–fed groups. We also examined virus titers in hearts from the high-dose L-NAME (3.7 mmol/L)–fed group. In each examination, 5 mice were used. The whole heart was weighed and homogenized in 1 mL of MEM. After centrifuging at 1500g for 10 minutes at 4°C, 0.1 mL of supernatant was inoculated into Vero cells for 60 minutes at 37°C in 5% CO2. Cells were then overlaid with 4 mL of medium containing MEM and 10% methylcellulose. After 4 days of incubation at 37°C in a humidified atmosphere containing 5% CO2, cells were stained with crystal violet, and plaques were counted with a microscope. The myocardial virus titer was expressed as log10 pfu/mg.
Effect of L-NAME Treatment on Prostaglandin Production in the Heart
Since proinflammatory prostaglandins play an important role in the early phase of inflammation and NO is reported to inhibit them,24 25 we measured tissue PGE2 levels in both nontreated and low-dose L-NAME–fed mice (1×105 pfu). In the L-NAME–fed group, L-NAME (0.37 mmol/L) feeding was started on the day of virus inoculation and was continued until death. On day 4 and day 7, 5 mice in each group were killed so that tissue PGE2 levels could be assessed. After excising the heart, the specimens were homogenized in 1 mL of PBS containing 1.8% indomethacin, frozen in liquid nitrogen, and stored at −80°C. The concentrations of PGE2 were determined by specific radioimmunoassay using the 125I-PGE2 assay system (Amersham). The cross-reactivity of other prostaglandins with the antisera was as follows: 6-ketoprostaglandin F1, 0.04%; thromboxane B2, 0.08%. The concentration was expressed as picograms per milligram tissue.
Survival curves of infected mice were obtained by the Kaplan-Meier method. The survival curves were evaluated by Mantel-Cox log-rank test. χ2 analysis was used to compare the mortality rate between the groups. Statistical analysis of body weight, heart weight, heart weight–to–body weight ratio, the pathological grade and percent necrotic area, blood pressure, virus titers, and prostaglandin production was performed by one-way ANOVA (Bonferroni). Differences were considered statistically significant at P<.05. Results are expressed as mean±SEM.
Mortality of the Infected Mice
Nontreated and low-dose L-NAME–fed mice began to die on day 5 or 6 after virus inoculation, and the majority of deaths occurred from day 6 to day 10. This is in accordance to our previous observations.9 All of these deceased mice showed signs of congestive heart failure, such as coat ruffling, weakness, and irritability before death. Macroscopic examination revealed pleural effusion and congestion of the lung and liver, suggesting the presence of congestive heart failure, in the deceased mice. On day 14, mice fed L-NAME (0.37 mmol/L) in drinking water exhibited a significantly lower mortality ratio compared with the nontreated mice (23.3% versus 50.0%, P<.05; Fig 1⇓).
On the other hand, when the infected mice were treated with high-dose L-NAME (3.7 mmol/L), ≈60% of the mice died on day 3, 80% of the mice were lost before day 4, and almost all mice died by day 7. The postmortem pathological examination of the mice that died on day 3 (n=10) revealed no distinct microscopic findings of myocardial necrosis or cellular infiltration in the heart. In addition, macroscopic examination of the whole body revealed no distinct abnormalities, such as bleeding, in any organ.
Ten nontreated, 10 low-dose L-NAME–fed, and 10 high-dose L-NAME–fed mice were killed on day 3; 15 nontreated, 13 low-dose L-NAME–fed, and all 6 surviving high-dose L-NAME–fed mice were killed on day 7; and 23 nontreated and 24 low-dose L-NAME–fed mice were killed on day 14. The inflammatory process of infected mice in these groups was in accordance with previous reports. On day 3, a few scattered small foci of myocyte necrosis were noted. The myocardial necrosis and cell infiltration were extensive on day 7. Infiltration of the inflammatory cells was decreased, and necrotic myocardium gradually changed to fibrosis and calcification on day 14, as was reported previously.26 27 Along with these histopathological changes, the infected mice decreased their body weight (infected mice [nontreated], 16.5±3.9 g; uninfected mice, 21.3±0.4 g; P<.05; on day 14). The heart weight of the infected mice showed a significant increase compared with that of the uninfected mice (infected mice, 0.11±0.02 g; uninfected mice, 0.09±0.04 g; P<.05; on day 14) (Table 1⇓). The infected hearts, with enlarged cardiac cavities, consisted of necrotic foci surrounded by infiltrating cells and interstitial edema.
As in the high virus titer–inoculated mice, mice inoculated with low virus titers (1×105 pfu) experienced high mortality after high-dose L-NAME treatment. Approximately 50% of the mice died by day 3, and only 10% of the mice survived on day 7. Examinations of 10 high-dose L-NAME–treated mice surviving on day 3 revealed the presence of small necrotic foci, but the area of necrosis was not different from that in the other two groups. There was no pulmonary congestion in any of three groups on day 3. The mice surviving on day 7 demonstrated the presence of necrosis and cell infiltration. The areas of necrosis and cell infiltration of the surviving mice were small compared with those in the other two groups (Fig 2⇓). However, because of the high mortality, it was not possible to collect sufficient data for quantitative histopathological analysis in high-dose L-NAME–fed mice; the use of surviving animals of limited number may bias the data analysis. Therefore, the quantitative analysis on day 7 was performed in only low-dose L-NAME–fed and nontreated mice. When the infected mice were treated with low-dose L-NAME (0.37 mol/L), the body weight was bigger and heart weight was smaller compared with the nontreated infected mice (Table 1⇑). In addition, macroscopic findings of heart failure, such as pleural effusion or congestion of the lung, were observed in 4 of the 23 nontreated mice, whereas none of the low-dose L-NAME–fed mice showed such findings.
Histopathological examination revealed a remarkable reduction in the percent necrotic area in the low-dose L-NAME–fed group compared with the nontreated infected group (no treatment, 22.4±6.8%; low-dose L-NAME, 11.9±4.6%; P<.05) (Figs 2⇑ and 3a⇓). Because the disease demonstrated the focal nature and because infiltrated cells were intermingled in the inflammatory lesions, the quantitative cell type identification in the inflammatory area in each animal was difficult. Therefore, we did not determine whether there was a change in the infiltrated cell types by low-dose L-NAME treatment. We evaluated the extent of inflammatory cell infiltration by semiquantitative analysis. The pathological grading revealed a significant reduction of the extent of cell infiltration on day 7 and on day 14 in low-dose L-NAME–fed mice (Fig 3b⇓).
There were no necrotic lesions or cell infiltration in the hearts of uninfected control mice. Likewise, neither necrosis, cell infiltration, nor fibrosis was detected in uninfected mice treated with low-dose or high-dose L-NAME. D-NAME treatment did not change the area of myocardial necrosis and the degree of cell infiltration. On the other hand, histopathological examination in the deceased mice in the “mortality protocol” revealed the presence of intensive myocardial necrosis and fibrosis in both low-dose L-NAME–fed and nontreated mice.
Blood Pressure in Mice
Blood pressure was low immediately after anesthesia and remained low for at least 2 hours compared with 24 hours later. Table 2⇓ shows the changes in mean blood pressure after virus inoculation and the effects of L-NAME treatment. High-dose L-NAME treatment for 14 days increased mean blood pressure in control (uninfected) mice (93±4 versus 122±4 mm Hg, P<.01), and low-dose L-NAME for 14 days also increased blood pressure significantly (93±4 versus 106±4 mm Hg, P<.01). In nontreated infected mice, blood pressure was decreased on days 7 and 14 after virus inoculation, probably because of myocardial damage and/or heart failure. Blood pressure of low-dose L-NAME–fed mice was significantly higher compared with that of nontreated mice on day 7 and day 14. On the other hand, on day 3, when high-dose L-NAME–fed mice began to die, there were no significant differences in blood pressure among three groups (nontreated, low-dose L-NAME–fed, and high-dose L-NAME–fed groups).
Myocardial Virus Titer
Virus content was not detected in the noninfected mice. No differences were found in the virus content on days 2, 3, 6, 10, and 14 between the nontreated and the low-dose L-NAME–fed groups. Even on day 6, when the virus replication in the heart is at its peak, there were no statistical differences in the virus titers between low-dose L-NAME–fed and nontreated mice (Table 3⇓). In the high-dose L-NAME–fed group, virus titers were examined only on day 3, because most of the animals died thereafter. On day 3, there were no statistical differences in virus titers between high-dose L-NAME–fed and nontreated groups. However, since we measured virus titers in mice surviving on day 3 (5 of 10 mice inoculated), we could not rule out the possibility that the titers might be increased in the deceased animals.
Tissue Prostaglandin Levels in the Heart
L-NAME treatment decreased the production of proinflammatory prostaglandins. We measured tissue PGE2 levels on day 4, the early stage of inflammation, and on day 7, when the inflammatory cell infiltration was most extensive. The tissue PGE2 levels in low-dose L-NAME–fed mice were significantly reduced compared with those in nontreated mice on days 4 and 7 (Fig 4⇓).
Recently, we and another group demonstrated the expression of iNOS in a murine model of CVB3-induced viral myocarditis.9 17 The histochemical study revealed that immunostaining for iNOS was present in the infiltrating cells, which were mainly macrophages and polymorphonuclear leukocytes. The induction of iNOS is likely due to proinflammatory cytokines in the process of viral myocarditis.26 We reported that the peak of iNOS expression in the heart was 8 days after virus inoculation, when myocardial necrosis and cell infiltration were most extensive. The prominent expression of iNOS suggests local production of NO in the heart with viral myocarditis. Because of its multiple biological actions, NO produced in the heart may exert either protective or detrimental action in the pathogenesis of viral myocarditis. In the present study, we found that the inhibition of NO synthesis by low-dose L-NAME markedly decreased the mortality rate in the murine model of CVB3-induced viral myocarditis. Along with the reduction of mortality rate, the area of myocardial necrosis and the degree of cell infiltration were significantly decreased in the hearts of mice treated with low-dose L-NAME.
As one of the mechanisms of myocardial destruction of viral myocarditis, the direct cytolytic effect of the virus on myocytes plays an important role in the early phase after virus inoculation.27 Viral replication causes myocardial necrosis with inflammation in the heart. Experimental studies in vitro have shown an inhibitory effect of NO on viral replication.28 The anti–viral-replication effect of NO is suggested to be mediated by its inhibitory action on viral ribonucleotide reductase.29 Therefore, it was possible that an inhibition of NO synthesis resulted in the worsening of myocardial damage. Indeed, the recent report of Lowenstein et al17 showed that mice with CVB3-induced viral myocarditis had increased myocardial virus titers and a higher mortality when treated with NOS inhibitors. However, in the present study, we found that low-dose L-NAME treatment had no effects on viral titers in the heart throughout the observation period of 2 weeks after virus inoculation. In accordance with the present study, Akaike et al30 reported that in mice with influenza virus–induced pneumonia, L-NMMA, a NOS inhibitor, significantly improved the survival rate without suppression of virus replications in the lung. The difference between the study of Lowenstein et al and ours seems at least in part due to the dose of NOS synthesis inhibitor given to the animal. In the study of Lowenstein et al, they fed mice relatively high concentrations of L-NAME (10 and 100 mmol/L), whereas we gave them a low concentration of L-NAME (0.37 mmol/L). Lowenstein et al found unchanged myocardial virus titers when the concentration of given L-NMMA was <10 mmol/L. In addition, a relatively high concentration of NO was shown to be required to inhibit viral replication.28 Therefore, although NO was produced in the heart, the concentration of NO produced in the heart might not be high enough to inhibit viral replication.
The difference in the dose also likely accounts for the discrepancy between the present study and that of Lowenstein et al17 involving the effect of L-NAME on the mortality of mice with viral myocarditis. Indeed, when we fed the infected mice high-dose L-NAME (3.7 mmol/L), almost all of them died by 7 days after virus inoculation. Although we found unchanged myocardial virus titers in high-dose L-NAME–treated mice on day 3 in spite of increased mortality, we could not rule out the possibility that there might occur increased viral replication between days 4 and 7.
There are several mechanisms that may contribute to the beneficial effects of low-dose L-NAME treatment on viral myocarditis in the present study. First, an improvement of tissue perfusion by NO synthesis inhibition must be taken into account. We found that even the low-dose L-NAME increased blood pressure. The nontreated infected mice showed reductions of blood pressure on days 7 and 14, which probably represented the hemodynamic deterioration due to myocardial damage and resultant heart failure. However, the low-dose L-NAME–fed infected mice exhibited significantly higher mean blood pressure than did the nontreated mice on days 7 and 14. Therefore, the low-dose L-NAME used in the present study likely served to improve tissue perfusion, particularly when the animals were in heart failure or shock state, and resulted in the improvement of survival rate.
Next, it is shown that NO produced in neutrophils or macrophages acts as a cytotoxic molecule in some circumstances. The induction of iNOS has been demonstrated in various types of autoimmune complex injury and inflammatory reactions.31 32 In those pathological conditions, NO may be involved in the production of tissue damage and edema.33 NO is shown to inhibit mitochondrial respiratory chain reaction, aconitase of the Krebs cycle, and synthesis of DNA by inactivating ribonucleotide reductase.34 Moreover, NO may combine with oxygen-derived free radicals to form peroxynitrite and further change to hydroxy radicals.35 These radicals are potent cytotoxic molecules and thereby may participate in the mechanisms of myocyte destruction.36 In the present study, L-NAME treatment decreased the area of myocardial necrosis and fibrosis. Therefore, it is possible that a large amount of NO produced in the heart plays a crucial role in myocardial destruction in viral myocarditis and that an inhibition of NO production results in the amelioration of myocardial damage.
In addition to the augmentation of the inflammatory and cytotoxic process, NO produced in the heart may decrease ventricular function and thus aggravate heart failure. Brady et al15 showed in isolated cardiac myocytes that endogenously produced NO from endothelium attenuated the contraction of myocytes. Moreover, NO produced by the cardiac myocyte itself has been shown to act as an autocrine factor to reduce myocardial contractility. Balligand et al37 reported an augmentation of inotropic response to β-adrenergic agonists by administration of the NO synthesis inhibitor L-NMMA. The improved survival rate by L-NAME observed in the present study may be partially related to the augmentation of cardiac function by this drug. Although we did not measure cardiac function in the present study, the preserved blood pressure in low-dose L-NAME–fed mice may indicate improved cardiac function, and the improved cardiac function may also be related to the reduced myocardial damage by NO synthesis inhibition.
On the other hand, NO has been demonstrated to augment the production of proinflammatory prostaglandins in the tissue with inflammation, and NOS inhibitors reduce them. NO may exaggerate the inflammatory process by the generation of additional proinflammatory prostaglandin, which enables inflammatory cells to reach the lesion of inflammation.24 25 In the present study, we found reduction in inflammatory cell infiltration in the inflammatory area and decreased tissue PGE2 levels by low-dose L-NAME treatment. Recently, Salvemini et al38 used the rat carrageenan-stimulated air pouch model and reported that the anti-inflammatory mechanisms of NO synthesis inhibitors are partly mediated by the inhibition of prostaglandin production and cellular infiltration. Although the reduced prostaglandin production may represent the result of decreased inflammation, it is possible that the beneficial anti-inflammatory effects of low-dose L-NAME treatment are in part mediated by the inhibition of proinflammatory prostaglandin.
Finally, we found increased mortality in high-dose L-NAME–fed mice. It seemed that NO synthesis inhibition resulted in biphasic effects depending on the dose used. We measured blood pressure in these animals on day 3, when they began to die, and found that blood pressure values were not different from those of control mice. Therefore, the high-dose L-NAME–treated mice were neither in excess hypertensive condition nor in shock state on day 3, nor did the high mortality seem to be the result of increased myocardial damage. Indeed, we found no pulmonary congestion and no lethal necrotic foci in mice treated with high-dose L-NAME on day 3. Thus, although we cannot completely exclude the possibility of increased viral replication, the high mortality may have resulted from either the toxic effect of this agent or yet-unknown mechanism(s). Further studies to clarify the mechanism of high mortality induced by high-dose L-NAME treatment are needed.
Our results suggest that NO can be regarded as a common important modulating factor of myocardial damage in various pathological conditions mediated by immune and inflammatory processes. Furthermore, recent clinical studies demonstrated induction of iNOS in the heart of patients with heart failure, and locally produced cytokines such as tumor necrosis factor-α are suggested to be responsible for the induction.39 NO produced in the heart may also be involved in pathophysiology other than viral myocarditis.
Selected Abbreviations and Acronyms
|D-NAME||=||Nω-nitro-d-arginine methyl ester|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
This study was supported in part by a grant-in-aid for Scientific Research to Dr Kawashima (C 06670717) and to Dr Hayashi (B 07457050) from the Ministry of Education, Science, and Culture, Japan.
- Received June 23, 1997.
- Accepted July 5, 1997.
- © 1997 American Heart Association, Inc.
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