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J Am Coll Cardiol, 1999; 33:1400-1407 © 1999 by the American College of Cardiology Foundation |
a Department of Cardiovascular Medicine, Kyoto University, Kyoto, Japan
Manuscript received June 22, 1998; revised manuscript received November 5, 1998, accepted December 22, 1998.
Reprint requests and correspondence: Dr. Akira Matsumori, Department of Cardiovascular Medicine, Kyoto University, 54 Kawaracho, Shogoin, Sakyo-ku, Kyoto 606-8397, Japan.
amat{at}kuhp.kyoto-u.ac.jp
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Abstract |
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This study was designed to examine the effects of pimobendan in a murine model of viral myocarditis in relation to proinflammatory cytokine production and nitric oxide (NO) synthesis by inducible NO synthase (iNOS) in the heart.
BACKGROUND
Pimobendan has been recently confirmed to improve both acute and chronic heart failure. Since the modulation of myocardial necrosis and contractile dysfunction by various proinflammatory cytokines may be partially mediated by the production of nitric oxide, the effects of pimobendan on the production of proinflammatory cytokines and NO were investigated in an animal model of viral myocarditis involving heart failure.
METHODS
DBA/2 mice were inoculated with the encephalomyocarditis virus. To observe its effect on survival up to 14 days, pimobendan (0.1 mg/kg or 1 mg/kg) or vehicles were given from the day of virus inoculation (day 0) orally once daily. The effects of pimobendan on histological changes, cytokine production, NO production and iNOS gene expression in the heart were studied in mice treated either with pimobendan, 1 mg/kg or with vehicles only, and sacrificed seven days after virus inoculation.
RESULTS
The survival of mice improved in a dose-dependent fashion such that a significant difference (p < 0.02) was found between the higher-dose pimobendan group (20 of 30 [66.7%]) and the control group (11 of 30 [36.7%]). Histological scores for cellular infiltration (1.1 ± 0.1 vs. 2.0 ± 0.0, p < 0.001), intracardiac tumor necrosis factor (TNF)-
(18.2 ± 1.8 vs. 35.8 ± 4.2 pg/mg heart, p < 0.001) and interleukin (IL)-1ß (9.3 ± 1.2 vs. 26.6 ± 7.1 pg/mg heart, p < 0.01) were significantly lower in the mice given pimobendan versus those of the control mice. Interleukin-6 levels (7.1 ± 0.8 vs. 9.2 ± 1.9 pg/mg heart) were also lower in the mice treated with pimobendan. Furthermore, intracardiac NO production was significantly (p < 0.001) less in the pimobendan group (0.165 ± 0.004 nmol/mg heart) than in the control group (0.291 ± 0.051 nmol/mg heart), and intracardiac iNOS gene expression in the mice given pimobendan was 74% lower than it was in the control animals (p < 0.01).
CONCLUSIONS
These findings suggest that the beneficial effects of pimobendan in viral myocarditis are partially mediated by the inhibition of both proinflammatory cytokine production and NO synthesis by iNOS.
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Recent studies have revealed the roles played by various cytokines in diverse cardiovascular disorders (13). The levels of circulating tumor necrosis factor (TNF)-, interleukin (IL)-1ß, IL-6 and other proinflammatory cytokines have been found to be elevated in patients with myocarditis and acute myocardial infarction (14–17). Furthermore, circulating TNF-alpha has been reported to be increased in patients with chronic congestive heart failure, including in the context of myocarditis, transplant rejection and dilated cardiomyopathy (18–20).
In a murine model of encephalomyocarditis (EMC) virus-induced myocarditis, the majority of mice die of congestive heart failure due to severe myocarditis in the acute stage (21). The severity in the course of the disease appears to correlate with the degree of intracardiac production of proinflammatory cytokines (22). Recent studies have shown that several proinflammatory cytokines cause myocardial contractile dysfunction and that their negative inotropic effects may be mediated by nitric oxide (NO) (23,24). We previously demonstrated that pimobendan strongly inhibited the production of NO in cultured macrophages stimulated by lipopolysaccharide (LPS) (25). Thus, in the present study, we examined the effects of pimobendan in a murine model of viral myocarditis induced by EMC virus in relation to proinflammatory cytokine production and gene expression of inducible nitric oxide synthase (iNOS) in the heart.
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Methods |
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Drug preparation. Pimobendan (UD-CG 115 BS) was supplied by Nippon Boehringer Ingelheim Co., Ltd., Kawanishi, Japan. The drug was prepared as an oral suspension in 0.25% methylcellulose solution in concentrations of 120 µg/mL and 12 µg/mL, and stored at 4°C.
Protocol 1—survival studies. Since, in this model, most mice die of congestive heart failure within 14 days after EMC virus inoculation (21), the survival was observed up to 14 days in this study. Pimobendan was administered in doses of 0.1 mg/kg or 1 mg/kg daily for 14 days from the day of EMC virus inoculation while control mice received vehicles only. Thirty mice were randomly assigned to each group.
Protocol 2—histochemical and pathological studies.
Since we had demonstrated in earlier studies that the expression of intracardiac TNF- messenger RNA (mRNA) is maximal at seven days after virus inoculation in the same murine model, the effects of pimobendan on the production of TNF-, other proinflammatory cytokines (IL-1ß and IL-6) and NO in the heart was examined on day seven. On day 0 the mice were randomly assigned to receive pimobendan in a dose of 1 mg/kg orally once daily (n = 7 or 5) or vehicles only (n = 5). The animals were sacrificed by cervical dislocation under general anesthesia, seven days after EMC virus inoculation. Following measurements of body and heart weights, the hearts were divided into two sections along their short axis at the midleft ventricular level. The apical sections were used to determine intracardiac levels of cytokines and NO and the basal sections were used for histological examination. For the measurements of iNOS gene expression, the mice were randomly assigned to receive pimobendan in a dose of 1 mg/kg orally once daily (n = 6) or vehicles only (n = 5), and the hearts were harvested in the same manner.
Histological examination. The hearts were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The extent of cellular infiltration and myocardial necrosis was graded by two blinded observers and was scored as follows: 0 = no lesions; 1+ = lesions involving <25% of the myocardium; 2+ = lesions involving 25% to 50% of the myocardium; 3+ = lesions involving 50% to 75% of the myocardium and 4+ = lesions involving 75% to 100% of the myocardium. The scores assigned by the two observers were averaged.
Assay of intracardiac cytokines.
Frozen sections of hearts were mixed with 1.5 ml of PBS/0.05% NaN3 and homogenized with an ultrasonic liquid processor (ASTRASONTM Model XL2020, Misnox Inc., Farmingdale, New York). Supernatants containing cytosolic protein were used to detect the intracardiac cytokines with commercially available enzyme-linked immunosorbent assay (ELISA) kits (TNF- and IL-6: GENZYME Co., Cambridge, Massachusetts; IL-1ß: BioSource International Co., Camarillo, California). The procedures were performed according to the manufacturer’s recommendations. The detection limits of TNF-, IL-1ß and IL-6 assays were 15 pg/mL, 7 pg/mL and 5 pg/mL, respectively. Final results were expressed as pg/mg of heart tissue.
Measurement of intracardiac NO production. Since nitrite/nitrate are endproducts of NO, their production in heart tissue was calculated from the levels of intracardiac nitrite/nitrate measured by the Griess reaction (26,27). Frozen sections of hearts were homogenized with 1.5 ml of PBS as described above. After centrifugation, the supernatant was mixed with nitrate reductase (0.25 U/ml) and incubated for 15 min at 37°C in the presence of 5 mM NADPH. Fifty µl of the samples were incubated with 25 µl of 1% sulfanilamide and 25 µl of 0.1% N-1-naphthyl-ethylenediamine dihydrochloride in 5% phosphoric acid for 10 min. The absorbance at 540 nm (OD540) was measured with a microplate reader (Multiskan BICHROMATIC, Labsystems, Finland). Nitrite/nitrate concentrations were calculated by comparison with OD540 of standard solutions of sodium nitrite prepared in PBS. The final results were expressed as nmol/mg of heart tissue.
Measurement of iNOS mRNA levels using the competitive reverse-transcriptase polymerase chain reaction (RT-PCR). Total RNA was extracted from the hearts by the guanidinium thiocyanate-phenol-chloroform-isoamylalcohol procedure. The RNA concentration was measured spectrophotochemically. Five µg of total RNA was subjected to first strand cDNA synthesis in a 40 µL reaction mixture using murine leukemia virus reverse transcriptase (Gibco BRL, Rockville, Maryland). The reaction mixture was incubated at 37°C for 60 min, heated to 70°C for 5 min to denature the reverse-transcriptase then cooled on ice for 3 min. Sixty µL of water was added to each sample.
Quantitative competitive PCR was developed as described previously (28). Using a commercially available kit (PCR MIMICTM Construction Kit, CLONETECH Laboratories Inc., Palo Alto, California), nonhomologous DNA fragment with iNOS or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene-specific primer templates was constructed to be 148 or 184 nucleotides shorter than each PCR product generated from native iNOS or G3PDH cDNA, respectively. The primer sites for PCR were located at nucleotides 2943 to 2967, and 3416 to 3440 of iNOS cDNA and 51 to 75, and 816 to 840 of G3PDH cDNA. The sense primer for iNOS was 5' CCC TTC CGA AGT TTC TGG CAG CAG C 3' and the antisense primer was 5' GGC TGT CAG AGC CTC GTG GCT TTG G 3'. The sense primer for G3PDH was 5' TGA AGG TCG GTG TGA ACG GAT TTG G 3' and the antisense primer was 5' TCA GAT GCC TGC TTC ACC ACC TTC T 3'. One µL of the samples and 1 µL of two-fold serial dilutions of the nonhomologous DNA fragment was added to 23 µL of the PCR master mix, which contained thermostable Taq polymerase (PERKIN ELMER Japan Co., Tokyo, Japan), the gene-specific primers, dNTP, PCR buffer and [-32P]-dCTP. The two-fold serial dilutions of constructed DNA, for example, were 1.0, 5.0 x 10–1, 2.5 x 10–1, 1.25 x 10–1, 6.25 x 10–2 and 3.125 x 10–2 attomoles/µL (1 attomole = 6 x 105 molecules). After having heated the PCR mixture to 95°C for nine min to activate thermostable Taq polymerase, PCR was performed for 35 cycles, each consisting of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min. Ten µL of each PCR product was separated by electrophoresis on a 5% polyacrylamide gel. The gels were dried, autoradiographed with Fuji imaging plates (Fuji Photo Film Co., Ltd., Tokyo, Japan) and analyzed with the use of a Fujix BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.). The log of the ratio of iNOS or G3PDH target radioactivity to the nonhomologous DNA fragment radioactivity was plotted against the log of the number of the nonhomologous DNA fragment molecules added to each PCR reaction tube. The number of iNOS or G3PDH cDNA was calculated from these plots. The values obtained were expressed as attomoles/µg of total RNA. Each value of iNOS cDNA was divided by that of the corresponding G3PDH cDNA to be normalized. Finally, iNOS cDNA levels, which were found to be equivalent to iNOS mRNA levels, were expressed as percentages of G3PDH cDNA levels.
Statistical analysis.
Survival was analyzed by the Kaplan-Meier method. The 
-square test was used for the final survival rates. Student t test was used for cytokine levels, nitrite/nitrate concentration levels, iNOS mRNA levels, body weight, heart weight, heart weight to body weight ratio and histological scores. Data are expressed as mean ± SEM. Statistical significance was defined as p < 0.05.
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level of the pimobendan 1 mg/kg group (18.0 ± 1.8 pg/mg heart, n = 7) was significantly less (p < 0.001) than that of the control group (35.8 ± 4.2 pg/mg heart, n = 5). The intracardiac IL-1ß level of the pimobendan 1 mg/kg group (9.3 ± 1.2 pg/mg heart, n = 7) was also significantly less (p < 0.01) than that of the control group (26.6 ± 7.1 pg/mg heart, n = 5). Although the intracardiac IL-6 level in the pimobendan 1 mg/kg group (7.1 ± 0.8 pg/mg heart, n = 7) was also less than that in the control group (9.2 ± 1.9 pg/mg heart, n = 5), the difference did not reach statistical significance (p = 0.23).
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Discussion |
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We have previously studied the effects of PDE III inhibitors, pimobendan, vesnarinone and amrinone on the production of NO in cultured macrophages stimulated by LPS (25). Pimobendan most strongly inhibited NO production. Thus, in the present study, the effects of pimobendan were investigated in a murine model of viral myocarditis in the perspective of the production of proinflammatory cytokines and iNOS gene expression in the heart. The doses of pimobendan chosen for the experiments were 1 mg/kg/day and 0.1 mg/kg/day. A previous pharmacokinetic study in rats who had received a comparable oral dose of pimobendan (31) suggests that its plasma and heart tissue concentrations in the present study were close to the concentrations found effective in our previous in vitro studies (25,32). A dose of 1 mg/kg/day in the present study is approximately ten times higher than the dose used in the treatment of chronic heart failure in humans and thus, may seem to have been inappropriately high. However, on the basis of body surface area, a dose of 1 mg/kg in mice is equivalent to 0.08 mg/kg in humans, which is within the range used clinically (33). Pimobendan improved the 14-day survival of the animals in a dose-dependent manner. On histological examination, myocardial cellular infiltration was significantly reduced in mice given pimobendan in a dose of 1 mg/kg, and myocardial necrosis tended to be reduced. Since both cytokines and NO are known to act mainly in an autocrine or paracrine fashion, it is important to measure their production at the level of a particular tissue. The productions of TNF- and IL-1ß in the heart tissue were reduced significantly in the pimobendan 1 mg/kg group, and IL-6 showed a similar tendency. Furthermore, intracardiac NO production and iNOS gene expression were also significantly reduced by pimobendan.
Proinflammatory cytokines and NO in myocarditis and the failing heart.
Several clinical studies have described the participation of proinflammatory cytokines in the pathogenesis of cardiac diseases. The levels of circulating proinflammatory cytokines such as TNF-, IL-1 and IL-6 are elevated in patients with myocarditis (14). It has also been reported that levels of circulating IL-1, IL-6 and IL-8 are elevated in patients with acute myocardial infarction (15–17). A recent report from our laboratory has shown that, in the same murine model of viral myocarditis, the intracardiac expression of TNF-, IL-1ß, IFN-
and IL-2 genes was increased at three days and reached maximal levels at seven days after virus inoculation (22). The degree of their expression correlated with the severity in the course of the disease, suggesting that the overproduction of these proinflammatory cytokines by viral infection may aggravate the disease. This is supported by the recent reports that the overexpression of TNF- in the heart caused severe myocarditis and cardiomegaly in transgenic mice (34) and that IL-1ß as well as TNF- alone promoted coxsackie virus B3-induced myocarditis in resistant mice (35). We previously showed that treatment with anti-TNF- antibody had improved both mortality and myocardial damage in the acute stage of this myocarditis model (36), indicating that the overproduction of TNF- aggravates viral myocarditis.
Some experimental studies have indicated that these proinflammatory cytokines may contribute to myocardial contractile dysfunction. Infusion of TNF- reversibly impaired left ventricular systolic and diastolic function in conscious dogs (37). Tumor necrosis factor-, IL-6 and IL-2 inhibited contractility of isolated hamster papillary muscles in a concentration-dependent and reversible manner (23). Tumor necrosis factor- exerted a direct negative inotropic effect in both the human (38) and feline (39) left ventricle and in isolated cardiac myocytes. Interleukin-1ß also depressed myocardial contractility in the isolated perfused rat heart (40). In addition to cardiac contractile dysfunction, both TNF- and IL-1ß are known to cause neonatal rat cardiac myocyte hypertrophy in vitro (41,42). Overexpression of both IL-6 and IL-6 receptor also caused ventricular myocardial hypertrophy in the transgenic mice (43). Myocardial hypertrophy is considered to compensate the failing heart at first but to be profoundly related to ventricular remodeling leading to heart failure.
Although most of the signaling pathways of cardiac contractile dysfunction related to proinflammatory cytokines are still unclear, recent evidence suggests that the direct negative inotropic effect of these cytokines may be mediated by NO (23,44,45). Some experimental studies have shown that IL-1ß and IFN- induce iNOS in both adult rat ventricular myocytes (46) and intracardiac microvascular endothelial cells (47), suggesting that the overproduction of NO in myocytes and endothelial cells by proinflammatory cytokines may depress myocyte contractility in an autocrine or paracrine fashion.
Inhibition of NO production in myocarditis. We have shown here that both NO production and the expression of iNOS gene in the heart were also significantly decreased by pimobendan. We have previously reported the effect of NO inhibition in the same model (48). Immunohistochemical studies showed that macrophages and endothelium-like cells stained positively for iNOS protein in the myocardium of virus-inoculated mice but negatively in uninfected mice. Furthermore, L-NMMA, an inhibitor of NOS, decreased both cellular infiltration and myocardial necrosis significantly. These results indicate a beneficial effect of NOS inhibition in this model. This is supported by another recent study in which the inhibition of NO synthesis by a relatively specific iNOS inhibitor decreased myocardial inflammation and necrosis in a rat autoimmune myocarditis (49). Opposite observations have been reported of the promotion of viral replication and myocardial damage (50) or of increased mortality (51) in murine coxsackie virus B3-induced myocarditis by NOS inhibition. Although these reports appear to contradict our results, it is likely that different degrees of NO inhibition cause different responses: excessive inhibition of NO may fail to suppress viral replication, resulting in aggravation of myocarditis, whereas more moderate inhibition of NO may salvage myocardial damage and contractile dysfunction. The degree of NO inhibition by pimobendan might be appropriate to improve the disease in the present model.
Mechanism of action of pimobendan. The mechanism by which pimobendan reduces the production of proinflammatory cytokines and iNOS gene expression in the heart is unclear. In our previous study, various PDE inhibitors differentially inhibited the production of cytokines by human peripheral blood mononuclear cells stimulated by LPS (31). Interestingly, most PDE inhibitors inhibited cytokine production, though not 8 Br-cAMP. In addition, we studied the effects of various PDE III inhibitors on the production of NO in LPS-activated macrophages (25). Although pimobendan strongly inhibited NO production, 8 Br-cAMP did not suppress, but increased, NO production. Thus, the inhibitory effects of pimobendan on the production of proinflammatory cytokines and iNOS gene expression cannot be explained by an increase in cAMP.
In the earlier in vitro study, pimobendan decreased the production of TNF- and IL-6 and increased IL-1ß production, whereas, in the present study, IL-1ß production and TNF- were both significantly reduced by pimobendan. Since different stimuli cause different patterns of cytokine induction in vitro and in vivo, it is hypothesized that there may be differences in the mode of cytokine induction between the in vitro model and the present in vivo model where viral infection is the main cause of the disease.
Conclusions. In conclusion, this study showed a therapeutic effect of pimobendan in the acute stage of viral myocarditis, mediated in part by inhibition of the inflammatory response, of the production of proinflammatory cytokines and of the synthesis of NO by iNOS. Pimobendan may be of great value in the treatment not only of viral myocarditis with heart failure but also of other cardiac diseases in which the overproduction of proinflammatory cytokines and NO play a significant pathogenetic role.
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Footnotes |
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in severe congestive cardiac failure. Br Heart J. 1993;70:141–143 production. J Clin Invest. 1994;94:1212–1217[Medline]
antibody on the murine model of viral myocarditis induced by encephalomyocarditis virus. Circulation. 1994;89:846–851 in conscious dogs. J Clin Invest. 1992;90:389–398[Medline]
in the adult mammalian heart. J Clin Invest. 1993;92:2303–2312[Medline]
gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest. 1995;96:1042–1052[Medline]
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