Inhibitory effects of vesnarinone in the progression of myocardial damage in experimental autoimmune myocarditis in rats

Abstract

Objectives: Vesnarinone, a positive inotropic and immunomodulatory agent, diminishes nitric oxide (NO) levels by suppressing the induction of inducible NO synthase (iNOS) expressed in cytokine-stimulated macrophages and cardiomyocytes. We examined whether vesnarinone exerts inhibitory effects on the progression of myocardial damage in experimental autoimmune myocarditis in rats through suppression of iNOS. Methods: Myocarditis was induced in 30 Lewis rats by injection of porcine cardiac myosin and vesnarinone was orally administered to 20 of the 30 rats. On day 21 after immunization (the climax of inflammation), the hemodynamics were examined and the severity of myocarditis was evaluated by determining the area ratio (%) [affected/entire area] of myocardial lesions in histological sections. Levels of serum CK-MB, NOx (NO₂⁻+NO₃⁻), TNF-α and IL-1β, and cyclic GMP, iNOS mRNA, TNF-α and IL-1β in heart tissues were determined. Expression of iNOS and TNF-α protein were examined by immunohistochemical methods. Results: Histopathological examination revealed extensive myocardial destruction and massive infiltration of inflammatory cells in the vesnarinone-untreated rats. The area ratio of the lesions in the treated rats was significantly lower than that in the untreated ones. Levels of CK-MB, NOx, cyclic GMP, cytokines and iNOS mRNA were significantly lower in the vesnarinone-treated rats. Infiltrating macrophages and cardiomyocytes in the untreated rats showed much higher levels of expression of iNOS and TNF-α than those in the vesnarinone-treated rats. Conclusions: Vesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of iNOS induced by cytokines.

Myocarditis, Vesnarinone, Nitric oxide, Inducible nitric oxide synthase, Proinflammatory cytokines

Time for primary review 27 days.

1 Introduction

There is accumulating evidence to support the view that proinflammatory cytokines play a crucial role in the pathogenesis of viral myocarditis and dilated cardiomyopathy [1,2], although the mechanism by which myocardial damage/cell death occurs is still unclear. A number of studies have demonstrated that serum and myocardial levels of cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1β are elevated in patients with myocarditis and cardiomyopathy [3], myocardial infarction [4] and congestive heart failure [5]. TNF-α has been found to contribute to myocardial damage and/or apoptotic death of cardiomyocytes [3] and myocardial/cardiac dysfunction in animal models [6,7]. IL-1β is also known to suppress cardiac function [8]. It has been shown that the negative inotropic effect of these cytokines is mediated largely by nitric oxide (NO) [9] and cytokines trigger production of an inducible NO synthase (iNOS) in a variety of cells, including cardiomyocytes, macrophages, endothelial cells, and vascular smooth muscle cells, leading to production of massive amounts of NO; this process may be responsible for the suppressive effect of cytokines on myocardial contractility.

Vesnarinone is a quinolinone derivative with positive inotropic properties [10–12] and it is useful clinically in the treatment of heart failure [13]. Recently, several investigators have suggested that vesnarinone may act as an immunomodulator, providing new therapeutic possibilities for the treatment of viral myocarditis [14] and for the suppression of rejection of cardiac allografts through its lymphocyte-suppressive properties [15]. Furthermore, in vitro studies have shown that vesnarinone suppresses TNF-α production by spleen cells from mice with acute viral myocarditis [16] and it inhibits the induction of NOS in J774 macrophages and cardiomyocytes [17]. Recently, we have reported that excess amounts of NO produced by iNOS appear to contribute to the progression of myocardial damage in autoimmune myocarditis in rats [18]. In this autoimmune myocarditis model, IL-1 appears early, whereas TNF-α and iNOS are present later, during the period of peak inflammation [19], suggesting that these cytokines are involved in the development of myocardial damage. Considering these observations together, it seems possible that vesnarinone may exert its beneficial effect in patients with heart failure in part by inhibiting the induction of NO synthase [20,21]. Therefore, we investigated whether vesnarinone is effective in the treatment of myocarditis through its inhibitory effect on the induction of proinflammatory cytokines and iNOS, and suppression of NO production.

2 Methods

2.1 Induction of autoimmune myocarditis

Autoimmune myocarditis was induced in rats as previously described [18,22]. The protocol for these experiments was approved by the committee in charge of animal welfare of our hospital (Tokyo Medical and Dental University and Tokyo Women’s Medical University). Thirty-five female 7-week-old Lewis rats were used. Thirty rats were each administered 1 mg of porcine heart myosin (10 mg/ml) (Sigma Chemical Co., St. Louis, MO) mixed with an equal volume of Freund’s complete adjuvant (FCA) by injection into the footpad on days 1 and 7. On days 2 and 5, each of the rats was administered 0.1 ml of a Bordetella pertussis suspension (Nacalai Tesque, Kyoto, Japan), prepared in 0.9 ml of saline solution, via the caudal vein.

2.2 Experimental design

Ten of thirty rats were sacrificed on day 21 (Group A). 10 of the other 20 rats were administered vesnarinone orally every day starting the day before the first sensitization with porcine cardiac myosin until the day of sacrifice (day 21) (Group B-0). The other 10 rats were administered vesnarinone orally every day starting on day 14 until the day of sacrifice (day 21) (Group B-14). Group C consisted of 5 control rats administered saline but not myosin, and were sacrificed on day 21. After sacrificing the rats, blood samples were collected promptly from the heart chamber to measure the concentration of vesnarinone, CK-MB, NOx (NO₂⁻+NO₃⁻), and various cytokines (TNF-α, IL-1β). The hearts were then removed and divided to obtain specimens for histopathological examination, immunohistochemical analyses, and measurement of the levels of cyclic GMP, iNOS mRNA, and various cytokines (TNF-α, IL-1β) in the heart tissues. The various treatment group are summarized in Table 1.

Table 1

Summary of the various treatment groups

Groupa	n	Myosin	Vesnarinone
A	10	+	−
B-0	10	+	day 0–21b
B14	10	+	day 14–21c
C	5	−	−

a

Rats were treated with myosin except Group C.

b

In Group B-0, rats were administered vesnarinone orally every day starting the day before the first sensitization with myosin until the day of sacrifice (day 21).

c

In Group B-14, rats were administered vesnarinone orally every day starting on day 14 until the day of sacrifice (day 21).

2.3 Hemodynamic measurements

Hemodynamic tests were performed after the administration of sodium pentobarbital (50 mg/kg i.p.) by the previously described method [23]. In brief, on day 21, the jugular vein was cannulated with a polyethylene catheter connected to a Statham transducer (P10EZ, Gould, Inc., Cleveland, OH). The catheter was advanced into the right ventricle. The external right carotid artery was exposed and cannulated with a micromanometer-tipped catheter (PR249, Miller Instruments, Houston, TX). Measurements were thus obtained of both the ventricular systolic and end-diastolic pressures and positive dp/dt of the left ventricle, aortic pressure, and heart rate by ECG monitoring.

2.4 Histopathological study

Transverse sections through the middle portions of heart specimens sliced into three equal portions were prepared, fixed in phosphate-buffered 10% formalin, embedded in paraffin, cut into sections 3 μm thick, and stained with hematoxylin-eosin. The area of the entire heart and that of regions affected by myocarditis, i.e., regions showing infiltration by inflammatory cells and myocardial necrosis, were determined using a personal computer (Apple Computer, Inc.; software: EM, Rise Co., Sendai, Japan), and the area ratio (%) [affected/entire area] was calculated. These data were determined by two blind observers and the inter- or intra-observer variance was less than 5%.

2.5 Agent

Vesnarinone was a gift from Otsuka Pharmaceutical Co. (Tokushima, Japan). To decide the dosage of vesnarinone to be employed, rats without myocarditis were orally administered vesnarinone as a component of the diet at various dosages for 21 days. Then, the serum concentrations of vesnarinone were measured by a high-performance liquid chromatographic method as previously described [24]. At the dosage level of 100 mg/Kg, the mean serum level was similar to the serum concentration observed in humans receiving vesnarinone therapy [12]. Therefore, vesnarinone was orally administered as a component of the diet at this dosage level (100 mg/Kg/day) in our further experiments.

2.6 Immunohistochemical study

Tissue specimens were fixed in phosphate-buffered 10% formalin and embedded in paraffin. As primary antibodies, anti-tumor necrosis factor-a (anti-TNF-α polyclonal IgG (R-19); 1:300 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-iNOS antibody (NOS2 (M-19); 1:300 dilution, Santa Cruz Biotechnology Inc.) were used. Sections were incubated with primary antibodies overnight at 4°C. The phosphate-buffered saline (PBS)-washed sections were then incubated with goat anti-rabbit IgG-peroxidase conjugate (1:200 dilution, BML, Nagoya, Japan) for 60 minutes at RT in the case of iNOS or rabbit Immunogloblins/AP (DAKO A/S, Glostrup, Denmark) in the case of TNF-α. In the case of iNOS, peroxidase activity was detected in Tris-buffered saline solution containing 3,3′-diaminobenzidine tetrahydrochloride (DAB) and 0.1%. In the case of TNF-α, sections were detected using BCIP/NBT substrate system (DAKO A/S). Sections were then examined by light microscopy.

2.7 Measurements of the serum concentration of vesnarinone

The concentrations of vesnarinone in serum were determined by a high-performance liquid chromatographic method as previously described [24] at Otsuka Pharmaceutical Co. (Tokushima, Japan).

2.8 Determination of serum CK-MB

Serum levels of CK-MB, as a marker of myocardial injury, were determined in a Type II Chemical Analyzer (Ciba Corning Inc., Tokyo, Japan) using Chemilumi CK-MB Magic Lite (Ciba Corning, Inc.) as the reagent.

2.9 Determination of serum NOx (NO₂⁻+NO₃⁻)

Serum levels of NOx (NO₂⁻+NO₃⁻) were determined by the Cd-Cu method using the TCI-NOX1000 system (Tokyo Kasei Kogyo Co., Tokyo, Japan) [25]. In principle, NO₂⁻ was reduced to NO₃⁻ using a high-performance Cd-Cu reduction column and the total NO₃⁻ content was determined by high-performance liquid chromatography using a NOx assay system. In this assay system, the absorption at 546 nm was determined.

2.10 Determination of cyclic GMP

Specimens of freshly isolated rat hearts were extracted in ice-cold 0.1 N HCl for 30 min and centrifuged at 10,000 g for 20 min at 4°C. Supernatants were stored at –80°C and measured in a simple assay using a cGMP ELISA Kit (Funakoshi Co., Tokyo, Japan).

2.11 Northern blot analysis of iNOS mRNA

Northern blot analysis of murine macrophage iNOS mRNA (homology with rat, 97%) was performed as previously described [26]. In brief, total RNA from rat heart tissues was isolated in guanidine thiocyanate and centrifuged through a 5.7 M CsCl cushion. RNA was then size-fractionated through a l.4% agarose gel in 0.7 M formaldehyde and 20 mM morpholinopropanesulfonic acid/5 mM sodium acetate/1 mM EDTA. Northern blot hybridization was performed with hybridization buffer containing 50% formamide, 5X Denhardt’s solution, 100 mg/ml salmon sperm DNA, and 5X SSPE (0.75 M NaCl/0.05 M NaH₂PO₄/0.005 M EDTA). The cDNA clone for rat iNOS (1,500 bp) were generously provided by Dr. Robert Star (Southwestern Medical Center, Dallas, TX, USA). ³²P-labeled cDNA probes were prepared by the random primer method [27]. The membranes (Magnagraph Nylon; Micron Separations Inc., Westborough, MA) were washed twice with 5X SSPE/10% sodium dodecyl sulfate (SDS) at room temperature, twice with 1 X SSPE/10% SDS, and once with 0.1X SSPE/10% SDS at 60°C for 15 minutes each time. Autoradiography was performed using Fuji RX film and an intensifying screen at −80°C.

2.12 Assay of cytokines (TNF-α, IL-1β) in serum and heart tissue

Heart tissue specimens were homogenized in PBS and centrifuged to obtain the supernatant. In each instance, the concentration of total protein in the supernatant was determined. TNF-α and IL-1β levels in serum and the supernatants of heart tissues were determined by means of specific ELISA assay kits (Otsuka Pharmaceutical Co., Tokushima, Japan).

2.13 Statistical analysis

Results are expressed as mean±SD. Affected area, NOx, CK-MB, cyclic GMP, cytokines, and hemodynamic parameters were compared by one-way ANOVA test with the use of the StatView IV statistical software (Abacs Concepts, Inc., Berkeley, CA) on a Macintosh computer. A value of P<0.05 was considered statistically significant.

3 Results

3.1 Hemodynamics

In Group A (vesnarinone-untreated rats with myocarditis), significant weight loss was observed compared to Group B-0 and B-14 (vesnarinone-treated rats with myocarditis) and C (control rats without myocarditis). Furthermore, both left and right ventricular end-diastolic pressures were elevated, while systolic left ventricular and mean aortic pressures were markedly decreased. A substantial decrease in positive dp/dt of the left ventricle was also evident in Group A, indicating a global impairment of left ventricular function. The values obtained for all five parameters including left and right ventricular EDP, systolic left ventricular pressure, mean aortic pressure, and positive dp/dt of the left ventricle in Group B-0 and B-14 were similar to those in Group C (Table 2).

Table 2

Summary of hemodynamics in experimental ratsa

Group	n	BW, g	HW/BW, g/g	HR, bpm	Pressure, mm Hg				+dp/dt, mm Hg/s	Ao (mean), mm Hg
					RV		LV
					SP	EDP	SP	EDP
A	10	190±10b	4.4±0.4b	322±28	36±7b	5.8±2.0c	105±11b	16±5c	4003±444c	94±10b
B-0	10	264±10	3.6±1.0	320±21	27±10	2.1±1.5	129±10	8±5	6998±301	110±17
B-14	10	263±10	3.3±0.8	311±23	25±6	1.9±0.8	136±17	7±4	7030±255	109±9
C	5	262±11	3.3±0.4	318±30	24±6	1.5±1.6	131±10	5±3	7033±392	112±14

a

n, values indicate the number of rats in each group; BW, body weight; HW, heart weight; HR, heart rate, RV, right ventricle; SP, systolic pressure; EDP, end-diastolic pressure; LV, left ventricle; Ao, aortic pressure. Values are mean±SD.

b

P<0.05 vs. Group B-0, B-14, and C.

c

P<0.01 vs. Group B-0, B-14, and C.

3.2 Histopathology

Tissue specimens from rats in Group A exhibited marked infiltration by inflammatory cells including lymphocytes, macrophages, neutrophils, and giant cells, and extensive necrosis of cardiac muscle cells (Figs. 1a and 2a). In contrast, those from rats in Group B-0 (Figs. 1b and 2b), or B-14 (Fig. 2c) exhibited only focal infiltration by inflammatory cells, and necrosis was limited to cardiac myocytes that were in contact with the inflammatory cells. Neither infiltration by inflammatory cells nor myocyte necrosis was noted in the control group (Group C) (Fig. 2d).

Fig. 2

Microscopic pictures of the hearts in Groups A, B-0, B-14 and C, and affected area ratio (%). Tissue specimens from rats in Group A exhibited marked infiltration by inflammatory cells, including lymphocytes, macrophages, neutrophils, and giant cells, and extensive damage to cardiomyocytes (a; original magnification, ×100). Those from rats in Group B-0 (b; original magnification, ×100) or B-14 (c; original magnification, ×100) exhibited only focal infiltration by inflammatory cells, and necrosis was limited to cardiomyocytes that were in contact with the inflammatory cells. No infiltration by inflammatory cells or damage to cardiomyocytes was noted in Group C (d; original magnification, ×100). The myocarditis-affected area ratios in Group A (n=10, Ves(−)), B-0 (n=10, Ves(+; day 0–21), B-14 (n=10, Ves(+; day 14–21)), and C (C) were 53±11*, 4.5±2.0, 5.9±3.0 and 0%, respectively. The value in Group B-0 or B-14 was significantly low compared to that in Group A. *P<0.001 vs. Group B-0, B-14 and C.

Fig. 1

Representative histology of the whole transverse section of the heart. The affected lesion in Group A is widely observed in both ventricles (a; original magnification, ×20), but only focal infiltration by inflammatory is noted in Group B-0 (b; original magnification, ×20).

3.3 Affected area ratio

As shown in Fig. 2, the myocarditis-affected area ratio in Groups A, B-0, B-14 and C was 53±11, 4.5±2.0, 5.9±3.0 and 0%, respectively. The affected area was significantly larger in Group A than those in Groups B-0, B-14 and C.

3.4 Immunohistochemistry

In heart tissues from rats in Group A, lymphocytes, macrophages, neutrophils, giant cells, myocytes, vascular endothelial cells and vascular smooth muscle cells in inflammatory lesions stained positive for iNOS (Fig. 3a). In tissue specimens from rats in Group B-0 (Fig. 3d) and B-14, most of the focally infiltrating inflammatory cells and a number of cardiac muscle cells were iNOS-negative. Group C exhibited no staining for iNOS (Fig. 3f). As shown in Fig. 3, in tissue specimens from rats in Group A, myocytes (Fig. 3b) as well as lymphocytes (Fig. 3c), macrophages, neutrophils in inflammatory lesions and endothelial cells stained positive for TNF-α. In tissue specimens from rats in Group B-0 (Fig. 3e) and B-14, most of the focally infiltrating inflammatory cells, and cardiac muscle cells were TNF-α-negative, but endothelial cells were TNF-α-positive. In the control group (Group C), only endothelial cells exhibited positive staining for TNF-α (Fig. 3g).

Fig. 3

Immunohistochemical study for iNOS and TNF-α. (a) In tissue specimens from rats in Group A, lymphocytes, macrophages, neutrophils, giant cells, myocytes, vascular endothelial cells and vascular smooth muscle cells in inflammatory lesions stained positive for iNOS (original magnification, ×200). (d) In tissue specimens from rats in Group-0, most of the focally infiltrating inflammatory cells and cardiomyocytes were iNOS-negative (original magnification, ×100). (f) Group C exhibited no positive staining for iNOS (original magnification, ×100). In tissue specimens from rats in Group A, myocytes (b, original magnification, ×200) as well as infiltrating inflammatory cells (i.e. lymphocytes, macrophages and neutrophils) (c, original magnification, ×400) and endothelial cells in inflammatory lesions stained positive for TNF-α. (e) In tissue specimens from cats in Group B, most of the focally infiltrating inflammatory cells and cardiac muscle cells were TNF-α-negative (original magnification, ×100). (g) In Group C, only endothelial cells exhibited positive staining for TNF-α (original magnification, ×100).

3.5 Serum concentration of vesnarinone

In Group B-0, serum concentrations in the vesnarinone-treated rats were within the range of 7.1 to 13.8 μg/ml with a mean of 9.4 μg/ml. On the other hand, in Group B-14, serum concentrations of vesnarinone were within the range of 6.3 to 11.0 μg/ml with a mean of 8.2 μg/ml.

3.6 Determination of serum CK-MB

Serum levels in Groups A, B-0, B-14 and C were 71±10, 15±9, 23±14 and 9±4 IU/L, respectively (Fig. 4). There was a significant difference between Groups A and other groups.

Fig. 4

Determination of serum NO_x (NO₂⁻+NO₃⁻), CK-MB and cyclic GMP. NO_x (NO₂⁻+NO₃⁻) levels in Groups A (Ves(-)), B-0 (Ves(+; day 0–21)), B–14 (Ves(+; day 14-21)) and C (C) were 90±13*, 19±10, 26±13 and 14±6 nmol/ml, respectively. There was a significant difference between Groups A and B-0, or B-14 (*P<0.001 vs. Group B-0, B-14). CK-MB levels in Groups A (Ves(−)), B-0 (Ves(+; day 0–21)), B-14 (Ves(+; day 14–21)) and C (C) were 71±10*, 15±9, 23±14 and 9±4 IU/L, respectively. There was a significant difference between Groups A and B-0, or B-14 (*P<0.001 vs. Group B-0, B-14). Levels of cGMP in heart tissues were significantly higher in Group A (n=10) vs. Group B-0 (n=10), Group B-14 (n=10), or Group C (n=5) (23±5.0* vs. 6.1±2.1, 8.8±3.0, or 3.8±1.9 pmol/mg protein, respectively, *P<0.001 vs. Groups B-0, B-14, and C).

3.7 Determination of serum NO_x (NO₂⁻+NO₃⁻)

Serum NOx levels in Groups A, B-0, B-14 and C were 90±13, 19±10, 26±13 and 14±6 nmol/ml, respectively (Fig. 4). There was a significant difference between Groups A and other groups.

3.8 cGMP levels of heart tissue

Levels of cGMP in heart tissues were significantly higher in Group A than in Group B-0, Group B-14, or Group C (23±5.0 vs. 6.1±2.1, 8.8±3.0, or 3.8±1.9 pmol/mg protein, respectively) (Fig. 4).

3.9 Northern blot analysis

The levels of iNOS mRNA were markedly up-regulated in Group A than those in Group B-0, while no iNOS transcript was detected in Group C (Fig. 5).

Fig. 5

Northern blot analysis for iNOS in heart tissues. Northern blot analysis reveals a distinct band of 1,400 bp that corresponded to the size of murine macrophage iNOS mRNA. The levels of iNOS mRNA are much higher in Group A [2] than in Group B-0 [3], although no iNOS transcript is detected in Group C [1].

3.10 Serum and heart tissue levels of cytokines

As shown in Fig. 6, serum levels of TNF-α and IL-1β in Group A (998±169 and 1205±230 pg/ml, respectively) were much higher than those in Group B-0 (301±59, 599±80 pg/ml, respectively) and in Group C (79±13, 189±50 pg/ml, respectively). Levels of TNF-α and IL-1β in heart tissues in Group A (13.2±2.1 and 4.0±0.5 pg/mg protein, respectively) were also significantly higher than those in Group B-0 (9.7±2.2 and 2.0±0.4 pg/mg protein, respectively) and in Group C (8.2±0.7 and 1.7±0.4 pg/mg protein, respectively).

Fig. 6

Serum and heart tissue levels of TNF-α and 1L-1β. In serum levels of TNF-α and IL-1β, there was a significant difference between Groups A (Ves(−)) and B-0 (Ves(+)), and C (C). *P<0 001, **P<0.01, ***P<0.05. In levels of TNF-α and IL-1β in heart tissues, there was a significant difference between Groups A and B-0 or C. **P<0.01.

4 Discussion

There are several interesting findings in the present study. First, oral treatment with vesnarinone from day 14 (early-stage myocarditis) inhibited the development of myocardial damage with improvement of cardiac dysfunction in rats with myosin-induced autoimmune myocarditis. Second, the levels of proinflammatory cytokines such as TNF-α and IL-1β in serum and cardiac tissues were reduced in the vesnarinone-treated rats with induced myocarditis. Third, decreased levels of serum NOx and cyclic GMP, and cardiac iNOS protein and mRNA were observed in the vesnarinone-treated rats. These data suggest that vesnarinone may prove to be useful in the treatment of myocarditis by attenuating NO production through suppression of the production of iNOS and cytokines.

With respect to the inhibitory effect of vesnarinone on production of cytokines, Matsui et al. have shown in vitro that vesnarinone suppresses the production of TNF-α by cultured murine spleen cells stimulated by treatment with bacterial lipo-polysaccharide (LPS) [14]. Furthermore, Matsumori et al. have noted that vesnarinone inhibits the production of TNF-α and IFN-γ by LPS-stimulated cells in whole blood from patients with heart failure [13]. Furthermore, Hattori et al. have reported that vesnarinone inhibits NO synthase by inhibiting the induction of NOS in the mouse macrophage cell line J774 and in cultured rat cardiomyocytes stimulated by treatment with LPS or LPS+IFN-γ [17]. Considered together with these previous reports, our data indicating that vesnarinone significantly inhibits the production of TNF-α and IL-1β resulting in decreased levels of these cytokines in both serum and tissues and further indicating that vesnarinone inhibits the production of serum NOx and myocardial iNOS protein seem reasonable.

In our myocarditis model, it is characteristic that a focal inflammatory reaction in the myocardium initially occurs, induced mainly by macrophages on 14 days after immunization with cardiac myosin, and subsequently infiltration of inflammatory cells such as macrophages and CD4+ T cells leads to myocardial cell destruction. These cells release proinflammatory cytokines, extending the area of myocardial damage, and these cytokines trigger the production of various other factors including excess amounts of NO. Therefore, it is postulated that vesnarinone breaks this vicious cycle by inhibiting iNOS production induced by cytokines released by infiltrated macrophages, leading to less myocardial damage/cell death caused by NO in myosin-induced myocarditis.

Recently, Ikeda U et al. have reported that vesnarinone increases NO synthase in cultured cardiomyocytes stimulated by treatment with cytokines, at least partially through a cAMP-dependent pathway [28]. In their study, however, the dose of vesnarinone used was higher (more than 30 μM) than that employed clinically, suggesting that vesnarinone at high dosage levels could act as an enhancer of cardiac NO production, leading to deterioration of myocardial contractility, tissue damage, and heart failure. In contrast, the serum concentration of vesnarinone in the present study was quite similar to that in humans receiving vesnarinone therapy (5 to 10 μg/ml; 13 to 26 μM). In fact, an increase in mortality was noted in patients with heart failure who were administered a high dose of vesnarinone (120 mg/day), whereas six months of therapy at 60 mg/day resulted in lower morbidity and morality and improved the quality of life [12]. Most recently, however, it is reported that vesnarinone is associated with a dose dependent increase in mortality in patients with severe heart failure [29]. This discrepancy from our data may be caused by the difference in the pathogenesis of cardiac diseases and in the term of medication.

In addition to the anti-cytokine and anti-NO effects, another possible explanation for the improvement observed with vesnarinone is that it increases the intracellular concentration of cyclic AMP through inhibition of phosphodiesterase. In this regard, however, the precise mechanisms by which vesnarinone inhibits the development of myocardial inflammation and damage in animals with autoimmune myocarditis are still unclear.

In conclusion, we have demonstrated in the present study that oral vesnarinone treatment has clinically and histopathologically beneficial effects and has inhibitory actions on the development of autoimmune myocarditis. As we learn more about the pathophysiological and pathogenic role of cytokines in myocarditis, it should be possible to design better and more targeted pharmacologic agents. Furthermore, investigation of inotropic agents which are effective in suppressing the production of NO may be helpful in developing new immunosuppressive agents. Vesnarinone may therefore be of value in preventing the development of other diseases in which increased expression of cytokines or the production of NO plays a major role in the pathogenesis. We believe that vesnarinone may provide a new therapeutic strategy for the management of infections or immunological disorders of the heart.

Acknowledgements

This study was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, a Research Grant for Diseases from the Ministry of Health and Welfare of Japan, a grant from the Japan Private School Promotion Foundation and a grant from the Japan Research Promotion Society for Cardiovascular Diseases.