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Cardiovascular Research 1998 38(1):192-197; doi:10.1016/S0008-6363(97)00317-9
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Effects of vesnarinone on nitric oxide synthesis in rat cardiac myocytes

Kenji Kurosaki, Uichi Ikeda*, Yoshikazu Maeda, Masahisa Shimpo, Shuichi Ueno and Kazuyuki Shimada

Department of Cardiology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan

* Corresponding author. Tel.: +81 (285) 442111 ext. 3556; fax: +81 (285) 445317; e-mail: uikeda@jichi.ac.jp

Received 29 May 1997; accepted 11 November 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The purpose of this study was to investigate the effects of vesnarinone on nitric oxide (NO) synthesis in cardiac myocytes. Methods: We measured the accumulation of nitrite, a stable oxidation product of NO, and the expression of inducible NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. Results: Incubation of the cultures with interleukin-1β (IL-1β; 10 ng/ml) and tumor necrosis factor {alpha} (TNF{alpha}; 10 ng/ml) caused a marked increase in nitrite production. Although vesnarinone by itself showed no effect on nitrite accumulation, it enhanced cytokine-induced nitrite production by cardiac myocytes in a dose-dependent manner. The effect of vesnarinone was completely abolished in the presence of NG-monomethyl-L-arginine or actinomycin D. The vesnarinone-induced nitrite production was accompanied by increased iNOS protein expression. In the presence of dibutyryl-cAMP, cytokine-induced nitrite accumulation was further increased, but the stimulatory effect of vesnarinone on nitrite accumulation was diminished. The effect of vesnarinone was also inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner. Conclusions: These findings indicate that vesnarinone increases NO synthesis in cytokine-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

KEYWORDS Vesnarinone; Nitric oxide; Cardiac myocyte; Myocyte; Cytokine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Nitric oxide (NO), a free-radical gas, is now considered an important intracellular messenger in cardiovascular, immune, and neural systems [1, 2]. This messenger causes various biological actions including vasodilation, inhibition of platelet aggregation, and cytotoxic or cytostatic actions in many kinds of cells. NO is synthesized from L-arginine by three isoenzymes expressed either constitutively (neuronal, type I cNOS; endothelial, type III cNOS) or following stimulation by cytokines (inducible, type II iNOS) [2, 3]. iNOS generates the sustained release of large amounts of NO in endotoxin- and cytokine-treated neutrophils, hepatocytes, vascular smooth muscle cells and cardiac myocytes. Recent studies showed that iNOS gene expression occurred frequently in failing human cardiac myocytes of patients with dilated cardiomyopathy, ischemic heart disease and valvular heart disease [4, 5].

Many inotropic agents, stimulating β-adrenergic receptor or inhibiting phosphodiesterase (PDE) III, have dramatic short-term beneficial effects in patients with heart failure. However, long-term treatment with these agents results in an unfavorable outcome. Vesnarinone, a quinolinone derivative, is an oral inotropic agent that augments myocardial contractility with little effect on the heart rate or myocardial oxygen consumption [6, 7]. The mechanisms of action associated with the inotropic properties of vesnarinone in animals include a decrease in the delayed outward and inward rectifying potassium currents [8], an increase in intracellular sodium caused by the prolonged opening of sodium channels [9], and an increase in the inward calcium current attributable to the inhibition of PDE III [10, 11]. Recently, this agent has also been shown to improve the mortality in patients with heart failure [12]. The purpose of this study is to determine the effects of vesnarinone on the synthesis of NO, another modulator of cardiac function, in cardiac myocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Cardiac myocytes were prepared from ventricles of 1-day-old Sprague–Dawley rats as described previously [13]. Briefly, after dissociation with 0.25% trypsin, cell suspensions were washed with Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and centrifuged at 500 g for 10 min. The centrifuged cells were then resuspended in 10% FBS containing DMEM. For selective enrichment of cardiac myocytes, the dissociated cells were preplated for 1 h, during which nonmyocytes readily attached to the bottom of the culture dishes. The resulting suspensions of myocytes were plated onto 24-well dishes at a density of 0.5x106 cells/well. Thymidine (0.6 mg/ml) was added during the first 72 h to prevent proliferation of nonmyocytes. Using this method, we routinely obtained enriched cultures containing >95% myocytes, as assayed by immuno-fluorescence staining with an anti-myosin heavy chain antibody [14].

The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedure) Act, 1986, published by Her Majesty's Stationery Office, London.

2.2 Measurement of nitrite levels
Cell were incubated in 0.5% FBS containing DMEM, and nitrite in the culture medium was measured by mixing 0.5 ml of the medium with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) [15]. The absorbance at 550 nm was measured, and nitrite concentration was determined by using a curve calibration from sodium nitrite standards. After washing, the cells were dissolved in 0.2 ml 1% sodium dodecyl sulfate (SDS) and used for protein assay (BCA protein assay kit; Bio-Rad assay, Hercules, CA) with bovine serum albumin as a standard. Nitrite levels were corrected for protein levels, and data are shown as nmol/mg protein.

2.3 Assay for iNOS protein
The expression of iNOS protein was analyzed by immunoblotting with an anti-iNOS antibody as described previously [16]. Briefly, cells cultured in 100-mm dishes were lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 µM leupeptin, 1 µM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride and 1 M dithiothreitol, and sonicated. The homogenates were then centrifuged at 100 000 g for 20 min, and the supernatants (60 µg protein) were subjected to 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with the anti-mouse iNOS antibody for 2 h, followed by peroxidase-labeled anti-mouse IgG for 1 h. Peroxidase-labeled proteins were detected using the ECL detection system (Amersham Int., Bucks, UK) on X-ray film.

2.4 Reagents
Human recombinant interleukin-1β (IL-1β), interferon-{gamma} (IFN-{gamma}) and vesnarinone were gifts from Otsuka Pharmaceuticals (Tokushima, Japan). Tumor necrosis factor {alpha} (TNF{alpha}) was purchased from Genzyme Corp. (Cambridge, MA). A monoclonal anti-mouse iNOS antibody, which cross-reacts against rat iNOS, was obtained from Transduction Lab. (Lexington, KY). Lipopolysaccharide (LPS), NG-monomethyl-L-arginine (L-NMMA), actinomycin D and N2,2'-O-dibutyryl cAMP (db-cAMP) were purchased from Sigma (St. Louis, MO). Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS) was purchased from Biolog Life Science Institute (Bremen, FRG).

2.5 Statistical analysis
Data are expressed as means±SEM of four samples, which represented at least three separate experiments. Differences were analyzed by one-way ANOVA combined with Scheffe's test, and values of P<0.05 were considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of vesnarinone on nitrite production
Accumulation of nitrite in the medium represents the summation of NOS activity during the time period studied, since NO secreted by cells is rapidly decomposed to the more stable products nitrite and nitrate. As shown in Fig. 1, IL-1β (10 ng/ml) plus TNF{alpha} (10 ng/ml) stimulated the accumulation of nitrite in cardiac myocytes in a time-dependent manner. This cytokine-induced nitrite accumulation was significantly increased in the presence of vesnarinone (100 µM). After a 24 h incubation, the level of cytokine-induced nitrite accumulation in the presence of vesnarinone was about 1.5–2-fold that in the absence of vesnarinone.


Figure 1
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  		Fig. 1 Effects of cytokines and vesnarinone on nitrite production by cultured neonatal rat cardiac myocytes. Myocytes in DMEM containing 0.5% FBS were exposed to IL-1β (10 ng/ml)+TNF{alpha} (10 ng/ml) (closed circles), IL-1β+TNF{alpha}+vesnarinone (100 µM) (open circles) or vehicle (open squares) for 24 h. Nitrite accumulation in the culture medium was measured as described in Section 2, and the values were normalized to the protein content per well. Data are means±SEM of four samples. *P<0.05 compared with cytokine-stimulated cells.

 
As shown in Fig. 2, incubation with vesnarinone for 24 h increased nitrite production by cytokine-stimulated myocytes dose-dependently (1–100 µM), while vesnarinone by itself did not affect the basal level of nitrite production (data not shown).


Figure 2
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  		Fig. 2 Dose-dependent effects of vesnarinone on cytokine-stimulated nitrite production by cardiac myocytes. Myocytes were exposed to various concentrations of vesnarinone (1–100 µM) in the presence of IL-1β (10 ng/ml) and TNF{alpha} (10 ng/ml) for 24 h. Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. Data are means±SEM of four samples. *P<0.05 compared with control samples without vesnarinone.

 
Previously, Hattori et al. [17]reported that vesnarinone inhibited NO synthesis in LPS plus IFN-{gamma}-stimulated cardiac myocytes. As shown in Fig. 3, we thus investigated the effect of vesnarinone on NO synthesis in these cells, and found that vesnarinone also enhanced nitrite production in a dose-dependent manner.


Figure 3
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  		Fig. 3 Dose-dependent effects of vesnarinone on LPS plus IFN-{gamma}-stimulated nitrite production by cardiac myocytes. Myocytes were exposed to various concentrations of vesnarinone (1–100 µM) in the presence of LPS (30 µg/ml) and IFN-{gamma} (100 units/ml) for 24 h. Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. Data are means±SEM of four samples. *P<0.05 compared with control samples without vesnarinone.

 
Simultaneous incubation with the NOS inhibitor L-NMMA (1 mM) or the RNA synthesis inhibitor actinomycin D (5 µg/ml) for 24 h completely inhibited vesnarinone- and cytokine-induced nitrite production (data not shown).

3.2 Effects of vesnarinone on iNOS protein expression
We then examined whether or not vesnarinone augmented cytokine-induced NO production at the level of iNOS protein. As shown in Fig. 4, unstimulated cardiac myocytes expressed no detectable iNOS protein, whereas exposure to cytokines for 24 h clearly induced its expression. Co-incubation with vesnarinone for 24 h further augmented cytokine-induced iNOS protein expression.


Figure 4
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  		Fig. 4 Effects of vesnarinone on iNOS protein accumulation. Cells were incubated for 24 h with IL-1β (10 ng/ml) plus TNF{alpha} (10 ng/ml) with or without vesnarinone (100 µM). Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis with the anti-iNOS antibody. The iNOS protein band with a molecular mass of about 125-kD is the band above the 116.5-kD marker. Lane 1, control; lane 2, IL-1β plus TNF{alpha}; lane 3, IL-1β plus TNF{alpha} plus vesnarinone; lane 4, positive control (mouse macrophage iNOS).

 
3.3 Involvement of cAMP
We also investigated the mechanism of the stimulatory effect of vesnarinone on NO synthesis. It has been reported that vesnarinone increases intracellular cAMP concentrations of ventricular myocytes [11], and we previously observed that cAMP upregulates iNOS expression in cardiac myocytes [18]. Thus, we speculated the involvement of a cAMP-dependent pathway in the effect of vesnarinone on NO synthesis. As shown in Fig. 5, addition of db-cAMP (1 mM) increased the level of nitrite accumulation in IL-1β plus TNF{alpha}-treated cells. Addition of vesnarinone to the cultures significantly increased cytokine-induced nitrite accumulation, but its effect was not additive or synergistic in the presence of db-cAMP.


Figure 5
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  		Fig. 5 Effects of db-cAMP on cytokine- and vesnarinone-induced nitrite accumulation in cardiac myocytes. Cells were incubated for 24 h with cytokines (10 ng/ml IL-1β plus 10 ng/ml TNF{alpha}) or cytokines plus 100 µM vesnarinone (VES) in the presence or absence of 1 mM db-cAMP. Nitrite accumulation in the culture medium was measured. Data are means±SEM of four samples. *P<0.05, ns; not significant.

 
Furthermore, as shown in Fig. 6, the effect of vesnarinone was significantly inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner, while the cytokine-induced nitrite accumulation was not affected by Rp-8-Br-cAMPS.


Figure 6
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  		Fig. 6 Effects of Rp-8-Br-cAMPS on nitrite accumulation in cardiac myocytes. Cells were incubated for 24 h with IL-1β (10 ng/ml) plus TNF{alpha} (10 ng/ml) and various concentrations of Rp-8-Br-cAMPS (10–7–10–5 M) in the presence or absence of vesnarinone (100 µM). Nitrite accumulation in the culture medium was measured, and the values were normalized to the protein content per well. Data are means±SEM of four samples. *P<0.05 compared with cytokine-stimulated cells without vesnarinone and Rp-8-Br-cAMPS.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This study was designed to examine whether or not vesnarinone modulates NO production by cytokine-stimulated cardiac myocytes. We measured the accumulation of nitrite, a stable oxidation product of NO, in the medium to indicate the amount of NO synthesis. Vesnarinone alone had no effect on nitrite production; it markedly augmented nitrite production by cytokine-stimulated myocytes in a time- and dose-dependent manner. Its effect was reduced in the presence of L-NMMA or actinomycin D, suggesting that the upregulation of nitrite production by vesnarinone is likely due to the enhanced induction of iNOS. Indeed, Western blot analysis revealed that vesnarinone further increased cytokine-induced iNOS protein levels.

Previously, Hattori et al. [17]noted that vesnarinone dose-dependently inhibited NO synthesis induced by LPS plus IFN-{gamma} in rat cardiac myocytes. A possible reason for the discrepancy between their findings and ours could be that we used the primary culture of neonatal rat cardiac myocytes, while they used the neonatal rat cardiac myocytes at passage 15–20. In our culture system, enriched cultures containing more than 95% cardiac myocytes were routinely obtained [14]. However, their cultured myocytes at passage 15–20 probably did not keep the characteristics as cardiac myocytes nor grew without contamination of non-cardiac myocytes. Another possibility is that we used IL-1β plus TNF{alpha}-treated myocytes, while they used LPS plus INF-{gamma}-treated myocytes. However, we observed that vesnarinone also enhanced nitrite accumulation in our cultured myocytes stimulated with LPS plus INF-{gamma} (Fig. 3).

Vesnarinone increases intracellular concentrations of cAMP through PDE III inhibition resulting in a positive inotropic effect [11]. It has been shown that cAMP enhances iNOS mRNA stability in cardiac myocytes [19]and that cAMP-elevating agents, such as forskolin, isoproterenol, and adrenomedullin, augment cytokine-induced NO production in cardiac myocytes [18, 20]. The causal relationship between the production of cAMP and augmentation of NO synthesis by vesnarinone in rat cardiac myocytes is evidenced by the findings that the cAMP analogue db-cAMP increased cytokine-induced NO production and that the effect of vesnarinone on cytokine-induced NO production was not additive or synergistic in the presence of db-cAMP (Fig. 5). Furthermore, the effect of vesnarinone was inhibited by Rp-8-Br-cAMPS (Fig. 6). These findings suggest that vesnarinone augments cytokine-induced NO production, at least partially through a cAMP-dependent pathway.

Previous studies have noted high levels of iNOS activity in endomyocardial biopsy specimens as well as increased plasma levels of nitrite in patients with dilated cardiomyopathy [21, 22]. High plasma levels of cytokines have also been reported in patients with heart failure [23, 24]. Very recently, Satoh et al. [25]reported that iNOS was consistently coexpressed with TNF{alpha} in myocardial tissues of patients with dilated cardiomyopathy. Cytokine-induced NO can moderate myocardial contractility [26–30]. Hosenpud et al. [26]demonstrated that IL-1β caused a negative inotropic effect on canine hearts in vivo through NO production. Roberts et al. [27]reported that IL-1β showed a suppressive effect on the beating rate of cultured rat cardiac myocytes. Enhanced NO generation by IL-1β leads to sustained reduced myocardial contractility, presumably via activation of soluble guanylate cyclase to generate cGMP, which has been shown to decrease myocardial contractility by decreasing cytoplasmic Ca2+ concentrations [31]. Furthermore, lethal effects of cytokine-induced NO on cardiac myocytes have been noted [32].

Although 60 mg of vesnarinone was shown to improve both the quality of life and the prognosis of patients with heart failure, there was an increase in mortality in the group of patients treated with high-dose (120 mg) vesnarinone [12]. The therapeutic serum concentrations of vesnarinone in humans are about 13 to 26 µM. In the present study, vesnarinone at more than 30 µM significantly enhanced nitrite accumulation, suggesting that high concentrations of vesnarinone could act as an enhancer of cardiac NO production and deteriorate myocardial contractility and cell viability in heart failure. However, cytoprotective effects of NO on cardiovascular diseases have also been shown [33], and it might be difficult to apply the observed findings in neonatal rat cardiocytes to the adult human heart, and further studies are necessary to determine if the effects of vesnarinone on NO synthesis reported here contribute to the outcome of heart failure.

Time for primary review 33 days.


   Acknowledgements
 
We thank Toshiko Kanbe for her technical assistance. This study was supported by the Ministry of Education, Science, Sports and Culture Japan (#8670821) and Mitsukoshi Grant-in-Aid 1996.


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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