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. 2011 Feb 11;7(2):154-67.
doi: 10.7150/ijbs.7.154.

Olmesartan, an AT1 antagonist, attenuates oxidative stress, endoplasmic reticulum stress and cardiac inflammatory mediators in rats with heart failure induced by experimental autoimmune myocarditis

Vijayakumar Sukumaran  1 , Kenichi Watanabe, Punniyakoti T Veeraveedu, Narasimman Gurusamy, Meilei Ma, Rajarajan A Thandavarayan, Arun Prasath Lakshmanan, Ken'ichi Yamaguchi, Kenji Suzuki, Makoto Kodama
Affiliations

Olmesartan, an AT1 antagonist, attenuates oxidative stress, endoplasmic reticulum stress and cardiac inflammatory mediators in rats with heart failure induced by experimental autoimmune myocarditis

Vijayakumar Sukumaran et al. Int J Biol Sci. .

Abstract

Studies have demonstrated that angiotensin II has been involved in immune and inflammatory responses which might contribute to the pathogenesis of immune-mediated diseases. Recent evidence suggests that oxidative stress may play a role in myocarditis. Here, we investigated whether olmesartan, an AT(1)R antagonist protects against experimental autoimmune myocarditis (EAM) by suppression of oxidative stress, endoplasmic reticulum (ER) stress and inflammatory cytokines. EAM was induced in Lewis rats by immunization with porcine cardiac myosin, were divided into two groups and treated with either olmesartan (10 mg/kg/day) or vehicle for a period of 21 days. Myocardial functional parameters measured by hemodynamic and echocardiographic analyses were significantly improved by the treatment with olmesartan compared with those of vehicle-treated rats. Treatment with olmesartan attenuated the myocardial mRNA expressions of proinflammatory cytokines, [Interleukin (IL)-1β, monocyte chemoattractant protein-1, tumor necrosis factor-α and interferon-γ)] and the protein expression of tumor necrosis factor-α compared with that of vehicle-treated rats. Myocardial protein expressions of AT(1)R, NADPH oxidase subunits (p47phox, p67phox, gp91phox) and the expression of markers of oxidative stress (3-nitrotyrosine and 4-hydroxy-2-nonenal), and the cardiac apoptosis were also significantly decreased by the treatment with olmesartan compared with those of vehicle-treated rats. Furthermore, olmesartan treatment down-regulated the myocardial expressions of glucose regulated protein-78, growth arrest and DNA damage-inducible gene, caspase-12, phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-JNK. These findings suggest that olmesartan protects against EAM in rats, at least in part via suppression of oxidative stress, ER stress and inflammatory cytokines.

Keywords: Experimental autoimmune myocarditis; endoplasmic reticulum stress; inflammation; olmesartan; oxidative stress.

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Conflict of interest statement

Conflict of Interests: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1
Effects of olmesartan on cellular infiltration and inflammatory cytokines in rats with EAM induced by autoimmune myocarditis. [1A] Histopathology in the heart (H&E-staining) at ×200 fold magnification. (a) Histopathology in a control (group N) (grade 0). (b) Representative histopathology in rat with myocarditis treated with vehicle (group V), the myocardial inflammation was diffused and exceeding 50% of the transverse section (grade 3). (c) Representative histopathology in rat with myocarditis treated with olmesartan (group Olm-10), showed a small focus of cellular infiltration (grade 1). [1B-F] Myocardial messenger RNA expression levels of IL-1β [1B], MCP-1 [1C], TNF-α [1D], IFN-γ [1E] and protein expression of TNF-α [1F], NF-κB [1G] in rats with EAM was determined by quantitative RT-PCR and Western blot. The mRNA expression level of each sample was expressed relative to the expression level of GAPDH gene. [1F-1G] Representative Western blots showing specific bands for TNF-α, NF-κB and GAPDH as an internal control. Equal amounts of protein sample (30 μg) obtained from whole ventricular homogenate were applied in each lane. The mean density value of TNF-α, NF-κB was expressed as a ratio relative to that of GAPDH. Data are mean ± SEM of 4 to 6 rats. Group N, age-matched untreated rats; group V, EAM rats administered with vehicle; group Olm-10, EAM rats treated with olmesartan (10 mg/kg/day). The values are mean ± SEM. *P<0.05, **P<0.01 vs group N; ##P<0.01 vs group V.
Figure 2
Figure 2
Effects of olmesartan on cardiac fibrosis and oxidative stress in rats with EAM induced by autoimmune myocarditis. [2A] Azan-Mallory staining for fibrosis of the cross-sectional tissue slices of hearts. Fibrosis is indicated by the blue area as opposed to the red myocardium (×200). [2A1] Myocardial protein expression of osteopontin (OPN). Representative Western blots showing specific bands for OPN and GAPDH as an internal control. Equal amounts of protein sample (30 μg) obtained from whole ventricular homogenate were applied in each lane. The mean density value of OPN was expressed as a ratio relative to that of GAPDH. [2B, C, D] Immunohistochemistry of 4-hydroxy-2-nonenal (4-HNE), 3-nitrotyrosine (3-NT) and angiotensin-II [counterstained with hematoxylin; ×400]. Bar graph shows quantitative analysis of 4-HNE [2B1] and Ang-II positive cardiomyocytes [2D1] in groups N, V and Olm-10. Group N, age-matched untreated rats; group V, EAM rats administered with vehicle; group Olm-10, EAM rats treated with olmesartan (10 mg/kg/day). The bar value indicates 20 μm. The values are mean ± SEM. *P<0.05 and **P<0.01 vs group N; #P<0.05, ##P<0.01 vs group V.
Figure 3
Figure 3
Effects of olmesartan on myocardial protein expressions of AT1R, p47phox, p67phox, and gp91phox. [3A-D] Representative Western blots showing specific bands for AT1R, p47phox, p67phox, gp91phox and GAPDH as an internal control. Equal amounts of protein sample (30 μg) obtained from whole ventricular homogenate were applied in each lane. These bands are representative of four separate experiments. 3A-D, Densitometric data of protein analysis. The mean density value of AT1R, p47phox, p67phox and gp91phox was expressed as a ratio relative to that of GAPDH. Each bar represents mean ± SEM of 4 to 6 rats. Group N, age-matched untreated rats; group V, EAM rats administered with vehicle; group Olm-10, EAM rats treated with olmesartan (10 mg/kg/day). The values are mean ± SEM. **P<0.01 vs group N; #P<0.05, ##P<0.01 vs group V.
Figure 4
Figure 4
Effects of olmesartan on myocardial protein expressions of GRP78, CHOP, caspase-12 and apoptosis. [4A-C], Representative Western blots showing specific bands for GRP78, CHOP, caspase-12 and GAPDH as an internal control. Equal amounts of protein sample (30 μg) obtained from whole ventricular homogenate were applied in each lane. These bands are representative of three separate experiments. 4A-C, Densitometric data of protein analysis. The mean density value of GRP78, CHOP and caspase-12 was expressed as a ratio relative to that of GAPDH. [4D] Myocardial tissue sections stained for TUNEL-positive apoptotic nuclei (indicated by arrows) in the hearts of myosin-immunized rats (×400). [4D1] Bar graph shows quantitative analysis of TUNEL positive cells. Each bar represents mean ± SEM of 4 to 6 rats. Group N, age-matched untreated rats; group V, EAM rats administered with vehicle; group Olm-10, EAM rats treated with olmesartan (10 mg/kg/day). The values are mean ± SEM. **P<0.01 vs group N; #P<0.05, ##P<0.01 vs group V.
Figure 5
Figure 5
Myocardial expressions of phospho-p38 MAPK, phospho-JNK family proteins. [5A-B] Representative western blots showing specific bands for phospho-p38 MAPK, phospho-JNK, p38 MAPK and JNK as an internal control. Equal amounts of protein sample (30 μg) obtained from whole ventricular homogenate were applied in each lane. These bands are representative of four separate experiments. 5A-B, Densitometric data of protein analysis. The mean density value of phospho-p38 MAPK and phospho-JNK was expressed as a ratio relative to that of p38 MAPK and JNK. Each bar represents mean ± SEM of 4 to 6 rats. Group N, age-matched untreated rats; group V, EAM rats administered with vehicle; group Olm-10, EAM rats treated with olmesartan (10 mg/kg/day). The values are mean ± SEM. **P<0.01 vs group N; #P<0.05 vs group V.
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