Attenuation of virus-induced myocardial injury by inhibition of the angiotensin II type 1 receptor signal and decreased nuclear factor-kappa B activation in knockout mice
Kanjo Yamamoto, MD*,
Tetsuo Shioi, MD, PhD*,
Koji Uchiyama, MD*,
Tadashi Miyamoto, MD, PhD*,
Shigetake Sasayama, MD, PhD, FACC* and
Akira Matsumori, MD, PhD, FACC*,*
* Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan
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Figure 1 Fourteen-day cumulative survival after encephalomyocarditis virus (EMCV) inoculation in Ang-II type I-receptor (AT1R) mice (open circles) and wild-type mice (solid circles). Survival was significantly higher in AT1R knock-out mice (n = 20 each). *p < 0.05.
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Figure 2 Myocardial histopathologic scores for myocardial necrosis (A) and cellular infiltration (B) on day 6 after encephalomyocarditis inoculation in surviving mice (n = 8 each). The extent of myocardial necrosis and cellular infiltration was less in knock-out (KO) mice than in wild-type (WT) mice. *p < 0.01.
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Figure 3 Myocardial cytokine mRNAs after encephalomyocarditis virus (EMCV)-induced myocarditis inoculation. Inoculation with EMCV significantly increased the expression of TNF-alpha (A) and IL-1-beta (B) in wild-type (WT) mice, but not in knock-out (KO) mice (n = 10 each). *p < 0.01 vs. WT with PBS inoculation. #p < 0.01 vs. KO with EMCV inoculation.
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Figure 4 Activity of NF-kB in heart tissue after encephalomyocarditis virus (EMCV)-induced myocarditis inoculation. (A) Representative autoradiogram of electrophoretic mobility shift assay (EMSA) experiment. A supershifted band was observed with 0.3 µg of anti-p65 antibody (arrowhead). Lane 1 = WT with EMCV inoculation; lane 2 = lane 1 with a 100-fold excess of cold probe; lane 3 = lane 1 with anti-p65 antibody; lane 4 = without nuclear protein (negative control). (B) Representative EMSA experiment. From each sample, equal amounts of nuclear protein were obtained and mixed in each group (n = 8 each). The experiments were performed using 8 µg of the mixed proteins. Lane 1 = WT with phosphate buffered saline (PBS) inoculation; lane 2 = knock-out (KO) with PBS inoculation; lane 3 = wild-type (WT) with EMCV inoculation; lane 4 = KO with EMCV inoculation. (C) Densitometric analysis of the results expressed as arbitrary units of mean values ± SEM of eight animals from each group. The mean value of nuclear factor-kappa B activities in the WT group with PBS inoculation is represented as 1. *p < 0.01 vs. WT with PBS inoculation. #p < 0.01 vs. KO with EMCV inoculation.
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Figure 5 Significant attenuation of proinflammatory cytokine expression and nuclear factor-kappa B (NF-kB) activity in the heart of wild-type (WT) mice (n = 9 each) with viral myocarditis by Ang-II type 1-receptor (AT1R) blockade with candesartan (10 mg/kg per day). Myocardial cytokine mRNAs of TNF-alpha (A) and interleukin-1-beta (IL-1-beta) (B). (C) Densitometric analysis of the results expressed as arbitrary units of mean values ± SEM. Mean NF-kB activity in the infected WT mice treated with the vehicle is represented as 1. *p < 0.01. **p < 0.05.
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Figure 6 Effects of intravenous Ang-II injection on nuclear factor-kappa B (NF-kB) activity and expression of proinflammatory cytokines in heart tissue. (A) Representative electrophoretic mobility shift assay experiment. From each sample, equal amounts of nuclear protein in heart tissue were obtained and mixed (n = 5 in each group); 8 µg of the mixed proteins was used for the experiments. Lane 1 = wild-type (WT) with phosphate buffered saline (PBS) injection; lane 2 = knock-out (KO) with PBS injection; lane 3 = WT with Ang-II injection; lane 4 = KO with Ang-II injection. (B) Densitometric analysis of the results expressed as arbitrary units of mean values ± SEM (5 animals from each group). Mean NF-kB activity in the WT group with PBS injection is represented as 1. At 30 min after the injection of Ang-II, NF-kB activity was significantly increased in WT but not KO mice. (C, D) Gene expression of proinflammatory cytokines in heart tissue harvested 90 min after the injection of Ang-II or PBS. The cytokine mRNAs were corrected by the amount of GAPDH mRNA. At 90 min after the injection of Ang-II, the expressions of TNF-alpha (C) and IL-1-beta (D) were significantly increased in WT but not KO mice (n = 5 each). *p < 0.01 vs. WT with PBS injection. #p < 0.01 vs. KO with Ang-II injection.
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