LCZ696 ameliorates doxorubicin-induced cardiomyocyte toxicity in rats

Figures

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  Figure 1. Effect of valsartan and LCZ696 on the serum cardiac troponin T levels in doxorubicin-treated rats. (A) On day 11, DOX significantly increased the serum cardiac troponin T level, which did not differ from that in the DOX + VAL and DOX + LCZ groups. n = 15 in each group. (B) On day 18, the increased serum cardiac troponin T level in the DOX group was significantly reduced in the DOX + LCZ group, but not in the DOX + VAL group. n = 10–15 in each group. Data are expressed as the mean ± standard error. *p < 0.05 versus control and #p < 0.05 versus DOX. MDA, malondialdehyde; DOX, doxorubicin; VAL, valsartan; LCZ, LCZ696.

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  Figure 2. Effect of valsartan and LCZ696 on serum malondialdehyde (MDA) levels and oxidative stress in rat hearts treated with doxorubicin. (A) On day 11, DOX significantly increased the serum MDA level which was significantly reduced by LCZ, but not VAL. n = 15 in each group. (B) On day 18, DOX significantly increased the serum MDA level, which was not reduced by LCZ and VAL. n = 10–15 in each group. (C) Representative DHE staining on day 18 in each group. (D) Oxidative stress in the cardiac tissue was measured based on the fluorescence intensity of DHE. LCZ, but not VAL, significantly reduced the DOX-induced oxidative stress. Five random fields from three random samples per group were re-evaluated for the analysis. (E) Real-time PCR to check for the expression of p47phox day 18. DOX significantly increased p47phox mRNA expression. This increase was significantly reduced by LCZ, but not VAL. Data are presented relative to the control group. n = 10–15 in each group. (F) Representative Western blot images of the expression of p47phox in the cardiac tissue on day 18. (G) Protein expression of p47phox as measured by western blotting. DOX significantly increased the level of p47phox which was…

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  Figure 3. Effect of valsartan and LCZ696 on activation of AMPK and the expression of Bax, Bcl-2, and cleaved caspase3 in rat hearts treated with doxorubicin. (A) Representative western blot images of the expression of phosphorylated- and total-AMPK in the cardiac tissue on day 18. (B) The ratio of p-AMPK/AMPK as measured by western blotting. DOX significantly decreased the ratio of p-AMPK/AMPK, which LCZ significantly increased, but not VAL. (C) Representative western blot images of the expression of Bax and Bcl-2 in the cardiac tissue on day 18. (D) The ratio of p-AMPK/AMPK as measured by western blotting. DOX significantly increased the ratio of Bax/Bcl-2, which LCZ significantly reduced, but not VAL. (E) Representative western blot images of the expression of cleaved caspase-3 in the cardiac tissue on day 18. (F) Protein expression of cleaved caspase-3 as measured by western blotting. DOX significantly increased the level of caspase-3, which LCZ significantly reduced, but not VAL. Data are expressed as the mean ± standard error. *p < 0.05 versus control and #p < 0.05 versus p-AMPK; phosphorylated adenosine monophosphate-activated protein kinase; DOX, doxorubicin; VAL, valsartan; LCZ,…

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  Figure 4. Effect of valsartan and LCZ696 on morphological changes in rat heart treated with doxorubicin. (A) Representative hematoxylin and eosin and Masson’s trichrome staining on day 18 in each group. (B) DOX significantly decreased the cross-sectional area of cardiomyocytes compared to the control group, whereas the increase in the cross-sectional area of cardiomyocytes by VAL and LCZ was not significant. (C) Representative Masson’s trichrome staining of intestinal fibrosis on day 18 in each group. (D) The percentage of intestinal fibrotic area was significantly increased by DOX. LCZ, but not VAL significantly reduced the percentage of intestinal fibrosis induced by DOX. (E) Representative Masson’s trichrome staining of perivascular fibrosis on day 18 in each group. (F) The percentage of perivascular fibrotic area was significantly increased upon DOX treatment. LCZ, but not valsartan, significantly reduced the DOX-induced percentage perivascular fibrotic area. Data are expressed as the mean ± standard error. Scale bars, 50 μm. Five random fields from three random samples per group were evaluated for the analysis. *p < 0.05 versus control and #p < 0.05 versus DOX. DOX, doxorubicin; VAL,…

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  Figure 5. Effect of valsartan and LCZ696 on the mRNA expression of collagen, tumor necrosis factor-alpha (TNFa), and atrial natriuretic peptide (ANP) in the rat heart, and on serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels in DOX-treated rats. (A) Real-time PCR to check for the expression of COL1A1 mRNA on day 18. The DOX-induced increase in the mRNA levels of COL1A1 was significantly suppressed by LCZ, but not VAL. n = 10–15 in each group. (B) Real-time PCR analysis of TNFa mRNA on day 18. The DOX-induced increase in the mRNA levels of TNFa was not reduced by VAL or LCZ significantly. n = 10–15 in each group. (C) Real-time PCR to check for the expression of ANP mRNA on day 18. The DOX-induced increase in the mRNA levels of ANP was significantly suppressed by VAL or LCZ. n = 10–15 in each group. (D) Serum NT-proBNP level was significantly increased upon DOX treatment. LCZ, but not VAL, significantly reduced the serum NT-proBNP levels that were increased upon DOX treatment. n = 10–15 in each group. Data are expressed as the mean ± standard error. *p < 0.05 versus control and #p < 0.05 versus DOX. DOX, doxorubicin; VAL, valsartan; LCZ, LCZ696; COL1A1, collagen type I alpha…

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  Figure 6. Effect of LCZ696 on doxorubicin-induced mitochondrial oxidative stress and cytotoxicity in H9c2 cells. (A) Cells were stained for superoxide using MitoSOX, a fluorescence indicator of mitochondrial superoxide, and were assayed via flow cytometry. Cells were pre-treated with 20 μM VAL and LCZ for 12 h, and then treated with 5 µM of doxorubicin for 6 h. (B) The mean fluorescence intensity was measured through flow cytometry in individual cells and plotted. LCZ effectively attenuated DOX-induced superoxide generation. Data are expressed as the mean ± standard error. n = 3 in each group. †p < 0.01 versus DOX and **p < 0.05 versus DOX + VAL. (C) Cell viability was measured with MTS assay and expressed as the percentage of control. DOX markedly decreased cell viability. Pre-treatment with 10 μM and 20 μM LCZ ameliorated cytotoxicity, whereas pre-treatment with VAL did not inhibit DOX-induced cytotoxicity. Each point represents the mean ± standard error. n = 5 in each group. ‡p < 0.05 versus control and §p < 0.05 versus DOX treatment alone. DOX, doxorubicin; VAL, valsartan; LCZ, LCZ696; MFI, mean fluorescence intensity.