Cardioprotective effect of nicorandil on isoproterenol induced cardiomyopathy in the Mdx mouse model

Figures

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  Fig. 1. Schematic of the 10 day treatment protocol

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  Fig. 2. Histologic assessment of myocardial fibrosis. Frozen mouse heart tissue was sectioned and stained with Masson’s trichrome stain to assess myocardial fibrosis (blue tissue coloration). a Representative images of Masson’s trichrome stained heart tissue, with groups identified as follows: 1: Vehicle, 2: Nicorandil, 3: Isoproterenol, 4: Isoproterenol + Nicorandil. Images are centered in the interventricular septum (IVS) and areas corresponding to right ventricle (RV) and left ventricle (LV) are labeled. b Quantification of percent fibrosis on Masson’s trichrome staining, represented as percentage of total tissue area ± SEM. Isoproterenol led to an increase in percent fibrosis. Nicorandil was protective against isoproterenol-induced fibrosis. Number of samples for each experimental group: vehicle n = 10, Nicorandil n = 10, Isoproterenol n = 14, Isoproterenol + Nicorandil n = 14. SEM, standard error of the mean. (*p < 0.05 vs vehicle)

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  Fig. 3. Reactive oxygen species gene expression. Quantitative RT-PCR was performed on mouse cardiac tissue to quantify mRNA expression of select genes involved in reactive oxygen species formation, including NOX2, NOX4, SOD2, and Sirtuin 3. Expression of NADPH Oxidase (NOX) 4 and NOX 2 were increased with isoproterenol stress. Treatment with nicorandil prevented this change in gene expression. (n = 6, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001)

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  Fig. 4. Reactive oxygen species protein expression. Western blot was performed on mouse cardiac tissue to assess protein expression of selected proteins involved in reactive oxygen species production and inflammation. Relative protein expression is shown in part a, and cropped representative immunoblot images are shown in part b. the vinculin loading control is shown for each separate western blot that is shown in the figure. Note that NFkB and TNFα were assessed using different regions of the same western blot membrane. Full-length blots are presented in Additional files 1, 2, 3, 4: Figs. 1–4 (n = 2, *p ≤ 0.05, **p ≤ 0.01)

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  Fig. 5. Xanthine oxidase activity: Xanthine oxidase activity was assessed in cardiac tissue by fluorometric assay kit (Cayman Chemical). Nicorandil led to a decrease in xanthine oxidase activity with concomitant isoproterenol treatment protocol (n = 4, *p < 0.05 vs vehicle)