Pharmacological Inhibition of S-Nitrosoglutathione Reductase Reduces Cardiac Damage Induced by Ischemia–Reperfusion

Figures

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  Figure 1. Impact of S-nitrosoglutathione reductase (GSNOR) inhibitor on hemodynamic parameters in a model of cardiac ischemia–reperfusion. Effect of GSNOR inhibitor (C2) at 3 different concentrations (0.1, 1 and 10 µmol/L) and controls (vehicle) on (A), left ventricular developed pressure, (B), cardiac contractility evaluated as dP/dtmax, (C), coronary perfusion pressure, and (D), cardiac frequency. The shaded area corresponds to the period of time (45 min) that the isolated heart was in ischemia by stopping the perfusion. Asterisk (*) indicates p < 0.05, compared to the other groups, by 2-way ANOVA with multiple comparisons followed by Newman Keules as post-hoc analysis. n = 5 in each group.

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  Figure 2. Impact of GSNOR inhibitor on cardiac damage in ischemia–reperfusion. (A), levels of creatine kinase release in the coronary effluent of Langendorff perfused hearts in the presence of increasing concentrations of the GSNOR inhibitor C2 and controls (vehicle). (B), representative images of hearts stained with TTC after being submitted to a protocol of ischemia–reperfusion, under control conditions or in the presence of the GSNOR inhibitor C2 at 0.1, 1 and 10 µmol/L. Right, graph depicting the analysis of the infarcted area. Asterisk (*) indicates p < 0.05 and **, p < 0.005 compared to vehicle (control), by two-way ANOVA (creatine kinase) and one-way ANOVA with multiple comparisons for infarct size analysis. n = 5 in each group.

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  Figure 3. Impact of GSNOR inhibitor on global cardiac S-nitrosylation. Analysis of S-nitrosylated proteins from cardiac extracts of hearts submitted to ischemia reperfusion, treated with vehicle (control) or with the GSNOR inhibitor C2, 1 µmol/L. (A), left—representative images of a gel stained with Coomasie blue for total proteins submitted to the biotin switch process; right—a parallel Western blot of the same samples submitted to the biotin switch, probed with an anti-biotin antibody. (B), densitometric analysis for the intensity of the bands for anti-biotin (n = 3 in each group). (C), quantification of GSNOR enzymatic activity in control hearts and those treated with the GSNOR inhibitor C2, 1 µmol/L (n = 3 in each group). Asterisk (*) indicates p < 0.05.

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  Figure 4. Impact of GSNOR inhibitor on cardiac mitochondrial S-nitrosylation. Analysis of mitochondrial S-nitrosylated proteins from cardiac extracts of hearts submitted to ischemia reperfusion, treated with vehicle (DMSO) or with the GSNOR inhibitor C2, 1 µmol/L. The upper panels show representative Western blot images for S-nitrosylated proteins from hearts under control conditions or those treated with C2. The lower graph depicts the analysis of the levels of S-nitrosylation of the indicated mitochondrial proteins. Asterisk (*) indicates p < 0.05 vs. control, n = 5 in each group, except for cytochrome C (n = 3).

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  Figure 5. Expression of nitric oxide synthase (NOS) isoforms. Western blot analysis for the expression of inducible (iNOS) and endothelial (eNOS) NOS in the rat heart after ischemia–reperfusion. The panels show the levels of the NOS isoforms iNOS and eNOS in cardiac homogenates of rat hearts submitted to the ischemia–reperfusion protocol in the absence or presence of the GSNOR inhibitor C2 (1 µmol/L). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as load control. Densitometric units of each corresponding isoform were normalized by the levels of GAPDH in each heart. n = 3 in each group.