Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation, Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

Figures

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  Figure 1. Activation of PPARβ/δ improves cardiac recovery and reduces arrhythmias and infarct size following I/R. (A) Left ventricular developed pressure (LVDP, systolic minus diastolic pressure) was measured during equilibration and post-ischemic period; (B) Infarct size (IS), expressed in % of risk area (LV). (C) Ectopic activity was evaluated as total number of extrasystoles (ES) during the post-ischemic period. (D) Incidence of ventricular tachycardia expressed in percentage. Data are presented as mean ± SEM, n = 3–12 per group, * p < 0.05 and *** p < 0.001. GW, pretreated with agonist GW0742; GSK/GW, pretreated with antagonist GSK0660 and agonist GW0742.

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  Figure 2. Administration of PPARβ/δ agonist GW0742 results in upregulation of PPARβ/δ and of its target genes Angptl4 and Mcad. Relative mRNA expression of PPARβ/δ (A), Angptl4 (B) and Mcad (C) was normalized to β-actin. Data are presented as mean ± SEM, n = 3–4, * p < 0.05 and ** p < 0.01. GW, pretreated with agonist GW0742; GSK/GW, pretreated with antagonist GSK0660 and agonist GW0742.

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  Figure 3. Activation of PPARβ/δ upregulates myocardial antioxidant enzymes SOD2, catalase and UCP3 and ameliorates I/R-induced oxidative stress. Relative mRNA expression of (A) mitochondrial SOD2, (B) catalase and (C) UCP3 was determined by qPCR and normalized to β-actin. (D) Quantification of protein carbonyls. (E) Representative blot of 4-HNE mediated protein adducts formation (upper panel) and quantification by densitometric analysis (lower panel). Data are shown as mean ± SEM, n = 3–5, * p < 0.05 and *** p < 0.001. GW, pretreated with agonist GW0742; GSK/GW, pretreated with antagonist GSK0660 and agonist GW0742.

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  Figure 4. PPARβ/δ activation upregulates mitochondrial ALDH2 activity and expression leading to decreased infarct size after I/R. (A) ALDH2 enzymatic activity. Data are shown as mean ± SEM, n = 4–12 per group. (B) mRNA expression of ALDH2 was determined by qPCR and normalized with β-actin. Data are shown as mean ± SEM, n = 4–6 per group. (C) Infarct size expressed as % of risk area (LV) was determined in the presence or absence of ALDH2 inhibitor cyanamide (CYA). Data are shown as mean ± SEM, n = 5–8 per group. * p < 0.05, ** p < 0.01 and *** p < 0.001. GW, pretreated with agonist GW0742; GSK/GW, pretreated with antagonist GSK0660 and agonist GW0742; CYA, ALDH2 inhibitor cyanamide.

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  Figure 5. PPARβ/δ activation preserves mitochondrial energy production and function in I/R. (A) PGC-1α mRNA expression was normalized to β-actin. (B) Citrate synthase activity. (C) IDH2 mRNA expression was normalized to β-actin. (D) ATP content expressed as μmoles/g of cardiac tissue. Data are shown as mean ± SEM, n = 3–5, * p < 0.05 and ** p < 0.01. GW, pretreated with agonist GW0742; GSK/GW, pretreated with antagonist GSK0660 and agonist GW0742.

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  Figure 6. Graphical representation of the proposed effect of PPARβ/δ activation on cardiac antioxidant defense and mitochondrial function during myocardial I/R. PPARβ/δ, peroxisome proliferator activated receptor β/δ; PGC-1α, peroxisome proliferator activated receptor gamma cofactor 1 alpha; IDH2, isocitrate dehydrogenase 2; CS, citrate synthase; ATP, adenosine triphosphate; SOD2, superoxide dismutase 2; CAT, catalase; UCP3, uncoupling protein 3; ALDH2, mitochondrial aldehyde dehydrogenase 2; 4-HNE, 4-hydroxy-2-nonenal.

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  Figure 7. Experimental design for drug administration and induction of ischemia—reperfusion (I/R) in isolated rat hearts. GW, PPARβ/δ-specific ligand GW0742; GSK, PPARβ/δ antagonist GSK0660; CYA, ALDH2 inhibitor cyanamide.