Kynurenic Acid: A Novel Player in Cardioprotection against Myocardial Ischemia/Reperfusion Injuries

Figures

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  Figure 7. In vitro and in vivo experimental design of protocols and groups. (A) In vitro: H9C2 control cells underwent no intervention; cells from the hypoxia/re-oygenation (H/R) groups were submitted to 4 h 50 min of hypoxia followed by 2 h of reoxygenation and were either treated with a vehicle (DMSO) or with kynurenic acid (KYNA 1 µM) 10 min pre hypoxia, pre + per hypoxia, or pre + per + post hypoxia. Cell death quantification as well as mitochondrial membrane potential were performed two hours after reoxygenation. (B) In vivo: The sham animals underwent no injection and no left anterior descending coronary artery (LAD) ligation; the MI group underwent 40 min of ischemia followed by 2 h of reperfusion. Then, 10 min before coronary artery occlusion, a vehicle (NaOH 1 M) or KYNA (300 mg/kg) was administrated intraperitoneally. Infarct size assessment using 2,3,5-triphenyltetrazolium chloride (TTC) staining was realized after 2 h of reperfusion; tissue sampling was performed after either 15 min or 2 h of reperfusion.

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  Figure 1. Histograms showing (A) relative cell death and (B) percentage of cells with conserved mitochondrial membrane potential in control cells that did not undergo hypoxia/reoxygenation (H/R), and cells that underwent H/R, either vehicle (DMSO) or KYNA (1 µM) treated. KYNA treatment was performed either 10 min pre hypoxia in DMEM medium, pre + per hypoxia, or pre + per + post hypoxia for KYNA (n = 6–9 for relative cell death and n = 3–7 for mitochondrial membrane potential). Data are expressed as mean ± SEM; * p <0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Scatter dot plot showing (A) area at risk (AAR) as a percentage of the total left ventricle (LV) and (B) area of necrosis (AN) as a percentage of AAR after 2 h of reperfusion (n = 6–7). Data are expressed as mean ± SEM; * p < 0.05.

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  Figure 3. Mitochondrial respiratory chain complex, PGC1α and AMPKα protein expression by means of Western blot and mitochondrial respiratory chain complex activity by spectrophotometry. (A) Representative immunoblots and histograms showing quantification of protein expression for the mitochondrial respiratory chain complex in non-ischemic (sham), ischemic + vehicle (MI), and ischemic + kynurenic acid (MI + KYNA) left ventricle (LV) samples after 2 h of reperfusion (n = 5–10). β-actin or GAPDH was used as loading control. (B) Histograms showing complex over citrate synthase activity ratio (I/CS, II/CS, III/CS, IV/CS) or complex ratio activity (I/II, III/I, IV/I) in sham, MI, and MI + KYNA LV samples after 2 h of reperfusion (n = 5–10). (C) Representative immunoblots and histograms showing quantification of protein expression for AMPKα and PGC1α in sham, MI, and MI + KYNA LV samples after 2 h of reperfusion (n = 5–10). Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. Foxo3α, Akt and ERK1/2 phosphorylation levels by means of Western blot. (A) Representative immunoblots and histogram quantification of protein expression levels for p-FOXO3α, FOXO3α and (B) p-ERK1/2, ERK1/2, p-Akt, Akt, in non-ischemic (sham), ischemic + vehicle (MI), and ischemic + kynurenic acid (MI + KYNA) left ventricle samples following 15 min of reperfusion (n = 6–9). GAPDH was used as the loading control. Data are expressed as mean ± SEM. * p < 0.05, *** p < 0.001.

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  Figure 5. Antioxidant markers mRNA and protein expression, respectively, by means of RT-qPCR and Western blot. (A) SOD1, 2, 3, and catalase mRNA expression in non-ischemic (sham), ischemic + vehicle (MI), and ischemic + kynurenic acid (MI + KYNA) left ventricle samples following 2 h of reperfusion (n = 6–11). (B) Representative immunoblots and histogram quantification of protein expression for SOD1, 2, 3 and catalase in the sham, MI, and MI + KYNA groups following 2 h of reperfusion (n = 6–9). GAPDH was used as the loading control. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. Mitophagy protein markers protein expression levels using Western blot. Representative immunoblots and histogram quantification of protein expression for (A) p62 and (B) PARK2 in non-ischemic (sham), ischemic + vehicle (MI), and ischemic + kynurenic acid (MI + KYNA) left ventricle samples after 2 h of reperfusion (n = 5–7). GAPDH was used as the loading control. Values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01.