Figure 3.

Knockout of iPLA₂β protects the heart from I/R injury. (a) Overview of the targeting strategy to create a Pla2g6^−/− mouse model (C57BL/6N) by CRISPR/Cas9-mediated genome engineering. (b) Knockout (KO) mice were genotyped by agarose gel electrophoresis. The size of the PCR product of Pla2g6^−/− mice was 650 bp, whereas the PCR product of the wild-type (WT, Pla2g6^+/+) mice was 573 bp. (c) Absence of iPLA₂β mRNA in KO mice. Total RNA was isolated from ventricular tissues of WT and iPLA₂β KO mice, respectively. Real-time PCR was conducted to assess iPLA₂β at the mRNA level. n = 4–5 for each group. (d) Confirmation of iPLA₂β deletion in iPLA₂β-KO mice. Total proteins were extracted from the ventricular tissues of wild-type (WT) and iPLA₂β-KO mice. Western blotting was conducted to assess iPLA₂β at protein level. n = 5 for each group. (e,f) Knockout of iPLA₂β protected the heart from I/R injury. WT and iPLA₂β-KO mice at 12–14 weeks of age were subjected to myocardial ischemia for 30 min, followed by 24 h of reperfusion. Then, 2,3,5-triphenyltetrazolium chloride (TTC) staining was conducted to detect and quantify the injured cardiac tissue. The white region depicts the ischemic infarct zone; the pink/red area is the border zone; and the blue region represents the remote zone. The relative ratio of the infarct region (IF) to the area at risk (AAR, ischemic and border) was compared between WT and iPLA₂β-KO mice. The relative ratio of AAR to the left ventricle did not differ between WT and KO groups, suggesting that I/R surgery was performed similarly across genotypes. n = 4 per group. (g) Representative photographs of heart sections stained with terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) from WT and KO mice after myocardial I/R (left). Scale bar = 25 μm. Quantification is shown on the right. n = 10–11 per group. Data are represented as mean ± SEM. All experiments were independently replicated in triplicate. * p < 0.05.