Prolylcarboxypeptidase Mitigates Myocardial Ischemia/Reperfusion Injury by Stabilizing Mitophagy

Figures

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  FIGURE 1. PRCP–Ang-(1–7)/BK-(1–9) changes in rat hearts after ischemia/reperfusion (I/R). (A) Western blot analysis of PRCP protein expression in the myocardium; (B) quantification of myocardial PRCP activity; (C–E) quantifications of myocardial Ang II (C), Ang-(1–7) (D), and BK-(1–9) levels (E) with and without overexpression (Ad) or shRNA knockdown (sh). n = 6 in each group; *p < 0.05 and **p < 0.01 vs. sham; #p < 0.05 and ##p < 0.01 vs. adenovirus-mediated empty vector (Ad-Con); &p < 0.05 and &⁣&p < 0.01 vs. scramble shRNA (sh-Con).

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  FIGURE 2. Effects of PRCP on I/R-induced left ventricular dysfunction and apoptosis. (A) M-mode echocardiogram showing left ventricular dimensions after myocardial I/R; (B) quantifications of left ventricular fractional shortening before ischemia (n = 20) and after reperfusion (n = 8∼10); (C) representative TUNEL staining (dark brown) for apoptosis with nuclear hematoxylin counterstaining (blue) in six groups of rats (scale bar: 100 μm); (D) quantification of TUNEL-positive staining (n = 6 in each group); (E) quantification of caspase-3 activity (n = 6 in each group). *p < 0.05 and **p < 0.01 vs. sham; #p < 0.05 and ##p < 0.01 vs. Ad-Con; &p < 0.05 and &⁣&p < 0.01 vs. sh-Con.

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  FIGURE 3. Effect of PRCP on myocardial I/R-induced mitophagy. Representative transmission electron microscopy (TEM) images of mitochondria and mitophagosomes with PRCP overexpression and knockdown in the ischemic myocardium (A) and reperfused myocardium (F) (scale bar on top panels: 1 μm; scale bar on bottom panels: 0.2 μm; blue arrows: mitophagosomes; yellow arrows: completely vacuolated mitochondria). (B,G) Representative Western blot analysis of LC3, PINK1, Parkin, and COX IV protein expressions in the myocardium; quantifications of LC3-II/LC3-I ratio (C,H) and PINK1 (D,I) and Parkin (E,J) protein expressions relative to COX IV (a mitochondrial marker). n = 6 in each group; *p < 0.05 and **p < 0.01 vs. sham; #p < 0.05 vs. Ad-Con; &p < 0.05 and &⁣&p < 0.01 vs. sh-Con.

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  FIGURE 4. Effects of Ang-(1–7) and BK-(1–9) on cardiomyocyte mitophagy after hypoxia/reoxygenation (H/R). (A) Western blot analysis of LC3-II/LC3-I ratio and PINK1 and Parkin protein expressions relative to COX IV as well as AMP-dependent protein kinase (AMPK) and Akt phosphorylation in rat cardiomyocytes under normal oxygen or hypoxia and treated with vehicle, Ang-(1–7), Ang-(1–7) + A779, or Ang-(1–7) + PD123319; (B) under hypoxia and treated with vehicle, BK-(1–9) or BK-(1–9) + HOE140; (C) under normal oxygen or H/R and treated with vehicle, Ang-(1–7), Ang-(1–7) + A779 or Ang-(1–7) + PD123319; and (D) under H/R and treated with vehicle, BK-(1–9), or BK-(1–9) + HOE140. n = 6 in each group. (A,C) *p < 0.05 and **p < 0.01 vs. normal oxygen; #p < 0.05 and ##p < 0.01 vs. vehicle; &p < 0.05 and &⁣&p < 0.01 vs. Ang-(1–7). (B,D) *p < 0.05 and **p < 0.01 vs. vehicle; #p < 0.05 and ##p < 0.01 vs. BK-(1–9).

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  FIGURE 5. Signaling mechanisms involved in mitophagy stabilized by Ang-(1–7) and BK-(1–9). (A) Western blot analysis of LC3-II/LC3-I ratio and PINK1 and Parkin protein expressions relative to COX IV in rat cardiomyocytes under hypoxia and treated with vehicle, Ang-(1–7), dorsomorphin, or Ang-(1–7) + dorsomorphin; (B) under hypoxia and treated with vehicle, BK-(1–9), or BK-(1–9) + dorsomorphin; (C) under H/R and treated with vehicle, Ang-(1–7), BYL719, or Ang-(1–7) + BYL719; and (D) under H/R and treated with vehicle, BK-(1–9) or BK-(1–9)+ BYL719. n = 6 in each group; *p < 0.05 and **p < 0.01 vs. vehicle; #p < 0.05 and ##p < 0.01 vs. Ang-(1–7) or BK-(1–9).

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  FIGURE 6. PRCP–Ang-(1–7)/BK-(1–9) modifies the mitophagy pathway in maintaining survival homeostatic response to I/R injury.