Nitric oxide mediates cardiac protection of tissue kallikrein by reducing inflammation and ventricular remodeling after myocardial ischemia/reperfusion

Abstract

We assessed the role of nitric oxide (NO) and the kinin B2 receptor in mediating tissue kallikrein's actions in intramyocardial inflammation and cardiac remodeling after ischemia/reperfusion (I/R) injury. Adenovirus carrying the human tissue kallikrein gene was delivered locally into rat hearts 4 days prior to 30-minute ischemia followed by 24-hour or 7-day reperfusion with or without administration of icatibant, a kinin B2 receptor antagonist, or N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. Kallikrein gene delivery improved cardiac contractility and diastolic function, reduced infarct size at 1 day after I/R without affecting mean arterial pressure. Kallikrein treatment reduced macrophage/monocyte and neutrophil accumulation in the infarcted myocardium in association with reduced intercellular adhesion molecule-1 levels. Kallikrein increased cardiac endothelial nitric oxide synthase phosphorylation and NO levels and decreased superoxide formation, TGF-beta1 levels and Smad2 phosphorylation. Furthermore, kallikrein reduced I/R-induced JNK, p38MAPK, IkappaB-alpha phosphorylation and nuclear NF-kappaB activation. In addition, kallikrein improved cardiac performance, reduced infarct size and prevented ventricular wall thinning at 7 days after I/R. The effects of kallikrein on cardiac function, inflammation and signaling mediators were all blocked by icatibant and L-NAME. These results indicate that tissue kallikrein through kinin B2 receptor and NO formation improves cardiac function, prevents inflammation and limits left ventricular remodeling after myocardial I/R by suppression of oxidative stress, TGF-beta1/Smad2 and JNK/p38MAPK signaling pathways and NF-kappaB activation.

Figure 1

Myocardial infarct size at 1 day after I/R. (A) Representative photomicrographs show immunohistochemical identification of human tissue kallikrein in the infracted area of rats at 1 and 7 days after I/R (5 and 11 days after local gene delivery). Original magnification is 400×. (B) Representative photomicrographs show ventricular infarct size stained with TTC. (C) Quantitative analysis of infarct size from I/R-injured cardiac sections. Data are expressed as mean ± SEM., n=6-7. *P<0.01 vs. other groups.

Figure 1

Myocardial infarct size at 1 day after I/R. (A) Representative photomicrographs show immunohistochemical identification of human tissue kallikrein in the infracted area of rats at 1 and 7 days after I/R (5 and 11 days after local gene delivery). Original magnification is 400×. (B) Representative photomicrographs show ventricular infarct size stained with TTC. (C) Quantitative analysis of infarct size from I/R-injured cardiac sections. Data are expressed as mean ± SEM., n=6-7. *P<0.01 vs. other groups.

Figure 2

Ventricular remodeling at 7 days after I/R. (A) Representative photomicrographs show ventricular sections stained with Masson’s trichrome. (B) Quantitative analysis of ventricular infarct size. (C) Left ventricular wall thickness. Data are expressed as mean ± SEM., n = 5-8. *P<0.01 vs. other I/R groups. Original magnification is 10×.

Figure 3

Effects of kallikrein on myocardial inflammation at 1 day after I/R. (A) Representative photomicrographs show ventricular sections stained with ED-1 antibody. Original magnification is 400×. (B) Quantitative analysis of monocyte/macrophage number. (C) Quantitative analysis of neutrophil number. (D) Western blot analysis of ICAM-1 levels. Data are expressed as mean ± SEM., n = 6-7. *P<0.01 vs. other I/R groups.

Figure 3

Effects of kallikrein on myocardial inflammation at 1 day after I/R. (A) Representative photomicrographs show ventricular sections stained with ED-1 antibody. Original magnification is 400×. (B) Quantitative analysis of monocyte/macrophage number. (C) Quantitative analysis of neutrophil number. (D) Western blot analysis of ICAM-1 levels. Data are expressed as mean ± SEM., n = 6-7. *P<0.01 vs. other I/R groups.

Figure 4

Effects of kallikrein on cardiac eNOS phosphorylation, NOx levels and superoxide formation at 1 day after I/R. (A) Western blot analysis of phosphorylated and total eNOS. (B) Cardiac NOx levels. (C) Superoxide formation. Data are expressed as mean ± SEM., n=6-7. *P<0.01 vs. other groups.

Figure 5

Effects of kallikrein on MAPK phosphorylation and NF-κB translocation at 1 day after I/R. Western blot analysis of (A) cardiac TGF-β levels and Smad2 phosphorylation, (B) JNK and p38MAPK phosphorylation, and (C) IκB-α (Ser 32) phosphorylation. (D) Nuclear NF-κB-DNA EMSA.

Figure 5

Effects of kallikrein on MAPK phosphorylation and NF-κB translocation at 1 day after I/R. Western blot analysis of (A) cardiac TGF-β levels and Smad2 phosphorylation, (B) JNK and p38MAPK phosphorylation, and (C) IκB-α (Ser 32) phosphorylation. (D) Nuclear NF-κB-DNA EMSA.

Figure 5

Effects of kallikrein on MAPK phosphorylation and NF-κB translocation at 1 day after I/R. Western blot analysis of (A) cardiac TGF-β levels and Smad2 phosphorylation, (B) JNK and p38MAPK phosphorylation, and (C) IκB-α (Ser 32) phosphorylation. (D) Nuclear NF-κB-DNA EMSA.