p47phox-Dependent Oxidant Signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-Induced Cardiac Hypertrophy and Apoptosis

Abstract

The p47^phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47^phox in the heart remains unclear. In this study, we used wild-type (WT) and p47^phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47^phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47^phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47^phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47^phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis.

Keywords: Angiotensin II; apoptosis; cardiac hypertrophy; knockout mice; p47phox; redox-signalling.

Figure 1

Development of hypertension and cardiac hypertrophy in AngII-infused WT and p47^phox KO mice. (A) Systolic blood pressure. (B) Diastolic blood pressure. Day 0: day of minipump implantation. Day 14: day of minipump removal. (C) Heart weights. (D) Heart weight (HW, mg)/body weight (BW, g) ratio. (E) Left panels: Representative images of cardiomyocyte sizes on the cross-sections of left ventricular tissues. The cardiomyocytes membranes were labelled with WGA-FITC (green). Right panel: Statistical analysis of cardiomyocyte cross-sectional areas (µm²). n = 9 mice per group. Statistical analyses were performed using two-way ANOVA. * p < 0.05 for AngII values versus saline values in the same genetic group; ^† p < 0.05, for p47^phox KO AngII values versus WT AngII values.

Figure 2

Cardiac ROS production. (A) Levels of O₂^●− production measured by lucigenin-chemiluminescence. Left panel: Representative examples of kinetic measurements of O₂^●− production by WT heart homogenates. NADPH (0.1 mM) was added at 10 min. Tiron (10 mM) was added at 30 min to scavenge O₂^●−. Right panel: Differences in NADPH-dependent O₂^●− production measured between 10–30 min shown in the left panel. (B) The effects of different enzyme inhibitors on the levels of O₂^●− by AngII-infused WT heart homogenates. L-NAME: (NG-nitroarginine methyl ester), NOS inhibitor; Rotenone: mitochondrial respiratory chain inhibitor; Oxypurinol, xanthine oxidase inhibitor; DPI: (diphenyleneiodonium), flavoprotein inhibitor; SOD: (superoxide dismutase). (C) Cardiac H₂O₂ production detected by amplex red assay. (D) In situ detection of reactive oxygen species production by DHE fluorescence. Left panel: Representative images of DHE staining on cardiac sections; right panel: Quantification of DHE fluorescence intensity. n = 9 per group. Statistical comparisons were done using one-way ANOVA for inhibitor assay, and two-way ANOVA for the rests (panels A, C, D). * p < 0.05 for indicated AngII values versus saline values in the same genetic group; ^† p < 0.05 for p47^phox KO AngII values versus WT AngII values.

Figure 3

Expressions of isoforms of the catalytic subunit of Nox (i.e., Nox1. Nox2, Nox4) and other subunits of Nox (p47^phox, p22^phox, p67^phox and rac1) in murine hearts. Left panels: Representative Western blot images. β-actin detected in the same samples were used as loading controls. Right panels: Optical densities (OD) of Western blot bands were quantified and normalised to the levels of β-actin detected in the same samples. n = 9 per group. Statistical comparisons were made using two-way ANOVA. * p < 0.05 for AngII values versus saline values in the same genetic group; ^† p < 0.05 for p47^phox KO AngII values versus WT AngII values.

Figure 4

Expressions of protein kinase C alpha (PKCα), Proto-oncogene tyrosine-protein kinase Src (C-Src) and p47^phox phosphorylation in AngII-infused murine hearts. (A) Western blots. Left panels: Representative images. β-actin detected in the same samples were used as loading controls. Right panels: Optical densities (OD) of Western blot bands were quantified and normalised to the levels of β-actin detected in the same samples. (B) Confocal immunofluorescence of cardiac sections. Left panel: Representative immunofluorescence images. Cardiomyocyte cell membrane was labelled by WGA-FITC (green) and p47^phox phosphorylation was identified using phos-p47^phox specific antibody (Cy3, red). Nuclei were labelled by DAPI (blue). Right panel: Quantification of phos-p47^phox fluorescence intensity. n = 9 hearts per group. Data were presented as Mean ± SD. Statistical comparisons were made using two-way ANOVA. * p < 0.05 for AngII values versus saline values in the same genetic group.

Figure 5

AngII-induced activation of mitogen-activated protein kinase kinase 3/6 (MKK3/6), mitogen-activated protein kinases (i.e., ERK1/2, p38MAPK and JNK) and Akt (also called protein kinase B) in murine hearts. Left panels: Representative immunoblotting images. The total protein bands of each molecule in heart homogenates were pre-tested for equal loading. Right panels: Quantification of the optical densities (OD) of phos-protein bands expressed as phosphorylated/total (P/T) protein ratio. n = 9 mice per group. Data were presented as Mean ± SD. Statistical comparisons were made using two-way ANOVA. * p < 0.05 for AngII values versus saline values in the same genetic group. ^† p < 0.05 for p47^phox KO AngII values versus WT AngII values.

Figure 6

Activation of apoptosis signal-regulating kinase 1 (ASK1), p53 and phosphorylation of H2A histone family member X (γH2AX) in murine hearts. (A) Western blots for the expressions of phos-ASK1, p53 and γH2AX. Optical densities of protein bands were quantified and normalised to the levels of β-actin detected in the same samples. (B) Confocal immunofluorescence detection of Nox2 (Cy3 labelled, red) and phos-ASK1 expressions (FITC labelled, green) in the cardiac sections. (C) Confocal immunofluorescence detection of γH2AX expression (Cy3-labelled, red) in the cardiac sections. The cardiomyocyte membrane was labelled by WGA-FITC (green), and the nuclei were labelled by DAPI (blue). The specific fluorescent densities were quantified. n = 9 mice per group. Data were presented as Mean ± SD. Statistical comparisons were made using two-way ANOVA. * p < 0.05, for AngII values versus saline values in the same genetic group. ^† p < 0.05 for p47^phox KO AngII values versus WT AngII values.

Figure 7

Schematic illustration of p47^phox redox-signalling pathways examined.