Disruption of Nrf2 Synergizes with High Glucose to Cause Heightened Myocardial Oxidative Stress and Severe Cardiomyopathy in Diabetic Mice

Abstract

High glucose-induced oxidative stress is a major contributing mechanism to the development of diabetic cardiomyopathy. Nrf2 is an emerging critical regulator of cellular defense against oxidative damage. The role of Nrf2 in diabetic cardiomyopathy was investigated in vivo. Streptozotocin (STZ) induced diabetes in Nrf2 knockout (KO) mice that rapidly progressed to severe conditions with high mortality within two weeks of injection; whereas, in wild type (WT) mice, diabetes was less severe with no death. Severe myocardial lesions were observed in diabetic KO mice that had high, sublethal levels of blood glucose including: (a) irregular myocardial arrangements, myofibrillar discontinuation, and cell death; (b) reduced electron density, discontinuation of myocardial fibers, and mitochondrial damage; and (c) markedly reduced contractility of the cardiomyocytes to β-agonist stimulation. Parallel to severe cardiomyopathy, the diabetic KO hearts showed: (a) increased apoptosis as revealed by TUNEL and PARP1 cleavage assays; (b) infiltration of granulocytes and macrophages as well as fibrosis indicating robust inflammatory response; and (c) heightened oxidative stress as evidenced by increased levels of 8-hydroxydeoxyquanine, free malondialdehyde, and 3-nitrotyrosine. Increased oxidative stress in the KO hearts was attributed to decrease or loss of the basal and induced expression of Nrf2-dependent cytoprotective genes. Our findings demonstrate that loss of Nrf2 function synergizes with high glucose to cause heightened oxidative stress in the heart leading to severe diabetic cardiomyopathy.

Figure 1

Increased sensitivity of Nrf2 KO mice to STZ-induced diabetic lesions. Eight-week-old male WT and Nrf2 KO mice were treated with 0.1 M citrate buffer (n = 8), STZ (150 mg/kg body weight, n = 8), or STZ plus insulin (n = 4). Tail blood glucose level was monitored for diagnosis of diabetic conditions. Mortality was monitored daily for 14 days. (A) Mortality at day 14. (B) Time curve of percent survival. In (B), mortality was observed only in the group of KO+STZ.

Figure 2

Myocardial structural damage. Hearts from Nrf2 WT and Nrf2 KO mice were collected 14 days after STZ treatment. Diabetic condition was confirmed by blood glucose detection (≥250 mg/dL). (A) H&E staining: Paraffin-embedded heart tissue (transverse section) was stained with H&E and examined under light microscope (40X). Apparently damaged areas were located inside of circles and arrows. Bar = 20 μm. (B) Ultrastructural damage: Micrographs of transmission electron microscopy of control and diabetic heart samples. Bar = 2 μm.

Figure 3

Ventricular myocyte contractility. AMVM were isolated from diabetic mice two weeks after STZ injection. Contractility was measured as percent shortening of ventricular myocytes in the presence of b-adrenergic agonist isoproterenol at 100 nM. Data represent means ± standard deviations from 12 - 15 individual myocytes isolated from eight separate myocyte preparatioins. **, p<0.01.

Figure 4

Myocardial apoptosis. (A) Paraffin sections were stained for apoptosis using the DeadEnd Fluorometric TUNEL assay (green, upper panel). DAPI (blue, lower panel) was used to stain for nuclei. Magnification = 40 X. (B) PARP cleavage was detected by immunoblotting using anti-PARP. Upper panel, PARP protein and cleavage product; lower panel, actin as loading control.

Figure 5

Inflammatory infiltration and fibrosis. (A) Granulocyte infiltration. Paraffin sections of heart tissues were stained for Naphthol AS-D chloroacetate esterase activity. Increased esterase-positive granulations (red and brown) shown by arrows indicate granulocyte infiltration. Magnification = 40 X. (B) Quantitative data of experiment (A). (C) Macrophage infiltration. Mac-2-positive macrophages were shown by fluorescent staining using an anti-Mac-2 antibody of paraffin sections of mouse hearts. DAPI staining was used to show the nuclei. (D) Quantification of (C). (E) Fibrosis. Paraffin heart sections were stained with picrosirius red. Increased collagen formation (red) indicates fibrosis. Magnification = 40X. (F) Quantification of (E).*, p<0.05; **, p<0.01.

Figure 6

Oxidative stress and damage. (A) Oxidative DNA damage. The 8-OHdG levels in Nrf2 WT and KO heart tissues were examined by immunofluorescent staining with an anti-8-OHdG antibody. (B) Lipid peroxidation. Nrf2 WT and KO heart tissues were homogenized and MDA production was measured. (C) Protein tyrosine nitrosation. Nrf2 WT and KO heart homogenates were immunoblotted with an anti-3NT antibody. Anti-sarcomeric acting was used as loading control. (D) Quantification of experiment (C).*, p<0.05; **, p<0.01.

Figure 7

Induction of cytoprotective genes. Total RNA was prepared from hearts of control and diabetic Nrf2 WT and KO mice. Expression of mRNA of Nqo1, Nrf2, and Action was expressed as fold change over control.**, p<0.01.