Metallothionein Suppresses Angiotensin II–Induced Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation, Nitrosative Stress, Apoptosis, and Pathological Remodeling in the Diabetic Heart
Guihua Zhou, MD, PhD*,¶,
Xiaokun Li, MD, PhD¶,
David W. Hein, PhD ,
Xilin Xiang, MD, PhD*,
James P. Marshall, BS*,
Sumanth D. Prabhu, MD*, ,||,* and
Lu Cai, MD, PhD*,, ,¶,*
* Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky
Department of Radiation Oncology, University of Louisville School of Medicine, Louisville, Kentucky
|| Medical Service, Louisville Veterans Affairs Medical Center, Louisville, Kentucky
¶ Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical College, Wenzhou, China.
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Figure 1 MT Prevents Ang II–Induced Apoptosis, 3-NT Formation, and NOX Membrane Translocation in the Hearts of Mice (n  5) That Were Given 1 mg/kg of Ang II at 7 or 24 h Before the Experiments
Apoptosis was detected by terminal transferase dUTP nick end labeling assay (A) and Western blotting activated caspase-3 (B). All 3-nitrotyrosine (3-NT) formation (bands from 30 to 60 kDa) (C and D) and nicotinamide adenine dinucleotide phosphate oxidase (NOX) p47phox membrane translocation (E) were detected by Western blotting assay. *p < 0.05 versus corresponding control. Ang II = angiotensin II; CAT-TG = catalase-overexpressing transgenic; MT = metallothionein; MT-TG = cardiac-specific, metallothionein-overexpressing transgenic; WT = wild type.
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Figure 2 MT Protects Cardiomyocyte Apoptotic Death From a 24-h Exposure to Ang II (100 nmol/l) and Role of NOX-Dependent Peroxynitrite Generation in Ang II Apoptotic Effect
(A) Deoxyribonucleic acid (DNA) fragmentation by enzyme-linked immunosorbent assay. (B) 3-NT formation by Western blotting. (C) Fluorescent imaging of peroxynitrite in the cultured cardiomyocytes exposed to Ang II for 6 h. Top row, phase contrast images; bottom row, fluorescent images. (D) Cardiomyocytes exposed to Ang II for 24 h with or without 1-h pre- and coincubation with urate (peroxynitrite inhibitor), MnTMPyP (superoxide inhibitor), L-NAME (nitric oxide synthase inhibitor), and apocynin (NOX inhibitor). (E) Western blotting for the membrane translocation of NOX p47phox induced by a 12-h Ang II exposure. Results are presented as relative to control with pooled results from 3 separate experiments with triple samples for each. *p < 0.05 versus control; #p < 0.05 versus Ang II alone. L-NAME = NG-nitro-L-arginine methyl ester; MnTMPyP = Mn(111) tetrakis 1-methyl 4-pyridylporphyrin pentachloride; other abbreviations as in Figure 1.
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Figure 3 Effects of Ang II on Blood Pressure, Cardiac Histopathology, and Inflammatory Factors
Mice (n 5) were given angiotensin (Ang) II at 0.5 mg/kg body weight for 2 weeks, and 1 month after the first dosing of Ang II, mean aortic blood pressure (MAP), left ventricular end-systolic pressure (LV ESP), and left ventricular end-diastolic pressure (LV EDP) (A) were measured with cardiac morphological examination by hematoxylin and eosin staining (B) and messenger ribonucleic acid (mRNA) expression of tumor necrosis factor (TNF)- and endothelin (ET)-1 by real-time polymerase chain reaction (C and D). *p < 0.05 versus corresponding control.
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Figure 4 Chronic Exposure to Ang II–Induced Cardiac Apoptosis
Mice (n 5) were given Ang II at 0.5 mg/kg body weight for 2 weeks, during which the hearts were collected at 1 and 2 weeks for terminal transferase dUTP nick end labeling assay (A), Western blotting of activated caspase-3 (B), and colocalization of activated caspase-3 with cardiomyocytes (-sarcomeric actin) with immunofluorescent staining (C). *p < 0.05 versus corresponding control. Abbreviations as in Figure 1.
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Figure 5 MT Prevents Ang II–Induced Cardiac 3-NT Formation and PAI-1 Expression
Mice (n 5) were given Ang II at 0.5 mg/kg for 2 weeks, and then the hearts were collected within the 2 weeks during (indicated by 0.5 m) and 1, 3, 6 months after Ang II administration for measuring 3-NT by Western blotting (A), and PAI-1 mRNA (B) and protein expression (C) by real-time polymerase chain reaction and immunohistochemical staining, respectively. *p < 0.05 versus corresponding control. mRNA = messenger ribonucleic acid; PAI-1 = plasminogen activator inhibitor-1; other abbreviations as in Figure 1.
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Figure 6 MT Prevents Ang II–Induced Fibrosis and Cardiac Hypertrophy
Animal treatment and tissue sampling were the same as in Figure 5. Cardiac fibrosis was examined via CTGF expression with western blotting (A) and Sirius red staining for collagen (B). (C) Atrial natriuretic peptide (ANP) messenger ribonucleic acid (mRNA); (D) - and β-MHC protein. *p < 0.05 versus corresponding control. CTGF = connective tissue growth factor; other abbreviations as in Figure 1.
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Figure 7 MT Prevents Ang II–Induced Nitrosative Damage, Apoptosis, Granulocyte Infiltration, and Fibrotic Responses in Diabetic Hearts
One month after streptozotocin treatment, diabetic mice were given Ang II at 0.5 mg/kg for 2 weeks. Mice (n 5) were then sacrificed 12 h after the 2-week Ang II exposure to measure serum and cardiac Ang II levels by enzyme-linked immunosorbent assay (A and B), cardiac 3-NT accumulation (C), caspase-3 activation (D), CTGF up-regulation (F), and granulocyte infiltration by Naphthol AS-D chloracetate esterase staining (E). *p < 0.05 versus corresponding control. Abbreviations as in Figures 1 and 6.
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Figure 8 Schematic Illustration for the Protection of MT Against Ang II Pathogenic Effects on the Heart
iNOS = inducible nitric oxide synthase; NO = nitric oxide; ONOO = peroxynitrite; other abbreviations as in Figures 1 and 2. Figure illustration by Rob Flewell.
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