This Article Abstract Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (34) Citing Articles via Google Scholar Google Scholar Articles by Cai, L. Articles by Kang, Y. J. Search for Related Content PubMed PubMed Citation Articles by Cai, L. Articles by Kang, Y. J.

Attenuation by Metallothionein of Early Cardiac Cell Death via Suppression of Mitochondrial Oxidative Stress Results in a Prevention of Diabetic Cardiomyopathy

Lu Cai, MD, PhD^_*,,,_*, Yuehui Wang, MD, PhD^_*, Guihua Zhou, MD, PhD^_*, Teresa Chen, PhD, Ye Song, MD, PhD^_*, Xiaokun Li, MD, PhD^,_* and Y. James Kang, DVM, PhD^_*,

^_* Department of Medicine, the University of Louisville, Louisville, Kentucky
Department of Pharmacology and Toxicology, the University of Louisville, Louisville, Kentucky
School of Pharmaceutical Sciences, Wenzhou Medical College, Wenzhou, China.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Diabetic model and diabetes-induced cardiac apoptosis. Whole-blood glucose (A) and glycated hemoglobin (Hb-A1c) (B) were measured for diabetic mice 14 days after streptozotocin (STZ) treatment. Cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Panel C presents the representatives of TUNEL staining images in different groups from diabetic mice 14 days after STZ treatment and panel D presents combined results for the quantitative analysis of TUNEL-positive cells in the cardiac tissues of the diabetic mice from day 7 to day 21 after STZ treatment. Data were presented as mean values ± SD. For each group, at least 5 mice were used. One-way analysis of variance (ANOVA) was used followed by the post-hoc Tukey multiple comparison test. *p < 0.05 versus corresponding control. MT-TG = cardiac-specific, metallothionein-overexpressing transgenic mice, WT = wild-type mice.

View larger version (99K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Caspase-3 activation in the diabetic hearts. Activation of caspase-3 was measured by enzymatic assay for its activity (A), Western blotting assay for the protein expression of its active isoform (B), and double staining for its localization (C). Cardiac tissues for both assays were collected from diabetic mice 14 days after STZ treatment. In panel C, "-SA" indicates staining for cardiomyocytes using -sarcomeric actin; "Csp-3" indicates the specific staining for active isoform of caspase-3; and "overlay" indicates the overlapping of these 2 specific stainings. These images in panel C are the representatives of each group including more than 3 mice with 2 sections from each mouse. Data were presented as mean values ± SD. In each group, at least 5 mice were included. One-way ANOVA was used and was followed by the post-hoc Tukey multiple comparison test for the data from WT mice in A. A 2-tailed unpaired Student t test was used for the rest of the data. *p < 0.05 versus corresponding control. Abbreviations as in Figure 1.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Mitochondrial cytochrome c release to cytosol was measured by Western blotting assay in the cytosolic and mitochondrial fractions from the hearts of control and diabetic mice 14 days after STZ treatment. Gel was a representative of 2 experiments using 3 hearts as 1 sample to isolate the cytosolic and mitochondrial proteins. A 2-tailed unpaired Student t test was used. *p < 0.05 versus corresponding control. Abbreviations as in Figure 1.

View larger version (99K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Diabetes-induced cardiac protein nitration and lipid peroxidation. Panel A presents the double staining for cardiomyocytes by -sarcomeric actin (labeled by "-SA") and protein nitration by 3-NT. These images in panel A are the representatives of each group including more than 3 mice with 2 sections from each mouse. For panel B, cardiac tissues were collected from control and diabetic mice (at lease 5 mice for each group) 14 days after STZ treatment, and lipid peroxidation was measured by thiobarbituric acid-reactive substance (TBARS) assay. Data were presented as mean values ± SD. A 2-tailed unpaired Student t test was used. *p < 0.05 versus corresponding control. Abbreviations as in Figure 1.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Changes of glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (Gpx), and glutathione reductase (GR) contents or activity in the diabetic hearts and MT’s effect. The GSH and GSSG contents were measured by high-performance liquid chromatography with a dual electrochemical method from the cytosolic (A) and mitochondrial (B) fractions of the control and diabetic tissues from WT and MT-TG diabetic mice. The Gpx and GR activities (C) were measured by biochemical assay using commercially available kits and visible spectrometer. Data were presented as mean values ± SD. Animal numbers are 4 to 5 in each group. A 2-tailed unpaired Student t test was used except for the right panel of B, for which, because the variables seriously violated the assumptions for normality and homogeneity of variances, a Welch t test was used. *p < 0.05 versus corresponding control; #p < 0.05 versus WT control. Other abbreviations as in Figure 1.

View larger version (107K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Diabetes-induced ultrastructural changes and fibrotic effect of the heart. Ultrastructural evaluation was performed by electron microscopic examination (A) for the hearts of diabetic mice 6 months after STZ treatment. Inset in panel A are the scores for the semi-quantitative analysis as described in the Materials and Methods section. Fibrosis was evaluated by Sirius-red staining of collagen in the hearts of diabetic mice 6 months after STZ treatment (B). The semi-quantitative analysis was performed by a computer-image analysis system as described in Materials and Methods. Data were presented as mean values ± SD. In each group, at least 5 mice were included. A 2-tailed unpaired Student t test was used. *p < 0.05 versus corresponding control. Abbreviations as in Figure 1.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Animal survival rate. Animal survival rate until 4 months was observed by 3 separate experiments with at least 10 mice for each group. Data were presented as mean values ± SD. A 2-tailed unpaired Student t test was used. *p < 0.05 versus corresponding control (time 0). Abbreviations as in Figure 1.