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J Am Coll Cardiol, 2006; 48:1688-1697, doi:10.1016/j.jacc.2006.07.022
(Published online 26 September 2006). © 2006 by the American College of Cardiology Foundation |
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* 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.
Manuscript received April 7, 2006; revised manuscript received June 15, 2006, accepted June 19, 2006.
* Reprint requests and correspondence: Dr. Lu Cai or Dr. Xiaokun Li, 511 South Floyd Street, MDR 533, Louisville, Kentucky 40202. (Email: L0cai001{at}louisville.edu).
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Abstract |
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BACKGROUND: Our previous study showed that cardiac apoptosis as a major early cellular response to diabetes is induced by hyperglycemia-derived oxidative stress that activates a mitochondrial cytochrome c-mediated caspase-3 activation pathway. Metallothionein (MT) as a potent antioxidant prevents the development of diabetic cardiomyopathy.
METHODS: Diabetes was induced by a single dose of streptozotocin (STZ) (150 mg/kg) in cardiac-specific, metallothionein-overexpressing transgenic (MT-TG) mice and wild-type (WT) controls. On days 7, 14, and 21 after STZ treatment, cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activation. Cardiomyopathy was evaluated by cardiac ultrastructure and fibrosis in the diabetic mice 6 months after STZ treatment.
RESULTS: A significant reduction in diabetes-induced increases in TUNEL-positive cells, caspase-3 activation, and cytochrome c release from mitochondria was observed in the MT-TG mice as compared to WT mice. Cardiac protein nitration (3-nitrotyrosine [3-NT]) and lipid peroxidation were significantly increased, and there was an increase in mitochondrial oxidized glutathione and a decrease in mitochondrial reduced glutathione in the WT, but not in the MT-TG, diabetic mice. Double staining for cardiomyocytes with alpha sarcomeric actin and caspase-3 or 3-NT confirmed the cardiomyocyte-specific effects. A significant prevention of diabetic cardiomyopathy and enhanced animal survival were observed in the MT-TG diabetic mice as compared to WT diabetic mice.
CONCLUSIONS: These results suggest that attenuation of early-phase cardiac cell death by MT results in a significant prevention of the development of diabetic cardiomyopathy. This process is mediated by MT suppression of mitochondrial oxidative stress.
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Cardiac apoptosis as a comprehensive consequence of cardiac responses to various stresses causes a loss of contractile tissue, which initiates a cardiac remodeling and fibrosis; therefore, the cardiac apoptosis has been identified as a pivotal cause of various cardiomyopathies (8–10). We, as well as others, have shown that cardiac apoptosis increases in diabetic animals and patients (11–14). It remains to be determined whether the increased cardiac apoptotic cell death plays an important role in the development of diabetic cardiomyopathy (7).
Mitochondrial cytochrome c release and activation of caspase-3 play an important role in diabetes-induced cardiac cell death (14,15). Among apoptotic stimuli, reactive oxygen species (ROS) or reactive nitrogen species (RNS) play a critical role in the mitochondrial cytochrome c release and caspase-3 activation (7,8,14–17). A significant increase in ROS and RNS was found in cardiac cells exposed to high levels of glucose in vitro (14) and in isolated cardiomyocytes from diabetic mice (13,18). We have further identified an increased accumulation of superoxide with an increase in protein nitration (measured by 3-nitrotyrosine [3-NT]) in the hearts of diabetic mice (19). Therefore, we hypothesize that diabetes-derived oxidative and/or nitrosative damage causes an early-stage cell death that activates a series of cardiac remodeling responses, leading to morphological and functional abnormalities, and eventually to cardiomyopathy at the late stage (7).
Metallothioneins (MTs), a group of intracellular metal-binding proteins with high cysteine contents, are able to efficiently protect cells or tissues from oxidative damage (20–22). By functional examination, the preventive effect of MT against diabetes-induced cardiac injury has been demonstrated in our recent studies (19,23) and also in other studies (18,24). In the present study, we used cardiac-specific, MT-overexpressing transgenic (MT-TG) mice to determine (1) whether MT can prevent diabetes-induced cardiac apoptosis at early stage via suppression of oxidative stress, and (2) whether MT’s attenuation of diabetes-induced early-stage cardiac apoptosis can result in a prevention of the development of cardiomyopathy at late stage.
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Materials and methods |
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Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Cardiac sections in paraffin were prepared as before (14) and were processed for a TUNEL assay using an ApopTag in situ detection kit from Intergen (Purchase, New York). Mouse testicular tissue was used as positive control. For negative control, terminal deoxynucleotidyltransferase was omitted from the reaction mixture. The apoptotic cell death was quantitatively analyzed by counting the TUNEL-positive cells randomly selected from 5 fields from each of the 3 slides from each mouse.
Detection of cardiac caspase-3. Caspase-3 activation was measured by Western blotting, based on a published method (15). Briefly, cardiac tissue homogenates were lysed and fractionated electrophoretically on sodium dodecyl (lauryl) sulfate polyacrylamide gel electrophoresis (15% gradient gels), and proteins were transferred to a nitrocellulose membrane. The membrane was blocked with a 5% non-fat, dried milk for 1 h and subsequently incubated with the primary antibody against the active form of caspase-3 (Sigma, St. Louis, Missouri) at 1:500 overnight at 4°C. After the unbound antibodies with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20 (Bio-Rad Laboratories, Hercules, California) were removed, the membrane was incubated with the secondary antibody for 2 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase protein expression was probed as an internal control.
Caspase-3 activation was also examined by caspase-3 activity as described before (14). Briefly, fresh heart tissues were homogenized with Telfon homogenizer in an extract buffer, and the homogenate was centrifuged at 20,000 g for 30 min. The supernatant was diluted with an assay buffer and incubated at 37°C with 200 µmol/l caspase-3 substrate I (Ac-DEVD-pNA [N-acetyl-asp-glu-val-asp-pNA], CalBiochem, La Jolla, California). Cleavage of the substrate was monitored at 405 nm using a microplate reader and recorded in 10-mm intervals for 2 h.
The localization of the activated caspase-3 was determined by double stains for cardiomyocyte and caspase-3, on the basis of a published study (25). Briefly, cardiac tissues were fixed with 75% alcohol and embedded in paraffin. Tissue sections at 5-µm thickness were deparaffinized and rehydrated in a graded alcohol series. Sections were incubated with cocktail primary antibodies overnight at 4°C. The cocktail primary antibodies included a mouse anti-
-sarcomeric actin monoclonal antibody (Abcom Inc., Cambridge, Massachusetts) and rabbit anti-active caspase-3 polyclonal antibody (Becton Dickinson, Franklin Lakes, New Jersey), both at a final dilution of 1:50. After being washed with phosphate-buffered saline, sections were incubated with cocktail secondary antibodies, fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG + IgM + IgA (Abcam Inc., Cambridge, Massachusetts) at a dilution of 1:40 and Cy3-conjugated goat anti-rabbit IgG H & L (Abcom Inc.) at a dilution of 1:100, for 1 h in darkness at room temperature. Sections were examined using confocal laser scanning microscopy (Zeiss LSM510, Carl Zeiss Meditec Inc., Dublin, California) at emission of 500 to 530 nm and 550 to 560 nm, after excitation at 488 and 543 nm, respectively. Laser power and detection gains were set such that signals from single-stained controls would not appear in adjacent channels. Negative controls were realized on specimens incubated either with nonimmune serum or with the incubation buffer alone, followed by the second conjugated antibody.
Detection of mitochondrial cytochrome c release. Cytosolic and mitochondrial proteins were prepared for Western blotting assay to detect cytochrome c content, as previously described (14). Densitometry analysis for each band was performed using the computer program as described above, and quantitative data are presented as relative to control.
Measurements of mitochondrial GSH, GSSG, Gpx, and GR. To obtain sub-cellular fractionations (cytosolic and mitochondrial), heart tissue (3 mouse hearts were pooled as 1 sample) was homogenized with cold HMS (220 mmol/l D-Mannitol, 70 mmol/l sucrose, 2 mmol/l K-HEPES, pH 7.4) and centrifuged at 4°C for 5 min at 600 x g. The supernatants were further centrifuged at 7,000 x g for 10 min. The heavy membrane fraction (mitochondria) was washed once again in HMS by centrifugation at 600 x g for 5 min to remove any contaminating particles and then recovered by centrifugation at 7,000 x g for 10 min. The isolated mitochondria were resuspended in incubation buffer (250 mmol/l sucrose, 10 mmol/l K-HEPES, pH 7.2, 2 mmol/l KH2PO4, 5 mmol/l sodium succinate, 2.5 mmol/l EGTA). The cytosol and mitochondria were then frozen immediately for GSH and oxidized glutathione (GSSG) assay. Glutathione and GSSG contents were simultaneously quantified by high-performance liquid chromatography with dual electrochemical detection (26).
The mitochondrial proteins were also used to measure Gpx and GR using the Glutathione Peroxidase Cellular Activity Assay Kit and the Glutathione Reductase Assay Kit (Sigma, St. Louis, Missouri). Both GR and Gpx activities were calculated using the extinction coefficient of 6.22 mmol/l–1 cm–1, and expressed as nmol/l of nicotinamide adenine dinucleodtide phosphate consumed per minute per milligram of sample protein (22).
Assay for lipid peroxidation. Cardiac lipid peroxidation was quantified by measuring thiobarbituric acid-reactive substance (TBARS) as described previously (21). The TBARS concentrations were expressed as nmol/g tissue.
Double staining for cardiomyocytes and protein nitration.
To determine whether protein nitration occurs in the cardiomyocytes, double stains for cardiomyocytes by -sarcomeric actin and protein nitration by 3-NT were performed using confocal laser scanning microscopy (25), as previously described except for the primary antibody (i.e., using a rabbit anti-3-NT polyclonal antibody at 1:50 dilution [Chemicon Inc., Temecula, California]).
Ultrastructural examination by electron microscopy. The hearts subjected to electron microscopic examination were fixed in situ by vascular perfusion with saline for 10 min, followed by a Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer, pH 7.4) for 15 min. The in situ fixative perfusion procedure and semi-quantitative analysis were described in our previous studies (19,22).
Sirius-red staining of collagen. Tissue sections of 5 µm were used for Sirius-red staining of collagen using 0.1% Sirius-red F3BA and 0.25% fast green FCF. The sections stained for Sirius red then were assessed for the proportion of fibrosis (collagen) using a computer-assisted image-analysis system as described in our previous study (23).
Statistical analysis.
Data were collected from repeated experiments and are presented as mean ± SD. Comparisons between 2 groups with normality and homogeneity of variances were performed by 2-tailed unpaired Student t test. Alternatively, comparisons between groups with abnormality and heterogeneity of variances were performed by Welch t test. One-way analysis of variance (ANOVA) was used, followed by the post-hoc Tukey multiple comparison test to analyze data for WT mice when the insulin-treated diabetic group was included (Figs. 1 and 2A).
Disclaimer concerning type 1 error rate is a limitation of this study, since no correction for multiple comparisons was applied. The SPS 9.1 system (SAS Institute Inc., Cary, North Carolina) was used for all statistical tests. Differences were considered to be significant at p < 0.05.
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Results |
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To confirm the inhibitory effect of MT on diabetes-induced cardiac apoptosis, caspase-3 activation, another important indication of apoptosis, was measured by its activity and the content of its active form using enzymatic and Western blotting assays (Fig. 2). Enzymatic assay showed a significant increase in caspase-3 cleavage activity in the WT diabetic heart, but not in the insulin-treated WT diabetic or MT-TG diabetic heart (Fig. 2A). Consistent with the activity assay, Western blotting analysis also showed an increase in active isoform of caspase-3 in the hearts of WT diabetic mice, but not in the hearts of MT-TG diabetic mice, as shown in Figure 2B. To define whether apoptosis occurs in cardiomyocytes, double stains for cardiomyocytes using -sarcomeric actin and active isoform of caspase-3 were examined under confocal laser scanning microscopy (Fig. 2C). Double staining clearly showed that the activated caspase-3 are localized in cardiomyocytes (left panel), whereas such staining was not observed in the hearts of MT-TG mice (right panel) or insulin-treated WT diabetic mice (data not shown).
In a previous study, we found that mitochondrial cytochrome c release is one of the critical upstream events leading to caspase-3 activation and apoptosis in the diabetic heart and cardiac cells exposed to high levels of glucose (14). In the present study, a significant cytochrome c release from mitochondria to cytosol in the WT diabetic mice, but not in the MT-TG diabetic mice, was also detected by Western blotting assay (Fig. 3). This indicates that MT inhibits mitochondrial cytochrome c release, resulting in a prevention of caspase-3 activation and apoptotic cell death induced by diabetes.
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Attenuation of animal mortality in the MT-TG diabetic mice. Diabetes causes an increase in mortality at late stage in diabetic patients (1–3), probably because of multiple organ dysfunctions including cardiomyopathy. As shown in Figure 7, WT diabetic mice showed a significant decrease in the survival rate, an effect significantly prevented in the MT-TG diabetic mice. Although the mortality may not directly indicate the cardiac effect, the improved animal survival rate in MT-TG diabetic mice would relate to MT prevention of diabetic cardiomyopathy under the conditions of this study, because cardiac MT overexpression is the only difference between WT and MT-TG mice.
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Discussion |
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The loss of cardiac myocytes is a major cause of the compromised cardiac function (8–10). Apoptosis of cardiac myocytes and endothelial cells occurs in the hearts of diabetic patients and STZ-induced diabetic animals (11–17). The present study showed that an average incidence of apoptotic cell death, detected by TUNEL-positive cells, in the WT diabetic heart from days 7, 14, and 21 after STZ treatment is 163 of 106 cells (Fig. 1D). This incidence is in agreement with our previous study using the same mouse model (14). Kajstura et al. (13), using mice, showed that diabetes induced an apoptotic cell death about 4 times as currently observed. Fiordaliso et al. (12), using rats, showed a similar incidence of cardiac apoptosis (about 160 of 106 cells at day 10 after STZ treatment) to the current observation. The difference for the incidence of cardiac apoptosis in our own studies and those of Kajstura et al. (13) is unknown, but we used a strain of mice (FVB) which may be different from mice used by Kajstura et al.
Cell death as a comprehensive consequence of abnormal cell metabolisms and gene expressions at an early stage in the heart in response to diabetes may thus be a critical cause of diabetic cardiomyopathy (4,7,15). This assumption was supported by a previous study showing that insulin-like growth factor 1 (IGF-1) overexpression inhibits cardiac cell death in diabetic mice along with a preservation of cardiac function (13), and by the present study, which shows that overexpressing MT in the heart prevents diabetes-induced early cell death, leading to a prevention of late cardiomyopathy.
In the study with IGF-1 overexpression (13), however, the cardiomyopathy was examined by cardiac function only at 7 and 30 days after STZ treatment, and there was also no information about cardiac structural changes. Diabetic cardiomyopathy is a chronic manifestation at late stage, which should be diagnosed by morphological and functional observation in a relative long time period (2,3,7). In a recent study we demonstrated the preservation of cardiac function in MT-TG diabetic mice, whereas WT-diabetic mice showed a significant cardiac dysfunction at 6 months after diabetic onset (19). In the present study, under the same conditions as the previous study (19), we provided further evidence that cardiac cell death occurred at early stage from day 7 to day 21 after STZ treatment. Inhibition of the early-phase cell death in the heart of MT-TG diabetic mice correlates with a significant prevention of ultrastructural abnormalities and fibrosis in the hearts of diabetic mice 6 months after STZ treatment. This finding is consistent with our recent study using a different transgenic mouse model that systemically overexpresses the MT gene to demonstrate the prevention of MT from diabetes-induced cardiac fibrosis (23). The preservation of structural normality and prevention of fibrosis in the heart by MT, observed in the present study, may be the basis of cardiac functional normality of MT-TG diabetic mice 6 months after STZ treatment, observed in our previous study (19). Furthermore, the preservation of the cardiac structure and function in the MT-TG diabetic mice may be responsible for the high animal survival rate (Fig. 7).
Oxidative damage caused by ROS and RNS is related directly to multiple complications of diabetes (27), including cardiomyopathy (11–15). A recent study (17) further indicated that mitochondrial oxidative stress such as GSH depletion is the direct cause for cardiac cell death under the diabetic condition. We show that MT significantly inhibits mitochondrial GSH depletion, cardiac protein nitration, and lipid peroxidation, along with a significant prevention of cardiac cell death in the hearts of diabetic mice, thus suggesting the cause–effect relationship of the mitochondrial oxidative stress and cardiac cell death. Combined with our recent study (19) showing that attenuation of early oxidative damage such as protein nitration (likely belonging to mitochondrial proteins according to the molecular size) in the MT-TG diabetic heart resulted in a significant prevention of diabetes-caused cardiac dysfunction observed 6 months after STZ treatment, it strongly suggests that oxidative stress is a critical cause of early-phase cardiac cell death.
Metallothionein cooperation with GSH to maintain cellular redox status has been implicated (28,29). We have shown that there is no significant change in the total GSH, Gpx, and GR levels in the hearts of MT-TG mice compared to WT mice under physiological conditions (22). However, GSSG contents in both cytosolic and mitochondrial portions were significantly lower in MT-TG hearts than those in WT hearts even under physiological conditions (i.e., in control groups; Figs. 5A and 5B), suggesting the prevention of oxidization of GSH under physiological conditions. We recently confirmed the existence of MT disulfide bonds in the MT-TG hearts under physiological conditions and further increased formation of MT disulfide bonds under oxidative stress conditions (30), suggesting the interaction of MT with ROSs or RNSs. In our previous study (28), it was shown that in the presence of a high concentration of MT, the primary antioxidant defense against ROS and/or RNS would shift from GSH to MT; therefore, there was no increase in mitochondrial GSSG in the hearts of MT-TG diabetic mice even though there was a significant increase in GSSG in the cardiac mitochondria of WT diabetic mice. In the hearts of WT diabetic mice, the increased GSSG that was associated with increased Gpx activity (Fig. 5) may cause the adaptive increase in mitochondrial GR activity in the hearts of WT diabetic mice (Fig. 5). However, how diabetes causes Gpx activation remains unclear on the basis of the present study. We have demonstrated the overproduction of superoxide in the hearts of WT diabetic mice 2 weeks after STZ treatment (19). The overproduced superoxide may be converted to hydrogen peroxide, which may in turn stimulate the cells to increase Gpx activity in order to compensate.
In summary, the present study, in combination with our recent studies (14,19,23), demonstrates that diabetes causes cardiac mitochondria cytochrome c release-dependent apoptosis at early stage. Metallothionein significantly inhibits diabetes-caused early cardiac apoptosis through suppression of mitochondrial oxidative stress (i.e., GSH depletion) leading to a significant prevention of the development of diabetic cardiomyopathy. Although the precise mechanisms underlying how MT protects from diabetes-caused mitochondrial GSH depletion remain to be explored, the clinical implication of MT’s cardiac prevention from diabetes is critically important, since zinc supplementation given to diabetic mice significantly prevented the development of diabetic cardiomyopathy through cardiac MT induction (31).
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Acknowledgments |
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