Inhibition of Superoxide Generation and Associated Nitrosative Damage Is Involved in Metallothionein Prevention of Diabetic Cardiomyopathy
http://www.100md.com
糖尿病学杂志 2005年第6期
1 Department of Medicine, University of Louisville, School of Medicine, Louisville, Kentucky
2 Department of Pharmacology and Toxicology, University of Louisville, School of Medicine, Louisville, Kentucky
ABSTRACT
The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor- for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.
Previous studies have shown that oxidative stress is critically involved in the pathogenesis of diabetic cardiomyopathy (1), a leading cause for mortality in both type 1 and type 2 diabetic patients (2eC4). The pathological changes in the heart were found to be associated with increased myocardial cell death, predominately in the mode of apoptosis and accumulation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) (5eC8). The roles of ROS and RNS in the diabetic complications of multiple organ systems were extensively documented (1,8eC13), although the exact mechanisms of ROS/RNS-induced pathogenesis have not been fully understood.
Superoxide overproduction in the organ systems is an important feature of diabetic complications (10eC12). One risk of the superoxide generation is related to its interaction with nitric oxide to form peroxynitrite (11eC15), which is a potent oxidant that causes nitrosative stress in the organ systems. A significant increase in serum and tissue 3-nitrotyrosine (3-NT), a by-product of the reaction between peroxynitrite and proteins, has been found in diabetic patients (6,15). Studies using experimental animal models and in vitro cultured cells have demonstrated that peroxynitrite is an important causative agent in diabetes-caused cardiovascular injury (16eC19).
Therefore, we hypothesized that inhibition of myocardial nitrosative stress would lead to suppression of diabetic cardiomyopathy. Recent studies have shown that overexpression of metallothionein, a small molecular weight protein that has been shown to protect the heart from injury under various oxidative stress conditions (20eC24), protects the heart from diabetes-induced damage (25,26), probably through suppression of oxidative stress (27,28). However, the mechanisms of metallothionein inhibition of diabetes-induced oxidative stress in the heart are largely unknown. Although we have demonstrated that metallothionein directly interacts with peroxynitrite to prevent peroxynitrite-induced lipid and DNA damage (29), whether the peroxynitrite formation is also involved in the pathogenesis of diabetic cardiomyopathy in vivo and whether metallothionein prevents peroxynitrite-induced cardiac damage in vivo remain unknown.
The present study, therefore, was undertaken to address the role of superoxide generation and associated nitrosative damage in the pathogenesis of diabetic cardiomyopathy in a mouse model of type 1 diabetes induced by streptozotocin (STZ). Furthermore, an attempt was made to understand the inhibitory action of metallothionein on the nitrosative cardiac damage in vivo.
RESEARCH DESIGN AND METHODS
Diabetes model.
MT-TG mice were produced from FVB mice and have been well characterized (20). Both MT-TG+ mice (heterozygotes) and MT-TGeC littermates (wild type) were kept in the same cages with free access to rodent diet and tap water and used for experiments. All animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association for Accreditation of Laboratory Animal Care.
Eight-week-old male mice were given a single dose of STZ (150 mg/kg body wt i.p.; Sigma, St. Louis, MO) dissolved in sodium citrate buffer (pH 4.5). Whole-blood glucose obtained from the mouse tail vein was detected using a SureStep complete blood glucose monitor (LifeScan, Milpitas, CA) 2 and 3 days after STZ treatment. STZ-treated mice with glucose levels higher than 12 mmol/l were considered diabetic, and mice serving as controls were given the same volume of sodium citrate (5). To eliminate the effects of STZ on cardiotoxicity, insulin-treated diabetic mice were used. For these mice, when hyperglycemia was diagnosed 2 or 3 days after STZ treatment, insulin was immediately given three times (8-h interval) using Humulin U (Eli Lilly, Indianapolis, IN) at a concentration of 10 units · dayeC1 · mouseeC1 to maintain the blood glucose levels between 5.6 and 11.2 mmol/l with an average of 7.6 ± 1.1 mmol/l, until they were killed.
Histopathological and immunohistochemical examination by light microscopy.
Heart tissues were fixed with 10% neutral and embedded in paraplast (23,30). Tissue sections of 5 e thickness were stained by hematoxylin-eosin and examined under a light microscope. The 3-NT as a marker of peroxynitrite-induced damage was detected by immunohistochemical stain for two sections of each mouse of five mice in each group (6,30).
Ultrastructural examination by electron microscopy.
The hearts 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 (20,23). The detail procedures of fixation, rinsing, postfixation, dehydration, and embedding for the tissue samples were described previously (20,23). Ultrathin sections were obtained with a LKB ultramicrotome, stained with uranyl acetate and lead citrate and observed with a Philip transmission electron microscope.
Measurements of serum creatine phosphokinase and lipid peroxidation.
Whole blood was collected from the dorsal vena cava of the anesthetized animals. Serum was prepared using a serum separator apparatus (Becton Dickinson, Rutherford, NJ) to measure creatine phosphokinase (CPK) activity following the instructions provided in the kit (Sigma). Serum lipid peroxidation was measured by a thiobarbituric acideCreactive substance assay, as described previously (29).
Assessment of left ventricle performance.
General measures of cardiac performance were done by in situ left ventricle (LV) hemodynamic analysis, as described previously (31). Mice were anesthetized using sodium pentobarbital (60 mg/kg i.p.). A midline incision (1eC2 cm) in the neck external to the trachea and a small opening in the trachea were made for the insertion of a PE-100 catheter to ensure a patent airway. The rostra end of the artery was clamped to occlude blood flow from the heart. A small incision was then made in the artery for the insertion of a hand-stretched, fluid-filled PE-50 catheter, which was connected to a transducer and a computer recording system. The catheter was then slowly advanced through the common carotid artery, through the ascending aorta and into the left ventricle. The animal was allowed to stabilize for 20eC30 min before recording of the waveform for up to 2 h. At the end of each experiment, the chest was opened to confirm the presence of catheter inside the left ventricle.
Measurement for superoxide.
Superoxide generation in the heart was examined by two methods: fluorescence labeling of superoxide specific staining (32) and a cytochrome c reduction assay for NADPH-dependent superoxide generation (33). Dihydroethidine is oxidized to ethidine (red fluorescence) selectively by superoxide, but not by other ROS or RNS such as hydrogen peroxide, hydroxyl radicals, or peroxynitrite. Dihydroethidine at 10 mg/kg was injected via a tail vein 1 h before tissue harvest, as described previously (32), and cryostat sections of heart were cut at 5 e and mounted on glass slides. The fluorescence was detected with a Nikon 2000S fluorescent microscope.
For cytochrome c reduction assay, the measurement of NADPH-dependent superoxide generation, cardiac tissues were homogenized and centrifuged at 800g for 10 min. The supernatant was incubated in the presence of 30 eol/l succinylated ferricytochrome c and 1 mmol/l NADPH (both from Sigma). The change in absorbance at 550 nm was measured. The difference in the amount of reduced succinylated ferricytochrome c in the presence or absence of 0.3 mg/ml superoxide dismutase (SOD) (Sigma) was used to estimate the amount of superoxide generation by using an absorbance coefficient 21.1 mmol/leC1 cmeC1 (33).
Primary cultures of neonatal cardiomyocytes.
Primary cultures of neonatal mouse cardiomyocytes were prepared by a modification of the method published previously (22,34). Briefly, hearts from 1- to 3-day-old wild-type and MT-TG mice were minced and dissociated with 0.15% trypsin. Dispersed cells were plated for 2 h in 100-mm dishes with minimum essential medium plus 10% bovine calf serum to remove noncardiomyocytes that attached to the plate within the incubation time. The myocytes that remained in the suspension were plated at a density of 500 cells/mm2 with minimum essential medium plus 10% bovine calf serum, 0.1 mmol/l bromodeoxyurindine, and 20 eol/l arabinosylcytosine. The medium was replaced 24 h later with fresh medium. On day 3, the wild-type and MT-TG cardiomyocytes were incubated with 30 e/ml lipopolysaccharide (LPS) (Sigma) plus 100 ng/ml tumor necrosis factor (TNF-) (PreproTech, Rocky Hill, NJ) (LPS/TNF-) for 48 h, based on previously published studies (35eC38). In some experiments, wild-type cardiomyocytes were coincubated with 100 eol/l peroxynitrite scavenger urate (Sigma) or 50 eol/l SOD mimic Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP) (Calbiochem, La Jolla, CA) with LPS/TNF-.
Detection of 3-NT by Western blot.
Heart tissues were homogenized in lysis buffer using homogenizer. Cardiomyocytes were sonicated in lysis buffer. The lysis buffer contains 2% SDS, 10% glycerol, and 62.5 mmol/l Tris (pH 7.0). Tissue or cell proteins were collected by centrifuging at 12,000g at 4°C in a Beckman GS-6R centrifuge for 10 min. The protein concentration was measured. The sample, diluted in loading buffer and heated at 95°C for 5 min, was then subjected to electrophoresis on 10% SDS-PAGE gel at 120 V (5). After electrophoresis of the gel and transfer of the proteins to nitrocellulose membrane, the membranes were rinsed briefly in Tris-buffered saline, blocked in blocking buffer (5% milk and 0.5% BSA) for 1 h, and washed three times with Tris-buffered saline with Tween containing 0.05% Tween 20. The membranes were incubated with rabbit anti-NT polyclonal antibody (Chemicon, Temecula, CA) at a dilution of 1:1,000 for 2 h and then washed as above and reacted with secondary horseradish peroxidaseeCconjugated antibody for 1 h. Antigen-antibody complexes were then visualized using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ).
Measurement of lactate dehydrogenase activity.
Cytotoxicity for primary cultures of cardiomyocytes exposed to LPS/TNF- was assessed by spectrometric measurement of lactate dehydrogenase (LDH) activity in the culture media of cultured cells, and LDH activity was presented as optical density (OD) value at 490 nm, as described in the assay instruction (Promega, Madison, WI).
Statistical analysis.
Data were collected from repeated experiments and are presented as means ± SE. One-way ANOVA and Student’s t test were used for statistical analysis. Differences were considered to be significant at P < 0.05.
RESULTS
Diabetes-induced cardiac structural and functional abnormalities and metallothionein prevention.
Diabetes was developed in both MT-TG and wild-type mice on day 3 after STZ treatment, as characterized by multiple systemic changes (Table 1). There was no significant difference between MT-TG and wild-type mice in the incidence and manifestations of the STZ-induced diabetes. Serum thiobarbituric acideCreactive substance significantly increased in both wild-type and MT-TG diabetic mice, suggesting that systemic oxidative stress exists in both kinds of diabetic mice. Insulin-treated diabetic (diabetes/insulin) mice have significantly reduced glucose levels, compared with diabetic mice, and no systemic diabetes-related changes (Table 1).
There were significant differences in the histopathological and ultrastructural changes in the heart between the wild-type and MT-TG diabetic mice. Disorganized array of the myocardial structure, foci blooding and cell necrosis, and myofibrillar discontinuation were observed in the heart of the wild-type diabetic mice, but not in the heart of control or insulin-treated wild-type diabetic mice (Fig. 1A) or in MT-TG diabetic mice. Under the electron microscope (Fig. 1B), the hearts of the wild-type control and insulin-treated wild-type diabetic mice did not show any abnormalities. Only the heart of wild-type diabetic mice displayed increased numbers of lipid droplets and glycogen particles around mitochondria and scattered mitochondrial damage (swelling and disrupted cristae). However, the mitochondrial damage was significantly prevented in the heart of the MT-TG diabetic mice, although lipid droplets remained observable. These results indicated that the histopathological and ultrastructural changes were directly related to diabetes-associated pathogenesis rather than STZ toxicity, as documented in other studies (5,30,39), and metallothionein significantly prevented diabetes-induced cardiac toxicities.
To further evaluate the myocardial injury caused by diabetes, serum CPK was measured (Fig. 1C). Serum CPK levels were not changed in both wild-type and MT-TG diabetic mice on days 3eC7 after STZ treatment, but significantly increased in the wild-type diabetic mice on day 14 after STZ treatment. This phenomenon implies that the increase in serum CPK levels was not directly related to STZ toxicity, rather than related to diabetes. More importantly, the diabetes-induced increase in serum CPK levels did not occur in the MT-TG diabetic mice. It should be mentioned that CPK is not a measurement specifically for cardiac muscle injury because skeletal muscle injury also releases it to serum; however, it may indicate the cardiac injury in the present case because metallothionein in the MT-TG mice was overexpressed only in the cardiac myocytes. If the measured CPK was derived from skeletal muscle, it would not be protected in the MT-TG diabetic mice. The fact that there was no change in serum CPK levels in the MT-TG diabetic mice may indicate that the cardiac injury was the predominant source of serum CPK in the current experimental model.
The prevention of early structural damage (2 weeks after STZ treatment) in the MT-TG diabetic mice would result in a significant prevention of cardiomyopathy. Therefore, left ventricular functional changes were assessed by left ventricular hemodynamic analysis in diabetic mice 2 weeks (early stage) (Table 2) and 6 months (late stage) (Table 3) after STZ treatment. The heart rate was significantly increased in wild-type diabetic mice both 2 weeks and 6 months after STZ treatment and was not changed in insulin-treated diabetic or MT-TG diabetic mice. The assessment of left ventricular function changes induced by diabetes further revealed that significant defects occurred in the wild-type diabetic mice: decreased maximum dP/dt, a measurement of the mechanical ability of the heart to generate force for ejection of blood from the left ventricle; increased left ventricular end diastolic pressure (LVEDP), an index of the ventricular wall compliance; and increased , an index of the stiffness of the left ventricle (Tables 2 and 3). These changes were not observed in the MT-TG diabetic mice. Furthermore, the left ventricular minimum diastolic pressure (LVMDP), although not significantly changed in wild-type diabetic mice 2 weeks after STZ treatment, was significantly increased in the wild-type diabetic mice 6 months after STZ treatment (Table 3), suggesting the development of significant diastolic dysfunction in the wild-type diabetic mice. The increased LVMDP was not observed in the MT-TG diabetic mice (Table 3).
MT inhibition of diabetes-induced 3-NT and superoxide generation in the heart.
To determine the possible involvement of nitrosative damage in the pathogenesis of diabetic cardiomyopathy and the effect of metallothionein, 3-NT formation was assessed both qualitatively and quantitatively in the heart of wild-type and MT-TG diabetic mice 2 weeks after STZ treatment. As shown in Fig. 2A, immunohistochemical staining for 3-NT formation showed a strongly positive signal in the heart of wild-type diabetic mice, but a very weak signal in the heart of MT-TG diabetic mice or insulin-treated wild-type diabetic mice. To validate the antibody specificity, two sections were stained using the 3-NT antibody and preincubated overnight with nitrotyrosine (nitrated-BSA; Alpha Diagnostic International, San Antonio, TX). These two sections did not show 3-NTeCpositive signals (data not shown). Quantitative analysis by Western blot method further confirmed a significant increase in 3-NT formation in the heart of wild-type diabetic mice, which was significantly inhibited both in MT-TG or insulin-treated wild-type diabetic mice (Fig. 2B). A control membrane for 3-NT antibody specificity was also performed by blocking 3-NT antibody with nitrotyrosine and did not show the positive signals (data not shown). In addition, the significant increase in 3-NT formation in the wild-type diabetic heart and its prevention in the MT-TG diabetic heart was also evident in diabetic mice 4 weeks after STZ treatment (Fig. 2C).
To explore whether the inhibitory effect of metallothionein on 3-NT formation results from direct inhibition of the interaction between peroxynitrite and proteins and/or from the inhibition of the reaction between superoxide and nitric oxide, immunofluorescent staining for superoxide was performed using fluorescent superoxide probe (dihydroethidine) and showed a significant increase in superoxide generation in the heart of wild-type diabetic mice 2 weeks after STZ treatment, but not in the heart of wild-type control or insulin-treated wild-type diabetic mice (Fig. 3A). The diabetes-induced superoxide formation in the heart of the wild-type diabetic mice and its inhibition in the heart of MT-TG diabetic mice or insulin-treated wild-type diabetic mice were confirmed by quantitative measurement using cytochrome c reduction assay, a measurement of NADPH-dependent superoxide generation (Fig. 3B).
Urate and MnTMPyP inhibition of 3-NT formation in the LPS/TNF-eCtreated cardiomyocytes.
The in vivo results suggested that the suppression of superoxide and 3-NT formations in the MT-TG diabetic heart was accompanied by significant prevention of diabetes-induced cardiomyopathy. However, there is no direct link of the prevention of peroxynitrite formation and associated nitrosative damage to the inhibition of cardiomyopathy. In addition, although we have demonstrated the preventive effect of metallothionein on peroxynitrite-induced DNA and protein damage in a cell-free system (29), whether metallothionein exerts the same effect on intracellular peroxynitrite-induced nitrosative damage remains unclear. To this end, the primary cultures of neonatal cardiomyocytes from MT-TG and wild-type mice were exposed to LPS/TNF- for intracellularly generating superoxide and nitric oxide (35eC38). LPS/TNF- was selected not only because it is a well-defined intracellular peroxynitrite generation system, but also because we have found the increase in systemic and cardiac TNF- levels in the diabetic mice (30) and TNF- also play a critical role in various cardiomyopathy including diabetic cardiomyopathy (7,37,38). The results in Fig. 4 show that MT-overexpressing cardiomyocytes were resistant to the LPS/TNF-eCinduced cytotoxicity, detected by cell morphology (Fig. 4A) and medium LDH activity (Fig. 4B). The protective action of metallothionein was associated with the inhibition of LPS/TNF-eCinduced 3-NT formation in the cells (Fig. 4C), as observed in the in vivo studies (Fig. 2). Importantly, when the wild-type cardiomyocytes were treated with LPS/TNF- for 24 h in the presence of a peroxynitrite-specific scavenger urate or a cell-permeable SOD mimic MnTMPyP, the LPS/TNF-eCinduced 3-NT formation and cytotoxicity both were abolished (Figs. 5A and B), as observed in the MT-overexpressing cardiomyocytes (Fig. 4).
DISCUSSION
Studies aiming for mechanistic insights into the metallothionein inhibition of diabetic cardiomyopathy are important undertakings. This study focused on the effect of metallothionein on peroxynitrite-induced damage in the mouse model of type 1 diabetes induced by STZ. The results obtained clearly showed that peroxynitrite-induced protein damage, as measured by 3-NT, was involved in the diabetic cardiomyopathy. The formation of 3-NT most likely resulted from the overproduction of superoxide in the diabetic heart, which in turn reacts with nitric oxide to produce peroxynitrite, the causative agent for the formation of 3-NT. That the increased superoxide was responsible for the formation of 3-NT was revealed by the fact that suppression of superoxide formation both in vivo and in vitro resulted in reduced formation of 3-NT.
The involvement of oxidative and/or nitrosative stress in the pathogenesis of diabetic cardiomyopathy has been implicated in experimental animal studies and in patients (5eC913-16,40,41). The overproduction of superoxide has been previously recognized as an important contributor to diabetic vascular complications; however, superoxide alone cannot be considered as a strong oxidant toward most types of biological molecules (18). Recent studies indicate that peroxynitrite, which results from reaction of superoxide and nitric oxide, may play a central role in the pathogenesis of diabetic vascular complications (15eC19,40), because tyrosine nitration changes the structure and function of the proteins (18,40eC43). The direct evidence for the causative effect of peroxynitrite-caused protein nitration on diabetic nephropathy has been documented by two recent animal studies (44,45). In the present study, we are unable to identify the proteins that are nitrated in the heart of wild-type diabetic mice; however, based on the molecular size (30 kDa; Fig. 2), the nitrated proteins may belong mainly to mitochondrial proteins (42,43). Studies showed that high susceptibility of mitochondrial proteins, including energy production-related proteins (succinyl-CoA:3-oxoacid CoA-transferase and creatine kinase) and apoptosis-related protein, voltage-dependent anion channel-1 (molecule weight, 32 kDa), to tyrosine nitration in the heart from STZ- and alloxan-induced diabetic rats may be predominantly responsible for the mitochondrial and eventually myocyte dysfunction, leading to cardiomyopathy (41eC43).
In a recent study (6), by immunohistochemical staining, 3-NT as an index of peroxynitrite-induced protein nitration was significantly increased along with endothelial and myocyte cell death in the heart of diabetic patients, suggesting the possible association of peroxynitrite-induced protein nitration with cardiomyopathy. Metallothionein as a potent antioxidant significantly protects the heart from diabetes-induced damage in a spontaneously developed (26,28) and STZ-induced (25; present study) type 1 diabetic mouse model. In the STZ-induced diabetic mouse model, we further demonstrated that 3-NT was significantly increased in the heart of wild-type diabetic mice, but not MT-TG diabetic mice, 2 and 4 weeks after STZ treatment by Western blot assay. The innovative finding of the present study is that inhibition of superoxide and 3-NT formation is accompanied by a significant protection from diabetes-induced cardiac diastolic dysfunction observed 6 months after STZ treatment, indicating the involvement of nitrosative damage in the pathogenesis of diabetic cardiomyopathy. To confirm this in vivo observation, we used primary cultures of cardiomyocytes treated with LPS/TNF- in the presence of the cell-permeable SOD mimic MnTMPyP or urate, a peroxynitrite specific scavenger, to dissect the direct effect of superoxide or peroxynitrite on LPS/TNF-eCinduced cytotoxicity. The direct link between peroxynitrite-induced nitration and cardiomyocyte cytotoxicity was then defined because both urate and MnTMPyP significantly prevented 3-NT and cytotoxicity.
The metallothionein inhibition of peroxynitrite-induced protein nitration, shown by an increase in 3-NT formation, could result from its direct interaction with peroxynitrite (29), from its inhibition of superoxide generation (46,47), or from both. The results obtained from the in vivo study showed that both the formation of 3-NT and the NADPH-dependent generation of superoxide were inhibited to the same extent, suggesting that metallothionein inhibition of superoxide generation may be responsible for the decreased formation of 3-NT; in particular, metallothionein almost completely abolished the NADPH-dependent generation of superoxide in the heart. Based on the present study, we still do not know how metallothionein prevents the NADPH-dependent superoxide generation.
The use of LPS/TNF- to treat the cultured cells to produce peroxynitrite-induced damage, rather than to use 3-morpholinosydnonimine (SIN-1) for the same purpose is important. Several studies (48,49) have used SIN-1 to treat cultured cells to produce peroxynitrite-induced damage. These studies have observed that although SIN-1 caused cell injury, the formation of 3-NT in the cell was not observed. Addition of SIN-1 to the medium causes an immediate formation of peroxynitrite extracellularly (29). This leads to a direct damage to the cell membrane and cell death, without the intracellular process. In the present study, an obstacle is to develop an in vitro model to define the role of peroxynitrite in hyperglycemia-induced cardiomyopathy in vivo. Although the occurrence of peroxynitrite and its "footprint" of 3-NT were observed in the tissue of diabetic animals or patients (6,15,44,45) and in the high-glucose perfused hearts (16), there is no evidence that shows the peroxynitrite generation in cultured cardiomyocytes by exposure to high levels of glucose (17). We also failed to induce 3-NT formation in the neonatal cardiomyocytes exposed to 22.5 mmol/l for 48 or 72 h (data not shown). Under in vivo conditions, endothelial cells in the heart play a critical role in the formation of peroxynitrite due to hyperglycemia (6,15,44,45). It has been shown that if endothelial cells (mostly cell lines) were exposed to high levels of glucose for several days, a significant induction of peroxynitrite and 3-NT was observed (19,40,50). Therefore, the lack of peroxynitrite formation in the cultured cardiomyocytes may result from the omission of endothelial cells from the cultures. Thus, to mimic the role of peroxynitrite in vivo, we adapted the established intracellular peroxynitrite formation model by exposing the cardiomyocytes to LPS/TNF- (35eC38).
The use of the mouse model of type 1 diabetes induced by STZ has been criticized for the possible nonspecific effect of STZ. However, this should not be a major concern in the present study. We have used insulin-treated STZ-induced diabetic mice with a range of blood glucose levels (<8 mmol/l) as controls, as in our previous study (5). Our results demonstrated that only the diabetic mice with persistent high levels of blood glucose developed significant cardiomyopathy (Fig. 1; Table 2). In addition, we also found that serum CPK was not significantly increased in the wild-type diabetic mice until 2 weeks after STZ treatment (Fig. 1), at which time hyperglycemia has been well established. Therefore, these results indicated that cardiac toxicity is related directly to diabetes, rather than STZ toxicity. Several studies (6,16,42,44,45) and the results obtained here have shown that superoxide generation and associated nitrosative damage indeed play a critical role in the pathogenesis of diabetic cardiomyopathy. Metallothionein prevents diabetic cardiomyopathy at least in part through suppression of diabetes-caused superoxide generation and the associated nitrosative cardiac damage.
ACKNOWLEDGMENTS
This work was supported in part by Jewish Hospital Foundation Grant JHF 010808, Philip Morris USA Grant PM020187, American Diabetes Association Grant ADA020667 (to L.C.), and National Institutes of Health Grants HL63760 and HL59225 (to Y.J.K.). Y.J.K. is a Distinguished University Scholar of the University of Louisville.
We greatly appreciate the technological assistance of Yibo Du and Don Mosley.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
3-NT, 3-nitrotyrosine; CPK, creatine phosphokinase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; LVEDP, left ventricular end-diastolic pressure; LVMDP, left ventricular minimum diastolic pressure; MnTMPyP, Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride; MT-TG, metallothionein-overexpressing transgenic; OD, optical density; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN-1, 3-morpholinosydnonimine; SOD, superoxide dismutase; STZ, streptozotocin; TNF-, tumor necrosis factor-
REFERENCES
Cai L, Kang YJ: Oxidative stress and diabetic cardiomyopathy. Cardiovasc Toxicol 1: 181eC193, 2001
Zimmet P, Alberti KG, Shaw J: Global and societal implications of the diabetes epidemic. Nature 414: 782eC787, 2001
Trost S, LeWinter M: Diabetic cardiomyopathy. Curr Treat Options Cardiovasc Med 3: 481eC492, 2001
Francis GS: Diabetic cardiomyopathy: fact or fiction Heart 85: 247eC248, 2001
Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ: Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetes 51: 1938eC1948, 2002
Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P: Myocardial cell death in human diabetes. Circ Res 87: 1123eC1132, 2000
Cai L, Kang YJ: Cell death and diabetic cardiomyopathy. Cardiovasc Toxicol 3: 219eC228, 2003
Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P: IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin IIeCmediated oxidative stress. Diabetes 50: 1414eC1424, 2001
Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, Packer L: The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17: 189eC212, 2001
Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M: Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A 97: 12222eC12226, 2000
Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787eC790, 2000
Yamagishi SI, Edelstein D, Du XL, Brownlee M: Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction. Diabetes 50: 1491eC1494, 2001
Desco MC, Asensi M, Marquez R, Martinez-Valls J, Vento M, Pallardo FV, Sastre J, Vina J: Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes 51: 1118eC1124, 2002
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813eC820, 2001
Ceriello A, Quagliaro L, Catone B, Pascon R, Piazzola M, Bais B, Marra G, Tonutti L, Taboga C, Motz E: Role of hyperglycemia in nitrotyrosine postprandial generation. Diabetes Care 25: 1439eC1443, 2002
Ceriello A, Quagliaro L, D’Amico M, Di Filippo C, Marfella R, Nappo F, Berrino L, Rossi F, Giugliano D: Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat. Diabetes 51: 1076eC1082, 2002
Esberg LB, Ren J: Role of nitric oxide, tetrahydrobiopterin and peroxynitrite in glucose toxicity-associated contractile dysfunction in ventricular myocytes. Diabetologia 46: 1419eC1427, 2003
Ceriello A: New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy. Diabetes Care 26: 1589eC1596, 2003
Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA: Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells: role of peroxynitrite. J Biol Chem 278: 34003eC34010, 2003
Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN: Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest 100: 1501eC1506, 1997
Kang YJ: The antioxidant function of metallothionein in the heart. Proc Soc Exp Biol Med 222: 263eC273, 1999
Wang GW, Schuschke DA, Kang YJ: Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity. Am J Physiol 276: H167eCH175, 1999
Zhou Z, Kang YJ: Immunocytochemical localization of metallothionein and its relation to doxorubicin toxicity in transgenic mouse heart. Am J Pathol 156: 1653eC1662, 2000
Kang YJ, Zhou ZX, Wang GW, Buridi A, Klein JB: Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases. J Biol Chem 275: 13690eC13698, 2000
Cai L, Kang YJ: Metallothionein prevents diabetic cardiomyopathy. Toxicol Sci 60: 13, 2001
Liang Q, Carlson EC, Donthi RV, Kralik PM, Shen X, Epstein PN: Overexpression of metallothionein reduces diabetic cardiomyopathy. Diabetes 51: 174eC181, 2002
Kang YJ, Cai L: Metallothionein suppression of diabetic cardiomyopathy by inhibition of hyperglycemia-induced oxidative stress. Free Radic Biol Med 31 (Suppl. 1): 74, 2001
Ye G, Metreveli NS, Ren J, Epstein PN: Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production. Diabetes 52: 777eC783, 2003
Cai L, Klein JB, Kang YJ: Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage. J Biol Chem 275: 38957eC38960, 2000
Song Y, Song Z, Zhang L, McClain CJ, Kang YJ, Cai L: Diabetes enhances lipopolysaccharide-induced cardiac toxicity in the mouse model. Cardiovasc Toxicol 3: 363eC372, 2003
Li Y, Gu Y, Song Y, Zhang L, Kang YJ, Prabhu SD, Cai L: Cardiac functional analysis by electrocardiography, echocardiography and in situ hemodynamics in streptozotocin-induced diabetic mice. J Health Sci 50: 356eC365, 2004
Zhou Z, Wang L, Song Z, Saari JT, McClain CJ, Kang YJ: Abrogation of nuclear factor-kappaB activation is involved in zinc inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production and liver injury. Am J Pathol 164: 1547eC1556, 2004
Zhou Z, Sun X, James KY: Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress. Exp Biol Med 227: 214eC222, 2002
Deng XF, Rokosh DG, Simpson PC: Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes: comparison with rat. Circ Res 87: 781eC788, 2000
Asagiri K, Nakatsuka M, Konishi H, Noguchi S, Takata M, Habara T, Kudo T: Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts. J Obstet Gynaecol Res 29: 49eC55, 2003
Bachschmid M, Thurau S, Zou MH, Ullrich V: Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation. FASEB J 17: 914eC916, 2003
Klein JB, Wang GW, Zhou Z, Buridi A, Kang YJ: Inhibition of tumor necrosis factor-alpha-dependent cardiomyocyte apoptosis by metallothionein. Cardiovasc Toxicol 2: 209eC218, 2002
Aikawa R, Nitta-Komatsubara Y, Kudoh S, Takano H, Nagai T, Yazaki Y, Nagai R, Komuro I: Reactive oxygen species induce cardiomyocyte apoptosis partly through TNF-alpha. Cytokine 18: 179eC183, 2002
Akula A, Kota MK, Gopisetty SG, Chitrapu RV, Kalagara M, Kalagara S, Veeravalli KK, Gomedhikam JP: Biochemical, histological and echocardiographic changes during experimental cardiomyopathy in STZ-induced diabetic rats. Pharmacol Res 48: 429eC435, 2003
Zou MH, Shi C, Cohen RA: High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptoreCmediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes 51: 198eC203, 2002
Turko IV, Marcondes S, Murad F: Diabetes-associated nitration of tyrosine and inactivation of succinyl-CoA:3-oxoacid CoA-transferase. Am J Physiol Heart Circ Physiol 81: H2289eCH2294, 2001
Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, Murad F: Protein tyrosine nitration in the mitochondria from diabetic mouse heart: implications to dysfunctional mitochondria in diabetes. J Biol Chem 278: 33972eC33977, 2003
Turko IV, Murad F: Quantitative protein profiling in heart mitochondria from diabetic rats. J Biol Chem 278: 35844eC35849, 2003
Chander PN, Gealekman O, Brodsky SV, Elitok S, Tojo A, Crabtree M, Gross SS, Goligorsky MS: Nephropathy in Zucker diabetic fat rat is associated with oxidative and nitrosative stress: prevention by chronic therapy with a peroxynitrite scavenger ebselen. J Am Soc Nephrol 15: 2391eC2403, 2004
DeRubertis FR, Craven PA, Melhem MF, Salah EM: Attenuation of renal injury in db/db mice overexpressing superoxide dismutase: evidence for reduced superoxide-nitric oxide interaction. Diabetes 53: 762eC768, 2004
Thornalley PJ, Vasak M: Possible role for metallothionein in protection against radiation-induced oxidative stress: kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 827: 36eC44, 1985
Hussain S, Slikker W Jr., Ali SF: Role of metallothionein and other antioxidants in scavenging superoxide radicals and their possible role in neuroprotection. Neurochem Int 29: 145eC152, 1996
Sharma SK, Ebadi M: Metallothionein attenuates 3-morpholinosydnonimine (SIN-1)-induced oxidative stress in dopaminergic neurons. Antioxid Redox Signal 5: 251eC264, 2003
Li X, Chen H, Epstein PN: Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J Biol Chem 279: 765eC771, 2004
Quagliaro L, Piconi L, Assaloni R, Martinelli L, Motz E, Ceriello A: Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells: the role of protein kinase C and NAD(P)H-oxidase activation. Diabetes 52: 2795eC2804, 2003(Lu Cai, Jianxun Wang, Yan)
2 Department of Pharmacology and Toxicology, University of Louisville, School of Medicine, Louisville, Kentucky
ABSTRACT
The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor- for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.
Previous studies have shown that oxidative stress is critically involved in the pathogenesis of diabetic cardiomyopathy (1), a leading cause for mortality in both type 1 and type 2 diabetic patients (2eC4). The pathological changes in the heart were found to be associated with increased myocardial cell death, predominately in the mode of apoptosis and accumulation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) (5eC8). The roles of ROS and RNS in the diabetic complications of multiple organ systems were extensively documented (1,8eC13), although the exact mechanisms of ROS/RNS-induced pathogenesis have not been fully understood.
Superoxide overproduction in the organ systems is an important feature of diabetic complications (10eC12). One risk of the superoxide generation is related to its interaction with nitric oxide to form peroxynitrite (11eC15), which is a potent oxidant that causes nitrosative stress in the organ systems. A significant increase in serum and tissue 3-nitrotyrosine (3-NT), a by-product of the reaction between peroxynitrite and proteins, has been found in diabetic patients (6,15). Studies using experimental animal models and in vitro cultured cells have demonstrated that peroxynitrite is an important causative agent in diabetes-caused cardiovascular injury (16eC19).
Therefore, we hypothesized that inhibition of myocardial nitrosative stress would lead to suppression of diabetic cardiomyopathy. Recent studies have shown that overexpression of metallothionein, a small molecular weight protein that has been shown to protect the heart from injury under various oxidative stress conditions (20eC24), protects the heart from diabetes-induced damage (25,26), probably through suppression of oxidative stress (27,28). However, the mechanisms of metallothionein inhibition of diabetes-induced oxidative stress in the heart are largely unknown. Although we have demonstrated that metallothionein directly interacts with peroxynitrite to prevent peroxynitrite-induced lipid and DNA damage (29), whether the peroxynitrite formation is also involved in the pathogenesis of diabetic cardiomyopathy in vivo and whether metallothionein prevents peroxynitrite-induced cardiac damage in vivo remain unknown.
The present study, therefore, was undertaken to address the role of superoxide generation and associated nitrosative damage in the pathogenesis of diabetic cardiomyopathy in a mouse model of type 1 diabetes induced by streptozotocin (STZ). Furthermore, an attempt was made to understand the inhibitory action of metallothionein on the nitrosative cardiac damage in vivo.
RESEARCH DESIGN AND METHODS
Diabetes model.
MT-TG mice were produced from FVB mice and have been well characterized (20). Both MT-TG+ mice (heterozygotes) and MT-TGeC littermates (wild type) were kept in the same cages with free access to rodent diet and tap water and used for experiments. All animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association for Accreditation of Laboratory Animal Care.
Eight-week-old male mice were given a single dose of STZ (150 mg/kg body wt i.p.; Sigma, St. Louis, MO) dissolved in sodium citrate buffer (pH 4.5). Whole-blood glucose obtained from the mouse tail vein was detected using a SureStep complete blood glucose monitor (LifeScan, Milpitas, CA) 2 and 3 days after STZ treatment. STZ-treated mice with glucose levels higher than 12 mmol/l were considered diabetic, and mice serving as controls were given the same volume of sodium citrate (5). To eliminate the effects of STZ on cardiotoxicity, insulin-treated diabetic mice were used. For these mice, when hyperglycemia was diagnosed 2 or 3 days after STZ treatment, insulin was immediately given three times (8-h interval) using Humulin U (Eli Lilly, Indianapolis, IN) at a concentration of 10 units · dayeC1 · mouseeC1 to maintain the blood glucose levels between 5.6 and 11.2 mmol/l with an average of 7.6 ± 1.1 mmol/l, until they were killed.
Histopathological and immunohistochemical examination by light microscopy.
Heart tissues were fixed with 10% neutral and embedded in paraplast (23,30). Tissue sections of 5 e thickness were stained by hematoxylin-eosin and examined under a light microscope. The 3-NT as a marker of peroxynitrite-induced damage was detected by immunohistochemical stain for two sections of each mouse of five mice in each group (6,30).
Ultrastructural examination by electron microscopy.
The hearts 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 (20,23). The detail procedures of fixation, rinsing, postfixation, dehydration, and embedding for the tissue samples were described previously (20,23). Ultrathin sections were obtained with a LKB ultramicrotome, stained with uranyl acetate and lead citrate and observed with a Philip transmission electron microscope.
Measurements of serum creatine phosphokinase and lipid peroxidation.
Whole blood was collected from the dorsal vena cava of the anesthetized animals. Serum was prepared using a serum separator apparatus (Becton Dickinson, Rutherford, NJ) to measure creatine phosphokinase (CPK) activity following the instructions provided in the kit (Sigma). Serum lipid peroxidation was measured by a thiobarbituric acideCreactive substance assay, as described previously (29).
Assessment of left ventricle performance.
General measures of cardiac performance were done by in situ left ventricle (LV) hemodynamic analysis, as described previously (31). Mice were anesthetized using sodium pentobarbital (60 mg/kg i.p.). A midline incision (1eC2 cm) in the neck external to the trachea and a small opening in the trachea were made for the insertion of a PE-100 catheter to ensure a patent airway. The rostra end of the artery was clamped to occlude blood flow from the heart. A small incision was then made in the artery for the insertion of a hand-stretched, fluid-filled PE-50 catheter, which was connected to a transducer and a computer recording system. The catheter was then slowly advanced through the common carotid artery, through the ascending aorta and into the left ventricle. The animal was allowed to stabilize for 20eC30 min before recording of the waveform for up to 2 h. At the end of each experiment, the chest was opened to confirm the presence of catheter inside the left ventricle.
Measurement for superoxide.
Superoxide generation in the heart was examined by two methods: fluorescence labeling of superoxide specific staining (32) and a cytochrome c reduction assay for NADPH-dependent superoxide generation (33). Dihydroethidine is oxidized to ethidine (red fluorescence) selectively by superoxide, but not by other ROS or RNS such as hydrogen peroxide, hydroxyl radicals, or peroxynitrite. Dihydroethidine at 10 mg/kg was injected via a tail vein 1 h before tissue harvest, as described previously (32), and cryostat sections of heart were cut at 5 e and mounted on glass slides. The fluorescence was detected with a Nikon 2000S fluorescent microscope.
For cytochrome c reduction assay, the measurement of NADPH-dependent superoxide generation, cardiac tissues were homogenized and centrifuged at 800g for 10 min. The supernatant was incubated in the presence of 30 eol/l succinylated ferricytochrome c and 1 mmol/l NADPH (both from Sigma). The change in absorbance at 550 nm was measured. The difference in the amount of reduced succinylated ferricytochrome c in the presence or absence of 0.3 mg/ml superoxide dismutase (SOD) (Sigma) was used to estimate the amount of superoxide generation by using an absorbance coefficient 21.1 mmol/leC1 cmeC1 (33).
Primary cultures of neonatal cardiomyocytes.
Primary cultures of neonatal mouse cardiomyocytes were prepared by a modification of the method published previously (22,34). Briefly, hearts from 1- to 3-day-old wild-type and MT-TG mice were minced and dissociated with 0.15% trypsin. Dispersed cells were plated for 2 h in 100-mm dishes with minimum essential medium plus 10% bovine calf serum to remove noncardiomyocytes that attached to the plate within the incubation time. The myocytes that remained in the suspension were plated at a density of 500 cells/mm2 with minimum essential medium plus 10% bovine calf serum, 0.1 mmol/l bromodeoxyurindine, and 20 eol/l arabinosylcytosine. The medium was replaced 24 h later with fresh medium. On day 3, the wild-type and MT-TG cardiomyocytes were incubated with 30 e/ml lipopolysaccharide (LPS) (Sigma) plus 100 ng/ml tumor necrosis factor (TNF-) (PreproTech, Rocky Hill, NJ) (LPS/TNF-) for 48 h, based on previously published studies (35eC38). In some experiments, wild-type cardiomyocytes were coincubated with 100 eol/l peroxynitrite scavenger urate (Sigma) or 50 eol/l SOD mimic Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP) (Calbiochem, La Jolla, CA) with LPS/TNF-.
Detection of 3-NT by Western blot.
Heart tissues were homogenized in lysis buffer using homogenizer. Cardiomyocytes were sonicated in lysis buffer. The lysis buffer contains 2% SDS, 10% glycerol, and 62.5 mmol/l Tris (pH 7.0). Tissue or cell proteins were collected by centrifuging at 12,000g at 4°C in a Beckman GS-6R centrifuge for 10 min. The protein concentration was measured. The sample, diluted in loading buffer and heated at 95°C for 5 min, was then subjected to electrophoresis on 10% SDS-PAGE gel at 120 V (5). After electrophoresis of the gel and transfer of the proteins to nitrocellulose membrane, the membranes were rinsed briefly in Tris-buffered saline, blocked in blocking buffer (5% milk and 0.5% BSA) for 1 h, and washed three times with Tris-buffered saline with Tween containing 0.05% Tween 20. The membranes were incubated with rabbit anti-NT polyclonal antibody (Chemicon, Temecula, CA) at a dilution of 1:1,000 for 2 h and then washed as above and reacted with secondary horseradish peroxidaseeCconjugated antibody for 1 h. Antigen-antibody complexes were then visualized using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ).
Measurement of lactate dehydrogenase activity.
Cytotoxicity for primary cultures of cardiomyocytes exposed to LPS/TNF- was assessed by spectrometric measurement of lactate dehydrogenase (LDH) activity in the culture media of cultured cells, and LDH activity was presented as optical density (OD) value at 490 nm, as described in the assay instruction (Promega, Madison, WI).
Statistical analysis.
Data were collected from repeated experiments and are presented as means ± SE. One-way ANOVA and Student’s t test were used for statistical analysis. Differences were considered to be significant at P < 0.05.
RESULTS
Diabetes-induced cardiac structural and functional abnormalities and metallothionein prevention.
Diabetes was developed in both MT-TG and wild-type mice on day 3 after STZ treatment, as characterized by multiple systemic changes (Table 1). There was no significant difference between MT-TG and wild-type mice in the incidence and manifestations of the STZ-induced diabetes. Serum thiobarbituric acideCreactive substance significantly increased in both wild-type and MT-TG diabetic mice, suggesting that systemic oxidative stress exists in both kinds of diabetic mice. Insulin-treated diabetic (diabetes/insulin) mice have significantly reduced glucose levels, compared with diabetic mice, and no systemic diabetes-related changes (Table 1).
There were significant differences in the histopathological and ultrastructural changes in the heart between the wild-type and MT-TG diabetic mice. Disorganized array of the myocardial structure, foci blooding and cell necrosis, and myofibrillar discontinuation were observed in the heart of the wild-type diabetic mice, but not in the heart of control or insulin-treated wild-type diabetic mice (Fig. 1A) or in MT-TG diabetic mice. Under the electron microscope (Fig. 1B), the hearts of the wild-type control and insulin-treated wild-type diabetic mice did not show any abnormalities. Only the heart of wild-type diabetic mice displayed increased numbers of lipid droplets and glycogen particles around mitochondria and scattered mitochondrial damage (swelling and disrupted cristae). However, the mitochondrial damage was significantly prevented in the heart of the MT-TG diabetic mice, although lipid droplets remained observable. These results indicated that the histopathological and ultrastructural changes were directly related to diabetes-associated pathogenesis rather than STZ toxicity, as documented in other studies (5,30,39), and metallothionein significantly prevented diabetes-induced cardiac toxicities.
To further evaluate the myocardial injury caused by diabetes, serum CPK was measured (Fig. 1C). Serum CPK levels were not changed in both wild-type and MT-TG diabetic mice on days 3eC7 after STZ treatment, but significantly increased in the wild-type diabetic mice on day 14 after STZ treatment. This phenomenon implies that the increase in serum CPK levels was not directly related to STZ toxicity, rather than related to diabetes. More importantly, the diabetes-induced increase in serum CPK levels did not occur in the MT-TG diabetic mice. It should be mentioned that CPK is not a measurement specifically for cardiac muscle injury because skeletal muscle injury also releases it to serum; however, it may indicate the cardiac injury in the present case because metallothionein in the MT-TG mice was overexpressed only in the cardiac myocytes. If the measured CPK was derived from skeletal muscle, it would not be protected in the MT-TG diabetic mice. The fact that there was no change in serum CPK levels in the MT-TG diabetic mice may indicate that the cardiac injury was the predominant source of serum CPK in the current experimental model.
The prevention of early structural damage (2 weeks after STZ treatment) in the MT-TG diabetic mice would result in a significant prevention of cardiomyopathy. Therefore, left ventricular functional changes were assessed by left ventricular hemodynamic analysis in diabetic mice 2 weeks (early stage) (Table 2) and 6 months (late stage) (Table 3) after STZ treatment. The heart rate was significantly increased in wild-type diabetic mice both 2 weeks and 6 months after STZ treatment and was not changed in insulin-treated diabetic or MT-TG diabetic mice. The assessment of left ventricular function changes induced by diabetes further revealed that significant defects occurred in the wild-type diabetic mice: decreased maximum dP/dt, a measurement of the mechanical ability of the heart to generate force for ejection of blood from the left ventricle; increased left ventricular end diastolic pressure (LVEDP), an index of the ventricular wall compliance; and increased , an index of the stiffness of the left ventricle (Tables 2 and 3). These changes were not observed in the MT-TG diabetic mice. Furthermore, the left ventricular minimum diastolic pressure (LVMDP), although not significantly changed in wild-type diabetic mice 2 weeks after STZ treatment, was significantly increased in the wild-type diabetic mice 6 months after STZ treatment (Table 3), suggesting the development of significant diastolic dysfunction in the wild-type diabetic mice. The increased LVMDP was not observed in the MT-TG diabetic mice (Table 3).
MT inhibition of diabetes-induced 3-NT and superoxide generation in the heart.
To determine the possible involvement of nitrosative damage in the pathogenesis of diabetic cardiomyopathy and the effect of metallothionein, 3-NT formation was assessed both qualitatively and quantitatively in the heart of wild-type and MT-TG diabetic mice 2 weeks after STZ treatment. As shown in Fig. 2A, immunohistochemical staining for 3-NT formation showed a strongly positive signal in the heart of wild-type diabetic mice, but a very weak signal in the heart of MT-TG diabetic mice or insulin-treated wild-type diabetic mice. To validate the antibody specificity, two sections were stained using the 3-NT antibody and preincubated overnight with nitrotyrosine (nitrated-BSA; Alpha Diagnostic International, San Antonio, TX). These two sections did not show 3-NTeCpositive signals (data not shown). Quantitative analysis by Western blot method further confirmed a significant increase in 3-NT formation in the heart of wild-type diabetic mice, which was significantly inhibited both in MT-TG or insulin-treated wild-type diabetic mice (Fig. 2B). A control membrane for 3-NT antibody specificity was also performed by blocking 3-NT antibody with nitrotyrosine and did not show the positive signals (data not shown). In addition, the significant increase in 3-NT formation in the wild-type diabetic heart and its prevention in the MT-TG diabetic heart was also evident in diabetic mice 4 weeks after STZ treatment (Fig. 2C).
To explore whether the inhibitory effect of metallothionein on 3-NT formation results from direct inhibition of the interaction between peroxynitrite and proteins and/or from the inhibition of the reaction between superoxide and nitric oxide, immunofluorescent staining for superoxide was performed using fluorescent superoxide probe (dihydroethidine) and showed a significant increase in superoxide generation in the heart of wild-type diabetic mice 2 weeks after STZ treatment, but not in the heart of wild-type control or insulin-treated wild-type diabetic mice (Fig. 3A). The diabetes-induced superoxide formation in the heart of the wild-type diabetic mice and its inhibition in the heart of MT-TG diabetic mice or insulin-treated wild-type diabetic mice were confirmed by quantitative measurement using cytochrome c reduction assay, a measurement of NADPH-dependent superoxide generation (Fig. 3B).
Urate and MnTMPyP inhibition of 3-NT formation in the LPS/TNF-eCtreated cardiomyocytes.
The in vivo results suggested that the suppression of superoxide and 3-NT formations in the MT-TG diabetic heart was accompanied by significant prevention of diabetes-induced cardiomyopathy. However, there is no direct link of the prevention of peroxynitrite formation and associated nitrosative damage to the inhibition of cardiomyopathy. In addition, although we have demonstrated the preventive effect of metallothionein on peroxynitrite-induced DNA and protein damage in a cell-free system (29), whether metallothionein exerts the same effect on intracellular peroxynitrite-induced nitrosative damage remains unclear. To this end, the primary cultures of neonatal cardiomyocytes from MT-TG and wild-type mice were exposed to LPS/TNF- for intracellularly generating superoxide and nitric oxide (35eC38). LPS/TNF- was selected not only because it is a well-defined intracellular peroxynitrite generation system, but also because we have found the increase in systemic and cardiac TNF- levels in the diabetic mice (30) and TNF- also play a critical role in various cardiomyopathy including diabetic cardiomyopathy (7,37,38). The results in Fig. 4 show that MT-overexpressing cardiomyocytes were resistant to the LPS/TNF-eCinduced cytotoxicity, detected by cell morphology (Fig. 4A) and medium LDH activity (Fig. 4B). The protective action of metallothionein was associated with the inhibition of LPS/TNF-eCinduced 3-NT formation in the cells (Fig. 4C), as observed in the in vivo studies (Fig. 2). Importantly, when the wild-type cardiomyocytes were treated with LPS/TNF- for 24 h in the presence of a peroxynitrite-specific scavenger urate or a cell-permeable SOD mimic MnTMPyP, the LPS/TNF-eCinduced 3-NT formation and cytotoxicity both were abolished (Figs. 5A and B), as observed in the MT-overexpressing cardiomyocytes (Fig. 4).
DISCUSSION
Studies aiming for mechanistic insights into the metallothionein inhibition of diabetic cardiomyopathy are important undertakings. This study focused on the effect of metallothionein on peroxynitrite-induced damage in the mouse model of type 1 diabetes induced by STZ. The results obtained clearly showed that peroxynitrite-induced protein damage, as measured by 3-NT, was involved in the diabetic cardiomyopathy. The formation of 3-NT most likely resulted from the overproduction of superoxide in the diabetic heart, which in turn reacts with nitric oxide to produce peroxynitrite, the causative agent for the formation of 3-NT. That the increased superoxide was responsible for the formation of 3-NT was revealed by the fact that suppression of superoxide formation both in vivo and in vitro resulted in reduced formation of 3-NT.
The involvement of oxidative and/or nitrosative stress in the pathogenesis of diabetic cardiomyopathy has been implicated in experimental animal studies and in patients (5eC913-16,40,41). The overproduction of superoxide has been previously recognized as an important contributor to diabetic vascular complications; however, superoxide alone cannot be considered as a strong oxidant toward most types of biological molecules (18). Recent studies indicate that peroxynitrite, which results from reaction of superoxide and nitric oxide, may play a central role in the pathogenesis of diabetic vascular complications (15eC19,40), because tyrosine nitration changes the structure and function of the proteins (18,40eC43). The direct evidence for the causative effect of peroxynitrite-caused protein nitration on diabetic nephropathy has been documented by two recent animal studies (44,45). In the present study, we are unable to identify the proteins that are nitrated in the heart of wild-type diabetic mice; however, based on the molecular size (30 kDa; Fig. 2), the nitrated proteins may belong mainly to mitochondrial proteins (42,43). Studies showed that high susceptibility of mitochondrial proteins, including energy production-related proteins (succinyl-CoA:3-oxoacid CoA-transferase and creatine kinase) and apoptosis-related protein, voltage-dependent anion channel-1 (molecule weight, 32 kDa), to tyrosine nitration in the heart from STZ- and alloxan-induced diabetic rats may be predominantly responsible for the mitochondrial and eventually myocyte dysfunction, leading to cardiomyopathy (41eC43).
In a recent study (6), by immunohistochemical staining, 3-NT as an index of peroxynitrite-induced protein nitration was significantly increased along with endothelial and myocyte cell death in the heart of diabetic patients, suggesting the possible association of peroxynitrite-induced protein nitration with cardiomyopathy. Metallothionein as a potent antioxidant significantly protects the heart from diabetes-induced damage in a spontaneously developed (26,28) and STZ-induced (25; present study) type 1 diabetic mouse model. In the STZ-induced diabetic mouse model, we further demonstrated that 3-NT was significantly increased in the heart of wild-type diabetic mice, but not MT-TG diabetic mice, 2 and 4 weeks after STZ treatment by Western blot assay. The innovative finding of the present study is that inhibition of superoxide and 3-NT formation is accompanied by a significant protection from diabetes-induced cardiac diastolic dysfunction observed 6 months after STZ treatment, indicating the involvement of nitrosative damage in the pathogenesis of diabetic cardiomyopathy. To confirm this in vivo observation, we used primary cultures of cardiomyocytes treated with LPS/TNF- in the presence of the cell-permeable SOD mimic MnTMPyP or urate, a peroxynitrite specific scavenger, to dissect the direct effect of superoxide or peroxynitrite on LPS/TNF-eCinduced cytotoxicity. The direct link between peroxynitrite-induced nitration and cardiomyocyte cytotoxicity was then defined because both urate and MnTMPyP significantly prevented 3-NT and cytotoxicity.
The metallothionein inhibition of peroxynitrite-induced protein nitration, shown by an increase in 3-NT formation, could result from its direct interaction with peroxynitrite (29), from its inhibition of superoxide generation (46,47), or from both. The results obtained from the in vivo study showed that both the formation of 3-NT and the NADPH-dependent generation of superoxide were inhibited to the same extent, suggesting that metallothionein inhibition of superoxide generation may be responsible for the decreased formation of 3-NT; in particular, metallothionein almost completely abolished the NADPH-dependent generation of superoxide in the heart. Based on the present study, we still do not know how metallothionein prevents the NADPH-dependent superoxide generation.
The use of LPS/TNF- to treat the cultured cells to produce peroxynitrite-induced damage, rather than to use 3-morpholinosydnonimine (SIN-1) for the same purpose is important. Several studies (48,49) have used SIN-1 to treat cultured cells to produce peroxynitrite-induced damage. These studies have observed that although SIN-1 caused cell injury, the formation of 3-NT in the cell was not observed. Addition of SIN-1 to the medium causes an immediate formation of peroxynitrite extracellularly (29). This leads to a direct damage to the cell membrane and cell death, without the intracellular process. In the present study, an obstacle is to develop an in vitro model to define the role of peroxynitrite in hyperglycemia-induced cardiomyopathy in vivo. Although the occurrence of peroxynitrite and its "footprint" of 3-NT were observed in the tissue of diabetic animals or patients (6,15,44,45) and in the high-glucose perfused hearts (16), there is no evidence that shows the peroxynitrite generation in cultured cardiomyocytes by exposure to high levels of glucose (17). We also failed to induce 3-NT formation in the neonatal cardiomyocytes exposed to 22.5 mmol/l for 48 or 72 h (data not shown). Under in vivo conditions, endothelial cells in the heart play a critical role in the formation of peroxynitrite due to hyperglycemia (6,15,44,45). It has been shown that if endothelial cells (mostly cell lines) were exposed to high levels of glucose for several days, a significant induction of peroxynitrite and 3-NT was observed (19,40,50). Therefore, the lack of peroxynitrite formation in the cultured cardiomyocytes may result from the omission of endothelial cells from the cultures. Thus, to mimic the role of peroxynitrite in vivo, we adapted the established intracellular peroxynitrite formation model by exposing the cardiomyocytes to LPS/TNF- (35eC38).
The use of the mouse model of type 1 diabetes induced by STZ has been criticized for the possible nonspecific effect of STZ. However, this should not be a major concern in the present study. We have used insulin-treated STZ-induced diabetic mice with a range of blood glucose levels (<8 mmol/l) as controls, as in our previous study (5). Our results demonstrated that only the diabetic mice with persistent high levels of blood glucose developed significant cardiomyopathy (Fig. 1; Table 2). In addition, we also found that serum CPK was not significantly increased in the wild-type diabetic mice until 2 weeks after STZ treatment (Fig. 1), at which time hyperglycemia has been well established. Therefore, these results indicated that cardiac toxicity is related directly to diabetes, rather than STZ toxicity. Several studies (6,16,42,44,45) and the results obtained here have shown that superoxide generation and associated nitrosative damage indeed play a critical role in the pathogenesis of diabetic cardiomyopathy. Metallothionein prevents diabetic cardiomyopathy at least in part through suppression of diabetes-caused superoxide generation and the associated nitrosative cardiac damage.
ACKNOWLEDGMENTS
This work was supported in part by Jewish Hospital Foundation Grant JHF 010808, Philip Morris USA Grant PM020187, American Diabetes Association Grant ADA020667 (to L.C.), and National Institutes of Health Grants HL63760 and HL59225 (to Y.J.K.). Y.J.K. is a Distinguished University Scholar of the University of Louisville.
We greatly appreciate the technological assistance of Yibo Du and Don Mosley.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
3-NT, 3-nitrotyrosine; CPK, creatine phosphokinase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; LVEDP, left ventricular end-diastolic pressure; LVMDP, left ventricular minimum diastolic pressure; MnTMPyP, Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride; MT-TG, metallothionein-overexpressing transgenic; OD, optical density; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN-1, 3-morpholinosydnonimine; SOD, superoxide dismutase; STZ, streptozotocin; TNF-, tumor necrosis factor-
REFERENCES
Cai L, Kang YJ: Oxidative stress and diabetic cardiomyopathy. Cardiovasc Toxicol 1: 181eC193, 2001
Zimmet P, Alberti KG, Shaw J: Global and societal implications of the diabetes epidemic. Nature 414: 782eC787, 2001
Trost S, LeWinter M: Diabetic cardiomyopathy. Curr Treat Options Cardiovasc Med 3: 481eC492, 2001
Francis GS: Diabetic cardiomyopathy: fact or fiction Heart 85: 247eC248, 2001
Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ: Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome c-mediated caspase-3 activation pathway. Diabetes 51: 1938eC1948, 2002
Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P: Myocardial cell death in human diabetes. Circ Res 87: 1123eC1132, 2000
Cai L, Kang YJ: Cell death and diabetic cardiomyopathy. Cardiovasc Toxicol 3: 219eC228, 2003
Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P: IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin IIeCmediated oxidative stress. Diabetes 50: 1414eC1424, 2001
Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, Packer L: The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17: 189eC212, 2001
Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M: Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A 97: 12222eC12226, 2000
Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787eC790, 2000
Yamagishi SI, Edelstein D, Du XL, Brownlee M: Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction. Diabetes 50: 1491eC1494, 2001
Desco MC, Asensi M, Marquez R, Martinez-Valls J, Vento M, Pallardo FV, Sastre J, Vina J: Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes 51: 1118eC1124, 2002
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813eC820, 2001
Ceriello A, Quagliaro L, Catone B, Pascon R, Piazzola M, Bais B, Marra G, Tonutti L, Taboga C, Motz E: Role of hyperglycemia in nitrotyrosine postprandial generation. Diabetes Care 25: 1439eC1443, 2002
Ceriello A, Quagliaro L, D’Amico M, Di Filippo C, Marfella R, Nappo F, Berrino L, Rossi F, Giugliano D: Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat. Diabetes 51: 1076eC1082, 2002
Esberg LB, Ren J: Role of nitric oxide, tetrahydrobiopterin and peroxynitrite in glucose toxicity-associated contractile dysfunction in ventricular myocytes. Diabetologia 46: 1419eC1427, 2003
Ceriello A: New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy. Diabetes Care 26: 1589eC1596, 2003
Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA: Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells: role of peroxynitrite. J Biol Chem 278: 34003eC34010, 2003
Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN: Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest 100: 1501eC1506, 1997
Kang YJ: The antioxidant function of metallothionein in the heart. Proc Soc Exp Biol Med 222: 263eC273, 1999
Wang GW, Schuschke DA, Kang YJ: Metallothionein-overexpressing neonatal mouse cardiomyocytes are resistant to H2O2 toxicity. Am J Physiol 276: H167eCH175, 1999
Zhou Z, Kang YJ: Immunocytochemical localization of metallothionein and its relation to doxorubicin toxicity in transgenic mouse heart. Am J Pathol 156: 1653eC1662, 2000
Kang YJ, Zhou ZX, Wang GW, Buridi A, Klein JB: Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases. J Biol Chem 275: 13690eC13698, 2000
Cai L, Kang YJ: Metallothionein prevents diabetic cardiomyopathy. Toxicol Sci 60: 13, 2001
Liang Q, Carlson EC, Donthi RV, Kralik PM, Shen X, Epstein PN: Overexpression of metallothionein reduces diabetic cardiomyopathy. Diabetes 51: 174eC181, 2002
Kang YJ, Cai L: Metallothionein suppression of diabetic cardiomyopathy by inhibition of hyperglycemia-induced oxidative stress. Free Radic Biol Med 31 (Suppl. 1): 74, 2001
Ye G, Metreveli NS, Ren J, Epstein PN: Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production. Diabetes 52: 777eC783, 2003
Cai L, Klein JB, Kang YJ: Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage. J Biol Chem 275: 38957eC38960, 2000
Song Y, Song Z, Zhang L, McClain CJ, Kang YJ, Cai L: Diabetes enhances lipopolysaccharide-induced cardiac toxicity in the mouse model. Cardiovasc Toxicol 3: 363eC372, 2003
Li Y, Gu Y, Song Y, Zhang L, Kang YJ, Prabhu SD, Cai L: Cardiac functional analysis by electrocardiography, echocardiography and in situ hemodynamics in streptozotocin-induced diabetic mice. J Health Sci 50: 356eC365, 2004
Zhou Z, Wang L, Song Z, Saari JT, McClain CJ, Kang YJ: Abrogation of nuclear factor-kappaB activation is involved in zinc inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production and liver injury. Am J Pathol 164: 1547eC1556, 2004
Zhou Z, Sun X, James KY: Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress. Exp Biol Med 227: 214eC222, 2002
Deng XF, Rokosh DG, Simpson PC: Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes: comparison with rat. Circ Res 87: 781eC788, 2000
Asagiri K, Nakatsuka M, Konishi H, Noguchi S, Takata M, Habara T, Kudo T: Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts. J Obstet Gynaecol Res 29: 49eC55, 2003
Bachschmid M, Thurau S, Zou MH, Ullrich V: Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation. FASEB J 17: 914eC916, 2003
Klein JB, Wang GW, Zhou Z, Buridi A, Kang YJ: Inhibition of tumor necrosis factor-alpha-dependent cardiomyocyte apoptosis by metallothionein. Cardiovasc Toxicol 2: 209eC218, 2002
Aikawa R, Nitta-Komatsubara Y, Kudoh S, Takano H, Nagai T, Yazaki Y, Nagai R, Komuro I: Reactive oxygen species induce cardiomyocyte apoptosis partly through TNF-alpha. Cytokine 18: 179eC183, 2002
Akula A, Kota MK, Gopisetty SG, Chitrapu RV, Kalagara M, Kalagara S, Veeravalli KK, Gomedhikam JP: Biochemical, histological and echocardiographic changes during experimental cardiomyopathy in STZ-induced diabetic rats. Pharmacol Res 48: 429eC435, 2003
Zou MH, Shi C, Cohen RA: High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H2 receptoreCmediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes 51: 198eC203, 2002
Turko IV, Marcondes S, Murad F: Diabetes-associated nitration of tyrosine and inactivation of succinyl-CoA:3-oxoacid CoA-transferase. Am J Physiol Heart Circ Physiol 81: H2289eCH2294, 2001
Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, Murad F: Protein tyrosine nitration in the mitochondria from diabetic mouse heart: implications to dysfunctional mitochondria in diabetes. J Biol Chem 278: 33972eC33977, 2003
Turko IV, Murad F: Quantitative protein profiling in heart mitochondria from diabetic rats. J Biol Chem 278: 35844eC35849, 2003
Chander PN, Gealekman O, Brodsky SV, Elitok S, Tojo A, Crabtree M, Gross SS, Goligorsky MS: Nephropathy in Zucker diabetic fat rat is associated with oxidative and nitrosative stress: prevention by chronic therapy with a peroxynitrite scavenger ebselen. J Am Soc Nephrol 15: 2391eC2403, 2004
DeRubertis FR, Craven PA, Melhem MF, Salah EM: Attenuation of renal injury in db/db mice overexpressing superoxide dismutase: evidence for reduced superoxide-nitric oxide interaction. Diabetes 53: 762eC768, 2004
Thornalley PJ, Vasak M: Possible role for metallothionein in protection against radiation-induced oxidative stress: kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 827: 36eC44, 1985
Hussain S, Slikker W Jr., Ali SF: Role of metallothionein and other antioxidants in scavenging superoxide radicals and their possible role in neuroprotection. Neurochem Int 29: 145eC152, 1996
Sharma SK, Ebadi M: Metallothionein attenuates 3-morpholinosydnonimine (SIN-1)-induced oxidative stress in dopaminergic neurons. Antioxid Redox Signal 5: 251eC264, 2003
Li X, Chen H, Epstein PN: Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J Biol Chem 279: 765eC771, 2004
Quagliaro L, Piconi L, Assaloni R, Martinelli L, Motz E, Ceriello A: Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells: the role of protein kinase C and NAD(P)H-oxidase activation. Diabetes 52: 2795eC2804, 2003(Lu Cai, Jianxun Wang, Yan)