Chinese Medical Journal 2009;122(21):2580-2586
Attenuation of myocardial apoptosis by alpha-lipoic acid through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy

LI Chun-jun,  ZHANG Qiu-mei,  LI Ming-zhen,  ZHANG Jing-yun,  YU Pei,  YU De-mi

LI Chun-jun (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

ZHANG Qiu-mei (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

LI Ming-zhen (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

ZHANG Jing-yun (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

YU Pei (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

YU De-mi (The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China)

Correspondence to:YU De-min,The Ministry of Health Key Laboratory of Hormone and Development, Department of Endocrinology, Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China (Tel: 86-22-23086606. Fax:86-22-23086999. E-mail:Li_chunjun@126.com)
Keywords
alpha-lipoic acid; myocardial apoptosis; mitochondrial oxidative stress; diabetic cardiomyopathy
Abstract

Background  Cardiac failure is a leading cause of the mortality of diabetic patients. In part this is due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy. Oxidative stress is widely considered to be one of the major factors underlying the pathogenesis of the disease. This study aimed to test whether the antioxidant α-lipoic acid (α-LA) could attenuate mitochondrion-dependent myocardial apoptosis through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy.
Methods  A rat model of diabetes was induced by a single tail intravenous injection of streptozotocin (STZ) 45 mg/kg. Experimental animals were randomly assigned to 3 groups: normal control (NC), diabetes (DM) and DM treated with α-LA (α-LA). The latter group was administered with α-LA (100 mg/kg ip per day), the remainder received the same volume vehicle. At weeks 4, 8, and 12 after the onset of diabetes, cardiac apoptosis was examined by TUNEL assay. Cardiomyopathy was evaluated by assessment of cardiac structure and function. Oxidative damage was evaluated by the content of malondialdehyde (MDA), reduced glutathione (GSH) and the activity of manganese superoxide diamutase (Mn-SOD) in the myocardial mitochondria. Expression of caspase-9 and caspase-3 proteins was determined by immunohistochemistry and mitochondrial cytochrome c release was detected by Western blotting.
Results  At 4, 8, and 12 weeks after the onset of diabetes, significant reductions in TUNEL-positive cells, caspase-9,-3 expression, and mitochondrial cytochrome c release were observed in the α-LA group compared to the DM group. In the DM group, the content of MDA in the myocardial mitochondria was significantly increased, and there was a decrease in both the mitochondrial GSH content and the activities of Mn-SOD. They were significantly improved by α-LA treatment. HE staining displayed structural abnormalities in diabetic hearts, while α-LA reversed this structural derangement. The index of cardiac function (±dp/dtmax) in the diabetes group was aggravated progressively from 4 weeks to 12 weeks, but α-LA delayed deterioration of cardiac function (P <0.05).
Conclusions
 Our findings indicate that the antioxidant α-LA can effectively attenuate mitochondria-dependent cardiac apoptosis and exert a protective role against the development of diabetic cardiomyopathy. The ability of α-LA to suppress mitochondrial oxidative damage is concomitant with an enhancement of Mn-SOD activity and an increase in the GSH content of myocardial mitochondria.

Cardiac failure is a leading cause of death in diabetic patients. Accumulated evidence indicates that heart failure in diabetes is, at least in part, due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy, which is distinct from coronary arteriosclerosis. This was first proposed by Rubler et al1 in 1972 based on postmortem findings of heart failure in diabetic patients free of coronary artery disease. Recent studies have suggested that massive ventricular myocyte loss, compensatory hypertrophy of the remaining myocytes and reproductive fibrosis play a key role in the development of diabetic cardiomyopathy in rodents2 and humans.3 Mitochondria are the major source of reactive oxygen species (ROS) because these organelles continuously generate superoxide, by a product of electron transport, so it is not unexpected that mitochondria have been shown to be a primary target of damage in diabetes.4 The mitochondrial apoptic pathway appears to play an important role in diabetes-induced myocardial cell apoptosis, and among the apoptotic stimuli, ROS and/or reactive nitrogen species (RNS) play a critical role in the mitochondrial cytochrome c release and caspase-3 activation.5,6 Recent studies have shown that antioxidant has been shown to protect the heart from injury under various oxidative stress conditions, protects the heart from diabetes induced damage, probably through suppression of oxidative stress.7-9 Therefore we hypothesized that suppression of mitochondrial oxidative stress might attenuate the cardiac apoptosis against diabetic cardiomyopathy. α-Lipoic acid (α-LA) was a free radical scavenger and a potent natural antioxidant, which is useful in the prevention of cardio-vascular diseases.9,10 To test this, we studied the effects of α-LA treatment on markers of the mitochondrial apoptic pathway in the cardiac tissue of diabetic animals.

METHODS

Diabetic rat model
Male Wistar rats weighing 200 to 250 g were purchased from the Department of Experimental Animal in Peking (China). They were housed individually under a 12-hour light-dark cycle at 21°C. The rats were provided a standard rat chow and tap water ad libitum. The experimental and feeding protocols were approved and in accordance with the laws and regulations controlling experiments on live animals in China and the Asian Convention for the Protection of Vertebrate Animals used in Experimental and Other Scientific Purposes. Rats were rendered diabetic by a single tail intravenous injection of 45 mg/kg body weight streptozotocin (Sigma, USA) dissolved in 1 ml sodium citrate buffer at 4°C. Rats were considered diabetes if the random plasma glucose concentration (assayed using hexokinase method) was greater than 16.7 mmol/L 7 days after STZ treatment. Seventy-two experimental animals were randomly assigned to 3 groups: normal control (NC), diabetes (DM) and treatment with α-LA (α-LA), every group with 24 rats. In the treatment group, diabetic rats were given intraperitoneal injections of α-LA (100 mg/kg per day) for 12 weeks, starting at the onset of diabetes. Rats in each group were sacrificed at three time points: 4, 8 and 12 weeks from the onset of diabetes.

Cardiac function
Before sacrifice, animals were anesthetized with chloral hydrate (100 mg/kg, ip). An electrocardiogram was performed and then the carotid artery was cannulated to measure arterial systolic pressure and diastolic pressure. Subsequently the cannula was advanced into the left ventricle to evaluate ventricular pressures (left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP)) and the rate of pressure rise and delay (±dp/dtmax) (BL-420E+ multifunction experiment system, Taimeng Technology Limited Company Sichuan, China).

Tissue preparation
The heart was excised from the chest, trimmed of atria and large vessels and weighed. First a mid left ventricular (LV) section was cut perpendicularly to its longitudinal axis and fixed in phosphate-buffered 20% formaldehyde. Histological paraffin-embedded sections (5 µm) were then prepared. The rest of heart was used to extract mitochondria.

Interstitial collagen and cardiac structure
LV sections were stained with Masson to measure interstitial fibrosis. Interstitial collagen was quantified at a final magnification of 200× with a microscope (Olympus, America) connected to a video camera (Nikon, Japan). The resulting image was processed on Biomas Image-analysis System (Taimeng, Sichuan, China). The content of interstitial collagen (expressed as the fractional area of the entire cross-section) was averaged on nine fields selected across the wall thickness in the septum and free wall. Cardiac structure was displayed by staining with hematoxylin and eosin (HE).

TUNEL assay
The sections were processed for TUNEL (Roche; Mannheim, Germany) assay. The ApopTag in situ detection kit from Intergen (Purchase, Germany) was used in accordance with the manufacturer′s instructions. Briefly, the slides were treated with H2O2 and incubated with a reaction mixture containing TdT and digoxigenin-conjugated dUTP for 1 hour at 37°C. Labeled DNA was visualized with peroxidase-conjugated anti-digoxigenin antibody using 3,3′-diaminobenzidine (DAB) as the chromogen. Rat testicular tissue was used as positive control. For the negative control, TdT was omitted from the reaction mixture.

Caspase-3, -9 immunohistochemical staining
Briefly, after deparaffinization and rehydration, slides of heart tissues were treated with 3% H2O2 in phosphate buffered saline (PBS) for 10 minutes and with 5% normal sera in PBS for 20 minutes. Sections were incubated overnight at 4°C with polyclonal rabbit anti-active caspase-3 (Boshide, Wuhan, China) at 1:75 dilution, then incubated for 20 minutes in biotinylated goat anti-rabbit IgG (Boshide, Wuhan, China), followed by incubation with horseradish peroxidase (HRP)-streptavidin for 20 minutes. The antibody binding sites were visualized through reaction with DAB-H2O2 solution. Finally, sections were counterstained with 0.5% hydrochloric acid alcohol.

Detection of mitochondrial cytochrome c release
Subcellular fractionation and Western blotting analysis were used to detect cytochrome c content in the cytosol and mitochondria. An increase in the cytosol with a concomitant decrease in mitochondria was indicative of cytochrome c release from mitochondria. Briefly, heart was excised and washed in cold PBS and then homogenized in cold lysis buffer supplemented with 250 mmol/L sucrose, 3 mmol/L HEPES and 0.5 mmol/L EDTA (pH 7.4). The homogenate was centrifuged twice at 750 ×g at 4°C for 10 minutes. The supernatant from the second centrifugation at 750 ×g was removed to fresh tubes and centrifuged twice at 10 000 ×g at 4°C for 10 minutes. The supernatant of the 10 000 ×g spin was removed to clean tubes (the supernatant of this spin is the cytosolic light membrane fraction). The 10 000 ×g mitochondrial pellet was resuspended and lysed in lysis buffer. The protein concentration was assayed with an aliquot of each, and the remaining was boiled in 2× SDS sample buffer. The protein samples from each fraction were separated via SDS/PAGE and subsequently transferred to nitrocellulose filters for Western blotting. Filters were probed using purified mouse anti-cytochrome c monoclonal antibodies (Santa Cruz, USA) and subsequently exposed to secondary HRP-conjugated IgG. Antigen-antibody complexes were then visualized through reaction with DAB-H2O2 solution.

Measurements of mitochondrial GSH, Mn-SOD and MDA
The content of MDA in the myocardium mitochondrion was assayed by thiobarbituric acid, and the content of GSH, and the activities of SOD in the myocardium mitochondrion were measured by spectophotometer assays. All steps were used according to the manufacturer′s instructions (Jiancheng Limited Company, Nanjing, Jiangsu, China).

Statistical analysis
All statistical analyses were performed on a personal computer with the software program SPSS 11.5. Data were collected from repeated experiments and were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Student′s Newman Keuls test (SNK) was used for statistical analysis. Differences were considered to be significant at P <0.05.

RESULTS

Induction of diabetes
At sacrifice, blood glucose concentrations were all significantly increased in the DM group compared to NC group at all three time points. However blood glucose concentrations in diabetic rats were not significantly affected by α-lipoic acid treatment compared with untreated diabetic rats. The body weights of diabetic rats were all lower than that of control animals at 4, 8 and 12 weeks (–33%, –24%, –43%, respectively). The body weights of diabetic rats were not significantly affected by α-lipoic acid treatment, but exhibited an upward trend (Table).


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Table. Glucose and body weight of the patients

Cardiac function of animals
Each group of animals had comparable systolic blood pressure (SBP) and diastolic blood pressure (DBP). However diabetic rats were more vulnerable to arrhythmia, but α-lipoic acid significantly reduced the incidence rate of arrhythmia. Heart weight to body weight ratios remained constant in the NC group ((2.69±0.10), (2.65±0.32) and (2.62±0.16) mg/g at 4, 8 and 12 weeks, respectively), but the DM group had higher heart weight to body weight ratios at each point (P <0.01), and this was normalized at 4 and 8 weeks. At 12 weeks, α-LA group ((3.06±0.23) mg/g) did not differ from DM group ((3.25±0.25) mg/g, P >0.05). At 4 weeks, the DM group was characterized by a decrease in –dp/dtmax and an increase in LVEDP. At 8 weeks, cardiac diastolic function was further depressed. At 12 weeks, cardiac systolic function was also impaired, DM group showed an increase in LVSP and a decrease in +dp/dtmax. At all three time points, α-lipoic acid significantly improved cardiac function impaired (Figure 1).


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Figure 1. Effects of diabetes on LVEDP (A), –dp/dtmax (B), LVSP (C) and +dp/dtmax (D) in NC, DM and α-LA groups at 4, 8, 12 weeks. Eight rats per group. Data presented as mean±SD. *P <0.05 vs NC, P <0.05 vs DM (ANOVA and SNK).

Interstitial collagen and cardiac structure
Interstitial collagen content remained constant in the NC group at 4, 8, 12 weeks. However cardiac collagen contents were higher in DM (1.67-, 2.26- and 2.60 fold vs NC, P <0.001) but decreased by α-LA (–26%, –22% and –23% vs DM, P <0.01, Figure 2). HE staining of the heart tissues showed that compared to NC, diabetic hearts displayed structural abnormalities. More importantly, structural abnormalities induced by diabetes were significantly ameliorated by α-lipoic acid treatment (Figure 2E).


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Figure 2. Effects of diabetes on cardiac structure and fibrosis (A–D, HE staining, original magnification ×200). Tissue A was collected from normal control, B from diabetic rats after 4 weeks, C from diabetic rats after 12 weeks and D from diabetic rats treated by α-lipoic acid after 12 weeks. Figure E shows left ventricular area. Interstitial collagen density was expressed as percentage of tissue. Eight rats per group. Data presented as mean±SD. *P <0.05 vs control, P <0.05 vs DM.

Cardiomyocyte apoptosis
The extent of cardiomyocyte apoptosis remained at a constant low level in the NC group at 4, 8 and 12 weeks. However TUNEL-positive cardiomyocytes were dramatically raised in diabetic rats (approximately 10-fold vs NC, at all three time points, P <0.001), α-LA treatment significantly suppressed this apoptosis at every time point (–72.7%, –56.2%, –52.4%, P <0.01, Figure 3).


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Figure 3. Quantitative analysis of apoptotic cells in the heart of diabetic rats. In TUNEL detection of apoptotic cells, the positive nuclei were dying brown indicated by the arrow. These example slides were processed for TUNRL staining (200×). Tissue A was collected from diabetic rats, B from diabetic rats treated by α-lipoic acid. There were eight rats per group.

Mitochondrial oxidative stress parameter
We measured reduced GSH, Mn-SOD and MDA to assess oxidative damage in the myocardium mitochondria and to determine whether α-LA protects against mitochondrial oxidative stress damage. At all three time points, the content of GSH in the myocardial mitochondria of DM showed 35%, 40% and 72% decrease compared to NC (respectively all P <0.05), but this was increased by α-LA treatment (+44%, 45% and 127% vs DM, P <0.01, Figure 4A). At 4 weeks, there was no significant difference in the activities of Mn-SOD in the myocardial mitochondria among the groups, but at 8 weeks and 12 weeks, the activities of Mn-SOD in DM showed a significant reduction (–57% and 74% vs NC, P <0.001). Again this was increased by α-LA treatment (+83% and 137% vs DM, P <0.01, Figure 4B). The content of MDA in the myocardial mitochondria from DM rats obviously increased at all three time points (2.26-, 3.28- and 5.54-fold vs NC, P <0.001), and this was reversed by α-LA treatment (35%, 49% and 63% vs DM, P <0.01, Figure 4C).


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Figure 4. Effects of diabetes on marker of mitochondrial oxidative stress and the protective role of α-lipoic acid. The change of mitochondrial GSH is displayed in Figure A, Mn-SOD in Figure B and MDA in Figure C. There were eight rats per group. Data are presented as mean ± SD. *P <0.05 vs control, P <0.05 vs DM.

Marker of mitochondrial apoptic pathway
To further clarify the molecular basis of increased apoptosis in the heart of diabetic rats, we studied the mitochondria-dependent apoptosis pathway. We examined the expression levels of caspase-9, the major initiator caspase in mitochondria-dependent apoptosis pathway. Caspase-3 is another important indication of apoptotic cell death. To determine whether caspase-3 activation is mediated by mitochondrial cytochrome c release, we used a Western blotting method to detect the cytochrome c translocation from mitochondria to cytosol.

Figure 5 shows that cytosolic concentrations of cytochrome c were gradually increased with time accompanied by decreased mitochondrial concentrations from 4 weeks to 12 weeks in diabetic rats (Figure 5 A–D). Concomitant with these changes was an increased expression of caspase-9 (Figure 5E) and caspase-3 (Figure 5F). All of these results suggest that diabetes- induced apoptosis in the heart is likely mediated, at least in part, by the cytochrome c-mediated caspase-3 activation pathway. At three time points, α-lipoic acid significantly reduced mitochondrial cytochrome c release, which was associated with down-regulation expression of caspase-9 and caspase-3.


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Figure 5. Effects of diabetes on mitochondria-dependent apoptosis pathways. Mitochondrial cytochrome c release was measured by Western blotting (A and B) of cytosol and mitochondria in NC, α-LA, and DM. The quantitative analysis of cytochrome c release from mitochondria into cytosol are displayed in C and D. The expression level of caspase-9, -3 are displayed in E and F. There were eight rats per group. Data presented as mean values ±SD. *P <0.05 vs control, P <0.05 vs DM.

DISCUSSION

Considerable evidence indicates that increased oxidative stress and induction of apoptosis may play an important role in the development of cardiovascular complications in diabetes mellitus.11 Numerous studies also indicate that antioxidative treatment may prevent the development of diabetic cardiomyopathy.12,13

Accumulated evidence showed that apoptotic cell death is a key element in the pathogenesis and progression of various cardiac diseases, including ischemia reperfusion, toxic exposure, myocardial infarction and arrhythmias.14,15 Apoptosis of cardiac myocytes may contribute to progressive pump failure, arrhythmias and cardiac remodelling. And it has recently been shown that apelin improves cardiac function by inhibiting cardiomyocyte apoptosis.16 In the present study, we found that diabetic rats exhibited an significant increase in cardiac myocyte apoptosis, were more vulnerable to arrhythmia (the most serous being bigeminal rhythm), had higher heart weight to body weight ratios, and had impaired cardiac function and structural abnormalities. Furthermore, antioxidant α-lipoic acid effectively attenuated cardiac apoptosis and exerted a protective role against the development of diabetic cardiomyopathy. Alpha-lipoic acid can neutralize reactive oxygen species (ROS scavenger) as well as reducing the oxidized forms of other antioxidants due to its low redox potential. It is soluble in water as well as in fats, which is a unique feature among antioxidants.17 And it has recently been shown that lipoic-acid-mediated elevation of cellular defense is accompanied by an increased resistance to ROS-elicited cardiac cell injury in vitro.18 So we chose it as our experiment antioxidant.

Oxidative damage caused by ROS and RNS is related directly to multiple complications of diabetes,11 including cardiomyopathy.19-22 Indeed Ghosh et al,23 using STZ induced diabetic rats, showed that mitochondrial oxidative stress such as GSH depletion is the direct cause of cardiac cell apotosis in diabetes. Cai et al,24 using cardiac-specific metallothionein (MT) transgenic mice, showed that MT significantly inhibits mitochondrial GSH depletion, cardiac protein nitration, and lipid peroxidation, and is associated with a significant prevention of cardiac cell death in the hearts of diabetic mice. We show that α-LA could attenuate mitochondrial oxidative damage and this was accompanied by an enhanced SOD activity and increased GSH content, both of which resulted in a decline in cardiomyocyte apoptosis. This suggests a cause-effect relationship between mitochondrial oxidative stress and cardiac cell apoptosis.

The molecular mechanisms responsible for apoptosis have been elucidated in recent years. A family of intracellular proteases, the caspases, is responsible directly or indirectly for the terminal morphologic and biochemical changes of apoptosis. Although multiple pathways for activating caspases may exist, two important apoptotic pathways have been extensively studied in mammalian cardiac tissues. The first involves signaling via the TNF family receptors, also called death receptors. The second is often called the mitochondrial apoptotic program. During apoptosis, mitochondrial permeability transition increases, and the release into the cytosol of pro-apoptotic factors (procaspases, the caspase activator cytochrome c, and caspase-independent factors such as apoptosis-inducing factor) leads to the apoptotic phenotype. However the detailed mechanisms by which diabetes induces myocardial cell death are poorly understood. Diabetes-induced myocardial cell apoptosis is thought to occur through mitochondrial cytochrome c release and the activation of caspase-3 pathway.4 So using myoblast H9c2 cells, direct exposure to high levels of glucose induces significant apoptotic cell death, detected by TUNEL assay and Dapi nuclear staining,4 and caspase-3 activation, with concomitant mitochondrial cytochrome c release, was observed in cells exposed to high levels of glucose.

To further clarify the molecular basis of increased apoptosis in the heart of diabetic rats, we studied the mitochondrion-dependent apoptosis pathway. We found that hyperglycemia induced mitochondria oxidative damage which triggered mitochondrial cytochrome c release, along with significant up-regulation of caspase-9 and caspase-3 activation and cell apoptosis. Furthermore, mitochondrial cytochrome c release was significantly suppressed by antioxidative treatment, and down-regulation of caspase-9, -3. These changes were accompanied by decreasing cardiac cell apoptosis. This suggested that the mitochondrial apoptotic program play an important role in hyperglycemia induced myocardial apoptosis and that it may be triggered by mitochondrial oxidative damage.

In conclusion, hyperglycemia could induce myocardial apoptosis, which may be an important mechanism contributing to progressive cardiac failure and the development of diabetic cardiomyopathy. Under diabetic conditions, myocardial apoptosis was mediated, at least in part, by activation of the cytochrome c-activated caspase-3 pathway, which may be triggered by mitochondrial oxidative stress. The antioxidant α-LA attenuated mitochondrial oxidative damage by enhancing SOD activity and increasing GSH content, and a decrease in cardiomyocyte apoptosis dependent on mitochondrion. This may have important implications for the clinical management of diabetes in humans, and antioxidatives may be an important therapeutic option for preventing cardiomyopathy in diabetes mellitus.

Acknowledgments: We greatly appreciate the technological assistance from Professor YAN Yu-qin, ZHANG Ming-fang and WANG Yong-ming. We also greatly appreciate Professor Gareth Denyer for his careful language polishing.

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  1. Metabolic Disease Hospital Foundation and University Scholar of the Tianjin Medical University,No. 2006ky43;