Oxidative Stress As A Common Mediator for Apoptosis Induced-Cardiac Damage in Diabetic Rats

Abstract

Abstract:Aim:

To investigate the possible role of oxidative stress as a common mediator of apoptosis and cardiac damage in diabetes.

Materials and Methods:

This experimental work was conducted on 5 groups of Wistar rats. Group I was the control group. Diabetes type 1 was induced in other groups (by streptozotocin) and animals received insulin or vitamin E (300 mg /kg body weight), both insulin and vitamin E, or no treatment for 4 weeks according to their group. At the end of the study, serum and cardiac tissues were examined for biochemical parameters of cardiac function, oxidative stress and apoptosis. Electron microscopy pictures of cardiac tissue were also evaluated for signs of cardiac damage

Results:

Markers of oxidative stress, apoptosis, inflammation as well as manifestations of cardiac damage as assessed by electron microscopy were significantly decreased in rats treated with both insulin and vitamin E when compared with untreated diabetic rats or rats treated with either insulin or vitamin E alone

Conclusion:

Administration of both vitamin E and insulin was effective in reducing markers of oxidative stress and apoptosis and improving parameters of cardiac function in experiments animals. Antioxidants might prove beneficial as an adjuvant treatment in addition to insulin in type 1 diabetes associated with manifestations of cardiac complications

Key Words: Diabetes, vitamin E, Wistar rats, diabetic cardiomyopathy, apoptosis, oxidative stress, cardiac enzymes.

INTRODUCTION

Diabetes represents a serious risk factor for the development of cardiovascular complications such as coronary heart disease, peripheral arterial disease, hypertension, stroke, cardiomyopathy and nephropathy [1]. Identifying risk factors that may lead to diabetes type 2, such as metabolic syndrome (MetS) provides the base for life modification and/or pharmacological intervention in order to prevent cardiovascular complications[2]. Among other factors, increased oxidative stress has been implicated as a possible mechanism for such complications [3].

In diabetes the circulating free radicals may contribute to progression of heart disease and possibly mediate the process of apoptosis [4], a state where increased oxidative stress is documented [5]. Recent reports provide evidence that high ambient glucose can promote apoptosis in vitro, suggesting potential cellular damage as a result of hyperglycemia in diabetes [6]. Though oxidative stress-induced apoptosis was postulated to occur in cases of myocardial infarction [7] it is uncertain whether apoptosis occurs in cardiac muscle during the course of diabetes. Levrand et al. [8] postulated that Peroxynitrite (ONOO) triggers apoptosis in cardiomyocytes in vitro and in the myocardium in vivo. ONOO is a strong biological oxidant and nitrating species formed from the near-diffusion-limited reaction of the free radicals nitric oxide and superoxide anion [9]. It has been documented that ONOO formation represents a major mechanism of myocardial injury in various cardiac pathologies including myocardial infarction, chronic heart failure and cardiomyopathy associated with diabetes [10].

ONOO may cause myocardial cytotoxicity through direct oxidative damage to lipids, proteins and DNA [11], activation of metalloproteinases [12], and the nitration of tyrosine residues within proteins [13]. ONOO acts as a potent signaling molecule in cardiomyocytes, activating all members of the MAP kinase family [14], and inhibiting the activation of the transcription factor nuclear factor kappa B [15]. One major pathway of ONOO dependent myocardial cytotoxicity relies on oxidative DNA damage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), which consumes cellular nicotinamide dinucleotide (NAD) and adenosine triphosphate (ATP), leading to cell necrosis [16].

However, Levrand et al. [8] showed that ONOO exerts potent proapoptotic effects in cardiomyocytes in vitro and in the myocardium in vivo, characterized by the activation of caspase-3 and the cleavage of PARP. The authors added that ONOO may represent a major effector of cardiomyocyte apoptosis that may cause myocardial damage and dysfunction in several cardiac pathologies.

On another level, antioxidant administration has been reported to show beneficial effects on parameters of oxidative stress and cardiovascular functions in experimental diabetes [17].

Haidara et al. [18] showed that administration of antioxidants, vitamin E or C, may potentially ameliorate endothelial dysfunction and reduce thrombotic tendency in rats with streptozotocin (STZ)-induced DM associated with hypertension.

The aim of the present study was to assess the possible contribution of apoptosis as a mechanism for oxidative stress-induced injury in the myocardium in STZ-induced diabetic rats. We also evaluated the effects of administration of insulin and/or the antioxidant vitamin E on biochemical parameters of oxidative stress and apoptosis, as well as on the histological manifestations of damage in cardiac muscle.

MATERIALS AND METHODS

The experiments were conducted in the Faculty of Medicine, Cairo University.

Experimental Animals

50 male albino rats, weighing 170-200 g were used. They were kept in the animal house of Kasr Al-Aini Faculty of Medicine, Cairo University. The rats had free access to standard rat chow and water. They were kept at 22 ± 1ºC temperature at 12 h dark-light cycles.

Rats were randomly divided into 5 groups (each, n=10). Control group (Group 1) received an intraperitoneal (i.p.) injection of 0.1 mol/L sodium citrate buffer (pH 4.5). All other groups (Groups II, III, IV and V) received a single i.p. injection of STZ, 65 mgKg-1 body weight [19], freshly dissolved in 0.1 M citrate buffer (pH 4.5). Diabetes mellitus was verified by measuring blood glucose levels (after overnight fast) with the use of glucose oxidase reagent strips (Lif3 scan, Milpitas, CA, USA). Rats having blood glucose level ≥ 300 mg/ dL were considered diabetic [20].

Group II diabetic rats received no treatment during the course of the study, while group III animals (Group III, INS) received 1 unit of insulin, injected subcutaneously (s.c.) every day for 4 weeks. Group IV (E300): diabetic group receiving vitamin E 300 mgKg-1 body weight intramuscularly (i.m.) 3 times per week for 4 weeks [21]. Group V (I±E300): Diabetic rats received insulin (1 U), s.c., daily, and vitamin E im 3 times a week for 4 weeks.

The study period lasted for 4 weeks, a period which has been proved to induce detectable diabetic complications in kidneys, skeletal muscles, heart and retina [22].

At the end of 4 weeks, retro-orbital blood samples were obtained under anesthesia, using 40 mgKg-1 body weight sodium thiopentone i.p. after an overnight fast. Samples were allowed to clot for 20 min and then centrifuged at 14000 rpm for 10 min for serum separation which was kept at -80 ºC until time of assay of cardiac enzymes. Samples from the left ventricles were removed and prepared for detection of malondialdehyde (MDA), glutathione peroxidase(GPX),cyclic guanosine monophosphate (cGMP), gene expression of annexin V, induced nitric oxide synthase (iNOS) and electron microscopic studies of cardiac tissues.

Chemicals

STZ (Trade name Zanosar) was purchased from Sigma chemical company, St. Louis Missouri, USA, in the form of 1 g vials. The drug was dissolved in 0.1 M sodium citrate (pH adjusted to 4.5). Insulin (Act rapid HM) was purchased from Nordisk Company, in the form of ampoules 100 IU /ml. Vitamin E was purchased from Pharco Pharmaceutical Company in the form of ampoules 250 mg dissolved in arachis oil.

Measurements

Biochemical Parameters

1-. Detection of Annexin V and iNOS Gene Expression

About 30 mg of each heart tissue was homogenized in RNA lysis buffer which contains mercaptoethanol then centrifuged at 14000 rpm for 10 min. The supernatant was frozen at -80ºC until examined for gene expression of annexin V and iNOS by RT-PCR.

RNA Extraction:

RNA was extracted from heart homogenate using SV-total RNA isolation system kit (Promega, Madison, USA) according to manufacturer’s recommendation and the extracted RNA was measured spectrophotometrically at 280 nm.

Reverse Transcriptase and Polymerase Chain Reaction (RT-PCR):
cDNA was prepared from RNA as follows:

About 20µg of mRNA was heated at 70 ºC for 5 min with 50 pmol of reverse primer of selected gene (annexin V, iNOS) before adding 5 XRT buffer (50 mM Tris CL, pH8.3. 10 mM dNTPS and 200 units of murine leukemia virus reverse transcriptase in a final volume up 36µL) .RT reaction was carried for 2 h at 37 ºC.

Polymerase Chain Reaction (PCR)

5 µL of cDNA was subjected to PCR under the conditions specified below; PCR reaction was carried by adding 50 pmol of each of forward and reverse primer specific to each gene as detailed later.

10 mM dNTPS, 2-5 unit TACL,PCR 10x buffer (containing 100 mM Tris HCL pH 8.3 , KCL 10 mM to final volume 50 µL):

Primer Sequence Cycling Condition
1-  Annexin V 94 °C ® 1 min
Sense: 5- GTC TCC ACC CAC TTA 60 °C ® 1 min
GTC TAA GTT-3- 72 °C ® 1 min
Anti sense: Extension
5-CCC TGC CAA TGA ACG CTG 72 °C ® 1 min
CCA-3-  
2- iNOS 94 °C ® 30 sec
Sense: 5-GTG AGG ATG AAA ACA 57 °C ® 45 sec
TGG- 3- 72 °C ® 45 sec
Antisense Extension
5-ACC TGC AGG 72 °C ® 8 min
TTG GAC CA- 3-  

Agarose Gel Electrophoresis

The amplified PCR product of selected gene were electrophoresed on 1.5 % Agarose gel and were UV visualized after staining with ethidium bromide. UV illuminated gel were photographed. A densitometry system using a Standard DNA of known concentration gene Gel, documentation system was used for analysis (Syngene, Cambridge, UK), Figs. (1 & 2).

Fig. (1).

An agarose gel electrophoresis showing products of annexin gene expression; Lane M: PCR marker; Lane 1: gene product in control group. Lane 2&3: gene product in diabetic group. Lane 4: gene product in diabetic group receive insulin. Lane 5: gene product in diabetic group receive vitamin E. Lane 6: gene product in diabetic group receive insulin and vit E

Fig. (2).

An agarose gel electrophoresis showing product of iNOS gene expression. Lane M: PCR marker. Lane 1: gene product in control group. Lane 2, 3&4: gene product in diabetic group. Lane 5: gene product in diabetic group receive insulin. Lane 6: gene product in diabetic group receive vitamin E. Lane 7: gene product in diabetic group receive insulin and vit E

Fig. (3).

Electron micrograph of an ultrathin section in the ventricular muscle of control rats showing that the nucleus of a cardiac myocyte (n) appears oval with extended euchromatin. Z lines (Z) are easily demarcated. Rows of mitochondria (m) are abundant between the myofibrils. A blood capillary (C) is seen (Original mag. X 3100)

Fig. (4).

Electron micrograph of an ultrathin section in the ventricular muscle of group II (diabetic rats) showing that mitochondria with dense matrix (m) appear irregularly dispersed. Myofibrils appear widely separated with rarefied cytoplasm of cardiac myocyte (r). Note widening of the intercellular space (W). A blood capillary (C) in the intercellular space shows an endothelial cell (E) in its wall and RBCs in its lumen (R). (Original mag. X 5000).

Fig. (5).

Electron micrograph of an ultrathin section in the ventricular muscle of group II (diabetic rats) showing one rarefied cardiac myocyte (R) with marked myofibrillar lysis. Another myocyte shows rarefied myofibrils (arrows) and mitochondria with dense matrix (m) (Original mag. X 3000).

Fig. (6).

Electron micrograph of an ultrathin section in the ventricular muscle of group II (diabetic rats) showing margination of the nuclear chromatin of a nucleus of a cardiac myocyte (arrows). Myo-fibrils appear completely rarefied (r). Most mitochondria show dense matrix (m) while others show disrupted cristea (arrowhead) (Original mag. X 4000).

Fig. (7).

Electron micrograph of an ultrathin section in the ventricular muscle of group II (diabetic rats) showing that the nucleus of a cardiac myocyte (N) has irregular nuclear outline (arrows) with margination of its chromatin. Cardiac myocytes are rarefied with abundant vacuoles (V) and dispersed mitochondria with dense ma-trix (m) (Original mag. X 3000).

Fig. (8).

). Electron micrograph of an ultrathin section in the ventricular muscle of group III (diabetic rats receiving insulin) showing margination of nuclear chromatin of a cardiac myocyte (arrows). Myofibrils are rarefied (r). Mitochondria (m) are irregularly dis-persed (Original mag. X 3000).

Fig. (9).

Electron micrograph of an ultrathin section in the ventricular muscle of group IV (diabetic rats receiving vitamin E) showing that most of the myofibrils appear to be forming regular striations yet some of them appear interrupted in certain areas (arrows). Most myofibrils show clear Z lines (Z). Mitochondria (m) with dense matrix are detected between the myofibrils. Sarcolemma appears irregular (short arrows). A blood capillary (C) is seen (Original mag. X 3000).

Fig. (10).

Electron micrograph of an ultrathin section in the ventricular muscle of group V (diabetic rats receiving insulin and vitamin E) showing more regular striations of myofibrils with clear Z lines (Z). Rows of mitochondria (m) are longitudinally arranged between the myofibrils. A fibroblast (F) can be observed in the intercellular space between the cardiac myocytes (Original mag. X 5000).

2). Measurement of MDA

MDA was measured in cardiac tissue homogenate after precipitation of protein by the addition of trichloracetic acid (TCA) then thiobarbituric acid (TBA). TBA reacted with MDA to form thiobarbituric acid product which was measured at 532 nm according [23]. The level of peroxidation was expressed as the amount of MDA in nmol/mg protein.

3). Glutathione Peroxidase (GPX)

GPX was assessed in tissue homogenate using a method based on GSH oxidation by cymene hydroxyl catalyzed by GSH-Px activity (using Wak-Cheme, cat. No: Wak-FR-GPX 80. Germany) according to the manufacturer’s instructions. Absorbance was measured at 340 nm [24].

4). Cyclic Guanosine Monophosphate (cGMP) Assay

Frozen samples stored in 0.1 normal HCL were grounded with a stainless steel mortar, then homogenized and centrifuged at 600g, at 4 ºC for 10 min. The supernatant was used for the cGMP assay by ELISA kit (R&D System, Minnepolis, MN, USA) according to manufacturer’s recommendation [25].

5). Serum Cardiac Enzymes

Creatine kinase (CK) activity was determined using a kit provided by Randox [26].

Ultrastructural Studies

Specimens from the ventricles of all animals were processed according to the method of Jans and de-jong [27]. Semi-thin sections “1 µm thickness” were cut by 2 KB ultra-microtome and stained with 1 % toluidine blue for observation. Ultra-thin sections “60-100 nm” thickness were prepared and stained with uranyl acetate and lead citrate to be examined under JOEL EM 1005 transmission electron microscope using an accelerated voltage of 60 KV.

This research was funded and approved by the ethical committee of Kasr Al-Aini Faculty of Medicine, Cairo University.

Statistical Analysis

The results are presented as mean ± SD. Comparison were made using paired and unpaired t test one-way ANOVA as required. When a significant F was obtained, multiple comparison post tests were used to determine which groups were significantly different. P ≤ 0.05 was considered significant.

RESULTS

Table(1) shows the effects of diabetes on biochemical parameters of cardiac injury. There is significant increase of serum CK activity in the untreated diabetic group II (P < 0.001) compared with controls. Treatment with either vitamin E or insulin alone significantly decreased the activity of this enzyme compared with the untreated diabetic group (P < 0.05). Treatment with vitamin E and insulin decreased this enzyme back to control levels.

Table 1.

Creatine Kinase (CK) Activity (U /L Serum) in the Studied Groups 4 Weeks After Induction of Diabetes (n=10 in Each Group)

Groups Control Diabetic INS E 300 I±E300
CK 2.9 ± 0.6 6.0 ± 1.2* 3.4 ± 0.8■ 2.8 ± 0.9■ 1.9 ± 0.8 ■

Results are mean ±SD; INS= Insulin, E=300: vitamin E 300 mg, I+E=300: insulin 1U sc +Vitamin E 300 mg. CK= Creatine kinase.*Significant with control (P < 0.001);

■ Significant with DM (P < 0.01).

Table 2.

The Levels of Oxidative Markers MAD (nmol/mg Protein) and the Antioxidant GPX (µ unit/mg Tissue) in the HeartTissues of Studied Groups and Their Ratio GPX/MDA 4 Weeks After Induction of Diabetes (n=10 in Each Group)

Groups Control Diabetic INS E 300 I±E300
MDA 0.1± 0.0 0.3 ±0.1* 0.2± 0.1* ■ 0.1 ±0.0■ 0.1 ± 0.0■
GPX 2.1 ± 0.5 .1 ±0.0* 1.7 ± 0.4■ 1.5 ± 0.5■ 1.6 ± 0.5■
GPX/MDA 19.6± 6.7 0.3± 0.1* 9.0± 3.1* 15.0 ± 7.1■ 15.6 ± 3.2■

Results are mean ±SD; INS= Insulin, E=300: vitamin E 300 mg, I+E=300: insulin 1U sc +Vitamin E 300 mg. MDA= malondialdehyde, GPX= glutathione peroxidase.*Significant with control (P < 0.001);

■ Significant with DM (P < 0.01).

Table 3.

Annexin V Levels (μg /mg Protein) in the Heart Tissues of Studied Groups 4 Weeks After Induction of Diabetes (n=10 in Each Group)

Groups Control Diabetic INS E 300 I±E300
Annexin 403 ± 97 1043 ± 138* 564 ± 108■* 657± 138■* 424 ± 66 ■

Results are mean ±SD;.*Significant with control (P < 0.001); ■ Significant with diabetic (P < 0.01).

Table 4.

The Levels of iNOS (μg /mg Protein) and cGMP (n mol /mg Protein) in the Heart Tissues of Studied Groups 4 Weeks After Induction of Diabetes (n=10 in Each Group)

Groups Control Diabetic INS E 300 I±E300
iNOS 169 ± 29 451 ± 106* 277 ± 64*■ 356 #x00B1; 78* 231 ± 58■
cGMP 1.3 ± 0.4 3.6± 0.6* 2.6± 0.5*■ 2.3±0.8*■ 1.4 ± 0.4■

Results are mean ±SD.

Ins= Insulin, E=300: vitamin E 300mg, I+E300: insulin 1U sc +Vitamin E 300 mg, iNOS: inducible nitric oxide synthase, cGMP= cyclic guanosine monophosphate.

*Significant with control (P < 0.001);■ Significant with DM (P < 0.01).

Table(2) shows that MDA, an oxidative stress marker, was significantly elevated (P < 0.01) in the diabetic untreated group compared with controls. MDA significantly decreased in insulin treated rats when compared with the untreated group (P< 0.01). Treatment with vitamin E and or insulin decreased MDA significantly below levels in the untreated diabetic group and back to control levels

On the other hand, GPX, which assesses the antioxidant state in tissues, was significantly decreased in untreated diabetic groups compared with controls. Treatment with vitamin E and insulin increased GPX back to control levels.

The antioxidant status in cardiac tissues is better indicated by the ratio of GPX/MDA. This ratio was decreased in the diabetic untreated group and the group only receiving insulin. However, the ratio was increased to the control value in groups treated with either vitamin E alone or vitamin E with insulin

Table (3) shows that annexin V levels were significantly elevated (P < 0.001) in the untreated diabetic (group II) cardiac tissues compared with the controls. However, levels were decreased significantly (P < 0.001) in diabetic rats receiving insulin (group III) when compared with untreated diabetic rats. Diabetic animals treated with both insulin and vitamin E showed significant decrease of annexin V in comparison with the untreated diabetic group (P < 0.01) and diabetic rats receiving only vitamin E (P < 0.01) .

Table (4) shows the effects of diabetes on the expression of the pro-inflammatory parameters and iNOS. There was a significant increase in both iNOS and cGMP in cardiac tissues of the untreated diabetic group (P < 0.001). Neither insulin alone, nor vitamin E decreased them to control values; however; in the group treated with both insulin and vitamin E, values returned to control levels

Ultrastructural Electron Microscopic Examination

Electron microscopic examination of ultra-thin sections of the left ventricular myocardium of the control group (group I) revealed normal histological structure of cardiac myocytes Fig. (3). Ventricular sections obtained from untreated rats (group II) showed myofibrillar lysis in the form marked degeneration, disruption and rarefaction of the myofibrils (Figs. 4, 5). Nuclei of cardiac myocytes exhibited several degenerative changes where some nuclei showed peripherally condensed marginated chromatin (Fig. 6). In addition several cardiac myocytes exhibited marked cytoplasmic vacuolations (Fig. 7).

The ultrastructure of ventricular sections obtained from group III (diabetic rats which received insulin) revealed rarefaction of myofibrils with wide dispersion of mitochondria. Nuclei exhibited condensation of nuclear chromatin (Fig. 8). The diabetic group which received vitamin E 300 mg showed that most of the myofibrils formed regular striations with clear Z-lines yet striations appeared interrupted in some sites. Mitochondria with dense matrix substance were present between the myofibrils. Sarcolemma appeared markedly irregular (Fig. 9).

The ultrastructure of ventricular ultrathin sections of the diabetic group which received both insulin and vitamin E 300 mg showed more regular striations of myofibrils with clear Z-lines. Sarcomeres could be easily detected. Rows of mitochondria appeared longitudinally arranged between the myofibrils (Fig. 10).

DISCUSSION

Patients with diabetes type 1 or 2, have a 2-8 fold increased risk of developing cardiovascular diseases such as myocardial infarction, congestive heart failure, cerebrovascular and peripheral arterial diseases [28]. There is growing evidence that oxidative stress associated with diabetes mellitus may promote endothelial dysfunction, hypertension [29], thromboembolism and cardiomyopathy [30]. In both clinical and experimental models of diabetes, reactive oxygen species (ROS)-induced oxidation is considered to be a key factor in causing cardiac injury [31].

High glucose has been postulated to generate ROS and nitrogen species in numerous cell types. Generation of superoxide by high glucose is well described and arises principally via the mitochondrial electron transport chain. [32]. Another source of glucose-induced oxidative stress is via the polyol pathway where glucose is reduced to sorbitol by aldose reductase in a process that consumes NADPH. This will impair the NADPH-dependent generation of glutathione, an essential cellular antioxidant [33]

Increased ROS generation increases the activity of nuclear factor kappa-B (NF-kB) in various cell types including endothelial [34], mesangial [35], and vascular smooth muscle cells [36]. This process is dependent on protein kinase C (PKC) activation [37,38].

Our study showed that diabetic cardiomyopathy may occur in untreated diabetic rats due to long standing hyperglycemia which was demonstrated by structural and functional changes such as increased CK activity and myocardial damage assessed by electron microscopic studies. Assessment of oxidative stress revealed increased MDA content, decreased GPX and GPX/MDA ratio.

Yoon et al. [39] postulated that H2O2 induces an increase in apoptosis signal regulating kinase-1 which cause down regulation of antiapoptotic Bcl-2, disruption of the mitochondrial membrane potential and activation of caspase cascade [40]. High glucose also causes a 2 fold increase in Bax expression, which induced cytochrome C release which in turn stimulates apoptosis activating factor, caspase 9 and caspase 3 [41].

There is evidence that the incidence of apoptosis increases in heart of patients with diabetes [42] and STZ-induced diabetic rats [43]. In this study, increased apoptosis in diabetic cardiac muscles was evident by the increased levels of annexin V in heart tissues of untreated diabetic rats. Annexin V can be used as an apoptotic marker in the heart [44]. Annexin is mainly located in cardiomyocytes. However, it could be relocated to interstitial tissues in ischemic and failing hearts or it could be externalized and exhibit a pro-apoptotic effect in cardiomyocytes [45]. There is a significant increase of plasma annexin V concentration in patients with acute myocardial infarction which could reflect the severity of myocardial damage [46].

It was found that early apoptosis can be assessed and imaged with annexin V scintigraphy in rats [47,48], based on its ability to identify extracellular phosphatidyl-serine, which arises during apoptosis [49].

Annexin V was originally discovered as an antithrombotic activity in vivo and it links apoptosis to thrombosis and haemostasis [50]. It is now accepted that cell surface exposure of phosphatidylserine (PS) is an integral part of the apoptotic process. Once committed to die the cell quickly exposes PS at its surface while maintaining the integrity of the plasma [51,52]. PS on the apoptotic cell is thought to serve primarily for the clearance of the dying cell [53]. Annexin V has a high affinity for these surfaces in the complex environment of the tissue and likely form an antithrombotic shield, which reduces the prothrombotic risk associated with apoptosis [54]

Our results also showed that iNOS protein of the heart is elevated in untreated diabetic rats suggesting that inflammation could play a role in the pathogenesis of diabetic cardiomyopathy in type 1 diabetes. Cheng et al. [55] and others [56] also found that the activity of iNOS was 3 fold higher in the heart of diabetic rats relative to controls. In addition, they found, that selective inhibition of iNOS restored cardiovascular response to noradrenalin. Apoptosis has been postulated to be involved in the cardiac damage associated with diabetes, sepsis, and dilated cardiomyopathy [57] which are all associated with an enhanced generation of ONOO within the myocardium [11]. On the basis of these findings, Levrand et al. [8] propose that ONOO may represent a major oxidant species involved in the process of cardiomyocyte apoptosis in these cardiac diseases. These data imply that ONOO-dependent oxidant stress is instrumental in activating proapoptotic signals (caspase-3 and PARP cleavage). The authors added that PARP cleavage as a consequence of ONOO generation was secondary to the activation of caspase-3, but additional mechanisms may be implicated as well. PARP can also be cleaved in the nucleus of cardiomyocytes through the action of matrix metalloproteinase 2 (MMP-2) [58], known to be activated by ONOO [12]. Furthermore, Bojunga et al. [59] showed that antioxidative treatment was capable of reversing changes in NO-cGMP system and may therefore be an important option for preventing vascular damage in diabetes mellitus. Haidara et al., also [30] found that vitamin E was able to modulate the blood pressure and lipid profile in STZ-induced diabetic rats

Vitamin E is an important non-enzymatic natural lipid-soluble chain breaking antioxidant in tissue, red cells and plasma [60,61]. It protects against lipid peroxidation by acting directly with a variety of oxygen radicals to form a relatively innocuous tocopherol radical [62].

Vitamin E prevents H2O2 – induced apoptosis in remote non-infarcted myocardial cells with prevention of mitochondrial cytochrome C release and activation of caspase 3 [63]. These findings indicated that antioxidant vitamins reduce myocyte apoptosis mediated via inhibition of mitochondrial pathway [21]

Vitamin E also seems to inhibit the release of inflammatory mediators from activated monocytes [64] as well as reducing smooth muscle proliferation and platelet aggregation [65].

The present study demonstrates that administration of insulin and vitamin E to diabetic rats reduces oxidative stress and apoptosis with preservation of cardiac function as assessed by the cardiac enzymes and electron microscopy.

Yoshida et al. [66] suggested a similar effect in the eye lens as they found that combined treatment of vitamin E and insulin was useful in preventing the development and progression of diabetic cataract. Economides et al. [67] did not recommend the use of high dosage of vitamin E in diabetic patients because of their worsening effect on the endothelial or left ventricular function. The dose of vitamin used in this study was based on previous studies [68,69]. The dose is 3-10 times the current recommended dietary allowance but is within human therapeutic range based on body weight [70]. Our findings suggest that the dose of vitamin E (300 mgKg-1 body weight) chosen is both clinically relevant but also pharmacologically sufficient to produce antioxidant effects in the experimental setting [21]

CONCLUDING REMARKS

Our results demonstrated that STZ-induced diabetes in rats leads to functional and structural changes in the heart which include oxidative stress and apoptosis. The changes were significantly ameliorated by administration of insulin and vitamin E which abrogate oxidative stress and produce a cardioprotective effect. The combination of both forms of treatment decreased CK activity and myocardial damage, thus suggesting a strategy which could reduce cardiovascular complications in diabetes mellitus.

REFERENCES

1
Haidara MA, Mikhailidis DP, Rateb MA, et al. Evaluation of the effect of oxidative stress and vitamin E supplementation on renal function in rats with streptozotocin-induced Type 1 diabetes J Diabetes Complications 2008.
2
Kakafika AI, Liberopoulos EN, Karagiannis A, Athyros VG, Mikhailidis DP. Dyslipidaemia, hypercoagulability and the metabolic syndrome Curr Vasc Pharmacol 2006; 4: 175-83.
3
Haidara MA, Yassin HZ, Ammar H, Rateb MA, Zorkani MA. Role of oxidative stress in cardiovascular complication in diabetes mellitus Curr Vasc Pharmacol 2006; 4: 215-27.
4
Bassege E, Sommer O, Schwemmar M, Bunger R. Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species Am J Physiol 2000; 279: H 2431.
5
Horie K, Miyata T, Maeda K, et al. Immunohistochemical co-localization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions.cImplication for glycoxidative stress in the pathogenesis of diabetic nephropathy J Clin Invest 1997; 100: 2995-3004.
6
Allem DA, Harwood S, Varagunam M, Raftery MJ, Yaqoob MM. High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases FASEB J 2003; 17: 908-10.
7
Buttke TM, Sandstorm PA. Oxidative stress as a mediator of apoptosis Immunol Today 1994; 15: 7-10.
8
Levrand S, Vannary-Bouchiche C, Pesse B, Pacher P, Fehl F, Waeber B. Peroxynitrite is a major trigger of cardiomyocyte apoptosis in vivo and in vitro Free Radic Biol Med 2006; 41(6): 886-95.
9
Beckman JS, Koppenol WH. Nitric oxide superoxide and peroxynitrite The good the bad and ugly Am J Physiol 1996; 271: C1424-37.
10
Ferdinandy P, Schulz R. Nitricoxide superoxide and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning Br J Pharmacol 2003; 138: 532-43.
11
Pacher P, Schulz R, Liaudet L, Szabo C. Nitrosative stress and pharmacological modulation of heart failure Trends Pharmacol Sci 2005; 26: 302-10.
12
Wang W, Sawicki G, Schulz R. Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2 Cardiovasc Res 2002; 53: 165-74.
13
Borbely A, Toth A, Edes I, et al. Peroxynitrite-induced alpha-actinin nitration and contractile alterations in isolated human myocardial cells Cardiovasc Res 2005; 67: 225-33.
14
Pesse B, Levrand S, Feihl F, et al. Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21(Ras) in H9C2 cardiomyocytes J Mol Cell Cardiol 2005; 38: 765-75.
15
Levrand S, Pesse B, Feihl F, et al. Peroxynitrite is a potent inhibitor of NF-{kappa}B activation triggered by inflammatory stimuli in cardiac and endothelial cell lines J Biol Chem 2005; 280: 34878-87.
16
Jagtap P, Szabo C. Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors Nat Rev Drug Discov 2005; 4: 421-40.
17
Bursell SE, Clermont AC, Aiello LP, et al. High-dose vitamin E supplementation normalizes retinal blood flow and creatinine clearance in patients with type 1 diabetes Diabetes Care 1999; 22: 1245-12451.
18
Haidara MA, Khlousy H, Ammar H, Kassem L. Impact of a-tocopherol and vitamin C on endothelial markers in rats with STZ-induced diabetes Med Sci Mon 2003; 9: 214-7.
19
Haidara MA, Ibrahim MI, Sit El, Banat A, El-Tuwjery A. Effect of a-tocopherol on glucose uptake and contractility in rat skeletal muscles Med Sci Mon 2003; 9(5): BR 214-217.
20
Halliwell B, Vitamin E. and treatment and prevention of diabetes, a case for controlled clinicl trial Singapore Med J 2002; 43(9): 479-48.
21
Fuzhong Q, Chen Y, Ravish P, Weimin L, Erdan D, Vitamin CE. attenuates apoptosis , B-adrenergic receptor desensitization and sarcoplasmic reticular Ca2 ATPase down regulation after myocardial infarction Free Radic Biol Med 2006; 40: 1827-42.
22
Knoll K, Pietruz J, Liang M. Tissue-specific transcription responses in rats with early streptozotocin-induces diabetes Physiol Genomics 2005; 21: 222-9.
23
Yoshioka T, Kawata k, Shimata T. Determination of malondialdhyde Am J Obstet Gynecol 1979; 135-45.
24
Pagalia DE, Valantine WN. Studies on quantitative and qualitative characterization of erythrocyte GPX J Lab Clin Med 1967; 70: 158.
25
Chinkers M, Garbers DL, Chang MS, et al. Membrane form of guanylate cyclise are an atrial natriuretic peptide receptor Nature 1989; 338: 78-83.
26
Stein W, Bohner J. Need the small laboratory still uses the plasma creatin kinase aspartate aminotransferase ratio Clin Chem 1985; 31: 1910.
27
Jans SW, de-jong F. Differential expression and localization of annexin V in cardiac myocytes during growth and hypertrophy Mol Cell Biochem 1998; 178: 229-36.
28
Grundy S, Benjamin I, Buke G, Chait A, Eckel R, Howard B. Diabetes and cardiovascular disease A statement for health care professionals from the American Heart Association Circulation 1999; 100: 1134-46.
29
Haidara MA, Desoky A, Khlousy H, Sebaee H. The mechanism underlying the development of hypertension in STZ-induced diabetic rats Prog Med Res 2004; 2: 30.
30
Cai L, Kang YJ. Cell death and diabetic cardiomyopathy Cardiovasc Toxicol 2003; 3: 219-28.
31
Brownlee M. Biochemistry and molecular cell biology of diabetic complications Nature 2001; 214: 813-20.
32
Chung SS, Ho EC, Lam KS, Chung SK. Contribution of poloyl pathway to diabetes-induced oxidative stress J Am Soc Nephrol 2003; 14: S233-6.
33
Du X, Sttocklauser FK, Rosen P. Generation of reactive oxygen intermediates, activation of NF-Kappa B and induction of apoptosis in human endothelial cells by glucose role of nitric oxide synthase Free Radic Biol Med 1999; 27: 752-63.
34
Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB. Role of high glucose -induced nuclear factor -kappa B activation in monocyte chemoattractant protein-1 expression by mesangial cells J Am Soc Nephrol 2002; 13: 894-902.
35
Hattori Y, Hattori S, Sato N, Kasai K. High glucose-induced nuclear factor kappa B activation in vascular smooth muscle cells Cardiovasc Res 2000; 46: 188-97.
36
Ramana KV, Friedrich B, Bhatnagar A, Srivastava SK. Aldose reductase mediates cytotoxic signals of hyperglycemia and TNF-alpha in human lens epithelial cells FASEB J 2003; 17: 315-7.
37
Kowlur RA, Koppolu P, Charkabarti S, Chen S. Diabetes-induced activation of nuclear transcriptional factor in the retina and its inhibition by antioxidants Free Radic Res 2003; 37: 1169-80.
38
Ca L, Wang I, Zhou Y, et al. Attenuation by metallothionein of early cardiac death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy J Am Coll Cardiol 2006; 48: 1688-97.
39
Yoon SO, Kim MM, Park SJ, Chung AS. Selenite suppresses hydrogen peroxide-induced apoptosis through inhibition of ASK1/JNK and activation of P13-K/Akt pathway FASEB J 2002; 16: 111-3.
40
Fujita R, Ueda H. Protein Kinase C-mediated cell death mode switch induced by high glucose Cell Death Differ 2003; 10: 1336-47.
41
Hare JM. Oxidative stress and apoptosis in heart failure progression Circ Res 2001; 98: 198-200.
42
Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A. Myocardial cell death in human diabetes Circ Res 2000; 87: 1123-30.
43
Lu C, Yuehui W, Guihua A, Teresa C, Ye S, Xiaokun L. Attenuation by metallothionine of early cardiac cell death via suppression of mitochondrial oxidative stress results in prevention of diabetic cardiomyopathy J Am Coll Cardiol 2006; 48: 1688-97.
44
Gerke VX, Moss S. Annexin from structure to function Physiol Rev 2002; 82: 331-71.
45
Camors E, Monceau V, Charlemagne D. Annexin and Ca2± handling in the heart Cardiovasc Res 2005; 65: 793-802.
46
Matusda R, Kaneko N, Kikuchi M, et al. Clinical significance of measurement of plasma annexin V in patients in the emergency room Resuscitation 2003; 57: 171-7.
47
Bennink RJ, Avan-denhoff H, Van-Henert FJ, de-Bruin KM, Annexin V. imaging of acute doxorubicin cardiotoxicity (apoptosis) in rats J Nucl Med 2004; 45: 842-8.
48
Murkami Y, Takamatsu H, Taki J. 18 F -labelled annexin V: PET tracers for apoptosis imaging Eur J Nucl Med Mol 2004; 31: 469-74.
49
Thimister P, Hofstra L, Lime IH, Bergsma HH. In vivo detection of cell death in the area at risk in acute myocardial infarction J Nucl Med 2003; 44: 391-6.
50
Cardio-Villa M, Arap W, Pasqualini A. Alpha V beta 5 inergrin-dependent programmed cell death triggered by a peptide mimic of annexin V Mol Cell 2003; 11: 1151-62.
51
Verhoven B, Schlegel RA, Williamson P. Mechanism of phosphatidyserine exposure, a phagocyte recognition signal on apoptotic T lymphocyte J Exp Med 1995; 182: 1597-601.
52
Freyssinet JM, Toti F, Hugel B, Gidon JC, Kunzelmann C, Meyer D. Apoptosis in vascular disease Thromb Haemost 1999; 82: 727-35.
53
Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM. The role phosphatidylserine recognition of apoptotic cells by phagocytes Cell Death Differ 1998; 5: 551-62.
54
Rand JH. Antiphospholipid antibody-mediated disruption of the annexin V antithrombotic shield a throbogenic mechanism for the antiphospholipid syndrome J Autoimmun 2000; 15: 107-11.
55
Cheng X, Cheng XS, Kuo KH, Pang CC. Inhibition of iNOS augments cardiovascular action of noradrenalin in STZ-induced diabetes Cardiovasc Res 2004; 64: 298-307.
56
Baydas G, Cantan H, Turkoglu A. Comparitive analysis of the protective effects of melatonin and vitamin E on STZ-induced diabetes J Pineal Res 2002; 32: 225-30.
57
Kumar D, Jugdutt BI. Apoptosis and oxidants in the heart J Lab Clin Med 2003; 142: 288-97.
58
Kwan JA, Schulze CJ, Wang W, et al. Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro FASEB J 2004; 18: 690-692 .
59
Bojuna J, Dresar MB, Usadel KH, Kusterer K, Zeuzem S. Antioxidant treatment reverses imbalances of nitric oxide isoform expression and attenuates tissue-cGMP activation in diabetic rats Biochem Biophys Res Commun 2004; 316(3): 771-80.
60
Sen CK, Packer LT. Homeostasis and supplements in physical exercise Am J Clin Nutr 2000; 72: 653-95.
61
Ghatak A, Bar MJ, Agarwal A, Goel N. Oxy free radical system in heart failure and therapeutic role of oral vitamins Int Cardiol 1996; 57: 119-27.
62
Rimm E, Stampfer M, Asceris A, Willet W, Vitamin E. consumption and risk of coronary disease in men N Engl J Med 1993; 328: 1450-60.
63
Han H, Long H, Wang H, Wang Y, Zhang Y, Wang Z. Progressive apoptotic cell death triggered by transient oxidative insult result H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanism Am J Physiol Heart Circ Physiol 2004; 286: H2169-2182.
64
Devaraj S, Jialal I. The effect of a-tocopherol supplementation on monocyte function J Clin Invest 1996; 98: 756-63.
65
Flohe R, Kelly F. European perspective of vitamin E Am J Clin Nutr 2002; 76: 703-16.
66
Yoshida M, Kimora H, Kyuki K, Blto M. Combined effect of vitamin E and insulin on cataracts of diabetic rats fed a high cholesterol diet Biol Pharm Bull 2004; 27: 338-44.
67
Economides PA, Khaodhir L, Caslli A, Caballero AE, Keenan H, Burssel Se. The effect of vitamin E on endothelial function of micro-and macrocirculation and left ventricular function in type 1 and type 2 diabetic patients Diabetes 2005; 54: 204-11.
68
Shite J, Qin F, Mao W, Kawai H, Stevens SY, Liang C. Antioxidant vitamins attenuate oxidative stress and cardiac dysfunction in tachycardia-induced cardiomyopathy J Am Coll Cardiol 2001; 38: 1734-40.
69
Qin F, Shite J, Liang C. Antioxidants attenuate myocyte apoptosis and improve cardiac function in CHF association with changes in MAPK pathways Am J Physiol Heart Circ Physiol 2003; 285: H 822- H832.
70
Butterworth CE. Vitamin safety a current appraisal 1994 update Vitam Nutr inform Serv 1995; 5: 1-10.