Carvedilol improved diabetic rat cardiac function depending on antioxidant ability

1. Introduction 

Diabetes is the largest morbidity of patients with heart failure and being a major risk factor for cardiovascular disease [1]. Oxidative stress has been associated with the pathogenesis of chronic diabetic complications including cardiomyopathy [2]. The ability of antioxidants to inhibit these injuries has raised the possibility of newer therapeutic treatment for diabetic heart diseases. Recently, Bcl-2 gene has been focused because it was involved in preventing oxidant-induced cell death and in decreasing oxygen radical production [3].

Carvedilol is a nonselective beta-adrenoceptor and selective alpha1-adrenoceptor blocker. So far, it has been widely used in the treatment of heart failure, hypertension with or without diabetes [3]. β-blockers have been always reluctant to be used on the treatment of diabetes, because they often brought negative effects on the glucose and lipid metabolisms. But it has been reported carvedilol had no negative effects including insulin sensitivity besides glucose and lipid metabolisms [4], [5]. In thus, we selected carvedilol to be observed in the diabetic rats. It was demonstrated that carvedilol has antioxidant and free radical scavenger properties. In physicochemical, biochemical, and cellular assays carvedilol inhibited the formation of reactive oxygen radicals and lipid peroxidation, scavenged oxygen free radicals, and prevented the depletion of endogenous anti-oxidants [6]. It has been suggested that carvedilol may provide greater benefit than traditional β-blockers in chronic heart failure because of its antioxidant actions that synergize with its nonspecific β- and α-blocking effects.

However, whether carvedilol can improve cardiac function in diabetic animal model and this protection via antioxidant pathway is not clear. Therefore, in the present study we investigated the effect of carvedilol on cardiac function, activities of oxidant and antioxidant enzymes and Bcl-2 mRNA expression in the diabetic rat hearts.

3. Results 

3.1. Weight and blood glucose level 

There was no significant deference in six groups at the initial experiment (Fig. 1A). After 5 weeks, animals injected with STZ had lost weight compared with normal controls (167±12g versus 363±56g, p<0.0001), and carvedilol treatment alleviated the weight loss significantly in diabetes (247±66g, 255±60g versus 167±12g). There was no significant change in healthy rats treated with carvedilol (Fig. 1B).

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Fig. 1. Changes in body weight in normal controls (C), normal treated with small dose of carvedilol (CS), normal treated with large dose of carvedilol (CL), untreated diabetic (D), diabetic treated with a small dose of carvedilol (DS), diabetic treated with a large dose of carvedilol (DL). A: the bars indicate the body weight at the initial of experiment. B: the bars indicate the body weight at the 5th weekend of experiment after treated with or without carvedilol administration [11].

Three days after injection of STZ, the average blood glucose levels were over than 20mmol/l in the diabetic group and remained during the experimental period of 5 weeks. In the diabetic rats, blood glucose levels had no obvious variation after 5 weeks of treatment with small or large doses of carvedilol (Fig. 2). Level of blood glucose had no significant change in healthy rats treated with carvedilol (Fig. 2).

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Fig. 2. Changes in blood glucose level in normal controls (C), normal treated with small dose of carvedilol (CS), normal treated with large dose of carvedilol (CL), untreated diabetic (D), diabetic treated with a small dose of carvedilol (DS), diabetic treated with a large dose of carvedilol (DL). The upper bars in every group indicated the glucose level at the 5th weekend of experiment, whereas the nether bars displayed the glucose level at the initial of experiment. Each bar represents the mean±S.D.

3.2. Hemodynamics 

We performed hemodynamic parameters at the 5th weekend for two reasons. One was that this term was the early diabetic impairment, and our original intention was just to observe the effects on the diabetic rats from carvedilol as early as they were. The other reason was that chronic treatment duration with carvedilol administration should not be less than 4 weeks according data [9], [10]. Heart rate (HR) in untreated diabetic rats significantly decreased (362±49 versus 426±45, p=0.0024). Carvedilol treatment reduced HR slightly without significance in diabetic rats (326±38 versus 362±49, p=0.0604, Fig. 3A). There were reduced HR in control treated large dosage carvedilol, this reducing seemed dose-related (348±46 versus 426±45, p=0.0018, Fig. 3A). Left ventricular systolic pressure (LVSP) had no significant increase in diabetic rats (106±15 versus 98±14, p=0.1202), but was decreased by small carvedilol and large carvedilol treatment (106±15 versus 94±11, p=0.0151; 106±15 versus 96±13, p=0.0448, Fig. 3B). Left ventricular developed pressure (LVDP), and ±dP/dtmax were significantly lower in untreated diabetes (LVDP: 82±14 versus 49±9, p<0.0001; +dP/dtmax: 4890±690 versus 3236±590, p<0.0001; −dP/dtmax: 4698±646 versus 3014±685, p<0.0001, Fig. 3D–F), and left ventricular end diastolic pressure (LVEDP) was substantially elevated compared with normal control rats (5±1 versus 12±2, p<0.0001, Fig. 3C). ±dP/dtmax and LVDP in diabetic rats treated with large dosage of carvedilol were significantly elevated (+dP/dtmax: 3236±590 versus 3835±730, p=0.0455; −dP/dtmax: 3711±730 versus 3014±685, p=0.0117; LVDP: 62±12 versus 49±9, p=0.0088, Fig. 3), whereas LVEDP were declined (9±2 versus 12±2, p=0.0037, Fig. 3). Nevertheless, there were significant differences compared with control untreated carvedilol.

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Fig. 3. Homodynamic parameters of normal controls (C), normal treated with small dose of carvedilol (CS), normal treated with large dose of carvedilol (CL), untreated diabetic (D), diabetic treated with a small dose of carvedilol (DS), diabetic treated with a large dose of carvedilol (DL). Each bar represents the mean±S.D. A: heart rate; B: LVSP; C: LVEDP; D: LVDP; E: +dP/dtmax; F: −dP/dtmax.

3.3. Antioxidant system evaluation in cardiac muscle 

Treatment with carvedilol increased activities of CAT, SOD and GSH-Px enzymes significantly as well as reduced the level of MDA in healthy and diabetic groups. The level of MDA in the heart was increased markedly in untreated diabetic rats compared with the rats in the control group (P<0.0001, Table 1), whereas SOD and GSH-Px activities were lower in diabetic untreated rats significantly (Table 1). Treatment with carvedilol returned the MDA level and the activities of enzymes to normal. CAT activities were increased in diabetic untreated rats significantly (Table 1). Moreover, large dosage of carvedilol administration enhanced this rise.

Table 1. Biochemical parameters in normal controls (C), normal treated with small dose of carvedilol (CS), normal treated with large dose of carvedilol (CL), untreated diabetic (D), diabetic treated with a small dose of carvedilol (DS), diabetic treated with a large dose of carvedilol (DL) at the 5th weekend of experiment

		MDA (nmol/g protein)	CAT (U/g protein)	GSH-Px (U/g protein)	SOD (U/g protein)
	I-C (n=7)	7.624±0.81	0.1724±0.04	0.4951±0.07	0.3905±0.04
	II-CS (n=8)	5.426±0.73	0.1903±0.07	0.5507±0.06	0.4561±0.05
	III-CL (n=7)	4.857±0.72	0.2508±0.09	0.5933±0.07	0.5528±0.04
	IV-D (n=7)	14.771±0.86	0.2621±0.08	0.4054±0.07	0.2199±0.06
	V-DS (n=7)	6.290±0.89	0.2498±0.03	0.4444±0.05	0.3772±0.05
	VI-DL (n=7)	5.924±0.88	0.3454±0.06	0.4924±0.07	0.4290±0.05
	p-Value
	I–II	0.0071	0.7816	0.0591	0.0219
	I–III	0.0031	0.0453	0.0329	0.0065
	II–III	0.0047	0.0585	0.1590	0.0038
	I–IV	<0.0001	0.0187	0.0422	<0.0001
	IV–V	<0.0001	0.7797	0.1179	0.0023
	IV–VI	<0.0001	0.0301	0.0101	<0.0001
	V–VI	0.0745	0.0258	0.0449	0.0474

3.4. RT-PCR assay 

Expression of Bcl-2 mRNA was compared between untreated and carvedilol-treated cardiomyocytes. Administration of carvedilol significantly increased the expression of Bcl-2 mRNA in healthy groups and diabetic groups. The levels of mRNA encoding for GAPDH were not found to be significantly different between each group. The expression of Bcl-2 mRNA was very slow in untreated diabetic cardiomyocytes compared with healthy groups. The expression was significantly upregulated in carvedilol-treated diabetic groups compared with untreated diabetic groups. And these increases were dose-related change (Fig. 4).

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Fig. 4. Semiquantitative RT-PCR analysis of Bcl-2 mRNAs in the heart tissue of normal controls (C), normal treated with small dose of carvedilol (CS), normal treated with large dose of carvedilol (CL), untreated diabetic (D), diabetic treated with a small dose of carvedilol (DS), diabetic treated with a large dose of carvedilol (DL). Each bar represents the mean±S.D.; n=5 for each group. *P<0.05 vs. C, **P<0.01 vs. C, ##P<0.01 vs. D, ++P<0.01 DS vs. DL.

4. Discussions 

Our results showed that carvedilol treatment did not affect blood glucose level significantly (Fig. 1, Fig. 2). Carvedilol was shown to have antioxidant actions in healthy volunteers treated with small and moderate doses (6.25–25mg/d) [12]. We selected corresponsive small and large doses in healthy and diabetic rats (1 and 10mg/kg/d).

Oxidative stress often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2, which is an integral mitochondrial membrane protein, blocks apoptosis induced by a wide array of death signals [13]. Evidently, it is via antioxidant pathways that Bcl-2 prevents apoptosis. Furthermore, Bcl-2-overexpressing cells exhibit elevated expression of antioxidant enzymes and higher levels of cellular GSH compared with the control cells transfected with the vector alone [14]. Carvedilol have been investigated to inhibit cardiomyocyte apoptosis following ischemia and reperfusion, which was related to the increased Bcl-2 mRNA expression [15]. Spallarossa et al. have observed that carvedilol pre-treatment blunted both the decrease of Bcl-2 and the increase of Bax-alpha mRNA expression of cardiomyocytes induced by doxorubicin, lead to reducing free radical release and apoptosis [16]. Our results displayed that the expression of Bcl-2 was significantly upregulated in carvedilol-treated diabetic hearts compared with untreated diabetic groups, as well as in control treated groups. This upregulation was more obviously in large carvedilol-treated groups. In addition, this upregulation was concomitant with antioxidant enzymes positive changes both control and diabetes treated with carvedilol. Increased oxidative stress and altered antioxidant pool have been implicated in clinical and experimental type 1diabetes [17]. We found that the level of MDA and the activities of CAT, but not GSH-Px and SOD, in the cardiac tissue of diabetic rats significantly increased compared with the control rats. MDA, a routine index of lipid peroxidation, increased in diabetes mellitus, which implies that hyperglycemia induces peroxidative reactions in lipids. The increase of CAT activity in diabetic heart tissue suggests increased oxidative stress due to chronic exposure to H2O2, which may be an important mediator for any possible tissue damage in STZ-induced diabetes [18]. Both SOD and GSH-Px were decreased in diabetic untreated rats in the present study. The level of MDA was reduced markedly, but the activity of GSH-Px, CAT and SOD in the cardiac tissue was elevated by carvedilol treatment both control and diabetic rats. We found that CAT activity increased in carvedilol groups. The increased CAT activity may be an adaptive response to the increased oxidative stress in STZ-induced diabetes heart. SOD catalyzes the conversion of superoxide radicals to H2O2, which protects the cell against the toxic effects of superoxide radicals. The activity of GSH-Px is also responsible for metabolizing lipid peroxides. The increased activities of CAT, SOD and GSH-Px in the diabetic-treatment hearts maybe reason for higher expression of Bcl-2. In addition, the rise of activities is more obvious in large carvedilol group.

Our results also showed that administration of carvedilol in diabetic rats improved heart systolic and diastolic ability partly based on the improvement of anti-oxidative enzymes. LVDP and +dP/dtmax were elevated, closed to the normal, whereas reduced LVEDP and −dP/dtmax indicated diastolic function improved in diabetic rats with carvedilol administration. Reactive oxygen species (ROS) was able to impair contractile function by disrupting excitation–contraction coupling processes in vitro, notably trans-sarcolemmal calcium fluxes and intracellular calcium cycling [19], which also depressed mitochondrial respiration and reduced ATP generation, thereby causing a rise in resting tension and contractile dysfunction in isolated muscle preparations [20]. These contractile changes can be ameliorated by antioxidant treatment [20], [21]. Dalla Libera et al. have observed that carvedilol at the dose of 2mg/kg per day was able to prevent the myofibrillar protein oxidation and improved force production on isolated muscles, while bisoprolol (0.1mg/kg) did it only partially [22]. Data also have displayed carvedilol inhibited mitochondrial permeability transition in myocytes ischemic cardiac mitochondria, and these were related to antioxidant activity independent of its beta-adrenoceptor blocking effects [23], [24]. Schwarz et al. have reported that carvedilol (1mg/kg) did not reduce infarct size after 60min of coronary occlusion in rabbits similar to its hydroxylated analogue BM-91.0228, but reduce apoptosis after 60min as the same degree as its analogue [25]. Therefore, we conferred it is through reducing oxidative stress instead of its beta-adrenoceptor blocking effects that carvedilol improved the early diabetic heart function.

In healthy treated groups, carvedilol did not exhibit positive effect on the cardiac function although it upregulated expression of Bcl-2 mRNA and increased activities of antioxidant enzymes. It is difficult logically to display curative effect on the good condition.

5. Conclusion 

In summary, oxidative stress was involved in the early diabetic cardiac dysfunction that not only led to reduced activities of antioxidant enzymes but also subdued cardiac dysfunction. Carvedilol treatment upregulated expression of Bcl-2 mRNA, recovered activities of antioxidant enzymes and improved cardiac function resultantly. Our study indicated that carvedilol was benefit to hearts of early diabetic rats due to its antioxidant property partly at least.