Bexarotene enhances heart structure and function in diabetic rats by inhibiting ventricular remodeling and cardiomyocyte apoptosis

Purpose: To investigate the influence of bexarotene (Bex) on cardiac structure and function in streptozotocin (STZ)-induced diabetes mellitus (DM) rats, and the mechanism of action involved. Methods: Four groups of Sprague Dawley rats (n = 40) were used: normal control, DM, DM+ Bex (10 mg/kg/day), and DM+ Bex (20 mg/kg/day) (n = 10). The DM rat model was established by intraperitoneal injection of STZ. Cardiac structure and function of rats were determined and compared. Whole heart and left ventricle were weighed. The protein expressions of Bcl2 and Bax in rat myocardial tissue were determined using Western blotting. Results: Compared to control group, there was significant reduction in the levels of IVSd (inlet ventricular septal defect) and LVPWd (left ventricle posterior wall in diastole) in DM group, but significant increase in these parameters in DM +Bex (20 mg/kg/day) group, relative to DM-treated rats (p < 0.05). Moreover, there were higher expression levels of Bcl2 and Bax in DM group, when compared with normal control, but Bcl2/Bax ratio was significantly lower (p < 0.05). Furthermore, Bcl2 and Bax levels in DM + Bex (20 mg/kg/day) group were significantly lower than those in DM group, while Bcl2/Bax ratio increased significantly (p < 0.05). Conclusion: Bexarotene improves the cardiac structure of DM rats by lowering blood glucose, and by inhibiting ventricular remodeling and cardiomyocyte apoptosis. These findings may be beneficial in the development of new anti-DM drugs.


INTRODUCTION
Diabetes mellitus (DM) is associated with high blood glucose levels as a result of impairment of insulin production or insulin effect. It causes ocular damage as well as dysfunctions in the kidney, heart, blood vessels and nervous system. In severe cases, DM results in electrolyte complications and disorders in acid-base balance [1]. The incidence of DM is increasing year by year, and cardiovascular complications are the main causes of increased mortality and decreased quality of life in DM patients. In the development of DM cardiomyopathy, the body undergoes apoptosis through complex molecular mechanisms and signaling pathways, leading to severe and deleterious changes in myocardial structure which may degenerate to heart failure. Thus, an important strategy in the treatment of DM is to prevent and control its complications with cardiovascular diseases [2,3].
Studies have shown that retinoid acid receptor (RAR) agonists inhibit cardiomyocyte hypertrophy by regulating certain cell signaling pathways, thereby mitigating cardiac hypertrophy. Retinoid X receptor (RXR), a member of the nuclear hormone receptor superfamily which includes α, β and γ subtypes, is involved in regulating cell differentiation, proliferation, and immune function. Studies have shown that RXRα has a unique role in the receptor family through its interaction with many other nuclear receptors [4]. It has been reported that the RXR-specific agonist, bexarotene, significantly inhibited STZ-induced atherosclerosis in apoE -/mice [5].
This study was carried out to investigate the effect of bexarotene on the structure and function of DM rats, using various imaging methods so as to provide potential therapeutic targets for the prevention of DM complications.

Animals and grouping
Eight-week-old SPF rats (n = 48) weighing 180 ± 20 g were purchased from Beijing Vitallihua, and kept at 22 ± 2 ℃ and 55 ± 5 % humidity in an environment with 12 h light/day. Drinking water and feed were liberally provided.  [6].

Animal studies
Blood glucose level in the fasted state was determined using glucose oxidase. Whole heart and left ventricle masses were determined gravimetrically. Serum creatinine level was determined with picric acid method, while liver function was determined enzymatically. Cardiac structure and function were evaluated through echocardiography, using the parameters of aortic sinus diameter (systolic phase), left atrial anteroposterior diameter (systolic phase), interventricular septum diastole (IVSd), left ventricular posterior wall diastolic (LVPWd), left ventricular EF and FS. Changes in myocardial tissue structure were determined histologically with H & E staining, while western blotting was used to assay the protein expressions of Bcl2 and Bax in rat myocardial tissue.

Statistical analysis
Data are presented as mean ± SD. Multicomponent comparison was performed using one-way analysis of variance (ANOVA), while least significant difference (LSD) method was used for comparison between two groups. Statistical analysis was performed using SPSS19.0 software package. Values of p < 0.05 were considered statistically significant.

Influence of bexarotene on blood glucose in STZ-induced DM rats
As shown in Table 1, before treatment with bexarotene, the fasting blood glucose level of DM group was higher than that of normal control group (p < 0.05). There were no significant changes in blood glucose levels in the DM and DM + Bex (10 mg/kg/day) treatment groups at 2, 4, and 8 weeks, when compared with their corresponding values before treatment (p > 0.05). However, in the DM+ Bex (20 mg/kg/day) treatment group, blood glucose levels at 4 and 8 weeks after drug administration were significantly lower than the blood glucose level before treatment (p < 0.05). Table 2 shows that rats in the DM group had lower body weight, lower weight of left ventricular tissue, and lower heart weight than normal control rats (p < 0.05). Rat weight, and heart and left ventricular weights in the DM + Bex (20 mg/kg/day) treatment group of rats were markedly increased, relative to DM group rats (p < 0.05). However, liver function and serum creatinine levels were comparable between diabetic rats in the two Bex treatment groups, and normal control rats (p > 0.05). Table 3 shows that relative to control group, no appreciable differences were seen in AO (aortic root), left atrium, LVEDs, LVEDd, LVEF and LVFS in the DM group, DM + Bex (10 mg/kg/day) treatment group, and DM + Bex (20 mg/kg/day) treatment group (p > 0.05). Moreover, AO, LA, LVEDs and LVFS were comparable among the three treatment groups (p > 0.05). In contrast, IVSd and LVPWd in DM rats were significantly decreased, relative to control values, while IVSd and LVPWd levels in rats treated with DM+Bex (20 mg/kg/day) were significantly increased, relative to DM rats (p > 0.05).

Effect of bexarotene on myocardial structure of STZ-induced DM rats
The results showed that cardiomyocytes from normal rats were arranged neatly, with uniform size of the nucleus and clear structural boundaries and clear horizontal stripes (Figure 1  A). In contrast, the myocardial cells from the DM group were irregularly arranged; the cell sizes were uneven, with blurred structural boundaries and unclear horizontal stripes (Figure 1 B). In the DM + Bex (10 mg/kg/day) treatment group, a more regular arrangement of cardiomyocytes, uniform cell size, and clear horizontal stripes were evident (Figure 1 C) A and B show that the expression levels of Bax and Bcl2 in diabetic rats were markedly increased, when compared with normal rats, and the Bcl2/Bax ratio was significantly lower (p < 0.05). The levels of Bcl2 and Bax in DM + high dose Bex treatment rats were markedly less than the corresponding levels in DM rats, and Bcl2/Bax ratio was significantly elevated (p < 0.05).

DISCUSSION
Diabetes mellitus (DM) is a disease caused by chronic, persistent hyperglycemia due to absolute or relative lack of insulin secretion. Epidemiological investigations have shown that the incidence of cardiovascular disease in DM patients is 2 to 3 times higher than that in non-DM patients. Diabetes mellitus (DM) is one of the major risk factors for heart failure [7]. Chronic hyperglycemia is the most important factor for the microvascular and cardiovascular complications of DM. Hyperglycemia affects the structure and function of cardiomyocytes through various signal transduction systems, leading to cardiac insufficiency [8]. Echocardiography is usually used in the clinical evaluation of myocardial structure and function in DM patients. However, due to its low sensitivity, echocardiography is not able to evaluate early alterations in myocardial structure and function in diabetic patients. Studies have shown that RXRα inhibits cell proliferation, delays cell growth, and inhibits cell protein synthesis through multiple pathways [9]. In the present study, the RXRα agonist bexarotene was used to treat STZinduced DM rats, and its mechanism of inhibiting ventricular remodeling and cardiomyocyte apoptosis was further explored, resulting in good results.
Disorders in peripheral and cardiac glycolipid metabolism constitute the major causative factors associated with impairments in structure and function in DM. Studies have shown that hyperglycemia increases the expression of advanced glycation end-products and their receptors; activates nuclear transcription factors, alters myocardial contractility, changes structural proteins, increases myocardial stiffness, and induces myocardial interstitial fibrosis [10,11]. Left ventricular hypertrophy is a pathological change in most heart diseases and an important risk factor for many cardiovascular diseases. There are significant changes in cardiac structure at the early stages of DM, but as the disease progresses, there is a gradual enlargement of the left atrium and left ventricle [12].
In this study, conventional echocardiography was employed for determination of lesions in myocardial morphology and function in DM rats and the effect of bexarotene on DM cardiomyopathy. The results showed that the fasting blood glucose level was higher in diabetic rats than in normal rats. Blood glucose levels at the 4th and 8th weeks after treatment in the DM + Bex (20 mg/kg/day) treatment group were significantly lower than that before treatment. Rat weight, whole heart weight and left ventricular weight of DM group were significantly lower than those of the control group.
Levels of IVSd and LVPWd in the DM group were markedly reduced, relative to control, and IVSd and LVPWd levels in the DM + Bex (20 mg/kg/day) treatment group were significantly increased, relative to DM group. These results suggest that left ventricular weight loss is one of the important features of DM cardiomyopathy, and that bexarotene not only restored the left ventricular weight, but also improved cardiac systolic function in DM cardiomyopathy rats. The different doses of bexarotene used in the study did not produce significant hepato-renal toxicity, indicating the safety of the treatment at low doses. The results from histology also confirmed changes in myocardial structure in DM cardiomyopathy, as manifested in the disordered cell arrangement, uneven cell size, and blurring of structural boundary and horizontal grain. Moreover, the results of this study showed that bexarotene mitigated myocardial remodeling in DM rats. This may be an important mechanism for the improvement of cardiac structure and function in DM rats. The anti-oxidative stress, anti-inflammatory and anti-proliferative effects of bexarotene may be responsible for its mitigation of cardiac remodeling [13].
Apoptosis of cardiomyocytes is one of the important mechanisms involved in myocardial ischemia-reperfusion injury in DM. The Bcl2 and Bax are important factors that regulate the mitochondrial apoptosis pathway. The Bcl2 protein is a membrane-bound protein located in mitochondria and lysosomes. Being an antiapoptotic protein, it plays an important role in the regulation of apoptosis [14]. The function of Bax gene is opposite to that of Bcl2 gene; Bax forms heterodimers which inactivate Bcl2 protein, thus promoting cell apoptosis. During myocardial ischemia and ischemia-reperfusion, Bcl2 protein expression is significantly down-regulated, while and Bax gene expression is significantly upregulated, suggesting that Bcl2 and Bax are involved in the regulation of apoptosis in myocardial ischemia-reperfusion injury [15,16].
The results of this study indicate that bexarotene significantly down-regulated the expressions of Bcl2 and Bax proteins, inhibited cardiomyocyte apoptosis, and up-regulated the relative ratio of Bcl2/Bax, suggesting that inhibition of cardiomyocyte apoptosis may be a mechanism involved in the bexarotene-induced improvement of cardiac structure and function in a rat model of STZ-induced DM.

CONCLUSION
Changes in left ventricular structure and function in diabetic cardiomyopathy occur earlier than changes in EF and FS values. Bexarotene significantly mitigates impairments in heart structure and function in diabetic rats via a mechanism related to reduction in blood glucose, inhibition of ventricular remodeling, and suppression of apoptosis of cardiomyocytes. These findings may be beneficial in the development of new anti-DM drugs.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the author(s) named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors all authors read and approved the manuscript for publication. Guo Xi conceived and designed the study, Ying Ma, Yunfeng Li, Yongqiang Ren, Guo Xi collected and analysed the data while Ying Ma wrote the manuscript.

Open Access
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