Int J Clin Exp Med 2016;9(6):10775-10783www.ijcem.com /ISSN:1940-5901/IJCEM0023146

Original ArticleAtorvastatin attenuates myocardial apoptosis and cardiac remodeling, and improves cardiac function after acute myocardial infarction in diabetic rats by further enhancing the activation of hepatocyte growth factor/c-Met pathway

Zaiyong Zhang1*, Guangdong Yan2*, Wenzhu Zhang1, Jianhao Li1, Guoqin Chen1, Handong Lei1, Weijie Liang1, Shanjun Zhao1, Yulan Zhang3

1Department of Cardiovascular, Panyu Central Hospital (Cardiovascular Institute of Panyu District), Guangzhou, P. R. China; 2Department of Cardiovascular, Guangzhou Red Cross Hospital, Guangzhou, P. R. China; 3Department of Ultrasound, Guangdong Women and Children Hospital, Guangzhou, P. R. China. *Equal contributors.

Received January 3, 2016; Accepted March 22, 2016; Epub June 15, 2016; Published June 30, 2016

Abstract: To investigate the effect of atorvastatin on myocardial cell apoptosis, ventricular remodeling and cardiac function after acute myocardial infarction (AMI) in diabetic rats, and to explore whether the effect is mediated by hepatocyte growth factor (HGF)/c-Met signaling pathway. Diabetes in 70 male Sprague-Dawley rats was induced by intraperitoneal injection of streptozotocin (65 mg/kg). After 8 weeks, AMI was induced by the ligation of the left anterior descending coronary artery in the diabetic rats, and 32 surviving rats were randomly divided into AMI control group (n = 16) and atorvastatin intervention group (n = 16; 20 mg/kg/day). Similar surgical procedure was completed in sham group (n = 11) without coronary ligation. Atorvastatin was given daily by lavage from the first day after AMI. Two weeks later, the cardiac function, pathological changes of myocardial tissues, myocardial cell apoptosis, and the expression of HGF and c-Met were compared among groups using ultrasonography, hematoxylin and eosin staining and Masson staining, TUNEL assay, quantitative real-time polymerase chain reaction and im-munohistochemical staining. AMI significantly reduced cardiac function, increased collagen volume fraction (CVF) and myocardial apoptotic index, and up-regulated the expression of HGF and c-Met at mRNA and protein levels in AMI control group (P < 0. 05). The cardiac function was improved, and CVF and myocardial apoptotic index were reduced by treatment with atorvastatin, which also up-regulated the expression of HGF and c-Met (P < 0. 05). The present study demonstrates that atorvastatin significantly attenuates myocardial apoptosis and cardiac remodeling, and improves cardiac function after AMI in diabetic rats by further enhancing the activation of HGF/c-Met pathway.

Keywords: Diabetes mellitus, acute myocardial infarction, cardiac function, hepatocyte growth factor, c-Met, statins, atorvastatin

Introduction

Acute myocardial infarction (AMI) and second-ary heart failure severely threaten the life qual-ity and health of people, especially patients with diabetes mellitus (DM). DM patients have enhanced death rates caused by AMI and sec-ondary heart failure, with poorer prognosis than patients without DM [1, 2]. It is reported that the deterioration of heart function after AMI is mainly correlated with ventricular remod-eling induced by myocardial fibrosis and myo-cardial cell apoptosis [3]. Statins not only regu-late lipid levels, but also inhibit inflammatory

responses and oxidative stress, improve vascu-lar endothelial function, and stabilize athero-sclerotic plaques [4, 5]. In addition, statins are shown to inhibit ventricular remodeling and deterioration of heart function after AMI [6]. However, few studies are focused on the effect of statins on myocardial cell apoptosis, ventric-ular remodeling and heart function in patients with both DM and AMI.

The mechanisms by which statins improve heart function are still unclear, but hepatocyte growth factor (HGF) pathway is considered as one of the most important signal pathways that

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affect myocardial fibrosis, cell apoptosis and ventricular remodeling [7]. HGF exerts its effect against inflammation, myocardial fibrosis, and myocardial cell apoptosis by binding to its receptor c-Met in the heart and hence, facilitat-ing ventricular remodeling and heart function [7]. However, it is still unclear whether the effect of statins on ventricular remodeling and heart function is correlated with HGF/c-Met sig-naling pathway. In the present study, we inves-tigate the effect of statins on myocardial cell apoptosis; ventricular remodeling and heart function in rats with both DM and AMI, and test whether the effect is related with HGF/c-Met signaling pathway.

Materials and methods

Animals

A total of 70 male Sprague-Dawley rats (8 weeks, 200-250 g) were purchased (license No. SCXK (GD) 2008-0002; Guangdong Medical Laboratory Animal Center, Guangzhou, China) and bred under 20-25°C in a humidified room (humidity, 40-55%) with natural light-dark cycle and free access to food and water. To construct rat model for DM, the rats were injected intra-peritoneally with streptozotocin (65 mg/kg; dis-solved in 0.1 mmol/L sodium citrate buffer, pH 4.5; BaomanBio, Shanghai, China). At 3 days, 7 days, 30 days and 8 weeks after injection with streptozotocin, blood glucose level was tested using a portable blood glucose meter (Bayer, Leverkusen, Germany). Rats with blood glucose level ≥ 16.7 mmol/L, polyuria, polydipsia, poly-phagia, marasmus and gray hair were included in the present study. Eight weeks of DM induc-tion, rat model with AMI was constructed by the ligation of the left anterior descending coronary artery in DM rats. In the sham operation group of 11 rats, a line was threaded at the left coro-nary artery but not ligated. After AMI operation, 32 rats survived and were divided randomly into two groups, including AMI control group (n = 16) and atorvastatin intervention group (n = 16). At 24 h after operation, atorvastatin inter-vention group was lavaged with atorvastatin (20 mg/kg/day; dissolved in 3 ml saline), while AMI control group and sham operation group were lavaged with the same volumes of saline. All groups were lavaged every day for a total duration of 2 weeks. Two weeks later, 9 rats in sham operation group, 10 rats in AMI control group, and 12 rats in atorvastatin intervention group survived and were used for subsequent examinations. All animal experiments were con-

ducted according to the ethical guidelines of the Central Hospital of Panyu District, Guang- zhou, China.

Ultrasonography

After treatment for 2 weeks, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg). ATL-HDI5000 Doppler echocardiography instrument (10 MHz probe; Philips, Amsterdam, Netherlands) was used to determine the left ventricular ejection fraction (LVEF), left ventricular fractional short-ening (LVFS), left ventricular end-diastolic diam-eter (LVDd) and left ventricular end-systolic diameter (LVDs). Mean values were calculated from five consecutive cardiac cycles.

Detection of blood lipid

After ultrasonography, 3 mL blood was drawn from the heart ventricle of the rats. Then, an automatic biochemistry analyzer (7150; Hita- chi, Tokyo, Japan) was used to measure serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

Hematoxylin and eosin (HE) staining

To observe histopathological changes of myo-cardial tissues after AMI in DM rats, paraffin-embedded specimens were cut into 4 μm sec-tions, and baked at 60°C over night. Then, the specimens were deparaffinized with xylene and rehydrated, followed by washing with flowing water. Specimens were then placed in hema-toxylin for 2 min, followed by a brief rinse in water. Slides were placed in eosin solution for 30 sec, followed by serial rinsing with ethanol and xylene, before coverslips were applied. The stained tissues were examined under a micro-scope (BX51; Olympus, Tokyo, Japan) with a magnification of 100×.

Masson staining

Using Masson staining, myocardial collagens were stained blue, and myocardial fibers were stained red. Under a microscope (BX51; Olympus, Tokyo, Japan) of 100× magnification, five fields were randomly selected next to the infarction sites. The images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The myocar-dial collagen volume fraction (CVF) was calcu-lated using the equation: CVF = collagen area/(collagen area + myocardial area).

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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Myocardial cell apoptosis was labeled using TUNEL assay kit (Roche, Basel, Switzerland). Normal cell nucleus was presented blue under a microscope, while apoptotic cell nucleus was shown as brown yellow granules (positive stain-ing). Under a magnification of 400×, five fields were selected from each sample for the calcu-lation of apoptotic index by averaging the ratios of positive nuclei number/total nuclei number.

Quantitative real-time polymerase chain reac-tion (qRT-PCR)

Total RNA was extract from 100 mg tissues using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) was used as internal reference. The primers were: HGF, 5’-GAGAGAGGCGAGGAGAAACG-3’ (upstream) and 5’-GTGTAGCCCCAGCCGTAAAT-3’ (down-stre-am); c-Met, 5’-ACGTACGGTGTCTCCAGCAT -3’ (upstream) and 5’-CTGGAGACACAGGATAGG- AA-3’ (downstream); GAPDH, 5’-CTCAGTTGCT- GAGGAGTCCC-3’ (upstream) and 5’-ATTCGA- GAGAAGGGAGGGCT-3’ (downstream) (Gene- Pharma, Shanghai, China). After cDNA synthe-sis by reverse transcription (Thermo Fisher Scientific, Waltham, MA, USA), qRT-PCR was

software (Media Cybernetics, Rockville, MD, USA). Five fields were randomly selected (200×) to measure the integral absorbance that repre-sented protein expression.

Statistical analysis

All results were analyzed using SPSS 13.0 sta-tistical software (IBM, Armonk, NY, USA). Data were expressed as means ± standard devia-tion. Intergroup differences were compared using one-way analysis of variance. In case of homogeneity of variance, LSD test was per-formed. In case of heterogeneity of variance, Dunnett’s T3 test was employed. Differences with P < 0.05 were considered statistically significant.

Results

AMI decreases LVEF and LVFS but increases LVDd and LVDs, while treatment with atorvas-tatin partially alleviates these changes

To study the heart function of the rats, ultraso-nography was performed. The data showed that the LVEF and LVFS values in AMI control group and atorvastatin intervention group were significantly lower than that in sham operation group (P < 0.05), and their LVDd and LVDs were

Table 1. Comparison of echocardiographic parameters (means ± standard deviations)

Parameter Sham operation group (n = 9)

AMI control group (n = 10)

Atorvastatin intervention group (n = 12)

LVEF (%) 83.29 ± 5.83 38.56 ± 6.36* 51.51 ± 612*,#

FS (%) 50.22 ± 3.97 21.64 ± 6.17* 28.38 ± 6.09*,#

LVDd (mm) 5.75 ± 0.60 7.79 ± 0.56* 7.02 ± 0.69*,#

LVDs (mm) 2.53 ± 0.41 6.10 ± 0.71* 5.33 ± 0.74*,#

Note: *, P < 0.05 compared with sham operation group; #, P < 0.05 compared with AMI control group.

performed using Rotor-Gene PCR System (R1011122; Qia- gen, Hilden, Germany). Relative expression of HGF and c-Met mRNA was calculated against GAPDH.

Immunohistochemical staining

To measure the expression of HGF and c-Met proteins in myo-cardial tissues, immunohisto-chemistry was used. Tissues were fixed using formalin, paraf-fin-embedded, and made into slices of a thickness of 4 μm. After dewaxing, the slices were rehydrated and stained using HGF polyclonal primary antibody (1:100; Signalway Antibody, College Park, MD, USA) or c- Met polyclonal primary antibody (1:150; Signalway Antibody, Col- lege Park, MD, USA). Brown staining indicated positive ex- pression. The images were ana-lyzed using Image-Pro Plus 6.0

Table 2. Comparison of body weight, blood sugar level and se-rum lipid level (means ± standard deviation)

Test item Sham operation (n = 9)

AMI control (n = 10)

Atorvastatin intervention

(n = 12)Body weight (g) 234.8 ± 14.3 226.4 ± 17.0 228.7 ± 13.9Blood glucose (mmol/L) 22. 5 ± 3.6 22.9 ± 2.8 21.7 ± 3.1TC (mmol/L) 1.96 ± 0.38 2.04 ± 0.32 1.89 ± 0.34TG (mmol/L) 1.26 ± 0.37 1.18 ± 0.29 1.10 ± 0.27LDL-C (mmol/L) 0.73 ± 0.25 0.66 ± 0.28 0.71 ± 0.21HDL-C (mmol/L) 0.45 ± 0.13 0.47 ± 0.15 0.51 ± 0.18

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significantly higher than that in sham operation group (P < 0.05). In addition, the LVEF and LVFS values in atorvastatin intervention group were significantly higher than that in AMI control

group, respectively (P < 0.05), but its LVDd and LVDs were significantly lower than that in AMI control group, respectively (P < 0.05) (Table 1). The results suggest that AMI decreases LVEF

Figure 1. Pathological changes of myocardial tissues. A. Images of tissues in sham operation, AMI control and ator-vastatin intervention groups taken after hematoxylin & eosin (HE) staining (×100) and Masson staining (×100). B. Quantification of collagen volume fraction determined by Masson staining. The data were presented as means ± standard deviation. *, P < 0.05 compared with sham operation group; #, P < 0.05 compared with AMI control group.

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and LVFS but increases LVDd and LVDs, while treatment with atorvastatin partially alleviates these changes.

AMI and atorvastatin intervention do not alter the body weight, blood glucose level or serum lipid level of rats

To test how AMI and atorvastatin intervention affect the basic physiological parameters of the rats, body weight, blood glucose level and serum lipid level were measured. Two weeks later, the body weight, blood glucose level and serum lipid level were not significantly different among the three groups (P > 0.05) (Table 2). The result indicates that AMI and atorvastatin intervention do not affect the basic physiologi-cal parameters of the rats.

Treatment with atorvastatin partially reduces the degree of pathological changes induced by AMI

To examine the pathological changes of myo-cardial tissues, we used HE staining and Masson staining. HE staining showed that myo-cardial cells in sham operation group had ordered arrangement, good alignment, no bro-ken filaments, uniform intercellular gaps, and little inflammatory cell infiltration. Myocardial cells in AMI control group showed larger infarc-tion areas, reduced number of myocardial cells, broken filaments, disordered structure and abundant inflammatory cell infiltration. Com- pared with AMI control group, the myocardial cells in atorvastatin intervention group had less characteristic change, alleviated structural

Figure 2. Myocardial cell apoptosis. A. TUNEL assay of myocardial tissue (×400). B. Quantification of myocardial apoptotic index. The data were expressed as means ± standard deviation. *, P < 0.05 compared with sham opera-tion group; #, P < 0.05 compared with AMI control group.

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disorder, and decreased inflammatory cell infil-tration (Figure 1A). Masson staining showed that the degrees of myocardial fibrosis (CVF) in AMI control group and atorvastatin intervention group were significantly higher than that in sham operation group, but the degree of myo-cardial fibrosis in atorvastatin intervention group was significantly reduced compared with AMI control group (P < 0.05) (Figure 1A and 1B). These results suggest that treatment with atorvastatin partially reduces the degree of pathological changes induced by AMI.

Treatment with atorvastatin partially inhibits myocardial cell apoptosis induced by AMI

To detect myocardial cell apoptosis, TUNEL assay was employed. The data showed that sham operation group had only a few apoptotic myocardial cells, while AMI control group and atorvastatin intervention group had significant-ly more apoptotic myocardial cells (P < 0.05) (Figure 2A and 2B). In addition, treatment with atorvastatin significantly reduced the apoptotic index compared with AMI control group (P < 0.05) (Figure 2B). The result indicates that treatment with atorvastatin partially inhibits myocardial cell apoptosis induced by AMI.

Treatment with atorvastatin further increases the mRNA levels of HGF and c-Met in myocar-dial tissues that are already enhanced by AMI

To measure the levels of HGF and c-Met mRNA in myocardial tissues, qRT-PCR was carried out. The data showed that HGF and c-Met mRNA expression in AMI control group and atorvas-tatin intervention group were significantly high-

already elevated by AMI

To determine the protein expression of HGF and c-Met, immunohistochemistry was used. The data showed that HGF and c-Met protein expression in AMI control group and atorvas-tatin intervention group were significantly high-er than that in sham operation group (P < 0.05). In addition, HGF and c-Met protein expression in atorvastatin intervention group was signifi-cantly higher than that in AMI control group (P < 0.05) (Figure 4). The result indicates that treat-ment with atorvastatin further enhances the protein levels of HGF and c-Met in myocardial tissues that are already elevated by AMI.

Discussion

The deterioration of heart function was further aggravated in patients with DM after AMI. Backlund et al. show that DM promotes myo-cardial apoptosis, myocardial fibrosis and con-nective tissue growth factor expression that are induced by AMI, and aggravates cardiac remodeling after AMI [8]. Our results demon-strate that treatment with atorvastatin attenu-ates myocardial fibrosis, myocardial cell apop-tosis and cardiac remodeling, and improves cardiac function after AMI in diabetic rats. It is reported that statins may induce or even aggra-vate DM [9], but experimental and clinical results show that the benefits of statins still far outweigh their disadvantages. However, the mechanisms by which statins inhibit ventricular remodeling after AMI are still unclear. It is shown that HGF has protective effects on both heart and kidney. HGF is a multifunctional fac-

Figure 3. HGF and c-Met mRNA expression in myocardial tissues. Quantita-tive real-time polymerase chain reaction was used to measure mRNA expres-sion. The data were expressed as means ± standard deviation. *, P < 0.05 compared with sham operation group; #, P < 0.05 compared with AMI control group.

er than that in sham opera-tion group (P < 0.05). In addition, HGF and c-Met mRNA expression in atorvas-tatin intervention group was significantly higher than that in AMI control group (P < 0.05) (Figure 3). The re- sult suggests that treatment with atorvastatin further in- creases the mRNA levels of HGF and c-Met in myocardial tissues that are already en- hanced by AMI.

Treatment with atorvastatin further enhances the protein levels of HGF and c-Met in myocardial tissues that are

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tor derived from mesenchymal cells that can promote mitosis of cells. It is a dimer formed by an α chain and a β chain. Its receptor is proto-oncogene c-Met, which first binds to α chain and then binds to β chain. Subsequently, c-Met

receptor tyrosine phosphorylation is induced to exert its bioactivity [10].

Guo et al. report that HGF improves ventricular remodeling and cardiac function by inhibiting

Figure 4. HGF and c-Met protein expression in myocardial tissues. A. Immunohistochemical staining of myocardial tissues for the detection of HGF and c-Met protein expression (×200). B. Quantification of HGF and c-Met protein expression by measuring integral absorbance. The data were expressed as means ± standard deviation. *, P < 0.05 compared with sham operation group; #, P < 0.05 compared with AMI control group.

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myocardial cell apoptosis and promoting angio-genesis [11]. Komamura et al. show that HGF improves hypertension and the corresponding arterial stiffness and heart failure in rats with hypertension [12]. In fact, HGF signaling path-way plays important roles in the acting mecha-nisms of cardiovascular drugs [13]. A clinical study shows that increased blood level of HGF in AMI patients is correlated with ventricular remodeling and cardiac function, and that high-er blood level of HGF usually indicates poorer prognosis [14]. Therefore, HGF/c-Met signaling pathway has become an important protective mechanism in the heart. However, there has been no report on whether statins affect cardi-ac remodeling via HGF/c-Met signaling path-way. The present study demonstrates that mRNA and protein expression of HGF and c-Met are both elevated in AMI control group com-pared with sham operation group, suggesting that HGF/c-Met signaling pathway is activated after AMI. In addition, treatment with atorvas-tatin further enhances the mRNA and protein expression of HGF and c-Met, suggesting that atorvastatin may improve cardiac function and exert heart protective effect by activating HGF/c-Met signaling pathway.

By now, HGF gene therapy has been used in the treatment for heart failure after AMI. It is report-ed that transplanted cells that are transfected with HGF better improve cardiac remodeling and heart function after AMI [15]. Yuan et al. show that treatment by HGF that is transported by micro bubbles reduces left ventricular mass and myocardial collagen content, and increas-es capillary density [16]. A previous study shows that simvastatin improves renal function by inhibiting renal fibrosis via increased HGF expression in the kidney [17]. Cantoni et al. find that the expression of HGF and its receptor was enhanced in human mesenchymal stem cells after the intervention by atorvastatin [18]. These reports are consistent with our results in the present study. However, statins are also found to reduce HGF secretion in vitro by osteo-sarcoma cells from mice [19], suggesting that statins may have distinct effects on HGF expression in cells from different tissue types at different pathological status.

The mechanism by which statins affect HGF signaling pathway is not clear yet. A study shows that angiotensin II and transforming growth factor are important inhibitors for the production of HGF [20]. The expression of angiotensin II and transforming growth factor is

significantly enhanced after heart failure and AMI and hence, down-regulating the production of HGF. A study shows that statins significantly inhibit the increases in angiotensin II and trans-forming growth factor, and further up-regulate HGF expression [21]. However, it still needs fur-ther studies to understand how statins affect HGF signaling pathway. In conclusion, the pres-ent study demonstrates that atorvastatin sig-nificantly attenuates myocardial apoptosis and cardiac remodeling, and improves cardiac func-tion after AMI in diabetic rats by further enhanc-ing the activation of HGF/c-Met pathway.

Acknowledgements

This work was supported by Guangzhou Medical and Health Science and Technology General Guidance Project (No. 2015A012080020) and Master & Doctoral Research Funds of Panyu Central Hospital.

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Wenzhu Zhang, Department of Cardiovascular, Panyu Central Hos- pital, 8 East Fuyu Road, Guangzhou 511400, Guang- dong Province, P. R. China. Tel: 86-20-34859342; Fax: 86-20-34859340; E-mail: zaiyong@sina.cn

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