Cell Biochem Biophys (2015) 71:735–740 DOI 10.1007/s12013-014-0257-1
ORIGINAL PAPER
Reduced Apoptosis After Acute Myocardial Infarction by Simvastatin Ke-qin Luo • Hui-bao Long • Bing-can Xu
Published online: 11 October 2014 Ó Springer Science+Business Media New York 2014
Abstract To observe the effect of simvastatin in patients with acute myocardial infarction in rabbits against myocardial apoptosis, and to explore its possible mechanism. Male New Zealand white rabbits were randomized into three groups, including the myocardial infarction group (12 rabbits), the simvastatin treatment group (15 rabbits), and the sham group (12 rabbits). In the simvastatin treatment and myocardial infarction groups, the rabbits received myocardial infarction surgeries. While in the sham group, loose knots were tied in the left anterior descending coronary artery branches. The simvastatin treatment group was given simvastatin by oral gavage 24 h after surgery. Parameters, which included left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction, and left ventricular mass index, were recorded in these three groups. Edge myocardial infarction and myocardial cell apoptosis were analyzed using TUNEL assay, and Bcl-2, Bax, and Caspase-3 protein levels were detected by Western blot. Acute myocardial infarction model was successfully established in rabbits by ligation of the left anterior descending coronary artery. Compared with the myocardial infarction group, left ventricular end-diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were significantly reduced and left ventricular ejection fraction (LVEF) increased in the simvastatin treatment group. Compared with the sham group, LVEDD and LVESD were significantly increased and LVEF decreased in the simvastatin treatment group. All the differences were statistically significant (P \ 0.05). Left ventricular mass index in the K. Luo (&) H. Long B. Xu Emergency Department, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen Univeristy, Guangzhou 510210, Guangdong, China e-mail: [email protected]
simvastatin treatment group was statistically lower than the myocardial infarction group. Compared with the sham group, left ventricular mass index in both the simvastatin treatment and myocardial infarction groups was significantly increased. The differences of the above comparisons were statistically significant (P \ 0.05). Compared with the sham group, the apoptosis rate of the myocardial infarction group and the simvastatin treatment groups was significantly increased as shown by TUNEL assay, however, the apoptosis rate of the simvastatin treatment group was significantly lower than that of the myocardial infarction group. All the differences among above comparisons were statistically significant (P \ 0.05). Bcl-2 levels significantly increased in the simvastatin treatment group compared with the myocardial infarction group, but Bcl-2 levels in both groups were significantly lower than the sham group. However, Bax protein levels showed inverse expression with Bcl-2. Meanwhile, Caspase-3 protein expression showed similar trend with Bcl-2. Simvastatin can improve cardiac function after myocardial infarction and reduce apoptosis of myocardial cells, possibly by decreasing Bax and Caspase-3 expression and increasing the expression level of Bcl-2. Keywords Simvastatin Acute myocardial infarction Myocardial cells Apoptosis
Introduction With the rapid social and economic development, the incidence of acute myocardial infarction is on the rise, increasingly becoming a serious disease that threatens the health, life quality and lives of mankind [1, 2]. Traditional studies suggested that acute myocardial infarction led to
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myocardial necrosis. However, accumulating evidence also implied that apoptosis plays an important role in the development of myocardial infarction disease [3, 4]. Apoptosis is the process of programed cell death, during which cells actively die in response to some stimulations or some signals [5, 6]. The myocardial ischemia can powerfully induce the apoptosis of cardiomyocytes. Studies have shown that apoptosis in myocardial infarct area was significantly higher than that in the non-infarcted area, therefore, myocardial apoptosis in myocardial infarction has received increasing attention [7, 8]. Numerous studies show that statin-like drugs can effectively improve left ventricular remodeling after myocardial infarction to improve cardiac function, which may mechanistically attenuate myocardial apoptosis [9, 10]. In this study, we employed the rabbit model of acute myocardial infarction to explore the impact of simvastatin on myocardial apoptosis in the hope that our results can provide experimental basis for better clinical treatment of patients with acute myocardial infarction.
Materials and Methods Experimental Animals and Treatments Male New Zealand white rabbits were purchased from Nanjing Qinglongshan animal breeding farms. Rabbits of 2.0 ± 0.2 kg body weights were randomized into three groups: the myocardial infarction group (12 rabbits), the simvastatin treatment group (15 rabbits), and the sham group (12 rabbits). The simvastatin treatment group and the myocardial infarction group were subjected to myocardial infarction surgeries. Loose knots were tied in the left anterior descending coronary artery branches in the sham group. The simvastatin treatment group received oral gavage of simvastatin 5 mg/(kg day) 24 h after surgeries, the other two groups received only saline.
Cell Biochem Biophys (2015) 71:735–740
Pathology Two weeks after surgery, ear vein injection of KCL was used to kill the rabbits. Skin and sternum were cut to remove the heart. Ventricular weight was measured after saline flush, infarct tissue was preserved in -80 °C freezer and part of the tissue was fixed in paraformaldehyde, paraffinembedded, sectioned before pathological examination. TUNEL Assay TUNEL assays were performed with the infarct tissue sections according to manufacturer’s instructions in TUNEL kit (Roche). Apoptotic cardiomyocytes were stained brown. Positive cells were counted with 5 fields under high magnification. Western Blot Proteins were extracted according to manufacturer’s instructions in the Western blot kit. Briefly, 9 volumes of extraction buffer was mixed with 1 volume of cell pellet and pipetted evenly. Supernatant was aliquoted for protein quantification. b-actin was used as loading control, film was scanned for band analysis, relative target protein level = integrated optical density (IOD) of target protein protein/IOD of b-actin. Statistical Analysis The data were analysed with SPSS11.0 statistics suite. Student t test was used for the comparisons between two groups and one-way ANOVA was used among groups, P \ 0.05 was considered statistically significant.
Results
Myocardial Infarction Surgery in Rabbits
Myocardial Infarction Rabbit Model was Successfully Established
Rabbits were anesthetized with sodium pentobarbital through ear vein injection, followed by injection of lidocaine and keep the natural breathing state. After anesthesia, rabbits fixed on the operating table in supine position, skin and sternum were cut to expose the heart, and the integrity of both sides of the pleura was preserved. Longitudinal pericardial incision was performed to fully expose the left anterior descending coronary artery, and with 5–0 lines LAD ligation, successful ligation leads to weakened heartbeat and apical darkening. Chest was stitched layer by layer followed by routine treatment of penicillin to prevent infection.
The myocardial infarction surgeries were performed with rabbits. By comparing the electrocardiography (ECG) before and after surgery, ECG ST-segment elevation and pathologic Q waves appeared after surgeries, which indicated successful establishment of acute myocardial infarction model in the rabbits. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) by echocardiogram compared with the myocardial infarction group, LVEDD and LVESD were significantly reduced and LVEF increased in the simvastatin treatment group. Compared with the sham
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Table 1 Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) Sham
Myocardial infarction
Simvastatin treatment
LVEDD (mm)
11.45 ± 0.18
15.38 ± 0.43
13.21 ± 0.17
LVESD (mm)
7.91 ± 0.13
11.47 ± 0.57
8.38 ± 0.28
LVEF ( %)
0.77 ± 0.01
0.47 ± 0.04
0.55 ± 0.03
group, LVEDD and LVESD were significantly increased and LVEF decreased in the simvastatin treatment group (Table 1; Fig. 1a, b and c). The differences of the above comparisons were statistically significant (P \ 0.05). Left Ventricular Mass Index Comparison Left ventricular mass index in the simvastatin treatment group was statistically lower than the myocardial infarction group. Compared with the sham group, left ventricular mass index in both the simvastatin treatment and myocardial infarction groups were significantly increased (Table 2; Fig. 2a, b). The differences of the above comparisons were statistically significant (P \ 0.05). The Apoptosis Analysis of the Myocardial Infarction Compared with the sham group, the apoptosis rate of the myocardial infarction group and the simvastatin treatment groups was significantly increased as shown by TUNEL assay, however, the apoptosis rate of the simvastatin treatment group was significantly lower than that of the myocardial infarction group (Fig. 3a–d). All the differences among above comparisons were statistically significant (P \ 0.05).
Table 2 Indexes of left ventricle weight
Left ventricular weight (g)
Sham
Myocardial infarction
Simvastatin treatment
3.11 ± 0.23
3.91 ± 0.22
3.52 ± 0.37
Body weight (kg)
2.16 ± 0.13
2.14 ± 0.13
2.13 ± 0.11
Left ventricular mass index (g/kg)
1.31 ± 0.11
1.78 ± 0.23
1.45 ± 0.24
Bcl-2, Bax, and Caspase-3 Expression Levels by Western Blot Bcl-2 levels significantly increased in the simvastatin treatment group compared with the myocardial infarction group, but Bcl-2 levels in both groups were significantly lower than the sham group. However, Bax protein levels showed inverse expression with Bcl-2. Bax expression was evidently lower than the myocardial infarction group, but Bax expression in both the myocardial infarction and the simvastatin treatment groups were significantly higher than the sham group. Meanwhile, Caspase-3 levels in both the myocardial infarction and the simvastatin treatment groups were significantly higher than the sham group. And Caspase-3 expression in the simvastatin treatment group is lower than that in the myocardial infarction group (Fig. 4a– d).
Discussion Several procedures have been proposed to establish animal models of myocardial infarction, such as drug injection, coronary artery ligation, and coronary artery balloon occlusion [11, 12]. However, recent reports found some defects of the drug injection model, which could result in
Fig. 1 Statistical analysis of LVEDD, LVESD and LVEF
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Fig. 2 Statistical analysis of indexes of left ventricle weight
increased myocardial oxygen consumption, thereby leading to myocardial ischemia induced by coronary spasm [13, 14]. Coronary artery balloon occlusion method, which requires surgeon’s experience, is only suitable to some large animals such as dogs, monkeys, etc., [15, 16]. In this study, we used coronary artery ligation to create a rabbit model of myocardial infarction, which displays several advantages like simple procedures and fewer complications. By ECG testing, our procedures effectively established rabbit model of myocardial infarction.
Fig. 3 a–c TUNEL analysis of apoptosis in myocardial infarction. d Statistical analysis of apoptosis
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Cell Biochem Biophys (2015) 71:735–740
Remodeling of the heart is a process of compensatory changes caused by a myocardial infarction, hypertension, and other heart diseases [17, 18]. The main manifestations of left ventricular remodeling are the changes in ventricular size and function. We examined parameters, such as LVEDD, LVESD, and LVEF, to determine left ventricle changes. In our study, myocardial infarction surgery significantly increased LVEDD and LVESD either with or without simvastatin treatment compared with the sham group, but LVEF was lower than the sham group. Compared with the myocardial infarction group, the simvastatin treatment group exhibited significantly LVEDD and LVESD, with LVEF significantly increased. All the differences were statistically significant. Our results showed that animals exhibited left ventricular function deterioration to different degrees after myocardial infarction surgeries. However, simvastatin treatment improved left ventricular function, indicating that simvastatin can effectively improve left ventricular chamber function after myocardial infarction. Besides parameters such as LVEDD, LVESD, and LVEF, left ventricular mass index is also a strong indicator of cardiac function. Our results also showed that despite the increase in left ventricular mass index after myocardial infarction, simvastatin treatment reduced left ventricular mass index significantly. These results highly suggested that simvastatin might improve
Cell Biochem Biophys (2015) 71:735–740
739
Fig. 4 a Western blot analysis of Bcl-2, Bax, Caspase-3 and GAPDH. b–d Statistical analysis of band intensity for Bcl-2, Bax, Caspase-3
myocardial infarction by reducing left ventricular hypertrophy. Previous studies indicated that myocardial apoptosis is the main mechanism for the pathogenesis of several primary heart diseases. Further, myocardial apoptosis after myocardial infarction is also important for its pathogenesis. After examination of myocardial apoptosis around myocardial infarction area, we found significantly elevated apoptosis rate in the myocardial infarction and the simvastatin treatment group than the sham group, but still significantly lower apoptosis rate in the simvastatin treatment group than the myocardial infarction group. Among the many factors and mechanisms associated with myocardial apoptosis, Bcl-2, Bax, and Caspase-3 have been at the center of research attention. We used Western blot to determine the protein levels of the three apoptotic factors and found that Bcl-2 increased significantly in the simvastatin treatment group over than the myocardial infarction group, however, Bcl-2 levels in both groups were lower than the sham group. Bax protein levels showed inverse changes with Bcl-2 expression. Caspase-3 expression displayed similar tendency to Bax protein expression, which indicated the important roles of the three apoptotic factors during myocardial apoptosis after myocardial infarction. In summary, during apoptosis after myocardial infarction, simvastatin improved cardiac function and reduced
apoptosis, however, the underlying mechanisms of reduced myocardial apoptosis by simvastatin warrant further investigations.
References 1. Ibrahim, A. W., Riddell, T. C., & Devireddy, C. M. (2014). Acute myocardial infarction. Critical Care Clinics, 30, 341–364. 2. Nakatsuma, K., Shiomi, H., Watanabe, H., Morimoto, T., Taniguchi, T., Toyota, T., et al. (2014). Comparison of long-term mortality after acute myocardial infarction treated by percutaneous coronary intervention in patients living alone versus not living alone at the time of hospitalization. The American Journal of Cardiology, 114(4), 522–527. 3. Shiraishi, J., Kohno, Y., Nakamura, T., Yanagiuchi, T., Hashimoto, S., Ito, D., et al. (2014). Clinical impact of thrombus aspiration during primary percutaneous coronary intervention in acute myocardial infarction with occluded culprit. Cardiovascular Intervention and Therapeutics,. doi:10.1007/s12928-014-0282-4. 4. Kim, J., Ghasemzadeh, N., Eapen, D. J., Chung, N. C., Storey, J. D., Quyyumi, A. A., et al. (2014). Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular death. Genome medicine, 6, 40. 5. Xiao, T. T., Wang, Y. Y., Zhang, Y., Bai, C. H., & Shen, X. C. (2014). Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling. PLoS One, 9, e88856. 6. Yang, C., Liu, Z., Liu, K., & Yang, P. (2014). Mechanisms of Ghrelin anti-heart failure: Inhibition of Ang II-induced cardiomyocyte
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Cell Biochem Biophys (2015) 71:735–740 apoptosis by down-regulating AT1R expression. PLoS One, 9, e85785. Lu, J., Sun, B., Huo, R., Wang, Y. C., Yang, D., Xing, Y., et al. (2014). Bone morphogenetic protein-2 antagonizes bone morphogenetic protein-4 induced cardiomyocyte hypertrophy and apoptosis. Journal of Cellular Physiology, 229, 1503–1510. Nam, S. W., Liu, H., Wong, J. Z., Feng, A. Y., Chu, G., Merchant, N., et al. (2014). Cardiomyocyte apoptosis contributes to pathogenesis of cirrhotic cardiomyopathy in bile duct-ligated mice. Clinical Science (Lond), 127, 519–526. Pastuszczak, M., Kotlarz, A., Mostowik, M., Zalewski, J., Zmudka, K., & Undas, A. (2010). Prior simvastatin treatment is associated with reduced thrombin generation and platelet activation in patients with acute ST-segment elevation myocardial infarction. Thrombosis Research, 125, 382–386. Chenot, F., Montant, P. F., Marcovitch, O., Blaimont, M., de Meester, A., & Descamps, O. S. (2007). Co-administration of ezetimibe and simvastatin in acute myocardial infarction. European Journal of Clinical Investigation, 37, 357–363. Waza, A. A., Andrabi, K., & Hussain, M. U. (2014). Protein kinase C (PKC) mediated interaction between conexin43 (Cx43) and K(ATP) channel subunit (Kir6.1) in cardiomyocyte mitochondria: Implications in cytoprotection against hypoxia induced cell apoptosis. Cellular Signalling, 26, 1909–1917. Shin, J. H., Lim, Y. H., Song, Y. S., So, B. I., Park, J. Y., Fang, C. H., et al. (2014). Granulocyte-colony stimulating factor reduces cardiomyocyte apoptosis and ameliorates diastolic dysfunction in Otsuka long-evans Tokushima Fatty rats. Cardiovascular drugs and therapy/sponsored by the International Society of Cardiovascular Pharmacotherapy, 28, 211–220.
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13. Sipkens, J. A., Krijnen, P. A., Hahn, N. E., Wassink, M., Meischl, C., Smith, D. E., et al. (2011). Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: A crucial role for nuclear p47(phox). Molecular and Cellular Biochemistry, 358, 229–239. 14. Xu, X. H., Xu, J., Xue, L., Cao, H. L., Liu, X., & Chen, Y. J. (2011). VEGF attenuates development from cardiac hypertrophy to heart failure after aortic stenosis through mitochondrial mediated apoptosis and cardiomyocyte proliferation. Journal of Cardiothoracic Surgery, 6, 54. 15. Boyle, A. J., Shih, H., Hwang, J., Ye, J., Lee, B., Zhang, Y., et al. (2011). Cardiomyopathy of aging in the mammalian heart is characterized by myocardial hypertrophy, fibrosis and a predisposition towards cardiomyocyte apoptosis and autophagy. Experimental Gerontology, 46, 549–559. 16. Ponce, N. E., Cano, R. C., Carrera-Silva, E. A., Lima, A. P., Gea, S., & Aoki, M. P. (2012). Toll-like receptor-2 and interleukin-6 mediate cardiomyocyte protection from apoptosis during Trypanosoma cruzi murine infection. Medical Microbiology and Immunology, 201, 145–155. 17. Verbrugge, F. H., Dupont, M., Rivero-Ayerza, M., de Vusser, P., Van Herendael, H., Vercammen, J., et al. (2012). Comorbidity significantly affects clinical outcome after cardiac resynchronization therapy regardless of ventricular remodeling. Journal of Cardiac Failure, 18, 845–853. 18. Choi, J. O., Kim, E. Y., Lee, G. Y., Lee, S. C., Park, S. W., Kim, D. K., et al. (2013). Predictors of left ventricular reverse remodeling and subsequent outcome in nonischemic dilated cardiomyopathy. Circulation Journal: Official Journal of the Japanese Circulation Society, 77, 462–469.
ORIGINAL PAPER
Reduced Apoptosis After Acute Myocardial Infarction by Simvastatin Ke-qin Luo • Hui-bao Long • Bing-can Xu
Published online: 11 October 2014 Ó Springer Science+Business Media New York 2014
Abstract To observe the effect of simvastatin in patients with acute myocardial infarction in rabbits against myocardial apoptosis, and to explore its possible mechanism. Male New Zealand white rabbits were randomized into three groups, including the myocardial infarction group (12 rabbits), the simvastatin treatment group (15 rabbits), and the sham group (12 rabbits). In the simvastatin treatment and myocardial infarction groups, the rabbits received myocardial infarction surgeries. While in the sham group, loose knots were tied in the left anterior descending coronary artery branches. The simvastatin treatment group was given simvastatin by oral gavage 24 h after surgery. Parameters, which included left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction, and left ventricular mass index, were recorded in these three groups. Edge myocardial infarction and myocardial cell apoptosis were analyzed using TUNEL assay, and Bcl-2, Bax, and Caspase-3 protein levels were detected by Western blot. Acute myocardial infarction model was successfully established in rabbits by ligation of the left anterior descending coronary artery. Compared with the myocardial infarction group, left ventricular end-diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were significantly reduced and left ventricular ejection fraction (LVEF) increased in the simvastatin treatment group. Compared with the sham group, LVEDD and LVESD were significantly increased and LVEF decreased in the simvastatin treatment group. All the differences were statistically significant (P \ 0.05). Left ventricular mass index in the K. Luo (&) H. Long B. Xu Emergency Department, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen Univeristy, Guangzhou 510210, Guangdong, China e-mail: [email protected]
simvastatin treatment group was statistically lower than the myocardial infarction group. Compared with the sham group, left ventricular mass index in both the simvastatin treatment and myocardial infarction groups was significantly increased. The differences of the above comparisons were statistically significant (P \ 0.05). Compared with the sham group, the apoptosis rate of the myocardial infarction group and the simvastatin treatment groups was significantly increased as shown by TUNEL assay, however, the apoptosis rate of the simvastatin treatment group was significantly lower than that of the myocardial infarction group. All the differences among above comparisons were statistically significant (P \ 0.05). Bcl-2 levels significantly increased in the simvastatin treatment group compared with the myocardial infarction group, but Bcl-2 levels in both groups were significantly lower than the sham group. However, Bax protein levels showed inverse expression with Bcl-2. Meanwhile, Caspase-3 protein expression showed similar trend with Bcl-2. Simvastatin can improve cardiac function after myocardial infarction and reduce apoptosis of myocardial cells, possibly by decreasing Bax and Caspase-3 expression and increasing the expression level of Bcl-2. Keywords Simvastatin Acute myocardial infarction Myocardial cells Apoptosis
Introduction With the rapid social and economic development, the incidence of acute myocardial infarction is on the rise, increasingly becoming a serious disease that threatens the health, life quality and lives of mankind [1, 2]. Traditional studies suggested that acute myocardial infarction led to
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myocardial necrosis. However, accumulating evidence also implied that apoptosis plays an important role in the development of myocardial infarction disease [3, 4]. Apoptosis is the process of programed cell death, during which cells actively die in response to some stimulations or some signals [5, 6]. The myocardial ischemia can powerfully induce the apoptosis of cardiomyocytes. Studies have shown that apoptosis in myocardial infarct area was significantly higher than that in the non-infarcted area, therefore, myocardial apoptosis in myocardial infarction has received increasing attention [7, 8]. Numerous studies show that statin-like drugs can effectively improve left ventricular remodeling after myocardial infarction to improve cardiac function, which may mechanistically attenuate myocardial apoptosis [9, 10]. In this study, we employed the rabbit model of acute myocardial infarction to explore the impact of simvastatin on myocardial apoptosis in the hope that our results can provide experimental basis for better clinical treatment of patients with acute myocardial infarction.
Materials and Methods Experimental Animals and Treatments Male New Zealand white rabbits were purchased from Nanjing Qinglongshan animal breeding farms. Rabbits of 2.0 ± 0.2 kg body weights were randomized into three groups: the myocardial infarction group (12 rabbits), the simvastatin treatment group (15 rabbits), and the sham group (12 rabbits). The simvastatin treatment group and the myocardial infarction group were subjected to myocardial infarction surgeries. Loose knots were tied in the left anterior descending coronary artery branches in the sham group. The simvastatin treatment group received oral gavage of simvastatin 5 mg/(kg day) 24 h after surgeries, the other two groups received only saline.
Cell Biochem Biophys (2015) 71:735–740
Pathology Two weeks after surgery, ear vein injection of KCL was used to kill the rabbits. Skin and sternum were cut to remove the heart. Ventricular weight was measured after saline flush, infarct tissue was preserved in -80 °C freezer and part of the tissue was fixed in paraformaldehyde, paraffinembedded, sectioned before pathological examination. TUNEL Assay TUNEL assays were performed with the infarct tissue sections according to manufacturer’s instructions in TUNEL kit (Roche). Apoptotic cardiomyocytes were stained brown. Positive cells were counted with 5 fields under high magnification. Western Blot Proteins were extracted according to manufacturer’s instructions in the Western blot kit. Briefly, 9 volumes of extraction buffer was mixed with 1 volume of cell pellet and pipetted evenly. Supernatant was aliquoted for protein quantification. b-actin was used as loading control, film was scanned for band analysis, relative target protein level = integrated optical density (IOD) of target protein protein/IOD of b-actin. Statistical Analysis The data were analysed with SPSS11.0 statistics suite. Student t test was used for the comparisons between two groups and one-way ANOVA was used among groups, P \ 0.05 was considered statistically significant.
Results
Myocardial Infarction Surgery in Rabbits
Myocardial Infarction Rabbit Model was Successfully Established
Rabbits were anesthetized with sodium pentobarbital through ear vein injection, followed by injection of lidocaine and keep the natural breathing state. After anesthesia, rabbits fixed on the operating table in supine position, skin and sternum were cut to expose the heart, and the integrity of both sides of the pleura was preserved. Longitudinal pericardial incision was performed to fully expose the left anterior descending coronary artery, and with 5–0 lines LAD ligation, successful ligation leads to weakened heartbeat and apical darkening. Chest was stitched layer by layer followed by routine treatment of penicillin to prevent infection.
The myocardial infarction surgeries were performed with rabbits. By comparing the electrocardiography (ECG) before and after surgery, ECG ST-segment elevation and pathologic Q waves appeared after surgeries, which indicated successful establishment of acute myocardial infarction model in the rabbits. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) by echocardiogram compared with the myocardial infarction group, LVEDD and LVESD were significantly reduced and LVEF increased in the simvastatin treatment group. Compared with the sham
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Table 1 Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) Sham
Myocardial infarction
Simvastatin treatment
LVEDD (mm)
11.45 ± 0.18
15.38 ± 0.43
13.21 ± 0.17
LVESD (mm)
7.91 ± 0.13
11.47 ± 0.57
8.38 ± 0.28
LVEF ( %)
0.77 ± 0.01
0.47 ± 0.04
0.55 ± 0.03
group, LVEDD and LVESD were significantly increased and LVEF decreased in the simvastatin treatment group (Table 1; Fig. 1a, b and c). The differences of the above comparisons were statistically significant (P \ 0.05). Left Ventricular Mass Index Comparison Left ventricular mass index in the simvastatin treatment group was statistically lower than the myocardial infarction group. Compared with the sham group, left ventricular mass index in both the simvastatin treatment and myocardial infarction groups were significantly increased (Table 2; Fig. 2a, b). The differences of the above comparisons were statistically significant (P \ 0.05). The Apoptosis Analysis of the Myocardial Infarction Compared with the sham group, the apoptosis rate of the myocardial infarction group and the simvastatin treatment groups was significantly increased as shown by TUNEL assay, however, the apoptosis rate of the simvastatin treatment group was significantly lower than that of the myocardial infarction group (Fig. 3a–d). All the differences among above comparisons were statistically significant (P \ 0.05).
Table 2 Indexes of left ventricle weight
Left ventricular weight (g)
Sham
Myocardial infarction
Simvastatin treatment
3.11 ± 0.23
3.91 ± 0.22
3.52 ± 0.37
Body weight (kg)
2.16 ± 0.13
2.14 ± 0.13
2.13 ± 0.11
Left ventricular mass index (g/kg)
1.31 ± 0.11
1.78 ± 0.23
1.45 ± 0.24
Bcl-2, Bax, and Caspase-3 Expression Levels by Western Blot Bcl-2 levels significantly increased in the simvastatin treatment group compared with the myocardial infarction group, but Bcl-2 levels in both groups were significantly lower than the sham group. However, Bax protein levels showed inverse expression with Bcl-2. Bax expression was evidently lower than the myocardial infarction group, but Bax expression in both the myocardial infarction and the simvastatin treatment groups were significantly higher than the sham group. Meanwhile, Caspase-3 levels in both the myocardial infarction and the simvastatin treatment groups were significantly higher than the sham group. And Caspase-3 expression in the simvastatin treatment group is lower than that in the myocardial infarction group (Fig. 4a– d).
Discussion Several procedures have been proposed to establish animal models of myocardial infarction, such as drug injection, coronary artery ligation, and coronary artery balloon occlusion [11, 12]. However, recent reports found some defects of the drug injection model, which could result in
Fig. 1 Statistical analysis of LVEDD, LVESD and LVEF
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Fig. 2 Statistical analysis of indexes of left ventricle weight
increased myocardial oxygen consumption, thereby leading to myocardial ischemia induced by coronary spasm [13, 14]. Coronary artery balloon occlusion method, which requires surgeon’s experience, is only suitable to some large animals such as dogs, monkeys, etc., [15, 16]. In this study, we used coronary artery ligation to create a rabbit model of myocardial infarction, which displays several advantages like simple procedures and fewer complications. By ECG testing, our procedures effectively established rabbit model of myocardial infarction.
Fig. 3 a–c TUNEL analysis of apoptosis in myocardial infarction. d Statistical analysis of apoptosis
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Cell Biochem Biophys (2015) 71:735–740
Remodeling of the heart is a process of compensatory changes caused by a myocardial infarction, hypertension, and other heart diseases [17, 18]. The main manifestations of left ventricular remodeling are the changes in ventricular size and function. We examined parameters, such as LVEDD, LVESD, and LVEF, to determine left ventricle changes. In our study, myocardial infarction surgery significantly increased LVEDD and LVESD either with or without simvastatin treatment compared with the sham group, but LVEF was lower than the sham group. Compared with the myocardial infarction group, the simvastatin treatment group exhibited significantly LVEDD and LVESD, with LVEF significantly increased. All the differences were statistically significant. Our results showed that animals exhibited left ventricular function deterioration to different degrees after myocardial infarction surgeries. However, simvastatin treatment improved left ventricular function, indicating that simvastatin can effectively improve left ventricular chamber function after myocardial infarction. Besides parameters such as LVEDD, LVESD, and LVEF, left ventricular mass index is also a strong indicator of cardiac function. Our results also showed that despite the increase in left ventricular mass index after myocardial infarction, simvastatin treatment reduced left ventricular mass index significantly. These results highly suggested that simvastatin might improve
Cell Biochem Biophys (2015) 71:735–740
739
Fig. 4 a Western blot analysis of Bcl-2, Bax, Caspase-3 and GAPDH. b–d Statistical analysis of band intensity for Bcl-2, Bax, Caspase-3
myocardial infarction by reducing left ventricular hypertrophy. Previous studies indicated that myocardial apoptosis is the main mechanism for the pathogenesis of several primary heart diseases. Further, myocardial apoptosis after myocardial infarction is also important for its pathogenesis. After examination of myocardial apoptosis around myocardial infarction area, we found significantly elevated apoptosis rate in the myocardial infarction and the simvastatin treatment group than the sham group, but still significantly lower apoptosis rate in the simvastatin treatment group than the myocardial infarction group. Among the many factors and mechanisms associated with myocardial apoptosis, Bcl-2, Bax, and Caspase-3 have been at the center of research attention. We used Western blot to determine the protein levels of the three apoptotic factors and found that Bcl-2 increased significantly in the simvastatin treatment group over than the myocardial infarction group, however, Bcl-2 levels in both groups were lower than the sham group. Bax protein levels showed inverse changes with Bcl-2 expression. Caspase-3 expression displayed similar tendency to Bax protein expression, which indicated the important roles of the three apoptotic factors during myocardial apoptosis after myocardial infarction. In summary, during apoptosis after myocardial infarction, simvastatin improved cardiac function and reduced
apoptosis, however, the underlying mechanisms of reduced myocardial apoptosis by simvastatin warrant further investigations.
References 1. Ibrahim, A. W., Riddell, T. C., & Devireddy, C. M. (2014). Acute myocardial infarction. Critical Care Clinics, 30, 341–364. 2. Nakatsuma, K., Shiomi, H., Watanabe, H., Morimoto, T., Taniguchi, T., Toyota, T., et al. (2014). Comparison of long-term mortality after acute myocardial infarction treated by percutaneous coronary intervention in patients living alone versus not living alone at the time of hospitalization. The American Journal of Cardiology, 114(4), 522–527. 3. Shiraishi, J., Kohno, Y., Nakamura, T., Yanagiuchi, T., Hashimoto, S., Ito, D., et al. (2014). Clinical impact of thrombus aspiration during primary percutaneous coronary intervention in acute myocardial infarction with occluded culprit. Cardiovascular Intervention and Therapeutics,. doi:10.1007/s12928-014-0282-4. 4. Kim, J., Ghasemzadeh, N., Eapen, D. J., Chung, N. C., Storey, J. D., Quyyumi, A. A., et al. (2014). Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular death. Genome medicine, 6, 40. 5. Xiao, T. T., Wang, Y. Y., Zhang, Y., Bai, C. H., & Shen, X. C. (2014). Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling. PLoS One, 9, e88856. 6. Yang, C., Liu, Z., Liu, K., & Yang, P. (2014). Mechanisms of Ghrelin anti-heart failure: Inhibition of Ang II-induced cardiomyocyte
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7.
8.
9.
10.
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