- Email: [email protected]
Imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens
IMMUNOLOGY, HEALTH, AND DISEASE Imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens Xue-Qin Hao,*1 Shou-Yan Zhang,†1 Xiang-Chao Cheng,‡2 Meng Li,§ Tong-Wen Sun,# Ji-Liang Zhang,* Wen Guo,* and Li Li* *Department of Pharmacy, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China; †Department of Cardiology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, China; ‡Department of Animal Science, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China; §Luoyang Entry-Exit Inspection and Quarantine Bureau, Luoyang 471003, China; and #Department of Integrated ICU, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China ABSTRACT This study explored the effect of imidapril on the right ventricular remodeling induced by low ambient temperature in broiler chickens. Twenty-four broiler chickens were randomly divided into 3 groups (n = 8), including the control group, low temperature group, and imidapril group. Chickens in the control group were raised at normal temperature, whereas chickens in the low temperature group and imidapril group were exposed to low ambient temperature (12 to 18°C) from 14 d of age until 45 d of age. At the same time, chickens in the imidapril group were gavaged with imidapril at 3 mg/kg once daily for 30 d. The thickness of the right ventricular wall was observed with echocardiography. The BW and wet lung weight as well as weight of right and left ventricles and ventricular septum were measured. Both wet lung weight index and right ventricular hypertrophy index were calculated. Pulmonary arterial systolic pressure was assessed according to echocardiography. The expression of ACE
and ACE2 mRNA in the right ventricular myocardial tissue was quantified by real-time PCR. Proliferating cell nuclear antigen-positive cells were detected by immunohistostaining. The concentration of angiotensin (Ang) II and Ang (1–7) in the right ventricular myocardial tissue was measured with ELISA. The results showed that right ventricular hypertrophy index, wet lung weight index, pulmonary arterial systolic pressure, expression of ACE mRNA in the right ventricular tissue, Ang II concentration, and the thickness of the right ventricular wall in the low temperature group increased significantly compared with those in the control group and imidapril group. The ACE2 mRNA expression increased 36%, whereas Ang (1–7) concentration decreased significantly in the low temperature group compared with that in the control group and imidapril group. In conclusion, imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens.
Key words: imidapril, broiler chicken, low temperature, right ventricular remodeling, pulmonary hypertension 2013 Poultry Science 92:1492–1497 http://dx.doi.org/10.3382/ps.2012-02671
INTRODUCTION Pulmonary arterial hypertension syndrome, also known as ascites syndrome, is a metabolic disorder found in modern broiler chickens that have insufficient pulmonary vascular capacity. Broiler chickens typically hatch at a weight of 40 g and can grow to 4 kg, doubling in weight almost 7 times within 8 wk. The extremely rapid early growth performance of broilers imposes proportional challenges to their developmentally immature ©2013 Poultry Science Association Inc. Received August 12, 2012. Accepted January 15, 2013. 1 These authors contributed equally to this article. 2 Corresponding author: [email protected]
pulmonary and cardiovascular systems (Wideman and Hamal, 2011). Ascites syndrome especially occurs under conditions such as high altitude or low ambient temperatures because of the higher metabolic rate and increased blood viscosity (Julian, 2000; Baghbanzadeh and Decuypere, 2008; van As et al., 2010). Pulmonary arterial hypertension is characterized by high pulmonary vascular resistance and vascular remodeling, which results in a striking increase in right ventricle (RV) afterload and subsequent failure (Franco, 2012). Ventricular remodeling, defined as changes in size, shape, and function of the heart in response to cardiac injury or increased load, correlates with the development and progression of heart failure (Ma et al., 2012; Zhou et al., 2012). The RV is in charge of pump-
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ing blood to the lungs for oxygenation. Right ventricular remodeling is associated with pulmonary arterial hypertension (Yang et al., 2009; Franco, 2012). Studies showed that RV function should be recognized as an important contributor and prognostic indicator of cardiopulmonary diseases (Franco, 2012). The process of ventricular remodeling is largely influenced by hemodynamic load, neurohumoral activation, and additional factors such as endothelin, cytokines, nitric oxide production, and oxidative stress (Ma et al., 2012; Zhou et al., 2012). Evidences have showed that angiotensin (Ang) II induces inflammation, leading to cardiac remodeling (Savoia and Volpe, 2011; Jia et al., 2012). On the other hand, the thioether-bridged, stabilized angiotensin-(1–7) analog cyclic ang-(1–7) exerts beneficial effect on the cardiac remodeling and endothelial function in rats with myocardial infarction (Capettini et al., 2012; Durik et al., 2012). The Ang II is a key effector peptide of the rennin-angiotensin system in regulating blood pressure, homeostasis, and cell proliferation. The Ang-(1–7) is a heptapeptide with antiproliferative and vasodilating properties, being a contradiction to Ang II. Imidapril is an ACE inhibitor that can prevent the formation of Ang II and the degradation of Ang-(1–7) and bradykinin, leading to a decrease in blood pressure and cell proliferation (Gallagher et al., 2011; Shrikrishna et al., 2012). Therefore, we hypothesize that imidapril might provide beneficial effect on the right ventricular remodeling. The present study was designed to explore the effect of imidapril on the right ventricular remodeling in broiler chickens.
MATERIALS AND METHODS Birds Broiler chickens at 13 d of age were purchased from Luoyang Chicken Alliance (Luoyang, China). All birds had ad libitum access to feed and water. Twenty-four chickens were randomly divided into 3 groups (n = 8): the control group, low temperature group, and imidapril group. Chickens in the control group were raised at normal temperature, whereas chickens in the low temperature group and imidapril group were exposed to low ambient temperature (12 to 18°C) from 14 d of age until 45 d of age. Meanwhile, chickens in the imidapril group were gavaged with imidapril (Tianjin Tanabe Pharmaceutical Co. Ltd., Tianjin, China; approved by the enterprise: H19990153) at 3 mg/kg once daily for 30 d. At the age of 45 d, chickens were killed and samples were collected. The present study was conducted in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (http://grants1.nih.gov/ grants/olaw/) and was approved by the local animal ethics committee at Henan University of Science and Technology.
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Transthoracic Echocardiography Transthoracic echocardiography was used to observe the ventricular wall thickness. Broiler chickens were anesthetized with pentobarbital (40 mg/kg). A layer of acoustic coupling gel was applied to the thorax, and 2-dimensional echocardiography was performed using a commercially available 12-MHz linear-array transducer system (IE33-S12-MHz, Philips, Hamburg, Germany). All echocardiograms were obtained by an experienced sonographer. Images were stored digitally using commercial software (Prosolv Cardiovascular, Indianapolis, IN,) and reported by a senior echocardiologist. Pulmonary arterial ultrasonic imaging was obtained, and pulmonary arterial systolic pressure was assessed. Pulmonary arterial systolic pressure (PASP) was determined by measuring the peak systolic pressure gradient of the RV to the right atrium, according to the simplified Bernoulli equation. By adding the mean right arterial pressure to the transtricuspid pressure gradient, one can predict the RV systolic pressure, which approximates the PASP (Arcasoy et al., 2003; Sciomer et al., 2005). The PASP was assessed by an experienced sonographer.
Evaluation of Wet Lung Weight Index At the age of 45 d, the BW and wet lung weight (wW) of chickens was measured. The wet lung weight index (LI) was calculated using the formula LI = wW/ BW.
Evaluation of Right Ventricular Hypertrophy Index Chickens were killed at 45 d of age. The right and left ventricle (LV) wall as well as ventricular septum (S) were weighed, and right ventricular hypertrophy index (RVHI) was calculated using the formula of RVHI = RV/(LV + S).
Real-Time PCR The expression of ACE and ACE2 mRNA in the RV tissue was assessed by real-time PCR. Total RNA was extracted from the RV tissue using Trizol (Roche Molecular Biochemicals, Mannheim, Germany) and was quantified by measuring absorbance at 260 nm. One microgram of total RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara Biological Engineering Dalian Co. Ltd., Dalian, China) using Thermo Hybaid Px2 thermal cycler (Thermo, Franklin, MA). β-Actin was used as an internal control. The PCR primers used were designed by Sangon Biotech Co. Ltd (Shanghai, China). Chicken nucleotide sequences for ACE, ACE2, and β-actin are as follows: chicken ACE (forward: 5′-AACACAGAGAACGGGGAGGT-3′; reverse: 5′-AG-
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GAAGACAAGTGCCAGTGC-3′); chicken ACE2 (forward: 5′-TTGCTTCACTTCTGGCTTCTC-3′; reverse: 5′-TCCTGGCTGTCTCCTCAGTTA-3′); chicken β-actin (forward: 5′-ACAATGGCTCCGCTATGTG-3′; reverse: 5′-CTTTTGCTCTGGGCTTCATC-3′). Each real-time PCR reaction was carried out in a total volume of 20 μL with SYBR Premix Ex Taq (Takara Biological Engineering Dalian Co. Ltd.) according to the following conditions: 30 s at 95°C, 40 cycles at 95°C for 5 s, 60°C (ACE, β-actin), 62°C (ACE2) for 20 s, using Lightcycler 2.0 Real-Time PCR Instrument (Roche, Rotkeuz, Switzerland). After amplification, a melting curve analysis was performed by collecting fluorescence data while increasing the temperature from 65 to 95°C over 300 s. The cycle threshold values were normalized to the expression levels of β-actin.
Statistical Analysis Values are expressed as means ± SD. One-way ANOVA and Tukey’s post hoc test were used for all analyses; P < 0.05 was considered significant. All analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL).
RESULTS Right Ventricular Wall Thickness Echocardiography showed that the right ventricular wall in the low temperature group was thicker than that in the control group (P < 0.01). This was alleviated by treatment with imidapril (P < 0.01; Figure 1).
ELISA
Assessment of PASP
The concentration of Ang II and Ang (1–7) in the RV tissue was measured with the ELISA method. Lung tissue was homogenized and then centrifuged, and the supernatant was collected. The Ang II and Ang (1–7) concentration was measured by using chicken Ang II and Ang (1–7) ELISA kits (Shanghai Xinran Industrial Co. Ltd., Shanghai, China) according to the instructions.
In the control group, no tricuspid regurgitation was observed. Therefore, the PASP could not be calculated. Nevertheless, it was assessed by the sonographer, who indicated that it was at the normal level (<30 mmHg). The PASP in the imidapril group was also at the normal level. The PASP in the low temperature group was high (>40 mmHg; Figure 2).
Immunohistostaining The expression or proliferating cell nuclear antigen (PCNA) was identified by the immunohistostaining SABC (streptavidin biotin complex) method. The right ventricular tissue was incubated in 4% paraformaldehyde for 24 h and embedded in paraffin wax. Sections (4 μm) were rinsed and rehydrated in PBS for 5 min. An immunohistochemical SABC detection kit (Wuhan Boster Bio-Engineering Co. Ltd., Wuhan, China) was used to identify PCNA. Briefly, endogenous peroxidase was inhibited by treatment with 3% H2O2 in PBS for 10 min. Then, blocking solution with 5% BSA was applied to the sections for 15 min at room temperature to avoid nonspecific binding of the biotinylated antibody. Antigen was repaired in boiling folic acid salt buffer (pH 6). Sections were incubated overnight at 37°C for 1 h with primary mouse anti-PCNA antibody (Beijing Biosynthesis Biotechnology Co. Ltd., Beijing, China). Labeling was identified by application of a rabbit anti-mouse IgG/HRP secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd.) at 37°C for 30 min. Peroxidase activity was visualized using a DAB kit (Wuhan Boster Biological Engineering Co. Ltd., Wuhan, China). The reaction was stopped by rinsing in PBS. Finally, the slides were dehydrated, mounted in aqueous-based mounting medium, and examined by light microscopy.
Wet Lung Weight Index The wet lung weight index in the low temperature group increased significantly compared with the control group (P < 0.01). However, it was reversed significant by imidapril treatment (P < 0.01; Table 1).
Right Ventricular Hypertrophy Index Right ventricular hypertrophy index in the low temperature group increased significantly compared with that of the control group (P < 0.01), which was reversed dramatically by imidapril treatment (P < 0.01) (Table 1).
Expression of ACE and ACE2 mRNA in RV Tissue Compared with that in the control group, the ACE mRNA expression in the RV tissue of the low temperature group increased significantly (P < 0.05). Imidapril can effectively reverse this change (P < 0.05) (Table 2). The ACE2 mRNA expression in the RV tissue of the low temperature group increased by 36% compared with that in the control group (P > 0.05). The ACE2 mRNA expression in the RV of the imidapril group was lower than that of the low temperature group (P < 0.01; Table 2).
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Figure 1. Imidapril reduced the right ventricular wall thickness induced by low ambient temperature in broiler chickens. (A) The right ventricular wall thickness of chickens was measured by echocardiography under anesthesia using pentobarbital (40 mg∙kg−1). (B) Histograms represent right ventricular wall thickness (mm). Data are presented as the mean ± SD (n = 3 in each group). **P < 0.01 compared with the control group and imidapril group (one-way ANOVA). Color version available in the online PDF.
Ang II and Ang (1–7) Concentration in RV Tissue The Ang II concentration in the RV tissue of the low temperature group was higher than that in the control
group (P < 0.01) and imidapril group (P < 0.01). The Ang (1–7) concentration was lower in the low temperature group than that in the control group (P < 0.01) and imidapril group (P < 0.01; Table 2).
Figure 2. Assessment of the pulmonary arterial systolic pressure (PASP) based on noninvasive transthoracic echocardiography. Pulmonary arterial systolic pressure was determined by measuring the peak systolic pressure gradient of the right ventricle to the right atrium, according to the simplified Bernoulli equation. By adding the mean right atrial pressure to the transtricuspid pressure gradient, one can predict the right ventricle systolic pressure, which approximates the PASP. The PASP was assessed by an experienced sonographer. In the control group, no tricuspid regurgitation was observed. Therefore, the PASP could not be calculated. Nevertheless, it was assessed by the sonographer that it was at the normal level (<30 mmHg). The PASP in the imidapril group was also at the normal level. The PASP in the low temperature group was high (>40 mmHg). Color version available in the online PDF.
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Hao et al. Table 1. The effect of imidapril on BW, wet lung weight index (LI), and right ventricular hypertrophy index (RVHI)1 Item Control Low temperature (12 to 18°C) Imidapril (3 mg/kg)
BW (kg)
LI
RVHI
2.0500 ± 0.3175 1.9878 ± 0.3940 2.1513 ± 0.3721
0.2100 ± 0.4504 0.3788 ± 0.1202** 0.2800 ± 0.3665
0.0050 ± 0.00043 0.0064 ± 0.00146** 0.0047 ± 0.00071
1Right ventricular hypertrophy is depicted as the increase in RVHI of the low temperature group compared with the control group and imidapril group. Data are presented as the mean ± SD (n = 8 in each group). **P < 0.01 compared with the control group and low temperature group (one-way ANOVA).
PCNA Expression in Right Ventricular Muscle Cells There was no viewable PCNA expression in right ventricular muscle cells in the control group, low temperature group, and imidapril group (Figure 3).
DISCUSSION Under normal conditions, the RV is a thin-walled, low-pressure pump that is poorly adapted to cope with a high afterload. Rapidly growing broiler chickens have a high metabolic requirement for oxygen that requires a high volume of blood flow through their lungs. The lungs of birds are rigid and fixed in the thoracic cavity. The small capillaries can expand only very little to accommodate increased blood flow. Because the weight percentage of LV to BW decreases as chickens grow, it is possible that pulmonary hypertension caused by insufficient lung capillary capacity or oxygen exchange area results in right ventricular failure and ascites in meat-type chickens (Julian, 1989). In patients with pulmonary arterial hypertension, elevated pulmonary vascular resistance often leads to a rise in RV afterload gradually, allowing a compensatory increase in RV mass, helping to maintain stroke volume and cardiac output. The RV mass is, therefore, an important measurement in pulmonary arterial hypertension patients (Blyth et al., 2011). Transthoracic echocardiography is a widely used safe tool in the evaluation of RV dysfunction (Franco, 2012). The RV free wall thickness can objectively reflect the development of the cardiac hypertrophy and remodeling in the RV (Yang et al., 2009). In this study, RVHI and right ventricular wall thickness was used to assess the right ventricular hypertrophy. According to the wet lung weight index and the noninvasive transthoracic echocardiography, pul-
monary arterial systolic pressure was also assessed. It was found in this study that low ambient temperature successfully induced pulmonary arterial hypertension and right ventricular hypertrophy, which could be effectively reversed by imidapril treatment. At the same time, Ang II decreased, whereas Ang (1–7) concentration increased in the imidapril group. These results suggest that the rennin-angiotensin system in the myocardial tissue is involved in the development of right ventricular remodeling in broiler chickens. We also noticed that ACE2 mRNA expression in the low temperature group increased by 36% compared with the control group and imidapril group. We hypothesize that this might be attributed to the compensatory mechanism that produces more Ang (1–7) to counteract the harmful action of Ang II. Proliferating cell nuclear antigen, a DNA polymerase-δ auxiliary protein, is a nuclear protein necessary for DNA synthesis and for cell cycle progression. When cells are in G0, PCNA mRNA levels are low but rapidly increase in the presence of growth factors, stimulating cells to divide. The induction of PCNA in myocytes may cause these cells to enter the cell cycle and undergo DNA synthesis and nuclear mitotic division. Because PCNA is expressed in the late G1 phase and in the S-phase of the cell cycle, positive PCNA staining indicates the activation of cells (Nakamura et al., 2010). In this study, we did not find the PCNA expression in myocardial cells of the RV, which suggests that PCNA might not be involved in the right ventricular remodeling induced by low ambient temperature in broiler chickens. In our previous pulmonary hypertensive rat model induced by monocrotaline, PCNA was expressed in myocardial cells (Yang et al., 2009). The difference in PCNA expression in myocardial cells might be due to the difference in models. The possible molecular mechanism needs further study.
Table 2. The effect of imidapril treatment on ACE/ACE2 mRNA expression and Ang II/Ang (1–7) concentration in the right ventricle tissue1 Group
ACE mRNA
ACE2 mRNA
Ang II (ng/L)
Ang (1–7; ng/L)
Control Low temperature (12 to 18°C) Imidapril (3 mg/kg)
9.95 ± 1.3 18.16 ± 8.8† 8.87 ± 4.9*
9.75 ± 3.98 13.23 ± 2.18 8.80 ± 3.54**
7.89 ± 1.38 14.25 ± 3.25‡ 5.97 ± 1.56**
48.21 ± 4.44 32.12 ± 3.45‡ 52.14 ± 15.61**
1Data
are presented as the mean ± SD (n = 8 in each group). †P < 0.05, ‡P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the low temperature group (one-way ANOVA).
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Figure 3. Effect of imidapril on proliferating cell nuclear antigen expression in right ventricular muscle cells of pulmonary hypertensive broiler chickens induced by low ambient temperature. Bar line = 20.0 µm. Color version available in the online PDF.
In conclusion, imidapril can inhibit right ventricular remodeling induced by low ambient temperature in broiler chickens.
ACKNOWLEDGMENTS This research was supported by Doctoral Scientific Research Foundation of Henan University of Science and Technology (Nº 09001575) and Project of Henan Science and Technology (Nº122300410234). We also express our thanks to Jian-Xiang Zhang from Third Military Medical University, Chongqing, China.
REFERENCES Arcasoy, S. M., J. D. Christie, V. A. Ferrari, M. S. Sutton, D. A. Zisman, N. P. Blumenthal, A. Pochettino, and R. M. Kotloff. 2003. Echocardiographic assessment of pulmonary hypertension in patients with advanced lung disease. Am. J. Respir. Crit. Care Med. 167:735–740. Baghbanzadeh, A., and E. Decuypere. 2008. Ascites syndrome in broilers: Physiological and nutritional perspectives. Avian Pathol. 37:117–126. Blyth, K. G., J. Kinsella, H. Nina, N. Hakacova, L. E. McLure, A. M. Siddiqui, G. S. Wagner, and A. J. Peacock. 2011. Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI. Pulm. Circ. 1:470–474. Capettini, L.S., F. Montecucco, F. Mach, N. Stergiopulos, R.A. Santos, and R.F. Da Silva. 2012. Role of renin-angiotensin system in inflammation, immunity and aging. Curr. Pharm. Des. 18:963–970. Durik, M., R. Van Veghel, A. Kuipers, R. Rink, M. Haas Jimoh Akanbi, G. Moll, A. H. Danser, and A. J. Roks. 2012. The effect of the thioether-bridged, stabilized angiotensin-(1–7) analogue cyclic ang-(1–7) on cardiac remodeling and endothelial function in rats with myocardial infarction. Int. J. Hypertens. 2012: 536426. http://dx.doi.org/10.1155/2012/536426. Franco, V. 2012. Right ventricular remodeling in pulmonary hypertension. Heart Fail. Clin. 8:403–412. http://dx.doi.org/10.1016/j. hfc.2012.04.005.
Gallagher, P. E., K. Cook, D. Soto-Pantoja, J. Menon, and E. A. Tallant. 2011. Angiotensin peptides and lung cancer. Curr. Cancer Drug Targets 11:394–404. Jia, L., Y. Li, C. Xiao, and J. Du. 2012. Angiotensin II induces inflammation leading to cardiac remodeling. Front. Biosci. 17:221–231. Julian, R. J. 1989. Lung volume of meat-type chickens. Avian Dis. 33:174–176. Julian, R. J. 2000. Physiological, management and environmental triggers of the ascites syndrome: A review. Avian Pathol. 29:519–527. http://dx.doi.org/10.1080/03079450020016751. Ma, L., R. Gul, J. Habibi, M. Yang, L. Pulakat, A. Whaley-Connell, C. M. Ferrario, and J. R. Sowers. 2012. Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the transgenic (mren2) rat. Am. J. Physiol. Heart Circ. Physiol. 302:H2341–H2351. Nakamura, H., M. Tokumoto, M. Mizobuchi, C.S. Ritter, J. L. Finch, M. Mukai, and E. Topolsky. 2010. Novel markers of left ventricular hypertrophy in uremia. Am. J. Nephrol. 31:292–302. Savoia, C., and M. Volpe. 2011. Angiotensin receptor modulation and cardiovascular remodeling. J. Renin Angiotensin Aldosterone Syst. 12:381–384. Sciomer, S., R. Badagliacca, and F. Fedele. 2005. Pulmonary hypertension: Echocardiographic assessment. Ital. Heart J. 6:840–845. Shrikrishna, D., R. Astin, P. R. Kemp, and N. S. Hopkinson. 2012. Renin-angiotensin system blockade: A novel therapeutic approach in chronic obstructive pulmonary disease. Clin. Sci. (Lond.) 123:487–498. van As, P., M. G. Elferink, A. M. Closter, A. Vereijken, H. Bovenhuis, R. P. Crooijmans, E. Decuypere, and M. A. Groenen. 2010. The use of blood gas parameters to predict ascites susceptibility in juvenile broilers. Poult. Sci. 89:1684–1691. http://dx.doi. org/10.3382/ps.2009-00430. Wideman, R. F., Jr., and K. R. Hamal. 2011. Idiopathic pulmonary arterial hypertension: An avian model for plexogenic arteriopathy and serotonergic vasoconstriction. J. Pharmacol. Toxicol. Methods 63:283–295. Yang, D. L., H. G. Zhang, Y. L. Xu, Y. H. Gao, X. J. Yang, X. Q. Hao, M. Su, X. Q. Wang, and X. H. Li. 2009. Galphaq-protein carboxyl terminus imitation polypeptide (GCIP)-27 inhibits right ventricular hypertrophy induced by monocrotaline in rats. Biol. Pharm. Bull. 32:376–381. Zhou, X., C. Li, W. Xu, and J. Chen. 2012. Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress, apoptosis, and inflammation. PLoS ONE 7:e40424.
and ACE2 mRNA in the right ventricular myocardial tissue was quantified by real-time PCR. Proliferating cell nuclear antigen-positive cells were detected by immunohistostaining. The concentration of angiotensin (Ang) II and Ang (1–7) in the right ventricular myocardial tissue was measured with ELISA. The results showed that right ventricular hypertrophy index, wet lung weight index, pulmonary arterial systolic pressure, expression of ACE mRNA in the right ventricular tissue, Ang II concentration, and the thickness of the right ventricular wall in the low temperature group increased significantly compared with those in the control group and imidapril group. The ACE2 mRNA expression increased 36%, whereas Ang (1–7) concentration decreased significantly in the low temperature group compared with that in the control group and imidapril group. In conclusion, imidapril inhibits right ventricular remodeling induced by low ambient temperature in broiler chickens.
Key words: imidapril, broiler chicken, low temperature, right ventricular remodeling, pulmonary hypertension 2013 Poultry Science 92:1492–1497 http://dx.doi.org/10.3382/ps.2012-02671
INTRODUCTION Pulmonary arterial hypertension syndrome, also known as ascites syndrome, is a metabolic disorder found in modern broiler chickens that have insufficient pulmonary vascular capacity. Broiler chickens typically hatch at a weight of 40 g and can grow to 4 kg, doubling in weight almost 7 times within 8 wk. The extremely rapid early growth performance of broilers imposes proportional challenges to their developmentally immature ©2013 Poultry Science Association Inc. Received August 12, 2012. Accepted January 15, 2013. 1 These authors contributed equally to this article. 2 Corresponding author: [email protected]
pulmonary and cardiovascular systems (Wideman and Hamal, 2011). Ascites syndrome especially occurs under conditions such as high altitude or low ambient temperatures because of the higher metabolic rate and increased blood viscosity (Julian, 2000; Baghbanzadeh and Decuypere, 2008; van As et al., 2010). Pulmonary arterial hypertension is characterized by high pulmonary vascular resistance and vascular remodeling, which results in a striking increase in right ventricle (RV) afterload and subsequent failure (Franco, 2012). Ventricular remodeling, defined as changes in size, shape, and function of the heart in response to cardiac injury or increased load, correlates with the development and progression of heart failure (Ma et al., 2012; Zhou et al., 2012). The RV is in charge of pump-
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ing blood to the lungs for oxygenation. Right ventricular remodeling is associated with pulmonary arterial hypertension (Yang et al., 2009; Franco, 2012). Studies showed that RV function should be recognized as an important contributor and prognostic indicator of cardiopulmonary diseases (Franco, 2012). The process of ventricular remodeling is largely influenced by hemodynamic load, neurohumoral activation, and additional factors such as endothelin, cytokines, nitric oxide production, and oxidative stress (Ma et al., 2012; Zhou et al., 2012). Evidences have showed that angiotensin (Ang) II induces inflammation, leading to cardiac remodeling (Savoia and Volpe, 2011; Jia et al., 2012). On the other hand, the thioether-bridged, stabilized angiotensin-(1–7) analog cyclic ang-(1–7) exerts beneficial effect on the cardiac remodeling and endothelial function in rats with myocardial infarction (Capettini et al., 2012; Durik et al., 2012). The Ang II is a key effector peptide of the rennin-angiotensin system in regulating blood pressure, homeostasis, and cell proliferation. The Ang-(1–7) is a heptapeptide with antiproliferative and vasodilating properties, being a contradiction to Ang II. Imidapril is an ACE inhibitor that can prevent the formation of Ang II and the degradation of Ang-(1–7) and bradykinin, leading to a decrease in blood pressure and cell proliferation (Gallagher et al., 2011; Shrikrishna et al., 2012). Therefore, we hypothesize that imidapril might provide beneficial effect on the right ventricular remodeling. The present study was designed to explore the effect of imidapril on the right ventricular remodeling in broiler chickens.
MATERIALS AND METHODS Birds Broiler chickens at 13 d of age were purchased from Luoyang Chicken Alliance (Luoyang, China). All birds had ad libitum access to feed and water. Twenty-four chickens were randomly divided into 3 groups (n = 8): the control group, low temperature group, and imidapril group. Chickens in the control group were raised at normal temperature, whereas chickens in the low temperature group and imidapril group were exposed to low ambient temperature (12 to 18°C) from 14 d of age until 45 d of age. Meanwhile, chickens in the imidapril group were gavaged with imidapril (Tianjin Tanabe Pharmaceutical Co. Ltd., Tianjin, China; approved by the enterprise: H19990153) at 3 mg/kg once daily for 30 d. At the age of 45 d, chickens were killed and samples were collected. The present study was conducted in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (http://grants1.nih.gov/ grants/olaw/) and was approved by the local animal ethics committee at Henan University of Science and Technology.
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Transthoracic Echocardiography Transthoracic echocardiography was used to observe the ventricular wall thickness. Broiler chickens were anesthetized with pentobarbital (40 mg/kg). A layer of acoustic coupling gel was applied to the thorax, and 2-dimensional echocardiography was performed using a commercially available 12-MHz linear-array transducer system (IE33-S12-MHz, Philips, Hamburg, Germany). All echocardiograms were obtained by an experienced sonographer. Images were stored digitally using commercial software (Prosolv Cardiovascular, Indianapolis, IN,) and reported by a senior echocardiologist. Pulmonary arterial ultrasonic imaging was obtained, and pulmonary arterial systolic pressure was assessed. Pulmonary arterial systolic pressure (PASP) was determined by measuring the peak systolic pressure gradient of the RV to the right atrium, according to the simplified Bernoulli equation. By adding the mean right arterial pressure to the transtricuspid pressure gradient, one can predict the RV systolic pressure, which approximates the PASP (Arcasoy et al., 2003; Sciomer et al., 2005). The PASP was assessed by an experienced sonographer.
Evaluation of Wet Lung Weight Index At the age of 45 d, the BW and wet lung weight (wW) of chickens was measured. The wet lung weight index (LI) was calculated using the formula LI = wW/ BW.
Evaluation of Right Ventricular Hypertrophy Index Chickens were killed at 45 d of age. The right and left ventricle (LV) wall as well as ventricular septum (S) were weighed, and right ventricular hypertrophy index (RVHI) was calculated using the formula of RVHI = RV/(LV + S).
Real-Time PCR The expression of ACE and ACE2 mRNA in the RV tissue was assessed by real-time PCR. Total RNA was extracted from the RV tissue using Trizol (Roche Molecular Biochemicals, Mannheim, Germany) and was quantified by measuring absorbance at 260 nm. One microgram of total RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara Biological Engineering Dalian Co. Ltd., Dalian, China) using Thermo Hybaid Px2 thermal cycler (Thermo, Franklin, MA). β-Actin was used as an internal control. The PCR primers used were designed by Sangon Biotech Co. Ltd (Shanghai, China). Chicken nucleotide sequences for ACE, ACE2, and β-actin are as follows: chicken ACE (forward: 5′-AACACAGAGAACGGGGAGGT-3′; reverse: 5′-AG-
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GAAGACAAGTGCCAGTGC-3′); chicken ACE2 (forward: 5′-TTGCTTCACTTCTGGCTTCTC-3′; reverse: 5′-TCCTGGCTGTCTCCTCAGTTA-3′); chicken β-actin (forward: 5′-ACAATGGCTCCGCTATGTG-3′; reverse: 5′-CTTTTGCTCTGGGCTTCATC-3′). Each real-time PCR reaction was carried out in a total volume of 20 μL with SYBR Premix Ex Taq (Takara Biological Engineering Dalian Co. Ltd.) according to the following conditions: 30 s at 95°C, 40 cycles at 95°C for 5 s, 60°C (ACE, β-actin), 62°C (ACE2) for 20 s, using Lightcycler 2.0 Real-Time PCR Instrument (Roche, Rotkeuz, Switzerland). After amplification, a melting curve analysis was performed by collecting fluorescence data while increasing the temperature from 65 to 95°C over 300 s. The cycle threshold values were normalized to the expression levels of β-actin.
Statistical Analysis Values are expressed as means ± SD. One-way ANOVA and Tukey’s post hoc test were used for all analyses; P < 0.05 was considered significant. All analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL).
RESULTS Right Ventricular Wall Thickness Echocardiography showed that the right ventricular wall in the low temperature group was thicker than that in the control group (P < 0.01). This was alleviated by treatment with imidapril (P < 0.01; Figure 1).
ELISA
Assessment of PASP
The concentration of Ang II and Ang (1–7) in the RV tissue was measured with the ELISA method. Lung tissue was homogenized and then centrifuged, and the supernatant was collected. The Ang II and Ang (1–7) concentration was measured by using chicken Ang II and Ang (1–7) ELISA kits (Shanghai Xinran Industrial Co. Ltd., Shanghai, China) according to the instructions.
In the control group, no tricuspid regurgitation was observed. Therefore, the PASP could not be calculated. Nevertheless, it was assessed by the sonographer, who indicated that it was at the normal level (<30 mmHg). The PASP in the imidapril group was also at the normal level. The PASP in the low temperature group was high (>40 mmHg; Figure 2).
Immunohistostaining The expression or proliferating cell nuclear antigen (PCNA) was identified by the immunohistostaining SABC (streptavidin biotin complex) method. The right ventricular tissue was incubated in 4% paraformaldehyde for 24 h and embedded in paraffin wax. Sections (4 μm) were rinsed and rehydrated in PBS for 5 min. An immunohistochemical SABC detection kit (Wuhan Boster Bio-Engineering Co. Ltd., Wuhan, China) was used to identify PCNA. Briefly, endogenous peroxidase was inhibited by treatment with 3% H2O2 in PBS for 10 min. Then, blocking solution with 5% BSA was applied to the sections for 15 min at room temperature to avoid nonspecific binding of the biotinylated antibody. Antigen was repaired in boiling folic acid salt buffer (pH 6). Sections were incubated overnight at 37°C for 1 h with primary mouse anti-PCNA antibody (Beijing Biosynthesis Biotechnology Co. Ltd., Beijing, China). Labeling was identified by application of a rabbit anti-mouse IgG/HRP secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd.) at 37°C for 30 min. Peroxidase activity was visualized using a DAB kit (Wuhan Boster Biological Engineering Co. Ltd., Wuhan, China). The reaction was stopped by rinsing in PBS. Finally, the slides were dehydrated, mounted in aqueous-based mounting medium, and examined by light microscopy.
Wet Lung Weight Index The wet lung weight index in the low temperature group increased significantly compared with the control group (P < 0.01). However, it was reversed significant by imidapril treatment (P < 0.01; Table 1).
Right Ventricular Hypertrophy Index Right ventricular hypertrophy index in the low temperature group increased significantly compared with that of the control group (P < 0.01), which was reversed dramatically by imidapril treatment (P < 0.01) (Table 1).
Expression of ACE and ACE2 mRNA in RV Tissue Compared with that in the control group, the ACE mRNA expression in the RV tissue of the low temperature group increased significantly (P < 0.05). Imidapril can effectively reverse this change (P < 0.05) (Table 2). The ACE2 mRNA expression in the RV tissue of the low temperature group increased by 36% compared with that in the control group (P > 0.05). The ACE2 mRNA expression in the RV of the imidapril group was lower than that of the low temperature group (P < 0.01; Table 2).
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Figure 1. Imidapril reduced the right ventricular wall thickness induced by low ambient temperature in broiler chickens. (A) The right ventricular wall thickness of chickens was measured by echocardiography under anesthesia using pentobarbital (40 mg∙kg−1). (B) Histograms represent right ventricular wall thickness (mm). Data are presented as the mean ± SD (n = 3 in each group). **P < 0.01 compared with the control group and imidapril group (one-way ANOVA). Color version available in the online PDF.
Ang II and Ang (1–7) Concentration in RV Tissue The Ang II concentration in the RV tissue of the low temperature group was higher than that in the control
group (P < 0.01) and imidapril group (P < 0.01). The Ang (1–7) concentration was lower in the low temperature group than that in the control group (P < 0.01) and imidapril group (P < 0.01; Table 2).
Figure 2. Assessment of the pulmonary arterial systolic pressure (PASP) based on noninvasive transthoracic echocardiography. Pulmonary arterial systolic pressure was determined by measuring the peak systolic pressure gradient of the right ventricle to the right atrium, according to the simplified Bernoulli equation. By adding the mean right atrial pressure to the transtricuspid pressure gradient, one can predict the right ventricle systolic pressure, which approximates the PASP. The PASP was assessed by an experienced sonographer. In the control group, no tricuspid regurgitation was observed. Therefore, the PASP could not be calculated. Nevertheless, it was assessed by the sonographer that it was at the normal level (<30 mmHg). The PASP in the imidapril group was also at the normal level. The PASP in the low temperature group was high (>40 mmHg). Color version available in the online PDF.
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Hao et al. Table 1. The effect of imidapril on BW, wet lung weight index (LI), and right ventricular hypertrophy index (RVHI)1 Item Control Low temperature (12 to 18°C) Imidapril (3 mg/kg)
BW (kg)
LI
RVHI
2.0500 ± 0.3175 1.9878 ± 0.3940 2.1513 ± 0.3721
0.2100 ± 0.4504 0.3788 ± 0.1202** 0.2800 ± 0.3665
0.0050 ± 0.00043 0.0064 ± 0.00146** 0.0047 ± 0.00071
1Right ventricular hypertrophy is depicted as the increase in RVHI of the low temperature group compared with the control group and imidapril group. Data are presented as the mean ± SD (n = 8 in each group). **P < 0.01 compared with the control group and low temperature group (one-way ANOVA).
PCNA Expression in Right Ventricular Muscle Cells There was no viewable PCNA expression in right ventricular muscle cells in the control group, low temperature group, and imidapril group (Figure 3).
DISCUSSION Under normal conditions, the RV is a thin-walled, low-pressure pump that is poorly adapted to cope with a high afterload. Rapidly growing broiler chickens have a high metabolic requirement for oxygen that requires a high volume of blood flow through their lungs. The lungs of birds are rigid and fixed in the thoracic cavity. The small capillaries can expand only very little to accommodate increased blood flow. Because the weight percentage of LV to BW decreases as chickens grow, it is possible that pulmonary hypertension caused by insufficient lung capillary capacity or oxygen exchange area results in right ventricular failure and ascites in meat-type chickens (Julian, 1989). In patients with pulmonary arterial hypertension, elevated pulmonary vascular resistance often leads to a rise in RV afterload gradually, allowing a compensatory increase in RV mass, helping to maintain stroke volume and cardiac output. The RV mass is, therefore, an important measurement in pulmonary arterial hypertension patients (Blyth et al., 2011). Transthoracic echocardiography is a widely used safe tool in the evaluation of RV dysfunction (Franco, 2012). The RV free wall thickness can objectively reflect the development of the cardiac hypertrophy and remodeling in the RV (Yang et al., 2009). In this study, RVHI and right ventricular wall thickness was used to assess the right ventricular hypertrophy. According to the wet lung weight index and the noninvasive transthoracic echocardiography, pul-
monary arterial systolic pressure was also assessed. It was found in this study that low ambient temperature successfully induced pulmonary arterial hypertension and right ventricular hypertrophy, which could be effectively reversed by imidapril treatment. At the same time, Ang II decreased, whereas Ang (1–7) concentration increased in the imidapril group. These results suggest that the rennin-angiotensin system in the myocardial tissue is involved in the development of right ventricular remodeling in broiler chickens. We also noticed that ACE2 mRNA expression in the low temperature group increased by 36% compared with the control group and imidapril group. We hypothesize that this might be attributed to the compensatory mechanism that produces more Ang (1–7) to counteract the harmful action of Ang II. Proliferating cell nuclear antigen, a DNA polymerase-δ auxiliary protein, is a nuclear protein necessary for DNA synthesis and for cell cycle progression. When cells are in G0, PCNA mRNA levels are low but rapidly increase in the presence of growth factors, stimulating cells to divide. The induction of PCNA in myocytes may cause these cells to enter the cell cycle and undergo DNA synthesis and nuclear mitotic division. Because PCNA is expressed in the late G1 phase and in the S-phase of the cell cycle, positive PCNA staining indicates the activation of cells (Nakamura et al., 2010). In this study, we did not find the PCNA expression in myocardial cells of the RV, which suggests that PCNA might not be involved in the right ventricular remodeling induced by low ambient temperature in broiler chickens. In our previous pulmonary hypertensive rat model induced by monocrotaline, PCNA was expressed in myocardial cells (Yang et al., 2009). The difference in PCNA expression in myocardial cells might be due to the difference in models. The possible molecular mechanism needs further study.
Table 2. The effect of imidapril treatment on ACE/ACE2 mRNA expression and Ang II/Ang (1–7) concentration in the right ventricle tissue1 Group
ACE mRNA
ACE2 mRNA
Ang II (ng/L)
Ang (1–7; ng/L)
Control Low temperature (12 to 18°C) Imidapril (3 mg/kg)
9.95 ± 1.3 18.16 ± 8.8† 8.87 ± 4.9*
9.75 ± 3.98 13.23 ± 2.18 8.80 ± 3.54**
7.89 ± 1.38 14.25 ± 3.25‡ 5.97 ± 1.56**
48.21 ± 4.44 32.12 ± 3.45‡ 52.14 ± 15.61**
1Data
are presented as the mean ± SD (n = 8 in each group). †P < 0.05, ‡P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the low temperature group (one-way ANOVA).
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Figure 3. Effect of imidapril on proliferating cell nuclear antigen expression in right ventricular muscle cells of pulmonary hypertensive broiler chickens induced by low ambient temperature. Bar line = 20.0 µm. Color version available in the online PDF.
In conclusion, imidapril can inhibit right ventricular remodeling induced by low ambient temperature in broiler chickens.
ACKNOWLEDGMENTS This research was supported by Doctoral Scientific Research Foundation of Henan University of Science and Technology (Nº 09001575) and Project of Henan Science and Technology (Nº122300410234). We also express our thanks to Jian-Xiang Zhang from Third Military Medical University, Chongqing, China.
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