Pulmonary Hypertension Syndrome in Fast-growing Broilers

Pulmonary Hypertension Syndrome in Fast-growing BroilersThis use up investigated variations of plasma angiotensin II and grammatical constituent reflectivity of renin, angiotensin converting enzyme ( pass with flying colors) and angiotensin II type 1 receptor (AT1R) in the snapper of pulmonary hypertensive chickens. Plasma angiotensin II was significantly increment at 28 years of years (P1R was developmentd at 12 and 42 geezerhood (P1R and champ facsimiles in the unexpended subject matter ventricle of the handle separateing were increased at eld 12 and 42 respectively comp ared to keep backs (P1R informational ribonucleic acids were con factorrly upregulated in gist of chickens developing pulmonary hypertension date commodious reduction of renin/ACE and elevation of AT1R in the discipline way ventricle of heart may involve in dilated cardiomyopathy.Key linguistic communication Renin-angiotensin system, Pulmonary hypertension, Broiler chicken.Introduct ionPulmonary hypertension syndrome (PHS, ascites) in fast-growing broilers is an grand blood pressure within the pulmonary circulation (Baghbanzadeh and Decuypere, cc8). Pulmonary hypertension initiates the incidental development of hypoxemia, obligation-sided congestive heart failure, central venous congestion, cirrhosis of the liver, and assembly of ascitic fluid into the abdominal cavity (Balog, 2003). It is commonly accepted that PHS in fast-growing broilers is a direct put up of righteousness atrioventricular valve insufficiency, ventricular volume overload and right ventricular dilation and failure (Baghbanzadeh and Decuypere, 2008). In PHS, a high vascular resistance repayable to an anatomically unequal to(predicate) pulmonary vascular capacity and excessive vascular tone reflecting an unbalance betwixt pulmonary vasoconstrictors and vasodilators has been demonstrated (Wideman et al., 2013). Many vaso sprightly elements are involved in the pathophysiology of PHS such as norepinephrine, thromboxane,endothelin, serotonin, nitric oxide, prostacyclin and angiotensin II (Teshfam et al., 2006, Hassanpour et al., 2009, Hassanpour et al., 2011, Wideman et al., 2013). Pathophysiologic alterations in the cellular and molecular levels of this syndrome have been noted (Kim and Iwao, 2000, Sato et al., 2012, Hassanpour et al., 2013a, Hassanpour et al., 2013b).Angiotensin II (Ang II) is the central active component of the reninangiotensin system (RAS) that plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the pathophysiology of hypertension, renal disorder and chronic heart failure (Dostal and Baker, 1999). This system is an ever-evolving endocrine system with considerable checks and balances on the production and catabolism of angiotensin peptides most likely due to the manifold puts of angiotensin (Putnam et al., 2012). In the RAS, a precursor peptide, angiotensinogen, is cleaved by renin to form the decapeptide angiotensin I. The dipeptidase angiotensin-converting enzyme (ACE) cleaves angiotensin I to form the octapeptide angiotensin II (Levy, 2004). Ang II, through the activation of specific Ang II types 1 and 2 receptors (AT1R AT2R), regulates cardiac contractility, cell communication, and impulse propagation. In addition, Ang II is involved in cardiac remodeling, growth, and apoptosis (Paul et al., 2006, Ferreira et al., 2008).The concept of a local RAS located in the heart with autocrine and paracrine roles has been sustain in mammals by many studies, particularly with the materialization that elements of the RAS and Ang receptors are present in cardiac tissue (De Mello and Danser, 2000). The object of this study was to determine plasma angiotensin and the relative steps of renin, ACE and AT1R mribonucleic acid carriage in the heart ventricles (right and left) of broiler chickens with pulmonary hypertension experimentally bring on by 3,5,3-l-triiodothyronine (T3). This endocrine increases metabolism via increasing itemize and size of mitochondria and arousal of the cell membrane enzyme Na+-K+ ATPase), thus, increases oxygen consumption and requirement (Griffin and Ojeda, 2000). The increased body demand for oxygen prompts an increase in cardiac offput. high-pitched cardiac output triggers an increase in lung arterial pressure, presumably be character of the first base compliance of the pulmonary vasculature (Balog, 2003).Materials and methodsBirds and wordsA total of 60 One-day old manly Ross 308 broiler chickens were assigned to a control or treatment group (30 birds per group). distributively group was randomly divided into three equal replicates of 10 chickens per pen. The chickens were reared for septet weeks on wood shavings under regular conditions and provided ad libitum access to pissing and a standard ration (Starter 12.6 MJ metabolisable energy (ME)/kg of diet, 230 g/kg crude protein (CP), Grower 13.2 MJ ME/kg of diet, 210 g/kg CP, Finisher 13.4 MJ ME/kg of diet, 190 g/kg CP formulated) to meet requirements for broilers . In the treatment group, T3 was include in the basal diets at a niggardliness of 1.5 mg T3/kg after day 6 of rearing period (Hassanpour et al., 2013a). Throughout the study mortality was recorded daily. Those broilers that died during the experimental period were examined for lesions of heart failure and ascites.The institutional Animal Care and Use Committee of Ahvaz and Shahrekord Universities approved all procedures utilize in this study.Assessment of right ventricular hypertrophyAt 12 and 42 d of age, six chickens from from each one group were selected at random, weighed and killed by decapitation. The heart was resected and right ventricle hypertrophy was estimated as described by Teshfam et al. (2006). The ratio of right ventricle to total ventricle (RV/TV) was calculated as an big businessman number of pulmonary hypertension. Chickens with an RV/TV ratio 0.28 were cla ssified as pulmonary hypertensive chickens (Wideman, 2001). The right and left ventricles of the heart were immediately flash-frozen in gas nitrogen and stored at -70C for subsequent ribonucleic acid analysis.RNA extraction and cDNA synthesisIn this experiment, frozen ventricular tissues which had been prepared in the aseptic condition, were homogenized in a liquid nitrogen bath. Total RNA from right (six samples from each group at 12 and 42 old age) and left ventricles (six samples from each group at 12 and 42 days) was extracted by a guanidine / phenol solution (RNx plus, Sinaclon Bioscience, Karaj, Iran). 100 mg of homogenized tissue was prepared in this solution. The homogenate was then mixed with chloroform. The resulting multifariousness was centrifuged (9000 rpm, 4C, 15 min), yielding an upper aqueous phase containing total RNA. Following 100% isopropanol precipitation, the RNA pellet was washed with 75% ethanol. The RNA samples were resuspended in DEPC-treated water. Total RNA was treated with RNase-free DNase (Sinaclon Bioscience, Karaj, Iran) to avoid intricacy of contaminating genomic DNA. RNA was evaluated by agarose gel (1.5%) electrophoresis to determine extracted RNA quality as indicated by discrete 18S and 28S rRNA bands. The come up and quality of RNA were stubborn by spectrophotometry. Only RNA of sufficient purity, having an absorbance ratio (A260/280) greater than 1.9, was considered for synthesis of cDNA.Total RNA was reverse transcribed into cDNA in a short time after extraction (less than 6 hours) exploitation M-MLV reverse transcriptase (Sinaclon Bioscience, Karaj, Iran) as described by Hassanpour et al. (2010). The reverse- organisation (RT) was done in a 20 l volume containing 2 g of extracted RNA, 200 ng random hexamer, 0.5 mM dNTP. This mixture was heated to 65C for 5 min, and 40 u of RNase inhibitor, RT original (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 10 mM DTT and 200 u M-MLV reverse transcriptase were added. This mixture was incubated for 5 min at 25C, followed by 50 min at 38C. The reverse transcription mix was heated to 75C for 15 min to modify the RNA and then stored at -20C.Quantitative real time PCR psychoanalysisIn this study, relative quantification of real time PCR was utilize to measure changes in a broker expression in response to T3 treatment. The levels of renin, AT1R, ACE and -actin transcripts were determined in the six samples of right and left ventricles from each group at 12 and 42 days by real-time reverse transcriptase (RT)-PCR development Eva-Green chemistry (Sinaclon Bioscience, Karaj, Iran). This method requires a suitable internal standard to control for variability between samples and to recipeise the input load of cDNA. -actin was used as an internal standard. Specific underseals of Renin, AT1R, ACE and -actin were designed with Primer-Blast (www.ncbi.nlm.nih.gov/tools/primer fervour/index.cgi?LINK_LOC=blastHome). The expected products of primers in PCR were checked i n Nucleotide-Blast (www.blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastnPAGE_TYPE=BlastSearchLINK_LOC=blasthome) which found no similarity with other chicken genes. Primers are listed in Table 1. PCRs were carried out in a real-time PCR cycler ( rotor Gene Q 6000, Qiagen, USA) in three replicates for each sample of ventricles. 1 l cDNA was added to 4 l colossus Hot Taq Eva-Green Ready Mix (Sinaclon Bioscience, Iran), 0.5 M of each specific primer in a total volume of 20 l. The thermal indite was 95C for 5 min, 35 cycles of 95C for 40 s, 60C for 35 s and 72C for 30 s. At the end of each phase, fluorescence was assessed by the real-time PCR cycler and used for duodecimal objectives. The no-template control and no-reverse transcriptase control were used to check contamination in the PCR reagents. Gene expression entropy were normalized to -actin. Data were analyzed using Rotor Gene- package, version 2.0.2 (build 4) (Qiagen, USA) and LinRegPCR software version 2012.0 (Amsterdam, Neth erland), to give the threshold cycle number and reaction efficiency (Ruijter et al., 2009). Relative transcript levels and fold changes in transcript abundance were calculated using efficiency adjusted Livak methodology (CT method) (Livak and Schmittgen, (2001).Measurement of angiotensin II in plasmaThe six chickens per group at 12, 21, 28, 35 and 42 days were selected for blood collection. Blood samples were placid from the brachial vein in heparinized syringes and centrifuged at 2,500g for 10 min to ascertain plasma. The total make out of Ang II plasma level was quantified by using a commercially available Ang II-EIA kit (catalog No. S-1133, Bachem Chemical Company, Germany) followers the manufacturers instructions. Plasma proteins was precipitated as follows before the use of this kit. Briefly, 1 mL plasma was mixed with 2 mL acetone and centrifuged (10000 rpm, 4C, 10 min). The supernatant was extracted with 4 mL petroleum benzine and left at direction for 30 min. After disc arding the ether phase, the aqueous, lower phase containing the angiotensin was evaporated to dryness at 40C. The dried extracts were redissolved in 0.25 mL bridle buffer (0.1 M Tris-HCl, (pH=7.4), 3 mg/mL bovine serum ovalbumin and 2 mg/mL neomycin sulfate) and stored at -20C prior to assay (Gray and Simon, 1985). The materials for protein precipitation were purchased from Sigma-Aldrich Chemical Co.The Ang II-EIA kit is an in vitro quantitative assay for detecting the angiotensin II peptide based on the principle of a competitive enzyme immunoassay (competitive binding to the Ang II antibody between biotinylated Ang II peptide and peptides in samples). This kit has intra-assay variation Statistical analysisData are equal as mean SE. Comparisons were made using an independent sample t-test between each treatment and its control. Statistical analysis was done using SPSS-16 software (SPSS Institute Inc.). All data were checked to have a normal distribution and log transformed if necessary. Any data requiring log version were back-transformed for presentation of data. P values less than 0.05 were considered significant.ResultsEstimation of right ventricular hypertrophyThe RV/TV ratio was greater in the treated groups at 42 days of age (0.303 0.021) than controls (0.215 0.017) (P=0.004), while this ratio was not significant at 12 days (control 0.154 0.014 treatment 0.171 0.012) (P=0.091). The increase of RV/TV ratio was 29% at 42 days. The clinical signs of ascites was apparent in the most treated chickens at the end of rearing period. behavior of renin, AT1R and ACE genes in the right and left ventriclesReal-time PCR results of renin, ACE and AT1R genes are shown in Figs. 13. The expression of -actin was detected in all samples. The renin, AT1R and ACE genes were expressed in the right and left ventricles of control and T3-treated broilers at 12 and 42 days of age. The relative descend of renin mRNA expression in the right ventricle of the treated gr oups was significantly increased at 12 days (15.5 fold) (P=0.009) and decreased at 42 days (4 fold) of age compared to controls (P=0.012 Fig. 1).The relative amount of ACE mRNA expression in the left ventricle of the treated group was significantly increased (9 fold) at 42 days of age compared to controls (P=0.008), but did not differ at 12 days of age (Fig. 2). In the right ventricle, the expression of this gene was increased (2.9 fold) at 12 days (P=0.031) while decreased (3 fold) at 42 days of age in the treated group compared to control (P=0.024).The relative amount of AT1R mRNA expression in the right ventricle of the treated group was significantly increased at 12 (5.9 fold) (P=0.036) and 42 (3.7 fold) (P=0.044) days of age compared to control. In the left ventricle of the treated group, the mRNA amount of this gene was only higher (3.9 fold) at 12 days of age than control (P=0.043 Fig. 3).Assessment of plasma angiotensin IIThe level of Ang II was deliberate in plasma samples of chickens at 12, 21, 28, 35 and 42 days of age. The amount of Ang II was significantly increased in T3-treated chickens only at 28 days of age when compared with control (P=0.041 Fig. 4).In this study, total mortality was 23.3% for treatment group and 3.3% for control group.DiscussionIn the present study, the effect of T3 hormone was observed at 42 days of age which increased cardiac index (i.e., RV/TV 0.28). According to Wideman (2001), this high cardiac index is associated with sustained pulmonary hypertension (significant high blood pressure of pulmonary artery and right ventricle). In T3-treated chickens of our experiment, cardiac index was not critically high to be noticed as pulmonary hypertension at 12 days of age. Thus, any alterations in cardiac RAS gene expression at this age were not related to this syndrome (Klein and Danzi, 2007, Vargas et al., 2012). Ang II, apart from its effect of elevating arterial pressure, exerts mitogenic and growth promoting effects on cardi ac myocytes both of these effects contribute to the development congestive heart failure (Varagic and Frohlich, 2002). In our experiment, the amount of plasma Ang II considerably was higher at 28 days of age and so at this time of rearing period could be critical in the incidence of PHS, as previously suggested by Hassanpour et al. (2011). However, our data showed that Ang II may be involved as an important factor in the induction of PHS, but its role in the development of this syndrome and heart dilation is not predominant, versus PHS in mammals (Wollert and Drexler, 1999). It must be noticed that thyrotoxicosis increases degradation of proteins far exceed synthesis (Decuypere et al., 2005). Thus, variation of Ang II amount during rearing period of chickens could be affected by excess T3.At 42 days of age, cardiac index was considerably high to cause heart failure and PHS. It is noticed that this stage could be associated with heart dilation, which may differ cardiomyocytes struct urally and functionally from hypertrophic stage (Lowes et al., 2002, Hassanpour et al., 2013a). Thus, alternations in the expression of mentioned genes in the heart ventricles, particularly in the right ventricle, which was more affected by PHS than the left ventricle, could be due to dilated cardiomyopathy.Renin mRNA has been detected in the heart of various species (Paul et al., 2006). Pieruzzi et al. (1995) described that volume overload of heart was able to increase renin mRNA in the rat heart. In contrast, Iwai et al. (1995) were unable to confirm these findings. In the present study, mRNA variations of this gene were not considerable in the left ventricle of the treated chickens while in the right ventricle, conspicuous increase (12 days) and decrease (42 days) were observed. The initial increase of renin mRNA may be influenced by volume overload of heart due to thyroid hormone while consequent decrease of this gene could be due to occurrence of the PHS. As previously mentione d, the end stage of PHS could be associated with dilated cardiomyopathy of the right ventricle in which cardiomyocytes are unable to contract properly. Apparently, this disability occurs in the expression of many genes (Ladenson et al., 1992, Lowes et al., 2002, Teshfam et al., 2006, Hassanpour et al., 2013b). A reduction of renin mRNA in the right ventricle may be due to negative compensatory feedback of cardiomycytes against high activation of general RAS (high plasma Ang II). It may be also noticed that T3 initially stimulates expression of genes (such as renin) and then, suppresses transcription in long time, similar to its effect on protein (Ruckebusch et al., 1991). Further, the elevation of ACE mRNA might be influenced by thyroid hormone and initial induction of hypertrophy in the heart ventricles, while the reduction of this transcript occurred in the dilated right ventricle at the end stage of PHS. Hao et al. (2013) reported an increase of ACE mRNA and concentration of Ang II in the right ventricular tissue of cold stress-chickens at 42 days of age. This apparent discrepancy between our results and study of Hao et al. (2013) could be due to different routes in the induction of PHS. Comparison of cardiac index in these two studies confirms that induction of PHS with T3 was more severe than cold stress. Probably, the right ventricular remodeling in the cold stress-chickens was not completely progressed. Thus, it could be dianoetic reason for increasing of ACE mRNA and Ang II in the hypertrophic right ventricle.The increasing of AT1R in the heart hypertrophy and heart failure has been confirmed (Barlucchi et al., 2001, Diniz et al., 2007) which is in relative agreement with our findings. Wollert and Drexler (1999) reviewed that AT-receptors-dependent signaling cascades potently shape cardiac myocyte function and growth. They also reported that cardiac hypertrophy in response to haemodynamic overload can occur independently of the AT-receptors.In concl usion, the gene expression of renin, ACE and AT1R was relatively upregulated in the heart of chickens developing PHS. The right ventricle of hearts from pulmonary hypertensive chickens showed considerable reduction of renin, ACE and elevation of AT1R which may be involved in dilated cardiomyopathy.