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To investigate the effect of T3-induced pulmonary hypertension on genes expression of heat shock proteins Hsp60, Hsp70 and Hsp90 during rearing, quantitative real time-PCR were performed in the heart ventricles.
The Right ventricle / total ventricle ratio (index of pulmonary hypertension) was increased in the treated groups at 12 and 42 days of age compared to controls; the difference was only significant at 42 days (P < 0.05).
The Hsp genes were expressed in the right and left ventricles of control and T3-treated broilers at 12 and 42 days of age. The relative amounts of Hsp60 and Hsp90 genes expression in the right ventricle of treated groups were significantly increased at 12 days and decreased at 42 days of age compared to controls (p < 0.05). Variations of Hsp60 and Hsp90 mRNAs in the left ventricle were not significant. The relative amount of Hsp70 mRNA expression in the right and left ventricles of treated groups was significantly decreased at 42 days of age compared to controls (p < 0.05). Hsp70 mRNA expression did not change in the right and left ventricles of heart at 12 days of age.
It is concluded that heat shock proteins (i.e., Hsp60 and Hsp90) gene expression were upregulated in the heart of chickens developing pulmonary hypertension syndrome, probably to delay pathological process of disease. Heart of pulmonary hypertensive chickens in the right ventricle finally was showed considerable reductions of Hsp60, Hsp70, and Hsp90, which provide evidence of a loss of compensatory responsiveness in dilated heart.
Key words: heat shock protein, pulmonary hypertension, broiler, thyroid hormone.
The heat shock proteins (Hsps) are well known as a family of endogenous, protective proteins. These molecules are located in the cytoplasm / nuclear (e.g., Hsp70 and Hsp90) or mitochondria (e.g., Hsp60) to maintain normal cellular function. The expression of Hsps is induced by a number of stressors, including handling, crating and transport of chickens, hyperthermia, hypertension, and oxidative stress (Al-Aqil, et al., 2013; Gupta and Knowlton, 2002; Zulkifli, et al., 2009). Signals such as reactive oxygen species (ROS), cytotoxic lysosomal enzymes, and cytoskeletal alterations could activate Hsps expression in the cell. Cells initiate a cascade of events that engage Hsps as molecular chaperones, to degrade damaged proteins or repair and facilitate the refolding, assembly, and stabilization of denatured proteins as a defense strategy to ensure cell viability (Benjamin and McMillan, 1998; Chen, et al., 2004; Lanneau, et al., 2008). Accumulative evidences indicated that Hsps suppressed pro-inflammatory cytokines, reduced oxidative bursts, repaired ion channels, protected toxic effect of nitric oxide, modulated immune-mediated injuries, and prevented apoptosis (Benjamin and McMillan, 1998; Snoeckx, et al., 2001). The cascade process of apoptosis could be inhibited by specific Hsps in special stages of this cascade (Latchman, 2001).
Pulmonary hypertension syndrome (PHS) is characterized by elevated pulmonary vascular resistance to an anatomically inadequate pulmonary vascular capacity, excessive vascular tone reflecting the dominance of pulmonary vasoconstrictors over vasodilators, and by vascular remodeling elicited by excessive hemodynamic stresses affecting the terminal pulmonary arterioles. Specific right ventricular hypertrophy has established the central role of elevated pulmonary arterial pressures in the pathogenesis of PHS (Wideman, et al., 2013). Right atrioventricular valve insufficiency, ventricular volume overload, and right ventricular dilation / failure are major subsequent pathogenesis of PHS. Factors such as hypoxia, adrenergic neurotransmitters, TxA2, ET-1, 5HT, respiratory damage or disease are able to increase the pulmonary vascular resistance leading to the pathophysiological progression of PHS (Hassanpour, et al., 2011; Wideman, et al., 2013).
The role of Hsps in cardioprotection has been studied. The induction of these stress proteins in the heart has been observed under various physiopathological disorders, including cardiac ischemia and hypertrophy (Cai, et al., 2010; Lakshmikuttyamma, et al., 2006; Snoeckx, et al., 2001). The objective of this study was to determine relative amounts of Hsp60, Hsp70 and Hsp90 mRNA expression in the right and left ventricles of heart in pulmonary hypertensive broiler chickens induced experimentally by 3,5,3´-l- triiodothyronine (T3).
MATERIALS AND METHODS
Birds and treatments
Sixty, day-old fast-growing chickens from Ross 308 breed were assigned into control and treatment (30 birds per group). The chicks were housed in pens of identical size (1 - 1 m) in a deep litter system, which wood shaving covered the floor. Each group was randomly divided into 3 equal replicates of 10 chicks per pen. Chicks were reared at standard condition for 6 weeks and provided free access to water and a basal diet. The basal diet was in mash form and was formulated for starter (1 to 10 d), grower (11 to 24 d) and finisher (25 to 42 d) broiler growth periods. The metabolisable energy and crude protein of diet in each stage were followed, starter: 29 MJ metabolisable energy (ME)/kg of diet, 220g/kg crude protein (CP); grower: 30 MJ metabolisable energy (ME)/kg of diet, 20g/kg CP; finisher: 31 MJ metabolisable energy (ME)/kg of diet, 18g/kg CP) (NRC, 1994) . For the treatment, T3 was included in the basal diets at a concentration of 1.5 mg/kg T3 after day 6 of rearing (Hassanpour, et al., 2009).
Preparation of heart samples
At 12 and 42 days of age, 6 chicks from each 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 index of pulmonary hypertension. Chickens with RV/TV ratio > 0.29 were accounted as pulmonary hypertensive chickens. The right and left ventricles of heart were immediately frozen in liquid nitrogen and stored at -70 °C for subsequent RNA analysis.
RNA extraction and cDNA synthesis
Total RNA of right and left ventricles of heart was extracted according to the acid guanidinium thiocyanate-phenol-chloroform single-step extraction protocol (Hassanpour, et al., 2009). Total RNA was treated with RNase-free DNase (Sinaclon Bioscience, Iran) to avoid amplification of contaminating genomic DNA. The purified RNA was evaluated by agarose gel electrophoresis. Only RNA samples representing an A260/A280 ratio >1.9 and showing integrity of the RNA by electrophoresis were used for synthesis of cDNA.
Total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Sinaclon Bioscience, Iran) as described by Hassanpour et al. (2009) . The reverse transcription mix was heated to 75 °C for 15 min to denature the RNA and then stored at -20 °C.
Quantitative real time PCR Analysis
The levels of Hsp60, Hsp70, Hsp90, and β-actin transcripts were determined by real time reverse transcriptase (RT)-PCR using Eva-Green chemistry (Sinaclon Bioscience, Iran). To normalize input load of cDNA between samples, β-actin was used as an endogenous standard. Specific primers of Hsp60, Hsp70, Hsp90, and β-actin were designed with Primer-Blast (www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). Primers are listed in table 1. PCRs were carried out in a real time PCR cycler (Rotor Gene Q 6000). 1 µl cDNA was added to the Titan Hot Taq Eva-Green Ready Mix (0.5 µM of each specific primer, and 4 µl of Titan Hot Taq Eva-Green Ready Mix) in a total volume of 20 µl. The thermal profile was 95 °C for 5 min, 35 cycles of 95 °C for 40 s, 62 °C for 45 s and 72 °C for 30 s. At the end of each phase, the measurement of fluorescence was done, and used for quantitative objectives. Data of genes expression were normalized to β-actin. Relative transcript levels were calculated using Rotor Gene Q software, version 2.0.2 (build 4) according to Livak and Schmittgen (2001).
Data are represented as mean ± SE. Comparisons were made for each left and right ventricle using Independent-Sample-t-test (SigmaStat software, Jandel Corp., San Rafael, CA).. P values less than 0.05 were considered statistically significant.
Assessment of right ventricle hypertrophy
The RV/TV ratio was increased in the treated groups at 12 and 42 days of age (12 days: 0.171±0.012; 42 days: 0.303±0.021) compared to controls (12 days: 0.154±0.014; 42 days: 0.215±0.017) but the difference was only significant at 42 days (P < 0.05). This increase was 9.9% at 12 days and 29% at 42 days.
Expression of Hsp genes in the right and left ventricles
Expression of Hsp60, Hsp70, and Hsp90 genes was studied using relative quantitative real time PCR in the right and left ventricles of pulmonary hypertensive broilers (induced by T3) at 12 and 42 days of ages during rearing. PCR results are shown in figures 1-3. The expression of β-actin was detected in all samples. The Hsp60, Hsp70, and Hsp90 genes were expressed in the right and left ventricles of control and T3-treated broilers at 12 and 42days of ages. Overall, The Hsp90 gene was considerably expressed more than other Hsps while the relative amounts of Hsp70 were less than others.
The relative amount of Hsp60 mRNA expression in the right ventricle of treated groups was significantly increased at 12 days and decreased at 42 days of age compared to controls (p < 0.05). Within each group of experiment, Hsp60 mRNA was only decreased in the right ventricle of treated groups during rearing (p < 0.05). Variations of Hsp60 mRNA in the left ventricle were not significant (Fig. 1).
The relative amount of Hsp70 mRNA expression in the right and left ventricles of treated groups was significantly decreased at 42 days of age compared to controls (p < 0.05). Hsp70 mRNA expression did not change in the right and left ventricles of heart at 12 days of age. Within each group of experiment, Hsp70 mRNA was decreased in the right and left ventricles of all groups during rearing (p < 0.05) (Fig. 2).
The relative amount of Hsp90 mRNA expression in the right ventricle of treated groups was significantly increased at 12 days and decreased at 42 days of age compared to controls (p < 0.05). Variations of Hsp90 mRNA in the left ventricle were not significant. Within each group of experiment, Hsp90 mRNA was increased in the left ventricle of all groups during rearing (p < 0.05) while in the right ventricle, it was only decreased in the treated group (p < 0.05) (Fig. 3).
This research investigated mRNA levels of Hsp90, Hsp70, and Hsp60 in the left and right heart ventricles of pulmonary hypertensive broiler chickens. Hypertrophy of the right ventricular wall and subsequent right ventricular dilation are resultant of pulmonary arterial pressure overload, and RV/TV ratio can represent this increased pressure load on the right ventricle. Then, this ratio was used as the most important index in determining right ventricular hypertrophy and progression of PHS (Hassanpour, et al., 2011; Wideman, 2001). At this experiment, according to this index, relative right ventricular hypertrophy was observed in T3-treated chickens at 12 days of age, which was not sign of PHS (RV/TV < 0.29). Thus, any alterations in Hsps gene expression at this age were not related to this syndrome; those are probably due to direct effect of T3 on the cardiomyocytes (Fazio, et al., 2004; Pantos, et al., 2001). Progress of right ventricular hypertrophy was considerable at 42 days, and RV/TV ratio (> 0.29) confirmed that PHS was experimentally induced at this age. It is noticed that this stage of PHS could be in association with heart dilation, which may differ cardiomyocytes structurally and functionally from hypertrophic cardiomyocytes. Thus, alternations in Hsp genes expression of heart ventricles specially right ventricle (more affected) in PHS, could be due to heart dilation.
It has been reported that high molecular Hsps such as Hsp60, Hsp70 and Hsp90 were upregulated immediately in the myocardium after coronary ligation of rats, while these Hsps (except for Hsp60) were not present after the development of congestive heart failure (Dohke, et al., 2006). This report is relatively agreed with our findings. In the present study, Hsp60 and Hsp90 were increased in the early stage of right ventricular hypertrophy (i.e. 12 days of age); However, It is not unclear whether Hsp70 did not differ at this early stage. Yu et al (2008) also reported that Hsp60, Hsp70 and Hsp90 mRNAs in the heart tissue of broilers were upregulated after 2 h of heat stress while reduced quickly with continued heat stress. Apparently, stimulated factors in gene expression of Hsps are time-dependent.
Evaluation of the cardiac expression of five Hsps, including Hsp60, Hsp70, Hsp72, Hsp90, and Hsp27 in human end-stage heart failure showed that only Hsp60 and Hsp27 were increased in dilated and ischemic cardiomyopathy, whereas Hsp70, Hsp72, and Hsp90 were not changed. In contrast to mentioned study, the present study showed that Hsp60, Hsp70, and Hsp90 were decreased in dilated heart of chickens. Cytoprotective functions of Hsps have been confirmed, and in the heart, transgenic overexpression of Hsps protected the heart from ischemic injury (Chen, et al., 2004). In the chickens, this protective function of Hsps were also suggested in different conditions by several studies (Al-Aqil, et al., 2013; Gu, et al., 2012; Hao, et al., 2012; Yan, et al., 2009). As Dokhe et al., (2006) suggested, it is possible that the failure to increase the levels of Hsps, represent a loss of compensatory responsiveness in the setting of heart failure, especially in the PHS of chickens with diminished Hsps gene expression. Diminished Hsps in pulmonary hypertension syndrome might precipitate apoptosis in cardiomyocytes (Gupta and Knowlton, 2002).
Previous studies determined that impaired nitric oxide is a crucial factor in the pathogenesis of chicken PHS (Hassanpour, et al., 2009; Tan, et al., 2007). Koundari et al. (2003) showed that interaction of Hsp90 and nitric oxide synthases could release signiï¬cant amounts of nitric oxide. Thus, impaired nitric oxide in chicken PHS might be due to diminished Hsps.
It has been determined that Hsp60 is located in cardiomyocytes and is rich in mitochondria. This mitochondrial Hsp60 plays a sensitive role in the preservation of mitochondrial integrity, function, and capacity for ATP generation (Yu, et al., 2008). It is thought that reduction of Hsp60 that reported in the present study, may involve in the dysfunction of mitochondria, which was previously described by Jarreta et al. (2000) in dilated cardiomyopathy.
It is concluded that heat shock proteins (i.e., Hsp60 and Hsp90) gene expression were upregulated in the heart of chickens dveloping pulmonary hypertension syndrome, probably to delay pathological process of disease. Heart of pulmonary hypertensive chickens in the right ventricle finally was showed considerable reductions of Hsp60, Hsp70, and Hsp90, which provide evidence of a loss of compensatory responsiveness in dilated heart.