Pulmonary hypertension (PH), a common complication of cardiopulmonary disease, is characterised by high pulmonary blood pressure, increased pulmonary vascular resistance, pulmonary vascular remodelling, right ventricular hypertrophy and right ventricular failure.1,2 Although the pathogenesis of PH remains unclear, hypoxia has been identified as one of the main factors.3 Right ventricular hypertrophy is a positive response to the early stages of PH, which increases cardiac output. However, chronic stimulation eventually results in decompensation and heart failure. The molecular pathways involved during that pathological progression have not been completely clarified. Several factors are thought to contribute to altered cardiac gene expression, which in turn triggers myocardial cell hypertrophy, apoptosis and fibrosis.4 One of the proteins thought to be involved in this process is phosphatase and tensin homologue on chromosome ten (PTEN).5
The tumour suppressor PTEN is a member of the phosphatase superfamily. It contains tyrosine and a phosphatase that is specific for both phosphorylated tyrosine and serine/threonine substrates and which also acts on dephosphorylated nonprotein substrates including phosphoinositide lipids, mRNA and complex carbohydrates.6,7 The phosphatase activity of PTEN is associated with several cellular processes, including growth, proliferation, survival and migration.8-10
PTEN has been identified as an antioncogene that is inactivated in breast, endometrial, glial, liver and prostate cancers.11-15 It has been recently shown that cardiac enlargement is associated with experimentally induced, cardiomyocyte specific, inactivation of PTEN in mice.5 The present study investigated the role of PTEN in the cardiac response to increased pulmonary vascular resistance in hypoxia induced PH rats.
All procedures undertaken in this study complied with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Healthy adult male Sprague Dawley rats weighing 200 to 250 g were given standard laboratory food and housed in a standard environment.
Pathogen free, adult male Sprague Dawley rats were randomly divided into an untreated control group and five hypoxic groups (n=12 per group). The rats in the hypoxic groups were exposed to normobaric hypoxia for 1, 3, 7, 14 or 21 days. Hypoxic conditions were obtained by placing rats in a ventilated Plexiglas chamber with the oxygen level maintained at (10.0±0.1)% with a mixture of nitrogen and oxygen gases.16 The chamber environment was monitored using an oxygen analyzer. Soda lime was used to absorb CO2. The chamber was opened every other day for 30 minutes to replenish food and water and cleaning the cages.
Haemodynamic measurement and right ventricle hypertrophy index
The rats were anaesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). For estimation of right ventricular hypertrophy, the right ventricular systolic pressure was measured via catheterization of pulmonary artery and a miniature pressure transducer (Millar Instruments, USA) and results recorded by a polygraph system (AD Instruments, Australia). The hearts were excised, dissected and weighed to determine right ventricular hypertrophy index (RVHI) as the ratio of right ventricle (RV) to left ventricle (LV) plus septum.17
Histomorphometry of right ventricle
The hearts were fixed with 10% buffered formalin (pH 7.4) and embedded in paraffin. Tissues were cut into 5 μm thick sections and stained with hematoxylin and eosin (measurement of cardiomyocyte cross sectional area) and picrosirius red (measurement of collagen volume fraction and quantified using image analysis software as previously described18,19).
Real time PCR analysis
The total RNA from each ventricle was isolated using Trizol reagent (Takara Biotechnology Inc., Japan). The RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara Biotechnology Inc.). The cDNA was amplified by real time fluorescent quantitative PCR and the 2ΔΔCT method using SYBR Premix Ex Taq (Takara Biotechnology Inc.) on an ABI StepOne System (Applied Biosystems, USA).
The following primers were used for PTEN: 5′AGCTTGTCCTCCCGTCGT3′ (forward primer) and 5′CCAGTCAGAGGCGCTAT3′ (reverse primer). The primers for β-actin were: 5′TCATCACTATCGGCAATGA3′(forward primer) and 5′CACTGTGTTGGCATAGAGGT 3′ (reverse primer) used as an endogenous control.
Western blotting analysis
Right and left ventricular homogenates were prepared in radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, China) containing a mixture of protease inhibitors. Equal amounts of protein (50 μg per lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) before being transferred to polyvinylidene difluoride membrane for Western blotting analysis. The primary rabbit polyclonal antibodies against PTEN, phosphorylated PTEN (p-PTEN) and β-actin (Santa Cruz Biotechnology Inc., USA) were used to probe the membranes with horseradish peroxidase conjugated goat antirabbit antibodies (Stressgen Biotechnologies Corporation, Canada) before detection with the ECL Plus chemiluminescence kit (Thermo Fisher Scientific Inc., USA). The luminescent signals were quantified using a Quantity One system (Bio-Rad Laboratories Inc., USA). Experiments were done in triplicate.
Statistical analysis was performed using SPSS 16.0 (SPSS Inc., USA). The results were expressed as mean±standard deviation (SD). Analysis of variance was used to analyze between group differences. Values of P <0.05 were considered statistically significant.
Pathological hypertrophy and right ventricular remodelling
Right ventricular systolic pressure significantly increased in a time dependent manner in rats exposed to hypoxia compared to that in control rats, reaching a maximum of (50.1±1.3) mmHg on day 21 compared with (23.8±0.8) mmHg in the control group (Figure 1A). There was a significant parallel increase in hypertrophy index from day 3 to day 21 of hypoxic exposure, reaching levels one and a half times higher than in the normoxic group on day 21 (Figure 1B). These findings are indicative of right ventricular hypertrophy.
Histologically, an increase in the right ventricular size was observed (Figure 2A) in hearts from hypoxic rats. The hypertrophy appeared as structural changes resulting from myocardial cell hypertrophy and interstitial fibrosis of right ventricle after hypoxic exposure for 14 days (Figure 2B and 2D). The cardiomyocyte cross sectional area and collagen volume fraction increased significantly compared with the control group (Figure 2C and 2E).
PTEN and phosphorylated PTEN levels in the right and left ventricle
Real time PCR results showed no statistically significant differences in relative PTEN transcript expression between hypoxic and control groups in the right or left ventricle (Figure 3). Likewise, Western blotting analysis showed no evidence of differences in PTEN protein expression (55 000) between the hypoxic and control groups in either ventricle (Figure 4A and 4B).
To analyze further the role of PTEN in the hypoxia induced right ventricular hypertrophy, p-PTEN protein expression levels were analyzed. As shown in Figure 4C, there was a time dependent increase in right ventricular p-PTEN expression after exposure to hypoxia. The between-group differences were statistically significant on days 3, 7, 14 and 21. No changes were seen in p-PTEN protein levels in left ventricular tissue.
Cardiomyocytic hypertrophy is a characteristic response to pulmonary pressure overload in chronic lung, heart and sleep diseases.5,20,21 Pathological right ventricular remodelling and hypertrophy are associated with the risk of dying. However, the roles of molecular pathways in this process are poorly understood. To clarify further the signal pathway in the basic pathological process, our study provides the documented evidence that PTEN is involved in the development of pathological right ventricular hypertrophy in response to PH. Over the three weeks of normobaric hypoxia, adult rats exhibited significant PH and right ventricular hypertrophy in a time dependent manner. We detected the expression and phosphorylation levels of PTEN in ventricles of the rats simultaneously. The mRNA and protein levels of PTEN in hypoxic rats remained unchanged, but the phosphorylation level of PTEN was increased in RV after hypoxic exposure. This trend was consistent with the findings for systolic pressure and hypertrophy index. Thus, p-PTEN participates in the process of pathological, right ventricular hypertrophy and remodelling in response to increased pulmonary vascular resistance.
Hypertrophy and hyperplasia of cardiomyocytes with increasing accumulation of extracellular matrix were found in RV of the rats further demonstrating that the structural remodelling of ventricle are pathological changes developing (hypoxic) PH. The pathological remodelling and hypertrophy is a result of an imbalance of hyperplasia and apoptosis in cells of the ventricular wall. It has been shown that PTEN is a downregulator of phosphoinositide 3-kinase (PI3K) signalling in myocardial, vascular smooth muscle and endothelial cells.22 This down-regulation is associated with several aspects of cellular function, including survival, growth, metabolism and apoptosis.
It has been reported recently that disruption of PTEN in the heart results in physiological hypertrophy, characterized by increased width and length of myocardial cells,5,18 however, there was no evidence of fibrotic changes or decompensation, even though Akt protein kinase B (PKB) was activated. Therefore, PTEN acts to prevent heart hypertrophy by blocking the growth factor signalling pathway.
However, other studies have proposed that PTEN plays a negative role in myocardial hypertrophy. Consistent with the key role of Akt/PKB in cell survival, increases in PTEN activity have been associated with enhanced apoptosis of cardiovascular cells.22 Apoptosis has been also associated with increased PTEN expression in neonatal cardiomyocytes.5 Another study23 suggested that increased basal phosphorylation of Akt/PKB increases glycogen synthase kinase-3β activity and contributes to decreased apoptosis in PTEN mutant mice.
Loss of PTEN has been also shown to be associated with enhanced expression levels of vascular endothelial growth factor-A (VEGF-A) and angiopoictin-2 (Ang-2) mediated via mammalian target of rapamycin (mTOR) pathway.24,25 Both of these factors are thought to be necessary for coronary angiogenesis as they increase coronary flow and thereby reduce ischaemic injury during the process of cardiomyocytic hypertrophy. This in turn protects against haemodynamic stress overload.23 In addition to regulating the balance between cardiomyocytic hypertrophy, growth, apoptosis and myocardial blood flow, PTEN might be also associated with myocardial energy metabolism in an attempt to protect myocardial cells against pathological hypertrophy.26,27
Several further studies have confirmed that loss of PTEN in heart muscle has a negative effect on the heart rate and basal myocardial contractility,28 both of which are associated with heart failure. However, the oxygen demand also decreases during this process. Perhaps then, negative inotropic effect increases resistance to ischaemic injury by reducing the myocardial oxygen consumption.
The data presented above indicate that multiple mechanisms are involved in the regulation of Akt signalling by PTEN in response to different stimuli. As a complement to findings reported using PTEN knock out models, our results indicate that right ventricular hypertrophy was not significantly correlated with changes in PTEN expression. However, it was related to increased levels of phosphorylated PTEN. Phosphorylation of PTEN changes its conformation thereby suppressing activity of PTEN by controlling the formation of complexes which include PTEN.29 Thus, changes in PTEN activity are involved in right ventricular hypertrophy. However, the pathways responsible for hypertrophy in hypoxia induced, hypertensive rats might be different from those activated therapeutically by medications such as isoprenaline and G protein-coupled receptor (GPCR) agonists, which positively regulate the PTEN mRNA and protein levels.30
A number of studies suggested that regulation of PTEN initiates or prevents cardiac hypertrophy via different downstream pathways in different cardiovascular physiological processes and diseases. As mentioned in the introduction, the primary objective of our study was to confirm the involvement of PTEN in pathological right ventricular hypertrophy in a hypoxic rat model. Our findings indicated that phosphorylation of PTEN was markedly upregulated in the hypertrophic right ventricular tissue of hypoxic rats. Future research should focus on investigating the role of the PTEN/Akt pathway and establishing whether PTEN inhibitors or activators would provide beneficial agents for preventing right ventricular hypertrophy caused by PH.
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