Impact of ATP synthase/coupling factor 6 in hypoxic pulmonary arterial hypertension: An experimental rat model

Background/aim Hypoxia-induced pulmonary arterial hypertension (PAH) is characterized by prostacyclin (PGI2) disorder, which manifests in the same manner as in monocrotaline (MCT)-induced PAH. Endogenous PGI2 inhibitor coupling factor 6 (CF6) is involved in MCT-induced PAH. This study aimed to explore the presence or absence of a correlation between hypoxia-induced PAH and CF6. Materials and methods This study was conducted between January 2019 and June 2020. A total of 135 male Wistar rats (aged 8 weeks and weighing 200–250 g) were randomly divided into five groups: (A) control, (B) 1 week of hypoxia, (C) 2 weeks of hypoxia, (D) 3 weeks of hypoxia, and (E) 4 weeks of hypoxia. CF6 expression in both lung tissue and blood samples from the lung vasculature and tail vein was measured by western blotting, immunohistochemistry, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. Results Hemodynamic and morphological changes in hypoxia-induced rats indicated PAH development. The results showed the presence of a correlation between the mRNA and protein levels of CF6 in lung tissue, activity of mitochondrial ATP synthase, and hypoxia time, and there was a significant increment in the group exposed to hypoxia for 4 weeks compared to the control group. The decrement expression of ATPase inhibitory factor 1 (IF 1) mRNA was consistent with the outcomes of ATP synthase activity in lung tissue in the 4 weeks of hypoxia group compared with the control group. However, the levels of CF6 and ATP synthase activity did not differ between blood samples from the lung vasculature and tail vein. Conclusion In hypoxia-induced PAH, CF6 showed downregulated expression in lung tissue, but not in pulmonary vasculature and circulation. Therefore, we speculated that CF6 and ATP synthase may play important roles in hypoxia-induced PAH.


Introduction
Pulmonary arterial hypertension (PAH), a fatal disease with sustained increased pulmonary vascular resistance and abnormal pulmonary vascular remodeling, eventually leads to right-sided heart failure and death. Pathological changes in PAH include destruction of pulmonary vascular cell homeostasis and extensive vascular disease.
Over the past several decades, significant progress has been made in the symptomatic relief of PAH, including the use of endothelin receptor antagonists, vasodilators, and phosphodiesterase inhibitors. However, these approaches do not improve the overall survival rate [1]. Benza et al. demonstrated that the five-year survival rates of patients has remained at 57% [2]. The high morbidity and mortality caused by PAH are still unacceptable [3], and new treatment regimens are needed.
The 6th World Symposium on Pulmonary Hypertension proposed that the hemodynamic definition of PAH should be changed to a mean pulmonary artery pressure (mPAP) of > 20 mmHg [4]. Pathological lesions typically present in PAH patients upon clinical presentation, such as pulmonary artery medial hypertrophy, adventitial thickening, or neointimal proliferation [5]. However, the mechanisms underlying these pathological lesions are not completely understood. Abnormal proliferation and secretion of endothelial cells in PAH increases the synthesis of vasoconstrictors and decreases the synthesis of vasodilators. PAH patients show enhanced levels of potent vasoconstrictors and impaired synthesis of vasodilatory factors, such as prostacyclin (PGI 2 ) [6,7].
Coupling factor 6 (CF6), an essential subunit of the stalk of mitochondrial Adenosine triphosphate (ATP) synthase, was recently identified as a novel endogenous inhibitor of PGI 2 [8]. This is important because the PGI 2 pathway is currently a major target for PAH treatment. CF6 can be released into the blood as a circulating vasoconstrictive peptide after vascular endothelial cells experience mechanical forces, stress, hypoxia, or high glucose levels [9,10]. In addition, the levels of CF6 have been shown to markedly increase in rats with lung disease [11,12]. Our previous experiments have also demonstrated that CF6 was increased in monocrotaline (MCT)-induced PAH and that the inhibition of CF6 alleviated PA remodeling in rats [13]. These results demonstrated the critical role of CF6 in the pathogenesis of MCT-PAH. Hypoxia-induced PAH is also characterized by PGI 2 disorder and PA vasoconstriction and remodeling; however, it is mechanically different from MCT-induced endothelial injury. As an endogenous PGI 2 inhibitor, CF6 may play an important role in hypoxiainduced PAH.
Our previous experiments showed that CF6 was markedly increased in MCT-induced PAH rat models and that the inhibition of CF6 alleviated PA remodeling. The pathophysiological mechanism of PAH caused by MCT differs from that observed in clinical settings. Therefore, we explored the potential role of ATP synthase-CF6 in hypoxia-induced PAH.

Animal models
The animal study was conducted between January 2019 and June 2020 [14]. One hundred and thirty-five male Wistar rats (aged 8 weeks and weighing 200-250 g) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in a standard animal room at a temperature of 21 ± 1 °C, humidity of 55% ± 5%, and 12-h light/dark cycle with free access to water and food. All experimental protocols and procedures used in this study were approved by the Animal Care Committee of Shandong University Affiliated with Qianfoshan Hospital (protocol number: S 030). This study was in accordance with the "Guidelines for the Care and Use of Laboratory Animals" from the National Institutes of Health (NIH Publications No. 8023, revised 1978).
Rats were randomly assigned to one of five possible groups according to the amount of time under hypoxic conditions: group A (n = 15) was the control group; group B (n = 30) included rats exposed to hypoxia (kept in normobaric hypoxia [10% O 2 ] by establishing a setpoint of 10% O 2 in the oxygen controller with N 2 tank and allowed the system to reach a steady state [XBS-03]) for 1 week; group C (n = 30) included rats exposed to hypoxia for 2 weeks; group D (n = 30) included rats exposed to hypoxia for 3 weeks; and group E (n = 30) included rats exposed to hypoxia for 4 weeks [15].
All animals were monitored daily until they developed symptoms of PH, such as weight loss and tachypnea. At weeks 1, 2, 3, or 4, animals were anaesthetized using sodium pentobarbital (30 mg/kg) and intubated using a small animal ventilator (HX-300S; Chengdu TME Technology Co., Ltd., China) at a rate of 60 breaths/min and a tidal volume of 1.1-1.3 mL/100 g. Hemodynamic, morphologic, and biochemical assessments were performed.

Echocardiography and hemodynamic measurements
The rats in the experimental groups were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg). Room temperature was maintained at approximately 25 °C. Cardiac function was evaluated using a Visual Sonics Vevo 770 echocardiographic machine (Visual Sonics, Toronto, Canada) equipped with a 14-MHz linear transducer. An echocardiographic expert (Y.J.) performed measurements in a blinded manner. Short-and long-axis B-dimensional parasternal views of both ventricles at the level of the papillary muscles were acquired to visualize the left ventricle (LV) and right ventricle (RV). Pulmonary artery pressure transduction was conducted with the right jugular vein using a 1.4-F Millar Mikro-Tip catheter transducer (Millar Instruments Inc., Houston, TX, USA) directed to the main pulmonary artery after insertion into the right ventricular outflow duct, although RV systolic pressure (RVSP) was detected with a power laboratory monitoring device (Miller Instruments). Hemodynamic values were accurately computed the LabChart 7.0 physiological data acquisition system (AD Instruments, Sydney, Australia).

Tissue processing and histology
After performing the above tests, cardiac arrest was induced using an overdose of sodium pentobarbital (160 mg/kg body weight). After thoracotomy, the entire lung was excised. The left lung was removed and frozen in liquid nitrogen, and the right lung was inflated with 0.5% low-melting agarose at a constant pressure of 25 cm H 2 O and then fixed in 10% formalin for 24 h. Next, the heart was isolated. The weight ratio of the right ventricle to the left ventricle plus the septum (RV/[LV + S]) was determined using Fulton's index [16].

Western blot
Western blot analysis was performed using a Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China) to isolate proteins, and protein concentration was measured using a bicinchoninic acid (BCA) protein assay reagent kit (Pierce) [17].

ELISA
After high-dose pentobarbital administration, the blood samples obtained from the tail vein and pulmonary vasculature were stored at room temperature for 1 h and were then centrifuged at 3000 rpm at 4 ℃ for 15 min. Plasma samples were collected and stored at −80 ℃. Lung tissue from the left lung was kept at 4 ℃ for 12 h and then at room temperature for 1 h. Lung tissue was rinsed with normal saline. After grinding, the homogenate was adjusted using PBS at a rate of 10%. The supernatant was centrifuged at 5000 × g at 4 ℃ for 15 min. CF6 levels in blood samples from the tail vein and pulmonary vasculature were measured using enzymelinked immunosorbent assay (ELISA) performed with a commercial enzyme immunoassay kit (EIA Assay Design, Inc.; Ray Biotech, Norcross, GA) according to the manufacturer's instructions. The amount of CF6 was expressed in picograms per milliliter of protein.

Determination of mitochondrial ATP synthase activity
Mitochondria were isolated from rat lung tissues using a mitochondrial isolation kit (Abcam, MA, USA, ab110168). ATP synthase activity was assayed spectrophotometrically using a microplate assay kit (Abcam, MA, USA, #ab109714) according to the manufacturer's instructions.

Immunohistochemistry
The right lung lobes were cut and processed as previously described by preparing standard formalin-fixed, paraffinembedded tissues for HE or regular immunohistochemical staining [22]. The tissue samples were sectioned at a thickness of 5 μm [23]. Immunohistochemical analysis involved incubation of the sections with the primary antibody, anti-α-SMA (1:200; Abcam), followed by incubation with a biotinylated secondary antibody, and use of the avidin-biotin complex technique with Vector Red substrate (Vector Laboratories, USA). All images were obtained using an Olympus (Olympus Corporation, Japan) LCX100 Imaging System and analyzed using ImageJ software (version 1.38x; National Institutes of Health) by an expert (L.Y.). In each lung section, small pulmonary arteries (PAs) (50-100 μm in diameter) were analyzed at × 40 magnification in a blinded manner. Medial wall thickness was expressed as the summation of two points of medial thickness/external diameter × 100 (%). Intraacinar (precapillary) PAs (20-30 μm in diameter, 25 vessels each) were assessed for occlusive lesions and rated as follows: grade 0, no evidence of neointimal lesions; grade 1, less than 50% luminal occlusion; and grade 2, more than 50% luminal occlusion [24]. There was no evidence of neointimal lesion formation in any PAs from control rats (all PAs were grade 0).

Statistics
The data are presented as mean ± standard error of the mean (SEM). The values from the two groups were compared using the unpaired t-test. Analysis of variance (ANOVA) was used to compare multiple groups. Subsequently, the Newman-Keuls test was performed. All analyses were performed using SPSS (version 17.0, SPSS Inc. Chicago, IL, USA). Results were considered statistically significant at p < 0.05.

Hemodynamic and morphologic changes in hypoxiaexposed PAH rats
After four weeks of hypoxia exposure, the skin of the rats lost its luster and became very rough, and the degree of dyspnea worsened. Pulmonary vascular remodeling was evaluated by measuring the wall thickness of pulmonary arterioles. After exposure to hypoxia, hemodynamic ( Figure 1a) and morphological (Figure 1b) changes were analyzed at 1, 2, 3, and 4 weeks. RVSP was significantly increased in the 3-and 4-week hypoxia exposure groups compared with that in the other study groups. There were no significant changes observed in the 1-or 2-week hypoxia exposure groups relative to the control group (p < 0.05, Figure 1c). The rats also developed the same increase in right ventricular hypertrophy after 3 weeks of exposure in both the 3-and 4 -week exposure group. This was confirmed by the RV/(LV + S) ratio (p < 0.05, Figure  1d).
The same trend was observed in morphological and hemodynamic changes. Hematoxylin-eosin staining indicated that the wall thickness of vessels was significantly increased in the hypoxia group in a time-dependent manner (Figure 2a). Immunohistochemical analysis revealed increased α-SMA immunostaining in the lung tissue of the hypoxia-induced group over time (Figure 2b). The ratios of vascular medial thickness (i.e. smooth muscle thickness) to outer diameter (total vessel wall thickness) of the small pulmonary arteries (diameter 50-100 μm) and the vascular occlusion score of the small pulmonary arteries (diameter 20-30 μm) in different groups also validated PAH (Figure 2c, Figure 2d).

CF6 level in hypoxia-induced PAH
The expression of CF6 in lung tissue and blood samples from the tail vein of hypoxia-induced PAH rats was examined. Western blotting results for CF6 showed that no significant change was observed in the first 3 weeks, but an almost half-fold change appeared in the 4-week hypoxia group compared with that in the control group (p < 0.05, Figure 3a). Western blot analysis of α-SMA confirmed a 1.7-fold change in CF6 during the development of hypoxia-induced PAH (Figure 3b). Reverse transcription Polymerase Chain Reaction (RT-PCR) was performed to further confirm the mRNA levels of CF6 and showed a significant decrease in the 4-week hypoxia group relative to the control group (p < 0.05, Figure 3c). The level of CF6 in lung tissue was downregulated in a time-dependent manner, and more than a half-fold change was observed in the 4-week hypoxia group relative to the control group (p < 0.05, Figure 3d). The level of CF6 in blood samples from the tail vein ( Figure 3e) and the lung vasculature (Figure 3f) was also analyzed to determine its expression. However, there were no significant difference in CF6 levels in blood samples or the lung vasculature in the 4-week hypoxia exposure group compared with those in the control group.

Mitochondrial ATP synthase activity and CF6 level
CF6 is an essential subunit of mitochondrial ATP synthase that is released into the blood to affect vascular function as a circulating vasoconstrictive peptide. Therefore, we analyzed ATP synthase activity to clarify the relationship between CF6 and mitochondrial ATP synthase. The mitochondrial ATP synthase activity in local lung tissue was downregulated in a time-dependent manner and showed a significant decrease only after 4 weeks of hypoxia (Figure 4a). No significant change in ATP synthase activity was observed in the circulatory level in blood samples from the tail vein (Figure 4b).

Local IF1 level in rats with hypoxia-induced PAH
IF1 is a key regulator of ATP synthase and its subunits, such as CF6. To investigate the influence of IF1 in hypoxic conditions, we examined the expression of IF1 mRNA. The IF1 mRNA expression in lung tissue was significantly lower after four weeks of hypoxia and showed a half-fold change in the 4-week hypoxia group relative to the control group (p < 0.05, Figure 4c). This result is consistent with the ATP synthase activity in lung tissue result, but is contrary to the ATP synthase activity in blood samples from the tail vein.  The mRNA level of lung CF6 was measured using RT-PCR. The level of CF6 in lung tissue (D), blood sample in tail vein, (E) and lung vasculature was measured using ELISA. Data are shown as the mean ± SEM, *p < 0.05 compared with control group (con, control; 1w, 1-week hypoxia group; 2w: 2-week hypoxia group; 3w: 3-week hypoxia group; 4w: 4-week hypoxia group).

Discussion
This study was designed to investigate the role of CF6 in different PAH types. Unlike the increased expression of CF6 in MCT-induced PAH, the expression of CF6 in hypoxia-induced PAH had a different profile. This interesting and unexpected finding demonstrated: 1) in hypoxic PAH, the gene and protein levels of CF6 in lung tissue were significantly lower; 2) no significant change in CF6 levels was observed in blood samples from the tail vein or lung vasculature in hypoxia-induced PAH; and 3) the local mitochondrial ATP synthase activity in the lung tissue showed a significant decrease only after four weeks of hypoxia. No significant changes in mitochondrial ATP synthase activity in blood samples from the tail vein were found.
Significant changes in hemodynamics and morphology confirmed the development of PAH in rats under hypoxic conditions. The expression of α-SMA in the lung tissue of rats exposed to hypoxia was significantly higher, indicating abnormal proliferation of the pulmonary vascular smooth muscle. The etiology of PAH is complex. Hypoxia-induced PAH is characterized by continuously increased pulmonary artery pressure, pulmonary vascular resistance, and RV hypertrophy [25]. Inflammation and smooth muscle cell proliferation may play key roles in this disease. The hypoxia animal model has been widely used to study PAH [26]. We successfully established a model of hypoxia-induced PAH, as demonstrated by the increase in RVSP and proliferation of pulmonary arterial smooth muscle cells (PASMCs).
At present, it is unclear whether the increase in CF6 levels in PAH is related to pulmonary constriction induced by PGI 2 inhibition. Studies have confirmed that CF6 levels are increased in MCT-induced PAH and appear to play an important role in the disease process [27]. This study found no indication of elevated CF6 levels in hypoxic PAH.
Endothelial cell injury is common in all metabolic diseases and MCT-induced diseases. Circulating levels of CF6 increase after endothelial injury. CF6 is produced in the cytoplasm in an immature form (amino acids 1-108) and released into the extracellular matrix in the mature form (amino acids 33-108) after enzymatic deletion of the signal peptide (amino acids 1-32) [28]. CF6 can bind to the subunit of ATP synthase, as ectopic ATP synthase, to accelerate proton import and induce intracellular acidosis [8,12]. Moreover, CF6 can increase blood pressure and enhance angiotensin II-induced vasoconstriction in small arterioles [9,10]. Considerable evidence suggests that CF6 plays an important role in many diseases such as hypertension [29], diabetes mellitus [30], acute myocardial infarction [31], and stroke [32]. In our previous study, local and circulating levels of CF6 were significantly increased in rats with MCT-induced PAH [27].
However, this study found no increase in CF6 expression in blood samples from the tail vein or lung vasculature in hypoxic PAH patients. The mechanism underlying hypoxic PAH is completely different from that of MCT-induced PAH. Hypoxic inhalation without direct cell damage showed no increase in CF6 levels. Because CF6 is localized to the surface of endothelial cells and is released mainly by endothelial cells, we can infer that hypoxia-induced PAH initially causes little injury or rupture of endothelial cells. Hypoxia is a strong stress signal, and the first response organ is the intratracheallung tissue pathway. The changes in CF6 found in the local lung tissue are rational. Oxygen supply closely influences ATP synthesis, and CF6 is an essential component of the stalk of mitochondrial ATP synthase, and it is also released from the cytoplasm to the cell membrane [10,33]. In this study, lung tissue CF6 levels may be modulated by hypoxic stimuli. Furthermore, because of negative regulation by the vasoconstrictor, CF6, we speculate that stress from hypoxia may suppress ATPase and its subunit expression. Our study did indeed indicate a decrease in ATP synthase activity. ATP synthase is a multi-subunit membranebound enzyme of the mitochondria that synthesizes ATP via the proton electrochemical gradient produced by the respiratory chain [34]. The activity of ATP synthase regulates oxidative phosphorylation, and cell death [35,36]. Mitochondrial ATP synthase is the fifth and final component of the oxidative phosphorylation chain [37]. ATP plays a crucial role in adenosine triphosphate (ATP) generation and acts as a chemical fuel for life processes [38]. However, the mechanism of CF6 decrease in hypoxic lung tissue may require further investigation.
ATPase IF1 is a physiological endogenous inhibitor of ATP synthase. IF1 inhibits both the synthase and hydrolase activity of the ATP synthase [39,40]. Walker et al. proposed that IF1 interacts with F1 of the ATP synthase enzyme to inhibit its function [41]. Many recent studies have indicated that IF1 plays important roles in normoxia, such as the inhibition of cell apoptosis, stabilization of the F1F0-ATPase structure, and inner mitochondrial membrane cristae structure [18][19][20]. In many cancers, IF1 is present at higher levels in the brain, colon, ovary and other organs [42-44]. However, the mRNA level of IF1 was downregulated under hypoxic conditions. This result was consistent with the expression of CF6. The codirectional regulation of ATPase-related subunits may allude to the influence of hypoxia on energy metabolism and that CF6 has little effect on hypoxic vasoconstriction.
The limitations of this study are as follows. First, our research lacks three IF1 antagonist interference analyses to confirm the role of IF1 in PAH. Second, other studies on the effect of CF6 in hypoxic PAH in rats must be further investigated. Third, the findings of this study were from rats and could not be projected completely to human beings.

Conclusions
CF6 showed downregulated expression in lung tissue, but not in the pulmonary vasculature or at the circulation level, in hypoxia-induced PAH. We speculate that hypoxic stress from the intratracheal-lung tissue pathway is different from the MCT-endothelial injury pathway in PAH. This study provides new insights into CF6 expression and the pathogenesis of PAH.