Role of PGC‐1α in fiber type conversion in the palatopharyngeus muscle of OSA patients

Abstract Background Obstructive sleep apnea (OSA) has a high incidence and is harmful to health. It is characterized by repeated collapse of the upper airway. However, the mechanism underlying upper airway collapse is unclear. Methods Patients with OSA and chronic tonsillitis were studied. Pathological changes in palatopharyngeus muscle were detected. The expression of peroxisome proliferator‐activated receptor‐γ co‐activator‐1α (PGC‐1α) and nuclear respiratory factor‐1 (NRF‐1) in muscles was detected by PCR and Western blotting. Immunofluorescence staining was used to detect the expression of type I and type II myofibril. Results The structure of the palatopharyngeus muscle was changed, and the expression of PGC‐1α and NRF‐1 was decreased in the OSA group compared with that in the control group. The expression of PGC‐1α, NRF‐1, and type I myofibril in C2C12 myoblasts was decreased by intermittent hypoxia exposure. The expression of type I myofibril was decreased when knocking down PGC‐1α. Conclusion OSA patients exhibited pathological damage in palatopharyngeus muscle. PGC‐1α was involved in the fiber type conversion in palatopharyngeus muscle caused by intermittent hypoxia.


| INTRODUC TI ON
Obstructive sleep apnea (OSA) has a high incidence of 2%-4% in the population. 1 Chronic intermittent hypoxia (CIH) is the main pathophysiological characteristic of OSA. Current studies have shown that OSA is caused by a combination of many pathological factors, and it is characterized by hypoxia and a series of secondary problems caused by the repeated collapse of the upper airway. 2 The mechanism underlying upper airway collapse is still unclear. Studying this mechanism is helpful for identifying key targets for controlling and treating upper airway collapse, which is of great significance for the prevention and treatment of OSA.
Skeletal muscle is composed of different muscle fiber types in a specific proportion. Muscle fibers can be divided into type I fibers and type II fibers according to the type of myosin heavy chain (MHC). Type I fibers, also called "slow oxidized fibers," contain myosin of slow muscle fibers (Myhc slow) and exhibit slow contraction speeds and strong fatigue resistance. Type II fibers are called "fast glycolytic fibers," and they contain Myhc fast and mainly obtain energy from anaerobic glycolysis. The tension and contraction of muscle fibers play important roles in maintaining the upper airway in an open position. 3 Previous studies have shown that in OSA, palatopharyngeus muscle exhibit pathological injuries, including muscle fibers atrophy, structural disorder, and type I fibers reduction, 4,5 which lead to changes in function of the palatopharyngeus muscle, collapse of the upper airway and exacerbation of hypoxia.
Peroxisome proliferator-activated receptorγ (PPARγ) is a ligandactivated nuclear transcription factor that belongs to the nuclear hormone receptor superfamily. Peroxisome proliferator-activated receptorγ co-activator-1α (PGC-1α) is a coactivator of PPARγ, which participates in the transcriptional regulation of PPARγ. It is a key factor in mitochondrial biosynthesis and highly expressed in skeletal muscle, especially in oxidized muscle fibers. Lin et al. showed that PGC-1α promoted skeletal muscle fiber conversion. 6 Studies have shown that hypoxia can down-regulate the expression of PPARγ in mouse pulmonary artery smooth muscle cells. 7 In addition, PGC-1α is a co-activator of PPARγ. Therefore, we speculated that hypoxia can also down-regulate the expression of PGC-1α in skeletal muscles. 8 Therefore, we will verify the pathological injuries of the palatopharyngeus muscle in OSA patients and explore the role of PGC-1α in the changes in airway dilator structure and conversion of muscle fiber types in patients with OSA. Exploring the pathophysiological mechanism underlying OSA provides a new perspective for the early clinical treatment of OSA.

| MATERIAL S AND ME THODS
Patients who were initially diagnosed with OSA and chronic tonsillitis and who underwent surgery in the Department of Otolaryngology Head and Neck Surgery at Sun Yat-sen Memorial Hospital of Sun Yat-sen University between August 2018 and August 2020 were included in this study. All patients underwent polysomnography (PSG).
According to the diagnostic criteria in the diagnostic and treatment guidelines for OSA, 9 apnea-hypopnea index (AHI) of ≥5 times each hour met the criteria for the diagnosis of OSA. The control group included patients with chronic tonsillitis without OSA. The study was approved by the hospital ethics committee, and all the enrolled patients signed an informed consent form.
Exclusion criteria: (1) patients with a history of oral, maxillofacial, and upper airway surgery; (2) patients with muscular diseases, such as systemic muscle weakness and muscular atrophy; (3) patients with systemic diseases involving muscles, such as vasculitis and connective tissue diseases; (4) patients who suffered cerebrovascular accidents or who had a history of radiotherapy or chemotherapy; and (5) patients with OSA who had been treated with CPAP, oral orthodontics or surgery in the past.
Tonsillectomies were performed in both groups, and two pieces of palatopharyngeal muscle tissue (approximately 4 × 4 × 3 mm in size) were collected. Avoid clamping or pulling the tissues and the tissue samples were stored in liquid nitrogen and glutaraldehyde.

| Immunohistochemistry
Tissues were embedded in paraffin and cooled on a −20°C refrigerated table. Then, the tissues were sliced with a paraffin slicer to a thickness of 4 μm. The tissues were collected on glass slides and placed in a 60°C oven for baking. Then, dewaxing, hydration, antigen retrieval, membrane rupture, and other treatments were carried out.
The cells were incubated with 10% goat serum to prevent nonspecific staining, and the following antibodies were added: rabbit antihuman PGC-1α antibody (Abcam), biotinylated goat anti-rabbit IgG (Kangwei), and DAB color liquid (Beyotime). Finally, the tissue sections were stained with hematoxylin, dehydrated, made transparent, sealed with neutral resin, and naturally dried.
The sections were viewed and photographed using a light microscope connected to a computer. Each sample was consecutively photographed with a 200× magnification objective. Myofiber morphology, muscle fiber arrangement, PGC-1α staining localization, and staining intensity were observed. The immunohistochemical results were semiquantitatively analyzed, 9 and 5 fields were randomly selected for scoring and measuring the intensity of cell staining at 200× magnification. The scoring system was as following: brown scored as 3 points, tan scored as 2 points, and light yellow scored as 1 point. No coloring scored as 0 points.

| Real-time quantitative PCR
Tissues were cut and homogenized, and RNA was extracted. The concentration and purity of the RNA and synthesized cDNA were determined according to the instructions of the Beyort™III cDNA Synthesis Kit (BeyoTime) (The primer sequences were shown in Table 1). With cDNA as the template and GAPDH as internal reference, the relative mRNA expression levels of PGC-1α and NRF-1 in the palatopharyngeus muscle of the OSA and control groups were assessed according to the instructions of the qPCR kit (Beyotime).

| Western blotting
Tissues were collected and transferred to a EP tube. After grinding the tissues, lysis solution was added to extract the total proteins. Kangweiji) was diluted, added to the membranes, and incubated for 1 h at room temperature. The membranes were exposed to ECL solution, and the densities of the obtained bands were quantified by NIH ImageJ. GAPDH used as the internal control.

| Electron microscopy
Tissues were immersed and fixed in glutaraldehyde (Alaaesar). Then, the tissues were cut into sections along the muscle fibers, fixed with 1% osmic acid (TED Pella, USA), dehydrated with ethanol and acetone, soaked with resin, embedded, and solidified with EP-812 (TED Pella). The samples were positioned, and semi-thin sections (70 μm thick) were generated with an ultrathin microslicer. The cells were stained with 2% uranium dioxide acetate (EMS, USA) and lead citrate (TED Pella). After film-making, the samples were observed and images were captured under a transmission electron microscope. When the C2C12 myoblasts had differentiated into muscular tubes, they were divided into 6 groups. Three groups were treated with intermittent exposure to hypoxia, and three groups (control)

| Cell culture, hypoxic culture, and transient transfection
were treated with normoxia. The intermittent hypoxia conditions were as follows: the CO 2 and O 2 concentrations were controlled by N 2 , and the cells were exposed to hypoxia treatments for 8 h each day. Each hypoxic treatment included culture under hypoxic conditions for 35 min and culture under normoxic conditions for 25 min.
The oxygen concentration was 5%, and the temperature, CO 2 concentration, and saturated humidity were the same as those used for the control group. 10 The cationic liposome method was used for transfection, and the procedure was performed according to the instructions of the LipofectamineTM 2000 kit (ReeboBio, Guangzhou). Cell qPCR and Western blotting followed the procedures described above (the primer sequences are shown in Table 2).

| Immunofluorescence staining
Paraformaldehyde (Beyotime) was used to fix the slides, and 0.5% Triton X-100 (Zhongshanqiao) was used for membrane permeabilization. The cells were incubated with 10% goat serum to prevent nonspecific staining, and the following antibodies were added: anti-slow skeletal myosin heavy chain antibody and anti-fast skeletal myosin heavy chain antibody. The sections were incubated with these antibodies at 4°C overnight. After washing, the goat anti-rabbit IgG (KangWeishiji) secondary antibody was added and incubated at room temperature for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) (KangWeishiji) was added dropwise and incubated to stain the nuclei. The sections were sealed with anti-fluorescence quenching agent, the sections were observed, and images were captured under a fluorescence microscope, and the densities of the images were quantified by NIH ImageJ. SPSS 20.0 statistical software was used for the data analysis. All data were tested for normality and analysis of variance.

| Statistical analysis
Immunohistochemical results were semiquantitatively analyzed by an integral comprehensive method according to the cell staining and NRF-1 expression in the palatopharyngeus muscle of OSA patients, and the decrease in PGC-1α expression was more pronounced in patients with severe OSA. We speculated that long-term hypoxia led to a decrease in PGC-1α expression and injury to the upper airway dilator muscle and exacerbated airway collapse and hypoxia.

| Protein expression of PGC-1α and NRF-1 in patients with OSA
Western blotting was used to detect the protein expression of PGC-1α and NRF-1 in the OSA and control groups. The gray values of the bands were measured by ImageJ for statistical analysis.
Compared with that in the control group, the protein expression of PGC-1α (t = 3.555, p = 0.002) and NRF-1 (t = 2.784, p = 0.012) in the OSA group was decreased (Figure 1).

| Changes in muscle fiber structure in the palatopharyngeus muscle of patients with OSA
To observe the structure of the palatopharyngeus muscle and the protein expression of PGC-1α in muscle fibers, we used immunohistochemical staining to observe muscle structure under a microscope. The brown color indicated positive PGC-1α protein staining. PGC-1α expression was observed in both nucleus and cytoplasm. In the control group, cells were darkly stained, mainly brown and tan, and neatly arranged. In the OSA group, cells were lightly stained, mainly light yellow, and exhibited disordered muscle fiber structure and increased connective tissue between cells. The immunohistochemistry results were semiquantitatively analyzed according to the methods described above. The results indicated that the expression of PGC-1α in the control group was higher than that in the OSA group (t = 8.321, p < 0.001) (Figure 2).

| The mRNA expression of PGC-1α and NRF-1 in C2C12 myoblasts cultured under intermittent hypoxic conditions
The qPCR was used to detect the mRNA expression of PGC-1α and NRF-1 in cells in the hypoxia group and control group (normoxia).
In the control group, the relative expression level of PGC-1α was 0.938 ± 0.138; however, it was 0.677 ± 0.084 in the intermittent hy-  intermittent hypoxia group (t = 2.791, p = 0.024). The difference was statistically significant (Figure 4).

| The protein expression of PGC-1α, NRF-1, and type I and type II myofibril in C2C12 myoblasts cultured under intermittent hypoxic conditions
The protein expression levels of PGC-1α and NRF-1 in the two groups were detected by Western blotting, and the expression of type I and type II myofibril was detected by immunofluorescence staining.
Results showed that the protein expression of PGC-1α and NRF-1 in the intermittent hypoxia group was lower than that in the control group ( Figure 5A). Compared with that in the control group (density: 0.231 ± 0.016), the expression of type I myofibril in the intermittent hypoxia group (density: 0.193 ± 0.006) was decreased(p = 0.001).

| DISCUSS ION
Previous studies have shown that in OSA, palatopharyngeus muscle exhibit pathological injuries, including muscle fibers atrophy and type I fibers reduction, 4  PGC-1α is an effective transcription activator of nuclear receptors and other transcription factors. PGC-1α is abundantly expressed in skeletal muscle, especially in type I muscle fibers. 11 Stimuli, such as hypoxia, cold, and starvation, can affect the expression of PGC-1α. 12 Previous studies have shown that hypoxia stimulation can downregulate the expression of PPARγ in smooth muscle, 7 and PGC-1α is its co-activator. We speculated that hypoxia may also down-regulate the expression of PGC-1α. Our study verified that the expression of inevitably leads to a decrease in the number of mitochondria. [13][14][15][16] The regulation of mitochondria by PGC-1α is mainly achieved by affecting the expression of NRF-1. The results of this study also found that the expression of NRF-1 is down-regulated in palatopharyngeus muscle of patients with OSA, but the relationship between the down-regulation of NRF-1 and PGC-1α expression and the conversion of palatopharyngeus muscle in patients with OSA remains to be further explored.

F I G U R E 1
Western blotting result showed that the protein expression of PGC-1α and NRF1 in OSA group was lower than that in control group F I G U R E 2 (A) Immunohistochemical staining was used to detect the changes in muscle fiber structure and PGC-1α expression in upper airway dilator in OSA group and control group (magnification ×200). In the OSA group, the morphology of muscle fibers was changed, exhibited disordered muscle fiber structure, and increased connective tissue between cells, and the cells were lightly stained. In the control group, muscle fibers were arranged neatly, the cell morphology was regular and the staining was uniform. (B) The immunohistochemistry results were semiquantitatively analyzed, and rhe results showed that the expression of PGC-1α in the OSA group was lower than that in the control group Studies have found that patients with OSA have disordered muscle structures, ruptured muscle filaments, motor nerve fiber edema, reduced mitochondrial numbers, and vacuolated mitochondria, which result in weakened muscle contractility. 17 Especially after a long period of muscle contraction, the decrease in contractility was larger in OSA patients than in normal subjects. This finding indicated that the anti-fatigue ability of the palatopharyngeal muscle is weakened in OSA patients. 4 In addition, the palatopharyngeal muscle in patients with OSA also had muscle fibers of different sizes and in- F I G U R E 4 mRNA expressions of PGC-1α and NRF-1 in the hypoxia group were lower than those in the control group group was higher than that in the control group. Immunofluorescence staining results showed that the expression of type I myofibril in the hypoxia group was lower than that in the control group (blue: nuclear staining, green: type I myofibril staining). (C) Immunofluorescence staining results showed that the expression of type II myofibril in the hypoxia group was higher than that in the control group (blue: nuclear staining, green: type II myofibril staining) suprapharyngeal constrictor during the expiration-inspiratory phase transition, thereby increasing pharyngeal collapsability. Therefore, those studies does not support the treatment of OSA by increasing upper airway collagen type I at the histological level. 20,21 The opening and closing of the upper airway is controlled by a complex interplay of pharyngeal muscles. The cause of increased pharyngeal structural collapse in OSA patients is unknown. Our study provides a preliminary idea into the mechanism of muscle fiber structural conversion, which still needs further exploration.

| CON CLUS ION
Pathological injuries were observed in the palatopharyngeus muscle of patients with OSA, including changes in the structure and arrangement of muscle fibers. The expression of PGC-1α and NRF-1 was down-regulated, which became more obvious as the disease worsened. In cell experiments, intermittent hypoxia treatment decreased the expression of PGC-1α, NRF-1, and type I myofibril.
When PGC-1α expression was knocked down, the expression of NRF-1 and type I myofibril was decreased. This result indicated that PGC-1α was involved in the conversion of palatopharyngeus muscle fiber types caused by intermittent hypoxia.

ACK N OWLED G M ENTS
This research was funded by the Natural Science Foundation of Guangdong Province (2017A030313669).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The article has no associated data.

O RCI D
Qian Cai https://orcid.org/0000-0001-8983-0657 F I G U R E 6 (A) PGC-1α expression was knocked down by siRNA. The result of WB showed that the protein expression of NRF-1 was down-regulated (GPADH was used as internal reference). (B) Immunofluorescence staining showed that the expression of type I myofibril was decreased compared with control group (NC) (blue: nuclear staining, green: type I myofibril staining). (C) The expression of type II myofibril was increased compared with control group (NC) (blue: nuclear staining, green: type II myofibril staining)