Effect of hypoxia-inducible factor 1 alpha inhibitor on immobilization-induced muscle fibrosis related to muscle contracture

Abstract

[Purpose] To test the effect of the hypoxia-inducible factor 1-alpha (HIF-1α) inhibitor, YC-1, on the upregulation of HIF-1α that leads to immobilization-induced muscle fibrosis underlying muscle contracture. [Materials and Methods] Rats were divided into control, immobilization (immobilization only), and YC-1 (immobilization and YC-1 injection) groups. The soleus muscles were the specimens. [Results] The HIF-1α protein expression in the control, immobilization, and YC-1 groups was 0.26 ± 0.11, 0.94 ± 0.28, and 0.66 ± 0.15, respectively. The expression of HIF-1α protein in the immobilization and YC-1 groups was significantly higher than in the control group and it was lower in the YC-1 group than in the immobilization group. There were strong positive correlations between HIF-1α protein expression and transforming growth factor (TGF)-β1 mRNA expression, TGF-β1 and α-smooth muscle actin (SMA) mRNA expressions, and between α-SMA mRNA expression and hydroxyproline content. A strong negative correlation was found between hydroxyproline content and range of motion on dorsiflexion at four-weeks. [Conclusion] Inhibition of HIF-1α may contribute to suppressing the overexpression of fibrosis-related molecules triggered by upregulation of HIF-1α, which may mitigate immobilization-induced muscle fibrosis related to muscle contracture.

INTRODUCTION

Muscle contracture is induced by immobilization due to cast fixation and excessive bed rest, which limits activities of daily living and interferes with rehabilitation¹⁾. The extensibility of skeletal muscles is reduced during muscle contracture because of immobilization-induced muscle fibrosis via collagen overexpression²⁾. Previous studies have indicated that hypoxia-inducible factor (HIF)-1α, a master transcription factor of oxygen homeostasis, has a key role in the progression of hepatic, renal, and pulmonary fibrosis^3,4,5). HIF-1α upregulation induces the overexpression of transforming growth factor-β1 (TGF-β1), a biomolecule that strongly promotes fibrosis^{6, 7)}, and the TGF-β1 overexpression progresses fibroblast differentiation into myofibroblasts, which are cells with high collagen-producing capacity⁸⁾. We previously found that HIF-1α upregulation leads to TGF-β1 overexpression in immobilized rat soleus muscles, promoting the differentiation of fibroblasts into myofibroblasts. These alterations trigger the progression of immobilization-induced muscle fibrosis⁹⁾. From those, HIF-1α may be a key target molecule for immobilization-induced muscle fibrosis. HIF-1α inhibitors are safe immunosuppressive agents with low nephrotoxicity and are used in many medical procedures, and these suppress the overexpression of TGF-β1 and lead to the mitigation of hepatic and pulmonary fibrosis^{10, 11)}. Another study indicated that HIF-1α inhibitors are essential in regulating knee joint contracture¹²⁾.

HIF-1α inhibitors also have side effects such as inhibiting macrophage migration and suppressing interleukin-1β (IL-1β) production¹³⁾. However, these side effects do not work to progress fibrosis caused by inflammation. In short, we hypothesized that HIF-1α inhibitor may reduce the HIF-1α upregulation, and this alteration may suppress the fibroblast differentiation to myofibroblast triggered by the TGF-β1 overexpression, resulting in mitigating immobilization-induced muscle fibrosis related to muscle contracture. Some patients are unable to receive early rehabilitation because of their poor general condition, while for others, rehabilitation is delayed because of cast fixation or bed resting. These patients are often exposed to long-term immobilization, the rehabilitation outcomes of muscle contracture triggered by immobilization-induced muscle fibrosis are unfavorable. If the HIF-1α inhibitor is administered when rehabilitation is impossible, it may suppress immobilization-induced muscle fibrosis and mitigate muscle contracture. The aim of this study was to test the effect of a HIF-1α inhibitor on HIF-1α upregulation leads to immobilization-induced muscle fibrosis underlying muscle contracture.

MATERIALS AND METHODS

Eight-week-old male Wistar rats (CLEA Japan Inc., Tokyo, Japan) were bred at the Research Center for Biomedical Models and Animal Welfare, Nagasaki University. The rats were housed in two per cage, maintained under a 12:12-hour light-dark cycle at 25°C, and had unrestricted access to food and water. The 19 rats were allocated to control (n=6) and experimental (n=13) groups. The control group was untreated throughout the study, while in the experimental group, ankle joint immobilization was performed as our previous report²⁾. For anesthesia, the rats in the experimental group received 2.0 mg/kg midazolam (Sandoz Pharma Co., Ltd., Tokyo, Japan), 0.375 mg/kg medetomidine (Kyoritu Pharma, Tokyo, Japan), and 2.5 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan). Both rat ankle joints were placed in full plantar flexion with the cast for four weeks to simulate human ankle full plantar flexion contracture. The soleus muscles were immobilized in a shortened position. The cast, which was applied from above the knee joint to the distal foot, was replaced as needed. The experimental groups were divided into an immobilization group (n=7), which was exposed to immobilization treatment alone, and a YC-1 group (n=6), which was exposed to immobilization treatment along with HIF-1α inhibitor injection. In the YC-1 group, rats were intraperitoneally injected with 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1; Abcam, Cambridge, UK) at 2 mg/kg once a day during the experimental period¹⁴⁾. The rats of the control and immobilization groups were administered the same volume of dimethyl sulfoxide (DMSO) during the experimental period. In the immobilization and YC-1 groups, the cast was removed weekly for ROM measuring, as described below. The protocol for animal experiments was reviewed and approved by the Ethical Review Committee at Nagasaki University (Approval No. 1404161137).

The range of motion (ROM) of ankle joint dorsiflexion was measured weekly under anesthesia during the experimental period. The angle (0–180°) between the fifth metatarsal-lateral malleolus line and the lateral malleolus-knee center line was used to define ROM. A force of 0.3 N was applied with a tension gauge (Shiro Industry, Osaka, Japan) to passively dorsiflex the ankle joint⁹⁾. The ankle dorsiflexion ROM was measured at 160° at baseline for all groups. The cast was removed for ROM measurement and then replaced with a new one. After the experimental period, the soleus muscles from both sides of all rats were harvested. The right soleus muscles were rapidly frozen in liquid nitrogen for biochemical analysis and the left soleus muscles were immersed in RNAlater^® (Ambion, Austin, TX, USA) immediately after being dissected for molecular biological analysis.

Western blotting was performed to determine the HIF-1α protein expression levels. Part of the right soleus muscle was minced and homogenized in lysis buffer, the homogenates were centrifuged at 12,000 × g for 20 minutes. The total protein content of the supernatant was then measured using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) and was adjusted to 2 mg/mL. Sodium dodecyl sulfate (SDS) buffer (Bio-Rad Laboratories, Hercules, CA, USA) and 2-mercaptoethanol were added to the supernatants and the samples were denatured at 50°C for 20 minutes. One-dimensional SDS-polyacrylamide gel electrophoresis was performed to separate the proteins according to their molecular weight. The proteins were transferred to polyvinylidene difluoride membranes after electrophoretic separation. The membranes were blocked with Tris-buffered saline (TBS)/casein blocker (Bio-Rad Laboratories) containing 0.05% Tween 20 for 1 hour, incubated overnight at 21°C with a monoclonal antibody of HIF-1α (1:500; Novus Biologicals, Centennial, CO, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; Abnova, Taipei, Taiwan) in TBS/casein blocker with 0.05% Tween 20. After several washes, the blots were incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:20,000; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour at 21°C. After several washes, ECL SelectTM Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) was used to visualize the bands, which were then imaged with an Image Quant LAS 500 system (GE Healthcare). The Scion imaging system (Scion, Frederick, MD, USA) was used to quantify HIF-1α and GAPDH band densities. Relative quantification of HIF-1α was performed based on the density of GAPDH as a loading control.

Part of the right soleus muscle was used for hydroxyproline measurements following a previously described method¹⁵⁾. The muscle tissues were immersed in 1.0 M PBS (pH 7.4) and subsequently homogenized, treated with 6 N HCl at 110°C for 15 hours to achieve hydrolysis, then dried using an evaporator (EZ-2, Ikeda Scientific, Tokyo, Japan). Subsequently, they were exposed to NaOH at 90°C for 1 hour, followed by mixing with a buffered chloramine-T solution and oxidation at 21°C. The chromophore was formed through the reaction with Ehrlich’s aldehyde reagent, and its absorbance was measured at 540 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, San Jose, CA, USA). The absorbance values were plotted against the concentration of the hydroxyproline standard, and the hydroxyproline content was normalized as the content per dry weight.

Left soleus muscles were subjected to real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from the muscle samples using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany), and then preparing complementary DNA (cDNA) using the QuantiTect^® Reverse Transcription Kit (Qiagen). The Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) was used for performing real-time RT-PCR. The cDNA content was normalized to 25 ng/µL and cDNA (0.2 µL) was applied into each well. Table 1 provides the specific primers used for TGF-β1, α-smooth muscle actin (SMA), a myofibroblast marker, and type I and III collagen. The Mx3005P Real-Time qPCR system (Agilent Technologies) was used to determine the threshold cycle (Ct), and the mRNA expression levels were calculated by the ΔΔCT method.

Table 1. Sequences of gene-specific primers.

Gene	Sequence		Gene Bank no.
	Forward	Reverse
TGF-β1	5′-AGAAGTCACCCGCGTGCTAAT-3′	5′-CACTGCTTCCCGAATGTCTGA-3′	BC076380.1
α-SMA	5′-CGGGCTTTGCTGGTGATG-3′	5′-GGTCAGGATCCCTCTCTTGCT-3′	BC158550.1
type I collagen	5′-ATCAGCCCAAACCCCAAGGAGA-3′	5′-CGCAGGAAGGTCAGCTGGATAG-3′	BC133728.1
type III collagen	5′-AGGCCAATGGCAATGTAAAG-3′	5′-TGTCTTGCTCCATTCACCAG-3′	BC087039.1
GAPDH	5′-CCATTCTTCCACCTTTGATGCT-3′	5′-TGTTGCTGTAGCCATATTCATTGT-3′	AB017801.1

TGF: transforming growth factor; SMA: smooth muscle actin; GAPDH: glyceraldehyde3-phosphate dehydrogenase.

All data are presented as means ± standard deviation. The ROM of dorsiflexion was assessed using two-way analysis of variance (ANOVA) followed by Scheffé’s method. Differences between groups for other parameters were assessed using one-way ANOVA, followed by Scheffé’s method. Differences were considered statistically significant at p<0.05. Associated examinations were performed using bivariate correlations, which were considered significant at p<0.05. Strong correlations were defined as those with coefficients of 0.70–1.00, moderate correlations as 0.40–0.69, and weak correlations as 0.10–0.39¹⁶⁾.

RESULTS

The relative HIF-1α protein expression level in the control group was 0.26 ± 0.07. The values for the immobilization and YC-1 groups were 0.94 ± 0.16 and 0.66 ± 0.08, respectively (Fig. 1). The expression of HIF-1α protein in the experimental groups was significantly higher compared to the control group, and it was significantly reduced in the YC-1 group compared to the immobilization group.

Fig. 1.

Quantification of HIF-1α protein levels. (A) Western blotting detection of HIF-1α and GAPDH. (B) The expression of HIF-1α protein. Open bar represent the control group (Con). Black bar represent the immobilization group (Im). Gray bar represent the YC-1 group. Data are presented as the mean ± standard deviation. *, Significant difference (p<0.05) compared with the control group. #, Significant difference (p<0.05) compared with the immobilization group.

In the control group, the TGF-β1 mRNA expression was measured at 0.82 ± 0.32. For the immobilization and YC-1 groups, the values were 2.71 ± 0.55 and 1.88 ± 0.40, respectively (Fig. 2A). The mRNA expression levels of α-SMA were 0.86 ± 0.29 in the control group, 3.50 ± 0.68 in the immobilization group, and 1.68 ± 0.49 in the YC-1 group (Fig. 2B). The TGF-β1 and α-SMA mRNA expression levels in the experimental groups were significantly higher than those in the control group, and significantly lower in the YC-1 group than the immobilization group.

Fig. 2.

TGF-β1 and α-SMA mRNA expression (A and B). Open bars represent the control group (Con). Black bars represent the Im group (Im). Gray bars represent the YC-1 group. Data are presented as the mean ± standard deviation. *, Significant difference (p<0.05) compared with the control group. #, Significant difference (p<0.05) compared with the immobilization group.

In the control group, hydroxyproline content was 3.94 ± 1.05 µg/mg dry weight, whereas the immobilization and YC-1 groups had contents of 12.81 ± 4.00 and 8.28 ± 2.00 µg/mg dry weight, respectively (Fig. 3A). In the control group, the mRNA expression of type I collagen was 0.92 ± 0.37, while it was 5.55 ± 0.71 in the immobilization group and 1.99 ± 0.66 in the YC-1 group (Fig. 3B). For type III collagen, the mRNA expression was 1.19 ± 0.36 in the control group, 5.89 ± 1.27 in the immobilization group, and 3.19 ± 0.78 in the YC-1 group (Fig. 3C). In comparison to the control group, hydroxyproline content and the mRNA expression of type I and III collagen were significantly increased in the experimental groups, the YC-1 group had significantly lower levels than the immobilization group.

Fig. 3.

Hydroxyproline content (A) and type I and III collagen mRNA expression (B, C). Open bars represent the control group (Con). Black bars represent the immobilization group (Im). Gray bars represent the YC-1 group. Data are presented as the mean ± standard deviation. *, Significant difference (p<0.05) compared with the control group. #, Significant difference (p<0.05) compared with the immobilization group.

The ankle dorsiflexion ROM in the control group was consistently maintained at 160° across all time points. In the immobilization and YC-1 groups, the ROM was 117.86 ± 4.66° and 116.67 ± 3.42°, 99.29 ± 4.01° and 101.25 ± 3.45°, 83.93 ± 3.18° and 92.08 ± 2.46°, and 75.36 ± 4.66° and 83.33 ± 2.58° at 1, 2, 3, and 4 weeks, respectively (Fig. 4). The ROM of dorsiflexion was significantly lower in the experimental group than the control group at all time points, and significantly higher in the YC-1 group than the immobilization group at 3 weeks.

Fig. 4.

Changes in the range of motion (ROM) of the ankle joint on dorsiflexion. Data are presented as the mean ± standard deviation. *, Significant difference (p<0.05) compared with the control group. #, Significant difference (p<0.05) compared with the immobilization group.

A bivariate correlation between the HIF-1α protein expression and the TGF-β1 mRNA expression indicated a strong positive correlation (Fig. 5A). There was a strong positive correlation between the TGF-β1 and α-SMA mRNA expressions (Fig. 5B) and between the α-SMA mRNA expression and the hydroxyproline content (Fig. 5C). There was a strong negative correlation between the hydroxyproline content and the ROM on dorsiflexion at 4-week (Fig. 5D).

Fig. 5.

Bivariate correlation analysis on the HIF-1α protein expression and the TGF-β1 mRNA expression (A), the TGF-β1 mRNA expression and the α-SMA mRNA expression (B), the α-SMA mRNA expression and the hydroxyproline content (C), the hydroxyproline content and the range of motion (ROM) on dorsiflexion at 4-week (D).

DISCUSSION

The HIF-1α protein expression level was significantly higher in the immobilization group than the control group, indicating that the rat soleus muscle was in a state of hypoxia in the immobilization group. HIF-1α levels increase in skeletal muscle under hypoxic conditions¹⁷⁾. Hypoxic changes are observed due to a decrease in blood flow, which is associated with a reduction in capillary density¹⁸⁾. Previous research has also found that the number of capillaries in the soleus muscle is reduced in the same rat ankle immobilization model used in this study¹⁹⁾. We surmised that similar lesions were induced, and that the rat soleus muscle in the immobilization group became hypoxic. The immobilization group showed a significant increase in TGF-β1 and α-SMA mRNA expression compared to the control group. The overexpression of HIF-1α leads to increased TGF-β1 expression levels, which induces the differentiation of fibroblasts to myofibroblasts^{20, 21)}. Myofibroblasts are responsible for collagen production and are crucial in fibrosis development and progression²²⁾. In this study, the hydroxyproline content and mRNA expression levels of type I and type III collagen were significantly higher in the immobilization group than the control group. The ROM of dorsiflexion was significantly lower in the immobilization group than the control group at all time points. From these results and the previous reports, we surmised that the number of myofibroblasts increased because of hypoxia-induced TGF-β1 overexpression, which in turn promoted the muscle contracture caused by immobilization-induced muscle fibrosis.

The HIF-1α protein expression level was significantly lower in the YC-1 group than the immobilization group. YC-1 accelerates HIF-1α degradation by targeting amino acids in the 720 to 780 region and YC-1 inhibits the synthesis of HIF-1α by inactivating the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway^{23, 24)}. In the present study, YC-1 may have suppressed the HIF-1α overexpression via the above mechanisms. The mRNA expression levels of TGF-β1 and α-SMA were significantly reduced in the YC-1 group compared to the immobilization group. Previous research has indicated that YC-1 suppresses the TGF-β1 overexpression caused by HIF-1α upregulation, resulting in mitigating the differentiation from fibroblasts to myofibroblasts⁶⁾. The hydroxyproline content and mRNA expression levels of type I and III collagen were significantly lower in the YC-1 group than the immobilization group. Previous studies have demonstrated that HIF-1α inhibitors mitigate fibroblast differentiation into myofibroblasts triggered by TGF-β1 upregulation and these effects suppress collagen overexpression²⁵⁾. The ROM of dorsiflexion was significantly higher in the YC-1 group than in the immobilization group after 3 and 4 weeks of immobilization. The associated examination results indicated strong positive correlations between the HIF-1α protein expression and the TGF-β1 mRNA expression, the TGF-β1 and α-SMA mRNA, and the α-SMA mRNA expression and the hydroxyproline content. There was a strong negative correlation between the hydroxyproline content and the ROM on dorsiflexion at 4 weeks. From these results, YC-1 may suppress HIF-1α upregulation, leading to inhibited fibroblast differentiation into myofibroblasts in response to TGF-β1 overexpression, resulting in the mitigation of muscle contracture caused by immobilization-induced muscle fibrosis.

Our previous research clarified that the IL-1β upregulation caused by macrophage accumulation led to immobilization-induced muscle fibrosis⁹⁾. HIF-1α inhibitor mitigates macrophage migration and IL-1β production¹³⁾, so HIF-1α inhibitor may suppress the above pathway of immobilization-induced muscle fibrosis. While it may be important to examine anti-fibrotic strategies other than HIF-1a inhibitors. Although a previous report indicated that collagenase was effective in suppressing fibrotic lesion, it only degrades existing collagen without addressing upstream pathways²⁶⁾. Collagenase also requires repeated injections due to rapid enzymatic degradation, often causing pain and localized tissue damage²⁷⁾, but HIF-1α inhibitor, administered systemically at optimized doses, avoids these procedural complications²⁸⁾. From these previous studies, HIF-1α inhibitor may be going to develop as new strategies for immobilization-induced muscle fibrosis. If animal studies and clinical trials in humans about HIF-1α inhibitors can be promoted, it will contribute to the development of innovative drug therapies for preventing immobilization-induced muscle fibrosis, which leads to muscle contracture.

This study has several limitations. YC-1 was the only HIF-1α inhibitor tested, and comparisons with other HIF-1α inhibitors were not performed. The dose of YC-1 was set at 2 mg/kg in accordance with a previous report¹⁴⁾; however, whether the effect of YC-1 occurs in a dose-dependent manner needs to be investigated. Additionally, the present study was unable to assess fibrosis based on histological analysis of the soleus muscles in all groups. Whether the fibrosis-inhibitory effect of YC-1 occurs in the perimysium and endomysium requires further examination. The changes in skin or joint capsule related to ROM were also not checked, it will need to search for fibrosis of these periarticular soft tissues as well. The cast was removed once a week for ROM measurements, and our model did not completely maintain continuous immobilization. Since the influence of temporary cast removal cannot be ruled out, a model in which is immobilized continuously for 4 weeks would be necessary.

In conclusion, HIF-1α inhibition may play a role in suppressing immobilization-induced muscle fibrosis and mitigating limited ROM. In the future, HIF-1α inhibitors should be evaluated for their side effects carefully, and then proceed to clinical trials for their use in humans.

Funding and Conflict of interest

This study was funded by the Japan Society for the Promotion of Science (KAKENHI, grant no. 16K12937). The funder had no role in the study design, data collection, analysis, publication decisions, or manuscript preparation. None of the authors have any conflicts of interest to disclose.