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. 2024 Jul 26;141(5):929–945. doi: 10.1097/ALN.0000000000005167

Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain–like Behavior in a Rat Model of Myofascial Trigger Points

Yu Liu 1, Feihong Jin 2, Lingwei Zhou 3, Xuan Li 4, Xiaoyue Li 5, Qinghe Chen 6, Shaozhong Yang 7, Jintang Sun 8, Feng Qi 9,✉
PMCID: PMC11463032  PMID: 39058323

Abstract

Background:

Myofascial trigger points (MTrPs) are the primary etiological characteristics of chronic myofascial pain syndrome. Receptor tyrosine kinases (RTKs) are associated with signal transduction in the central mechanisms of chronic pain, but the role of RTKs in the peripheral mechanisms of MTrPs remains unclear. The current study aimed to identify RTKs expression in MTrPs and elucidate the molecular mechanisms through which platelet-derived growth factor receptor-α (PDGFR-α) induces contraction knots and inflammatory pain-like behavior in a rat model of myofascial trigger points.

Methods:

MTrPs tissue samples were obtained from the trapezius muscles of patients with myofascial pain syndrome through needle biopsy, and PDGFR-α activation was analyzed by microarray, enzyme-linked immunosorbent assay, and histological staining. Sprague–Dawley rats (male and female) were used to investigate PDGFR-α signaling, assessing pain-like behaviors with Randall–Selitto and nest-building tests. Muscle fiber and sarcomere morphologies were observed using histology and electron microscopy. The PDGFR-α binding protein was identified by coimmunoprecipitation, liquid chromatograph mass spectrometer, and molecular docking. PDGFR-α–related protein or gene levels, muscle contraction, and inflammatory markers were determined by Western blot and reverse-transcription quantitative polymerase chain reaction.

Results:

PDGFR-α phosphorylation levels were elevated in the MTrPs tissues of individuals with trapezius muscle pain and were positively correlated with pain intensity. In rats, PDGFR-α activation caused pain-like behaviors and muscle contraction via the Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathway. JAK2/STAT3 inhibitors reversed the pain-like behaviors and muscle contraction induced by PDGFR-α activation. Collagen type I α 1 (COL1A1) binds to PDGFR-α and promotes its phosphorylation, which contributed to pain-like behaviors and muscle contraction.

Conclusions:

COL1A1-induced phosphorylation of PDGFR-α and the subsequent activation of the JAK2/STAT3 pathway may induce dysfunctional muscle contraction and increased nociception at MTrPs.

Phosphorylation of the receptor tyrosine kinase platelet-derived growth factor receptor-α (PDGFR-α) was increased in myofascial trigger point samples obtained from patients with chronic myofascial pain syndrome. In a rat model of myofascial trigger points, PDGFR-α activation caused pain-like behaviors and muscle contraction via the activation of the Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) signal transduction pathway. Collagen type I α 1 (COL1A1) protein, a major component of the extracellular matrix that is upregulated in multiple disease states, binds to and promotes phosphorylation of PDGFR-α.

Editor’s Perspective.

What We Already Know about This Topic

Myofascial trigger points are hyperirritable knots in the skeletal muscle and are hallmarks of myofascial pain syndrome

Receptor tyrosine kinases are high-affinity cell surface receptors and are implicated in pain pathomechanisms

The role of receptor tyrosine kinases in mediating pain via myofascial trigger points remains incompletely understood

What This Article Tells Us That Is New

Phosphorylation of the receptor tyrosine kinase platelet-derived growth factor receptor-α (PDGFR-α) was increased in myofascial trigger point samples obtained from patients with chronic myofascial pain syndrome

In a rat model of myofascial trigger points, PDGFR-α activation caused pain-like behaviors and muscle contraction via the activation of the Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) signal transduction pathway

Collagen type I α 1 (COL1A1) protein, a major component of the extracellular matrix that is upregulated in multiple disease states, binds to and promotes phosphorylation of PDGFR-α

Myofascial pain syndrome (MPS) is a prevalent form of chronic pain that affects approximately 30% of individuals worldwide.1 Myofascial trigger points (MTrPs), the primary etiological factor of MPS, are composed of abnormal contraction knots of muscles characterized as hyperirritable regions localized within taut bands (TBs) of skeletal muscle, which contribute to hyperalgesia, referred pain, and autonomic nervous system symptoms.2–4 The complex pathophysiological mechanisms and uncertain etiopathogenesis of MPS necessitate further advancements in treatment efficacy.5,6 Therefore, identification of potential molecular targets and mechanisms of MTrPs is imperative.

Receptor tyrosine kinases (RTKs) are integral transmembrane receptors that play a crucial role in the central mechanisms of chronic pain.7,8 RTKs are expressed in sensory neurons and facilitate the transition from acute to chronic pain.9–11 However, the involvement of RTKs in the mechanisms of chronic peripheral pain in MTrPs remains unclear. Therefore, additional insights are required to gain a more comprehensive understanding of the role of RTKs in MTrPs. One of the primary constraints in existing research on MTrPs is the scarcity of tissue samples from patients with MPS. In this study, we acquired clinical MTrPs tissues through biopsy and assessed the phosphorylation of RTKs. Our findings revealed that MTrPs are associated with the increased activity of a large number of RTKs. Of these, we chose to follow up on platelet-derived growth factor receptor-α (PDGFR-α) because its phosphorylation levels increased significantly. Notably, phosphorylation of PDGFR-β showed no difference, implying that PDGFR-α may play a crucial role in the development of MTrPs. PDGF/PDGFR plays an important role in signal transduction related to cell metabolism and muscle contraction.12,13 In the field of pain research, studies have demonstrated that activation of PDGF/PDGFR induced pain-like behaviors in rats in a dose-dependent manner,14 whereas inhibitors can relieve the pain and eliminate morphine analgesic tolerance.15,16 Furthermore, although the role of PDGFR-α in regulating vascular smooth muscle contraction has been established,17 its involvement in skeletal muscle contraction of MTrPs remains unclear. Notably, PDGFR-α has receptor and kinase activities that mediate signal transduction inside and outside of cells.18 The Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathway is known to induce chronic pain by releasing proinflammatory cytokines and could transduce intracellular signaling downstream of PDGFR-α.19,20 While evidence has demonstrated an association between the JAK2/STAT3 pathway and skeletal muscle metabolism, whether it is involved in the pathogenesis of MTrPs remains unclear.21

We hypothesize that PDGFR-α of the RTKs family contributes to MTrPs pathology, acting as a molecular switch via the JAK2/STAT3 pathway, with downstream effects on muscle contraction and inflammatory pain of MTrPs. In this study, we assessed the activity status of PDGFR-α at the clinical tissue level, investigated the upstream and downstream signaling pathways of PDGFR-α by establishing a rat animal model, and explored the impact of PDGFR-α on muscle contraction and inflammatory pain of MTrPs.

Materials and Methods

Clinical MTrPs Tissue Collection from the Upper Trapezius Muscle

This study was approved by the Qilu Hospital Ethics Committee (KYLL-202011-023) and registered in the Chinese Clinical Trial Registry (ChiCTR-CPR-15007329, October 2, 2015) by Feng Qi. Written informed consent was obtained from all patients. The inclusion criteria and biopsy procedures are detailed in the Supplemental Methods (https://links.lww.com/ALN/D626). Muscle samples from the control group were collected as previously described.22 Pain intensity was measured using a 10-point visual analog scale.

RTK s Phosphorylation Antibody Microarray Analysis

RayBio Human Phosphorylation Array Kit (No. AAH-PRTK-G1, Ray Biotech, China) was used to analyze RTKs phosphorylation in MTrPs tissue from the upper trapezius muscle. The tissue was incubated with blocking buffer through microarray and subsequently treated with biotin-conjugated anti-phosphor-tyrosine antibody and fluorescent dye-conjugated streptavidin. Signals were imaged using a laser scanner (GenePix 4000B Microarray Scanner, Mollecular Devices, USA).

Enzyme-linked Immunosorbent Assay

Blood samples from patients with MPS were tested for platelet-derived growth factor-AA (PDGF-AA) levels using a human PDGF-AA enzyme-linked immunosorbent assay (ELISA) kit (EK1696, Bosterbio, USA). Serum samples were added to the plates, incubated, and washed, and then the chromogen solution was added. Absorbance was measured at 450 nm.

Animal Models and Assessment of Mechanical Withdrawal Thresholds

All animal experiments were approved by the Animal Care and Use Committee of Shandong University (KYLL-2022ZM-175; Jinan, China) and conformed to the National Institutes of Health (Bethesda, Maryland) Guide for the Care and Use of Laboratory Animals. Six-week-old male and female Sprague–Dawley rats, weighing 200 to 250 g, were supplied by the Qilu Hospital of Shandong University Experimental Animals Laboratory. Rats were raised with a 12-h light and dark cycle and fed under standard conditions (24°C; 20 to 30% humidity). Four rats were allocated to each cage, and food and water were provided ad libitum. Experimental units were defined as single animals. The animal model was established according to widely used methods, which involved blunt strikes on the left gastrocnemius muscle and exercise for 8 weeks, followed by 4 weeks of rest (Figure S1, https://links.lww.com/ALN/D627).23–25 This model mimicked human MTrPs and exhibited similar characteristics. The location criteria for MTrPs include TBs, decreased mechanical withdrawal thresholds, and local twitch responses. Mechanical withdrawal thresholds were measured using a Randall–Selitto device with an 8-mm round head tip on the hind limb muscle. Five measurements were taken at 3-min intervals, with the highest and lowest values removed before averaging the remaining three. The procedure was performed by the same evaluator.

Animal Experimental Design

The primary outcome of this study was mechanical withdrawal thresholds, and the secondary outcomes included nest-building scores, c-fos protein levels, muscle fiber cross-sectional areas, sarcomere lengths, and related muscle contraction and inflammatory markers. The number of subjects for the experiments was selected based on previously published articles on mechanical withdrawal thresholds.26 Using a two-tailed test, with a statistical power of 85%, α = 0.05, the calculated sample size for each group was determined to be five rats. Animals that died before outcome measurements or failed to satisfy the MTrPs criteria were excluded. Otherwise, all collected data were included. The behavioral tests included 10 rats in each group and 8 rats otherwise.

Sprague–Dawley rats (n = 120) were randomly (random number table according to weight) assigned to one of the following groups: (1) wild type (WT), (2) MTrPs (12-week chronic stress), (3) WT plus PDGFR-α agonist (PDGF-A-chain homodimer, 110-13A, 20 μg/ml, Peprotech, USA), (4) MTrPs plus lentivirus-knockdown-PDGFR-α (LV-knockdown-PDGFR-α/control), (5) MTrPs plus JAK2/STAT3 inhibitor/control (AZD1480, GC12504, Glpbio, USA), (6) WT plus PDGF-A-chain homodimer plus AZD1480, (7) MTrPs plus lentivirus-overexpression-collagen type I α 1 (LV-overexpression-COL1A1/control), and (8) MTrPs plus LV-knockdown-COL1A1/control. The allocation of cage locations and treatment sequences was randomized. The investigators were unaware of the group assignments.

Intramuscular Injection of Agonists, Inhibitors, and LV in Rats

PDGF-A-chain homodimer and LV were used to activate and inhibit PDGFR-α, respectively. AZD1480, which inhibits JAK2/STAT3 expression, was administered into the MTrPs of the left gastrocnemius muscle of each rat. Additionally, 3 points (30 μl per point, per rat) were marked on the MTrPs and injected with micro syringes, which were left in the injection sites for 3 min or more before being slowly withdrawn. Short hairpin RNA (shRNA) LV for PDGFR-α knockdown (sh-PDGFR-α), lentiviral vector COL1A1 LV for overexpression, sh-COL1A1 LV, and negative control (empty vector and scrambled control) LVs were purchased from Jikai Gene Technology (Shanghai, China). The LV was injected intramuscularly (3 points × 10 μl) into unilateral gastrocnemius of each rat or into the TBs of MTrPs rats. Infection efficiency assays were conducted 14 days or more after virus injection.

Nest-building Behavior

The nest-building behavior of the rat was assayed by assessing nest quality after 24 h of exposure to a nestlet of tissue cotton (5 cm × 5 cm; mean weight, 2.5 g). After 24 h, the nest-building ability was rated on a 5-point scale.27

Hematoxylin and Eosin Staining

Standard hematoxylin and eosin (HE; C0105S, Beyotime) was used. The sections were viewed under a microscope (BX53, Olympus) equipped with a digital camera.

Immunohistochemistry and Immunofluorescence Staining

The primary antibodies used are listed in Table S1 (https://links.lww.com/ALN/D631). Fluorophore-conjugated secondary antibody (1:200, A23220, Abbkine, USA) was applied in addition to counterstaining with 4’,6-diamidino-2’-phenylindole (C1005, Beyotime, China) or diaminobenzidine (ZLI-9017, ZSGB-BIO, China). Images were captured using a laser-scanning confocal microscope (Nikon, Japan). Each rat was analyzed with an integrated optical density (IOD) of four images using Image-Pro Plus 6.0.

Western Blot

The primary antibodies used are listed in Table S1 (https://links.lww.com/ALN/D631). Goat antirabbit antibody was used as the secondary antibody (Bosterbio, BA1054, 1:10,000). Luminescence was used to visualize the immunoblots (ECL, 36208ES76, Yeasen Bioscience, China). Relative gray values were measured using ImageJ software (version 1.53).

Real-time Quantitative Polymerase Chain Reaction

Total RNA was extracted using TRIzol reagent (R0016, Beyotime), and cDNA was synthesized using an RNA Transcription Kit (R323-01, Vazyme, China). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using an SYBR Green RT-qPCR system (Q711-02, Vazyme). The primer sequences are listed in Table S2 (https://links.lww.com/ALN/D631).

Muscle Fiber Cross-sectional Area

Olympus CellSens Standard software was used to measure the cross-sectional area of the muscle fibers in three randomly selected microscope fields from the tissue sections of each rat. The top 15 results were analyzed.

Electron-microscopic Analysis

Transmission electron microscopy was used to observe sarcomeres and their ultrastructures in fresh tissue samples. The samples were dissected, fixed, dehydrated, embedded in resin, and examined by transmission electron microscopy (Hitachi, HT7800/HT7700). ImageJ software was used to analyze the length of the sarcomeres in the micrographs of each rat.

Coimmunoprecipitation and Liquid Chromatography–Mass Spectrometry Analysis

The coimmunoprecipitation (Co-IP) kit (P2179S, Beyotime) was used with Protein A/G beads and antibodies against PDGFR-α (ab203491, Abcam, United Kingdom) and COL1A1 (ab270993, Abcam) or rabbit IgG (A7016, Beyotime). Elution was performed the next day, and the supernatants were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide samples were identified using Shotgun liquid chromatography–mass spectrometry (LC-MS) and MASCOT 2.6 software to identify PDGFR-α–interacting proteins.

Molecular Docking

ZDOCK was used to study the relationship between PDGFR-α and COL1A1 by docking their protein structures downloaded from the Protein Data Bank database. The ZDOCK module identifies docking sites and calculates z-scores to predict interaction outcomes. The model with the highest z-score was selected for this study.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 8.2.1. All data are presented as mean ± SD. The Shapiro–Wilk test was used to assess normally distributed data. Correlations between phosphorylated PDGFR-α (p-PDGFR-α) levels and patient pain intensity were analyzed using Pearson correlation analysis. Student’s t test (two-tailed) was used for comparisons between the two groups with normal distributions, and nonnormal distributions were analyzed using the Mann–Whitney U test. Repeated-measures analysis of variance was used to analyze the mechanical withdrawal threshold data at different time points. The remaining data were analyzed using one- or two-way analysis of variance followed by Tukey post hoc test for multiple comparisons of means. P < 0.05 was considered statistically significant.

Results

Identification of Differentially Expressed RTKs at the MTrPs in the Upper Trapezius Muscle of Patients with MPS

Phosphorylation antibody microarray analysis was performed to identify highly expressed RTKs in MTrPs tissues. The results showed distinct differences in RTK expression between the control group and patients with MPS (fig. 1A). Among these, p-PDGFR-α expression was significantly increased in MTrPs tissue (879 ± 83.08 vs. 1,060 ± 96.84; units, fluorescent value; P = 0.0009) compared with control tissue, whereas no difference (338.8 ± 47.96 vs. 308.4 ± 51.67; P = 0.2290) was found in phosphorylated PDGFR-β expression between the groups. This discrepancy implied that p-PDGFR-α played a major role in MTrPs, prompting our focus on PDGFR-α. HE staining was performed to observe the morphology of MTrPs, which showed annular or enlarged muscle fibers of different sizes with centralized nuclei in the cross-sectional spaces (fig. 1B). Pearson correlation analysis showed a positive relationship between pain intensity and p-PDGFR-α expression (fig. 1C). ELISA results showed that PDGF-AA, a specific ligand for PDGFR-α, was upregulated in the sera of patients with MPS (3.74 ± 0.82 vs. 5.97 ± 0.98; units, ng/ml; P < 0.001; fig. 1D; Figure S2, https://links.lww.com/ALN/D628). Immunohistochemistry (IHC) demonstrated that ligand PDGF-AA expression was upregulated (1.51 ± 0.33 vs. 10.2 ± 1.57; units, IOD; P < 0.001), which supported the activation of p-PDGFR-α (fig. 1E). Furthermore, the cross-sectional area of muscle fibers increased (995.2 ± 166.5 μm2 vs. 1,398 ± 124.2 μm2; P < 0.001) in the MTrPs group, which was indicative of muscle contraction (fig. 1F). Immunofluorescence staining showed p-PDGFR-α was also increased (1.00 ± 0.10 vs. 1.44 ± 0.20; units, mean intensity; P < 0.001) in the MTrPs group (fig. 1G). These results suggest that PDGFR-α may be involved in the pathophysiological mechanism of MTrPs. Therefore, we focused our further investigation on PDGFR-α for more detailed studies.

Fig. 1.

Fig. 1.

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Receptor tyrosine kinases (RTKs) expression profiles of myofascial trigger points (MTrPs) tissue derived from upper trapezius muscle of myofascial pain syndrome (MPS) patients and control groups. (A) The upregulated RTKs members were validated by microarray analysis. MPS group = 11, Con group = 7. Platelet-derived growth factor receptor-α (PDGFR-α): Con, 879 ± 83.08; 95% CI, 802.2–955.8 versus MPS, 1,060 ± 96.84; 95% CI, 994.9–1,125; units, fluorescent value; mean ± SD; P < 0.001. PDGFR-β: Con, 338.8 ± 47.96; 95% CI, 294.5–383.2 versus MPS, 308.4 ± 51.67; 95% CI, 273.7–343.1; units, fluorescent value; mean ± SD; P > 0.05. (B) Representative microscopic images showing morphology of muscle fibers in different groups. The morphology of MTrPs showed that annular or enlarged muscle fibers (yellow arrows) of different sizes with centralized nuclei in cross-sectional spaces under microscopy (yellow arrows). Scale bars, 20 μm. (C) The relationship between pain intensity and expression level of phosphorylated PDGFR-α (p-PDGFR-α) was characterized by a significant positive correlation (r = 0.711; n = 11; P < 0.05). (D) Results from enzyme-linked immunosorbent assay (ELISA) showed that the level of serum platelet-derived growth factor-AA (PDGF-AA) was increased. Con, 3.74 ± 0.82; 95% CI, 2.96–4.5; versus MPS, 5.97 ± 0.98; 95% CI, 5.31–6.6; units, ng/ml; mean ± SD; P < 0.001. (E) Results from immunohistochemistry (IHC) showed that the expression of PDGF-AA (yellow arrows) was upregulated at MTrPs. Scale bars, 20 μm. Con, 1.51 ± 0.33; 95% CI, 1.16–1.85; versus MPS, 10.2 ± 1.57; 95% CI, 8.55–11.85; units, integrated optical density (IOD); mean ± SD; P < 0.001. (F) The cross-sectional area of muscle fibers was increased in MPS group. Con, 995.2 ± 166.5; 95% CI, 902.9–1,087; versus MPS, 1,398 ± 124.2; 95% CI, 1,330–1,467; units, μm2; mean ± SD; P < 0.001. (G) Representative fluorescence microscopic images showing expression of p-PDGFR-α (green) in different groups. Scale bars, 20 μm. Con, 1.00 ± 0.10; 95% CI, 0.89–1.11; versus MPS, 1.44 ± 0.20; 95% CI, 1.23–1.64; units, mean intensity; mean ± SD; P < 0.001. *P < 0.05; **P < 0.01; ***P < 0.001. ALK, anaplastic lymphoma kinase; EphA, ephrin receptor A; EphB, ephrin receptor B; LTK, leukocyte tyrosine kinase; TRKB, tyrosine kinase receptor B; ZAP70, zeta-chain-associated protein kinase 70.

Activation of PDGFR-α Induces Pain-like Behaviors, JAK2/STAT3 Pathway Activation, and Increased c-fos Protein Expression in Normal Rats

We considered whether upregulation of p-PDGFR-α was a contributor to pathophysiologic implications of MTrPs or was merely a bystander. Intramuscular injection of PDGF-A-chain homodimer was used to activate PDGFR-α in normal rats. After unilateral injection into the gastrocnemius muscle, the Randall–Selitto test was performed, which showed that the mechanical withdrawal threshold decreased in a time-dependent manner (407.7 ± 4.35g vs. 351.8 ± 31.81 g; P < 0.001), indicating behavioral pain in these rats. Moreover, results from the nest-building test showed that activation of PDGFR-α downgraded nest quality (3.8 ± 0.92 vs. 2.7 ± 0.95; units, scores; P < 0.05; fig. 2A). Two hours after PDGF-A-chain homodimer injection, samples were collected for Western blot (WB) and IHC assays. Results from WB and IHC analysis showed that, in contrast to that observed in controls, the activation of PDGFR-α increased protein levels of c-fos in the L4–L5 dorsal horn of spinal cords (IHC: 3.86 ± 1.08 vs. 17.53 ± 2.05; units, IOD; P < 0.001; WB: 0.21 ± 0.02 vs. 0.93 ± 0.46; units, relative gray values; P < 0.001; fig. 2, B and C). Images from electron microscopy revealed that sarcomere lengths were significantly decreased after activating PDGFR-α (2.13 ± 0.06 μm vs. 1.63 ± 0.05 μm; P < 0.001; fig. 2D). Similar findings were observed for the muscle fiber cross-sectional area (Figure S3, https://links.lww.com/ALN/D629). Results from the WB analysis showed that p-PDGFR-α, the JAK2/STAT3 pathway, and inflammation factors were also increased (P < 0.01; P < 0.001). Moreover, the muscle contraction biomarkers myosin light chain kinase (MLCK) and phosphorylated myosin light chain (p-MLC) were upregulated (P < 0.01, P < 0.001; fig. 2E; descriptive statistics can be found in the Supplemental Data, https://links.lww.com/ALN/D632). These results suggest that an activation in PDGFR-α may increase inflammatory factors and induce muscle contraction via the JAK2/STAT3 signaling pathway, which may then contribute to the pain-like behaviors observed in rats.

Fig. 2.

Fig. 2.

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Activation of platelet-derived growth factor receptor-α (PDGFR-α)–induced pain-like behaviors, Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) activation, and muscle contraction in normal rats. (A) Activation of PDGFR-α within gastrocnemius muscle decreased respectively mechanical withdrawal thresholds and the nest quality scores in Randall–Selitto and nest-building test. n = 10 rats per group for behavioral test and analysis. Randall–Selitto test on hour 2: wild-type (WT), 407.7 ± 4.35; 95% CI, 404.1–411.4; versus WT plus PDGF-A-chain, 351.8 ± 31.81; 95% CI, 325.2–378.4; units, g; mean ± SD; P < 0.001. Nest-building test: WT, 3.8 ± 0.92; 95% CI, 3.14–4.46; versus WT plus PDGF-A-chain, 2.7 ± 0.95; 95% CI, 2.02–3.38; units, scores; mean ± SD; P < 0.05. (B and C) Representative immunohistochemistry (IHC) images and Western blod (WB) results for showing expression of c-fos protein (yellow arrows) in L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 100 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantification were done for representative samples from each group. Three independent biologic replicate experiments were performed. IHC: WT, 3.86 ± 1.08; 95% CI, 2.72–4.90; versus WT plus PDGF-A-chain, 17.53 ± 2.05; 95% CI, 15.38–19.68; units, integrated optical density (IOD); mean ± SD; P < 0.001. Western blot (WB; normalized to GAPDH): WT, 0.21 ± 0.02; 95% CI, 0.19–0.22; versus WT plus PDGF-A-chain, 0.93 ± 0.46; 95% CI, 0.88–0.98; units, relative gray values; mean ± SD; P < 0.001. (D) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres and cross-sectional area of muscle fibers of different groups. Scale bars, 2 μm. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. Length of sarcomeres: WT, 2.13 ± 0.06; 95% CI, 2.10–2.16; versus WT plus PDGF-A-chain, 1.63 ± 0.05; 95% CI, 1.60–1.66; units, μm; mean ± SD; P < 0.001. Cross-sectional area of muscle fibers: WT, 1,572 ± 528; 95% CI, 1,280–1,865; versus WT plus PDGF-A-chain, 2,032 ± 246.7; 95% CI, 1,895–2,168; units, μm2; mean ± SD; P < 0.01. (E) Activation of PDGFR-α increased protein levels of JAK2/STAT3 pathway, inflammatory factors, and contraction biomarkers in rats. WB results for proteins with similar molecular weight were from the same samples and run in parallel in different concentrations gels. Three independent biologic replicate experiments were performed. n = 6 values per group were included in the analysis. Descriptive statistics of WB can be found in the Supplemental Data (https://links.lww.com/ALN/D632). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; kD, kilodalton; MLCK, myosin light chain kinase; MTrPs, myofascial trigger points; p-MLC, phosphorylated myosin light chain; TNF-α, tumor necrosis factor-α.

Knockdown of PDGFR-α or Inhibition of the JAK2/STAT3 Pathway Ameliorates Pain-like Behaviors, Inhibits Inflammatory Response, and Improves Muscle Contraction in MTrPs Rats

The results of the nest-building test showed that the ability to build a nest was impaired in MTrPs rats with lower nest scores (3.9 ± 1.00 vs. 2.2 ± 0.92; units, scores; P < 0.001). Mechanical withdrawal threshold was significantly decreased in the MTrPs group (391.9 ± 17.43 g vs. 360.8 ± 19.43 g; P < 0.001; fig. 3A). Results from WB analysis showed that high levels of p-PDGFR-α expression in MTrPs rats were observed compared with those in control rats (0.39 ± 0.03 vs. 0.91 ± 0.19; units, relative gray values; P < 0.01; fig. 3B). Morphological features similar to those of patients with MPS were observed in the MTrPs rat (fig. 3C). Results from IHC analysis revealed that p-PDGFR-α was significantly increased and expressed on the cell membrane (2.11 ± 1.02 vs. 38.31 ± 7.64; units, IOD; P < 0.001; fig. 3D). Next, the results from the nest-building test showed that PDGFR-α knockdown and JAK2/STAT3 inhibition rats exhibited improved nest scores (vector: 1.90 ± 0.88 vs. negative control [NC]-scramble: 1.80 ± 0.79 vs. knockdown-PDGFR-α: 2.9 ± 0.99; units, scores; P < 0.05; saline: 1.70 ± 0.67 vs. AZD1480: 2.60 ± 0.97; units, scores; P < 0.05; fig. 3E). Similar results were observed in the Randall–Selitto test (296.3 ± 32.22 g vs. 296.2 ± 23.34 g vs. 343.8 ± 29.59 g; P < 0.01; 295.2 ± 12.05 g vs. 375.1 ± 10.57 g; P < 0.001; fig. 3F). After LV infusion into MTrPs, the knockdown efficiency of PDGFR-α was examined by RT-qPCR and showed a 62% decrease compared with that in the control group, a trend similar to that observed from WB analysis (0.50 ± 0.07 vs. 0.58 ± 0.07 vs. 0.13 ± 0.03; units, relative gray values; P < 0.01; fig. 3G). Rat spinal cord tissues from the L4–L5 dorsal horn were collected after 2 h of AZD1480 treatment or 14 days of lentiviral infection with PDGFR-α-shRNA or nonsilenced-shRNA. Results from IHC analysis showed that c-fos protein expression in the L4–L5 dorsal horn of the spinal cord was decreased compared with that in MTrPs rats (WT: 5.61 ± 3.23 vs. MTrPs: 81.48 ± 4.69 vs. knockdown-PDGFR-α: 30.71 ± 7.56 vs. AZD1480: 24.14 ± 9.77; units, IOD; P < 0.001; fig. 3H). Results from the HE staining analysis showed that the cross-sectional area was significantly decreased in knockdown-PDGFR-α and JAK2/STAT3 inhibition rats compared with that in control rats (1,324 ± 393.6 μm2 vs. 2,432 ± 344.9 μm2 vs. 1,843 ± 356 μm2 vs. 2,015 ± 264.5 μm2; P < 0.001; P < 0.01; fig. 4A). Results from WB analysis showed that knockdown of PDGFR-α or inhibition of JAK2/STAT3 decreased levels of inflammatory factors (P < 0.01, P < 0.001) and downregulated MLCK and p-MLC compared with those in MTrPs rats (P < 0.001; fig. 4B; descriptive statistics can be found in the Supplemental Data, https://links.lww.com/ALN/D632). These findings demonstrated that knockdown of PDGFR-α ameliorated pain-like behaviors, decreased inflammatory factors, and improved muscle contraction and that this process likely occurred through the JAK2/STAT3 signaling pathway in MTrPs rats.

Fig. 3.

Fig. 3.

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Knockdown of platelet-derived growth factor receptor-α (PDGFR-α) or inhibition of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway ameliorates pain-like behaviors in myofascial trigger points (MTrPs) rats. (A) MTrPs model rats exhibits low-quality scores and mechanical withdrawal thresholds decreased. n = 10 rats per group for behavioral test and analysis. Nest-building test: wild-type (WT), 3.9 ± 1.00; 95% CI, 3.19–4.61; versus MTrPs, 2.2 ± 0.92; 95% CI, 1.54–2.86; units, scores; mean ± SD; P < 0.001. Randall–Selitto test in week 2: WT, 391.9 ± 17.43; 95% CI, 379.14–404.4; versus MTrPs, 360.8 ± 19.43; 95% CI, 346.9–374.7; units, g; mean ± SD; P < 0.001. (B) Western blot (WB) results showing the expression of phosphorylated PDGFR-α (p-PDGFR-α) within MTrPs in animal models. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): WT, 0.39 ± 0.03; 95% CI, 0.32–0.46; versus MTrPs, 0.91 ± 0.19; 95% CI, 0.43–1.39; units, relative gray values; mean ± SD; P < 0.01. (C) MTrPs rats model exhibits a highly similar morphology (yellow arrows) to MTrPs of MPS patients. (D) Representative immunohistochemistry (IHC) images for showing expression and localization (yellow arrows) of p-PDGFR-α in MTrPs of rats. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. IHC: WT, 2.11 ± 1.02; 95% CI, 1.04–3.18; versus MTrPs, 38.31 ± 7.64; 95% CI, 30.30–46.32; units, integrated optical density (IOD); mean ± SD; P < 0.001. Cross-sectional area: WT, 1,246 ± 223.2; 95% CI, 1,122–1,369; versus MTrPs, 2,636 ± 430.6; 95% CI, 2,398–2,875; units, μm2; mean ± SD; P < 0.001. (E and F) Representative behavioral images showing knockdown of PDGFR-α or inhibition of JAK2/STAT3 pathway increased nest scores and mechanical withdrawal thresholds of MTrPs rats. n = 10 rats per group for behavioral test and analysis. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 1.90 ± 0.88; 95% CI, 1.24–2.53; versus NC-scramble, 1.80 ± 0.79; 95% CI, 1.24–2.36; versus knockdown-PDGFR-α, 2.9 ± 0.99; 95% CI, 2.19–3.61; units, scores; mean ± SD; P < 0.05; saline, 1.70 ± 0.67; 95% CI, 1.22–2.18; versus AZD1480, 2.60 ± 0.97; 95% CI, 1.91–3.29; units, scores; mean ± SD; P < 0.05. Randall–Selitto test on hour 2: vector, 296.3 ± 32.22; 95% CI, 273.2–319.3; versus NC-scramble, 296.2 ± 23.34; 95% CI, 279.5–312.9; versus knockdown-PDGFR-α, 343.8 ± 29.59; 95% CI, 322.6–364.9; units, g; mean ± SD; P < 0.01. Saline, 295.2 ± 12.05; 95% CI, 285.2–305.3; versus AZD1480, 375.1 ± 10.57; 95% CI, 366.3–384.0; units, g; mean ± SD; P < 0.001. (G) Schematic representation of virus infection in MTrPs. Scale bar, 20 μm. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and WB analysis were used to validate the efficiency of PDGFR-α knockdown. n = 6 rats per group. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): vector, 0.50 ± 0.07; 95% CI, 0.43–0.58; versus NC-scramble, 0.58 ± 0.07; 95% CI, 0.51–0.66; versus knockdown-PDGFR-α, 0.13 ± 0.03; 95% CI, 0.11–0.16; units, relative gray values; mean ± SD; P < 0.01; RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.08; 95% CI, 0.92–1.08; versus NC-scramble, 0.94 ± 0.11; 95% CI, 0.83–1.06; versus knockdown-PDGFR-α, 0.38 ± 0.15; 95% CI, 0.22–0.54; units, 2-ΔΔCT; mean ± SD; P < 0.001. (H) Representative IHC images for showing expression of c-fos protein (yellow arrows) in L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. Experiments were repeated at least three times, and quantitation was done for representative samples from each group. n = 6 values per group were included in the analysis. IHC: WT, 5.61 ± 3.23; 95% CI, 2.22–8.99; versus MTrPs, 81.48 ± 4.69; 95% CI, 76.56–86.41; versus knockdown-PDGFR-α, 30.71 ± 7.56; 95% CI, 22.77–38.64; versus AZD1480, 24.14 ± 9.77; 95% CI, 13.89–34.40; units, IOD; mean ± SD; P < 0.001. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HE, hematoxylin-eosin staining; NC, negative control.

Fig. 4.

Fig. 4.

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Platelet-derived growth factor receptor-α (PDGFR-α) depends on Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway to induce pain-like behaviors and muscle contraction in myofascial trigger points (MTrPs) rats. (A) Representative hematoxylin and eosin (HE) staining images showing knockdown of PDGFR-α or inhibition of JAK2/STAT3 pathway decreased the cross-sectional area as compared with controls. Scale bars, 20 μm. Top 15 cross-sectional area values were analyzed from 6 rats per group. Wild-type (WT), 1,324 ± 393.6; 95% CI, 1,106–1,524; versus MTrPs, 2,432 ± 344.9; 95% CI, 2,241–2,363; versus knockdown-PDGFR-α, 1,843 ± 356; 95% CI, 1,646–2,040; versus AZD1480, 2,015 ± 264.5; 95% CI, 1,869–2,162; units, μm2; mean ± SD; P < 0.01; P < 0.001. (B) Knockdown of PDGFR-α or JAK2/STAT3 pathway within MTrPs suppresses inflammatory factors and contraction biomarkers expression. n = 8 rats per group. Three independent biologic replicate experiments were performed. Descriptive statistics of Western blot (WB) can be found in the Supplemental Data (https://links.lww.com/ALN/D632). (C) Inhibition of JAK2/STAT3 pathway rescued the pain-like behaviors. Nest-building test: PDGF-A-chain-homodimer, 2.10 ± 0.99; 95% CI, 1.39–2.81; versus PDGF-A-chain-homodimer plus AZD1480, 3.3 ± 0.95; 95% CI, 2.62–3.98; units, scores; mean ± SD; P < 0.05. Randall–Selitto test on hour 2: PDGF-A-chain-homodimer, 308.3 ± 14.11; 95% CI, 298.2–318.4; versus PDGF-A-chain-homodimer plus AZD1480, 357.2 ± 9.51; 95% CI, 350.4–364; units, g; mean ± SD; P < 0.001. (D) Cross-sectional area of muscles fibers, PDGF-A-chain-homodimer, 2,032 ± 246.7; 95% CI, 1,895–2,168; versus PDGF-A-chain-homodimer plus AZD1480, 1,408 ± 164.7; 95% CI, 1,317–1,499; units, μm2; mean ± SD; P < 0.001; and (E) contraction biomarkers expression resulting from PDGFR-α activation. n = 10 rats per group for behavioral test and analysis. Three independent biologic replicate experiments for WB were performed. Descriptive statistics of WB can be found in the Supplemental Data (https://links.lww.com/ALN/D632). (F) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres of different groups. Scale bars, 2 μM. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. WT, 2.40 ± 0.05; 95% CI, 2.37–2.43; versus MTrPs, 1.54 ± 0.06; 95% CI, 1.50–1.58; versus knockdown-PDGFR-α, 1.68 ± 0.05; 95% CI, 1.65–1.70; versus AZD1480, 1.83 ± 0.04; 95% CI, 1.81–1.86; mean ± SD; P < 0.001; PDGF-A-chain-homodimer, 1.67 ± 0.04; 95% CI, 1.65–1.70; versus PDGF-A-chain-homodimer plus AZD1480, 1.76 ± 0.07; 95% CI, 1.71–1.80; units, μm; mean ± SD; P < 0.01. (G) Representative immunohistochemistry (IHC) staining images showing c-fos protein expression of L4–L5 dorsal horn of spinal cords of different groups. Scale bars, 50 μm/20 μm. n = 8 rats per group and at least 4 images from 1 animal, and the quantifications were done for representative samples from each group. n = 6 values per group were included in the analysis. PDGF-A-chain-homodimer, 16.76 ± 2.21; 95% CI, 14.45–19.08; versus PDGF-A-chain-homodimer plus AZD1480, 9.25 ± 1.36; 95% CI, 7.83–10.68; units, integrated optical density (IOD); mean ± SD; P < 0.01. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; MLCK, myosin light chain kinase; p-MLC, phosphorylated myosin light chain; TNF-α, tumor necrosis factor-α.

JAK2/STAT3 Signaling Pathway Mediates Inflammation, Muscle Contraction, and Behavioral Anomalies Resulting from PDGFR-α Phosphorylation in MTrPs Rats

Next, to substantiate the hypothesis that pain-like behaviors and contraction induced by PDGFR-α were dependent on the JAK2/STAT3 signaling pathway, activation-PDGFR-α rats were treated with the JAK2/STAT3 inhibitor, AZD1480. AZD1480 treatment effectively ameliorated the pain-like and nest-building behaviors from this PDGFR-α agonist (Randall–Selitto test on hour 2: 308.3 ± 14.11 g vs. 357.2 ± 9.51 g; P < 0.001; nest-building test: 2.10 ± 0.99 vs. 3.3 ± 0.95; units, scores; P < 0.05) and decreased muscle fiber cross-sectional area (2,032 ± 246.7 μm2 vs. 1,408 ± 164.7 μm2; P < 0.001; fig. 4, C and D). As shown in figure 4E and Figure S3 (https://links.lww.com/ALN/D629), AZD1480 treatment markedly decreased MLCK and p-MLC expression and the muscle fiber cross-sectional area. Electron microscopy images showed that sarcomere length significantly increased after AZD1480 treatment (1.67 ± 0.04 μm vs. 1.76 ± 0.07 μm; P < 0.01; fig. 4F). Similar trends were observed for c-fos protein expression (16.76 ± 2.21 vs. 9.25 ± 1.36; units, IOD; P < 0.01; fig. 4G). These results demonstrated that this pharmacologic inhibition of the JAK2/STAT3 pathway significantly rescued the inflammation, muscle contraction, and pain-like behaviors resulting from PDGFR-α activation.

PDGFR-α Physically Interacted with COL1A1 in MTrPs Rats

The Co-IP and Shotgun LC-MS analysis was used to identify PDGFR-α–binding proteins in MTrPs rats. Based on the results of LC-MS (Supplemental Digital Content, https://links.lww.com/ALN/D630) and existing research, COL1A1 was identified. IHC and WB analyses showed that COL1A1 expression significantly increased (0.06 ± 0.01 vs. 1.05 ± 0.10; units, relative gray values; P < 0.001) in the extracellular space (fig. 5, A and B). Then, the Co-IP assay illustrated that PDGFR-α and COL1A1 could coprecipitate with each other in MTrPs rats. Moreover, results from WB analysis showed that COL1A1 increased Co-IP with PDGFR-α (fig. 5C). Analysis of molecular docking revealed that proteins PDGFR-α and COL1A1 formed a stable protein docking model. The z-score values and their best pose interactions were calculated; the highest z-score of PDGFR-α and COL1A1 was 1,284.318. As shown in figure 5D, PDGFR-α forms interaction forces with amino acid sites such as lysine (LYS) 35-aspartic acid (ASP) 846, glutamic acid (GLU) 55-threonine (THR) 894, and histidine (HIS) 845-arginase (ARG) 34 with COL1A1. These findings provide a rationale for the physical protein interactions. Finally, immunofluorescence results showed that PDGFR-α (green) colocalized with COL1A1 (red) expression (fig. 5E).

Fig. 5.

Fig. 5.

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Platelet-derived growth factor receptor-α (PDGFR-α) physically interacted with collagen type I α 1 (COL1A1) in myofascial trigger points (MTrPs) rats. (A and B) Representative immunohistochemistry (IHC) images and Western blot (WB) results for showing expression of COL1A1 of different groups. Scale bars, 100 μm. n = 8 rats per group and at least 4 images from 1 animal. Three independent biologic replicate experiments were performed. WB (normalized to GAPDH): wild-type (WT), 0.06 ± 0.01; 95% CI, 0.04–0.07; versus MTrPs, 1.05 ± 0.10; 95% CI, 0.94–1.15; units, relative gray values; mean ± SD; P < 0.001. (C) The interaction between PDGFR-α and COL1A1 identified was confirmed by immunoprecipitation in MTrPs rats. (D) Results of molecular docking showed that PDGFR-α forms interaction forces with amino acid sites such as LYS35-ASP846, GLU55-THR894, and HIS845-ARG34 with COL1A1. (E) Immunofluorescence (IF) results showed that PDGFR-α (green) colocalized with the COL1A1 (red) expression. n = 8 rats per group. For WB, n = 6 values per group were included in the analysis. Data are presented as mean ± SD. ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Overexpression/Knockdown of COL1A1 Induced/Ameliorated Pain-like Behaviors and Increased/Decreased Inflammatory Factors and Sarcomeres Length

Results from nest-building behaviors and the Randall–Selitto test showed that COL1A1-overexpression rats exhibited significantly decreased nest scores (3.90 ± 1.00 vs. 2.40 ± 0.84; units, scores; P < 0.01) and mechanical withdrawal thresholds (412.80 ± 16.02 g vs. 355.80 ± 28.76 g; P < 0.001; fig. 6A). Figure 6B shows the efficiency of COL1A1 overexpression (1.00 ± 0.06 vs. 2.81 ± 0.74; units, 2-ΔΔCT; P < 0.001). COL1A1 knockdown improved this behavior to some extent (nest-building test: 1.80 ± 0.79 vs. 2.10 ± 0.99 vs. 3.10 ± 0.74; units, scores; P < 0.01; P < 0.05; Randall–Selitto test: 306.40 ± 17.44 g vs. 302.40 ± 19.70 g vs. 341.20 ± 28.47 g; P < 0.01; fig. 6C). Results from WB analysis showed that COL1A1-overexpression rats exhibited significantly increased inflammatory factors (P < 0.001; P < 0.05) and contraction-related markers (P < 0.001; P < 0.01; descriptive statistics can be found in the Supplemental Data, https://links.lww.com/ALN/D632) and decreased sarcomere length (2.32 ± 0.04 μm; vs. 2.07 ± 0.05 μm; P < 0.001; fig. 6, D and E). In contrast, the opposite results were observed in the COL1A1-knockdown rats. As shown in figure 6E, similar trends were observed in sarcomere length.

Fig. 6.

Fig. 6.

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Overexpression/knockdown of collagen type I α 1 (COL1A1) induced/ameliorated pain-like behaviors and increased/decreased inflammatory factors. (A) Overexpression of COL1A1 in rats exhibits low-quality scores and mechanical withdrawal thresholds decreased. n = 10 rats per group for behavioral test and analysis. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 3.90 ± 1.00; 95% CI, 3.19–4.61; versus overexpression-COL1A1, 2.40 ± 0.84; 95% CI, 1.80–3.00; units, scores; mean ± SD; P < 0.01. Randall–Selitto test: vector, 412.80 ± 16.02; 95% CI, 401.40–424.30; versus overexpression-COL1A1, 355.80 ± 28.76; 95% CI, 335.20–376.30; units, g; mean ± SD; P < 0.001. (B) Schematic representation of virus infection in rats. Scale bar, 20 μm. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was used to validate the efficiency of COL1A1 overexpression. n = 6 rats per group. RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.06; 95% CI, 0.94–1.07; versus overexpression-COL1A1, 2.81 ± 0.74; 95% CI, 2.03–3.59; units, 2-ΔΔCT; mean ± SD; P < 0.001. (C) Knockdown of COL1A1 in myofascial trigger points (MTrPs) rats increased nest scores and mechanical withdrawal thresholds. n = 10 rats per group for behavioral test. RT-qPCR was used to validate the efficiency of COL1A1 knockdown. n = 6 rats per group. Behavioral test was performed at 14 days after lentivirus injection. Nest-building test: vector, 1.80 ± 0.79; 95% CI, 1.24–2.56; versus NC-scramble, 2.10 ± 0.99; 95% CI, 1.39–2.81; versus knockdown-COL1A1, 3.10 ± 0.74; 95% CI, 2.57–3.63; units, scores; mean ± SD; P < 0.01; P < 0.05. Randall–Selitto test: vector, 306.40 ± 17.44; 95% CI, 293.90–318.90; versus NC-scramble, 302.40 ± 19.70; 95% CI, 288.30–316.50; versus knockdown-COL1A1, 341.20 ± 28.47; 95% CI, 320.80–361.50; units, g; mean ± SD; P < 0.01. RT-qPCR (normalized to GAPDH): vector, 1.00 ± 0.10; 95% CI, 0.90–1.10; versus NC-scramble, 1.07 ± 0.34; 95% CI, 0.71–1.43; versus knockdown-COL1A1, 0.35 ± 0.17; 95% CI, 0.17–0.53; units, 2-ΔΔCT; mean ± SD; P < 0.001. (D) Overexpression/knockdown of COL1A1 within MTrPs increased/decreased inflammatory factors and contraction biomarkers expression. n = 8 rats per group. Three independent biologic replicate experiments were performed. Western blot (WB) results for proteins with similar molecular weight were from the same samples and run in parallel in different concentrations gels. Descriptive statistics of WB can be found in the Supplemental Data (https://links.lww.com/ALN/D632). (E) Representative transmission electron microscopy images and summary of data showing the length of sarcomeres of different groups. Scale bars, 2 μm. n = 8 rats per group and at least 4 images from 1 animal. One or two representative images per rat were included in the analysis. Vector, 2.32 ± 0.04; 95% CI, 2.29–2.34; versus overexpression-COL1A1, 2.07 ± 0.05; 95% CI, 2.04–2.10; mean ± SD; P < 0.001; vector, 1.51 ± 0.03; 95% CI, 1.49–1.54; versus NC-scramble, 1.56 ± 0.05; 95% CI, 1.53–1.59; versus knockdown-COL1A1, 1.70 ± 0.06; 95% CI, 1.66–1.75; units, μm; mean ± SD; P < 0.001; P < 0.01. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; IL-1β, interleukin 1β; kD, kilodalton; MLCK, myosin light chain kinase; p-MLC: phosphorylated myosin light chain; NC, negative control; TNF-α, tumor necrosis factor-α.

Discussion

This study examined the expression profiles of RTKs in MTrPs through human biopsies, offering insights into the pathophysiology of MTrPs. The disparate phosphorylation levels of PDGFR-α and β observed in RTKs profiles have prompted our investigation on PDGFR-α to elucidate its functions and mechanisms in MTrPs. Our data showed that p-PDGFR-α levels were significantly increased in MTrPs compared with human controls. Importantly, this upregulation produces significant pain and dysfunctional contraction promoting effects on MTrPs. This was supported by the finding that activation of PDGFR-α in normal rats induced a similar muscular phenotype via the JAK2/STAT3 pathway to that observed in MTrPs animal models. At the molecular level, COL1A1 was found to bind to PDGFR-α protein and promote its phosphorylation, which resulted in the release of inflammatory factors and pain-like behaviors at the functional level. These findings allowed us to propose the following paradigm for the regulation of inflammatory processes and dysfunctional contraction by COL1A1/PDGFR-α in the setting of MTrPs. We observed similar effects of COL1A1/PDGFR-α activation on pain-like behavior and the MTrPs phenotype in muscle tissue obtained from both male and female rats. This holds particular significance in light of the documented sex-specific susceptibility to chronic pain and enhances the applicability of our experimental results.28,29 Based on these findings, we concluded that COL1A1/PDGFR-α binding contributed to the initiation and sustainment of inflammatory pain and dysfunctional muscle contractions through activation of the JAK2/STAT3 pathway in the pathophysiology of MTrPs.

Recent studies have indicated the significance of RTKs, including anaplastic lymphoma kinase (ALK) and fms-like tyrosine kinase 3 families, in chronic pain conditions.8,30 For instance, nociceptors exhibit heightened excitability and neurite outgrowth under ALK overexpression, whereas the ALK inhibitor lorlatinib was found to be analgesic in mouse models of inflammatory and neuropathic pain. Furthermore, EphB2 regulates pathologic pain by modulating its interaction with the N-methyl-d-aspartate receptor in cortical and spinal cord neurons.31 Here, we describe RTKs family expression in MTrPs and focus on differentially expressed PDGFR-α, not PDGFR-β. Previous research has shown that intrathecal PDGFR-α activation induced thermal hyperalgesia and tactile allodynia in normal mice and that these effects were still observed for at least 1 week after injection,32 while suppression of PDGFR-α promotes recovery from pain-like behaviors in rats with bone cancer pain.33 The underlying mechanisms may include the elevation of PDGFR-α in tumor-infiltrating dendritic cells and the consequential activation of transcription factors to regulate the expression of genes associated with pain.34

PDGFR-α is a receptor protein and functions as a molecular switch distributed on cell membrane. Whether PDGFR-α can simultaneously bind to other proteins, except PDGF-AA ligands, in the pathophysiology of MTrPs remains unknown. Co-IP and LC-MS were used to explore potential PDGFR-α–binding proteins, which might influence PDGFR-α phosphorylation. Accordingly, COL1A1 was identified as a binding protein of PDGFR-α implicated in the pathophysiology of MTrPs. COL1A1, a major extracellular matrix component, is upregulated in multiple diseases, including malignant astrocytomas and lung cancer.35–37 Although a recent study has demonstrated that COL1A1 may become a therapeutic target for osteogenesis imperfecta mouse models to treat pain,38 the mechanisms require additional functional studies. Our data suggested that the phosphorylation of PDGFR-α by overexpression-COL1A1 effectively promoted inflammatory processes and dysfunctional contractions of muscle sarcomeres. Moreover, we predicted that the information of amino acid sites between PDGFR-α and COL1A1. COL1A1 appears to be a key target, because COL1A1 phosphorylates PDGFR-α, and PDGFR-α is speculated to play a key role in signal transduction and amplification.39–41 Other regulatory mechanisms may exist between COL1A1 and PDGFR-α; these should be investigated in subsequent studies.

The JAK2/STAT3 signaling pathway is activated by PDGFR-α in rats, contributing to MTrPs muscle contraction and inflammation. A large body of evidence has revealed the regulatory mechanisms of JAK2/Rho-kinase in smooth muscle contraction,42,43 and JAK2 promotes the activation of Rho-kinase.44 Our previous research reported that the ras homolog gene family (Rho-kinase), such as RhoA and ras-related C3 botulinum toxin substrate 1 (Rac1), was increased in the contraction knots of MTrPs.4 However, whether Rho-kinase is targeted downstream of JAK2/STAT3 signaling in MTrPs remains unclear. MLCK, an important target molecule downstream of Rho-kinase, is involved in muscle contraction through the phosphorylation of myosin light chain.45,46 Consistent with previous studies,47 we found that MLCK promoted MTrPs muscle contraction, presenting as increased cross-sectional area and decreased sarcomere length. However, the regulation of the MLCK/myosin light chain phosphatase balance was not fully elucidated. Similarly, the mechanism underlying Rho-kinase activity requires further investigation. The JAK2/STAT3 pathway is a classical signaling pathway related to inflammation. Results from this study showed that phosphorylation of PDGFR-α increased the protein levels of inflammatory factors via the JAK2/STAT3 pathway, which may explain PDGFR-α–mediated pain in MTrPs.

In conclusion, the findings of this study revealed that PDGFR-α activation, phosphorylated by COL1A1 via promotion of the JAK2/STAT3 pathway, plays an important role in the activation and release of inflammatory factors, coupled with dysfunctional muscle contractions induced by MTrPs. These results have biologic and clinical significance for the potential identification and treatment of MTrPs.

Acknowledgments

Support is gratefully acknowledged from the Research Center for Basic Medical Sciences, Qilu Hospital, Shandong University, Shandong, China.

Research Support

This study was supported by the National Natural Science Foundation of China (Shandong University, Jinan, China; No. 82272600), the Natural Science Foundation of Shandong Province (Jinan, China; No. ZR2022QH141), and the Fundamental Research Funds of Shandong University (Jinan, China).

Competing Interests

The authors declare no competing interests.

Supplemental Digital Content

Graphical abstract: Molecular mechanisms schematic, https://links.lww.com/ALN/D625

MTrPs biopsy: Biopsy procedures for MTrPs, https://links.lww.com/ALN/D626

Figure S1: Construction of the MTrPs animal model, https://links.lww.com/ALN/D627

Figure S2: Standard curve for the ELISA, https://links.lww.com/ALN/D628

Figure S3: Representative image of muscle fiber HE staining, https://links.lww.com/ALN/D629

COL1A1 (LC-MS): Shotgun LC-MS spectrum of COL1A1, https://links.lww.com/ALN/D630

Supplemental Tables: Primary antibody information and the sequences of primers used for RT-qPCR, https://links.lww.com/ALN/D631

Supplemental Data: Descriptive statistics of WB, https://links.lww.com/ALN/D632

Certificate of language editing: Language editing by Editage, https://links.lww.com/ALN/D633

Full blots of all WBs: Full unedited gel for WBs, https://links.lww.com/ALN/D634

Supplementary Material

aln-141-0929-s001.pdf (695.1KB, pdf)
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aln-141-0929-s002.pdf (366.4KB, pdf)
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aln-141-0929-s003.pdf (1.8MB, pdf)
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Footnotes

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org).

The article processing charge was funded by National Natural Science Foundation of China (No. 82272600).

Contributor Information

Yu Liu, Email: liuyu10254@qiluhospital.com.

Feihong Jin, Email: jinfeihong0321@163.com.

Lingwei Zhou, Email: 202215721@mail.sdu.edu.cn.

Xuan Li, Email: LeeX2021@outlook.com.

Xiaoyue Li, Email: 202320959@mail.sdu.edu.cn.

Qinghe Chen, Email: 202315608@mail.sdu.edu.cn.

Shaozhong Yang, Email: yszyang@163.com.

Jintang Sun, Email: sunjintang@sdu.edu.cn.

Feng Qi, Email: 198962001111@sdu.edu.cn.

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