The pathophysiological nature of sarcomeres in trigger points in patients with myofascial pain syndrome: A preliminary study

Abstract

Background: Myofascial pain syndrome (MPS) has a high global prevalence and is associated with myofascial trigger points (MTrPs) in taut bands or nodules. Little is known about the aetiology. The current study assessed the pathophysiological characteristics of MTrPs in MPS patients.

Methods: Biopsies of the trapezius muscle were collected from the MTrPs of MPS patients (MTrP group; n = 29) and from healthy controls (control group; n = 24), and their morphologies were analysed via haematoxylin-eosin (H&E) and Masson staining. A protein microarray was used to detect the receptor tyrosine kinase (RTK) family proteins. mRNA and long non-coding RNA (lncRNA) sequencing and analysis were conducted, and immunohistochemistry and Western blotting were used to examine the expression of EphB and Rho family proteins.

Results: Abnormally contracted sarcomeres showed enlarged, round fibres without inflammation or fibrosis. An lncRNA-mRNA network analysis revealed activation of muscle contraction signalling pathways in MTrP regions. Among RTK family proteins, 15 exhibited increased phosphorylation, and two exhibited decreased phosphorylation in the MTrP regions relative to control levels. In particular, EphB1/EphB2 phosphorylation was increased on the muscle cell membranes of abnormal sarcomeres. RhoA and Rac1, but not cell division control protein 42 (Cdc42), were activated in the abnormal sarcomeres.

Conclusions: EphB1/EphB2 and RhoA/Rac1 might play roles in the aetiology of abnormally contracted sarcomeres in MTrPs without inflammatory cell infiltration and fibrotic adhesion.

Significance: Contracted sarcomeres were found in MTrP regions, which is consistent with the MTrP formation hypothesis. EphB1/EphB2 and RhoA/Rac1 might play roles in the sarcomere contractile sites of MTrPs, which may be promising therapeutic targets.

FIGURE 1

Histology of human trapezius muscle in myofascial trigger points (MTrPs) samples and control samples. Muscle was stained with haematoxylin‐eosin (H&E) (a,b,e,f) and Masson stain (c,d,g,h) in MTrP samples (a–d) and control samples (e–h). Normal muscle cells were observed in the control group (e). Enlarged, round, contracted muscle (black arrow) was observed in MTrPs patients (a)

FIGURE 2

Receptor tyrosine kinase (RTK) phosphorylation expression in MTrPs. (a) RTK proteins with different phosphorylation levels between MTrP (n = 11) tissues and control group tissues (n = 7). (b–d) Levels of p‐Eph in MTrP and control group samples. The levels of p‐EphB1, p‐EphB2, and p‐EphB3 were significantly higher in MTrP patients than in controls. (e) Representative bands of p‐EphB expression in control and MTrPs muscle tissues. (f) Quantification of the average means optical density for p‐EphB in the control group and MTrP group. (g) Representative images of p‐EphB expression in control muscle tissues and MTrPs (×400). (h,i) IHC score of the total area (h) and single fiber (i) for p‐EphB in the control group and MTrP group. IHC staining score was formulated as integrated optical density/size. Data are presented as M ± SEM or as medians and interquartile ranges (25th and 75th percentiles). IHC, immunohistochemistry; MTrPs, myofascial trigger points. *p < 0.05, ****p < 0.0001

FIGURE 3

Long non‐coding RNA (lncRNA)‐mRNA network analysis of the muscle contraction signaling transduction (Pearson's coefficient > 0.99). Red ovals represent mRNAs, blue rectangles represent lncRNAs, a line represents the correlation

FIGURE 4

RhoA and Rac1, not Cdc42, transfer membrane activation in the contractile sarcomeres of MTrPs. (a–c) In the control group, Rho A, Rac1, and Cdc42 were expressed evenly in the muscle sarcomere. (d) In the MTrP group, in site A, the expression of RhoA in cytoplasm was decreased, with RhoA having migrated to the cell membrane, and the expression in site B was increased. (e) In the MTrP group, in site A, the expression of Rac1 in cytoplasm was decreased, with Rac1 having migrated to the cell membrane, and the expression in site B was decreased. (f) The expression of Cdc42 in sarcomeres of the MTrP group was uneven but have no consistency. (A) The abnormal contractile site; (B) the elongated site adjacent to site A. **p < 0.01

FIGURE 5

Schematic diagram of skeletal muscle contraction pathway mediated by RTKs. MLC, myosin light chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; p‐MLC, myosin light chain phosphorylation; RTKs, receptor tyrosine kinase; GPCR, G protein‐coupled receptor; PLC, phospholipase C; PKC, protein kinase C; SM, skeletal muscle