Induction of periostin-like factor and periostin in forearm muscle, tendon, and nerve in an animal model of work-related musculoskeletal disorder

Abstract

Work-related musculoskeletal disorders (WMSDs), also known as repetitive strain injuries of the upper extremity, frequently cause disability and impairment of the upper extremities. Histopathological changes including excess collagen deposition around myofibers, cell necrosis, inflammatory cell infiltration, and increased cytokine expression result from eccentric exercise, forced lengthening, exertion-induced injury, and repetitive strain-induced injury of muscles. Repetitive tasks have also been shown to result in tendon and neural injuries, with subsequent chronic inflammatory responses, followed by residual fibrosis. To identify mechanisms that regulate tissue repair in WMSDs, we investigated the induction of periostin-like factor (PLF) and periostin, proteins induced in other pathologies but not expressed in normal adult tissue. In this study, we examined the level of PLF and periostin in muscle, tendon, and nerve using immunohistochemistry and Western blot analysis. PLF increased with continued task performance, whereas periostin was constitutively expressed. PLF was located in satellite cells and/or myoblasts, which increased in number with continued task performance, supporting our hypothesis that PLF plays a role in muscle repair or regeneration. Periostin, on the other hand, was not present in satellite cells and/or myoblasts.

Figure 1

Forelimb muscles isolated from control, trained control (TC), or experimental rats [3-, 6-, 8-, or 12-week high-repetition, high-force (HRHF)] were cryosectioned and immunoreacted to periostin-like factor (PLF) antibody. (A) Normal control (Con) animals did not show PLF localization. (B) PLF was increased in TC animals. (C) Section from a 6-week HRHF time point on which primary antibody was omitted and which was used as a negative control (Neg Con). (D–G) HRHF animals (3-week, 6-week, and 8-week) showed increased localization of PLF in the muscle fibers, and muscle fibers appeared moth-eaten, with some fibers showing higher localization of PLF than others (asterisk). (H) Higher magnification of 12-week HRHF muscle showing the immunolocalization of PLF in muscle fibers as well as satellite-like cells (arrow).

Figure 2

Forelimb tendon isolated from control or experimental rats (3-, 6-, 8-, or 12-week HRHF) were cryosectioned and immunoreacted with PLF antibody. (A,B) No PLF was detected in the normal control or TC animal tendon (T) or nerve (N). (C,D) PLF was detected at high levels in the tendons of 3- and 12-week HRHF animals, and the shape of the fibroblast nuclei changed from elongated (C) to more-rounded and plump (D) and inset at higher magnification showing rounded nuclei). (E,F) High amounts of PLF were also seen in nerves from 3- and 12-week HRHF animals.

Figure 3

(A) Western blot analysis using protein samples from muscles for controls (C), TC, and 3-week–12-week HRHF animals showed an increase in PLF levels. (B) PLF increased significantly in task-performing animals compared with normal controls (NC) (^*p<0.05) and compared with TCs (^&p<0.05). PLF levels also increased significantly at week 6 compared with week 3 (^$p<0.05) and at week 12 compared with week 8 (^$$p<0.05). (C) Western blot analysis using protein samples from tendons for C, TC, and 3–12-week HRHF animals showed an increase in PLF levels. (D) PLF increased significantly in task-performing animals compared with normal controls (*p<0.05) and compared with TCs (^&p<0.05). Increase in PLF levels was also observed after each week of task performance and is denoted by ^$ symbols, where ^$ denotes increase in week 6 from week 3, ^$$ denotes increase in week 8 from week 6, and ^$$$ denotes increase in week 12 from week 8. Western blot data are the mean of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Forelimb muscles isolated from control (Con), TC, or experimental (3-, 6-, 8-, or 12-week HRHF) rats were cryosectioned and immunoreacted with periostin or PGP9.5 antibodies. (A,B) Periostin was not detected in tendon (T) or muscle (M) of control animals. (C,D) Periostin was present in tendons and muscles of TC animals. (E–H) Periostin was observed in tendons and nerves (N) of experimental animals. (I–L) The neuronal marker PGP9.5 (green) was used on 6-week HRHF sections to confirm that periostin (POSTN, red) was localized to axons in peripheral nerves in the forelimb. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear stain (K).

Figure 5

Western blot analysis using protein samples from C, TC, and 3–12-week HRHF animals for muscle (A,B) and tendon (C,D) showed an increase in periostin in experimental animals compared with C and TC animals. Periostin increased significantly in both muscle and tendons of experimental animals compared with normal controls (^*p<0.05) and TCs (^&p<0.05). Western blot data are the mean of three independent experiments.

Figure 6

Forelimb muscles isolated from control or experimental rats (12-week HRHF) were cryosectioned and immunoreacted with PLF, periostin (POSTN), or PAX7 (satellite cell–specific marker) antibody. (A) PAX7 (green) was detected in satellite cells and/or myoblasts; (B) PLF (red) was present in the muscle fibers and satellite cells and/or myoblasts in experimental animals. (C) DAPI was used as a nuclear stain. (D) Overlay of PLF and PAX7 cells showing co-localization of these two proteins in satellite cells and/or myoblasts, as indicated by white arrows. (E) PAX7 (green) was detected in satellite cells and/or myoblasts; (F) periostin (red) was present in the muscle fibers in experimental animals. (G) Overlay showed no colocalization of periostin and PAX7 in satellite cells and/or myoblasts, as indicated by white arrowheads. (H) A tissue section on which primary antibody was omitted was used as a negative control.

Figure 7

Muscle sections immunoreacted with PAX7 and counterstained with hematoxylin show PAX7-positive satellite cells and/or myoblasts as indicated by black arrows.

Figure 8

Thirty-six fields per section and three slides per group were counted to obtain number of satellite cells and/or myoblasts. Graph shows number of satellite cells and/or myoblasts per mm² against treatment groups. All experimental animals showed significant increase in number of satellite cells and/or myoblasts over weeks of task performance, compared with normal controls (^*p<0.05) and TCs (^&p<0.05).