Spatial and temporal changes in myogenic protein expression by the microenvironment after freeze injury

Abstract

Skeletal muscle has the remarkable capability to regenerate itself following injury. Adult myogenic stem cells (MSCs) are responsible for the repair and regeneration, and their activity is controlled by intrinsic and extrinsic factors. The aim of this study was to examine and compare the expression levels of Pax3, Pax7, MRF and p38 proteins during the course of regeneration and in different areas of the focal freeze-lesion damaged adult rat TA muscle. Using the focal freeze injury model, immunohistochemistry, laser-capture micro-dissection and Western blot analysis were performed. The results show that (1) in the severely damaged area, the focal freeze-lesion injury significantly activated Pax7 and myogenin expression within 7 days and down-regulated Pax3, MyoD and Myf-5 within 1 or 3 days, and (2) the level of the p38 protein was strongly and transiently up-regulated in the whole muscle on day 7 following injury, whereas the level of the pp38 protein was down-regulated within 3 days in the severely damaged and non-damaged areas. These findings indicate that the temporal (e.g. the time course of regeneration) and spatial (e.g. three zones created by the focal freeze-lesion) cues in a regenerating muscle have a significant impact on the activity of the adult MSCs.

Keywords: Pax; microenvironment; muscle regeneration; myogenic regulatory factor; myogenic stem cells.

Figure 1

Transverse sections of the adult TA muscle 3 days after focal freeze‐lesion injury, demonstrating three zones with different morphology and cellular components. (A) Zone 1 (Z1) represents the most severely damaged area. Zone 3 (Z3) contains intact myofibres and fibre integrity. Zone 2 (Z2) represents the transition area between zones 1 and 3. (B) An example showing that zone 2 has been sampled from a section using laser capture microscopy. Scale bars: 100 μm.

Figure 2

Western blot analyses of Pax3, Pax7, MyoD, Mrf‐5, myogenin, p38 and phospho‐p38 proteins in TA muscles 1, 3, 7 and 14 days following injury. (A) Representative Western blot bands for primary antibodies against Pax3, Pax7, MyoD, Myf‐5, myogenin, p38, phosphorylated p38 (pp38) and β‐actin proteins in uninjured contralateral (CL), freeze‐lesioned (FL) and untreated control (Nrm) muscles, 1, 3, 7 and 14 days following the focal freeze‐lesion injury. (B–G) The optical densities (OD) of Pax3, Pax7, MyoD, Myf‐5, p38 and pp38 protein levels in uninjured contralateral (CL) and freeze‐lesioned (FL) muscles were compared with the untreated control (Nrm) muscle using one‐way anova with Turkey's post hoc test (*P < 0.05). The comparison was not performed for myogenin as its OD was too low. n = 3 for each time point.

Figure 3

Immunohistochemical detection of Pax3, Pax7, MRFs and p38 proteins in the focal freeze‐lesioned TA muscle 3 days post injury. (A) Pax3⁺ profiles (arrows) were clustered in zone 2. (B) Pax7⁺ profiles (arrows) were observed in both zones 1 and 2. (C,D) MyoD⁺ (arrows) and phosphorylated p38⁺ (arrowheads) profiles were mainly distributed in zone 2, and some of them were co‐expressed (double arrowheads). (E) Myf‐5⁺ profiles (arrows) were detected in zone 2. (F) Myogenin⁺ profiles (arrows) were clustered in zone 2. Z1, Z2 and Z3: zones 1, 2 and 3, respectively. Scale bars: 10 μm.

Figure 4

Western blot analyses of Pax3, Pax7, MyoD, Myf‐5, myogenin, p38 and phosphorylated p38 in three zones of freeze‐lesion injured TA muscles. (A) Representative Western blot bands for primary antibodies against Pax3, Pax7, MyoD, Myf‐5, myogenin, p38, phosphorylated p38 (pp38) and β‐actin proteins in muscle samples from zones 1, 2 and 3 (respectively Z1, Z2, Z3) of the freeze‐lesioned muscle and from the untreated control (Nrm) muscle on day 3 post injury. (B–H) The optical densities (OD) of Pax3, Pax7, MyoD, Myf‐5, myogenin, p38 and phospho‐p38 protein levels in three zones were compared with the untreated control (Nrm) muscle using one‐way anova with Turkey's post hoc test (*P < 0.05). n = 3 for each time point.