Wnt7a promotes muscle regeneration in branchiomeric orbicularis oris muscle

Abstract

Background: The orbicularis oris muscle exhibits a deficiency in cleft lip patients. Compared with the somite-derived limb muscles, the regeneration performance of the branchiomeric orofacial muscle has seldom been investigated.

Objective: This study aims to explore the possibility of augmenting the orbicularis oris muscle through the stimulus of Wnt7a.

Methods: Adult rat orbicularis oris muscle and tibialis anterior muscle were injected with recombinant human Wnt7a protein. The muscles were harvested at different time points after Wnt7a delivery. Muscle regeneration-related activity, including cell proliferation, stem cell proportion, myofiber plasticity, and total fiber number, was examined.

Results: Adult rat orbicularis oris muscle and tibialis anterior muscle exhibit similar regeneration-related activities after Wnt7a administration. Recombinant human Wnt7a administration resulted in enhanced cell proliferation, stem cell expansion, and fiber type remodelling in rat orbicularis oris muscle. In addition, newly formed myofibers were detected, contributing to an increased total fiber number.

Conclusion: Wnt7a induces vigorous regeneration in rat orbicularis oris muscle. This study helps lay a foundation for developing biotherapies to combat orofacial muscle deficiency.

Keywords: Skeletal muscle; cell proliferation; myosin heavy chain; regeneration.

Figure 1

Rh-Wnt7a delivery to rat OOM. A, B. Top and lateral view of the injection field. The yellow dot indicates the port of entry, and the yellow dotted line indicates the oral commissure. The port of entry was 6 mm medial to the oral commissure. C. Schematic illustration of the study rationale. The TA muscle was harvested at three weeks after Wnt7a administration. The OOM was harvested at two, three, and five weeks after Wnt7a administration.

Figure 2

Regeneration-related activities in OOM and TA muscle. A-D. Immunofluorescence staining of Ki67 (red) and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate Ki67-positive cells. E-H. Immunofluorescence staining of Pax7 (red), laminin (green), and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate Pax7-positive nuclei. I-L. Immunofluorescence staining of emb-MyHC (red), laminin (green) and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate emb-MyHC positive myofibers. M-O. Quantification of Ki67 positive nuclei, Pax7-positive nuclei and emb-MyHC positive myofiber. Scale bar = 50 μm.For each group, n = 6. *P < .05; **P < .01; ***P < .001.

Figure 3

Wnt7a induces muscle resident cells’ proliferation in OOM. A-F. Immunofluorescence staining of Ki67 (red) and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. G-L. Immunofluorescence staining of laminin (green) and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate centrally-nucleated myofibers. M. Quantification of Ki67-positive nuclei, normalized by total cell count per field. N. Quantification of centrally-nucleated myofibers, normalized by total fiber number per field. Scale bar = 50 μm. For each group, n = 6. *P < .05; **P < .01.

Figure 4

Wnt7a increases satellite cell proportion. A-L. Immunofluorescence staining of Pax7 (red), laminin (green) and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate Pax7-positive nuclei. M. Quantification of Pax7-positive nuclei, normalized by total cell count per field. Scale bar = 50 μm. For each group, n = 6. ***P < .001.

Figure 5

Wnt7a induces fiber type switch in OOM. A-P. Immunofluorescence staining of MyHC-1/MyHC-2A/MyHC-2X/MyHC-2B (red), laminin (green) and DAPI (blue) in BSA-treated muscle groups and Wnt7a-treated groups. Q. Quantification of the proportion of MyHC-1, MyHC-2A, MyHC-2X and MyHC-2B positive myofibers in the OOM at 3 wk after injection. R. Quantification of the proportion of MyHC-1, MyHC-2A, MyHC-2X and MyHC-2B positive myofibers in OOM at 5 wk after injection. Scale bar = 50 μm. For each group, n = 6. *P < .05.

Figure 6

Wnt7a promotes myofiber hyperplasia. A-F. Immunofluorescence staining of emb-MyHC (red), laminin (green), and DAPI (blue) in BSA-treated and Wnt7a-treated muscle groups. White arrows indicate emb-MyHC positive myofibers. G. Quantification of emb-MyHC positive myofibers, normalized by total fiber number per field. H. Quantification of total fibermyofibers per field in BSA-treated and Wnt7a-treated muscle groups. I. Quantification of minimal fiber ferret in BSA-treated and Wnt7a-treated muscle groups. Scale bar = 50 μm. For each group, n = 6. *P < .05; **P < .01; ***P < .001.