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Introduction

The human body possesses ~640 skeletal muscles, which account for ~40% of the whole body mass (1). These skeletal muscles are responsible for body movement and fulfill the functions of locomotion, respiration, swallowing and speech. Although they share the common architectural traits of striated muscles, the skeletal muscles have distinct embryonic origins (2). Trunk and limb muscles derive from somites; the majority of head muscles, such as masseter muscle and pharyngeal muscles, derive from branchial arches; extraocular muscles derive from prechordal mesoderm (3–5).

The prototype for skeletal muscle study is somite-derived limb muscles, but these represent only a small proportion of the whole skeletal muscle population (1). Investigations into head muscles have revealed that they exhibit heterogeneity in comparison with their limb counterparts. During embryonic myogenesis, T-box 1 and pituitary homeobox 2 mediate the formation of head muscles, yet are absent in limb muscle development (4,6). Head muscles possess a visceral mesodermal origin and are closely associated with heart development in myogenesis (7,8). Mesoderm in the cardiopharyngeal field gives rise to both the branchiomeric muscles and part of the heart (3). In addition, masseter muscle was reported to regenerate less effectively compared with tibialis anterior (TA) muscle (9). These results point to the notion that head muscles and limb muscles are markedly different skeletal muscle populations.

TA muscle has been studied extensively, particularly its developmental origin, morphological characteristics and augmentation potential (9–11). Levator veli palatini (LVP) muscle, however, lacks investigation in spite of its critical role in fulfilling velopharyngeal function and its core status in cleft palate surgery (12–14). Plastic surgeons have long been searching for strategies to augment LVP muscle, in order to facilitate velopharyngeal closure and improve speech in patients with cleft palates (15–17).

Strategies for muscle augmentation have been sought for decades. Effective agents, such as insulin-like growth factor (IGF)-1, angiotensin and Wnt7a, have previously been proposed to increase myofiber diameter and enhance muscle function in atrophied or normal muscle (10,18–20). These studies were primarily performed in limb muscles like the TA muscle. Considering the general differences between limb and head muscle, combined with the knowledge gap for LVP muscle augmentation, the present study was performed for two reasons: i) To glean background information of LVP muscle, in comparison with TA muscle; ii) to investigate whether the two muscles exhibited different augmentation potential. In the present study, the differences between TA and LVP muscle were characterized in terms of muscle fiber composition, in situ stem cell population and activation level of the Wnt signaling pathway under basal conditions. Furthermore, it was investigated whether the two muscles responded differently to growth factor stimulus.

Materials and methods

Animals

All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee at Sichuan University (Chengdu, China). Adult male Sprague-Dawley rats (age, 10 weeks; weight, 280–300 g) were purchased from Chengdu Dashuo Experimental Animal Center (Chengdu, China). The animals were raised in a temperature- and humidity-controlled room (temperature, 21±2°C; relative humidity, 50±5%) on a 12-h light/dark schedule. Food and water were freely accessible. A total of 18 animals were used in the present study and were randomly allocated to the following three groups: i) comparison between TA muscle and LVP muscle under basal conditions (n=6); ii) intramuscular Wnt7a administration of TA muscle (n=6); iii) intramuscular Wnt7a administration of LVP muscle (n=6).

Intramuscular Wnt7a delivery

Recombinant human Wnt7a (R&D Systems, Inc., Minneapolis, MN, USA) was injected directly into the muscles. For each TA muscle (n=6), 75 µl Wnt7a (100 µg/ml) was injected and 75 µl PBS was injected to the TA muscle in the contralateral leg as control; for each LVP muscle (n=6), 25 µl Wnt7a (100 µg/ml) was injected and 25 µl PBS was injected to the LVP muscle on the contralateral side as control. Injection into LVP muscle was carried out via an intraoral procedure. Rats were sacrificed 3 weeks after treatment and muscles were harvested for analyses.

Muscle harvest

TA muscle was cut from tendon to tendon on the tibia anterior bone. LVP muscle was approached and isolated according to Carvajal Monroy et al (21), with small modifications. Briefly, a ventral incision extending from the mandibular symphysis to the clavicle was made and the subcutaneous tissue was separated to expose the salivary gland. After removal of the salivary gland, the digastric and sternocleidomastoid muscle was visible. The posterior belly of the digastric muscle was dissected to its origin to expose the stylohyoid muscle beneath it and the tympanic bulla. The stylohyoid muscle was cut at its junction to the hyoid and pulled laterally to visualize the LVP muscle with its tendon clearly attached to the tympanic bulla. The LVP muscle was carefully dissected from its origin in the tympanic bulla to its insertion in the soft palate.

Immunofluorescence analysis

Muscle samples were attached to the chuck using Tissue-Tek Optimal Cutting Temperature compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen in isopentane cooled with liquid nitrogen using Lawlor's method (22). Cryosections were made at 10 µm thickness and fixed in 0°C acetone (100%) for 20 min. The sections were dried at room temperature for 20 min and washed in 0.01 mol/l phosphate-buffered saline (PBS). Sections were then blocked with PBS containing 5% bovine serum albumin (Amresco, LLC, Solon, OH, USA), and 5% donkey serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h at 25°C, and subsequently incubated overnight at 4°C with primary antibodies. Following washing in PBS, sections were incubated for 1 h at 25°C with Alexa Fluor 488-conjugated (A-21206; 1:500) and 568-conjugated (A10037; 1:500) secondary antibodies (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After several washes in PBS, the nuclei were stained with DAPI. Images were captured with an Olympus BX63 fluorescence microscope (Olympus Corporation, Tokyo, Japan). The primary antibodies used were as follows: Rabbit anti-laminin polyclonal antibody (L9393; 1:500; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), mouse anti-Pax7 monoclonal antibody (PAX7; 1:5; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-Ki67 monoclonal antibody (ab1667; 1:500; Abcam, Cambridge, MA, USA), mouse anti-myosin heavy chain 1 (MyHC-1) monoclonal antibody (A4.840; 1:40; Developmental Studies Hybridoma Bank), mouse anti-MyHC-2A monoclonal antibody (SC-71; 1:20; Developmental Studies Hybridoma Bank), mouse anti-MyHC-2X monoclonal antibody (6H1; 1:5; Developmental Studies Hybridoma Bank) and mouse anti-MyHC-2B monoclonal antibody (BF-F3; 1:5; Developmental Studies Hybridoma Bank).

Western blot analysis

Muscle samples were minced and prepared in RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Tissues were incubated for 30 min on ice, followed by centrifugation at 12,000 × g for 10 min at 4°C. Total protein concentration in the supernatant was determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Protein extract (7 µl/lane) was loaded on 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin in 0.5% TBS-Tween-20 at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. The protein bands were visualized using an enhanced chemiluminescence system (G2014; Wuhan Goodbio Technology Co., Ltd., Wuhan, China). Densitometry values were normalized to the intensity of corresponding bands for GAPDH. Quantitative analysis of western blotting was performed using ImageJ version 1.50e (National Institutes of Health, Bethesda, MD, USA). Primary antibodies were used as follows: Rabbit polyclonal anti-Axin2 antibody (ab32197; 1:1,000; Abcam), sheep polyclonal anti-Vangl2 antibody (AF4815; 1:1,000; R&D Systems, Inc.), rabbit polyclonal anti-pS6 antibody (2211; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-pAkt antibody (9271; 1:1,000, Cell Signaling Technology, Inc.) and rabbit polyclonal anti-GAPDH antibody (ab9485; 1:1,000; Abcam). Secondary antibodies were used as follows: Goat anti-rabbit IgG (ab6721; 1:5,000; Abcam) and donkey anti-sheep IgG (ab97125; 1:5,000; Abcam).

Quantification and statistical analyses

Myofiber size and overall cell density were calculated using ImageJ 1.50e. Numbers of Pax7-positive nuclei, Ki67-positive nuclei and the percentage of each specific fiber type were manually counted with three fields of view selected for each sample type. All data were analyzed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and results are presented as the mean ± standard deviation. Student's t-test was used to evaluate statistical differences. P<0.05 was considered to indicate a statistically significant difference.

Results

Fiber type composition of TA and LVP muscle

Sections of muscle samples revealed that individual fibers in TA muscle were larger compared with LVP muscle (data not shown). With their corresponding markers immunostained, different types of muscle fibers were identified and quantified in TA and LVP muscle. The percentage of MyHC-1 fibers was significantly higher in LVP muscle compared with TA muscle (~25 vs. ~5%; Fig. 1A). The percentage of MyHC-2A fibers was significantly higher in LVP muscle compared with TA muscle (~60 vs. ~15%; Fig. 1B). The percentage of MyHC-2X fibers was significantly higher in LVP muscle compared with TA muscle (~40 vs. ~25%; Fig. 1C). By contrast, the percentage of MyHC-2B fibers was significantly higher in TA muscle compared with LVP muscle (~75 vs. ~5%; Fig. 1D). These results indicated that the vast majority of TA muscle is composed of type 2B fibers, while the majority of fibers in LVP muscle are types 2A and 2X.

Figure 1. Muscle fiber types in TA and LVP muscle. (A) Immunofluorescence staining of MyHC-1 (red) and laminin (green). Quantification of MyHC-1 is indicated on the right panel. (B) Immunofluorescence staining of MyHC-2A (red) and laminin (green), Quantification of MyHC-2A is indicated on the right panel. (C) Immunofluorescence staining of MyHC-2X (red) and laminin (green). Quantification of MyHC-2X is indicated on the right panel. (D) Immunofluorescence staining of MyHC-2B (red) and laminin (green). Quantification of MyHC-2B is indicated on the right panel. Scale bar=50 µm. ***P<0.001 as indicated. TA, tibialis anterior; LVP, levator veli palatini; MyHC, myosin heavy chain.

Satellite cells and centrally-nucleated myofibers in TA and LVP muscle

The number of nuclei per area was significantly higher in LVP muscle compared with TA muscle (Fig. 2A). The number of Pax7-positive nuclei, an indicator of satellite cells, was significantly higher in LVP muscle compared with TA muscle (~2.2% vs. ~0.3%; Fig. 2B). Satellite cells are resident stem cells in skeletal muscle tissue, wedged in the interspace between sarcolemma and basal lamina (23). When activated, satellite cells proliferate and differentiate either to merge into pre-existing fibers or form new fibers, distinguishing the involved myofibers as centrally-nucleated myofibers (24). In TA muscle, centrally-nucleated myofibers were only scarcely identified (~0.1% of total fibers), but they were observed significantly more frequently in LVP muscle (~1.5% of total fibers; Fig. 2C). These data suggested that LVP muscle possesses a broader stem cell pool and more active myonuclei turnover compared with TA muscle. Given that Wnt signaling is closely associated with satellite cell activity (25), further investigation was performed in the Wnt pathway.

Figure 2. Distribution of satellite cells and centrally-nucleated myofibers in TA and LVP muscle. (A) DAPI staining of muscle sections from TA muscle and LVP muscle. Quantification of total nuclei per field is indicated on the right panel. (B) Immunofluorescence staining of laminin (green), Pax7 (red) and DAPI (blue) in TA and LVP muscle. Quantification of Pax7-positive nuclei is indicated on the right panel. (C) Immunofluorescence staining of laminin (green) and DAPI (blue). Yellow arrowheads indicate centrally-nucleated myofibers. Quantification of centrally-nucleated myofibers is indicated on the right panel. Scale bar=50 µm. ***P<0.001 as indicated. TA, tibialis anterior; LVP, levator veli palatini.

Wnt signaling hubs in TA muscle and LVP muscle

In canonical Wnt pathways, Axin2 is a direct target gene of Wnt and its expression is a reliable marker for the activation of Wnt signaling (26). For non-canonical Wnt pathways, the expression of Vangl2 reflects the activation of Wnt/planar cell polarity pathway (27), while pAkt and pS6 are indicators of Wnt/Akt/mechanistic target of rapamycin pathway activation (10). No significant differences were identified in the expression of Axin2 between TA muscle and LVP muscle (Fig. 3). The expression level of Vangl2 was observed to be significantly higher in TA muscle compared with LVP muscle (Fig. 3). Similarly, pAkt was expressed at a significantly higher level in TA muscle compared with LVP muscle (Fig. 3). No significant differences were identified in the expression of pS6 between TA muscle and LVP muscle (Fig. 3). Based on these differences in the baseline level of Wnt hubs, it was investigated whether LVP and TA muscle responded differently to Wnt ligand stimulus.

Figure 3. Protein analysis of Wnt signaling hubs in TA and LVP muscle. Blots of Axin2, Vangl2, pAkt, pS6 and their densitometric analyses are indicated. All values were normalized to GAPDH levels. **P<0.01 as indicated. TA, tibialis anterior; LVP, levator veli palatini.

TA and LVP muscles respond differently to exogenous Wnt7a stimulus

Wnt7a has been demonstrated to be effective in expanding TA muscles (10,28), but to the best of our knowledge, has not been tested in LVP muscle before. At 3 weeks after a single injection of recombinant Wnt7a protein, TA and LVP muscles were harvested for comparison. After Wnt7a administration, the percentage of Ki67-positive nuclei, which indicated proliferative cells, increased significantly in TA and LVP muscle (Fig. 4A and B). The two muscle types exhibited a marked increase in centrally-nucleated myofibers after Wnt7a administration (Fig. 4C). Notably, in TA muscle, a significant increase in myofibers was observed after Wnt7a injection, leading to a five-fold increase in the number of myofibers and a significant decrease in the average myofiber size (Fig. 4C and D). In LVP muscle, however, the number and the average size of the myofibers stayed constant after Wnt7a administration (Fig. 4C and E).

Figure 4. Effect of exogenous Wnt7a stimulus on TA and LVP muscle. (A) Immunofluorescence staining of Ki67 (red) and DAPI (blue) in tissue sections from TA or LVP muscle treated with PBS or Wnt7a. (B) Quantification of Ki67-positive nuclei. (C) Immunofluorescence staining of laminin (green), and DAPI (blue) in tissue sections from TA or LVP muscle treated with PBS or Wnt7a. (D) Quantification of the number of myofibers per field with and without Wnt7a treatment. (E) Quantification of myofiber size with and without Wnt7a treatment. **P<0.01, ***P<0.001 as indicated. Scale bar=50 µm. TA, tibialis anterior; LVP, levator veli palatini.

Discussion

From the perspective of embryonic origin, TA and LVP muscle fall into different categories: TA muscle derives from somites (2), while LVP muscle derives from the fourth and sixth branchial arches (13). Apart from their differences in embryonic origin, the current study has characterized further heterogeneities between the two muscle types.

Until now, the predominant model of fiber types has been based on four major MyHC isoforms: MyHC-1, −2A, −2X and −2B (29). From type 1 to type 2B fibers, the shortening velocity gradually increases and resistance to fatigue decreases (29). The composition of different fiber types is closely associated with the muscle function. Specifically, TA muscle is more suitable for fast and powerful actions such as jumping and kicking, while LVP muscle is streamlined for slower contractions with longer durations in elevating and stretching the soft palate (30,31). Consequently, it was identified in the present study that TA muscle is largely composed of MyHC-2B myofibers, while LVP muscle primarily consists of MyHC-2A and MyHC-2X fibers. Hybrid fibers co-expressing different MyHC isoforms are common (29), since boundaries between different fiber types are not clear. This could explain why the sum of all four MyHC isoforms in the muscles was >100%. Apart from gleaning baseline information of fiber type composition in the two specific muscles, the data presented here could also be relevant to clinical practice. Fiber type composition can have profound impacts on the specific muscle's susceptibility to disease. For example, type 2 fibers are preferentially affected in Duchenne muscular dystrophy (DMD), and inducing type 1 fibers can ameliorate DMD (32).

Satellite cells are well-recognized resident stem cells within skeletal muscle tissue (23). It was revealed in the present study that LVP muscle maintains a higher satellite cell content compared with TA muscle. During muscle repair or regeneration, satellite cells undergo asymmetric division and differentiate into myogenic progenitors, either fusing into the existing myofibers or becoming de novo myofibers (33). In this process, the involved myofibers are distinguished as centrally-nucleated myofibers. It has long been speculated that satellite cells remain quiescent in their sublaminar niche until required for skeletal muscle repair (23,25,33). However, recent studies performed by Randolph et al (34) and Keefe et al (35) have demonstrated that satellite cells contribute nuclei to myofibers in adult muscles in sedentary mice, challenging this conventional view. Similarly, in the present study, satellite cell activity in 2-month-old rat skeletal muscle was detected under basal conditions, as evidenced by the presence of centrally-nucleated myofibers. It was also identified that the percentage of centrally-nucleated myofibers was significantly higher in LVP muscle compared with TA muscle. With regards to the core components in Wnt signaling, protein analyses revealed that Vangl2 and pAkt are expressed at a higher level in TA muscle compared with LVP muscle. These molecular differences indicated different Wnt pathway activation levels under basal conditions in TA muscle and LVP muscle. However, the mechanisms underlying the broader stem cell pool and more active myonuclei turnover in LVP are yet to be elucidated.

In the present study, it was further examined whether TA and LVP muscle responded differently to exogenous myogenic growth factor. Wnt7a was selected because it was less likely to induce resistance to growth hormone or cause hypoglycemia, when compared with IGF-1 (10). Previous studies by von Maltzahn et al (10,28) have indicated the key role of recombinant Wnt7a protein in augmenting mouse limb muscle, leading to significant myofiber hypertrophy and enhanced muscle function. Notably, there were two primary findings in the present study. First, Wnt7a induced hyperplasia rather than hypertrophy in TA muscle. This discrepancy may be attributed to the different postnatal growth patterns in muscle fibers between mice and rats. Typically, the number of myofibers remains constant after birth in mice, while the number and size of myofibers continue to increase for 10 weeks in rats (20,36). Secondly, TA and LVP muscle reflected different augmentation potential after Wnt7a delivery. In TA muscle, the number of myofibers increased significantly, a large proportion of which were observed to be centrally-nucleated myofibers. However, LVP muscle did not exhibit an increase in myofiber number, despite markedly upregulating the number of centrally-located myofibers. The two muscle types exhibited augmentation potential in response to Wnt7a stimulus, but had different responses. The reason for these differences requires further investigation.

LVP muscle was selected for investigation in the current study primarily due to its clinical significance. LVP muscle elevates the soft palate and serves a critical function in velopharyngeal closure. Reduced LVP muscle volume and compromised LVP muscle function in patients with cleft palate results in affected speech (12). Effective augmentation of LVP muscle via biological therapeutics may revolutionize the management of cleft palate.

The present study has certain limitations. The newly-formed myofibers with small diameters observed in TA muscle 3 weeks after Wnt7a administration were immature. To probe the augmentation effect, a longer observation time is required in order to assess the development of these new fibers. In addition, the notable discrepancies between the responses of TA and LVP muscle to Wnt7a administration highlights the requirement for more in-depth work in order to reveal the underlying mechanisms.

In conclusion, LVP muscle is distinct from TA muscle in its myofiber composition, in-situ stem cell population, Wnt signaling hub expression and augmentation potential. Therefore, therapeutics that are effective in TA muscle may not be effective in LVP muscle, and further studies are required in order to elucidate the differences between the muscle types.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China to Dr Jingtao Li (grant no. 81500829) and Professor Bing Shi (grant no. 81470729).

Glossary

Abbreviations

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References