Gαi2 regulates the adult myogenesis of masticatory muscle satellite cells

Abstract Although similar to trunk and limb skeletal muscles, masticatory muscles are believed as unique in both developmental origins and myogenesis. Gαi2 has been demonstrated to promote muscle hypertrophy and muscle satellite cell differentiation in limb muscles. However, the effect of Gαi2 on masticatory muscles is still unexplored. This study aimed to identify the role of Gαi2 in the proliferation and differentiation of masticatory muscle satellite cells, further exploring the metabolic mechanism of masticatory muscles. The proliferation rate, myotube size, fusion index of masticatory muscle satellite cells and Pax7, Myf5, MyoD, Tcf21 and Musculin expressions were significantly decreased by Gαi2 knockdown, while in cells infected with AdV4‐Gαi2, the proliferation rate, myotube size, fusion index and Tbx1 expression were significantly increased. Masticatory muscle satellite cells also displayed phenotype transformation as Gαi2 changed. In addition, Gαi2 altered myosin heavy chain (MyHC) isoforms of myotubes with less MyHC‐2A expression in siGαi2 group and more MyHC‐slow expression in AdV4‐Gαi2 group. In conclusion, Gαi2 could positively affect the adult myogenesis of masticatory muscle satellite cells and maintain the superiority of MyHC‐slow. Masticatory muscle satellite cells may have their unique Gαi2‐regulated myogenic transcriptional networks, although they may share some common characteristics with trunk and limb muscles.

developmental origin, masticatory muscles derive from the head paraxial mesoderm, while the trunk and limb muscles develop from somites. 5 In addition, masticatory muscles present smaller fibres, a higher percentage of hybrid fibres, and more type I, II and foetal myosin heavy chain (MyHC) isoforms compared to the trunk and limb muscles. 1 Furthermore, regulatory pathways controlling myogenesis in the masticatory muscles are distinct from those in the trunk and limb muscles. Previous studies discovered that Pax3 and Myf5 double mutant mice remained normal masticatory muscles but failed to develop trunk muscles. 6 In contrast, masticatory muscles were eliminated, whereas other skeletal muscles were unaffected in mice lacking both MyoR and capsulin. 7 It is widely believed that masticatory muscles have a huge regenerative capacity to keep their homeostasis, maintaining healthy muscle mass and function, due to the fundamental functions of muscle satellite cells. 8 Muscle satellite cells are quiescent myogenic cells, lying on the top of muscle fibres and generally keeping quiescent in adult skeletal muscles, but be able to activate and proliferate to regenerate, repair and remodel muscle tissues in responding to physiological and pathological changes. 9 Gαi2 is a subtype of G proteins which are a family of proteins acting as molecular switches inside cells. 10 Typical G protein-coupled receptors (GPCRs) such as β2-adrenergic receptors, can transduce signals through the Gαi-linked Gβγ complex in many cell types, independently of activating cyclic AMP through Gαs proteins. [11][12][13] Previous studies revealed that activated Gαi2 promoted skeletal myotube growth through PKC-GSK3β and mTOR-p70S6K pathways. 14 In addition, Gαi2 was also confirmed to induce the activation of limb muscle satellite cells via PKC-GSK3β pathway and HDAC inhibition. 15 However, the role of Gαi2 in masticatory muscles is still uncertain. This study aimed to identify the role of Gαi2 in the proliferation and differentiation of masticatory muscle satellite cells, further exploring the metabolic mechanism of masticatory muscles.
siRNA-mediated Gαi2 knockdown and adenoviral-mediated Gαi2 overexpression were conducted and the changes of cell proliferation rate, myotube size, fusion index, transcription factor expressions, cell phenotypes and MyHC isoforms were detected in this study. Primary masticatory muscle satellite cells were isolated from masseters of C57BL10 mice as previously reported. [16][17][18] In brief, mice were sacrificed by cervical dislocation, and masseters were dis-  After infection, the cells were cultured in GM2 or DM for another 48 h.

| Proliferation assay
The cell proliferation after Gαi2 knockdown or overexpression

| Myotube size analysis and fusion assay
To determine changes in myotube size and fusion index, myotubes For the measurement of myotube diameter, three pictures were taken in each well. Thirty largest myotubes from each picture were selected and measured by Image-Pro Plus 6.0. After dividing myotubes into thirds, the myotube diameter was calculated as the mean of distances between the midpoints of each portion. To analyse fusion index, the nuclei of 60-90 myotubes in each well were counted to obtain the average number of nuclei per myotube. After fixation with 4% PFA, cells were incubated with primary antibodies at 4°C overnight, nonspecific binding was blocked with 10% goat serum (Gibco) and the primary antibodies were visualized using appropriate species-specific 488 and 549 fluoro-chrome-conjugated secondary antibodies (1:100, Abbkine), stained with DAPI (Aspen), and mounted in Vectashield mounting medium (biosharp).

| RNA isolation and analysis
RNA was isolated with Total RNA kit I (OMEGA) and cDNA was prepared from 200 ng RNA according to the manufacturer's instructions (Thermo Fisher Scientific). The relative level of gene expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR) using a 7900 HT Fast Real-Time PCR System (Applied Biosystems). Primers used for detection are listed in Table 1. [19][20][21][22] All the MyHC types existing in mouse masseter were detected to explore the change of muscle fibre types ( Table 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Relative transcript abundance was normalized to the amount of genes in control group and quantitated by the 2 −ΔΔCT method.

| Statistical analysis
All the experiments in this study were independently performed three times. Data was expressed as mean ± standard deviation. Differences between two groups were evaluated using paired Student's t-test. pvalue < 0.05 was considered as statistically significant. Statistical analysis was conducted with SPSS 13.0 statistical software (IBM Corp).

| RE SULTS
Primary masticatory muscle satellite cells were successfully isolated, cultured and differentiated into long and multinuclear myotubes ( Figure 1).

| Gα i2 alters MyHC transition in masticatory muscle myotubes
The most accepted methods to classify muscle fibre types are based on specific myosin profiles, especially the MyHC isoform complement. 24 The mRNA expressions of different MyHC were tested when Gαi2 was suppressed or over-expressed to explore the possible role of Gαi2 in determining masticatory muscle fibre types.
In this study, we explored the possible role of Gαi2 in the prolif- preferred to predominantly maintain MyHC-2. 14,15 This deviation may be attributed to the difference in embryonic backgrounds of muscle-derived stems. In this study, masseter muscles deriving from cranial paraxial mesoderm were used to extract primary muscle satellite cells, while those two studies mentioned above used C2C12 cell line from thigh muscles and primary muscle satellite cells from hind limb muscles, which both belonged to limb muscles deriving from somites. 14,15 Another possible explanation is the different relationship between muscle fibre type and fibre size among different skeletal muscles. Slow-type fibres have a larger fibre cross-sectional area than the fast-type fibres in masseter muscles while the condition is reversed in the trunk and limb muscles. 42 Furthermore, MyHCα, also known as MyH6, predominantly expressed in cardiac muscles, 43 was found to be significantly upregulated by Gαi2 overexpression in masticatory muscles. These findings provided insights into a hypothesis that Gαi2 may participate in the regulation of lineage commitment among trunk and limb muscle satellite cells, masticatory muscle satellite cells and cardiac muscle satellite cells. Given the knowledge that trunk and limb muscles are primarily affected by critical illness myopathy (CIM), which is characterized by muscle wasting and myogenesis defect, while masticatory muscles and cardiac muscles are spared or less affected, Gαi2 may play roles in increasing masticatory muscles' resistance against CIM. 44 Further studies are needed to confirm this speculation and elucidate the underlying mechanisms.
In summary, our study demonstrated that

ACK N O WLE D G E M ENTS
This work was supported by grants from the National Natural Science Foundation of China (81200811 and 82001089).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.