Zhenghua Xiang, Institute of Neuroscience and Key Laboratory of Molecular Neurobiology, Ministry of Education, Neuroscience Research Centre of Changzheng Hospital, Second Military Medical University 200433 Shanghai, China. E: firstname.lastname@example.org Hongliang Zheng, Department of Otolaryngology & Head and Neck Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. E: email@example.com
In this study, single and double-labeling immunofluorescence histochemistry, Western blot and real-time polymerase chain reaction were used to study the expression of P2Y6 receptors in developing mouse skeletal muscle and during injury and repair. The results show that P2Y6 receptor immunoreactive (ir) cells were first detected in the dermamyotome at embryonic (E) day 9. The number and immunostaining intensity of the P2Y6 receptor-ir cells increased from E9 to E13, but decreased from E15 to postnatal day 60 in the developing skeletal muscle system. The expression levels of P2Y6 receptor protein and mRNA increased rapidly from 1 to 5 days after skeletal muscle injury and then decreased almost to the control level from 7 to 10 days, at the beginning of regeneration. P2Y6 receptor-immunoreactivity was mainly localized to the ends of single myoblasts and myotube processes in the developing and injury-repair skeletal muscle tissues. These data suggest that the P2Y6 receptor may be involved in the development and regeneration of skeletal muscle, especially in the migration and extension of the myoblast and myotube in developing and regenerating skeletal muscle.
Extracellular adenosine-5′-triphosphate (ATP) acts as a signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration (Burnstock, 2007). Recently, ATP, acting via P2X and P2Y receptors, has been shown to be involved in the process of muscle regeneration in rats (Ryten et al. 2002, 2004; Banachewicz et al. 2005). P2X1–7 receptors are intrinsic ligand-gated ion channels and activation of these receptors by ATP evokes a flow of cations across the plasma membrane, whereas P2Y receptors are G-protein-coupled receptors. There are two subgroups of G proteins which P2Y receptors are coupled with, Gq and Gi. P2Y1,2,4 and 6 receptors are linked to activation of phospholipase C (PLC), inositol lipid signaling and the mobilization of intracellular Ca2+, P2Y12, P2Y13 and P2Y14 receptors are coupled with Gi protein, resulting in inhibition of cAMP formation, and P2Y11 (only in humans) is coupled with Gs and Gq, resulting in increased cAMP (Burnstock, 2007). Within the family of P2Y receptors, P2Y1, P2Y12 and P2Y13 receptors respond to adenosine diphosphate (ADP). P2Y2 and P2Y4 receptors respond to both ATP and uridine 5’-triphosphate (UTP), and P2Y6 receptors to uridine 5’-diphosphate (UDP). The expression of specific P2X receptor subtypes during rat skeletal muscle development (Ryten et al. 2001, 2002, 2004) and in regenerating skeletal muscle (Ryten et al. 2004) has been demonstrated. Among these receptors, P2X5 and P2X6, and P2X2 and P2X5, have been found to be expressed in chick and rat skeletal muscle development, respectively (Meyer et al. 1999b; Bo et al. 2000, 2001; Ryten et al. 2001;). In contrast, there are few studies on the expression and function of P2Y nucleotide receptors in developing skeletal muscle. The expression of P2Y1 receptors was detected during the first 10 days of chick embryonic development (Meyer et al. 1999a). In rat embryonic skeletal muscle cells, Cheung et al. (2003) found early expression of the P2Y1 receptor, whereas P2Y2 receptor expression became gradually stronger in later development and P2Y4 receptor expression was high at both early and late embryonic days. C2C12 cells (a murine myoblast cell line) were reported to express P2Y1, P2Y4, P2Y6 and P2Y12 receptor mRNAs in myoblasts, but were expressed less in myotubes. In contrast, P2Y2 receptor mRNA was not detected in the myoblast, but high levels were detected in myotubes (Banachewicz et al. 2005). When we studied expression patterns of P2Y6 receptors in the early mouse embryo, high levels of P2Y6 receptor immunoreactivity were found in the dermamyotome. These findings suggest that P2Y6 receptors might be involved in some activities during the development and regeneration of skeletal muscle cells. Thus, in the present paper we have studied P2Y6 receptor expression in precursor cells of skeletal muscle from embryo day 9 to adult. The results showed that expression of P2Y6 receptors is down-regulated during development. After myotrauma, the expression of P2Y6 receptors was up-regulated rapidly in the early days and then down-regulated in the later days during regeneration. The expression pattern of P2Y6 receptors in the precursor cells and myotube suggested that P2Y6 receptors might be involved in the migration of the precursor cells and myotubes.
Materials and methods
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Second Military Medical University. Kunming mice at the prenatal stages of development (E 9, E11, E13, E15, E18), postnatal stages [postnatal day (P) 3, P10, P25, P60] and adult mice were used for the developmental study. Seventy-two male Kunming mice (25–35 g) received an extensive crush injury to the tibialis anterior muscle of the right leg as described in detail by a previous study (McGeachie & Grounds, 1987). The mice were divided into six groups –control, day 1, day 3, day 5, day 7 and day 10 after muscle injury. Four mice in each group were used for immunohistochemistry, Western blot and real-time RT-PCR, respectively. For the immunohistochemistry, the mice were anesthetized with sodium pentobarbitone and perfused through the aorta with a 0.9% NaCl solution and 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. The specimens were removed and refixed in 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, overnight. The specimen blocks were then transferred to 20% sucrose in phosphate-buffered saline (PBS) and kept in the solution until they sank to the bottom. Thereafter, the specimen blocks were rapidly frozen. Sections of 10 μm thickness were cut in a cryostat and thaw-mounted on gelatin-coated slides.
The following protocol was used for immunostaining of P2Y6 receptors. The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton-X-100, 0.4% sodium azide in 0.01 m PBS, pH7.2) for 30 min, followed by incubation with the P2Y6 antibody diluted 1 : 200 (goat polyclonal antibody; Santa Cruz) at room temperature. Subsequently, the preparations were incubated with Cy3-conjugated donkey anti-goat IgG diluted 1 : 400 for P2Y6 receptors. All the incubations and reactions were separated by 3 × 10 min washes in PBS.
The following protocol was used for double immunostaining of P2Y6 receptors with laminin-1 (a basement membrane marker) or α-sarcomeric actin (skeletal and cardiac muscle specific marker) or C-Met (a skeletal muscle satellite cell marker). The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution1 for 30 min, followed by incubation with different combinations of P2Y6 antibody diluted 1 : 200, laminin-1 diluted 1 : 400 (rabbit anti-rat; Abcam), α-sarcomeric actin (mouse-anti-rat; Abcam), C-Met diluted 1 : 100 (rabbit anti-rat; Boster) in antiserum solution 2 (1% normal bovine serum, 0.2% Triton-X-100, 0.4% sodium azide in 0.01 m PBS, pH 7.2), at room temperature. Subsequently, the preparations were incubated with fluorescein-labelled antibody (FITC)-conjugated donkey anti-goat diluted 1 : 400 for P2Y6, Cy3-conjugated donkey anti-rabbit IgG diluted 1 : 200 for laminin-1 and C-Met antibodies, and Cy3-conjugated donkey anti-mouse IgG in antiserum solution 2 for 2 h at room temperature. All the incubations and reactions were separated by 3 × 10 min washes in PBS.
Control experiments were carried out with the P2Y6 antibody preabsorbed with its peptide. No staining was observed in those preparations incubated with antiserum solutions preabsorbed with its peptide. A further negative control by omitting the primary antibody was also carried out. No staining was observed in those preparations.
Mice were deeply anesthetized by sodium pentobarbital (60 mg kg−1) and killed by decapitation. The injured muscle was rapidly removed and lysed with 20 mm Tris–HCl buffer, pH 8.0, containing 1% NP-40, 150 mm NaCl, 1 mm ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.1% mercaptoethanol, 0.5 mm dithiothreitol, and a mixture of proteinase and phosphatase inhibitors (Sigma). Protein concentration was determined by the BCA protein assay method using bovine serum albumin as standard (BCA protein assay kit from Beyotime). Protein samples (100 μg) from the colon were loaded in each lane, separated by SDS-PAGE (10% polyacrylamide gels) and then electrotransferred onto nitrocellulose membranes. The membranes were blocked with 10% non-fat dry milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with the P2Y6 antibody (Santa Cruz) diluted 1 : 200 or α-tubulin diluted 1 : 400 (loading control) in 2% bovine serum albumin (BSA) in PBS. The membranes were then incubated with alkaline phosphatase-conjugated goat anti-goat IgG (Beyotime) diluted 1 : 1000 in 2% BSA in PBS for 1 h at room temperature. The color development was performed with 400 μg mL−1 nitro-blue tetrazolium, 200 μg mL−1 5-bromo-4-chloro-3-indolyl phosphate and 100 mg mL−1 levamisole in TSM2 (0.1 m Tris–HCl2 buffer, pH 9.5, 0.1 m NaCl and 0.05 m MgCl2) in the dark. Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories).
Real-time quantitative PCR
Muscle tissues were homogenized in 1 mL of Trizol® reagent (Invitrogen Co., Carlsbad, CA, USA). Total RNA was extracted with chloroform, precipitated with isopropanol, and resuspended in 30 μL of RNAse-free water. The concentration of isolated total RNA was determined by measuring the optical density at A260 nm with an ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Reverse transcription of 1 μg of total RNA was performed to synthesize complementary DNA (cDNA) using the PrimerScriptTM RT reagent kit (TaKaRa Biotechnology Co. Ltd, Dalian, China) with the Geneamp PCR System 9700 (Applied Biosystems Inc., Foster City, CA, USA). Primers for the messenger RNA (mRNA) of interest were designed using the online software primer 3plus (Wageningen University and Research Centre, Gelderland, Netherlands). Nucleotide sequences for mouse P2Y6 receptors were obtained from GenBank, with β-actin as the calibrator housekeeping gene (internal control). The location and specific sequences of primers were chosen to exclude the detection of genomic DNA by placing one of the primers over a junction between two exons. The primer sequences for these target genes were as follows: (i) P2Y6 (NM_183168): forward primer 5′-ATC AGC TTC CTG CCT TTC C-3′, reverse primer 5′-CTG TGA GCC TCT GTA AGA GAT CG-3′, yielding a fragment of 214 bp; (ii) β-actin (NM_007393): forward primer 5′-AGC CAT GTA CGT AGC CAT CC-3′, reverse primer 5′-CTC TCA GCT GTG GTG GTG AA-3′, yielding a fragment of 228 bp.
Real-time quantitative PCR was performed using the reagent and protocol contained in the SYBR® Premix Ex Taq TM II kit (TaKaRa Biotechnology Co. Ltd, Dalian, China). Primers were used at a concentration of 20 mm. Reactions were run in a volume of 20 μL, containing 0.5 μL cDNA. The program was set up as follows: (i) 95 °C, 2 min; (ii) 95 °C, 15 s→62 °C, 20 s→72 °C, 10 s, for 40 cycles; (iii) 72 °C, 10 min. Data were collected and analyzed on the Rotor-Gene Q real-time PCR cycler (QIAGEN Pty Ltd, Doncaster, Victoria, Australia). The specificity of the PCR product was examined based on the melt curves of the reactions. Relative expression levels of target genes were analyzed using the 2−ΔΔCT quantitative method.
Images were taken with a Nikon digital camera DXM1200 (Nikon, Japan) attached to a Nikon Eclipse E600 microscope (Nikon). Images were imported into a graphics package (adobe photoshop).
Quantitative analysis for the P2Y6 receptor immunostaining in normal and injured muscle tissues of the adult mice and mice of different developmental ages was performed as follows: five random fields (each area 0.62 mm2) for one section were chosen and the number of positive cells was counted and expressed as the positive cell number per mm2. Five fields for each of the five sections from each of four mice were used. The mean number of positive cells per mm2 from each mouse was calculated and data expressed as the mean ± SE of the mean (n = number of mice). The ratio of P2Y6 to α-tubulin in Western blot and the relative transcript level in real-time PCR are also calculated and expressed as mean ± SE of the mean (n = number of mice). Statistical significance was tested by a one-way analysis of variance (anova) followed by an unpaired t-test. A probability of P < 0.05 was considered significant for each test.
Expression of P2Y6 receptors in developing muscle system
In the coronal section of embryo at E9, P2Y6 receptor immunoreactive (ir) cells were first detected in the dermamyotome, located at the ventrolateral side and under the epidermis. These P2Y6 receptor-ir cells formed a cell band in the dorsal to the ventral direction (Fig. 2A). The number of the P2Y6 receptor-ir cells and the intensity of the P2Y6 receptor immunostaining increased from E9 to E11 (Fig. 1A,B). P2Y6 receptor-ir cells were detected widely in the skeletal muscle system from E13 to E15. From E13 on, the strongly P2Y6-immunostained cells were mainly detected in the borders of the skeletal muscle bundles, although low to median immunostained cells were also detected in the medial part of the skeletal muscle bundles (Figs 2C,D and 3D1). At high power magnification, the distribution of immunostaining signals in the P2Y6 receptor-ir cells exhibited heteropolarity at these stages (E13–E18). As Figs 1C,D and 2D1 show, strong P2Y6 receptor immunostaining signals were detected at the sides near vertebrae/bone or the proximal/distal ends of the limb muscle mass and weak signals were detected in the medial part at E13 and E15. These P2Y6 receptor-ir asymmetrical cells were mainly detected in the border regions of the skeletal muscle bundle. Scattered P2Y6 receptor-ir cells were also detected in the tissues from E9 to E15. These scattered single P2Y6 receptor-ir cells also possessed the asymmetrical pattern of P2Y6 receptor localization (Fig. 2C), the P2Y6 receptor-ir signal being localized at both ends of some myoblasts (Fig. 2A,B). During the postnatal days, the number and immunostaining intensity of P2Y6 receptor-ir cells decreased dramatically and the asymmetrical pattern of P2Y6 receptor-ir disappeared at P3 (Fig. 1E). The P2Y6 receptor-ir cells were weak around some of the skeletal muscle cells at P25. The number and immunostaining intensity of P2Y6 receptor-ir cells were similar to those of adult mice (Fig. 1F). To confirm that the P2Y6 receptor-ir positive cells were skeletal muscle, the specific antibody for the skeletal muscle specific marker (α-sarcomeric actin) was used. The developing muscle bundle-like structures and scattered single cells with P2Y6 receptor-ir were also immunoreactive for α-sarcomeric actin (Fig. 2C,D1,D2,D3).
Expression of P2Y6 receptor mRNA and protein after skeletal muscle injury
The P2Y6 receptor-ir cells were just detectable around limited regions of skeletal muscle cells in normal conditions. To confirm whether the P2Y6 receptor-ir cells were satellite cells, the C-Met antibody was used. The results confirmed this, as P2Y6 receptor-ir cells were also immunoreactive for C-Met (Fig. 2E1,E2,E3). One day after muscle injury, the number and immunostaining intensity of P2Y6 receptor-ir cells significantly increased compared with the control group (Figs 1G and 3A). The majority of the cross-sections of the muscle cells in the injury area showed one to three P2Y6 receptor-ir cells. At 3 and 5 days after injury, the number and immunostaining intensity of the P2Y6 receptor-ir cells increased rapidly (Fig. 3B,C). The cell volume of the P2Y6 receptor-ir cells was also larger than that 1 day after injury. From 3 days, myotube-like P2Y6 receptor-ir cells appeared. In the longitudinal sections, the distribution pattern of the P2Y6 receptor-ir signals in single myotube-like structure was also asymmetrical, similar to those cells observed at E13–E18 (Figs 2F1 and 3E,F). At the proximal or distal parts of the P2Y6 receptor-ir myotube-like structure, the immunostaining signals were much more intense than in the median parts of the myotube-like structure. The ends of these positive myotube-like structures were conical in shape. In the longitudinal sections the majority of the cones at the proximal or distal part were orientated in one direction, towards the ends of the muscle mass (Fig. 3E,F). Most of the P2Y6 receptor-ir cells in the injured muscle were also reactive for the skeletal muscle specific marker (α-sarcomeric actin) (Fig. 2F1,F2,F3). The majority of the P2Y6 receptor-ir cells surrounded the injured muscle fibers and were circumvoluted by a basement membrane as shown by the laminin antibody (Fig. 2G), which also indicated that the majority of the P2Y6 receptor-ir cells in the injured areas were from skeletal muscle cells. The number of P2Y6 receptor-ir cells including the positive satellite cells, myoblasts and myotubes in the normal and different injured groups of adult mice are summarized in Fig. 4B. The number of P2Y6 receptor-ir cells in the skeletal muscle mass of hind legs from E15 to P60 mice were also quantitatively studied and are summarized in Fig. 4A. P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 and P2Y13 receptor antibodies, have also been used to immunostain the muscle tissues under normal and injury conditions (see Supporting Information Figs S1 and S2). We found that only expressions of P2Y1, P2Y4 and P2Y6 receptors were detected and that only the P2Y6 receptor changed significantly after injury.
Western blotting, performed on tissue extracts derived from the mouse tibialis anterior muscle, assessed the specificity of the polyclona P2Y6 receptor antibody. An immunoreactive band was detected at about 45 kDa. Preadsorption of the antiserum with the peptide antigen resulted in the absence of the band (Fig. 5A), indicating that the P2Y6 receptor antibody detected the appropriate antigen sequence.
Western blot analysis was used to detect P2Y6 receptor protein levels in the intact and injured tibialis anterior muscles of mice (Fig. 5B). Using the same antibody as for the immunohistochemistry, a single band of approximately 45 kDa was identified in the intact group (Fig. 5B, control lane). As shown in Fig. 5B, lanes 1d, 3d and 5d, the P2Y6 receptor protein levels increased significantly as compared with that of the control. In the 7-day group, the P2Y6 receptor protein level decreased compared with that of the 5-day group. By 10 days after injury, the P2Y6 receptor protein level had almost decreased to that of the control. Real-time PCR analysis was also used to detect P2Y6 receptor mRNA levels in the intact and injured tibialis anterior muscles of mice (Fig. 6). A similar pattern of P2Y6 receptor mRNA change was seen during the injury-repair process. The peak duration of P2Y6 receptor mRNA was 5 days after injury and by 10 days, the level of P2Y6 receptor mRNA was similar to that of the control group (Fig. 6).
In this study, using immunohistochemistry, Western blot and real-time PCR, we have shown that the P2Y6 receptor is expressed in developing skeletal muscles of the pre- and postnatal mouse, and in normal and injured skeletal muscle cells of the mouse. Specificity of P2Y6 antibodies on mouse skeletal muscle tissues was tested by Western blotting with normal and injured muscle tissue crude protein extracts. A single P2Y6 receptor reactive band of approximately 45 kDa was detected. The preabsorption of P2Y6 receptor antibodies with their relative peptides abolished nearly all immunoreactivity to these antibodies in skeletal muscle tissues. The molecular weight of the reactive band for the P2Y6 receptor was higher than that expected (a theoretical molecular weight of 37 kDa). Post-translational modification of the P2Y6 protein, by glycosylation for example, would result in the detection of proteins with different molecular weights.
The expression characterization of P2Y6 receptors during development and in injury and repair of adult mouse skeletal muscle implies that P2Y6 receptors might be involved in the development and regeneration of mouse skeletal muscle, such as the differentiation, proliferation, migration, fusion and extension of myoblasts and myotubes.
The distribution pattern of P2Y6 receptor-ir in a single cell is polar or asymmetrical. P2Y6 receptor-ir signals were mainly in one protrusion or both protrusions in these cells, where the immunostaining signals were very strong. It has been reported that after migration into the forming myotome, the round myoblasts start to elongate along the rostrocaudal axis, growing in the rostral and caudal directions at the same time, until they span the somite completely (Denetclaw et al. 1997; Gros et al. 2004). On the basis of P2Y6 receptor distribution patterns during early embryo development and regeneration after skeletal muscle injury, we suggest that P2Y6 receptors might be involved in the guidance of the myoblast and myotube migration and extension. Communication between myotubes and tendon cells is very important for myotube migration and, finally, attachment to the tendon cell (Schnorrer & Dickson, 2004). ATP and ADP have been reported previously to act as chemotatic factors. ATP binding to P2X4 receptors and ADP binding to P2Y12 receptors induces microglial chemotaxis via the PI3K pathway (Honda et al. 2001; Ohsawa et al. 2007). ATP or UTP binding to P2Y2 receptors guides neutrophil chemotaxis via the PKC-ERK pathway (Chen et al. 2006; Meshki et al. 2006).This suggests that the tendon precursors might release UDP, which guides the myoblasts and myotubes together.
Cell locomotion involves changes of the cell cytoskeleton, such as microtubule and actin, which play crucial roles in growth cone motility, axon outgrowth, and guidance (Dent & Gertler, 2003) and in the shape, interaction, movement and extension of myoblasts (Swailes et al. 2004; Guerin & Kramer, 2009). As mentioned previously, the signal pathways of UDP-activated P2Y6 receptors may be involved in cAMP-protein kinase A and Ca2+-dependent protein kinase C pathways. Thus these two pathways might take part in the regulation of the cytoskeleton in the protrusion of the myoblast and myotube during the development and regeneration of mouse skeletal muscle.
In summary, we show for the first time that P2Y6 receptors can be detected during the development, injury and regeneration processes of mouse skeletal muscle, using the methods of immunohistochemistry, Western blot and real-time PCR. Expression of the P2Y6 receptor was first detected in the dermamyotome at E9. The number and immunostaining intensity of the P2Y6 receptor-ir cells increased from E9 and decreased from E15 to adulthood. The expression levels of P2Y6 receptor protein and mRNA increased rapidly from 1 to 5 days after skeletal muscle injury and decreased almost to the control level at 10 days. The distribution pattern of P2Y6 receptors in developing skeletal muscle cells and myotubes was polar during the embryo days and regeneration after injury. These data suggest that P2Y6 receptors may be involved in the development of skeletal muscle and regeneration processes after skeletal muscle injury.
This work was supported by the National Natural Science Foundation of P. R. China (30970918 to Z. Xiang, 30772415 to H. Zheng), 973 program (20011CB504401 to Z. Xiang) and by the Science and Technology Commission of Shanghai Municipality (10XD1405500 to H. Zheng).
Z. Xiang, H. Zheng and G. Burnstock designed the research. D. Chen, W. Wang, W. Guo and Q. Yu performed the research. Z. Xiang, D. Chen and W. Wang analyzed data. Z. Xiang H. Zheng and G. Burnstock wrote the paper.