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Purinoceptor expression in regenerating skeletal muscle in the mdx mouse model of muscular dystrophy and in satellite cell cultures

Autonomic Neuroscience Institute, Royal Free & University College Medical School, Royal Free Campus; and
^* Molecular Tissue Repair Unit, Department of Surgery, Royal Free and University College Medical School, University College London, UK

¹ Correspondence: Autonomic Neuroscience Institute, Royal Free & University College Medical School, Royal Free Campus, Rowland Hill St., London NW3 2PF, UK. E-mail: g.burnstock{at}ucl.ac.uk

SPECIFIC AIMS

The aim of this study was to determine the expression and function of receptors for extracellular ATP in mammalian skeletal muscle regeneration, particularly in response to clinically relevant causes of muscle damage.

PRINCIPAL FINDINGS

1. Specific receptors for extracellular ATP are sequentially expressed in skeletal muscle regeneration in vivo in the mdx mouse model of muscular dystrophy and in myotube formation in vitro in rat myoblast cultures
The mdx mouse model of human Duchenne muscular dystrophy (DMD) was used as an example of a clinically relevant form of muscle damage and subsequent regeneration. In vivo muscle regeneration was the focus of this study, and a parallel study on normal rat skeletal myoblast cultures was conducted. The sequential expression of P2X₅, P2Y₁, and P2X₂ receptors was characteristic of muscle regeneration in vivo in the mdx mouse and myotube formation in vitro in normal (postnatal) rat myoblast cultures (Figs. 1 , 2 ).

View larger version (125K): [in this window] [in a new window]   Figure 1. A) Changes in the expression of the P2Y1, P2X5, and P2X2 receptors in adult mdx muscle. Sections of TA muscle from 2- to 3-month-old C57Bl/10 (control) and mdx mice were stained for P2Y1, P2X5, and P2X2 (black) and counterstained for nuclei (green). Immunoreactivity for P2Y1 was observed only in blood vessels in control muscle samples; many immunopositive mononucleated cells were present in adult mdx muscle. Similarly, while immunoreactivity for P2X5 or P2X2 was absent in muscle samples from adult control mice, P2X5-immunopositive cells were scattered throughout mdx muscle and strong immunoreactivity for P2X2 was observed on myotube and muscle fiber membranes (arrows). B) Sections of TA muscle from 3.5-wk-old mdx mice stained for P2Y1, P2X5, and P2X2 receptors (black) and counterstained for nuclei (green). The P2Y1 receptor was strongly expressed during the first phase of mdx muscle degeneration. P2X2 nor P2X5 receptors were not expressed at significant levels at this time. C) Sections of TA muscle from 5-wk-old mdx mice stained for P2Y1, P2X5, and P2X2 receptors (black) and counterstained for nuclei (green). Immunoreactivity for the P2X5 and P2X2 receptors was present at this age in mdx muscle, when the predominant process occurring is regeneration. Although the P2Y1 receptor was expressed at this time, immunoreactivity for this receptor appeared to be less widespread than in adult mdx muscle. Bars = 100 µm.

View larger version (37K): [in this window] [in a new window]   Figure 2. A) Sequential expression of P2 receptors in primary myoblast cultures. At day 1, myoblasts expressed only the P2Y1 and P2X5 receptors. By day 3 small myotubes had formed, which also expressed P2Y4 receptors. By day 5 there was no expression of P2Y1 on any cell type; P2X5 receptor expression was restricted to the tips of myotubes, which now expressed P2X2. On day 7, myotubes expressed only P2Y4 and P2X2 receptors. Bars = 100 µm. B) Coexpression of P2X2 and ACh receptors demonstrated using confocal microscopy. Day 5 myotubes were stained with Texas-Red-labeled -bungarotoxin to label ACh receptors (red), with P2X2 antibody (green), and with DAPI (blue). ACh receptors (red) and P2X2 receptors (green) were present in clusters across the myotube membranes. Triple staining for ACh receptors (red), P2X2 receptors (green), and DAPI (nuclear stain, blue) demonstrated colocalization of ACh receptors and P2X2 receptors on myotubes (yellow). However, some areas of myotube membrane were positive only for ACh receptors or P2X2 receptors. Bars = 50 µm.

Using immunohistochemistry it was possible to demonstrate that expression of specific purinoceptors in mdx skeletal muscle was associated with specific time points characterized by muscle degeneration or regeneration. Whereas skeletal muscle from 3.5-wk-old mdx mice (when muscle degeneration is the predominant process) was strongly immunopositive for P2Y₁, there was no immunoreactivity for P2X₂ and little immunostaining for the P2X₅ receptor. Double-staining experiments demonstrated that the P2Y₁ receptor was strongly expressed on leukocytes (as identified by immunoreactivity for CD11b); leukocyte infiltration could account for much of the observed increase in P2Y₁ receptor expression in damaged muscle. At 5 wk of age, when muscle regeneration was the predominant process occurring in mdx tibialis anterior skeletal muscle (similar changes occur at different times in other muscles), not only the P2Y₁ receptor, but also the P2X₅ and P2X₂ receptors, were strongly expressed (Fig. 1) .

A similar pattern of purinoceptor expression was observed during myotube formation in vitro (Fig. 2) . We found that postnatal mononucleated myoblasts in primary culture expressed P2X₅ and P2Y₁ receptor mRNA and protein. Expression of these receptor proteins on myoblasts could account for the P2X and P2Y-like responses detected by patch clamp recording upon addition of ATP. Subsequent formation of myotubes was characterized by expression of the P2Y₄ and P2X₂ receptors.

Thus, we demonstrate for the first time that specific receptors for extracellular ATP are expressed on skeletal muscle cells during muscle regeneration in vivo. The sequential expression of P2X₅, P2Y₁, and P2X₂ receptors was characteristic of muscle regeneration in vivo in the dystrophin-deficient mouse (mdx), a model of human Duchenne muscular dystrophy.

2. A subpopulation of activated satellite cells in mdx skeletal muscle expresses P2X₅ and P2Y₁ receptors

The ability of mammalian skeletal muscle to respond to pathological damage is largely attributed to the presence of a small population of self-renewing muscle precursors, termed skeletal muscle satellite cells. Normally quiescent, in response to muscle fiber injury the satellite cells are activated, proliferate, and ultimately fuse to repair existing muscle fibers and form new myotubes. Past research on mdx muscle suggests that although features of muscle regeneration are present continuously, they are most prominent at 4–5 wk of age, when activated satellite cells are present in the greatest numbers. Thus, the increase in P2X₅ receptor expression in mdx skeletal muscle from 5 wk of age onward strongly suggested a role for this receptor in muscle regeneration (Fig. 1) . Quantification of immunostaining demonstrated a >10-fold increase in the expression of the P2X₅ receptor in mdx muscle at 5 wk compared with age-matched control tissue and mdx muscle at 3.5 wk of age.

Immunolabeling of sequential muscle sections demonstrated that this marked increase in immunoreactivity was due to the expression of the P2X₅ receptor on a subpopulation of activated satellite cells (as identified by expression of myogenic transcription factors MyoD or myogenin). Some activated satellite cells were also positive for P2Y₁ (though fewer than for P2X₅). The absence of significant immunoreactivity for either receptor in mononucleated cells in control muscle samples suggested that purinoceptors were not expressed by quiescent satellite cells, only by activated satellite cells.

3. Coexpression of the P2X₂ and acetylcholine receptors on myotubes in vivo in the mdx mouse model of muscular dystrophy and in myotube formation in vitro in rat myoblast cultures
The absence of dystrophin in mdx muscle not only affects muscle architecture, but disrupts the relationship between motor nerve and muscle fiber. Abnormalities in the structure and function of neuromuscular junctions (NMJs) include NMJ fragmentation, changes in acetylcholine receptor function, and expression of embryonic-type acetylcholine receptors. These appear after the first postnatal month, following the onset of muscle damage and repair, and increase with age. Similarly, P2X₂ receptor expression in mdx muscle began only at 5 wk of age; immunoreactivity for this receptor was strongest in adult mdx muscle, when P2X₂-immunopositive clusters were detected across the cell membranes of myotubes and muscle fibers.

Coexpression of P2X₂ and acetylcholine receptors could account for some of the abnormalities observed in acetylcholine receptor activity in mdx muscle. In fact, our double-staining experiments demonstrated colocalization of P2X₂ and acetylcholine receptors in mdx muscle and myotube cultures (Fig. 2) . As reported earlier, acetylcholine receptors were expressed in clusters on the myotube membrane in aneural cultures; in many cases receptor clusters also contained P2X₂ receptors.

CONCLUSIONS AND SIGNIFICANCE

Many growth factors have been implicated in the regulation of muscle regeneration, including fibroblast growth factors, insulin-like growth factors and nitric oxide. In this study we explore the possibility that ATP might be involved in regulating muscle regeneration.

Past research demonstrates the expression and function of receptors for extracellular ATP in muscle development. More recently, the expression of specific P2X and P2Y receptor subtypes during skeletal muscle development has been demonstrated. P2Y₁, P2X₅, and P2X₆ are expressed in chick skeletal muscle development whereas expression of the P2X₂, P2X₅, P2X₆, P2Y₁, P2Y₂, and P2Y₄ receptors has been demonstrated in rat skeletal muscle development.

Whereas the P2Y₁ receptor has been implicated in the regulation of acetylcholine receptor and acetylcholinesterase expression during muscle development and on denervation, a role for the P2X₅ receptor in the regulation of myoblast activity and differentiation has been demonstrated in cultures.

We have used in vivo and in vitro models, immunohistochemistry, RT-PCR, and electrophysiology to provide significant and novel insights into the role of purinergic signaling in muscle pathology. The focus of this study, the mdx mouse, is a well-recognized model of human Duchenne muscular dystrophy, the most common and severe of the muscular dystrophies. By using this model and conducting a parallel study in culture, we demonstrated the expression and specific function of the P2X₂, P2X₅, and P2Y₁ receptors in muscle regeneration. These receptors were expressed on key cell types in the process of muscle regeneration. The P2X₅ and P2Y₁ receptors were expressed on activated satellite cells (myogenic stem cells). P2 receptors were expressed on regenerating myotubes and muscle fibers (P2X₂) and on infiltrating immune cells (P2Y₁).

Since ATP is known to be passively released upon cell damage, coreleased with acetylcholine from motor nerve terminals, and released from skeletal muscle fibers upon contraction, mdx muscle is expected to contain high levels of extracellular ATP. Hence, bearing in mind the known effects of extracellular nucleotides on myoblast activity, purinoceptor expression in mdx muscle is likely to be of functional significance in muscle regeneration. Furthermore, strong similarities in the specific subtypes and pattern of P2 receptor expression in regenerating muscle in vivo and in normal rat myoblast cultures suggest that P2 receptor expression is part of the normal response to muscle injury. Thus, this study provides the first evidence for a role for purinergic signaling in muscle regeneration in vivo and indicates that purinergic receptors may be therapeutic targets for the treatment of muscle diseases.

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the expression and potential functions of purinoceptors in skeletal muscle regeneration. Experiments in vitro and in vivo demonstrated the sequential expression of P2Y1, P2X5, and P2X2 receptors on key cell types during muscle regeneration. Upon muscle damage, the P2Y1 receptor was strongly expressed on infiltrating immune cells, followed by expression of P2X5 and P2Y1 receptors on activated satellite cells (myogenic stem cells). Myotubes and muscle fibers expressed P2X2 receptor protein. These findings suggest a role for purinergic signaling, particularly via the P2X5 receptor, in muscle regeneration.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1175fje; doi: 10.1096/fj.03-1175fje

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