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The T-tubule membrane ATP-operated P2X₄ receptor influences contractility of skeletal muscle

Dorianna Sandonà^*, Daniela Danieli-Betto, Elena Germinario, Donatella Biral, Tiziana Martinello^*, Antonella Lioy^*, Elena Tarricone^*, Stefano Gastaldello^* and Romeo Betto^,1

^* Department of Biomedical and Experimental Sciences and
Department of Human Anatomy and Physiology, University of Padova; and
Laboratory of Muscle Biology and Physiopathology, C.N.R. Neuroscience Institute, Padova, Italy

¹ Correspondence: Department of Biomedical Sciences, University of Padova, Padova 35121, Italy. E-mail: betto{at}bio.unipd.it

SPECIFIC AIM

It has recently been shown that the P2X₄ receptor plays a positive inotropic action in cardiac muscle, where it appears to have an important role in modulating the progression of heart failure. Stimulated by these findings, the aim of this study was to determine the expression, localization, and physiological significance of P2X₄ expression in skeletal muscle.

PRINCIPAL FINDINGS

Extracellular ATP stimulates a large number of biological responses, including neurotransmission in the central and peripheral nervous system, nociception, smooth muscle contraction, immune responses, and platelet aggregation. The nucleotide is actively released from cells via different mechanisms such as exocytosis, transporters, and stretch-activated channels. ATP exerts its action by stimulating specific receptors expressed in almost all mammalian tissues. Two families of ATP receptors have been described. The P2X family is composed of seven ionotropic receptors that, when activated, form nonselective channels mainly permeable to Ca²⁺. The P2Y family comprises eight metabotropic receptors that are functionally coupled to either phospholipase C or adenylate cyclase. Essential in controlling ATP signaling is a class of extracellular enzymes, the ectonucleotidases. These nucleotide-degrading enzymes terminate the nucleotide actions, generate new signaling molecules, and permit salvage of purines.

Much evidence suggests that all components of extracellular ATP signaling exist in skeletal muscle: 1) ATP is released from muscles during contractile activity; 2) a full set of enzymes able to degrade extracellular ATP is present at the surfaces of both sarcolemma and T-tubule membranes; and 3) transcripts of different purinergic receptors have been isolated from muscle RNA. Growing evidence shows the important role played by extracellular ATP in myogenesis and muscle regeneration, also indicated by studies on dystrophic muscles. However, little evidence of extracellular ATP physiological actions has been produced in adult skeletal muscle. The present work demonstrates the expression, specific localization, and possible physiological role of the ATP-receptor P2X₄ in skeletal muscle.

1. Expression of P2X₄ in rat skeletal muscles
Transcripts of P2X₄ have been found in almost all tissues, including skeletal muscle. Studies in the heart have demonstrated that P2X₄ is specifically expressed in cardiomyocytes, where it positively affects contractility and contributes to the inotropic response to extracellular nucleotides. The present work was undertaken to examine whether P2X₄ receptors are expressed by adult skeletal muscle fibers and to reveal possible functional roles of the receptors. RT-PCR analysis demonstrated the expression of the full-length receptor both in fast-twitch (glycolytic, fatigue-sensitive) and slow-twitch (oxidative, fatigue-resistant) muscle cells.

Western blot analyses revealed the presence of the 60 kDa P2X₄ protein band in total crude membrane preparations isolated from different rat tissues. Significant levels of the protein were evident in heart and skeletal muscle. N-glycosidase F treatment reduced the apparent molecular mass of P2X₄ receptor from 60 to 44 kDa, consistently with the presence of six putative N-linked glycosylation sites. Detailed analyses show that P2X₄ protein is expressed in developing hind-limb muscles (15-day-old rat) and in adult muscles. Apparently, the level of P2X₄ protein expression was higher in slow-twitch soleus muscle than in fast-twitch EDL muscle.

2. Cytolocalization of P2X₄
Immunofluorescence analysis of transverse cryostat sections from EDL and soleus muscles, using an antibody specific for the P2X₄ receptor, revealed clear intracellular staining of muscle fibers. The antibody also decorated neuromuscular junction, nerve axons, and the inner wall of blood vessels. All soleus fibers were stained by the P2X₄ antibody, while a mosaic distribution of staining was observed in the EDL muscle. Rat hind-limb muscles are composed of four fiber types (type 1, 2A, 2X, and 2B) with distinct contractile and metabolic properties. Colocalization immunofluorescence staining of the P2X₄ antibody with antibodies specific for the diverse myosin isoforms permitted us to establish that expression of P2X₄ was elevated in type 1, 2A, and 2X fibers but nearly absent in type 2B fibers. Contraction speed of muscle fibers is dependent on the pattern of myosin isoform expressed by the fiber. Type 1 myosin attributes to the fiber the slowest speed, type 2A and 2X the intermediate, and type 2B the fastest. The slow contracting rat soleus muscle is composed of type 1 and type 2A fibers; type 2X and 2B fibers are predominant in the fast EDL muscles. Type 1, 2A, and 2X fibers are characterized by high oxidative metabolism; type 2B are glycolytic. These properties are associated with fiber resistance or sensitivity to fatigue, respectively. P2X₄ expression was particularly elevated in slow-contracting fibers, which are rich in mitochondria and more resistant to fatigue.

The expression of P2X₄ receptor within the muscle fiber was unexpected for a cell surface receptor. Immunofluorescence confocal images showed, however, that the P2X₄ antibody produced very regular transverse staining in longitudinal sections of muscle fibers, consistent with T-tubule localization. Double staining with an antibody specific for the 1 subunit of DHP receptor, a marker of T-tubule membranes, revealed strict colocalization of the two epitopes. Thus, the ATP-operated P2X₄ receptor is localized into the T-tubule membranes of skeletal muscle, a site of excitation-contraction (EC) coupling. Stimulation of these receptors by ATP opens a nonselective cation channel that promotes Ca²⁺ entry.

3. ATP release from contracting skeletal muscle
The localization of P2X₄ receptor in the T-tubules suggests possible physiological Ca²⁺-dependent roles for this receptor in skeletal muscle. Since stimulation of P2X receptors is strictly dependent on the presence of ATP in the extracellular space, it was important to establish whether muscle fibers are able to release the nucleotide. Accordingly, rat flexor digitorum brevis (FDB) fibers were isolated and cultured according to established protocols. Electrical field stimulation was used to produce muscle contraction of FDB fibers in culture. Stimulation produced the immediate and substantial release of ATP, which reached maximum levels within seconds. The subsequent progressive fall of extracellular ATP levels to baseline values suggests the presence of extracellular enzymes on the fiber surface that are able to degrade released nucleotide.

4. P2X₄ receptor and skeletal muscle contractility
After finding that ATP is released as a consequence of each muscle contraction, we hypothesized that the nucleotide may exert autocrine and/or paracrine actions to muscle fibers through the activation of P2X₄ receptors and subsequent activation of downstream signaling pathways. The putative increased Ca²⁺ entry and resultant Ca²⁺-dependent activities could exert important effects on muscle contraction, especially during sustained activity. To test any possible effects of extracellular ATP, we used a low-frequency (0.05/s) stimulation protocol that produced a progressive potentiation of soleus twitch tension to values that were 20% higher than those at the beginning (Fig. 1 A). We speculated that potentiation in soleus muscle could be ascribed to the action of ATP on P2X₄ receptor.

View larger version (46K): [in this window] [in a new window]   Figure 1. Contractile responses of soleus muscle. Soleus muscle was isolated and immersed in a Ringer physiological solution at 30°C, then bubbled with 95% O2-5% CO2. The muscle was stimulated with supramaximal single pulses (0.5 ms duration, every 20 s for 43 min) and twitch tension output was recorded. A) Example of a soleus muscle stimulated in normal Ringer showing all twitch tension recordings. B) Soleus muscle stimulated in normal Ringer () and in Ringer containing 1U/mL hexokinase and 1U/mL apyrase (•). C) Soleus muscle stimulated in normal Ringer (), nominally Ca2+-free Ringer (•), and normal Ringer containing a mix of P2X inhibitors (10 µM PPADS, 10 µM suramin, and 50 µM reactive blue-2, ). D) Soleus muscle stimulated in normal Ringer () and in Ringer containing either 5 µM Zn2+, 10 µM ivermectin (IVM), and 100 µM ATP (•) or 1 µM 2meSATP ().

To investigate this hypothesis, soleus muscles were electrically stimulated in the presence of the ATP-degrading enzymes hexokinase and apyrase to prevent the action of released ATP on the receptor (Fig. 1B ). Experiments were also conducted in the absence of extracellular Ca²⁺ to avoid Ca²⁺ entry through P2X receptors. Last, a cocktail of P2X inhibitors (PPADS, suramin, and reactive blue-2) were used to prevent activation of the receptors (Fig. 1C ). These treatments abolished the rise of twitch tension produced by low-frequency stimulation of soleus muscle. In contrast, maneuvers directed to further potentiate twitch tension were ineffective. Contractile responses were not potentiatiated by addition of ATP co-agonists Zn²⁺ and ivermectin nor by the P2X-selective agonist 2-methylthio-ATP (Fig. 1D ). These results are explained by assuming that the receptor is likely fully activated by the natural agonist ATP, which we have shown is liberated during contractile activity.

CONCLUSIONS AND SIGNIFICANCE

The present work demonstrates for the first time that the P2X₄ receptor protein is expressed in rat skeletal muscle and that slow-twitch muscles have higher levels of receptor expression than fast-twitch hind-limb muscles. The present findings show that the receptor is localized in the T-tubule membranes, suggesting a putative role for extracellular ATP in the EC coupling process of skeletal muscle. In vitro experiments demonstrated that contracting muscle fibers release substantial amounts of ATP, indicative of possible autocrine/paracrine actions of released ATP during contractile events. The potentiation of soleus twitch tension normally produced by extended low-frequency (0.05 Hz) stimulation was abolished by treatments designed to prevent P2X₄ receptor activation, suggesting that the activation of P2X₄ receptors is concurrent with contractile activity.

Figure 2 illustrates a working model mechanism for the possible physiological roles of P2X₄ receptor. The T-tubule membranes are invaginations of sarcolemma and represent 80% of the plasma membrane of a skeletal muscle fiber. Upon nerve excitation, the action potential generated at the neuromuscular junction spreads over muscle fiber membrane and propagates into the interior through the T-tubules. Depolarization of T-tubules permits the release of Ca²⁺ from sarcoplasmic reticulum (SR), which is in close association with the T-tubules. T-tubule-mediated SR calcium release results in what is known as EC coupling of skeletal muscle. Thus, T-tubule membranes represent an essential element in the EC coupling process. Expression in the T-tubules of a previously undescribed ATP-operated cation channel in the form of the P2X₄ suggests that this receptor may participate in modulating muscle contraction. The P2X₄ receptor, when stimulated by ATP, is a nonselective cation channel with a relatively high Ca²⁺ permeability. As a consequence, repetitive contractions may lead to the rise of P2X₄-mediated Ca²⁺ influx, making available more Ca²⁺ to subsequent contractions. The P2X₄-mediated Ca²⁺ entry may stimulate Ca²⁺-dependent signaling pathways, which could further affect skeletal muscle contractility. P2X₄ activation may thus result in the potentiation of mechanical responses of skeletal muscle fibers expressing the receptor, particularly the oxidative fibers, where ATP-dependent receptor activation could in part explain their fatigue resistance.

View larger version (35K): [in this window] [in a new window]   Figure 2. Schematic model of possible mechanisms by which the contractile activity-dependent P2X4 activation affects skeletal muscle contractility. Upon nerve excitation (1), the action potential generated at the neuromuscular junction spreads over muscle fiber membrane and propagates into the interior through the T-tubules (TT). Depolarization of T-tubules activates the voltage-sensitive dihydropyridine receptor (DHPR), which mechanically stimulates (2) the sarcoplasmic reticulum (SR) Ca2+ release channel (RyR). Stimulation of RyR causes the large release of Ca2+ from the SR (3) and thus the onset of muscle contraction (4). Muscle contraction is followed by the release of ATP (5) from muscle fiber, which activates the P2X4 receptor localized in the T-tubule membranes (6). P2X4 activation determines Ca2+ entry into the cell (7), which may contribute to the refilling of SR (8) and/or activate Ca2+-dependent signaling pathways (9). The recurring activation of P2X4 that occurs during sustained activity may potentiate twitch tension by contributing to SR Ca2+ refilling (8), making more Ca2+ available for subsequent contractions. P2X4-mediated Ca2+ entry may activate Ca2+-dependent activities (9) able to indirectly stimulate skeletal muscle contractility, but it is also possible that P2X4-mediated stimulation of Ca2+-dependent activities may be part of the program controlling muscle gene expression (10).

The P2X₄ receptor can in fact be activated by ATP release during single contractions unrelated to fatigue. What is the effect of such "basal" stimulation of P2X₄? We hypothesize that purinergic stimulation could be an appropriate trigger of the so called excitation-transcription coupling of skeletal muscle. This process, through mechanisms still unknown, couples nerve activity to muscle gene expression. It is now well established that nerve electrical activity has a crucial role on skeletal muscle growth and fiber type differentiation. However, the initial events mediating the nerve-dependent activation of the process are not yet known, though they appear to be mostly mediated by calcium. Given that ATP is released during each contraction, the nucleotide stimulates muscle fibers at the same frequency of contractile activity and, as a result, regularly evokes intracellular calcium transients and activates downstream signaling pathways that could control skeletal muscle growth and fiber type determination.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3333fje; doi: 10.1096/fj.04-3333fje

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