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MUSCLE CELL BIOLOGY AND CELL MOTILITY

The COX-2 pathway regulates growth of atrophied muscle via multiple mechanisms

¹Department of Pharmacology and ²Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia

Submitted 18 October 2005 ; accepted in final form 23 January 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Loss of muscle mass occurs with disease, injury, aging, and inactivity. Restoration of normal muscle mass depends on myofiber growth, the regulation of which is incompletely understood. Cyclooxygenase (COX)-2 is one of two isoforms of COX that catalyzes the synthesis of prostaglandins, paracrine hormones that regulate diverse physiological and pathophysiological processes. Previously, we demonstrated that the COX-2 pathway regulates early stages of myofiber growth during muscle regeneration. However, whether the COX-2 pathway plays a common role in adult myofiber growth or functions specifically during muscle regeneration is unknown. Therefore, we examined the role of COX-2 during myofiber growth following atrophy in mice. Muscle atrophy was induced by hindlimb suspension (HS) for 2 wk, followed by a reloading period, during which mice were treated with either the COX-2-selective inhibitor SC-236 (6 mg·kg^–1·day^–1) or vehicle. COX-2 protein was expressed and SC-236 attenuated myofiber growth during reloading in both soleus and plantaris muscles. Attenuated myofiber growth in the soleus was associated with both decreased myonuclear addition and decreased inflammation, whereas neither of these processes mediated the effects of SC-236 on plantaris growth. In addition, COX-2^–/– satellite cells exhibited impaired activation/proliferation in vitro, suggesting direct regulation of muscle cell activity by COX-2. Together, these data suggest that the COX-2 pathway plays a common regulatory role during various types of muscle growth via multiple mechanisms.

cyclooxygenase-2; prostaglandins; myonuclear number; satellite cells; inflammation

We have shown previously that the cyclooxygenase (COX)-2 pathway regulates myofiber growth in mice following injury that causes extensive degeneration (3). COX-2 is one of two isoforms of COX that catalyzes the rate-limiting step in the synthesis of prostaglandins (PG), autocrine/paracrine signaling molecules that are synthesized in response to cytokines, growth factors, and cell injury, and are potent regulators of inflammation (12). Growth of regenerated myofibers was attenuated by the COX-2-selective inhibitor SC-236 as well as in COX-2^–/– mice (3). This effect was associated with the presence of fewer myoblasts within regenerating muscles, implicating adverse effects on myoblasts as a potential mechanism behind attenuated myofiber growth. Regenerating muscles from SC-236-treated mice and COX-2^–/– mice also exhibited decreased inflammation.

Whether COX-2 regulates myofiber growth under diverse physiological circumstances or specifically during muscle regeneration is unknown. Muscle regeneration involves the replacement of necrotic myofibers via de novo myofiber formation and involves a robust inflammatory response (14). Because myonuclei within existing myofibers are postmitotic, myofiber formation and subsequent growth depends on the activation, proliferation, differentiation, and fusion of muscle precursor cells (MPC). MPC, the majority of which lie between the basal lamina and sarcolemma and are called satellite cells (SC), are quiescent cells that become activated to reenter the cell cycle and proliferate by various growth factors and cytokines produced during muscle growth (6). In contrast to regeneration, restoration of muscle mass after atrophy involves the growth of existing myofibers. In mice, reloading of muscles that have been atrophied by unweighting typically causes minimal myofiber degeneration despite the occurrence of some myofiber membrane damage and inflammation (49). Growth of atrophied myofibers not only involves anabolic processes to facilitate myofiber growth but also the reversal of catabolic processes that were induced during the atrophy period (25). In addition to myonuclear loss, atrophy is also associated with the loss of MPC, and the remaining MPC exhibit impaired proliferation and differentiation (33). Thus the physiological state of atrophied muscle before reloading is distinct from that of degenerating muscle and may necessitate alternative mechanisms to facilitate regrowth.

In this study, we employed a mouse model of muscle atrophy and regrowth to test whether myofiber growth after atrophy is also regulated by COX-2. We show that treatment with the COX-2 inhibitor SC-236 attenuates myofiber growth in both the soleus and plantaris muscles after atrophy. We also provide evidence for the involvement of COX-2 in myonuclear addition and inflammation during growth in the soleus but not the plantaris, suggesting that multiple mechanisms underlie COX-2 function in muscle growth. Finally, in vitro evidence suggests that regulation of myonuclear addition by the COX-2 pathway may be attributable to direct regulation of muscle cells.

	MATERIALS AND METHODS

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Animals and drugs. Female BALB/c mice (10–11 wk) were purchased from Charles River Laboratories. Male COX wild-type (WT), COX-1^–/–, and COX-2^–/– mice (B6,129P2; 7–13 wk old) were purchased from Taconic Farms. The mice were housed under 12:12-h light-dark cycle conditions with food and water provided ad libitum. All animal protocols were approved by Emory University’s Institutional Animal Care and Use Committee. SC-236 was generously provided by Pfizer.

To induce muscle atrophy, mice were subjected to hindlimb suspension (HS) for 2 wk as described previously (32, 33). Mice were randomly assigned to several groups: HS alone, HS followed by a reloading period with drug or vehicle treatment, or control. After the HS period, mice in the HS group were euthanized, whereas mice in the reloading group were anesthetized briefly (100 mg/kg ketamine and 15 mg/kg xylazine) for removal of the tail harnesses and then allowed to resume normal cage activity for varying lengths of time. Control mice maintained normal cage activity throughout the experiment. Mice in the reloading group were treated with the COX-2 inhibitor SC-236 (6 mg·kg^–1·day^–1) or vehicle (95% polyethylene glycol, 5% Tween 20) in the drinking water starting 3 days before the reloading period. The selectivity of this dose for COX-2 is supported by both the manufacturer’s recommendations and our previous experiments (3).

Muscle collection and histological analyses. The mice were euthanized by CO₂ inhalation, and both the soleus and plantaris muscles were excised. For each mouse, muscles from one leg were snap-frozen in liquid nitrogen for biochemical analyses, and muscles from the contralateral leg were collected and prepared for histological and immunohistochemical analyses, as described previously (3, 17, 32). Briefly, the muscles were embedded in tissue freezing medium (Triangle Biomedical Sciences) and frozen in methylbutane cooled in liquid nitrogen. Serial 14-µm-thick sections were collected along the entire muscle length at 450-µm increments and stained with hematoxylin and eosin. Sections collected from the midbelly of the muscle were chosen for analysis. An image from the center of the section was captured, and the cross-sectional area (XSA) of each myofiber within this 307,200-µm² field was measured. The same images were also used for quantification of centrally nucleated fibers (CNF). All analyses and photography were performed on a Zeiss Axiovert microscope equipped with a video camera and Scion Image software version 1.63.

Immunohistochemistry. For immunohistochemical analyses, 8-µm-thick sections were collected along the entire length of each muscle. Sections collected from the midbelly of the muscle were chosen for analysis. Myonuclear number was determined as described (32, 33) by immunostaining the myofiber sarcolemma with a mouse anti-dystrophin antibody (1:400, Sigma), followed by Texas red-conjugated goat anti-mouse IgG (1:50, Jackson Immunoresearch). Nuclei were counterstained with 0.25 µg/ml 4',6'-diamidino-2-phenylindole (DAPI; Sigma). Myonuclei localized within the myofiber sarcolemma were counted for 100 myofibers in 4–5 fields per sample.

For immunostaining of inflammatory cells, sections were air dried for 30 min, fixed in ice-cold acetone for 10 min, and air dried for an additional 30 min. Sections were rehydrated in PBS for 5 min, and endogenous peroxidase activity was quenched with 0.3% H₂O₂ in PBS for 5 min. After being washed in PBS, the sections were incubated in block buffer (3% bovine serum albumin, 0.05% Tween 20, and 0.2% gelatin in PBS) for 30 min and then a rat monoclonal antibody against Mac-1 (1:15, BD Pharmingen) for 2 h. Sections were washed extensively with PBS and incubated with biotin-conjugated rabbit-anti-rat IgG (1:200, Vector Laboratories) for 30 min. After additional PBS washes, sections were incubated in horseradish peroxidase (HRP)-conjugated Avidin-D (1:1,000, Vector Laboratories) for 30 min. Slides were washed once with PBS and developed with 3-amino-9-ethylcarbazole substrate (AEC red, Vector Laboratories) for 4–8 min. No staining was observed in control slides that were incubated with a rat IgG isotype control antibody (BD Biosciences) instead of the anti-Mac-1 antibody. The mean number of inflammatory (Mac-1⁺) cells present in three entire sections was determined for each sample, and the area of the section was determined with the use of Scion Image software. The volume of each section was calculated as the product of the section area and thickness (8 µm), and the number of inflammatory cells was expressed per cubic millimeter (mm³).

For COX-2 immunostaining, sections were fixed as described for Mac-1 and washed with PBS. Sections were incubated in block buffer (5% normal donkey serum, 0.5% bovine serum albumin, and 0.5% Tween 20 in PBS) for 30 min and then a rabbit polyclonal antibody against COX-2 (1:250, Cayman Chemical) in block buffer for 1 h. After several washes in PBS, antibody binding was visualized using the tyramide amplification system (TSA kit, Perkin Elmer) according to the manufacturer’s protocol. Briefly, sections were incubated with biotin-conjugated donkey-anti-rabbit IgG (1:400, Jackson Immunoresearch) for 1 h, followed by HRP-conjugated streptavidin [1:100 in TNB buffer (Perkin Elmer)] for 30 min. Slides were washed with PBS and incubated in tertramethylrhodamine-tyramide (1:500) in amplification diluent for 5 min. The nuclei were counterstained with DAPI (0.25 µg/ml). No immunostaining was observed when the primary antibody was either replaced with a rabbit IgG control antibody (Jackson) or had been preadsorbed with an equal concentration of COX-2 blocking peptide (Cayman Chemical) for 90 min before application.

RNA isolation and real-time RT-PCR. Total RNA was isolated from soleus and plantaris muscles by homogenization in TRIzol Reagent (Life Technologies) following the manufacturer’s protocol. Total RNA (1 µg) was reverse transcribed in a 20–40 µl final reaction volume using random hexamers and Moloney murine leukemia virus-reverse transcriptase (Invitrogen). The reaction was incubated at 25°C for 10 min, 42°C for 50 min, followed by 72°C for 10 min to inactivate the reverse transcriptase. Real-time PCR was performed and results were analyzed using the iCycler iQ Real-Time Detection System and software (Bio-Rad) as described (3). Briefly, cDNA (2 µl from each sample) was amplified using primers specific for Mac-1 (3) in a 25-µl reaction containing the Mac-1 primer pair (400 nM each primer) and iQ SYBR Green Supermix (Bio-Rad). Samples were incubated at 95°C for 4 min, followed by 35–40 cycles (30 s each) of denaturation (95°C), annealing (55°C), and extension (72°C). SYBR green fluorescence was measured at the end of the extension step of each cycle. Reactions were run in duplicate or triplicate, and PCR product size was verified both by melt curve analysis and agarose gel electrophoresis at the conclusion of the PCR reaction. Mac-1 expression was quantified in reference to gene-specific standards as described (3).

Isolation and immunostaining of primary myoblasts and myofibers. Primary myoblast cultures were prepared from the hindlimb muscles of BALB/c mice (8–10 wk) based on methods described previously, except for omission of the Percoll gradient (3). Myoblasts were purified to 98% purity based on immunostaining for MyoD over 4–5 passages in selective growth media (39). For COX-2 immunostaining, cells were seeded onto dishes coated with entactin-collagen-laminin (Upstate Biotechnology) and immediately fixed in 3.7% formaldehyde for 10 min.

Single myofibers were isolated from the gastrocnemius muscles of two WT and COX-2^–/– mice, as previously described (31, 33). The gastrocnemius muscle was chosen for analysis based on high-myofiber yield. Briefly, the muscles were excised and digested with 0.1% collagenase (400 U/ml Type I, Worthington Biochemical) in digest media (DMEM; 4.5 mg/ml glucose, 25 mM HEPES, 100 U/ml penicillin G, and 100 µg/ml streptomycin) for 1.5 h at 37°C with gentle agitation. The muscles were further dissociated by trituration in a wide-bore serological pipette. Individual viable myofibers, which were distinguished easily from hypercontracted myofibers by their translucence, were collected using a fire-polished Pasteur pipette and cleared of tissue debris by 1–2 serial transfers to plates containing fresh digest media with 10% FBS. Myofibers were plated three per well in 24-well plates coated with growth factor-reduced Matrigel (BD Biosciences), centrifuged for 40 min at 1,100 g to facilitate adherence, and incubated in a humidified 5% CO₂ incubator. To quantify SC activation/proliferation, myofiber cultures were fixed either 48 or 72 h after muscle excision in 2% formaldehyde for 10 min. Phase-contrast microscopy determined the total number of cells that lay adjacent to each myofiber. Cells associated with myofibers that had detached or hypercontracted during the experiment were not analyzed. For Pax-7 immunostaining, a subset of myofibers was fixed immediately after being plated.

Similar protocols were used for both Pax7 and COX-2 immunostaining. All steps were carried out at room temperature unless otherwise noted. After being incubated in block buffer (Pax-7: 5% normal goat serum, 0.5% bovine serum albumin, 0.25% Triton X-100 in PBS; COX-2: TNB block buffer) for 1 h, the cells were incubated with an antibody against Pax-7 (neat hybridoma supernatant, Developmental Studies Hybridoma Bank) or COX-2 (1:200, Cayman Chemical) overnight at 4°C. Antibody binding was visualized using the tyramide amplification system (TSA kit) according to the manufacturer’s protocol. Briefly, cells were washed with PBS containing 0.2% Tween 20 (PBS-T) and then incubated in biotin-conjugated goat-anti-mouse for Pax-7 or biotin-conjugated donkey-anti-rabbit for COX-2 (1:500, Jackson) in PBS-T for 1 h. After further washes in PBS-T, cells were incubated in HRP-conjugated streptavidin (1:100 in TNB buffer) for 30 min, followed by fluorescein-tyramide for Pax-7 (1:300) or tertramethyl rhodamine-tyramide (1:500) in amplification diluent for 5–10 min. Nuclei were counterstained with DAPI (0.25 µg/ml). No staining was observed when the Pax-7 antibody was replaced with control mouse IgG (Serotec) or the COX-2 antibody had been preadsorbed with an equal concentration of COX-2 blocking peptide for 90 min before application.

Statistics. To determine the significance between two groups, comparisons were made using the Student’s t-test. Data from multiple groups were analyzed by one- or two-way ANOVA with the use of Prism version 4.0a (GraphPad Software) or SigmaStat version 2.03 (SPSS), respectively, followed by the Newman-Keuls post test. For all statistical tests, P < 0.05 was considered significant.

	RESULTS

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Atrophy and COX-2 protein expression in soleus muscle. Muscle atrophy was induced by subjecting mice to HS for 2 wk. Consistent with our previous results (32), the average myofiber XSA of the soleus decreased by 37% in response to HS (Fig. 1, A and C). This decrease in myofiber size was also associated with a 40% loss of myonuclei (Fig. 1B). After HS, muscle growth was induced by allowing mice to resume normal cage activity for up to 2 wk, a period that typically allows for full restoration of myofiber size and myonuclear number in BALB/c mice (32).

View larger version (89K): [in this window] [in a new window]   Fig. 1. Hindlimb suspension (HS) decreases soleus myofiber cross-sectional area (XSA) and myonuclear number. The average myofiber XSA (A) and myonuclear number (B) were determined for soleus muscles from control mice (C) and HS mice. Two weeks of HS treatment induced a 37% decrease in average XSA and a 40% decrease in myonuclear number. Data are means ± SE, n = 5. *P < 0.05. C: representative soleus sections stained with hematoxylin and eosin (H&E). Bar = 50 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Cyclooxygenase-2 (COX-2) protein expression in the soleus muscle. Representative sections of soleus muscles collected from control and HS mice and after 3 days (D3) of reloading immunostained for COX-2 (red). 4',6'-Diamidino-2-phenylindole (DAPI) was used to counterstain nuclei (blue). Inset, magnified merged image of a COX-2+ cell. No staining was observed when the primary antibody was preincubated with COX-2 blocking peptide before application (D3 + peptide). Bar = 30 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 3. SC-236 attenuates soleus myofiber growth. The average XSA of soleus myofibers was determined after 3, 7, and 14 days of reloading. Dotted lines indicate the average XSA of myofibers from control and HS mice. After 14 days of reloading, the myofibers from vehicle-treated (V) mice had recovered by 79%, whereas those from SC-236-treated mice recovered by only 48%. Data are means ± SE, n = 9–12. *P < 0.05 comparing vehicle-treated and SC-236 mice at the same time point.

View larger version (41K): [in this window] [in a new window]   Fig. 4. COX-2 regulates myoblast behavior both in vivo and in vitro. A: myonuclear number of soleus myofibers after 7 and 14 days of recovery with vehicle (V) or SC-236 (SC) treatment was determined. Dotted lines indicate the average myonuclear number of muscles from control and HS mice. No myonuclear addition occurred within 7 days of recovery, and SC-236 attenuated myonuclear addition from day 7 to day 14. Data are means ± SE, n = 5. *P < 0.05. B: treatment with SC-236 for the last 3 days of HS (HS+SC) had no effect on the average myonuclear number (measured immediately after the HS period) relative to vehicle treatment (HS+V). Data are means ± SE, n = 4–7. C: purified myoblasts were immunostained for COX-2 (red) and nuclei were counterstained with DAPI (blue). No staining was observed when the primary antibody was preadsorbed with COX-2 blocking peptide before application (+peptide). Bar = 50 µm. D–H: single myofibers were isolated from wild-type (WT) and COX-2–/– gastrocnemius muscles, and the number of myoblasts associated with each fiber was quantified after 48 and 72 h. Frequency distribution plots for both 48 (D) and 72 (E) h show that COX-2–/– myofibers were associated with fewer myoblasts than WT. n = 86–106 total myofibers from two WT or COX-2–/– mice. F: at 72 h, the average number of myoblasts per COX-2–/– myofiber was decreased 44% compared with WT. Data are means ± SE, n = 95–106 total myofibers from two WT or COX-2–/– mice. *P < 0.05. G: scatter plot showing that the number of Pax-7+ satellite cells resident on myofibers immediately after being plated did not differ between WT and COX-2–/– myofibers. Horizontal lines indicate mean values. n = 21–23 total myofibers. H: representative phase-contrast and fluorescent images of a myofiber immunostained for Pax-7 (green). DAPI was used to counterstain nuclei (blue).

SC-236 also attenuates myofiber growth in the plantaris. HS preferentially affects slow-twitch muscles such as the soleus compared with fast-twitch muscles (see Ref. 47 for review). In addition, the growth of different types of muscles often involves distinct cellular and molecular mechanisms (30, 32). To determine whether the regulation of atrophied myofiber growth by COX-2 is specific for the soleus or common to other muscles, we examined the effects of SC-236 on myofiber growth in the plantaris, a fast-twitch muscle. As shown in Fig. 5A, COX-2 protein was expressed in the plantaris under control, HS, and reloading conditions. Comparable staining was also observed after 1 and 2 wk of reloading (data not shown). Consistent with COX-2 expression in the soleus, immunoreactivity was observed in a punctate pattern throughout each section, and neither staining intensity nor the percentage of COX-2⁺ cells appeared to differ between samples.

View larger version (34K): [in this window] [in a new window]   Fig. 5. SC-236 attenuates plantaris myofiber growth after atrophy. A: representative sections of plantaris muscles collected from control and HS mice and after D3 reloading, immunostained for COX-2 (red). DAPI was used to counterstain nuclei (blue). No staining was observed when the primary antibody was preincubated with COX-2 blocking peptide before application (D3 + peptide). Bar = 30 µm. B: HS induced a 34% decrease in the average XSA of plantaris myofibers. Data are means ± SE, n = 5. *P < 0.05. C: average XSA of plantaris myofibers was determined after 3, 7, and 14 days of reloading. Dotted lines indicate the average XSA of myofibers from control and HS mice. After 14 days of reloading, muscles from vehicle-treated mice had recovered fully, whereas those muscles from SC-236-treated mice recovered only 55%. Data are means ± SE, n = 10–12. *P < 0.05 comparing vehicle-treated and SC-236 mice at the same time point.

Adverse effects of SC-236 on muscle growth are not associated solely with inflammation. In a previous study, we (3) showed that the COX-2 pathway regulates early stages of muscle regeneration after injury. The attenuated myofiber growth observed after SC-236 treatment and in COX-2^–/– mice was associated also with impaired inflammation, suggesting that the COX-2 pathway may regulate myofiber growth indirectly by modulating the inflammatory response to injury. Given that inflammation can also occur during reloading after HS (10, 11, 21, 34, 50), we sought to determine whether the effects of SC-236 on myofiber growth after atrophy were associated with adverse effects on inflammation.

We first examined the degree of myofiber degeneration incurred during reloading after HS. Myonuclei typically reside along the periphery of myofibers but are located centrally in myofibers that have undergone degeneration/regeneration in response to damage. As shown in Fig. 6A, sections from control and recovering muscles contained an average of 1–5% of CNF. In addition, the percentage of CNF in either vehicle- or SC-236-treated muscles during recovery did not increase significantly relative to control. Together, these results suggest that, in contrast to muscle regeneration, the amount of de novo myofiber formation induced during recovery from atrophy is minimal and is unaffected by SC-236 treatment.

View larger version (52K): [in this window] [in a new window]   Fig. 6. Myofiber regeneration and inflammation in soleus and plantaris muscles during reloading. A: scatter plot of the percentage of centrally nucleated fibers (CNF) observed in sections of soleus (SOL) and plantaris (PLAN) muscles from control mice and mice treated with vehicle or SC-236 for various reloading periods. Neither reloading nor SC-236 treatment significantly affected the mean percentage of CNF (indicated by horizontal lines). n = 11–26. B: Mac-1 mRNA levels in the soleus increased 19-fold on day 3 of recovery (Rec). HS, HS for 2 wk. Data are means ± SE, n = 4–5. *P < 0.05. C: representative soleus section (day 3) immunostained for Mac-1 (red) and a corresponding serial section stained with H&E. Asterisk indicates the same myofiber in both sections. D: SC-236 decreased the average number of Mac-1+ cells in the soleus on day 3 of recovery by 68%. Data are means ± SE, n = 5. *P < 0.05. E: Mac-1 mRNA levels in the plantaris increased 2-fold on day 3 of recovery. Data are means ± SE, n = 4–5. *P < 0.05.

Given that SC-236 attenuates myofiber growth in both the soleus and plantaris, we next determined whether SC-236 similarly attenuates inflammation during reloading in the plantaris. In contrast to the soleus, Mac-1 mRNA expression in the plantaris was induced only twofold after 3 days of recovery (Fig. 6E), and only an occasional Mac-1⁺ cell was observed within sections from either vehicle- or SC-236-treated plantaris muscles (data not shown). Thus, the attenuation of plantaris growth by SC-236 involves mechanisms other than the impairment of inflammation, thereby suggesting that the role of the COX-2 pathway in muscle growth is not associated solely with the inflammatory response.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In this investigation, we examined the involvement of the COX-2 pathway in regulating growth of atrophied myofibers in mice. The COX-2 inhibitor, SC-236, attenuated myofiber growth in both the soleus and plantaris muscle. This effect of SC-236 is consistent with that observed on the growth of newly formed myofibers during muscle regeneration (3), suggesting that COX-2 activity is important for myofiber growth in multiple physiological conditions. Attenuated myofiber growth in the soleus was associated with both decreased myonuclear addition and inflammation, whereas neither of these processes mediated the effects of SC-236 on plantaris growth, suggesting that regulation of muscle growth by the COX-2 pathway involves multiple mechanisms and may be muscle-type specific.

The adverse effects of SC-236 on soleus growth may be mediated, at least in part, by attenuated myonuclear addition (Fig. 4A). Because existing myonuclei are postmitotic, myonuclear addition depends on SC activation, proliferation, differentiation, and fusion. Thus attenuated myonuclear addition may reflect adverse effects of SC-236 on one or more of these processes. SC-236 may affect SC directly or indirectly. For example, SC-236 may affect SC indirectly by modulating inflammation, as supported by abundant evidence for functional interactions between muscle and inflammatory cells (4, 5, 7, 24, 29, 40, 41). Alternatively, the COX-2 pathway may have muscle cell-intrinsic functions, making SC directly susceptible to COX-2 inhibition. Our results provide the first evidence for the direct regulation of SC by the COX-2 pathway during the initial stages of muscle cell expansion. The decreased number of myoblasts associated with isolated COX-2^–/– myofibers after 48–72 h (Fig. 4, D and E) suggested SC activation and/or proliferation was impaired. Such a muscle cell-intrinsic role for COX-2 is consistent with other reports implicating PG in the direct regulation of myogenesis (8, 9, 18, 28, 36, 43, 44, 55, 56). Interestingly, the average number of myoblasts per myofiber increased by 7-fold for WT myofibers and 6-fold for COX-2^–/– myofibers from 48 to 72 h (data not shown), suggesting that COX-2 deficiency has minimal effects on myoblast proliferation. Similarly, we have observed that proliferation of COX-2^–/– myoblasts in bulk culture does not differ from WT (36). These results suggest that COX-2 activity is required during SC activation but not proliferation. Consistent with this possibility is that mitogenic activation of other cell types is associated with upregulation of COX-2 (20, 22, 37). However, our experiments do not rule out the possibility that COX-2 also regulates later stages of myogenesis in vivo because several reports (8, 9, 18, 28, 43, 55) have implicated PG in the regulation of differentiation and fusion. Furthermore, because plantaris growth does not require myonuclear addition (Ref. 32 and data not shown), attenuation of plantaris growth by SC-236 suggests that other mechanisms mediate COX-2-dependent functions in this muscle.

Inflammation in the soleus during reloading was decreased nearly 70% by SC-236 (Fig. 6D), suggesting that impaired inflammation may be one mechanism by which SC-236 attenuates soleus growth. Inflammation plays a critical role during various types of muscle growth (see Refs. 48 and 49 for reviews). In addition to their role in the phagocytosis of cellular debris, macrophages and neutrophils also secrete growth factors and chemoattractants that can modulate muscle protein synthesis, recruitment of additional inflammatory cells, as well as SC proliferation, differentiation, fusion, chemotaxis, and/or survival (4, 5, 7, 29, 41). Muscle regeneration in vivo is impaired by depletion of inflammatory cells (24, 40) and enhanced by factors that increase macrophage chemotaxis (24). Inflammation is not restricted to muscle regeneration but is also observed in the soleus during reloading after HS (49). Thus the interactions between inflammatory cells and muscle can influence the course of myofiber repair and growth. The recruitment of inflammatory cells is attributed to the release of complement factors and chemoattractants from damaged myofibers (48, 49). Whereas inflammation in muscle is often associated with the presence of myofiber necrosis (leading to the appearance of CNF), it can also be associated with myofiber membrane damage in the absence of necrosis (49), which has been observed after certain types of exercise (38). In our HS system, we observed an average of only 1–5% CNF in all samples, and the percentage of CNF did not increase significantly in muscles after reloading (Fig. 6A). Invasion of myofibers by inflammatory cells, which is indicative of necrosis (10), was not observed in any sample. Thus inflammation in the soleus may have been caused by myofiber membrane damage incurred during reloading in the absence of necrosis. In contrast to the soleus, however, inflammation did not occur in the plantaris muscle (Fig. 6E and data not shown) and therefore did not underlie the attenuation of plantaris growth by SC-236. Therefore, endogenous COX-2 expression in muscle in the absence of inflammation appears to be sufficient to modulate muscle growth.

The attenuation of myofiber growth, myonuclear addition, and/or inflammation by SC-236 in our system may be attributable in part to COX-2-independent mechanisms. Several studies have provided evidence for COX-independent actions of COX-2 inhibitors (see Ref. 46 for review). However, most COX-2-independent effects of COX-2 inhibitors have been observed using doses much higher than those required to inhibit PG synthesis. The decreased activation/proliferation observed in COX-2^–/– SC in vitro supports the conclusion that COX-2-dependent mechanisms regulate certain aspects of myofiber growth following atrophy.

The attenuation of plantaris myofiber growth by SC-236 was not due to effects on myonuclear addition or inflammation, suggesting the involvement of alternative mechanisms. Muscle atrophy is associated with myofibrillar protein degradation as well as decreased protein synthesis (see Ref. 25 for review). Recovery after atrophy involves the restoration of normal protein content via both increased protein synthesis and degradation of atrophy-associated proteins (25). Thus factors that regulate protein synthesis and/or degradation play a critical role in myofiber growth after atrophy. Several studies have implicated PG in the regulation of muscle protein synthesis and degradation both in vivo and in vitro. Ibuprofen, a nonselective inhibitor of both COX-1 and COX-2, decreased postexercise protein synthesis in humans (52) by decreasing PGF₂ levels (51). Whereas stretch-induced increases in myotube protein synthesis in vitro were associated with upregulation of both COX-2 and PGF₂ (53, 54), basal rates of protein turnover in myotubes were not influenced by PG (26). PGE₂ has been implicated in the regulation of muscle protein degradation but only in certain physiological conditions (2, 13, 15, 16, 42). Further examination of these pathways may elucidate the mechanisms behind SC-236-mediated attenuation of plantaris myofiber growth.

In summary, the COX-2 pathway regulates muscle growth in response to various physiological stimuli (3) and in phenotypically distinct muscles, implicating COX-2-derived PG as ubiquitous regulators of myofiber growth. Furthermore, our results suggest that administration of COX-2 inhibitors may be detrimental to muscle rehabilitation after atrophy. The importance of the COX-2 pathway in muscle growth warrants further research into the specific functions of COX-2-derived PG, which have not been defined. Of the five bioactive PG (TXB₂, PGE₂, PGF₂, PGD₂, and PGI₂), studies that have examined the functions of PGE₂ and PGF₂ in skeletal muscle are the most extensive. PGI₂ has been implicated in myofiber formation during development (27), but the potential function of this PG in adult muscle growth is unknown. PGD₂ is synthesized by necrotic muscle fibers in different types of muscular dystrophy (35), and its metabolite, 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂), has been implicated in the regulation of myoblast differentiation and MyoD expression in a muscle cell line (19). Classic PG signaling is mediated by specific G protein-coupled receptors, but signaling by PGI₂ and 15d-PGJ₂ can also be mediated by the PPAR family of receptors (12). Future studies with mice deficient in these receptors or in specific PG synthases will aid in the identification of the specific COX-2-derived PG that regulate myofiber growth, potentially leading to novel therapies for enhancing muscle growth and rehabilitation.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants AR-47314, AR-48884, and AR-051372 (to G. K. Pavlath). B. A. Bondesen was supported by American Heart Association predoctoral fellowship 0415084B.

	ACKNOWLEDGMENTS

 
We thank Pfizer Incorporated for the generous gift of SC-236.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. K. Pavlath, Emory Univ. School of Medicine, Dept. of Pharmacology, Rm. 5024, O. W. Rollins Research Bldg., Atlanta, GA 30322 (e-mail: gpavlat{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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