Effects of prostaglandins and COX-inhibiting drugs on skeletal muscle adaptations to exercise

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1 J Appl Physiol 115: , First published March 28, 2013; doi: /japplphysiol HIGHLIGHTED TOPIC Role of Inflammation in Skeletal Muscle, Connective Tissue, and Exertional Injuries: To Block or Not to Block? Effects of prostaglandins and COX-inhibiting drugs on skeletal muscle adaptations to exercise Todd A. Trappe and Sophia Z. Liu Human Performance Laboratory, Ball State University, Muncie, Indiana Submitted 16 January 2013; accepted in final form 18 March 2013 Trappe TA, Liu SZ. Effects of prostaglandins and COX-inhibiting drugs on skeletal muscle adaptations to exercise. J Appl Physiol 115: , First published March 28, 2013; doi: /japplphysiol It has been 40 yr since the discovery that PGs are produced by exercising skeletal muscle and since the discovery that inhibition of PG synthesis is the mechanism of action of what are now known as cyclooxygenase (COX)-inhibiting drugs. Since that time, it has been established that PGs are made during and after aerobic and resistance exercise and have a potent paracrine and autocrine effect on muscle metabolism. Consequently, it has also been determined that orally consumed doses of COX inhibitors can profoundly influence muscle PG synthesis, muscle protein metabolism, and numerous other cellular processes that regulate muscle adaptations to exercise loading. Although data from acute human exercise studies, as well as animal and cell-culture data, would predict that regular consumption of a COX inhibitor during exercise training would dampen the typical muscle adaptations, the chronic data do not support this conjecture. From the studies in young and older individuals, lasting from 1.5 to 4 mo, no interfering effects of COX inhibitors on muscle adaptations to resistance-exercise training have been noted. In fact, in older individuals, a substantial enhancement of muscle mass and strength has been observed. The collective findings of the PG/COX-pathway regulation of skeletal muscle responses and adaptations to exercise are compelling. Considering the discoveries in other areas of COX regulation of health and disease, there is certainly an interesting future of investigation in this re-emerging area, especially as it pertains to older individuals and the condition of sarcopenia, as well as exercise training and performance of individuals of all ages. PGE 2 ; PGF 2 ; acetaminophen; ibuprofen; sarcopenia PGS PRODUCED BY the cyclooxygenase (COX) enzyme are ubiquitous in human physiology and regulate numerous processes (105, 108), including muscle protein metabolism (78, 93, 106, 126, 128). As a result, PGs regulate adaptations to muscular exercise, and COX-inhibiting drugs can alter the acute and chronic responses to exercise. Because of the continuum of acute responses and chronic adaptations that exists from lowerintensity, longer-duration exercise to higher-intensity, shorterduration exercise, it is difficult to adopt a one-mechanismfits-all approach to PG and COX involvement in skeletal muscle exercise adaptations. This is also true when distinguishing between muscle injury and muscular exercise for health and performance. COX-inhibiting drugs are one of the most commonly consumed classes of drugs in the world. In the United States, the Address for reprint requests and other correspondence: T. Trappe, Human Performance Laboratory, Ball State Univ., Muncie, IN ( ttrappe@bsu.edu). COX inhibitors, acetaminophen, ibuprofen, and aspirin, are the top three consumed drugs of young, middle-aged, and older individuals, with 50 million individuals using each of these drugs during any given week (52, 129). Interestingly, two of these drugs (acetaminophen and aspirin) have been used for over 100 yr, originating in the 1800s (4). Understanding the role of PGs in skeletal muscle adaptation to activity is important because of the widespread use and potential influence of COX-inhibiting drugs, for better or worse, and so we can understand more completely the mechanisms that control muscle adaptation. The focus of this review will be on those studies that have used COX inhibition in human studies of skeletal muscle metabolism and adaptations to exercise. Distinction between studies using exercise paradigms that would be used for health benefits and those more focused on injury will be made when necessary and when appropriate animal and cell-culture studies will be discussed. A historical overview of the PG and COX pathway, with specific reference to skeletal muscle metabolism, will also be presented /13 Copyright 2013 the American Physiological Society 909

2 910 Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. PGs, THE COX PATHWAY, AND SKELETAL MUSCLE Historical context. PGs were discovered in the 1930s from extracts of the prostate gland and named by von Euler (136). Subsequent studies, over the next 40 yr, elucidated many of the tenets of PG structural biology and physiology, resulting in the Nobel Prize being awarded to Bergström, Samuelsson, and Vane in 1982 (10, 97, 135). There are numerous PGs (e.g., PGA-J) that are produced from arachidonic acid (53), which is liberated from the membrane of cells by PLA 2 (19, 32, 38) (Fig. 1). The enzyme PG G/H synthase, more commonly known as COX, is a dual-function enzyme that converts arachidonic acid to PGG 2 and then PGH 2 (105, 107), which is then rapidly converted to a specific PG (e.g., PGD 2, PGE 2, PGF 2, PGI 2 ) by PG synthases (70, 107, 137). Additionally, there are two well-known isoforms of COX COX-1 and COX-2 (44, 105, 107, 141) and a possible third isoform (an intron-retaining variant of COX-1, referred to as COX-1b or COX-3) may exist in some tissues (22, 86, 101, 138). PGs work in an autocrine and paracrine fashion through receptors specific to each PG, some of which have multiple isoforms (1, 17, 32, 62, 72, 112). PGs are relatively transient molecules with a half-life, typically, of only seconds to minutes (32). For example, 90% of PGE 2 and PGF 2 is removed from the blood in one pass through the pulmonary circulation (85). PGs are also potent in relatively small amounts (9, 32). As a result, circulating PG levels are typically low, and in response to stimulation, tissue production can be increased tremendously [e.g., 10 times the total resting tissue content can be generated every minute (135)]. The first studies of PG production and release by skeletal muscle in response to muscular work were published by Herbaczynska-Cedro and colleagues (41 43, 47) in 1974 and 1976 (Table 1). These studies were focused on identifying muscle-produced vasodilators and blood flow regulation during simulated exercise in dogs and showed that more than one PG was produced by exercising skeletal muscle (suggested as at least PGE 2 and PGF 2 ), which could be eliminated by the COX inhibitor indomethacin. These authors also speculated that the mechanism of the PG release was the distortion of the muscle cell membrane but showed that the PG release occurred during and after the exercise bouts. Soon thereafter, a study in humans using a static and dynamic exercise forearm model showed similar results (54). Subsequent arteriovenous studies by Nowak and Wennmalm (75) showed no net release or uptake of PGs across the leg at rest, but cycling exercise at 75% maximal oxygen consumption substantially increased net release of PGs from the leg. Whereas PGs were reported to be in resting human skeletal muscle as early as 1967 (51), Berlin et al. (11) showed in 1979, with radiolabeled arachidonic acid added to homogenates of skeletal muscle biopsy samples, that skeletal muscle could produce PGD 2, PGE 2, PGF 2, and PGI 2. PGE 2 was the predominant PG produced (11), but this could be shifted to PGF 2 if the assay environment was altered (74). Phospholipid Fig. 1. General schematic of the biosynthesis of the primary PGs in the PG/cyclooxygenase (COX) pathway and their associated receptors (53, 107, 108). The thicker arrows reflect conversions that are catalyzed by specific enzymes. Several other PGs are also made from the conversion (enzymatic or nonenzymatic) of those listed here: PGJ 2, 12 -PGJ 2, 15-deoxy- 12,14 -PGJ 2, and 11-epi-PGF 2 are derived from PGD 2; PGA 2, PGB 2, and PGC 2 are derived from PGE 2; 15-keto-PGF 2 is derived from PGF 2 ; 6-keto-PGF 1 and 6-keto-PGF 1 are derived from PGI 2. PGs are lipid molecules with a general chemical formula of C 20H 28 34O 3 6 and molecular weight range of Da. See text for specific references related to the numerous aliases that exist for the variants and isoforms of the enzymes and receptors and for further understanding of the nomenclature. Although not a PG, thromboxane A 2 is also derived from the enzymatic conversion of PGH 2. This review focuses on the 2 primary PGs that influence skeletal muscle protein turnover (PGF 2 and PGE 2), which are discussed in the text and presented in further detail (see Fig. 2). DP1 2, EP1 4, FP, and IP, PG receptors. PGD2 PGE2 Arachidonic Acid PGG2 PGH2 COX Enzyme PGF2α PGI2 Receptors DP1-2 EP1-4 FP IP

3 Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. Table 1. Noteworthy studies of PGs, skeletal muscle, and exercise adaptations 911 Author Year Species Key Findings Early studies Karim et al. (51) 1967 Humans PGs are located in skeletal muscle. Herbaczynska-Cedro and colleagues (41 43, 47) 1974, 1976 Dogs Skeletal muscle PG release increased during and after simulated exercise; eliminated with COX inhibitor. Kilbom and Wennmalm (54) 1976 Humans Forearm skeletal muscle PG release increased during and after static and dynamic exercise; reduced with COX inhibitor (indomethacin). Nowak and Wennmalm (75) 1978 Humans Leg skeletal muscle PG release increased during cycling exercise at 75% maximal oxygen consumption. Berlin et al. (11) 1979 Humans Skeletal muscle had the enzymatic capacity to produce PGD 2, PGE 2, PGF 2a, and PGI 2 from arachidonic acid. Young et al. (142) 1981 Monkeys Aging increased skeletal muscle PG production. Rodemann and Goldberg (93) 1982 Rats Arachidonic acid increased skeletal muscle PGF 2a and PGE 2, which increased muscle protein synthesis and degradation, respectively; reduced or eliminated with COX inhibitors. Palmer et al. (78) 1983 Rabbits Simulated exercise (intermittent stretching) increased skeletal muscle PGF 2a and protein Smith et al. (106) synthesis; reduced or eliminated with COX inhibitors. PGF 2a and protein synthesis levels correlated. Gibson et al. (33) 1991 Humans Reduced skeletal muscle PGF 2a levels associated with reduced muscle protein synthesis and type I and II muscle fiber size. Contemporary studies Trappe et al. (126, 128) 2001, 2002 Humans Increased skeletal muscle PGF 2a, PGE 2, and protein synthesis after resistance exercise; eliminated with over-the-counter, orally consumed COX inhibitors (acetaminophen and ibuprofen). Karamouzis et al. (49, 50) 2001 Humans Aerobic exercise increased intramuscular PGE 2; increased production with increased workload. Boushel et al. (15) 2002 Humans Confirmed findings of Karamouzis et al. (49, 50) and showed that an oral dose of COX inhibitor (indomethacin) reduced intramuscular PGE 2 during aerobic exercise by 90%. Höffner et al. (45) 2003 Humans An oral dose of COX inhibitor (aspirin) reduced intramuscular PGE 2 production at rest by nearly 90% within 1 h. Trappe et al. (123) 2006 Humans Both young and old individuals increased intramuscular PGF 2a in the hours after resistance exercise; no apparent effect of age on resting or postexercise levels. Mikkelsen et al. (67) 2008 Humans Local intramuscular low-dose COX inhibitor (indomethacin) delivery blocked PGE 2 production by 85% during and after resistance exercise. Burd et al. (18) 2010 Humans COX-2-specific inhibitor (celecoxib) did not eliminate or reduce the increase in skeletal muscle protein synthesis after resistance exercise. Paulsen et al. (79) 2010 Humans COX-2-specific inhibitor (celecoxib) did not influence intramuscular PGE 2 levels or satellite cell activity after resistance exercise. Petersen et al. (82) 2011 Humans COX inhibitor (ibuprofen) did not influence skeletal muscle protein synthesis 24 h after aerobic exercise in older osteoarthritic patients. Kreiner and Galbo (55) 2011 Humans Resting intramuscular PGE 2 levels 20 times higher than plasma. Standley et al. (110) 2013 Humans PGE 2 stimulated transcription of the skeletal muscle mass regulators IL-6 and muscle RING finger-1. COX inhibitor effects on chronic exercise adaptations Krentz et al. (56) 2008 Humans Duration: 6 wk; Training: resistance exercise 2 3 days/wk; Drug dose: ibuprofen 400 mg, 2 3 days/wk (training days); Participants: 24 yr, men and women, resistance exercise trained ( 6 yr); no effect on muscle mass or strength adaptations compared with placebo-consuming resistance exercise group Petersen et al. (81) 2011 Humans Duration: 12wk; Training: resistance exercise 3 days/wk; Drug dose: ibuprofen 1,200 mg/day; Participants: yr, men and women, knee osteoarthritis patients; no effect on muscle mass, increased muscle strength compared with placebo-consuming resistance exercise group Trappe et al. (125, 127) 2011, 2013 Humans Duration: 12wk; Training: resistance exercise 3 days/wk; Drug dose: acetaminophen 4 g/day or ibuprofen 1,200 mg/day; Participants: yr, men and women, healthy, untrained; enhanced muscle mass and strength gains 25 50% above placebo-consuming resistance exercise group Jankowski et al. (48) 2012 Humans Duration: 16wk;Training: resistance exercise 3 days/wk; Drug dose: acetaminophen 1 g, 3 days/wk (training days); Participants: 64 yr, men, healthy, untrained; no effect on fat-free mass or muscle-strength adaptations compared with placebo-consuming resistance exercise group COX, cyclooxygenase. The initial reports of PG regulation of skeletal muscle protein turnover were in the early 1980s when Rodemann and Goldberg (93) showed that arachidonic acid supplementation to rat muscle in vitro increased muscle protein synthesis and degradation, which could be replicated with PGF 2 and PGE 2 supplementation, respectively, and reduced or eliminated with COX inhibition. Arachidonic acid supplementation increased muscle production of both PGs, but about twice as much PGE 2 was synthesized compared with PGF 2. At the same time, Palmer and colleagues (78, 106) showed that with simulated exercise in rabbit muscle, PGF 2 was produced by intermittent muscle stretch ( 105%), which in turn, stimulated muscle protein synthesis ( 70%), both of which were reduced or eliminated by COX inhibition. In addition, the increase in

4 912 Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. muscle PGF 2 production and protein synthesis was sustained following the cessation of simulated exercise. Over the next yr following these seminal studies, many investigations in animal and cell-culture models built on these findings (5, 7, 34, 35, 39, 40, 64, 65, 77, 90, 94, 113, ). Although the animal- and cell-culture data were compelling with regard to PG and COX-inhibitor regulation of skeletal muscle protein turnover, very few studies in humans were completed during this time, and none focused on exercise. Gibson et al. (33) showed that women (mean age of 58 yr) with rheumatoid arthritis receiving steroid treatment (which lowers PG production via COX-independent PLA 2 inhibition; mean treatment time: 8 yr) had reduced muscle PGF 2 levels, muscle protein-synthesis rates, and type I and II muscle fiber size compared with a group of controls (mean age of 70 yr). In addition, the rheumatoid arthritis patients had elevated intramuscular PGE 2, resulting in 12 times more PGE 2 than PGF 2 compared with approximately three times in the controls. McNurlan et al. (65) showed that a single dose of the COX inhibitor indomethacin was unable to influence whole-body protein synthesis in the fed and fasted states, but given that muscle protein turnover constitutes 30% of whole-body turnover (71), it is unclear if there was an influence on muscle protein turnover (as they had shown in rats). Contemporary information. What is known about PG and COX-inhibiting drug regulation of muscle protein turnover with exercise in humans has been obtained since 2000 (Table 1). Several studies using animal models of simulated exercise and muscle growth have also been completed during this time period and have provided additional insight. Also during this time frame, the amount of basic information about the PG/ COX pathway has increased substantially, including characterization of the structure and enzymology of the PG synthases (107), leading the way for new drug development (8, 96). Much more has also been learned from continued investigation of the COX-specific inhibitors engineered in the 1990s and of many of the classic COX inhibitors in other areas of health and disease. Studies completed during the 1990s using isotopically labeled amino acids showed that muscle protein synthesis increased after resistance exercise for up to 48 h in humans (23, 58, 84), and the accumulation of these acute increases in muscle protein synthesis was generally understood to be, at least in part, the underlying basis of muscle hypertrophy. Perturbations (e.g., pharmaceutical, nutrition, a specific exercise regimen) that altered this response would alter the benefits of strength training. With the consideration of the aforementioned studies showing that PGF 2 was a regulator of muscle protein synthesis, it was realized that orally consumed COX inhibitors might interfere with the normal muscle proteinsynthesis response to resistance exercise. This interfering effect might be particularly important after damaging exercise that caused muscle soreness, which is, at least in part, a result of increases in intramuscular PGE 2 (3, 55, 66) and would further increase the likelihood of COX-inhibitor consumption. Indeed, this was shown to be the case when individuals consumed an over-the-counter dose of the COX inhibitor acetaminophen (4 g/day) or ibuprofen (1.2 g/day) in the 24 h following a single bout of excessive resistance exercise [10 14 sets of 10 eccentric (lowering) contractions; thus the work equivalent of 70 typical repetitions] that caused muscle soreness (126, 128). Muscle protein synthesis ( 76%) and intramuscular PGF 2 ( 77%) were increased significantly, 24 h after the exercise bout, and these increases were eliminated with the consumption of the COX-inhibiting drugs. Intramuscular PGE 2 was also increased ( 64%), 24 h after the exercise, and this increase was also eliminated with COX inhibition. This study presented some interesting initial findings regarding PG and COX regulation of skeletal muscle responses to exercise and raised several interesting questions. First, the continued, increased production of intramuscular PGF 2 and PGE 2, 24 h after a single bout of resistance exercise, considering the short half-life of PGs, highlighted that it would be important to understand the typical PG levels in human skeletal muscle and how they are affected by exercise (i.e., time course, magnitude, type of exercise). Second, considering the pharmacokinetic and metabolic complexities of orally consumed COX inhibitors, it was interesting that doses of the most commonly consumed COX inhibitors could apparently produce intramuscular drug levels that inhibited intramuscular COX to the extent that PGF 2 and PGE 2 production and muscle protein metabolism after exercise were inhibited. Given the large number of COX-inhibiting drugs available for human consumption, it followed that knowledge regarding the efficacy of these drugs and doses in relation to the COX isoforms found in skeletal muscle at rest and after exercise would be necessary. Third, the obvious question from this investigation was: what are the impacts of chronic COX-inhibitor consumption on exercisetraining adaptations? Whereas there is much yet to be discovered in this area, several studies have added to our understanding and started to address some of the key questions. Intramuscular PGs and exercise. Muscle biopsy-derived measures of PGs that regulate protein turnover show PGE 2 to be the dominant PG in young and old human muscle (11, 33, 51, 74, 126). Intramuscular PGE 2 levels are three to four times higher than PGF 2 at rest and after resistance exercise in young individuals, suggesting equivalent, relative increases in both PGs after exercise (126). This same ratio of PGE 2 to PGF 2 appears to hold in resting skeletal muscle of older individuals as well (33). However, studies in skeletal muscle of rhesus monkeys show that PG production is approximately twofold higher in older muscle compared with young (142). Several studies in humans using the microdialysis technique to sample the muscle interstitial fluid provide additional information on intramuscular levels of the PGs (primarily PGE 2 ). Few studies report both intramuscular and plasma levels of PGs, but it has been shown that resting vastus lateralis and trapezius intramuscular PGE 2 levels are 20 times higher than plasma levels (55), albeit in older individuals. However, the intramuscular levels reported in that study are similar to those reported in middle-aged and younger individuals (29, 49, 50). In addition, low-level muscular work does not increase PGE 2 levels (30, 50), whereas resistance and aerobic exercise stimulate the muscle to produce PGE 2 and/or PGF 2 during and/or after exercise (15, 49, 50, 67, 123, 126). Furthermore, increasing the workload during aerobic exercise increases the muscle production of PGE 2 (15, 49). Specifically, going from rest to cycling exercise at 100 W and 150 W increased intramuscular PGE 2 levels by 400% and 600%, respectively (49). No information is available on the possible workload effects on PG production following resistance exercise; however, there does

5 Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. not appear to be an age effect on PGF 2 production during the 24 h following resistance exercise (123). COX inhibitors and skeletal muscle COX. There are somewhat limited data on PG synthesis and muscle protein metabolism at rest or after exercise in response to oral consumption of over-the-counter COX inhibitors (82, 126, 128). Mikkelsen et al. (67) have shown that an intramuscular infusion of a relatively low dose of the prescription COX inhibitor indomethacin reduced PGE 2 levels in the muscle during and after resistance exercise by 85%. This same COX-inhibitor delivery approach during and for a few hours after resistance exercise did not influence muscle protein synthesis measured near the infusion site 24 h later (69). Furthermore, numerous studies of PG regulation of skeletal muscle blood flow at rest and in response to exercise have used over-the-counter- and prescription-strength COX inhibitors (15, 45, 100), and these studies provide some additional insight. For example, Höffner et al. (45) showed that a single oral dose of 1,000 mg acetylsalicylic acid (aspirin), an irreversible inhibitor of COX, inhibits resting skeletal muscle PGE 2 production by nearly 90% within 1 h. In addition, a single oral dose (100 mg) of indomethacin given to individuals, 16 h before exercise, eliminated 90% of the intramuscular PGE 2 production during aerobic exercise (15). Clearly, doses typical for human consumption can impact intramuscular PG production, but more data are needed in this area and in the context of muscle protein turnover. The efficacy of these over-the-counter and prescription COX inhibitors should also be considered in the context of the COX enzymes found in skeletal muscle (Table 2). That is, COX-inhibiting drugs are commonly classified based on their specificity toward the two main isoforms of COX COX-1 and COX-2 (24, 105). The aforementioned drugs that have been shown to reduce intramuscular PG production in humans (acetaminophen, ibuprofen, indomethacin, aspirin) are all generally considered to be nonspecific COX inhibitors (i.e., they block both COX-1 and COX-2 to some degree). Confusion can arise, however, if this classification is considered to hold across all tissues and physiological conditions. That is, different tissues under different stresses express different levels of the COX isoforms and have different cellular environments that appear to affect drug efficacy. In healthy individuals at rest and following exercise, human skeletal muscle expresses COX-1 almost exclusively at the transcript and protein level (18, 125, ). Interestingly, there is a relatively ignored variant of the COX-1 isoform that is the most abundant transcript in human skeletal muscle, and it is responsive to exercise (18, 125, 138). COX-2 transcript levels in healthy human skeletal muscle at rest or after exercise are very low, and similarly, the amount of detectable, enzymatically active COX-2 protein is questionable (18, 116, 125, 138). However, intramuscular COX-2 transcript levels do increase in response to COX-2-specific and nonspecific COX inhibitors (18, 69). The COX-3 (i.e., COX-1b) isoform in human skeletal muscle has been ruled out as a contributor to PG production (18, 125, 138). Animal models of muscle adaptation (13, 14, 27, 73, , 109) suggest that COX-2 plays a substantial role in PG production in skeletal muscle, but these models appear to be more reflective of muscle injury and not necessarily human exercise (18, 125). Overall, the COX-2 isoform in skeletal muscle appears to be more responsive to injury-related stimuli, which is consistent with the large induction of skeletal muscle COX-2 protein levels in humans with septic myopathy (87). Further support for this notion comes from two separate studies showing that a COX-2-specific inhibitor designed for human consumption did not influence the skeletal muscle responses to a single bout of eccentric resistance exercise (18, 79). Burd et al. (18) showed that the COX-2 inhibitor was unable to block the normal increase in muscle protein synthesis following exercise, as was shown previously with two different, nonspecific COX inhibitors (128). Similarly, Paulsen et al. (79) were unable to show an effect of the COX-2 inhibitor on intramuscular PGE 2 levels, satellite cell activity, or autologous-radiolabeled leukocyte accumulation. Chronic effects of COX inhibitors on exercise adaptations. Four recent studies (48, 56, 81, 125) provide the initial evidence as to whether chronic consumption of commonly consumed COX inhibitors negatively impacts chronic exercise adaptations (Table 1). Krentz et al. (56) showed in young men and women that 400 mg ibuprofen taken after six sets of biceps curls [three sets of eight to 10 concentric repetitions at 70% one repetition maximum (1RM) and three sets of four to six eccentric repetitions at 100% 1RM], 2 3 days/wk for 6 wk, did not influence muscle growth or strength adaptations. The discrepancy between the acute suppression of muscle protein synthesis in younger individuals by ibuprofen (128) and these chronic findings is not completely clear. The lower dose of ibuprofen (1,200 mg/day Table 2. COX enzymes in healthy human skeletal muscle in relation to exercise and COX-inhibiting drugs Isoform Variant(s) Comments COX-1 Variant 1 ( 1v1) Relatively abundant at the transcript and protein level at rest and after acute and chronic exercise. The protein product commonly believed to interact with nonspecific COX-inhibiting drugs. Acute exercise increases transcript levels; chronic exercise training increases transcript and protein levels. COX-1 Variant 2 ( 1v2) A truncated transcript of COX-1v1 (missing 111 bases from exon 9) that may not generate a functional protein product. Most abundant COX transcript but specific role in skeletal muscle unknown. Acute exercise and chronic exercise training increase transcript levels. COX-1 Variant b ( 1b) Also known as COX-3. An intron-1-retaining version of COX-1 with 3 splice variants: 1b 1, 1b 2, 1b 3. Apparently sensitive to common COX-inhibiting drugs in other tissues. Nondetectable or very low transcript levels at rest and nonresponsive to acute and chronic exercise. Unlikely involved in exercise adaptations or related COXinhibitor effects. COX-2 Very low or nondetectable transcript and enzymatically active protein levels at rest and after acute and chronic exercise. Although low, transcript levels increase with ingestion or infusion of COX-2-specific or nonspecific COX inhibitors after acute exercise, as well as with chronic exercise training. See text and related studies (18, 69, 105, 116, 125, 138) for further discussion of skeletal muscle COX.

6 914 Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. vs ,200 mg/wk); the somewhat short duration of training, limiting the time for the COX inhibitor to have an effect; and the possible influence on muscle protein breakdown are plausible explanations. Petersen et al. (81) recently showed that older patients with osteoarthritis (mean age 62 yr), taking ibuprofen (1,200 mg/day) and completing 12 wk of progressive resistance training, 3 days/wk (four to five sets of eight to 15 repetitions at 70 80% of 1RM), had no effect on muscle mass gains, but muscle strength was enhanced in those individuals consuming the COX inhibitor. The authors suggest that this effect on muscle function was related to the pain relief obtained from the drug consumption. These findings are corroborated by this same group s data showing that ibuprofen (1,200 mg/day) does not influence the muscle protein-synthesis response to exercise in older osteoarthritis patients (mean age 62 yr) (82). Interestingly, they also reported that 12 wk of training and taking ibuprofen did inhibit the increase in muscle satellite cell number induced with training in the placebo group (81). These results are in accordance with reports of COX-inhibiting drugs interfering with satellite cell activity after exercise, which is apparently mediated through COX-1 (61, 68, 79). These findings raise the question of whether satellite cells are necessary for muscle hypertrophy, at least the amount that is generally elicited with exercise-training paradigms used for health and wellness of older individuals and the treatment of sarcopenia. This general question has been debated recently (63, 76), and the answer is apparently not yet at hand (46, 60, 83). With the use of doses of acetaminophen (4 g/day) or ibuprofen (1.2 g/day) in healthy, older men and women (60 78 yr) completing resistance-exercise training 3 days/wk (three sets of 10 repetitions at 75% of 1RM/day) for 12 wk, Trappe et al. (125) unexpectedly showed an enhancement of muscle mass and strength gains of 25 50% over a placebo-consuming group. Follow-up studies on muscle biopsies obtained from these individuals (125, 127) and subsequent ex vivo studies (110) provide some mechanistic clarity about these unexpected COX-inhibitor effects (Fig. 2). It appears the COX inhibitors reduced the PGE 2 production (126) and resultant stimulation of intramuscular IL-6 and muscle RING finger protein-1 (MuRF-1) production (57, 88, 111), which increased net protein balance in response to each exercise bout throughout the exercise program. This hypothesis is based on the data that show that low-level increases in IL-6 acutely inhibit muscle protein turnover (131), chronically promote muscle atrophy (12, 37), and are associated with a reduction in muscle mass and functional independence in older individuals (6, 25, 28, 99), as well as the proteolytic nature of the ubiquitin ligase MuRF-1 (20, 98). The COX-inhibitor consumption also promoted an upregulation of the PGF 2 receptor in the muscle of the drug groups (127). This increase coupled with a general training increase in COX-1 and the PGF 2 -producing enzymes (PGF 2 synthase and PGE 2 -to-pgf 2 reductase) (125, 127) would make the muscle less susceptible to the same, daily COX-inhibiting drug doses and more sensitive to any PGF 2 that was produced following exercise. Whether these responses and muscle adaptations are specific to older individuals and any potential basal inflammatory state or exaggerated response following exercise (21, 26, 36, 80, 89, 117, 118, 124) is unclear and needs further investigation. Interestingly, COX-inhibitor consumption did not promote muscle growth in the nonexercising hamstring muscles, suggesting an exercise-loading and/or stretch-related mechanism. This nonexercise finding is somewhat in conflict with the data from Rieu et al. (92), showing that chronic consumption of ibuprofen limits sarcopenia through restoration of the muscle protein-synthesis response to feeding in older rats. The discrepancy between these studies is possibly due to the longer-term dosing of the animals (20% vs. 0.4% of the lifespan) and the higher dose of the drug (30 vs. 14 mg kg body wt 1 day 1 ). However, these collective findings have implications, not only for use of COX inhibitors during resistance-exercise training for the treatment of sarcopenia but also for the potential chronic use of low-level, longterm, exercise-independent consumption of COX inhibitors for Fig. 2. Schematic of the portion of the PG/ COX pathway involved in the regulation of skeletal muscle protein metabolism and adaptation. See text for specific references related to the enzymes, intermediates, and receptors of the COX pathway, as well as the studies that have delineated the factors and cellular processes regulated by the PGE 2 and PGF 2 receptors that influence skeletal muscle mass. See Trappe et al. (127) for nomenclature related to the PG synthases, reductase, and receptors. MuRF-1, muscle RING finger protein-1; PI3K/ERK/mTOR, phosphoinositide 3-kinase/ERK/mammalian target of rapamycin (62). IL-6 Protein synthesis PGE2 receptor MuRF-1 Protein degradation Muscle Atrophy PGE2 synthase Phospholipid PLA2 Arachidonic Acid COX PGH2 COX Inhibitors PGF2α synthase PGF2α receptor PI3K/ERK/mTOR growth signaling Protein synthesis Muscle Growth PGE2 PGE2 reductase PGF2α

7 the treatment of sarcopenia, as is promoted or contemplated for several other conditions, such as cardiovascular disease, dementia, and certain types of cancer (91, 95, 105, 130). It is interesting to note that acetaminophen is not commonly considered an nonsteroidal, anti-inflammatory drug because of its relative lack of COX-inhibitory or anti-inflammatory effect in many peripheral tissues, yet it is a potent analgesic, fever reducer, and COX inhibitor within the central nervous system (22, 31). Nonetheless, acetaminophen clearly inhibits PG synthesis in human skeletal muscle (126). Chronic acetaminophen consumption in animals has also been shown to influence skeletal muscle fiber size and glucose metabolism (139, 140). The basis for this tissue specificity is not clear but may be related to the cellular environment (16, 138). Finally, Jankowski et al. (48) showed recently that 16 wk of progressive resistance-exercise training, 3 5 days/wk (three sets of five to 12 repetitions at 60 80% 1RM coupled with stair-climbing and jumping exercises), combined with the COX-inhibitor acetaminophen (1,000 mg only on days of exercise, average of 3 days/wk) had no effect on fat-free mass or muscle-strength gains in older men (mean age 64 yr). The large difference in acetaminophen dosing (28 g/wk vs. 3 g/wk) likely explains the different responses in this study and those reported by Trappe et al. (125). Collectively, these chronic studies, albeit of a limited number, highlight three main points: 1) chronic consumption of commonly consumed COX inhibitors at over-the-counter doses during exercise training does not appear to interfere with the muscle mass and strength gains expected from typical resistance-exercise-training regimens; 2) there appears to be a threshold of the amount of drug that is needed to influence skeletal muscle metabolism and adaptation; and 3) there may be differences between the acute and chronic COX-inhibitor effects on muscle metabolism between younger and older individuals. It should also be noted that the animal studies in this area clearly show an interfering effect of COX inhibitors on muscle growth and adaptation (14, 59, 73, 109). The discrepancy between these studies and the aforementioned human exercisetraining and COX-inhibitor studies is most likely due to the animal studies not necessarily reflecting typical human exercise stimuli, as eluded to previously (18). The consideration of the stress placed on the muscle in the different models is also important in understanding the potential role of COX isoforms, as well as PG and inflammatory regulation of muscle adaptation. For example, typical and highly effective resistanceexercise programs in humans only need to load the muscle for a few minutes every 2 3 days (2, 115, , 125), resulting in 10 min of muscle loading/wk. Whole muscle-growth rates typical for these types of training paradigms are 0.5%/wk, and the highest reported muscle-growth rate reported in the literature is 1%/wk (114). These rates are in contrast to the animal models of hypertrophy some of which have almost constant loading and elicit edema that result in muscle growth of 25 40%/wk. Considering the potential use of the animal studies and particularly, the PG/COX-pathway genetically modified animals, development of appropriate animal exercise models could facilitate significant advancements in this area. Prostaglandins, COX Inhibitors, and Muscular Exercise Trappe TA et al. CONCLUDING REMARKS The research field of PG and COX-inhibitor regulation of health and disease has grown enormously over the last 80 yr of existence. That skeletal muscle responses and adaptations are regulated by PGs synthesized by the muscle that produced them is now firmly established. It is also clear that one of the most commonly consumed classes of drugs in the world COX inhibitors can alter several cellular processes that regulate skeletal muscle responses to acute exercise loading and chronic exercise training. There is much research yet to be done in this complex area to better understand the role of PGs and COX inhibitors, specifically as they pertain to older individuals and the condition of sarcopenia, as well as exercise training and performance of individuals of all ages. The PG/ COX-pathway research being conducted in other areas of health and disease will no doubt continue to add significantly to our understanding of this research area in skeletal muscle. ACKNOWLEDGMENTS The authors thank all who contributed to the PG and COX-related research associated with the authors. GRANTS Support for this research was provided by the National Institute on Aging (AG and AG-00831). DISCLOSURES The authors declare no conflicts of interest. 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