Point: Counterpoint: Estrogen and sex do / do not influence. post-exercise indices of muscle damage, inflammation and repair.

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1 Articles in PresS. J Appl Physiol (July 3, 2008). doi: /japplphysiol Point: Counterpoint: Estrogen and sex do / do not influence post-exercise indices of muscle damage, inflammation and repair. Point: Estrogen and sex do influence post-exercise indices of muscle damage, inflammation and repair Authors: Peter M. Tiidus & Deborah L. Enns Counterpoint: Estrogen and sex do not influence post-exercise indices of muscle damage, inflammation and repair Authors: Monica J. Hubal & Priscilla M. Clarkson 1 Copyright 2008 by the American Physiological Society.

2 Point: Estrogen and sex do influence post-exercise indices of muscle damage, inflammation and repair Peter M. Tiidus & Deborah L. Enns Department of Kinesiology & Physical Education Wilfrid Laurier University Waterloo ON Canada Correspondence: Peter M. Tiidus, Ph.D. Professor & Chair, Department of Kinesiology & Physical Education, Wilfrid Laurier University Waterloo ON Canada, N2L 3C ext (telephone), (fax) 2

3 Estrogen and sex influences due to difference in estrogen exposure have been consistently reported to attenuate damage and/or inflammation and to accentuate repair in a variety of tissues and organs (4, 13, 22). Consistent with these findings, rodent models have provided compelling evidence of the efficacy of estrogen in decreasing indices of post-exercise damage and inflammation in skeletal muscle (10, 29). In addition, we have recently demonstrated tantalizing indications of the potential for estrogen to accentuate factors involved in muscle repair (5, 6, 28). Although less consistent than the findings in rodents, human studies focusing on sex differences have also provided some evidence for positive estrogenic influences of skeletal muscle damage and repair (18, 23). This article will present an overview of the evidence for estrogen to: i. Ameliorate indices of postexercise muscle damage, ii. Diminish post-exercise muscle inflammatory responses and iii. Accentuate factors related to muscle repair, as well as the possible mechanisms by which estrogen may act. Estrogen diminishes indices of post-exercise muscle damage: Several lines of evidence, primarily from animal studies indicate that exposure to estrogen will diminish indices of muscle damage. In one of the few studies to directly examine histological evidence of muscle damage, Komulainen et al (3) reported that for up to 96 hours postdownhill running male rats exhibited earlier and greater indices of damage to muscle structural proteins and more muscle fiber swelling than female rats. They also noted significantly elevated levels of $-glucuronidase (ß-glu) activity in the muscles of male relative to female rats. $-glu is a lysosomal enzyme activated in response to muscle disruption and considered to be a quantitative indicator of muscle damage (17). Our laboratory has also reported that ovariectomized female rats with estrogen replacement 3

4 have significantly diminished elevations in muscle $-glu activities following downhill running relative to sham supplemented animals (5,6). Since skeletal muscle possesses both "- and $- estrogen receptors (9) we tested the hypothesis that estrogen influence on post-exercise muscle damage may manifest through receptor mediated mechanisms. Using the pure estrogen receptor blocker ICI 182,780 we have recently demonstrated that the ability of estrogen to attenuate exercise induced muscle damage, as quantified by reduced muscle $-glu activation is not receptor mediated and may instead be related to other characteristics of estrogen as discussed below (6). Although not a specific indicator of structural muscle damage, serum creatine kinase (CK) levels have been used as reliable semi-quantitative indicators specifically of sarcolemma membrane disruption (7). As noted by Clarkson and Sayers (3) pioneering studies by Amelink and colleagues in the 1980s conclusively established that estrogen will attenuate post-damage muscle CK release in male and ovariectomized female rats. The most compelling of these studies avoided possible in vivo confounds by demonstrating that isolated muscle from exercised male rats with prior estrogen exposure released CK into the surrounding medium significantly slower than untreated male rats (1). Studies examining gender differences in humans have also reported higher postexercise serum CK levels in males relative to females as well as a greater preponderance of high post-exercise CK responders in males (18, 23). Estrogen attenuates inflammation related muscle leukocyte infiltration and damage. The potential for estrogen to stabilize muscle membranes and diminish sarcolemma disruption may be a mechanism by which estrogen acts to reduce exercise induced muscle damage and inflammatory responses (27). We have proposed that 4

5 estrogen may act both as an antioxidant against exercise induced membrane phospholipid peroxidation and as a cholesterol like influence on membrane fluidity and stability. (26, 27) Findings from our laboratory have consistently demonstrated an estrogen-induced attenuation of post-damage inflammation related neutrophil infiltration of red and white skeletal muscles in male as well as ovariectomized female rats (6, 16, 29). We have suggested that the potential of estrogen to preserve sarcolemma stability following damaging exercise may help maintain muscle calcium homeostasis (27) which may, as we have demonstrated, result in attenuation of post-exercise muscle caplain activation (29). Both reductions in muscle sarcolemma disruption and attenuation of calpain activation could contribute to the ability of estrogen to attenuate post-exercise neutrophil infiltration in both red and white skeletal muscle of rats (27). In further support of this theory, we have recently demonstrated, with the use of an estrogen receptor blocker that the ability of estrogen to diminish post-exercise muscle neutrophil infiltration is not estrogen receptor-mediated and have suggested that this ability is instead mediated via direct estrogen action on muscle membranes (6). Neutrophils, while important in initiating tissue repair, are also responsible for significant post-exercise inflammation related muscle structural and oxidative damage (14). The attenuation of post-exercise neutrophil infiltration into skeletal muscle by estrogen may thus also be a factor in reducing post-exercise muscle damage progression. Indeed, several studies have reported reduced indices of oxidative damage in muscles of estrogen supplemented versus unsupplemented ovariectomized female rats following exercise or ischemia-induced disruption (13, 24). 5

6 Estrogen can enhance potential for skeletal muscle repair. Estrogen can also attenuate post-exercise macrophage infiltration of skeletal muscle (6, 11). Macrophage infiltration of skeletal muscle as well as muscle damage itself are both important for initiating post-damage muscle regeneration and satellite cell activation (25, 30). Hence it is possible that by delaying or diminishing post-exercise macrophage infiltration or reducing damage in skeletal muscle, estrogen may diminish muscle repair-related responses such as satellite cell activation (5, 6). Despite this possibility we have recently shown that estrogen supplementation will actually enhance activation and proliferation of satellite cells in muscles following downhill running in male and ovariectomized female rats (5, 6, 28). Although the mechanisms by which estrogen can enhance post-damage muscle satellite cell responses are not yet understood, we have demonstrated that by blocking muscle estrogen receptors we can completely block post-exercise muscle satellite cell activation and proliferation (Figure 1) (6). We speculated (5, 6) that the estrogen receptor- mediated phosphatidylinositol 3-kinase/protein kinase B (Akt) pathway which stimulates muscle growth and protein synthesis (20) or the estrogen-receptor induced induction of myoblast c-fos and egr-1 genes (8) may be involved in these effects. Alternatively, estrogen has also been shown to upregulate nitric oxide synthase (NOS) activity and nitric oxide (NO) levels in epithelial cells (4) and to influence epithelial inflammation via nitric oxide (NO) mediated mechanisms (15). Since NO is also a critical regulator of muscle satellite cell activation and proliferation (2), it is possible that estrogen may also act via muscle estrogen receptors to alter muscle NOS activity and NO levels and hence influence satellite cell responses to muscle damage (5, 6). 6

7 Application to human muscle damage and repair: The influence of estrogen on muscle damage, inflammation and repair may have its greatest relevance with postmenopausal females, as older females tend to suffer greater strength declines, impairments in muscle repair and rates of sarcopenia than older males (19, 21). Fortunately, studies have found that strength training significantly increases satellite cell numbers in skeletal muscles of all individuals, and especially those of older females (16). Furthermore, estrogen has been shown to enhance muscle recovery following periods of disuse atrophy (12). Thus, research examining countermeasures to protect skeletal muscle from the loss of estrogen-related protection in older females would be particularly relevant to preserving their health, strength and functional abilities. 7

8 References 1. Amelink GJ, Koot GJ, Erich WBM, Van Gijn J, Bar PR. Sex-linked variation in creatine kinase release and its dependence on oestrodiol can be demonstrated in an invitro rat skeletal muscle preparation. Acta Physiol Scand 138: , Anderson JE and Wozniak AC. Satellite cell activation on fibers: Modeling events in vivo. Can J Physiol Pharmacol 82: , Clarkson PM, Sayers SP. Gender differences in exercise-induced muscle damage. In Tarnopolsky M. ed. Gender differences in metabolism, Boca Raton, CRC Press 1999, pp Caulkin-Glaser T, Garcia-Cardena G, Sarrel P, Sessa WC, Bender JR. 17 Betaestradiol regulation of human endothelial cell basal nitric oxide release, independent of cytosolic Ca 2+ mobilization. Circ Res 81: , Enns DL, Tiidus PM. Estrogen influences satellite cell activation and proliferation following downhill running in rats. J Appl Physiol 104: , Enns DL, Iqbal S, Tiidus PM. Oestrogen receptors mediate oestrogen-induced increases in post-exercise rat skeletal muscle satellite cells. Acta Physiologica 193:, 2008 (pre-print published online April 29, 2008 doi: /j x) 7. Hortobagyi T, Denahan T. Variability in creatine kinase: methodological, exercise and clinically related factors. Int J Sports Med 10: 69-78, Kahlert S, Grohe C, Karas RH, Lobbert K, Neyses L, Vetter H. Effects of estrogen on skeletal myoblast growth. Biochem Biophys Res Commun 232: ,

9 9. Kalbe C, Mau M, Wollenhaupt K, Rehfeldt C. Evidence for estrogen receptor alpha and beta expression in skeletal muscle of pigs. Histochem Cell Biol 126: 83-87, Komulainen J, Koskinen S, Kalliokoski R, Takala T, Vihko V. Gender differences in skeletal muscle fibre damage after eccentrically biased downhill running in rats. Acta Physiologica Scandinavica 165: 57-63, McClung JM, Davis JM, Carson JA. Ovarian hormone status and skeletal muscle inflammation during recovery from disuse in rats. Exp Physiol 92: , McClung JM, Davis JM, Wilson MA, Goldsmith EC, Carson JA. Estrogen status and skeletal muscle recovery from disuse atrophy. J Appl Physiol 100: , Persky AM, Green PS, Stubley L, Howell CO, Zaulyanov L, Brazeau GA, Simpkins JW. Protective effects of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro. Proc Soc Exp Biol Med 223: 59-66, Pizza FX, Peterson JM, Baas JH, Koh TJ. Neutrophils contribute to muscle injury and impair its resolution after lengthening contractions in mice. J Physiol 562: , Prorock AJ, Hafezi-Moghadam A, Laubach VE, Liau JK, Ley K. Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol 284: H133-H140, Roth SM, Martel GF, Ivey FM, Lemmer JT, Tracey BL, Metter EJ, Hurley BF, Rogers MA. Skeletal muscle satellite cell characteristics in young and older men and women after heavy resistance strength training. J Gerontol A Biol Sci Med Sci 56: B240- B247,

10 17. Salminen A Kihlstrom M. Lysosomal changes in mouse skeletal muscle during repair of exercise injuries. Muscle and Nerve 8: , Sewright, KA, Hubal MJ, Kearns A, Holbrook M, Clarkson PM. Sex differences in response to maximal eccentric exercise. Med Sci Sports Exerc 40: , Sipila S, Taaffe DR, Cheng S, Puolakka J, Toivanen J, Suominen H. Effects of hormone replacement therapy and high-impact physical exercise on skeletal muscle in post-menopausal women: a randomized placebo-controlled study. Clin Sci 101: , Sitnick M, Foley AM, Brown MB, Spangenburg EE. Ovariectomy prevents the recovery of atrophied gastrocnemius skeletal muscle mass. J Appl Physiol 100: , Sorensen MB, Rosenfalck AM, Hojgaard L, Ottesen B. Obesity and sarcopenia after menopause are reversed by sex hormone replacement therapy. Obesity Res 9: , Sribnick EA, Swapan RK, Banik NL. Estrogen as a multi-active neuroprotective agent in traumatic injuries. Neurochem Res 29: , Stupka N, Lowther S, Chorneyko K, Bourgeois JM, Hogben C, Tarnopolsky MA. Gender differences in muscle inflammation after eccentric exercise. J Appl Physiol 89: , Stupka N, Tiidus PM. Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats. J Appl Physiol 91: ,

11 25. Tidball JG, Wheling-Henricks M. Macrophages promote muscle membrane repair and muscle fibre growth and regeneration during modified muscle loading in mice in vivo. J Physiol 578: , Tiidus PM. Can estrogens diminish exercise induced muscle damage? Can J Appl Physiol 20: 26-38, Tiidus PM. Influence of estrogen and gender on muscle damage and repair. Exerc Sport Sci Rev 31: 40-44, Tiidus PM, Deller M, Liu XL. Oestrogen influence on myogenic satellite cells following downhill running in male rats: A preliminary study. Acta Physiologica Scandinavica 184: 67-72, Tiidus PM, Holden D, Bombardier E, Zachowski S, Enns D, Belcastro A. Estrogen effects on post-exercise skeletal muscle neutrophil infiltration and calpain activity. Can J Physiol Pharmacol 79: , Vierck J, O Reilly B, Hossner K, Antonio J, Byrne K, Bucci L, Dodson M. Satellite cell regulation following myotrauma caused by resistance exercise. Cell Biol Int 24: ,

12 A Pax7 Expression (% Positive Fibers) 6 Sham Estrogen Soleus Estrogen + ICI 182, a,b a c a,b a c 0 Control 24 h 72 h B MyoD Expression (% Positive Fibers) 6 Sham Estrogen Estrogen + ICI 182, a a c a,b a c 0 Control 24 h 72 h C BrdU Incorporation (% Positive Fibers) 6 Sham Estrogen Estrogen + ICI 182, a,b Control 24 h 72 h a c a,b a c Figure 1 Effects of estrogen supplementation and ICI 182,780 administration on numbers of fibres positive for (a) Pax7, (b) MyoD and (c) BrdU satellite cell markers in rat soleus muscle 24 and 72 h following downhill running. Values are mean ± SEM. ap<0.05 compared with treatment-matched Control group. bp<0.05 compared with treatment matched Estrogen group. cp<0.05 compared with treatment-matched Sham and Estrogen groups. (6) 12

13 Counterpoint: Estrogen and Sex do not Significantly Influence Post-Exercise Indices of Muscle Damage, Inflammation and Repair Monica J. Hubal 1 and Priscilla M. Clarkson 2 1 Research Center for Genetic Medicine, Children s National Medical Center, Washington, DC 2 Department of Kinesiology, Totman Building, University of Massachusetts, Amherst, MA Correspondence to: Monica J. Hubal Research Center for Genetic Medicine Children s National Medical Center 111 Michigan Ave, NW Washington, DC Phone: Fax: mhubal@cnmcresearch.org 13

14 Exercise-induced muscle damage (EIMD) occurs following strenuous, novel exercise and is assessed directly via tissue analysis and characterized indirectly by muscle dysfunction, inflammation, delayed onset muscle soreness, and increased levels of muscle proteins (e.g. creatine kinase (CK), myoglobin) in circulation. Repair typically takes place during the 7-14 days following the initial exercise bout. There has been much debate over the past 20 years concerning the existence of sex differences in EIMD and repair, predicated on the assumption that estrogen levels affect skeletal muscle s response to damaging exercise (14, 28). Potential mechanisms by which estrogen levels could affect the EIMD response include estrogen s antioxidant properties and its potential to stabilize the membrane of the skeletal muscle cells during exercise. Either or both of these mechanisms would serve to protect females from EIMD and promote repair when compared with males. Indeed, animal studies have often demonstrated attenuated EIMD susceptibility in female or estradiol-supplemented animals, based largely on decreased levels of CK in the blood (1, 3). Animal studies (23) have also reported attenuations in the inflammatory process due to sex or estrogen status. However, other animal studies (17, 28) that used contractile deficits as the primary muscle damage marker have not found similar effects. In fact, Warren et al. (29) found that estradiol-supplemented ovariectomized mice demonstrated significantly greater contractile losses as compared to non-supplemented mice. In contrast to animal studies, the majority of human studies have reported similar responses for various EIMD markers when comparing men and women or groups with different estrogen levels (reviewed in 6-8). Direct muscle damage assessment: Two studies to date addressed the effect of sex on skeletal muscle ultrastructural properties following eccentric exercise in humans. Stupka et al. (24) found no differences between men (N=8) and women (N=8) in focal and extensive damage areas following unilateral eccentric leg exercise. A follow-up study (25) detected no effect of sex on the amount of Z-line streaming after a first bout of eccentric exercise. Although it may be beneficial to examine larger cohorts of men and women for direct markers of muscle damage in the future (given the variability associated with the biopsy method), these two studies indicate that ultrastructural changes in skeletal muscle following eccentric exercise are unaffected by sex. Muscle function: Several studies (4, 18, 20, 25) reported no sex differences in maximal isometric strength loss immediately following repetitive maximal eccentric contractions, while one study found that women demonstrate greater relative strength loss immediately after exercise (-57.8% in women vs % loss in men) (21). In a recent study (13), we found that patterns of relative isometric and eccentric torque losses during 50 maximal eccentric actions of the arm were remarkably similar between men (N=22) and women (N=24). We also found similar decrements in other contractile properties (i.e. work, rate of force production and half-relaxation time) throughout the exercise. Adding to this, 14

15 several studies that examined women of different estrogenic status (e.g. pre- vs. postmenopausal or oral contraceptive users vs. non-users) also demonstrated similar strength changes after both isometric (5) and eccentric (19, 26) exercise. Together, these data suggest that relative muscle fatigue during and immediately after exercise are unaffected by sex or estrogen levels. Studies that have examined recovery of strength following damaging exercise largely found no differences between men and women (20, 26). Some studies reported differences in contractile characteristics during recovery (4, 15), but these differences were based on statistical analysis of the change in absolute levels over time. Given the large baseline differences between men and women, it is important to account for these differences (or a surrogate such as lean body mass) in the analyses. For example, MacIntyre et al. (15) reported differences in concentric and eccentric torque recovery following 300 eccentric actions of the quadriceps. However, these differences were no longer significant when strength was normalized to body weight. Furthermore, although the recent Sewright et al. (21) paper demonstrated an acute difference in maximal isometric strength immediately post-exercise, the overall strength loss/recovery pattern (to 10d post-exercise) was not significantly different between men and women. Studies examining strength recovery in relation to estrogenic status have demonstrated mixed results, with two reporting no group differences in strength recovery in the 5d following exercise (5, 26) and one study suggesting a delay in recovery for oral contraceptive users (19). Muscle enzyme activity in blood: Women have lower resting CK levels than men (2), but consistent differences in the CK response to exercise have not been reported. Following downhill running, Sorichter et al. (22) found no sex differences in several different muscle proteins in the blood, including CK, myoglobin, myosin heavy chain fragments and troponin I. Eston et al. (12) also found no sex differences in CK following downhill running. After eccentric arm exercise, Sewright (21) found that women experienced a lower CK but not myoglobin elevations, while Stupka demonstrated lower CK values in women both before and after eccentric leg exercise (25). Studies of CK response to exercise in relation to estrogen levels in different groups of women have largely demonstrated no differences between groups (5, 16, 19, 26). Furthermore, while Arnett et al. (2) found an increased CK response in menarchal women vs. pre- or post-menarchal women following eccentric hamstring exercise, this effect was lost when the analysis was covaried for lean body mass. Pain/Soreness and Inflammation: Studies assessing pain or delayed onset muscle soreness measures typically have not found sex differences (10, 18, 21) or significant associations with estrogen (5, 19). One study that did report a difference in soreness patterns following eccentric exercise (15) looked only at soreness patterns over the first 24h post-exercise, while soreness levels typically peak several days following exercise. While overall inflammation (as assessed by swelling or circumference measures) has not typically demonstrated estrogenic effects (19, 26), alterations of certain components of the inflammatory process have been reported to be different between men and women, but these results have been inconsistent. MacIntyre et al. (15) reported higher numbers of neutrophils in women 2h post-exercise, while Stupka (24) 15

16 demonstrated an exercise-induced rise in circulating granulocytes only in men. In a follow up study, Stupka (25) reported no sex difference in neutrophil and macrophage numbers following a single bout of exercise, while women demonstrated a rise in neutrophil concentration following a repeated bout of exercise. Variability: While studies suggested that the average EIMD response may be similar between men and women, some data suggest differences in the variability of responses. For example, Sayers et al. (20) found that women had a greater incidence of profound post-exercise strength loss (>70%), while Sewright et al. (21) found that men had greater variability in CK levels. These findings may be in part due to interactions of sex or estrogen with genetic variability effects. Studies have reported associations between genetic mutations and variability in EIMD markers (9; 11), and a subset of these associations were sex-dependent. Genetic variability interactions might also explain, in part, apparent species differences in EIMD studies. Conclusion: The majority of data from human studies have indicated that sex and estrogen levels do not significantly affect exercise-induced muscle damage, as measured directly (i.e. ultrastructural damage) or as indicated by functional losses, muscle protein levels in the blood, pain or soreness. 16

17 References: 1. Amelink GJ, and Bar PR. Exercise-induced muscle protein leakage in the rat. Effects of hormonal manipulation. J Neurol Sci 76: 61-68, Arnett MG, Hyslop R, Dennehy CA, and Schneider CM. Age-related variations of serum CK and CK MB response in females. Can J Appl Physiol 25: , Bar PR, Amelink GJ, Oldenburg B, and Blankenstein MA. Prevention of exercise-induced muscle membrane damage by oestradiol. Life Sci 42: , Borsa PA, and Sauers EL. The importance of gender on myokinetic deficits before and after microinjury. Med Sci Sports Exerc 32: , Buckley-Bleiler R, Maughan RJ, Clarkson PM, Bleiler TL, and Whiting PH. Serum creatine kinase activity after isometric exercise in premenopausal and postmenopausal women. Exp Aging Res 15: , Clarkson PM, and Hubal MJ. Are women less susceptible to exercise-induced muscle damage? Curr Opin Clin Nutr Metab Care 4: , Clarkson PM, and Hubal MJ. Exercise-induced muscle damage in humans. Am J Phys Med Rehabil 81: S52-69, Clarkson PM, and Sayers SP. Gender differences in exercise-induced muscle damage. In: Gender differences in metabolism, edited by Tarnopolsky MA. Boca Raton, FL: CRC Press, 1999, p Clarkson PM, Hoffman EP, Zambraski E, Gordish-Dressman H, Kearns A, Hubal M, Harmon B, and Devaney JM. ACTN3 and MLCK genotype associations with exertional muscle damage. J Appl Physiol. 99(2):564-9, Dannecker EA, Hausenblas HA, Kaminski TW, and Robinson ME. Sex differences in delayed onset muscle pain. Clin J Pain 21: , Devaney JM, Hoffman EP, Gordish-Dressman H, Kearns A, Zambraski E, and Clarkson PM. IGF-II gene region polymorphisms related to exertional muscle damage. J Appl Physiol 102: , Eston RG, Lemmey AB, McHugh P, Byrne C, and Walsh SE. Effect of stride length on symptoms of exercise-induced muscle damage during a repeated bout of downhill running. Scand J Med Sci Sports 10: , Hubal MJ, Rubinstein SR, and Clarkson PM. Muscle function in men and women during eccentric exercise. Journal of Strength and Conditioning Research In press: Kendall B, and Eston R. Exercise-induced muscle damage and the potential protective role of estrogen. Sports Med 32: , MacIntyre DL, Reid WD, Lyster DM, and McKenzie DC. Different effects of strenuous eccentric exercise on the accumulation of neutrophils in muscle in women and men. Eur J Appl Physiol 81: 47-53, Miles MP, and Schneider CM. Creatine kinase isoenzyme MB may be elevated in healthy young women after submaximal eccentric exercise. J Lab Clin Med 122: , Moran AL, Nelson SA, Landisch RM, Warren GL, and Lowe DA. Estradiol replacement reverses ovariectomy-induced muscle contractile and myosin dysfunction in mature female mice. J Appl Physiol 102: , Rinard J, Clarkson PM, Smith LL, and Grossman M. Response of males and females to high-force eccentric exercise. J Sports Sci 18: ,

18 19. Savage KJ, and Clarkson PM. Oral contraceptive use and exercise-induced muscle damage and recovery. Contraception 66: 67-71, Sayers SP, and Clarkson PM. Force recovery after eccentric exercise in males and females. Eur J Appl Physiol 84: , Sewright KA, Hubal MJ, Kearns A, Holbrook MT, and Clarkson PM. Sex differences in response to maximal eccentric exercise. Med Sci Sports Exerc 40: , Sorichter S, Mair J, Koller A, Calzolari C, Huonker M, Pau B, and Puschendorf B. Release of muscle proteins after downhill running in male and female subjects. Scand J Med Sci Sports 11: 28-32, St Pierre Schneider B, Correia LA, and Cannon JG. Sex differences in leukocyte invasion in injured murine skeletal muscle. Res Nurs Health 22: , Stupka N, Lowther S, Chorneyko K, Bourgeois JM, Hogben C, and Tarnopolsky MA. Gender differences in muscle inflammation after eccentric exercise. J Appl Physiol 89: , Stupka N, Tarnopolsky MA, Yardley NJ, and Phillips SM. Cellular adaptation to repeated eccentric exercise-induced muscle damage. J Appl Physiol 91: , Thompson HS, Hyatt JP, De Souza MJ, and Clarkson PM. The effects of oral contraceptives on delayed onset muscle soreness following exercise. Contraception 56: 59-65, Tidball JG. Inflammatory processes in muscle damage and repair. Am J Physiol Regul Integr Comp Physiol 288: R , Tiidus PM. Can oestrogen influence skeletal muscle damage, inflammation, and repair? Br J Sports Med 39: , Warren GL, Lowe DA, Inman CL, Orr OM, Hogan HA, Bloomfield SA, and Armstrong RB. Estradiol effect on anterior crural muscles-tibial bone relationship and susceptibility to injury. J Appl Physiol 80: ,

19 REBUTTAL: Point: Estrogen and sex do influence post-exercise indices of muscle damage, inflammation and repair Peter M. Tiidus & Deborah L. Enns Department of Kinesiology & Physical Education Wilfrid Laurier University Waterloo ON Canada Correspondence: Peter M. Tiidus, Ph.D. Professor & Chair, Department of Kinesiology & Physical Education, Wilfrid Laurier University Waterloo ON Canada, N2L 3C ext (telephone), (fax) 19

20 Drs. Hubal and Clarkson agree that while animal studies generally support positive effects of estrogen on muscle damage, inflammation and repair, studies examining sex differences in humans have more varied results (4). Since, estrogen has also been clearly demonstrated to have protective and regenerative effects on a variety of other tissues in humans and animals (1, 3, 6, 8), the real issue is not whether estrogen can positively influence muscle damage and repair, but why this has not been clearly demonstrated in humans. There are several potential reasons for this. Most human studies involving muscle damage have focused only on sex differences which can be confounded by other sex based variables beyond differences in estrogen levels. Also, since previous training and length of exposure to various estrogen levels may affect membrane stability and numbers of estrogen receptors on muscle (2, 10) looking for differences in female responses only over the course of a brief estrus cycle may be spurious. Unlike animal studies, no human studies have examined controlled variations in estrogen levels alone (9). A more ecologically valid human model to demonstrate the positive effects of estrogen on muscle damage and repair is the pre- versus post-menopausal female. Studies have suggested that older females suffer greater strength declines and muscle damage with aging than males or younger females respectively (5, 7). Other factors confounding human results include the degree of muscle damage induced by exercise. It may be better to investigate damage and recovery rates in younger and older females following exercise related muscle sprains or contusions where protective and recovery effects of estrogen may be more evident. It is also more difficult to detect diffuse muscle damage from small biopsy samples of mixed fiber type in 20

21 humans relative to examining entire muscles from animals. Much of the human data is also based on changes in blood creatine kinase (CK) levels, which due to its high variability, may be responsible for some of these inherent inconsistencies. Due to less control over variables, and potentially less sensitive methods of measurement, it can be more difficult to detect protective and regenerative estrogen effects on muscle in humans than in animals. This does not mean that these effects don t exist or that they are not physiologically important. It does suggest that we have not yet conducted the most appropriately controlled experiments using the most relevant populations and measures necessary to unequivocally demonstrate the importance of estrogen in positively influencing muscle damage and repair. 21

22 References 1. Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson M. Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response. Am J Pathol 155: , Bar P, Amelink G, Oldenburg B, Blankenstein M. Prevention of exercise induced muscle membrane damage by oestradiol Life Sci 42: , Harada H, Pavlick KP, Hines IN, Hoffman JM, Bharwani S, Gray L, Wolf RE, Grisham MB. Effects of gender on reduced-size liver ischemia and reperfusion injury. J Appl Physiol 91: , Hubal M, Clarkson PM. Counterpoint: Estrogen and sex do not influence postexercise indices of muscle damage, inflammation and repair. J Appl Physiol??? 2008? 5. Phillips, SK, Rook KM, Siddle NC, Bruce SA,Woledge RC. Muscle weakness in women occurs at an earlier age than in men, but strength is preserved by hormone replacement therapy. Clin Sci 84: 95-98, Prorock AJ, Hafezi-Moghadam A, Laubach VE, Liau JK, Ley K. Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase. Am J Physiol Heart Circ Physiol 284: H133-H140, Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, Rogers MA. High-volume, heavy-resistance strength training and muscle damage in young and older women. J Appl Physiol 88: , Sribnick EA, Swapan RK, Banik NL. Estrogen as a multi-active neuroprotective agent in traumatic injuries. Neurochem Res 29: ,

23 9. Tiidus PM. Can oestrogen influence skeletal muscle damage, inflammation and repair? Br J Sports Med 39: , Wiik A, Gustafsson T, Esbjornsson M, Johansson O, Ekman M, Sundberg CJ, Jansson E. Expression of oestrogen receptor alpha and beta is higher in skeletal muscle of highly endurance-trained than of moderately active men. Acta Physiol Scand 184: ,

24 REBUTTAL: Counterpoint: Estrogen and Sex do not Significantly Influence Post- Exercise Indices of Muscle Damage, Inflammation and Repair: Rebuttal Monica J. Hubal 1 and Priscilla M. Clarkson 2 1 Research Center for Genetic Medicine, Children s National Medical Center, Washington, DC 2 Department of Kinesiology, Totman Building, University of Massachusetts, Amherst, MA Correspondence to: Monica J. Hubal Research Center for Genetic Medicine Children s National Medical Center 111 Michigan Ave, NW Washington, DC Phone: Fax: mhubal@cnmcresearch.org 24

25 Drs. Tiidus and Enns make a compelling case that estrogen and sex significantly influence some markers of exercise-induced muscle damage (EIMD) in animal models, specifically muscle enzyme efflux and inflammatory markers. However, central to our case in arguing against significant estrogen and sex differences on EIMD are the lack of differences in other muscle damage markers (specifically muscle function) and the lack of substantiation of the inflammatory marker differences in human studies. The exception to this is findings of sex differences in creatine kinase (CK) levels, which may or may not be attributable to estrogen levels. The preponderance of human studies of eccentric exercise effects shows no sex difference in soreness development and strength loss/recovery. In fact, our recent study (3) found that function during exercise is similar between sexes, and another study (4) showed a slight exacerbation of strength loss in women which would contradict the hypothesis that estrogen protects the muscle from EIMD. Our opponents mention that human studies focusing on gender differences have also provided some evidence for positive estrogenic influences of skeletal muscle damage and repair citing the Sewright et al. (4) and Stupka et al. (5) papers, the only two human studies cited in their article. Both the Sewright et al. and Stupka et al. studies noted a greater increase specifically in the CK response in men compared with women. However, Sewright et al. showed that many of the men in the study were high responders for CK. We have evidence that some of the variability in EIMD markers has a genetic basis, and we have found sexual dimorphisms in several genetic associations with muscle phenotypes. For example, we recently reported associations between IGF2 polymorphisms and muscle damage markers that were only significant in men (2). Drs. Tiidus and Enns suggest that the proposed estrogen-related protection of muscle may help older females preserve health and function. Although this is an intriguing notion, Arnett et al. (1) reported that post-menopausal women had lower CK values after eccentric exercise versus menarchal women. However, when the analysis was covaried for lean body mass, this effect was lost, thus suggesting that estrogen does not afford protection against EIMD in post-menopausal women. In summary, the data are strong that there is no sex difference in soreness or strength loss/recovery after eccentric exercise in humans. There may be a sex difference in the CK response, but its meaning is still elusive. 25

26 References: 1. Arnett MG, Hyslop R, Dennehy CA, and Schneider CM. Age-related variations of serum CK and CK MB response in females. Can J Appl Physiol 25: , Devaney JM, Hoffman EP, Gordish-Dressman H, Kearns A, Zambraski E, and Clarkson PM. IGF-II gene region polymorphisms related to exertional muscle damage. J Appl Physiol 102: , Hubal MJ, Rubinstein SR, and Clarkson PM. Muscle function in men and women during eccentric exercise. Journal of Strength and Conditioning Research Jun 9: [Epub ahead of print] 4. Sewright KA, Hubal MJ, Kearns A, Holbrook MT, and Clarkson PM. Sex differences in response to maximal eccentric exercise. Med Sci Sports Exerc 40: , Stupka N, Lowther S, Chorneyko K, Bourgeois JM, Hogben C, and Tarnopolsky MA. Gender differences in muscle inflammation after eccentric exercise. J Appl Physiol 89: ,