When skeletal muscle is damaged by unaccustomed exercise or other trauma, a characteristic reaction involving inflammatory responses, edema build up, and ultimate repair via satellite-cell activation and proliferation ensues. This complex cascade of events is regulated at each step by a myriad of factors that react locally and systemically to disruption of muscle membranes, ultrastructure, and metabolism induced by the insult to the muscle. These include the regulation of leukocyte mobilization, adhesion, and ultimate uptake into the damaged muscle, local and systemic inflammatory regulation by cytokines, and other inflammatory mediators, as well as factors influencing muscle satellite-cell activation and proliferation.
The past decade has seen a growing awareness of the potential for estrogen to influence the postdamage inflammatory response in various tissues. In particular, these effects of estrogen on brain, neural tissues, and cardiac muscle have been the most extensively characterized (15). Recent studies using animal models have also found that estrogen can influence indices of skeletal muscle damage (4) and leukocyte infiltration (11,13) after exercise or ischemia-reperfusion injury.
These findings open the door to numerous further questions regarding the potential influence of estrogen on skeletal muscle damage, inflammation, and repair mechanisms that remain unanswered. These include: (i) the physiological mechanism(s) by which estrogen may be able to influence muscle damage and inflammatory related responses, (ii) the implications of estrogen-altered inflammatory responses to muscle satellite-cell activation and ultimate repair, and (iii) the potential implications of estrogenic influence on skeletal muscle resistance to damage and robustness of muscle repair to human health. This paper reviews the current evidence for estrogen to influence muscle damage and inflammatory responses to insult and speculates briefly on the above questions.
INFLUENCE OF ESTROGEN ON SKELETAL MUSCLE DAMAGE
It is well documented that estrogen and sex play a significant role in muscle membrane stability. After muscle damage induced by eccentric or unaccustomed exercise, trauma, or ischemia-reperfusion injury, well-documented muscle membrane and structural disruption occur. Among the common features of muscle membrane disruption are a selective migration of muscle-specific proteins and polypeptides occurring both outward and inward across the muscle sarcolemma. The most commonly measured indicator of muscle membrane disruption has been creatine kinase (CK) migration into the circulation (2). A series of experiments by Amelink, Bär, and coworkers (reviewed in (2)) demonstrated that female and male rats supplemented with estrogen had significantly attenuated postexercise muscle CK loss in both in vivo and in vitro conditions. Figure 1 illustrates the effects of duration of estrogen treatment on immediate postexercise blood CK activity in male rats. It is significant that increased length of exposure to estrogen in male animals leads to increasing attenuation of muscle CK loss after exercise. Similarly, human females also have significantly attenuated muscle CK loss compared with males after exercise-induced muscle damage (10), as well as lower resting blood CK levels (2). It is clear from these and similar studies that estrogen has the ability to help maintain muscle membrane integrity consequent to muscle damage, however caused. As will be discussed, it is this potential influence on membrane stability that may be a key to the effect estrogen has on certain postdamage muscle inflammatory responses.
Relatively few studies have investigated whether estrogen can influence actual muscle ultrastructural damage brought about by exercise or eccentric contractions. In one of the few animal studies to examine this question, Komulainen et al. (4) compared hind limb muscles from male and female rats after downhill running exercise at time points up to 96 h postexercise. They noted that male animals exhibited greater disruption of muscle microarchitecture, and more histopathological disruption associated with loss of submembrane dystrophin, loss of desmin, actin disorganization, and muscle swelling than females. As further indirect indication of muscle damage, they reported twofold higher lysosomal enzyme (ß-glucuronidase) activity in male versus female muscle at 48 h postexercise. Komulainen et al. (4) suggested that the early loss of submembrane dystrophin seen in male but not female muscles, which preceded other muscle disruption, may indicate that postdamage muscle necrosis may originate as plasma membrane damage and that estrogen may act to diminish this.
Another interesting effect of estrogen on factors related to muscle damage is its ability to suppress heat shock protein 70 (HSP70) and HSP70 mRNA synthesis in skeletal muscle of male or ovariectomized female animals after running exercise (8). Normal female animals also produce little postexercise HSP70 compared with normal males, suggesting a lower incidence of damage-associated stimulation of HSP70 synthesis. HSPs are ubiquitous, rapidly inducible proteins that can act as chaperones for protein assembly. Their induction may be triggered by various types of stress, including muscle damage (8). HSPs may play an important role in protein synthesis during recovery from muscle damage and may enhance protection from subsequent damage. Greater stress or damage to muscle generally results in a greater production of HSPs. A diminished HSP response in female and estrogen-supplemented animals may indicate attenuation of HSP induction as a consequence of reduced muscle damage.
Some human studies (10) have failed to find histological evidence for a sex-based difference in postexercise muscle damage from biopsy samples. However, these studies tend to have smaller sample sizes and a wider variability in muscle damage than those seen in animal studies. These limitations may mask the sex differences seen in animal studies (10). Further, no studies have yet looked specifically at the potential of estrogen supplementation (outside of normal sex differences) in normal or ovariectomized animal models to influence skeletal muscle damage after exercise or other damaging events. Hence our knowledge of the ability of estrogen or, by extension, sex to influence muscle ultrastructural damage is far from complete.
Influence of Estrogen on Muscle Leukocyte Infiltration After Damage
Although there still are some discrepancies, particularly in studies involving humans, the majority of studies have reported that estrogen appears to have an inhibitory influence on postdamage leukocyte infiltration into skeletal muscle (9,11,13,14). Estrogen also appears to have similar inhibitory effects on postdamage leukocyte infiltration into cardiac muscle (12).
Leukocytes play important roles in muscle inflammatory responses and repair mechanisms after damaging insult. Neutrophils begin infiltration of muscle shortly after damage. Their primary role appears to be associated with removal of damaged tissue, although they are also responsible for some cytokine production. To facilitate degradation and removal of damaged tissue, neutrophils generate hypochlorous acid via a myeloperoxidase-mediated reaction and generate superoxide via nicotinamide adenine dinucleotide oxidase. Although their role in assisting in the elimination of damaged structures is important, the oxidizing reactions generated by neutrophils are also thought to be responsible for further “collateral” damage to healthy muscle tissue during the initial inflammatory period (3).
Macrophages are the other major leukocytes that invade muscle after damage. Two subpopulations of macrophages are involved in postdamage invasion of muscle. ED1+ macrophages infiltrate muscle within 12 h postdamage and are also important in removing damaged tissue and cytokine generation. The ED2+ macrophages tend invade muscle 24–48 h postdamage and their presence in muscle is crucial in stimulating satellite-cell activation and differentiation. Without the invasion of these macrophages, satellite cell–initiated repair of muscle would not occur and muscle healing would not take place (6).
We have repeatedly demonstrated that early leukocyte (primarily neutrophil) infiltration into damaged muscle is influenced by estrogen and sex. In 1999, Tiidus and Bombardier (14) reported that 24-h postexercise leukocyte infiltration into skeletal muscle was attenuated in male rats by 14 d of daily estrogen administration compared with sham males. Female rats also had significantly lower postexercise muscle leukocyte infiltration than males. In addition, the males that were supplemented with estrogen had similar postexercise leukocyte levels to the normal females. Subsequent studies from our laboratory also demonstrated similar effects of estrogen on ovariectomized female rats with or without estrogen supplementation. Stupka and Tiidus (11) reported attenuation on muscle neutrophil infiltration 2 h after ischemia-induced muscle damage in normal and ovariectomized estrogen–replaced rats, compared with ovariectomized sham rats (as determined by immunohistochemistry). Tiidus et al. (13) also found attenuation of muscle neutrophil infiltration at 1 h post–running exercise in ovariectomized estrogen replaced rats compared with ovariectomized shams.
Only one study has looked at longer-term effects of sex on postexercise muscle leukocyte infiltration. St. Pierre-Schneider et al. (9) reported that female mice had delayed macrophage infiltration into muscle, compared with male mice (peaking at 7 vs 5 d respectively) after running exercise. These relatively few studies involving animal models have thus consistently reported that estrogen and/or sex can attenuate or delay muscle leukocyte infiltration after damage to muscle.
Human studies have not been as consistent and have only focused on sex differences without manipulation of estrogen levels. Some studies (i.e., (10)) have reported that females have lower levels of muscle leukocyte infiltration after eccentric exercise, whereas other studies have not reported such a difference (i.e., (5)). Some of these inconsistencies may be explained by different exercise protocols and/or methods for determining muscle leukocyte content. Perhaps in humans, estrogen-related differences in postexercise muscle leukocyte infiltration may be more consistently seen in populations such as postmenopausal females with or without estrogen replacement therapy. This population is of particular interest because there may be significant effects of estrogen on skeletal muscle function and recovery that would have direct bearing on their health, physical activity, and quality of life. We are currently investigating this population.
Potential Mechanisms of Estrogen Influence on Muscle Neutrophil Infiltration
The mechanisms by which estrogen may influence muscle leukocyte infiltration are not yet known. The process of leukocyte capture, rolling, adhesion, and diapedesis into muscle is complex and potentially influenced by numerous factors including, adhesion molecules, selectins, and cytokines (4,9). Also, the means by which estrogen may influence any or all of the factors associated with muscle damage, inflammation, and repair are also largely unknown.
The mechanisms by which estrogen asserts its protective influence on other tissues, such as brain and neurons, have some limited data indicating that both estrogen receptor–mediated and non-receptor–mediated events may act to assert estrogen-mediated protection (15). Skeletal muscle is known to process small but significant levels of the membrane-bound estrogen receptor alpha (ERα). Hence it is possible that estrogen receptor–mediated events may be able to influence muscle damage, inflammation, and repair processes, but currently no data exist in this regard relative to skeletal muscle.
We have proposed that the attenuation of skeletal muscle neutrophil infiltration that occurs1–2 h postdamage may be associated primarily with estrogenic influence on membrane stability (13). Estrogen is known to influence membrane fluidity and function either by direct nonspecific interaction with phospholipids or by direct interactions with other membrane components (7). Belcastro et al. (1) have suggested that exercise-induced muscle damage that disrupts sarcolemma integrity will lead to a disruption of muscle calcium homeostasis and consequently an activation of calpain. Calpain is a nonlysosomal protease that is activated by increased cytosolic calcium, immediately consequent to muscle damage, and selectively degrades specific muscle proteins, producing neutrophil chemoattractive products via this degradation (1). Belcastro et al.(1) and others have provided some experimental evidence for calpain-mediated neutrophil invasion of skeletal muscle in the immediate postexercise time period. As previously noted, we have found that estrogen can inhibit muscle neutrophil infiltration 1–2 h after running or ischemia-reperfusion injury–induced damage in ovariectomized female rats (11,13).
Good evidence exists that estrogen can act as an antioxidant or as a direct membrane stabilizer (3). Estrogen can be directly incorporated into cell membranes, much in the same way as cholesterol, and may act in a similar manner to optimize membrane fluidity and polyunsaturated fatty acid arrangement. Thus, estrogen has the potential to be able to limit muscle membrane disruption after injury by direct interaction with membrane components. Diminished CK leakage from female and estrogen-treated muscle, which, as previously noted, increases with exposure time to estrogen (and thus presumably results in elevated membrane estrogen content) is one line of evidence for support of this suggestion. Further evidence from studies in which the estrogen-receptor blocker Tamoxifen failed to prevent the estrogen-induced inhibition of postexercise HSP synthesis also tend to support a non-receptor–mediated mechanism of estrogen influence on muscle (8).
Hence, as Figure 2 illustrates, estrogen may be able to inhibit the calpain-induced production of neutrophil chemoattractant peptides by diminishing calpain activation, and thereby limit the postdamage neutrophil infiltration. This may be attributable to the ability of estrogen to diminish damage-induced muscle membrane disruption, thereby limiting disruption to muscle calcium homeostasis and thereby indirectly inhibiting the upregulation of calpain activity. This scenario is attractive because it may, at least in part, be able to account for the influence of estrogen on skeletal muscle neutrophil infiltration during the immediate postdamage period. We have reported correlational evidence for this suggestion by demonstrating the ability of estrogen supplementation in ovariectomized female rats to co-inhibit both calpain activation and neutrophil infiltration into skeletal muscle 1 h after running exercise (see Fig. 3).
The potential mechanisms by which estrogen may inhibit later macrophage infiltration into skeletal muscle remain to be determined. Others have speculated that estrogen may be able to influence specific adhesion molecule expression (9). It is likely that estrogen has multifactoral influence on skeletal muscle leukocyte infiltration that requires further research to delineate.
Potential Influence on Muscle Damage and Repair?
These observations on estrogen inhibition of postdamage muscle leukocyte infiltration lead to questions as to the influence, if any, this may have on ultimate muscle repair. This has particular relevance to the health of older females relative to their estrogen-replacement status and its potential influence on muscle recovery after exercise, training, or injury. Based on its observed inhibition of leukocyte infiltration, estrogen has the theoretical potential either to enhance or to inhibit the rate of muscle repair during recovery. The invasion of muscle by macrophages, particularly the ED2+ macrophage subpopulation, is essential for muscle satellite-cell activation and satellite cell–mediated muscle repair (6). It has been documented that, without macrophage infiltration, muscle repair cannot occur. Hence, any delay or inhibition of postdamage macrophage infiltration may ultimately slow the rate of muscle repair and delay ultimate muscle recovery after damage (9).
Alternatively, because neutrophil infiltration into damaged tissue results in significant further collateral damage to previously uninjured tissue, limiting neutrophil infiltration by estrogen may reduce inflammation-related damage and thus speed healing (14). We are currently investigating these possibilities. However, evidence from other tissues, i.e., neural and cardiac tissue, tends to suggest that inhibition of neutrophil infiltration after damage by estrogen or other means leads to a reduction in inflammation associated tissue damage and a more rapid rate of repair (12,15).
SUMMARY AND FUTURE RESEARCH
Estrogen and sex have been shown to have a significant influence on muscle membrane stability and possibly on diminishing exercise-induced muscle damage. Estrogen also has an attenuating effect on postdamage leukocyte infiltration into skeletal muscle. It is possible that, in part, the ability of estrogen to protect muscle membranes from damage may indirectly cause the consequent downregulation of postdamage muscle calpain activation. By attenuating muscle calpain activation, estrogen may indirectly inhibit the production of neutrophil chemoattractant peptides and thereby inhibit the immediate postdamage infiltration of skeletal muscle by neutrophils. This and other mechanisms related to the ability of estrogen to delay postexercise macrophage infiltration have yet to be fully explored. The physiological consequences of estrogenic attenuation of postdamage muscle leukocyte infiltration are not yet known, but may theoretically inhibit or enhance muscle recovery. The potential for estrogen to influence muscle damage and repair may have important health and quality of life implications for populations such as postmenopausal females.
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