Medical Hypotheses 83 (2014) 758–765

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Exhaustive exercise – A near death experience for skeletal muscle cells? q Michael Behringer a,⇑, Johannes Montag a, Alexander Franz a, Molly L. McCourt a, Joachim Mester a, Kazunori (Ken) Nosaka b a b

Institute of Training Science and Sport Informatics, German Sport University Cologne, Germany School of Exercise and Health Sciences, Edith Cowan University, Joondalup, WA, Australia

a r t i c l e

i n f o

Article history: Received 9 June 2014 Accepted 5 October 2014

a b s t r a c t In sports medicine, muscle enzymes in the blood are frequently used as an indicator of muscle damage. It is commonly assumed that mechanical stress disrupts plasma membrane to an extent that allows large molecules, such as enzymes, to leak into the extracellular space. However, this does not appear to fully explain changes in muscle enzyme activity in the blood after exercise. Apart from this mechanically induced membrane damage, we hypothesize that, under critical metabolic conditions, ATP consuming enzymes like creatine kinase (CK) are ‘‘volitionally’’ expulsed by muscle cells in order to prevent cell death. This would put themselves into a situation comparable to that of CK deficient muscle fibers, which have been shown in animal experiments to be virtually infatigable at the expense of muscle strength. Additionally we expand on this hypothesis with the idea that membrane blebbing is a way for the muscle fibers to store CK in fringe areas of the muscle fiber or to expulse CK from the cytosol by detaching the blebs from the plasma membrane. The blebbing has been shown to occur in heart muscle cells under ischaemic conditions and has been speculated to be an alternative pathway for the expulsion of troponin. The blebbing has also been seen skeletal muscle cells when intracellular calcium concentration increases. Cytoskeletal damage, induced by reactive oxygen species (ROS) or by calcium activated proteases in concert with increasing intracellular pressure, seems to provoke this type of membrane reaction. If these hypotheses are confirmed by future investigations, our current understanding of CK as a blood muscle damage marker will be fundamentally affected. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Sarcolemma and enzyme efflux The sarcolemma is the muscle’s plasma membrane, which serves as a biological barrier, isolating intracellular components of the muscle fiber from its extracellular space. The sarcolemma is surrounded by the basement membrane (consisting of the inner basal lamina and the outer reticular lamina) and undercoated by a subsarcolemmal actin network [1]. Physiologically, the basal lamina and the underlying sarcolemma are tightly connected via major trans-sarcolemmal integrins and dystroglycan [2,3] and large molecules, such as enzymes, cannot permeate the membrane in normal conditions. Thus, if increased levels of intramuscular enzymes are q

The present paper was not supported by any grant.

⇑ Corresponding author at: Am Sportpark Muengersdorf 6, 50933 Cologne, Germany. Tel.: +49 (0)221 4982 3620; fax: +49 (0)221 4982 8180. E-mail address: [email protected] (M. Behringer). http://dx.doi.org/10.1016/j.mehy.2014.10.005 0306-9877/Ó 2014 Elsevier Ltd. All rights reserved.

found in the blood, it is considered indirect evidence of increased membrane permeability and/or membrane damage. It has been reported that the amount of damaged muscle tissue can be estimated by the magnitude of increases in intramuscular enzymes in the blood [4–6]. However, in some cases there are distinct discrepancies between the histological damage and the amount of enzyme efflux that cannot be satisfactorily explained [7].

Causes of muscle damage Trauma, extreme temperatures (high and low), myotoxins and diseases are well known stressors that induce injury to skeletal muscle cells [8–10]. All smooth, cardiac, and skeletal muscle fibers are affected by these stressors. While the former two are commonly damaged by hypoxia in the course of tissue infarction, skeletal muscle fibers are most frequently damaged by unaccustomed exercises consisting of lengthening (i.e. eccentric) contractions [11]. As this type of contraction is characterized by higher

M. Behringer et al. / Medical Hypotheses 83 (2014) 758–765

intramuscular tension, when compared with isometric or isotonic contractions [12], it has been speculated that mechanical forces are primarily responsible for the greater damage [13]. However, Yu et al. [14] found sarcolemma disruptions only in a minority of subjects following eccentric exercises, challenging the assumption that lengthening contractions always induces membrane clefts. Unfortunately, serum CK activity was not measured in the study by Yu et al., leaving it unclear if CK efflux is possible without distinct membrane lesions. Further, it was shown previously that exercises without high tension also induced changes in membrane permeability, provided that the exercise is prolonged at a high intensity, resulting in fatigue. For instance, Chen et al. [15] reported that CK significantly increased by about 6-times (from 800 U/l to 5300 U/l) in rats following a fatiguing swimming protocol, an activity known to lack eccentric contractions. Significant CK increases were also found in humans immediately and 24 h after an exhaustive 90 min swimming protocol [16]. Similar results were also reported following a 90 min cycling exercise [3], also known to lack mechanical impact and eccentric contractions. Although the magnitude of the increase in CK is much lower than that often seen after eccentric exercises (>10 times), these data indicate that the energy status of the muscle cells probably contribute to the amount of enzyme efflux independent from mechanically induced membrane damage [17,18]. Enzyme leakage as protective mechanism In this context we hypothesize that the enzyme leakage is also associated with protective mechanisms. To the best of the authors´ knowledge, only one study to date published potential benefits of enzyme leakage during strenuous exercises [19]. In this study, the authors suggested that AMP-activated protein kinase (AMPK) plays a major role in the process of creatine kinase (CK) expulsion from skeletal muscle cells during exercise in order to limit the ATP consumption of this enzyme. According to Baird et al. [19] this may prevent muscle failure and allows for relaxation and regeneration. The present work will expand on the idea presented by Baird et al. [19] and will examine potential avail and underlying mechanisms regarding exercise induced enzyme ‘‘expulsion’’ from myoplasm, with the focus of this study placed on CK. The hypothesis We hypothesize that CK expulsion from the cytosol serves as a last resort for muscle fibers to survive in highly energy demanding activities. We also assert that ‘‘membrane blebbing’’ is used to either store CK in fringe areas of the muscle fiber or, if necessary, to expulse enzymes from the cytosol by detaching the blebs from the muscle fiber in order to prevent cell death during exhaustive exercises. Thus, our hypothesis differs from current notion that CK is simply ‘‘lost’’ from muscle cells as a consequence of increased membrane permeability or membrane clefts. More precisely, ATP dependent systems such as the Na+/K+-ATPases are essential in maintaining cellular homeostasis. Failure of these systems due to ATP depletion would lead to membrane depolarization followed by an uncontrolled influx of calcium ions (Ca2+). This Ca2+ influx causes the activation of Ca2+-dependent proteases and phospholipases resulting in uncontrolled swelling ultimately leading to cell death [20]. Muscle fibers counteract this life threatening situation through the ‘‘volitional’’ expulsion of ATP consuming enzymes from the cytosol, rescuing the muscle fiber from apoptosis or necrosis. However, CK is important for ATP supply in muscle fibers, and though the cells escape complete exhaustion, consequent rigor state, and cell death, they do so at the expense of muscular performance. The present hypothesis may

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apply to both, concentric and eccentric exercises. However, sarcolemmal damage following eccentric exercise is blended with damaging mechanisms other than energy depletion. Therefore, in the present hypothesis, we focused on ‘‘pure’’ energy depleting exercises – i.e. without interference from high mechanical forces. In other words, the hypothesis does not necessarily apply for muscle damage induced by eccentric contractions, which is more mechanically oriented than metabolically oriented. Evaluation of the hypothesis CK – a key player in energy metabolism CK plays a key role in the energy metabolism of almost all cells by catalyzing the exchange of phosphate from phosphocreatine (PCr) to ADP and by connecting intracellular sites of ATP production with sites of ATP utilization via the CK/PCr energy shuttle [21]. During energy demanding exercises, the ATP concentration is initially held constant by CK while PCr breaks down to creatine (Cr) and inorganic phosphate (Pi) [22]. It was previously shown that Cr has little effect on muscle performance, whereas Pi substantially affects force production, Ca2+ sensitivity, and Ca2+ handling of the sarcoplasmic reticulum (SR) [22]. The importance of CK in the context of Pi accumulation becomes obvious when studying CK deficient (CK / ) mice. While these animals present an increased Pi level at rest, Pi does not appear to accumulate during fatigue and the rate of ATP depletion is lower, when compared to wild type mice [23]. The lack of Pi accumulation is thought to be responsible for the observation that muscle fibers of CK / mice are virtually infatigable. Although these animals are fatigue resistant, their muscle fibers are also characterized by a significantly lower power output when compared with those of wild-type mice [24]. Therefore, the increased fatigue resistance in these transgenic mice should not be confused with an improved contractile economy, which has been shown to be similar to that of muscle fibers from wild-type mice [25]. In summary lower intracellular CK concentrations seem to enable more contractions until muscular failure due to a lack of phosphate accumulation, while preserving intracellular ATP stores. It is our hypothesis that muscle fibers under energy demanding stress take on characteristics of CK / muscle fibers by releasing CK from the myoplasm. This CK expulsion will reduce power output but prevent the muscle fibers from aforementioned damaging consequences of a total ATP depletion. The fact that CK expulsion already occurs during exhausting exercises is supported by the data presented by Chen et al. [15], who found significantly reduced intracellular CK concentration immediately following a fatiguing swimming protocol in rats. Calcium dependent disturbances in membrane integrity Ca2+, which is markedly increased during fatiguing exercises, seems to be important in enabling enzymes to leave the cell. From eccentric contractions it is known that Ca2+-dependent activation of proteases and phospholipases induce sarcolemmal and cytoskeletal damages [26]. Ca2+ seems to enter the cell via stretch activated Ca2+ channels or through mechanically induced membrane lesions [27]. However, the ability of muscle fibers to clear Ca2+ from the myoplasm depends on the cell’s energy status [28,29]. This may explain disturbances of muscle cell integrity following fatiguing exercises even in the absence of an appreciable amount of mechanical loading [29]. More precisely, ion-motive ATPases are unable to keep up the cellular ionic homeostasis at low energy levels, resulting in an uncontrolled Ca2+ influx through voltage-gated Ca2+ channels [20] and sodium channels [30].

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Previously published studies on muscle fatigue indicated that neither voluntary nor electrically induced muscle fatigue was accompanied by a drop of cytoplasmic [ATP] below 60% of resting level [22]. Therefore, it seems doubtful if concentrations reached during muscle fatigue are low enough to trigger Ca2+ influx, as described above. However, as argued by Allen et al. [22] those results on cellular ATP level are based on whole muscles and muscle homogenates. Individual fibers, by contrast may reach much lower levels of ATP in consequence of fatiguing exercises. In type IIx fibers, for example, ATP level as low as 20% of the resting state was found following a 25 s maximal cycling exercise in humans [31]. Further, there may be intracellular sites at which high rates of ATP consumption are accompanied with a restricted diffusion, leading to a localized ATP depletion [22]. Apart from the ATP depletion, muscle fatigue resulting from repetitive contractions is also known to be associated with several stereotypical changes in other intracellular metabolites (Fig. 2). Some of these have been shown to reduce the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity or SR Ca2+ leakage, resulting in an increased cytosolic [Ca2+] at rest [22]. During prolonged exercises the intracellular Ca2+ concentrations might be large enough to activate Ca2+-dependent phospholipases [32] and proteases [26] that are capable of damaging the cytoskeleton and altering the membrane permeability of muscle fibers, respectively (Fig. 2). The latter, which may also be induced by chronic membrane depolarization as a consequence of extracellular potassium accumulation, contributes to an increased resting level of [Ca2+] by increasing the Ca2+ influx from the extracellular space [33]. Gissel et al. [34] found a 65% increase in total Ca2+ content after prolonged low-frequency stimulation (1 Hz, 240 min) in rat skeletal muscle fibers. They speculated that the release of lactate dehydrogenase (LDH) from these fibers was due to a partial loss in membrane integrity, induced by Ca2+-dependent proteases and phospholipases. Based on the facts that Ca2+ sensitivity of human calpain is slightly higher than that in rats, prolonged Ca2+ exposure (15 min) autolyzes calpain at comparatively low concentrations of 2.5 lM [35], and exercise increases the Ca2+ affinity of calpain [36], an activation of calpain during prolonged exercises seems reasonable. This assumption is challenged by a study of Murphy et al. [35], in which participants performed a prolonged cycling exercise until exhaustion (at 70% VO2 peak) and no evidence of calpain activation was found. However, these data were based on a small sample size of n = 3, which surely impedes the generalizability of the reported results. AMPK – the cellular energy sensor Adenosine monophosphate-activated protein kinase (AMPK) is known to be a sensor of the cellular energy status and limits unnecessary ATP-consumption under energy demanding situations. Combined with the fact that AMPK and CK strikingly colocalize in rat muscle fibers [37], it seems reasonable to assume that this enzyme is involved in the exercise-induced process of CK expulsion [19] and/or inactivation, as suggested by Baird et al. [19] (Fig. 2). According to the present hypothesis, a lack of AMPK would therefore be associated with a limited ability to inactivate or timely expulse CK from the cytosol. This would lead to an undamped muscle activity until cellular energy stores are depleted to a level that induces cellular apoptosis or even necrosis. This would result in an earlier decline in performance and pronounced muscle damage. In line with this assumption, Lantier et al. [38] recently reported that average running distance in muscle-specific AMPKa1a2 double-knockout (mdKO) mice was markedly decreased when compared to wild-type mice. While the average running speed of both groups did not differ, signs of degenerative

processes (i.e. centrally located nuclei and ultrastructural deterioration) were found more frequently in AMPK-deficient fibers. However, fatigue resistance of mdKO mice, tested as electrically induced repeated short tetani (100 Hz, 500 ms, every 2 s for 3 min), was reduced in the soleus but not tibialis muscles. The reasons for this outcome remain unclear. Further, some authors challenged the assumption that CK is inactivated by AMPK. Ingwall et al. [39] found the reaction velocities of CK and AMPK to be positively, not inversely, related only under normoxic conditions. These authors reported an inverse relationship under low pO2 states and indicated that AMPK velocity was surprisingly small under these circumstances. Therefore, the role of AMPK in regulation of CK activity and its expulsion from the cytosol remains unclear. Besides an AMPK-mediated CK inactivation by means of phosphorylation [19], we hypothesize that this enzyme may also somehow be involved in the induction of membrane blebbing (Fig. 2, dashed line). Are membrane blebs an ‘‘emergency exit’’ to prevent cell death? Besides membrane lesions, muscle cells may use an alternative mechanism to reduce myoplasmic CK concentration. From cardiac muscle cells it is known that hypoxia induces so called ‘‘membrane blebbing’’ [40]. According to Majno et al. [41], these blebs, which are initially reversible, are blister-like, fluid-filled structures that occur on the membrane surface during the early stages of ischemic conditions due to a failure of ionic membrane pumps (caused by ATP deficiency). The growth phase of membrane blebbing takes about 30 s while their retraction lasts about 2 min [42]. The subsarcolemmal actin network seems to be important in this context as membrane blebs develop when the cell membrane detaches from the underlying actin cortex or if the actin cortex is ruptured [42]. Since ATP is necessary to produce filamentous actin (F-actin) from its monomeric form (G-actin), ATP depletion has been hypothesized to induce blebbing via actin depolymerization and breakdown of the actomyosin network [43] (Fig. 2). In the context of our hypothesis, it can be speculated that CK does not actually need to leave the cell but that storing CK in those membrane protrusions during energy demanding activities may help the cell survive. Interestingly, in the 1980s Schmidt et al. already considered ‘‘blebbing to be a protective mechanism, which prolongs or guarantees the survival of the cells’’ [44]. When the energy demanding situation exceeds a certain time frame, it is possible that the blebs swell until they burst. If, however, the critical situation is reversed at an early stage, membrane blebs will be reabsorbed or are shed into the circulation [9,41]. Since bleb growth stops after detachment, it is assumed that their growth is primarily driven by intracellular pressure in form of hydrodynamic forces [45,46]. The pressure may be developed by the Ca2+ activated contraction of the actomyosin network of by the accumulation of metabolites that increase the cellular osmolarity, resulting in water influx [47,48]. However, according to Barros et al. [45], not only increased intracellular pressure but also hydrogen peroxide-induced membrane defects are needed to elicit membrane blebbing. Without membrane defects, hydrostatic pressure would only induce a homogeneously distributed cell swelling instead. In hepatocytes it was shown that the oxidative stress-induced membrane blebbing is mediated by the activation of the Ca2+-dependent protease calpain [49]. Interestingly, Blaser et al. [50] found free Ca2+ concentration to be significantly higher in regions of membrane protrusions, when compared to areas where no blebbing occured. Since resting Ca2+ concentration increases with the development of muscle fatigue, it seems likely that calcium activated proteases are involved if exercise-induced membrane blebbing actually occurs

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Fig. 1. Exercise-induced membrane blebbing. The developing sarcoplasm filled blebs might be used as ‘‘stock rooms’’ to temporarily store creatine kinase (CK) in fringe areas of the cell during energy demanding exercises (A). Upon recovery, the blebs will either be retracted after reformation of the actin cytoskeleton (i) or shed into the extracellular space as microparticles (MPs) (ii). Possibly, MPs are taken up by the same or other muscle cells (iii) (B). By contrast, if the situation of energy depletion persists, the blebs might grow until they burst, releasing their sarcoplasmic content (C). Black dots = creatine kinase, white ellipses = membrane-actin linker proteins.

(Fig. 1). Further, the membrane lesions may be amplified by CK leakage, as the latter is associated with a drop in the CK generated PCr, known to bind to and protect biological membranes against swelling [21]. The small size of fatigue-induced membrane lesions in concert with potent repair mechanisms may not only limit the negative consequences leading to cell death but provide a survival strategy, when used for removing energy demanding enzymes from the cytosol. However, this also suggests that there will be a threshold in terms of a critical size or amount of membrane lesions, at which the repair mechanisms of the cell are unable to reseal these sarcolemmal defects, or that the efficacy of membrane repair procedure is somehow compromised. For example, dysferlin-deficient muscle fibers are unable to rapidly repair membrane lesions and form blebs [51]. As a consequence, in dysferlin-deficient patients, even minimal physical activity will lead to a massive elevation in serum CK activity [52]. Interestingly, dysferlin appears to have a number of Ca2+ binding sites that are thought to be essential for its function [51]. This again points towards a crucial role of Ca2+ in the formation of membrane blebs. In order to reduce energy consumption, myofibrillar membrane blebs should predominantly be filled with energy consuming enzymes, while leaving energy producing units behind. However, exercise induced membrane blebbing has not been investigated yet, leaving it unclear, if those blebs are randomly filled with submembrane content. For that reason, it is currently impossible to estimate the minimum volume of blebs needed to be removed from the cell to achieve a meaningful decrease in cellular energy demand. Interestingly, following various types of experimental trauma, Jackson et al. {Jackson 1991 #462} reported proteins with 15, 17, 28, and 62,000 Da to be lost from muscle fibers in the largest amounts, while other proteins within the same range of molecular weight remained in the sarcoplasm. If any kind of selectivity applies to the content of membrane blebs remains to be determined by future research. Overall, it needs to be taken into account that blebbing likely also fulfils functions other than enzyme expulsion in damaged muscle cells. For example, it may act as a repair mechanism to remove damaged membrane fractions from the muscle fiber. However, we believe that

these different functions do not mutually exclude each other but rather go hand in hand.

Fatigue induced necrosis and its countermeasures Whether affected cells undergo apoptosis or necrosis when lethally injured, depends on the intracellular ATP concentration [53]. It seems that whenever ATP drops below a certain level, cells are not able to enter the energy consuming process of apoptosis and will undergo the unsystematic necrosis instead, which is associated with inflammation. However, in this context, Zong et al. [54] pointed out that there is a continuum of apoptosis and necrosis: while a cell-damaging stimulus at low doses will trigger apoptosis, higher doses of the same stimulus will induce necrosis. Since the entire myoplasm gets access to the extracellular space under necrosis, the unstructured cell death is likely associated with higher CK concentrations in blood, when compared to situations in which apoptosis is the predominant form of cell death. The fact that fatiguing exercise is capable of inducing necrosis of muscle fibers was previously shown in a study performed by Komulainen and Vihko [55]. The authors found necrotic muscle fibers and a significant release of CK 2, 4, and 48 h after a four hour uphill running protocol in rats. However, as reported by Vihko et al. [56], the number of necrotic fibers following fatiguing exercises was small and training seemed to protect muscle fibers from fatigue-induced cell death. This indicates that cellular mechanisms are effective in counteracting the ATP depletion. The fatigue-induced CK expulsion, as described here, may be only one of these mechanisms. For instance, MacIntosh et al. [57] proposed that phosphorylation of regulatory light chains (RLCs) increases the force production at a given Ca2+ concentration, which allows for a decrease in Ca2+ release without abandoning force production. This in turn preserves intracellular ATP stores due to the reduced amount of Ca2+ that needs to be pumped back into the sarcoplasmic reticulum (SR) by Ca2+-ATPases. Further these authors suggested that ATP depletion is avoided by decreasing the Ca2+ release (that triggers myosin ATPase activity) via regulation of the ryanodine receptor or by control of the membrane potential.

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Fig. 2. Possible mechanisms involved in CK expulsion from the cytosol. Muscle fatigue resulting from repetitive contractions is known to be associated with several stereotypical changes in intracellular metabolites, ROS production, and an extracellular potassium accumulation. An increased resting level of [Ca2+] by increasing the Ca2+ influx from the extracellular space or by a reduced activity of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) may activate Ca2+-dependent proteases [26] and phospholipases [32]. These enzymes are capable of damaging the cytoskeleton and altering the membrane permeability of muscle fibers, respectively. In concert with an increased intracellular pressure, the cytoskeletal damage induces membrane blebbing (see text for further explanation).

ROS as the vanguard of blebbing ROS may be involved in the cascade of blebbing by affecting the actin cytoskeleton [58,59], increasing AMPK activity [60], and/or decreasing the SERCA function (Fig. 2, dashed line) during muscle fatigue [61]. The influence of ROS may be exacerbated by the fact that Ca2+ uptake of mitochondria increases ROS production, which occurs if resting [Ca2+]i is increased (Fig. 2). It was previously shown that ROS production of neutrophils initiated membrane blebbing [62]. Neutrophils, which are rapidly mobilized after eccentric exercise, invade the damaged muscle tissue within several hours and remain present for up to 1 day [63]. Similar results were also shown after electrically stimulated contractions without eccentric components [64]. Contrary to the traditional point of view interpreting this invasion as a negative event for muscle fibers, it might be speculated in the framework of the present hypothesis that these cells are chemotactically ‘‘requested’’ by muscle fibers to support CK expulsion due to their ROSmediated effect on cell membranes [65]. In terms of a positive

feedback mechanism, blebs themselves have been speculated to induce monocyte chemotaxis [45]. Besides neutrophil ROS production, the intracellular formation of free radicals during strenuous exercises may also help to remove CK from the myoplasm via membrane blebs. Though the exact mechanisms of ROS formation during exercise still remain uncertain, it is clear that ROS are generated in active muscles [22]. However, it seems that free radical production occurs during exercise only if it is exhaustive – irrespective of its absolute intensity [66]. Goodman et al. [67] performed ultrastructural analyses of biopsies that were taken after an exhausting 21 km run, but did not find any evidence of muscle damage despite significant increases in serum CK activity. The authors concluded that this observation might be explained by ROS-induced membrane damage with increased permeability, but that it was unlikely the result of mechanical muscle damage. Interestingly, if free radical production of xanthine oxidase during exhaustive exercise is inhibited by allopurinol, the increases in plasma enzyme levels (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) after exhaustive exercise can be

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prevented [66,68]. These results clearly underline the crucial role of free radical production in the context of exercise-induced enzyme release. Fate and behavior of blebs It may also be of interest to investigate the fate and function of blebs after they have been shed into the extracellular space. It has been previously shown that microparticles (MPs) are able to transfer cytokines, receptors, RNA, and DNA that modulate the properties of target cells [69]. In a recently published article of our laboratory, it was shown that the uptake of endothelial MPs was associated with an improved protection of target cells against apoptosis [70]. In this context, it is conceivable that CK within muscular MPs is also of further use for the organism. That is, other cells or even the same cell, after recovery of its energy stores, may take up the CK molecule. But what would be the advantage of using blebs over small membrane disruptions to remove CK from myoplasm? Surrounding the enzymes with part of the cell membrane may possibly save ingredients from deactivation. That would hypothetically prolong the lifetime of CK in the extracellular space, which usually spans 22 h before inactivation and elimination [71]. Further, it may simplify the recycling of CK by a procedure that mounts the bleb to reabsorb its ingredients. Unfortunately, the fate and behavior of microparticles are largely unknown. However, in one previously published study it was shown that microvesicles not only bind to but fuse with activated platelets, resulting in an uptake of the bleb content into the cytoplasm of the target cell [72]. In order to verify the proposed hypothesis of the present paper, future work should not only investigate if ATP depletion goes along with an increased CK expulsion from myoplasm, but also the time course of these events. That is, if CK leakage occurs only after cessation of strenuous exercise, the proposed survival technique would be illogical. Further, it would be interesting to know if membrane blebbing, as described for cardiac muscle cells, occur in skeletal muscle fibers due to ATP depletion. Finally, the content of skeletal muscle blebs would have to be analyzed for the presence of CK. Consequences of the hypothesis and discussion Amendment to the interpretation of increased serum CK level Traditionally, the loss of membrane integrity is judged as a negative event compromising the homeostasis of muscle cells. However, as proposed by others, small and quickly repairable membrane disruptions should be interpreted as an alternative pathway for cells (apart from endo- and exocytosis) to transfer larger sized molecules across the lipid bilayer of membranes [73]. As speculated by Komulainen et al. [74] and others [7], quickly repairable membrane clefts may explain the mismatch between the histochemically detectable and the estimated amount of damaged muscle tissue based on CK blood levels. Using this pathway, muscle fibers are able to remove large molecules from the myoplasm without undergoing apoptosis or necrosis. In the case of CK, as hypothesized here, the small membrane lesions and/or membrane blebs may serve as an important mechanism to survive energy demanding activities. Implications for CK monitoring in sports medicine It needs to be taken into account that most physical activities are hybrid forms of different contraction types and differing amounts of metabolic needs. Both metabolic and mechanical

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stresses may induce membrane lesions by different mechanisms. However, we believe that high CK levels in the blood following strenuous exercises composed of eccentric and fatiguing elements, can at least be partially explained by enzyme expulsion from the muscle fibers. If the MPs shed from the sarcolemma do not collapse and membrane surrounded CK is undetectable by commonly used diagnostics, only those enzyme molecules released through small membrane lesions will contribute to the overall CK activity measured after fatiguing exercises. This may explain the greater CK increases frequently found after lengthening contractions, where CK leaks directly through large membrane clefts into the extracellular space. Since muscle cells will only benefit from the proposed mechanism of CK expulsion if it occurs simultaneously with the onset of muscle fatigue, the time course of CK increases in blood will likely differ from the well-known delayed CK peak in the blood 4–5 days following lengthening contractions. Data from studies on exhaustive, non-eccentric exercises point towards an early increase in blood and an early reduction of intracellular CK concentration [15] . However, to the best knowledge of the authors, no data are available on the time course of muscle damage markers following such exercises. In this context, Van der Meulen et al. [7] proposed that the early peak of enzyme concentrations in the blood may be the result of an increased membrane permeability due to an energy depletion of the cell, while the second peak is related to inflammatory responses. Therefore, the time course of changes in CK activity after exercise in the blood likely differs between a mechanical insult (e.g. high force eccentric exercise) and a metabolic insult (exhaustive exercise). While the former generally results in large, but delayed increases in CK activity, the latter is associated with small but early increases, which are already evident immediately after exercise. This could be a distinguished difference between the two causes of CK increase in the blood. However, further studies with multiple, closely spaced follow-up measurements are needed to elucidate the exact time course of muscle damage marker in blood after highly fatiguing exercises. If our hypothesis is confirmed by future research, the interpretation of increased CK levels in the blood would have to be fundamentally reevaluated. Increased CK activity in the blood would no longer be solely indicative of damaged but also substantially fatigued muscle fibers. Further, individual differences in the ability to expulse CK from muscle fibers during exhaustive exercises would affect the CK level in the blood and may therefore explain part of the well-known large inter-individual differences in CKresponse, commonly denoted as low and high-responder. Conflict of interest statement All authors declare that there are no financial and personal relationships with other people or organizations that could have inappropriately influenced (bias) the present work. The authors further state that no funding was received. References [1] Michele DE. Dystrophin–glycoprotein complex: post-translational processing and dystroglycan function. J Biol Chem 2003;278(18):15457–60. [2] Campbell KP. Skeletal muscle basement membrane–sarcolemma–cytoskeleton interaction minireview series. J Biol Chem 2003;278(15):12599–600. [3] Han R, Kanagawa M, Yoshida-Moriguchi T, Rader EP, Ng RA, Michele DE, et al. Basal lamina strengthens cell membrane integrity via the laminin G domainbinding motif of alpha-dystroglycan. Proc Natl Acad Sci USA 2009;106(31):12573–9. [4] Volfinger L, Lassourd V, Michaux JM, Braun JP, Toutain PL. Kinetic evaluation of muscle damage during exercise by calculation of amount of creatine kinase released. Am J Physiol 1994;266(2 Pt 2):R434–41. [5] Nosaka K, Sakamoto K. Changes in plasma enzyme activity after intramuscular injection of bupivacaine into the human biceps brachii. Acta Physiol Scand 1999;167(3):259–65.

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