**11. The production of free radicals**

et al., 1990). Some physiological evidence would show that the deficit of mytochondrial respiration inside the muscular fiber, cannot be considered the same way as an initial event in the onset of muscular damage; these affirmations are based on the fact that, a given level of production of force and /or mechanical power by the muscle, generated through an eccentric contraction, would result less costly than it is for the same level of production of force and /or generated power through a concentric or isometric contraction (Infante et al., 1964; Curtin and Davies, 1970; Bonde- Peterson et al., 1972) Despite this, it is the eccentric contraction the type of contractile muscular behavior which show higher harmful potential towards the integrity of the muscular structure (Asmussen, 1956; Armstrong et al., 1983; Newham et al., 1983; Armstrong, 1984; Ebbeling and Clarkson, 1989; Stauber, 1989). This lack of linking between metabolic cost and harmful event in the course of an eccentric contraction, would indicate, according to some authors, that the etiology of the muscular lesion would not lead to an insufficient production of ATP. Some authors have shown how there are no changes in levels of ATP, CP or in the pH after an injury, even though 24 hours after intense exercise we may register a significant increase in inorganic phosphate levels (Aldridge et al., 1986) At the same time, it is reasonable to expect that during a series of concentric contractions the muscular pH is lower than what it would be during an eccentric contraction. This could represent another indirect test of the fact that the lowering of the pH cannot, in itself, make up the initial factor of muscular damage. In this sense there exist experiments which show how, on isolated muscle, we may induct muscular damage also in the presence of neutral pH (between 7.3 and 7.6) with an average of 3mmol of lactate per liter (Duncan, 1987) Despite this it is of extreme importance to underline that these studies, and the consequential hypotheses, even though logical and rational, do not make up the indisputable test of the fact that the depletion of ATP or the lowering of the pH, are not implied in the process which carries to the muscular damage, but how rather they show that the muscular damage can come about also in absence of these assumptions of metabolic order (Armstrong et al., 1991). Particular attention must be placed on the fact that these specific situation of " metabolic unevenness", may be focal inside the fiber, reason for which in a well defined area of muscular fiber we may observe essential depletions of energetic phosphates and /or accumulation of lactate, which, on the contrary, are not observed in the rest of the muscular belly. So, even though definite demonstrations are missing of the fact that muscular damage recognizes its etiological cause in an insufficient mytochondrial respiration ratio, in bibliography there are not missing studies which speculate how the muscular damage, above all against the pure glycolytic fibers, at least on an animal model, is amenable to the contextual eccentric mechanism to a metabolic situation predispos‐ ing the damage itself (Liebere and Friden, 1988). In effect, a higher rational hypothesis in this sense is that which sees the intermittent anoxia, of which the muscle suffers during an intense series of eccentric contractions, as the cause of the drop in muscle pH to which follows a potential structural fragility situation both of the contractile tissue and of the connective tissue inside the muscle itself (Armstrong et al., 1991). So generally, a marginal fatigue may make up, at least from a theoretical point of view, a predisposing situation to muscular damage, even if a precise estimate of the role of fatigue in the harmful mechanism at the expense of the skeletal

muscle, is objectively difficult.

20 Muscle Injuries in Sport Medicine

Another consequence of the increase in metabolism during exercise is represented by the high production of free radicals (Packer, 1986; Jenkins, 1988; Matsunaga et al., 2003; Kon et al., 2008). Even though in many situations the increased production of free radicals is controlled by a wide variety of enzymes and of anti-oxidant molecules (Xu et al., 1997; Kon et al.,2008), in other circumstances this protective mechanism may result inefficient (Demopoulos, 1973b; Jenkins, 1988; Horakova et al., 2005). An uncontrollable production of free radicals may cause damage on a cellular level through an oxidation mechanism of phospholipids (Demopoulos, 1973; Blake et al., 1987) of DNA, (Cochrane et al., 1988), of carbohydrates (Blake et al., 1987) and of proteins (Tappel, 1973; Wolffe et al., 1986). The lipoperoxidation of the lipidic membrane may alter the normal permeability of the barrier of the sarcolemma (Quintanihla et al., 9182), allowing in such a way an abnormal molecular diffusion, in particular of Ca++ and of intra‐ muscular enzymes (Braughler, 1988); the inactivity of this enzyme can in fact perturb the homeostasis of the Ca++ inside the muscular fiber and cause, consequentially, the activity of a

series of cellular degradation processes. However, research which supports in an evident way the role of free radicals in the etiology of muscular lesion, is quite limited, above all if linked to an eccentric contraction as principal mechanical cause. One of the most convincing studies in this field is represented by that of Zerba in 1990 (Zerba et al., 1990), in which the authors showed how, in a murine model, an intraperitoneal injection of superoxide-dismutase limits, after the imposition of a series of eccentric contractions in situ, the drop in the value of P0 of the considered muscle. The treatment based on superoxide-dismutase was able to reduce the drop in the value of P0 for a period of three days following the eccentric exercise. Other experiments, carried out on animal models have further corroborated the hypothesis formu‐ lated by Zerba (Strosova et al., 2005; Kon et al., 2007), so for this it appears reasonable to extend this theoretical model also in a human field (Castilho et al., 1996; Close et al., 2005; Clanton, 2007; Kerkweg et al., 2007; Voss et al., 2008). There is not missing, however, in literature studies which deny the thesis that the administration of anti-oxidant agents may reduce muscular damage connected with high intensity exercise (Warren et al., 1990; Childs et al., 2001; Sacheck and Blumberg, 2001; Kerkweg et al., 2007). It is also important to remember some interesting experiences (Brooks et al., 2008) which underline the fact that the production of free radicals, on behalf of the skeletal muscle, and consequently their control and their regulation, are in function of precise physiological stimuli and how these parameters play a very important role in the field of physiological adaptation of the muscle during the contraction mechanism. These adaptations would include an optimization of the contraction mechanism, and in addition they would represent the beginning of adaptation processes and changes of gene expression regarding stress induced by the muscular contraction. Evidently these beneficial effects of the free radicals in the field of muscular contraction, contrast with contrary scientific evidence, which see the beginning and /or the cause of free radicals of a pathway of degenerative type which would appear fundamental, not only in the field of possible structural damage regard‐ ing the skeletal muscle, but also, more in general, in its aging process. This only apparent contradiction, underlines the necessity of deeper understanding in the role covered by the free radicals in the field of both physical exercise and the sarcopenia. Anyway, despite the relative lack of convincing and undisputable scientific evidence concerning the role of free radicals in the field of initial mechanisms, and /or predisposition of muscular lesion, it is without doubt legitimate to ask ourselves this question: is it reasonable to be able to support an increase in production of free radicals during an eccentric exercise? To answer this interesting and legitimate query, it is useful to remember that some studies (Brand and Lehninger, 1975) show how during an ischemic phenomena, in a model of ischemic /reperfusion damage, we can see in the cardiac muscle a destruction of the normal tight association between the elements of the chain of transport of the electrons. This would provide a particularly evident production of free radicals during the reperfusion phase, phase in which we may find high concentrations of O2 of the tissue(Hess et al., 1982; Arkhipenko et al., 1983; Faust et al., 1988; Fisher, 1988). It is possible to speculate that the high and specific muscular tensions which happen during of eccentric contraction, may alter the normal cytoskeletal structure, of which whose functions are to stabilize the position of the mitochondria (BIgland-Richie and Woods, 1976). The destruction of the cytoskeleton l could, in its turn, cause a disruption of the spatial configura‐ tion of the elements which compose the electron transport chain (Demopoulos, 1973a). This

structural disruption of the electron transport chain could lead to an excessive production of free radicals and so to a dramatic increase of the lipoperoxidation phenomena. So, in general, every disruption of the electron transport chain, may lead to an increase in the production of free radicals and potentially represent an initial mechanism in the field of the phenomena

Etiology, Biology and Treatment of Muscular Lesions

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23

If the initial events of harmful mechanism are of mechanical and metabolic nature, the immediately successive phases leading to the same harmful event, are characterized by an elevation of the levels of intracellular Ca++ in the injured area (Statham et al., 1976; Publicover et al., 1978; Kameyana and Etlinger, 1979; Baracos et al., 1984; Carpenter, 1989; Boobis et al., 1990). It is interesting to note how also in patients affected by muscular dystrophy and other muscular pathologies, we may find an increase in the intracellular levels of Ca++ (Jackson et al., 1985 ; Turner et al., 1988). The importance of maintaining the concentration of free cytosolic Ca++, is indirectly underlined by the number of the Ca++ transport mechanisms from the cytosolic compartment which the cell possesses (Gillis, 1985; Klug and Tibbits, 1988). There exists, in fact, at least seven membrane transport systems of Ca++. In the actual state of knowl‐ edge in the specific field, it would seem that there exists, until today, direct evidence of the fact that the elevation of the intracellular levels of Ca++ is involved in the mechanism of "exercise-induced muscle fiber injury" (Hall-Craggs, 1980; Steer and Mastaglia 1986; Childs et al., 2001), even if studies exist which show how in the condition of DOMS, they are present inside the muscle of high contextual levels of Ca++ to a same increased level of mitochondrial Ca++ (Duan et al., 1990a). The hypothesis that would justify an increase of intracellular levels, are essentially based on to the destruction of the sarcolemma found during the harmful event. The sarcolemma in fact represents a suitable barrier for the maintenance of concentration and of the electric gradient between the intra and extra cellular spaces; its destruction so permits the Ca++ to invade the intracellular space. The concentration of free extracellular Ca++ oscillates between 2 and 3 mmol.1-1 whereas that of cytosolic Ca++, in the muscle fiber at rest, is about 0.1

. So it evidently appears how, at the expense of Ca++, there exists and important gradient

between the intra and extra cellular space and that how each loss of normal permeability of the barrier, represented by the sarcolemma, may cause an important influx of Ca++ in the intracellular space. In experiments carried out on muscular fibers treated with saponin and incubated in Ca++ solution in concentration between 0.5 and umol- 1-1, we may observe a destruction of the myofibrils and a hyper-contraction of the sarcomeres. (Duncan, 1987). From the moment in which such concentrates enter in the same physiological range seen during an "*in vivo"* muscular contraction, this experimental data could induce us to believe that also during normal contractile activity the level of free cytosolic Ca++ could be high enough to start the degradation of the muscular ultrastructure. However, this event does not happen above all because the increase of the level of cytosolic Ca++ in the course of an *in vivo* muscular contraction is of transient type; In other words at the moment in which the Ca++ is released from the sarcoplasmatic reticulum in the course of the contraction itself, its level is readily

which we can name as "Exercise-induced muscle fiber injury".

**12. The loss of Ca++ homeostasis**

umol1-1

structural disruption of the electron transport chain could lead to an excessive production of free radicals and so to a dramatic increase of the lipoperoxidation phenomena. So, in general, every disruption of the electron transport chain, may lead to an increase in the production of free radicals and potentially represent an initial mechanism in the field of the phenomena which we can name as "Exercise-induced muscle fiber injury".

### **12. The loss of Ca++ homeostasis**

series of cellular degradation processes. However, research which supports in an evident way the role of free radicals in the etiology of muscular lesion, is quite limited, above all if linked to an eccentric contraction as principal mechanical cause. One of the most convincing studies in this field is represented by that of Zerba in 1990 (Zerba et al., 1990), in which the authors showed how, in a murine model, an intraperitoneal injection of superoxide-dismutase limits, after the imposition of a series of eccentric contractions in situ, the drop in the value of P0 of the considered muscle. The treatment based on superoxide-dismutase was able to reduce the drop in the value of P0 for a period of three days following the eccentric exercise. Other experiments, carried out on animal models have further corroborated the hypothesis formu‐ lated by Zerba (Strosova et al., 2005; Kon et al., 2007), so for this it appears reasonable to extend this theoretical model also in a human field (Castilho et al., 1996; Close et al., 2005; Clanton, 2007; Kerkweg et al., 2007; Voss et al., 2008). There is not missing, however, in literature studies which deny the thesis that the administration of anti-oxidant agents may reduce muscular damage connected with high intensity exercise (Warren et al., 1990; Childs et al., 2001; Sacheck and Blumberg, 2001; Kerkweg et al., 2007). It is also important to remember some interesting experiences (Brooks et al., 2008) which underline the fact that the production of free radicals, on behalf of the skeletal muscle, and consequently their control and their regulation, are in function of precise physiological stimuli and how these parameters play a very important role in the field of physiological adaptation of the muscle during the contraction mechanism. These adaptations would include an optimization of the contraction mechanism, and in addition they would represent the beginning of adaptation processes and changes of gene expression regarding stress induced by the muscular contraction. Evidently these beneficial effects of the free radicals in the field of muscular contraction, contrast with contrary scientific evidence, which see the beginning and /or the cause of free radicals of a pathway of degenerative type which would appear fundamental, not only in the field of possible structural damage regard‐ ing the skeletal muscle, but also, more in general, in its aging process. This only apparent contradiction, underlines the necessity of deeper understanding in the role covered by the free radicals in the field of both physical exercise and the sarcopenia. Anyway, despite the relative lack of convincing and undisputable scientific evidence concerning the role of free radicals in the field of initial mechanisms, and /or predisposition of muscular lesion, it is without doubt legitimate to ask ourselves this question: is it reasonable to be able to support an increase in production of free radicals during an eccentric exercise? To answer this interesting and legitimate query, it is useful to remember that some studies (Brand and Lehninger, 1975) show how during an ischemic phenomena, in a model of ischemic /reperfusion damage, we can see in the cardiac muscle a destruction of the normal tight association between the elements of the chain of transport of the electrons. This would provide a particularly evident production of free radicals during the reperfusion phase, phase in which we may find high concentrations of O2 of the tissue(Hess et al., 1982; Arkhipenko et al., 1983; Faust et al., 1988; Fisher, 1988). It is possible to speculate that the high and specific muscular tensions which happen during of eccentric contraction, may alter the normal cytoskeletal structure, of which whose functions are to stabilize the position of the mitochondria (BIgland-Richie and Woods, 1976). The destruction of the cytoskeleton l could, in its turn, cause a disruption of the spatial configura‐ tion of the elements which compose the electron transport chain (Demopoulos, 1973a). This

22 Muscle Injuries in Sport Medicine

If the initial events of harmful mechanism are of mechanical and metabolic nature, the immediately successive phases leading to the same harmful event, are characterized by an elevation of the levels of intracellular Ca++ in the injured area (Statham et al., 1976; Publicover et al., 1978; Kameyana and Etlinger, 1979; Baracos et al., 1984; Carpenter, 1989; Boobis et al., 1990). It is interesting to note how also in patients affected by muscular dystrophy and other muscular pathologies, we may find an increase in the intracellular levels of Ca++ (Jackson et al., 1985 ; Turner et al., 1988). The importance of maintaining the concentration of free cytosolic Ca++, is indirectly underlined by the number of the Ca++ transport mechanisms from the cytosolic compartment which the cell possesses (Gillis, 1985; Klug and Tibbits, 1988). There exists, in fact, at least seven membrane transport systems of Ca++. In the actual state of knowl‐ edge in the specific field, it would seem that there exists, until today, direct evidence of the fact that the elevation of the intracellular levels of Ca++ is involved in the mechanism of "exercise-induced muscle fiber injury" (Hall-Craggs, 1980; Steer and Mastaglia 1986; Childs et al., 2001), even if studies exist which show how in the condition of DOMS, they are present inside the muscle of high contextual levels of Ca++ to a same increased level of mitochondrial Ca++ (Duan et al., 1990a). The hypothesis that would justify an increase of intracellular levels, are essentially based on to the destruction of the sarcolemma found during the harmful event. The sarcolemma in fact represents a suitable barrier for the maintenance of concentration and of the electric gradient between the intra and extra cellular spaces; its destruction so permits the Ca++ to invade the intracellular space. The concentration of free extracellular Ca++ oscillates between 2 and 3 mmol.1-1 whereas that of cytosolic Ca++, in the muscle fiber at rest, is about 0.1 umol1-1 . So it evidently appears how, at the expense of Ca++, there exists and important gradient between the intra and extra cellular space and that how each loss of normal permeability of the barrier, represented by the sarcolemma, may cause an important influx of Ca++ in the intracellular space. In experiments carried out on muscular fibers treated with saponin and incubated in Ca++ solution in concentration between 0.5 and umol- 1-1, we may observe a destruction of the myofibrils and a hyper-contraction of the sarcomeres. (Duncan, 1987). From the moment in which such concentrates enter in the same physiological range seen during an "*in vivo"* muscular contraction, this experimental data could induce us to believe that also during normal contractile activity the level of free cytosolic Ca++ could be high enough to start the degradation of the muscular ultrastructure. However, this event does not happen above all because the increase of the level of cytosolic Ca++ in the course of an *in vivo* muscular contraction is of transient type; In other words at the moment in which the Ca++ is released from the sarcoplasmatic reticulum in the course of the contraction itself, its level is readily limited by the regulating proteins, in such a way that its level seems high only for a short amount of time, and too scarce to allow the activation of proteolytic enzymes (Robertson et al., 1981) ; in addition the proteolytic enzymes inside the fiber are in compartments and for this reason are not influenced by the increase of the level of Ca++ which happens during the stimulus- contraction cycle. So, the damage to the muscular membrane or to the sarcoplasmatic reticulum, may be caused by an increase of the concentrate Ca++ only in those compartments, inside the muscular fiber, where Ca++ is allowed to arrive in contact with the areas of degrading enzymes. (Duncan, 1987). So essentially, it would not be the absolute level of Ca++ which can represent an important starting factor of the process of muscular damage, rather than the temporary length of the magnitude of active movement of Ca++ through the muscular fiber (Duncan, 1987). In some experiments which simulated an injury, similar to that which can happen following an eccentric contraction obtained by using micro injections on the sarco‐ lemma, we observed the area of necrosis corresponding to the place of insertion, was literally "surrounded" by a sort of barrier, made up of hyper-contracted filaments, in which we could find an increase in the concentration of Ca++ (Armstrong et al.,1983b; Ogilvie et al., 1988). A similar mechanism may probably be observed also following an "exercise-induced muscle fiber injury" (Armstrong et al., 1983b; Kuipers et al., 1983; Ogilvie et al., 1988). Many muscular disease show an increase of the levels of intracellular Ca++, caused by the disturbance of the normal barrier permeability of the sarcolemma regarding the Ca++ itself. For example in muscles affected by the of Duchenne muscular dystrophy, the proteic degradation is directly linked to the increase in intracellular levels of Ca++ (Turner et al., 1988). Another example in which we can observe a high concentration of Ca++ is represented by the malignant hyperther‐ mia, in which a specific agent causes a prolonged increase of the concentration of intracellular Ca++ which, in its turn, provokes a massive and uncontrollable muscular contraction, whose consequence is an increase in body temperature which can reach 46° C (Cheah and Cheah, 1985). A second mechanism responsible for the elevation of free cytosolic levels of Ca++ is represented by the malfunction of the sarcoplasmatic reticulum. Apart from the fact that this happens, following an eccentric contraction which has caused muscular damage, represented by a flux of Ca++ from the extracellular space (Duan et al, 1990b) it would still seem certain that the malfunction on behalf of the sarcoplasmatic reticulum in re-absorbing Ca++, may contribute to the increase of its cytosolic concentration. In effect the sarcoplasmatic reticulum reduces its re-absorbing capacities of Ca++ reduced in the course of exercise, both in the case that the intensity of the latter is moderate or maximal (Byrd et al., 1999). However, there is no certain data which can enlighten us in regards to the different possible effects of eccentric, concentric or isometric exercise on the functionality of the sarcoplasmatic reticulum. In any case, it is plausible to put forward the hypothesis that the inhomogeneity of the sarcomeral length can negatively influence on the adjacent segments of the sarcoplasmatic reticulum itself (Arm‐ strong et al., 1991). Some experiments on isolated muscle would go into effect in this sense. When an isolated muscle is incubated with caffeine- a substance which stimulates the Ca++ inducing its release on behalf of the sarcoplasmatic reticulum - it is possible to observe a deterioration of the myofibril structure (Duncan,1987); in other respects also the incubation of isolated muscle in ruthenium red - substance which inhibits the Ca++-ATPase - is able to induce significant damage of the myofibril (Duncan et al., 1980). This experimental data witnesses the

fact that a loss in the homeostasis of Ca++, as in the case of muscular injury, could be, at least in part, due to a malfunction and/or and drop in the efficiency or normal re-absorbing machanisms of Ca++ on behalf of the sarcoplasmatic reticulum. Some authors, to this end, emphasize the fact that the mechanisms which cause the destruction of the membrane, are mainly responsible for the increase in levels of intracellular Ca++ inside the injured fibers (Armstrong et al. 1991), even if we have to admit the existence of other numerous factors able to perturb the homeostasis of the latter. For example some studies (Snowdowne and Lee, 1980; Lopez et al., 1985) would evidence the existence, inside the skeletal muscle, of "stretchsensitive calcium channels" ; so - from the moment the muscle during an eccentric contraction is mechanically elongated at the same time in which it is electrically active- the hypothesis appears more than plausible that these specific channels are involved in mechanisms which induce, during the eccentric contraction itself, the increase in intracellular Ca++ levels. Another mechanism which could be implied in the increase of intracellular Ca++ would be the pathway

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25

: Ca++, through which the mechanism of uptake and the release of Ca++ on cellular level is completed, (Allen et.al., 1989), even if in truth there isn't any unequivocal evidence of its involvement in the field of Ca++ overload mechanism observable in the injured skeletal muscle. It has also been shown how the inhibition of the acetylcholinesterase on a level of neuromuscular junctions, causes and influx of Ca++ inside the muscular area, contextual to contrac‐ tion of the fibers and local necrosis of the latter (Leonard and Salpeter, 1979). Also other similar experiments, which have induced an increase in the release of Ca++ on behalf of the sarcoplas‐ matic reticulum have permitted to observe an increase in the contraction of the fibers, together with a rapid process of destruction of the myofibril structure- which happen in less than 30 minutes- in addition to a drop of the intramuscular enzymes (Duncan, 1987). This data underlines the importance which the role of an increase in intracellular Ca++ levels could have in the field of construction of the theoretical model of "exercise-induced muscle fiber injury". One of the consequences of the elevation of the level of intracellular Ca++ is represented by the phenomena called "blebbing" which consists in the formation of cytoplasmic vesicular enlargements on the cellular surface. We retain that these alterations are to be put in relation with a possible disturbance of the relationship which runs between the cytoskeletal proteins (in particular actin and tubulin ) and the cell of the membrane (Orrenius et al., 1989). The "blebbing " phenomena is furthermore observable also outside the model represented by the skeletal muscle, like for example in the cells of the myocardium in the field of the model of "ischemia-reperfusion" (Ganote and Humphrey, 1985; Ariel et al., 2008). In the light of what we have already said, in the field of the theoretical model of "exercise-induced muscle fiber injury" we may think that, during exercise itself, an initial damage occurs, of probable mechanical nature, at the expense of the designated components for the maintenance of a correct permeability of the barrier regarding extracellular Ca++. This alteration would allow a massive diffusion, through the damaged membrane site, of Ca++, giving origin, in such a way, to the said phenomena of "Ca++ overload", whose consequence is represented by the annihi‐ lation of the tampon systems of the muscular fiber (like for example the Ca++ binding proteins, the functionality of the sarcoplasmatic reticulum and the mitochondria). Once the level of cytosolic Ca++ has reached a critic level, which remains for a sufficiently long level of time- and

of Na+

fact that a loss in the homeostasis of Ca++, as in the case of muscular injury, could be, at least in part, due to a malfunction and/or and drop in the efficiency or normal re-absorbing machanisms of Ca++ on behalf of the sarcoplasmatic reticulum. Some authors, to this end, emphasize the fact that the mechanisms which cause the destruction of the membrane, are mainly responsible for the increase in levels of intracellular Ca++ inside the injured fibers (Armstrong et al. 1991), even if we have to admit the existence of other numerous factors able to perturb the homeostasis of the latter. For example some studies (Snowdowne and Lee, 1980; Lopez et al., 1985) would evidence the existence, inside the skeletal muscle, of "stretchsensitive calcium channels" ; so - from the moment the muscle during an eccentric contraction is mechanically elongated at the same time in which it is electrically active- the hypothesis appears more than plausible that these specific channels are involved in mechanisms which induce, during the eccentric contraction itself, the increase in intracellular Ca++ levels. Another mechanism which could be implied in the increase of intracellular Ca++ would be the pathway of Na+ : Ca++, through which the mechanism of uptake and the release of Ca++ on cellular level is completed, (Allen et.al., 1989), even if in truth there isn't any unequivocal evidence of its involvement in the field of Ca++ overload mechanism observable in the injured skeletal muscle. It has also been shown how the inhibition of the acetylcholinesterase on a level of neuromuscular junctions, causes and influx of Ca++ inside the muscular area, contextual to contrac‐ tion of the fibers and local necrosis of the latter (Leonard and Salpeter, 1979). Also other similar experiments, which have induced an increase in the release of Ca++ on behalf of the sarcoplas‐ matic reticulum have permitted to observe an increase in the contraction of the fibers, together with a rapid process of destruction of the myofibril structure- which happen in less than 30 minutes- in addition to a drop of the intramuscular enzymes (Duncan, 1987). This data underlines the importance which the role of an increase in intracellular Ca++ levels could have in the field of construction of the theoretical model of "exercise-induced muscle fiber injury". One of the consequences of the elevation of the level of intracellular Ca++ is represented by the phenomena called "blebbing" which consists in the formation of cytoplasmic vesicular enlargements on the cellular surface. We retain that these alterations are to be put in relation with a possible disturbance of the relationship which runs between the cytoskeletal proteins (in particular actin and tubulin ) and the cell of the membrane (Orrenius et al., 1989). The "blebbing " phenomena is furthermore observable also outside the model represented by the skeletal muscle, like for example in the cells of the myocardium in the field of the model of "ischemia-reperfusion" (Ganote and Humphrey, 1985; Ariel et al., 2008). In the light of what we have already said, in the field of the theoretical model of "exercise-induced muscle fiber injury" we may think that, during exercise itself, an initial damage occurs, of probable mechanical nature, at the expense of the designated components for the maintenance of a correct permeability of the barrier regarding extracellular Ca++. This alteration would allow a massive diffusion, through the damaged membrane site, of Ca++, giving origin, in such a way, to the said phenomena of "Ca++ overload", whose consequence is represented by the annihi‐ lation of the tampon systems of the muscular fiber (like for example the Ca++ binding proteins, the functionality of the sarcoplasmatic reticulum and the mitochondria). Once the level of cytosolic Ca++ has reached a critic level, which remains for a sufficiently long level of time- and

limited by the regulating proteins, in such a way that its level seems high only for a short amount of time, and too scarce to allow the activation of proteolytic enzymes (Robertson et al., 1981) ; in addition the proteolytic enzymes inside the fiber are in compartments and for this reason are not influenced by the increase of the level of Ca++ which happens during the stimulus- contraction cycle. So, the damage to the muscular membrane or to the sarcoplasmatic reticulum, may be caused by an increase of the concentrate Ca++ only in those compartments, inside the muscular fiber, where Ca++ is allowed to arrive in contact with the areas of degrading enzymes. (Duncan, 1987). So essentially, it would not be the absolute level of Ca++ which can represent an important starting factor of the process of muscular damage, rather than the temporary length of the magnitude of active movement of Ca++ through the muscular fiber (Duncan, 1987). In some experiments which simulated an injury, similar to that which can happen following an eccentric contraction obtained by using micro injections on the sarco‐ lemma, we observed the area of necrosis corresponding to the place of insertion, was literally "surrounded" by a sort of barrier, made up of hyper-contracted filaments, in which we could find an increase in the concentration of Ca++ (Armstrong et al.,1983b; Ogilvie et al., 1988). A similar mechanism may probably be observed also following an "exercise-induced muscle fiber injury" (Armstrong et al., 1983b; Kuipers et al., 1983; Ogilvie et al., 1988). Many muscular disease show an increase of the levels of intracellular Ca++, caused by the disturbance of the normal barrier permeability of the sarcolemma regarding the Ca++ itself. For example in muscles affected by the of Duchenne muscular dystrophy, the proteic degradation is directly linked to the increase in intracellular levels of Ca++ (Turner et al., 1988). Another example in which we can observe a high concentration of Ca++ is represented by the malignant hyperther‐ mia, in which a specific agent causes a prolonged increase of the concentration of intracellular Ca++ which, in its turn, provokes a massive and uncontrollable muscular contraction, whose consequence is an increase in body temperature which can reach 46° C (Cheah and Cheah, 1985). A second mechanism responsible for the elevation of free cytosolic levels of Ca++ is represented by the malfunction of the sarcoplasmatic reticulum. Apart from the fact that this happens, following an eccentric contraction which has caused muscular damage, represented by a flux of Ca++ from the extracellular space (Duan et al, 1990b) it would still seem certain that the malfunction on behalf of the sarcoplasmatic reticulum in re-absorbing Ca++, may contribute to the increase of its cytosolic concentration. In effect the sarcoplasmatic reticulum reduces its re-absorbing capacities of Ca++ reduced in the course of exercise, both in the case that the intensity of the latter is moderate or maximal (Byrd et al., 1999). However, there is no certain data which can enlighten us in regards to the different possible effects of eccentric, concentric or isometric exercise on the functionality of the sarcoplasmatic reticulum. In any case, it is plausible to put forward the hypothesis that the inhomogeneity of the sarcomeral length can negatively influence on the adjacent segments of the sarcoplasmatic reticulum itself (Arm‐ strong et al., 1991). Some experiments on isolated muscle would go into effect in this sense. When an isolated muscle is incubated with caffeine- a substance which stimulates the Ca++ inducing its release on behalf of the sarcoplasmatic reticulum - it is possible to observe a deterioration of the myofibril structure (Duncan,1987); in other respects also the incubation of isolated muscle in ruthenium red - substance which inhibits the Ca++-ATPase - is able to induce significant damage of the myofibril (Duncan et al., 1980). This experimental data witnesses the

24 Muscle Injuries in Sport Medicine

above all if the latter stays high inside the specific compartments of the fiber - different degradation mechanisms start inside the injured muscle fiber which are represented by:

glycolitic fiber level (Sembrowich and Quintinskie, 1985). An excess in uptake of Ca++ on behalf of the mitochondria is accompanied by a contemporary uptake in phosphates causing, in such a way, a precipitation of calcium phosphate which can deposit itself in the intra-mitochondrial spaces (Gillis,1985). So, on the one hand, an increase in the level of mitochondrial Ca++, which stays in a nano-molar range, appears useful in stimulating the mitochondrial respiration, whereas on the other hand an accumulation of Ca++, in a micro-molar range, causes a depression of the respiratory functions on a level with the mitochondria itself (Wrogemann and Pena,

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The Ca++ dependent protease is of two types: type 1 and type 2: this division is based on the level of Ca++ necessary for their activation. The type 1 isoform is activated in presence of micromolar levels of Ca++, whereas the type 2 form needs quantities in milli- molars for its activation (Murachi et al., 1981). Unlike not for the lysosomal protease, this enzyme has it optimal pH in the field of neutrality. Its activation is associated with the degradation of particular structures inside the myocell and in particular in the degradation of the Z band (Bush et al., 1972; Ishiura et al., 1980), of the myofilaments (Daytona et al., 1976; 1979; Cullen and Fulthorpe, 1982) and of the A band (Friden et al., 1981; Newham et al., 1983; Ogilvie et al., 1988). All these alterations are observable in an injured muscle following eccentric exercise. Also the proteins of the cytoskeleton would represent a preferential underlayer for the action of the Ca++ dependent protease (Pontremoli and Melloni, 1986) To this end, there has been a hypothesis that the proteolysis of the vinculin (a protein of the cytoskeleton which anchors the cellular membrane to the cytoskeleton) on behalf of the Ca++ dependent protease, causes a fragility of the sarco‐ lemma of the myocardium cells in the course of the ischemic process (Steenbergen et al., 1987a).

Since the myofibrillar protein may be degraded by the proteolytic enzymes contained in the lysosomes of the muscular fibre (Schwartz and Bird,1977), it is reasonable to suppose that the lysosomial protease plays an important role in the field of the successive autogenic phase to the muscular damage. This supposition is corroborated by the evidence of a strong increase in the lysosomal protease, following exhaustive exercise in an animal model (Vinko et al., 1978). There is also evidence of the fact that the lysosomal enzymes are activated by the increase of

The phospholipase A2 (PLA2) uses the phospholipidic membrane as an underlayer for the production of arachidonic acid, prostaglandin – in particular the prostaglandin E2 (PGE2)-

1976; Hansford, 1985; McMillin and Madden, 1989).

**16. The lysosomal protease**

the level of intracellular Ca++ (Rodemann et al., 1982).

**17. The pathway of the phospholipase A2**

**15. The mechanism of the dependent Ca++ protease**


### **13. The mechanism of the reflex myofibrillar contraction**

The loss of the homeostasis of the Ca++ involves an uncontrollable contractionreflex (or not through the SNC) by the sarcomeres inside the injured area (Ogilvie et al., 1988). We need however to specify that the myofibrillar contraction reflex phenomena, does not have to be necessarily understood as a degradation phenomena in the strict meaning, like for example the enzymatic pathway could be. Even if some authors have put forward the hypothesis that this zone of concentration may make up a sort of barrier apt to block the degradation processes preventing the latter to extend to the sarcomeres adjacent to the injured zone (Carpenter and Karpati, 1989), we need to consider that this uncontrolled state of contraction of the sarcomeres may have serious consequences in the field of aggravation of structural damage. The first negative effect is represented by the local depletion of ATP following the endurance of the contraction itself, which would give origin to a vicious circle, and so, capable of auto sustain‐ ment, identifiable in "depletion of ATP- increase in levels of Ca++" and vice versa (Goodman, 1987). The second negative outcome of the mechanism of myofibrillar contraction reflex is made up of the fact that such a phenomena produces mechanical forces, inside the fibers able to damage further both the membrane and the same contractile components, contributing in such a way to further deterioration of the clinical situation (Armstrong et al., 1991).

### **14. The phenomena of mitochondrial Ca++ overload**

The mitochondria inside the muscular fiber have, among their tasks, also that which to react to "buffer", or to tampon mechanism, regarding the increase of the concentration of cytosolic Ca++. However the hypothesis is generally creditable that the uptake of Ca++ on a mitochondrial level is quite modest, and in any case insufficient to be able to consider as fundamental, or at least important, the role taken on the mitochondria itself in the field of the mechanism of relaxation of the muscular fiber. Even though we need to remember that the mitochondria, in particular pathologic situations, are capable of accumulating a large quantity of ions (Gillis, 1985). Between all the types of fibers, the oxidative ones show marked capacity of mitochon‐ drial buffering regarding the Ca++ which can exceed the registered ones by 2-3 times on a glycolitic fiber level (Sembrowich and Quintinskie, 1985). An excess in uptake of Ca++ on behalf of the mitochondria is accompanied by a contemporary uptake in phosphates causing, in such a way, a precipitation of calcium phosphate which can deposit itself in the intra-mitochondrial spaces (Gillis,1985). So, on the one hand, an increase in the level of mitochondrial Ca++, which stays in a nano-molar range, appears useful in stimulating the mitochondrial respiration, whereas on the other hand an accumulation of Ca++, in a micro-molar range, causes a depression of the respiratory functions on a level with the mitochondria itself (Wrogemann and Pena, 1976; Hansford, 1985; McMillin and Madden, 1989).
