Creatine Isoforms and Muscle Damage

Introduction

We have all experienced the pain and stiffness that comes with beginning an exercise program. So too have athletes when starting a new training regimen. This phenomenon is referred to as delayed onset muscle soreness, or DOMS, and is associated with muscle fiber injury incurred during novel activities. This is different than the transient pain fetl during exercise which is related to fatigue(16). DOMS is especially common after performance of eccentric contractions, which produce force while simultaneously lengthening the fibers involved. Along with this damage comes other related symptoms such as prolonged weakness, a decreased range of motion, and muscle protein leakage into the blood plasma(3,4,9). The side effects incurred with muscle damage can have profound effects on physical performance such as that seen in athletic competition. Questions important to the athlete need to be answered. Should elite caliber athletes train through the pain? Is it better to rest and recuperate? These are questions that previous research in the disciplines of both exercise science and biomechanics have tried to answer. And although successful in uncovering important findings in their respective fields they have been unable to correlate the data into practical applications for training. This is what this study, being carried out by Donna Goff, is aspiring to do. The purpose of this study was to examine how exercise induced muscle damage impacts upon both the morphology and functioning of the cells themselves. The study did this by looking at three variables, or criterion measures. By learning how these variables are interrelated we can see how their resulting dynamics impact upon level running gait. Subjective soreness (DOMS), protein leakage (plasma enzyme levels), and loss of function (biomechanics) are these criteria.

Literature Review Muscle Damage: Immediately following novel exercise, especially that which is eccentric in nature, evidence exists showing that fiber level injury has occurred (3,6). Because downhill running fits this description and is eccentric in nature inducing a CK response it is assumed to induce soreness. Muscle biopsies validate this theory with evidence that the contractile elements are being damaged(16). Examples of injury include sarcomeric structural damage such as Z-line streaming as well as leakage of intramuscular proteins into blood plasma (3,6,9,13). A closer microscopic examination reveals further damage to subcellular components (3). Some plasma enzymes include creatine kinase, myoglobin, and protein metabolites (3). CK and myoglobin levels correlate well with each other post activity indicating that exercise effects them both in the same way (6). These symptoms translate to a loss of muscular force production and the sensation of DOMS (3). The feeling of DOMS is associated with muscle shortening, swelling, and a loss in force production(8). There is also a sensation of pain, tenderness, and stiffness. DOMS begins 24-48 hrs. after exercise and peaks between 24-72 hrs(9,16). However, Lieber et al. found that structural changes to the fiber are present as soon as 5-15 mins. post exercise(14). One theory for the soreness associated with DOMS is that free nerve endings located in the connective tissue at the ends of fibers are stimulated by damage in both the tendons and muscle fibers(8,16). This manifests itself in a decreased resting angle which may be correlated with the amount of extension pain (8). After eccentric contractions the amount of connective tissue increases making them resistant to stretch and in turn decreases mechanical strain(13). The decrease in resting angle is both electrically silent and highly correlated with peak soreness(16). This happens along with a strength decrease which was demonstrated by Nosaka et al. who showed a 50% decrease in isometric force production which persists for long periods of time even after pain subsides(17). Lieber et al. proposed the possible presence of intermediate fibers which transmit force from the contractile proteins to the surrounding matrix(14). Without them strength will suffer. Despite the efforts of previous work on exercise induced muscle injury, no solid conclusion has been raised as to the mechanisms underlying the phenomenon(9). They appear to be intrinsic to the cells themselves and exclude the processes of self healing. This means that inflammation related to the nomadic journey of phagocytic cells into the injured site is not responsible for inducing morphological changes (3). An example is seen as fibers use proteolytic and degradative pathways that begin before the inflammatory reaction in respose to induced damage(3). Alternate explanations include the possible production of free oxygen radicals which are extremely reactive with the phospholipids making up cell membranes leaving them ineffective in holding isoenzymes(3,9,16). Armstrong proposed a four-stage theory of exercise induced muscle damage. The four stages are initial, autogenic, phagocytic, and regenerative and comprise the entire process of tissue rebuilding . The initial cause of the damage is the result of high "specific tensions" which are easily attainable through eccentric contractions (3). This would cause focal injury in and among fibers including mechanical failure in the sarcoplasm and filaments and is manifested in three lesion types: A-band disruption, Z-line dissolution, and clotted fibers (3). Byrnes and colleagues proposed the idea that a small pool of weak or degenerating fibers exist since a small percentage of fibers are effected (6). This could be due to inefficient motor unit recruitment patterns(8). The initial stage can be of either mechanical or metabolic origin. Physical stress placed upon the sarcolemma disrupts cell membrane permeability. This begins a sub-phase of Ca++ overload (4). If the overload is relatively small then the ATPase pumps can handle removing the excess Ca++ rather easily. However, if the concentration overwhelms the ability of these pumps to keep up then an "irreversible" injury can occur. This type of injury is characterized by cell necrosis brought on by processes for major damage being set in motion like a chain reaction(4,9,14). The autogenetic phase is the time period starting before phagocytes arrive and continues on during the inflammatory phase in which these cells become active at the damage site. Loss of Ca++ homeostasis within the fiber is thought to be the triggering catalyst that leads into the autogenetic phase of injury (3,4). The phagocytic phase commences 2-6 hrs. post trauma and continues on for several days. Lastly, the regenerative phase completes the cycle and returns fibers to a normal state. Once the repair is finished previously vulnerable cells are left better able to cope with stresses placed upon them. Ebbeling and Clarkson also wrote about the differences in metabolic and mechanical considerations. Previous feelings about metabolic possibilities delt with the issues of ischaemia and hypoxia. It was thought that ion imbalances, ATP deficiency, and accumulation of toxins were the cause of damage. However, now the feeling is that initial injury is caused by mechanical factors and complicated by metabolism(9,13). The initial damage is treated by an inflammatory response. As Ca++ builds up in the cell it induces chain reactions responsible for injury. The Ca++ levels then rise impairing ATP production(13). Some mechanical considerations pointed to the fact that during the eccentric phase of activity few fibers carry the workload. This translates to significant damage to the contractile apparatus despite eccentric tension's relatively cheap energy cost(9,13). Training the fibers damaged will improve their resistance to future injury. The adaptation is manifested in less soreness and reduced enzyme release in subsequent bouts of activity. Armstrong et al. found that initially weaker fibers are replaced with a stronger version to resist stress(4). Byrnes et al. demonstrated a "prophylactic effect" as a single exercise bout decreased soreness, CK response, and myoglobin counts in follow-up sessions for up to six weeks (6,17). It is believed that adaptation begins even before complete healing has occured(17). Possible explanations include eliminating weak fibers, improving cytoskeletal structure, and adapting neural responsibility(17). The reason is believed to be an increase in connective tissue making fibers less susceptible to later stresses. But is this really a good thing? Pain serves an important purpose. It lets the athlete know that weakness still exists and needs time to repair itself. Pain is detected by receptors which are free nerve endings that sense pain when stimulated by chemical, mechanical, and thermal stimuli(16). Two types of receptors exists and work together to produce accurate sensory descriptions of pain. Type III fibers quickly transmit feelings of sharp pain to the brain while its accomplice, Type IV fibers, sense dull aches slowly(16). If the athlete continues to train during a period of weakness they are opening themselves up for overuse injuries. In people there is a mismatch between intensity of soreness and peak swelling. DOMS begins 24-48 hrs. post exercise but cellular activity starts 3-4 days after(13). Since the affliction doesn't fit the symptoms other factors are thought to be responsible for the sensation of pain. Nosaka and Clarkson proposed that training with tissue damage may not generate a sufficient overload(17). Also, motor unit recruitment patterns may be altered and in this vulnerable state training is contradictory to the principle of specificity and may worsen present damage(17).

Because creatine kinase is mostly found in muscle tissue its presence in blood plasma is indicative of damage to the tissue(9). When measuring total CK in the blood it is important to consider that the enzyme level represents both release and clearance of the enzyme(9,2). Individual plasma enzyme respose is highly variable and related to subject fitness level as well as mode, intensity, and duration of exercise (4). These factors affect both response magnitude and time course of release following injury. This is where creatine kinase analysis, specifically the MM1 isoform of it, becomes important. CK analysis is an indirect method for assessing the extent and chronological order of fiber damage dynamics(9). Understanding the structure and function of creatine kinase can give us insite into the mechanisms behind injury. The structure of CK is composed of a "dimeric organization consisting of M and B subunits"(9). Three isoenzymes of CK exist and are specific to their tissue origin and become apparent when undergoing chromatographic and electrophoretic testing(2). CK-MM, which is the skeletal muscle form, accounts for 90-100% of total CK activity(2,9). It is this variation of the enzyme that is responsible for the rise in plasma CK following strenuous exercise(9). It is possible to seperate the three isoforms in a process called electrophoresis, or isoelectric focusing. The process works because of the "differing rates of migration in an electric field"(9). Once CK enters the plasma in the form of MM1 it is then converted into MM2 and then in turn to MM3(1,7,9). Because of this it is more appropriate to utilize MM1 as the true inicator of enzyme release rather than levels of total CK present(1,2,7). It has also been shown that MM1 parallels the onset of soreness(9). Release rates of CK into plasma are dependent on the type and intensity of exercise mode. After downhill running CK increases 3-6 hrs. post and reaches peak levels at 18- 24 hrs. after commencment of exercise bout(9). Apple et al. found that both the percentage of MM1 and the MM1/MM3 ratio increased as well at 6 hrs. post(2,7). This is different than local eccentric activity in which we see an increase at 48 hrs. but may not reach peak for seven days(9). It also appears that there is a significant difference between male and female CK responses. After a marathon Apple et al. identified a difference of 2552 U/g to 1598 U/g for men and women respecively(1). This could be due to a greater muscle mass being used to perform the activity. However, there was no difference in clearance rates between the sexes. A peak level for both sexes was found at 24 hrs. post for MM1 indicating leakage into the circulation(1). A small amount of MM1 is present at rest and is the result of normal functioning(7). When comparing CK with other enzymes we find interesting insites to CK release mechanisms. If membrane damage is the deciding factor in enzyme release then they should all have an identical time course for leakage. But they don't. This points to the possibility that CK is somehow different in its release mechanisms(18). It is interesting to look at differences the aging process imposes on CK as well. Manfredi et al. showed that after a comparable exercise bout older subjects had 90% focal damage in contrast to 5-50% by there younger counterparts(15). They believed this was true because of less muscle mass in the older subjects meaning more force was placed upon less area. However, Nosaka and Clarkson found no relationship between muscle mass and CK(18). They also found a decrease in the number of fibers associated with aging lessoning the efficiency of motor unit recruitment. This led to more damage but surprisingly CK levels were parallel in both groups(15). So it would appear that CK is not a good indicator of damage. CK seems to be more a result of damage than an accurate predictor of it(9). A useful tool for assessing clearance rates for CK isoforms is the MM1/MM3 ratio. It gives an index of enzyme release rate from damaged tissue(7). A value greater than one indicates a continued release of enzyme from the cell and less than one shows clearance is ongoing(1). This ratio may be the most accurate sign of enzyme release. The lymphatic system is the connection from the extracellular spaces to the blood. This is how the CK gets there. It is thought that because of the slow rate of lymph flow the time lapse between cessation of exercise and apparence of CK in plasma can be significant. Peak CK release is seen after ATP and CP are able to regenerate(9). Necrosis of tissue is believed to be associated with this phenomenon. An early release of CK indicates possible hypoxia and/or ischaemia(9). These two abnormalities are thought to affect membrane permeability and thus enzyme release. With exercise an energy reduction may incur permeability differences and damage in skeletal muscle. Hypoxia increases membrane permeability as well as Ca++ concentrations within the cell leading to fiber disruption. Ischaemia may also affect enzyme release as indicated by higher CK levels following bouts of isometric exercise(9). Variability in CK response between subjects is a confounding factor in muscle damage studies. After local eccentric exercise high plasma levels are found which are approximately 2,000 U/L. This is compared to studies which document levels in the range of 500-34,500 U/L(9). And despite expectations, there seems to be no correlation with fitness level and other factors. One theory is that some subjects may release CK inhibitors into the blood from injured sites. Another proposes a genetic component, and lastly the idea of the "prophylactic effect" may help find an explanation(9,18). Nosaka and Clarkson showed indicators of damage such as swelling and ROM parallel CK activity. CK also correlated with other enzymes(18). They concluded that the variability in CK response was due to the "change in signal intensity in MR images"(18).

Biomechanical Adaptations: An eccentric muscle activation is the controlled lengthening of the muscle under tension. During a gait cycle, or stride, most leg muscles work eccentrically to support the body against gravity and to absorb shock. When running downhill eccentric contractions become even more important as they need to resist their own acceleration of COM. As a result of damage a loss of fuctioning is incurred. Manifestations of this damage include performance decrements such as "loss of force production, reduced stimulated tetanic muscle tension, and a decreased ROM at the associated joint"(19). Performance may also be worsened by physiological tremor and proprioceptive decrements(19). One effect of eccentric exercise brought on by downhill running is the loss of strength for several days after. The long term losses in force production is thought to be a result of damage to the sarcoplasmic reticulum(10). This would impair Ca++ return to the cells repressing the ability to generate force. Sarcomeres are shorter near muscle ends. If they are pulled apart by eccentric lengthening overlap between contractile proteins is reduced, making them less efficient in creating cross bridges(10). The degree to which strength decreases is related to muscle length during the eccentric exercise. The shorter the length, the less change in force production. Muscle soreness is also greater when exercising at longer lengths(10). Therefore length and magnitude are important determinants in strength changes. DOMS appears to have little effect on the kinematics of the lower extremity during gait. However, soreness incurred through exercise causes a loss in functional muscle strength, stiffness, and an increase in enzyme levels in the blood plasma(11). Because tenderness occurs almost immediately post activity, it is felt tenderness originates in connective tissue(10). And although present with palpation and movement, DOMS is not felt at rest(20). With DOMS there may be a decrease in economy of movement, impairment of glycogen repletion, and/or changes in biomechanics of movement(20). These differences put the athlete at an increased risk of injury. Running downhill requires eccentric contractions of the legs for the purpose of slowing down the body's center of mass(11). This requirement is much less for level running gait which only needs to fulfill the swing and stance phases of gait. In general, eccentrics are responsible for shock absorption or braking in the direction of gravity(20). Altered gait patterns can have profound effects on shock attenuation abilities of the lower extremities. Knee and hip flexion and ankle dorsiflexion actions aid in shock attenuation during the initial portion of the support phase. DOMS in the quadriceps tranlates to decreased flexion at the hip and knee and a loss of shock attenuation ability. To compensate, the ankle increases dorsiflexion angle during support(11). Loss of this property leads to increased demands being placed upon the rest of the body meaning it’s only a matter of time before injury occurs. During normal locomotion the extensors of the lower limb contract eccentrically during each stride to decelerate the COM after foot strike(10). In downhill running the same action occurs only to a greater degree. However, in downhill running, changes in peak flexion angles are greater which causes a lengthening of the muscle. At this longer length the fibers are more open to injury. “As the strain on the lengthening muscle simultaneously increases with the degree of decline the potential for DOMS related damage is significant”(10). With the increased negative work requirement during downhill running more damage is the result. Adaptation to this greater damage is a reduction in stride length and knee ROM(12). Runners having DOMS adapt by shortening the stride length and reducing the excursion of their lower leg(12). Recovery from DOMS takes several days. Measures for total stride, stride length, stance period, stride period, and mechanical work and power aren't affected by DOMS. Therefore overall stride isn't affected by the presence of DOMS(11). Previous training reduces soreness, cell structural damage, and performance decrements(10). However, the regimen must consist of an eccentric component as concentric training does little in the way of eliciting adaptation. After a single bout of activity the soreness response was repressed for up to six weeks post(10). Strength loss and DOMS have different mechanisms for their existence. Clarkson et al. found that training also improves motor unit recruitment to avoid vulnerable fibers as well as distribute forces over more units avoiding severe injury(9). When exercise results in soreness to the muscles specific to exercise what should the athlete do? Is it better to continue training or to rest? There are two main proposed reasons why rest may be a better alternative. First, training at less than perfect neuromuscular functioning is contrary to the principle of specificity(11). An example is as swelling reduces the ROM around the associated joint altering kinematics of joint and surrounding segment motion(20). Second, chances of injuring oneself increase with altered movement patterns that come with soreness and stiffness(11). The damage caused by downhill running translates to level running adaptations that make lower limb injury a greater possibility. The sensation of pain serves an important purpose. As long as the athlete is able to distinguish between transient uncomfortable feeling and the pain of injury he can adjust training accordingly. However, DOMS can cause changes that affect performance. The phenomenon of natural splinting would indicate that rest is the best choice during the healing process(20). Recommendations for dealing with DOMS include taking time to rest, being mindful of the eccentric component involved in novel activities, and using the "repeat bout" training technique(20)

Reference List

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