It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.

Exercise-induced muscle injury: a calpain hypothesis.

This study investigated intramuscular triacylglycerol (IMTG) and glycogen utilisation, pyruvate dehydrogenase activation (PDHa) and acetyl group accumulation during prolonged moderate intensity exercise. Seven endurance-trained men cycled for 240 min at 57 % maximal oxygen consumption (V(O2,max)) and duplicate muscle samples were obtained at rest and at 10, 120 and 240 min of exercise. We hypothesised that IMTG utilisation would be augmented during 2-4 h of exercise, while PDHa would be decreased secondary to reduced glycogen metabolism. IMTG was measured on both muscle samples at each time point and the coefficient of variation was 12.3 +/- 9.4 %. Whole body respiratory exchange ratio (RER) decreased from 0.89 +/- 0.01 at 30 min to 0.83 +/- 0.01 at 150 min and remained low throughout exercise. Plasma glycerol and free fatty acids (FFAs) had increased compared with rest at 90 min and progressively increased until exercise cessation. Although plasma glucose tended to decrease with exercise, this was not significant. IMTG was reduced at 120 min compared with rest (0 min, 15.6 +/- 0.8 mmol kg(-1) d.m.; 120 min, 12.8 +/- 0.7 mmol kg(-1) d.m.) but no further reduction in IMTG was observed at 240 min. Muscle glycogen was 468 +/- 49 mmol kg(-1) d.m. at rest and decreased at 120 min and again at 240 min (217 +/- 48 and 144 + 47 mmol kg(-1) d.m.). PDHa increased above rest at 10 and 120 min, but decreased at 240 min, which coincided with reduced whole body carbohydrate oxidation. Muscle pyruvate and ATP were unchanged with exercise. Acetyl CoA increased at 10 min and remained elevated throughout exercise. Acetylcarnitine increased at exercise onset but returned to resting values by 240 min. Contrary to our first hypothesis, significant utilisation of IMTG occurred during the first 2 h of moderate exercise but not during hours 2-4. The reduced utilisation of intramuscular fuels during the last 120 min was offset by greater FFA delivery and oxidation. Consistent with the second hypothesis, PDHa decreased late in moderate exercise and closely matched the estimates of lower carbohydrate flux. Although the factor underlying the PDHa decrease was not apparent, reduced pyruvate provision secondary to diminished glycolytic flux is the most likely mechanism.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2002.018820

Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man

Intensive training for and competition in endurance events like the marathon are accompanied by injury to fibres in the active skeletal muscles. Evidence for the injury comes from the increases in intramuscular enzymes and myoglobin found in the blood following the exercise, from the subjective sensation of soreness in the muscles in the post-exercise period, and from direct histological examination of samples of the damaged muscles. Histological studies demonstrate that some muscle fibres undergo degenerative changes following the exercise; the necrosis is accomplished by macrophages and other phagocytic cells that invade the injured cells and the adjacent interstitium. Following the degeneration the fibers appear to be regenerated so that there is not a net loss of fibres. Precisely what initiates the cellular damage is not known, but hypotheses suggested include, 'metabolic overload' and 'mechanical strain'. Eccentric contractions are known to cause the greater amount of damage in muscles, which suggests that high local tensions in fibres may be more important than metabolic considerations in the aetiology of the injury. Training reduces the magnitude of the damage that occurs in response to a given exercise task, although competitors in endurance events may demonstrate chronic muscle injury because of increasing training intensities. Other than training, there is no compelling evidence that any drug treatment or preventative measures will lessen this form of injury.

Muscle damage and endurance events.

Skeletal muscle adapts to the stress of endurance and sprint exercise and training. There are 2 main types of skeletal muscle fibre — slow twitch (ST) and fast twitch (FTa, FTb, FTc). Exercise may produce transitions between FT and ST fibres. Sprint training has decreased the proportion of ST fibres and significantly increased the proportion of FTa fibres, while endurance training may convert FTb to FTa fibres, and increase the proportion of ST fibres (i.e. FTb → FTa → FTc → ST). However, the high proportion of ST fibres documented for elite endurance athletes may be simply the result of natural selection. ST fibres function predominantly during submaximal exercise, whereas FT fibres are recruited as exercise intensity approaches VO2max and/or glycogen stores are depleted. Long distance runners have greater ST and FT fibre areas than untrained controls. However, doubt remains as to whether the ST or FT fibre area is greatest in endurance athletes. Increases in FT fibre area seem to occur during the first 2 months of training, whereas ST fibre areas appear to increase after 2 to 6 months of training. Sprint training leads to the preferential use of FT fibres and male, but not female sprinters have larger FT fibres than untrained controls. Mitochondrial proteins and oxidative enzymes, as opposed to VO2maxare important determinants of the duration of endurance exercise. Endurance training increases intramuscular glycogen stores in both FT and ST fibres and produces a ‘glycogen-sparing’ effect which is characterised by an increased free fatty acid (FFA) metabolism. The activity of glycogen synthase is also increased by endurance training. Sprint training increases glycogen concentrations similarly in all fibre types, reduces the rate of glycogen utilisation at submaximal workloads and allows supramaximal workloads to be maintained for longer periods of time. During endurance exercise the pattern of glycogen depletion varies between muscle fibre types and between muscle groups. Glycogen stores in ST fibres are utilised initially, followed by stores in FTa then FTb fibres. Sprint activities are associated with a much greater rate of glycogen depletion. However, it is unlikely that glycogen depletion causes fatigue during sprinting. Sprint work is associated with a preferential depletion of glycogen from FTb then FTa and ST fibres. Endurance training appears to increase triglyceride stores adjacent to mitochondria and ST fibres have greater triglyceride stores than FT fibres. Endurance exercise is associated with a preferential use of triglycerides from ST fibres and endogenous triglycerides may account for over 50% of the total lipid oxidised during exercise. The oxidation of fat is unlikely to be a significant factor in sprinting tasks. Skeletal muscle has an increased capacity to form alanine from pyruvate after endurance training and leucine oxidation may also be enhanced. The largest increase in amino acid metabolism during exercise occurs from intra-rather than extramuscular sources. The pool of free amino acids is used by the glucose-alanine cycle and during BCAA oxidation. However, prolonged physical activity reduces the amino acids available for these metabolic pathways, suggesting that the use of protein as an energy substrate is limited. In contrast, short term exercise is associated with high plasma alanine levels and thus, it is likely that BCAA oxidation increases during sprinting. Glycolytic and oxidative enzyme responses may be significantly altered by both endurance and sprint training. Endurance training may increase phosphofructokinase (PFK), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activity, whereas sprint training may increase PFK, phosphorylase, lactate dehydrogenase and glyceraldehyde dehydrogenase activity. Creatine phosphate (CP) activity and ATP levels are higher in FT than ST fibres. Endurance training reduces CP and ATP depletion at submaximal workloads, but also increases CP and ATP concentrations. Superior sprinters are able to utilise phosphagens quickly and more completely than lesser competitors over distances up to 80m, but this may result from genetic predisposition rather than training. Extreme and prolonged training may produce skeletal muscle fibre type conversion. Additionally, acute and chronic exercise alter skeletal muscle substrate, metabolism and phosphagen profiles thus influencing physical performance and sporting success. Obviously, such skeletal muscle changes are important to coaches and athletes wishing to design training programmes to maximise the performance of a specific motor activity.

Acute and chronic responses of skeletal muscle to endurance and sprint exercise. A review.

Excessive exercise may lead to skeletal muscle cell damage with degradation of cellular components and leakage of intracellular enzymes. Calcium has repeatedly been proposed to be involved in these processes. Studies have shown that the resting level of cytoplasmic Ca 2+ increases up to threefold during long-term low-frequency stimulation. We have shown that electrical stimulation produces a marked increase in Ca 2+ uptake and Ca 2+ content in rat skeletal muscle, both in vivo and in vitro. Continuous stimulation for 240 min at 1 Hz results in an increased release (18-fold) of lactate dehydrogenase (LDH) from extensor digitorum longus (EDL) muscle. This was associated with an increased total Ca 2+ content (185%), was augmented at high [Ca 2+ ] o and suppressed at low [Ca 2+ ] o . The release of LDH may reflect partial loss of sarcolemmal integrity as a result of degradation of membrane components by Ca 2+ -activated enzymes (e.g. calpain or phospholipase A 2 ). After cessation of stimulation the increased release of LDH continues for at least 120 min. This is associated with an up to sevenfold increase in 45 Ca uptake. The increased permeability to Ca 2+ may further activate calpain and phospholipase A 2 and accelerate the loss of membrane integrity. Stimulation-induced uptake of Ca 2+ and release of LDH is most pronounced in EDL (mainly composed of fast-twitch fibres at variance with soleus which is mainly composed of slow-twitch fibres). This may account for the observation that prolonged exercise leads to preferential damage to fast-twitch fibres. We hypothesize that excessive exercise may lead to an intracellular accumulation of Ca 2+ and increased cytoplasmic Ca 2+ causing activation of self-accelerating degradative pathways leading to muscle damage.

Excitation-induced Ca2+ influx and skeletal muscle cell damage.

In order to ascertain the effects of long-term exercise training and long-term exhaustive exercise on mitochondrial 45Ca2+ uptake and related variables in rat skeletal muscle, female rats were randomly divided into three groups: sedentary-rested (SR), trained-rested (TR), and trained-exhausted (TE). The trained groups were exercised five times per week on a treadmill for 22 weeks. At the conclusion of the training period, the TE group was exercised to exhaustion following their daily 1 h run. The 45Ca2+ uptake and endogenous mitochodrial Ca2+ content of skeletal muscle followed stepwise increases of approximately 25% and 50%, respectively, across the groups, suggesting that long-term exercise induces the mitochondria to play an important role as a Ca2+ uptake buffer. A 75--83% reduction in 45Ca2+ binding in the TE group suggests a selective loss and partial saturation of membrane phospholipids with exhaustive exercise. The TE group had a two-fold greater content of mitochondrial Mg2+ than did the rested groups. It is speculated that the mitochondria accumulate Mg2+ during acute exercise to maintain the functional integrity of the membrane, thus offsetting the deleterious effects of excessive Ca2+ uptake

Calcium uptake in skeletal muscle mitochondria. II. The effects of long-term chronic and acute exercise.

Mitochondrial and sarcoplasmic reticular 45Ca2+ uptake and Ca2+-ATPase activity were determined in skeletal muscle from exercise trained and non-trained rats at rest or following short-term exhaustive exercise. In trained rats exercised to exhaustion, mitochondrial 45Ca2+ uptake was significantly depressed when compared to non-trained rats at rest. Ca2+-ATPase activity of sarcoplasmic reticulum from trained rats exercised to exhaustion was significantly increased as compared to trained rats at rest. These data suggest that the disruptive influence of Ca2+ accumulation in mitochondria isolated following exhaustive exercise may be diminished as a result of training.

Effects of exercise training and exhaustion on 45Ca uptake by rat skeletal muscle mitochondria and sarcoplasmic reticulum.

15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

Female Wistar rats were used to determine the effects of the chelating agents, EDTA and EGTA, on the in vitro 45Ca2+ accumulation by mitochondria isolated from the skeletal muscle of fatigued animals. The rats were divided into three groups: sedentary-rested (SR), trained-rested (TR), trained-exhausted (TE). The trained groups were exercised on a treadmill for 1 h daily, five times a week, for 22 weeks. At the conclusion of the training program, the TE group was rapidly exercised to exhaustion immediately following their daily 1-h run. In the TR group EDTA reduced 45Ca2+ binding while both EDTA and EGTA appeared to increase mitochondrial Ca2+ and Mg2+ content. In the TE group, EDTA reduced endogenous mitochondrial Ca2+ and Mg2+ content, while both EDTA and EGTA increased 45Ca2+ binding. Since chelating Ca2+ and Mg2+ from the membrane may affect the structure and function of the mitochondria, it is suggested that the use of chelating agents during the isolation of mitochondria from the skeletal muscle of trained rats be viewed with caution.

Charlotte A. Tate

Papers

Calcium uptake in skeletal muscle mitochondria. II. The effects of long-term chronic and acute exercise.

Effects of exercise training and exhaustion on 45Ca uptake by rat skeletal muscle mitochondria and sarcoplasmic reticulum.

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

Calcium uptake in skeletal muscle mitochondria. I. The effects of chelating agents on the mitochondria from fatigued rats.