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CELLULAR METABOLISM

µ-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

¹Department of Zoology, La Trobe University, Victoria; ²School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria, Australia

Submitted 15 June 2005 ; accepted in final form 16 August 2005

	ABSTRACT

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µ-calpain and calpain-3 are Ca²⁺-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2–200 µM Ca²⁺. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca²⁺ concentration levels ([Ca²⁺]_i; 1 µM) than the levels at which µ-calpain activation occurs. We have demonstrated the Ca²⁺-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca²⁺] levels (0, 2.5, 10, and 25 µM). Autolysis of calpain-3 was found to occur across a [Ca²⁺] range similar to that for µ-calpain, and both calpains displayed a seemingly higher Ca²⁺ sensitivity in human than in rat muscle homogenates, with 15% autolysis observed after 1-min exposure to 2.5 µM Ca²⁺ in human muscle and almost none after 1- to 2-min exposure to the same [Ca²⁺]_i level in rat muscle. During muscle activity, [Ca²⁺]_i may transiently peak in the range found to autolyze µ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% O_{2 peak} until fatigue, n = 3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca²⁺]_i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.

Ca²⁺-dependent proteases; proteolysis

µ-Calpain is one of the ubiquitous forms of calpain {the other ubiquitous calpain is m-calpain or calpain-2, which requires millimolar levels of intracellular Ca²⁺ concentration ([Ca²⁺]_i) for activation}. The amount of Ca²⁺ required for the activation of µ-calpain remains controversial, having been reported to occur over a wide [Ca²⁺] range (2–200 µM) (5, 14, 22, 23). Even though the reported [Ca²⁺] required for µ-calpain activation varies widely, it is well accepted that once activated, the Ca²⁺ requirement for µ-calpain activity is dramatically decreased (0.5–2 µM) (5, 10, 15). Significantly, in most cases, µ-calpain has been analyzed after isolation and purification, and consequently it is unclear whether the reported [Ca²⁺] required for activation would be relevant to the enzyme in vivo. Calpain-3, the more recently identified, predominantly muscle-specific calpain, is even more poorly understood. Researchers who have conducted experiments with recombinant calpain-3 have reported that it requires relatively low [Ca²⁺] (1 µM or less) to be activated (8), but at present no data are available regarding the Ca²⁺-dependent autolysis or activation of full-length, native calpain-3.

One current view is that full-length (80 kDa) µ-calpain is an inactive proenzyme and that its activation involves autolysis to a 78-kDa and then to a 76-kDa protein (5), with both of these autolysed isoforms being proteolytically active µ-calpain. Investigators in other studies have claimed that µ-calpain can be active before it has been autolyzed (10, 23). Although it may be unresolved whether autolysis is required for activation, Goll et al. (14) pointed out that this question may functionally be moot, given that activation and autolysis often go hand in hand. Similarly, in the case of calpain-3, it is uncertain whether it must be autolyzed to become proteolytically active (26) or whether it can be activated without autolysis. Autolysis of the 94-kDa, full-length calpain-3 results in the formation of 60-, 58-, and 56-kDa proteins (17, 31). Whether the Ca²⁺ sensitivity of calpain-3 changes once it has been autolyzed, and how these different autolyzed calpain-3 proteins are related to the activity of calpain-3, is complicated and currently not understood.

Given that factors such as ionic strength (20, 21) and pH (21) have been shown to influence the activity of at least µ-calpain, we thought it important to examine the proteins under conditions similar to the physiological milieu. In addition, a number of intracellular myoplasmic factors have been associated with an increased Ca²⁺ sensitivity of calpain. These include phospholipids (e.g., phosphatidylinositol) (27) and the molecular chaperone UK114 (22). In the present study, we have addressed some of these concerns by determining the [Ca²⁺] range within which autolysis of endogenous µ-calpain and calpain-3 occurs in skeletal muscle homogenates. We have found that calpain-3 was in fact only slightly more Ca²⁺ sensitive than µ-calpain and that both proteases were autolyzed in human muscle homogenates exposed to 2.5 µM Ca²⁺ for 1 min. This higher-than-expected Ca²⁺ sensitivity of µ-calpain suggests that there are indeed factors present in the whole muscle that were absent in previous biochemical assays that determined µ-calpain activity.

In rat skeletal muscle, calpainlike activity was reported to increase after level treadmill running (4), although this finding was revealed using a high [Ca²⁺] (800 µM free Ca²⁺) and the researchers in that study did not separate the activity of µ-calpain from that of m-calpain or calpain-3. Investigators in another study did isolate the Ca²⁺ dependence of µ-calpain and m-calpain activity in response to exercise, but the calpains may have been autolyzed partially during the purification process, making it difficult to interpret the physiological relevance of the findings (7). Nevertheless, given those findings and the fact that, during exercise, [Ca²⁺]_i may transiently increase to levels that are able to activate µ-calpain and calpain-3, we hypothesized that µ-calpain and calpain-3 might be autolysed during vigorous exercise in humans. Researchers in two prior studies measured µ-calpain and calpain-3 mRNA after exercise in humans, but neither the proteins nor their autolytic products were examined (12, 16). In the present study, we have examined for the first time the effect of exercise on the autolysis of µ-calpain and calpain-3 in human skeletal muscle.

	METHODS

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General. All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise stated. Experiments were conducted at room temperature (23 ± 2°C; mean ± SD).

Human muscle biopsies. Human muscle samples were collected from the vastus lateralis muscle using the percutaneous needle biopsy technique modified for suction. Samples obtained before and immediately after exhaustive, prolonged cycling exercise (performed at 70% O_{2 peak}) were collected from three endurance-trained males, and samples harvested before and immediately after 30-s "all-out" sprint cycling were obtained from eight untrained males. All samples were rapidly frozen in liquid nitrogen and stored at –80°C until being analyzed. The human muscle samples analyzed in the present study were spare tissues originally collected for other completed research projects and approved by The Human Research Ethics Committees at Deakin University. All subjects gave written informed consent.

Animals. Adult Long-Evans hooded rats aged 26–52 wk were killed using an overdose of halothane as approved by the Animal Ethics Committee at La Trobe University. The extensor digitorum longus (EDL) muscles were removed, blotted dry on filter paper, and placed into Ringer solution (containing no Ca²⁺) kept at room temperature until the muscle samples were chopped into small pieces and weighed for immediate preparation of muscle homogenates (within 2 h).

Ca²⁺ dependence of µ-calpain and calpain-3 autolysis. Rat EDL and human skeletal muscle samples were used to determine the Ca²⁺ dependence of µ-calpain and calpain-3 autolysis. Muscle samples were homogenized (10:1 wt/vol) in solutions very heavily Ca²⁺-buffered with EGTA and BAPTA to produce final [Ca²⁺] of <10 nM (denoted as 0 µM), 2–3 µM (2.5 µM), 8–12 µM (10 µM), and 20–30 µM (25 µM). These solutions were composed of (in mM) 126 K⁺, 36 Na⁺, 18, hexamethylethylene-diaminetetraacetic acid (HDTA²; Fluka, Buchs, Switzerland), 1 free Mg²⁺ (10.3 total Mg²⁺), and 60 HEPES, 50–44 EGTA, and 0–6 BAPTA (to produce 50 mM total Ca²⁺ buffering) was included because of its ability to bind Ca²⁺ rapidly. The pH of the solutions was initially set at 7.2 to obtain a final pH of 7.1 after homogenization of the muscle. A Ca²⁺-sensitive electrode was used to test one example of the muscle homogenates at each of the [Ca²⁺] used, to experimentally verify the calculated final [Ca²⁺]. The addition to the homogenates of 2,5-di-(tert-butyl)-1,4-hydroquinone (1 µM) to block any Ca²⁺ reuptake by the sarcoplasmic reticulum (SR) had no effect on calpain autolysis (data not shown). SDS was added to a final concentration of 4% at 1 min (human) and at 1 or 2 min (rat), and homogenates were then incubated at 4°C for 20–40 min, after which they were spun at 3,000 g for 5 min. Subsequently, the supernatant was collected, which was then added (2:1 vol/vol) to SDS loading buffer (0.125 M Tris·HCl, 10% glycerol, 4% SDS, 4 M urea, 10% mercaptoethanol, and 0.001% bromophenol blue; pH 6.8). Samples were heated to 95°C for 4 min and stored at –20°C until being subjected to Western blot analysis.

Time course of µ-calpain and calpain-3 autolysis. We also examined the autolysis of µ-calpain and calpain-3 in rat EDL muscles exposed to various [Ca²⁺] for different lengths of time. EDL muscles from three different rats were cut into nine portions. They were treated as detailed above at [Ca²⁺] of 0, 2.5, 10, and 25 µM with the addition of SDS to halt the effect of the increased [Ca²⁺] at 1 (n = 2), 2 (n = 1), 15 (n = 3) or 60 min (n = 3). The data for the 1- and 2-min samples were pooled.

Effect of exhaustive exercise on µ-calpain and calpain-3 autolysis in humans. To determine whether µ-calpain or calpain-3 was autolysed as a consequence of exhaustive exercise, human muscle samples obtained before and after exercise were homogenized in a 10:1 dilution (wt/vol) of 0.4 M Tris·HCl, pH 6.8, and 25 mM EGTA ([Ca²⁺] <10 nM). There was no difference between samples extracted in the Tris-based buffer and those extracted in the K⁺-based solutions detailed above (data not shown). After homogenization, 4% SDS was added and samples were treated as detailed above.

Western blot analysis of µ-calpain and calpain-3 in muscle homogenates. Muscle protein, prepared as described above, was separated on an 8% SDS-PAGE gel and transferred onto nitrocellulose membranes. Membranes were exposed to either mouse anti-µ-calpain (MAb clone 15C10; Sigma) or mouse anti-calpain-3 (MAb 12A2; Novocastra, Newcastle upon Tyne, UK), after which goat anti-mouse horseradish peroxidase (HRP, 1:5,000 or 1:20,000 dilution; Bio-Rad Laboratories, Hercules, CA) was added to the membranes. Bands were visualized using either Opti-4CN substrate or Immun-Star HRP substrate (Bio-Rad) and densitometry performed using Quantity One software (Bio-Rad). µ-calpain was visualized as an 80-kDa protein that autolysed to proteins of 78 and 76 kDa (5). Calpain-3 was observed as a 94-kDa protein that autolysed to proteins of 60, 58, and 56 kDa when activated (8, 17, 29–31). Data for Western blot analysis are presented as the density of a given band relative to the density of the total bands representing µ-calpain or calpain-3 for a given sample. The total density of the bands did not change appreciably, regardless of the degree of autolysis. As we reported previously, the 12A2 anti-calpain-3 MAb shows, in addition to protein bands at 94 and 58 kDa, a band at 82 kDa in rat and toad muscle (32), representing an unknown protein. The 12A2 anti-calpain-3 MAb used in the present study is specific for a unique insertion sequence of calpain-3; however, it is evident that this unknown 82-kDa protein was not calpain-3, given that it is not detected with other calpain-3 antibodies (18). We also found that the protein was not Ca²⁺ sensitive, because exposure of muscle homogenates to 5 mM Ca²⁺ for 60 s did not result in any change in the visualized protein band (data not shown). In agreement with a previous report (3), we did not detect this band in human muscle.

Statistics. The effects of exercise were analyzed using a paired, two-tailed Student's t-test. Time- and Ca²⁺-dependent data were analyzed using one-way ANOVA. Statistics were analyzed using GraphPad version 4.01 software. Data are expressed as means ± SE unless otherwise indicated. Statistical significance was accepted at P < 0.05.

	RESULTS

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Ca²⁺ dependence of µ-calpain and calpain-3 autolysis. Both µ-calpain and calpain-3 were detected in human and rat skeletal muscle samples. For both calpains, there was a small difference (2 kDa) in mobility between the human samples and the rat samples, with µ-calpain appearing slightly smaller and calpain-3 appearing slightly larger in the rat compared with the human samples (see Fig. 1). To simplify matters, we refer to the human and rat proteins as if they had been the same size in the context of this study. When muscle samples were homogenized in a buffer containing a high EGTA concentration, full-length µ-calpain (80 kDa) and calpain-3 (94 kDa) were the predominant bands observed using Western blot analysis (Fig. 1, A and B, lanes 1 and 5). Autolysis of µ-calpain is shown as the increasing appearance of the bands at 78 and 76 kDa as the 80-kDa band decreases in intensity. Calpain-3 autolysis is shown as the disappearance of the 94-kDa protein band and the appearance of bands at 60, 58, and 56 kDa. For the purpose of quantifying the amount of autolysis, the various autolytic products of the given calpain (e.g., 60-, 58-, and 56-kDa bands in the case of calpain-3) are pooled to show the total amount of autolysis. In the Western blot histogram showing data for one human and one rat EDL muscle homogenate, autolysis of both µ-calpain and calpain-3 is shown in both the human muscle (Fig. 1, A and B, lanes 2–4) and the rat EDL muscle (Fig. 1, A and B, lanes 6–8), although it is apparent that autolysis of both calpains was more sensitive to Ca²⁺ in the human homogenate than in the rat homogenate (Figs. 1 and 2). Figure 2 shows the relative amounts of full-length and autolyzed µ-calpain and calpain-3 found when homogenates (n = 3, human and rat) were exposed for 1 min to [Ca²⁺] levels over the physiological range (0, 2.5, 10, and 25 µM). In the human samples, some autolytic products were apparent even in association with 0 µM Ca²⁺, in which a high free EGTA concentration was used to try to keep the [Ca²⁺] levels in the homogenate low at all times (Fig. 2, A and B; see also DISCUSSION). Additional 23% autolysis of µ-calpain and 12% autolysis of calpain-3 were observed when the muscles were exposed to 2.5 µM Ca²⁺ for 1 min. The amount of autolysis occurring within 1 min increased as the [Ca²⁺] was increased, with 28% and 30% autolysis of µ-calpain and calpain-3 occurring, respectively, when the muscle homogenates were exposed to 10 µM Ca²⁺ and 46% and 49% autolysis of µ-calpain and calpain-3, respectively, when the muscle homogenates were exposed to 25 µM Ca²⁺ (Fig. 1, A and B, lanes 2–4, and Fig. 2, A and B). Furthermore, when the data for each human muscle sample were examined individually, it was found that the amount of autolysis invariably increased progressively as the [Ca²⁺] level increased. In rat EDL muscle, there was little autolysis (2–5%) of either µ-calpain or calpain-3 in homogenates prepared in high EGTA concentration (i.e., exposure to 0 µM Ca²⁺). There was a progressive increase in the amount of autolysis of µ-calpain as Ca²⁺ increased (Fig. 2C). Compared with 0 µM Ca²⁺, 1-min exposure to 2.5, 10, and 25 µM Ca²⁺_i level resulted in a further 11%, 21%, and 28% autolysis of µ-calpain, respectively. Relative to the 0 µM Ca²⁺ case for calpain-3 (Fig. 2D), no apparent additional autolysis occurred when the muscle was exposed to 2.5 µM Ca²⁺ for 1 min, but 9% and 43% autolysis was observed when the muscle was exposed to 10 and 25 µM Ca²⁺, respectively.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Effect of increasing Ca2+ concentration ([Ca2+]) on the autolysis of µ-calpain and calpain-3 proteins in skeletal muscle. Representative Western blot histograms for µ-calpain (A) and calpain-3 (B) in human and rat skeletal muscles are shown. The same samples are shown in both blots. Lanes 1–4 correspond to equal amounts of human muscle homogenates from the same human muscle tissue sample, and lanes 5–8 correspond to equal amounts of rat extensor digitorum longus (EDL) muscle homogenates. Muscle was homogenized in physiological solutions (see METHODS), either in the presence of EGTA alone (nominally 0 µM Ca2+) (lanes 1 and 5) or with 2.5 µM Ca2+ (lanes 2 and 6), 10 µM Ca2+ (lanes 3 and 7), or 25 µM Ca2+ (lanes 4 and 8). The protein was denatured by addition of SDS to the homogenate, effectively halting the exposure to Ca2+ after 1 min (human) or 2 min (rat). Full-length µ-calpain (80 kDa) and its autolysed fragments at 78 and 76 kDa are evident in various samples. Full-length calpain-3 at 94 kDa and lysed calpain-3 at 56 and 58 kDa are shown. The band at 82 kDa in the lanes containing the rat homogenates probed with calpain-3 antibody was observed previously in homogenates from rat, mouse, hamster, and chicken skeletal muscle but has not been observed in human muscle (3) (see METHODS). Molecular mass is shown at left.

View larger version (38K): [in this window] [in a new window]   Fig. 2. [Ca2+] dependence of µ-calpain and calpain-3 autolysis in skeletal muscle. Muscle homogenates were prepared in physiological solutions (see METHODS) in the presence of 0, 2.5, 10, or 25 µM Ca2+. Samples were subjected to Western blot analysis (see Fig 1) and means ± SE for each band were calculated as the density of the band in terms of the percentage of the total density of all bands (i.e., full-length and autolysed µ-calpain or calpain-3 bands) for the given sample (see text). Ca2+ exposure for 1 min caused the disappearance of full-length µ-calpain (80 kDa) and the appearance of autolysed µ-calpain (78 and 76 kDa) in both human (A; n = 3) and rat (C; n = 3) skeletal muscle homogenates, and similarly caused the disappearance of full-length calpain-3 (94 kDa) and the appearance of autolysed calpain-3 at 58 and 56 kDa in the human (B; n = 3) and rat (D; n = 3) homogenates. In every case, one-way ANOVA indicated that the presence of Ca2+ significantly affected the amount of full-length calpain. P < 0.05.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Time and [Ca2+] dependence of µ-calpain and calpain-3 autolysis in rat skeletal muscle. Rat EDL muscle homogenates were prepared in physiological solutions (see METHODS) in the presence of either 0 µM Ca2+ for 1 min (control, Con) or 2.5, 10, or 25 µM Ca2+ for 1, 15, or 60 min. µ-Calpain data are shown in left graphs in A–C, and calpain-3 data are shown in right graphs in D–F. In general, the amount of full-length protein (see RESULTS) decreased and the amount of autolyzed proteins increased with longer exposure to various [Ca2+]. Muscle homogenates maintained in the high EGTA concentration (exposure to 0 µM Ca2+) for up to 60 min showed no more autolysis of either µ-calpain or calpain-3 than the control. Values are means ± SE with 3 independent samples used for every condition. One-way ANOVA showed a significant effect of exposure time in every case. P < 0.05.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effect of exercise on µ-calpain and calpain-3 proteins in human skeletal muscle. Western blot analysis showing bands for µ-calpain (A) and calpain-3 (B) in human skeletal muscle from 3 of 11 study subjects before (pre) and after exercise (post). The same samples are shown in each blot. Subject 1 performed 30-s "all-out" sprint bicycle exercise, and subjects 2 and 3 cycled at 70% O2 peak until exhaustion. Molecular mass is indicated at left. Summarized data from Western blot histograms are graphed, showing the effect of 30-s all-out sprint cycling exercise (C) and 70% O2 peak endurance exercise (D) on the autolysis of µ-calpain and calpain-3. Data are representative of 8 subjects for the sprint exercise and 3 subjects for the endurance exercise. There was no significant difference in the amount of autolysis with regard to exercise in any case (paired Student's t-tests).

	DISCUSSION

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Ca²⁺ dependency of µ-calpain and calpain-3 autolysis. Using human muscle homogenates, we found some autolysis of calpain-3 with 1-min exposure to as little as 2.5 µM Ca²⁺. These data are the first to be published regarding the Ca²⁺ dependence of native calpain-3 in human skeletal muscle. It had previously been hypothesized that calpain-3 required a substantially lower [Ca²⁺]_i than µ-calpain for autolysis activation, although that idea was based on data from calpain-3 variants (8) and estimates for µ-calpain activation covering an extremely large [Ca²⁺] range (2–200 µM). We report herein that the difference between the Ca²⁺ sensitivity of autolysis of the calpains is relatively small in both human and rat muscle, because even though calpain-3 is autolyzed to a slightly greater extent than µ-calpain by 2.5 µM Ca²⁺ after long exposure (15–60 min) (Fig. 3), µ-calpain is in fact autolyzed proportionately more than calpain-3 during the first minute of exposure at that [Ca²⁺] (Figs. 2 and 3). This estimate of the [Ca²⁺]_i required for autolysis of native µ-calpain is lower than that described in most previous investigations of the activation of µ-calpain using purified µ-calpain, and in fact it is substantially lower than that reported in a recent biochemical study, in which a sensitive substrate was used and detailed kinetic analysis determined the EC₅₀ of [Ca²⁺] required for autolysis and activation to be 200 µM (5). Clearly, the analysis of whole muscle preparations in solutions that mimic the intracellular environment (i.e., similar ionic strength, pH) is important for obtaining physiologically relevant values. A further important point is that the muscle homogenates are still likely to contain UK114, a molecular chaperone that has been shown to reduce the Ca²⁺ dependence of µ-calpain autolysis (22), which is presumably not present during experiments with purified µ-calpain. The presence of UK114 or perhaps endogenous kinases and other physiological conditions in our muscle homogenates likely explains why we obtained a lower Ca²⁺ dependence for µ-calpain autolysis than biochemical measurements in previous studies in which researchers measured autolysis and/or activity of µ-calpain. It should be noted, however, that on the basis of our experiments, we cannot rule out the possibility that some key endogenous factor in the muscle was in some way altered during the preparation of the homogenates, which thereby might have influenced the Ca²⁺ dependence of the calpains. In addition, our muscle homogenate experiments were conducted at room temperature, potentially producing Ca²⁺ dependence values different from those prevailing at the normal in vivo temperature of 37°C (24). Regardless of these limitations, our findings have been produced using more physiologically relevant conditions than those used in previous studies and hence offer novel and likely relevant information about the regulation of µ-calpain and calpain-3 in skeletal muscle in vivo.

µ-Calpain and calpain-3 autolysis and exercise. In human muscle biopsies obtained before and after exercise, we always detected some autolysis of both µ-calpain and calpain-3, even though the [Ca²⁺] was kept low (<10 nM) during the homogenization procedure by using a high EGTA concentration. We do not know at which point in time this autolysis occurred, possibly in vivo during the biopsy procedure or during the preparation of the homogenates, even though high EGTA concentration was present. Importantly, this small amount of autolysis is not of concern, given that there was no further autolysis as a result of exercise. We also note that there was no further autolysis of µ-calpain or calpain-3 when muscle homogenates were kept in a high EGTA concentration for up to 60 min.

The exercise protocols used in the current study resulted in what is referred to as high-frequency fatigue (sprint) and/or metabolic fatigue (sprint and endurance) (2). In these types of fatigue, the muscle fails to produce adequate force because of the buildup of K⁺ in the T-tubules, inhibiting transmission of the action potential (high-frequency fatigue), or because of the buildup of intracellular metabolites or depletion of substrates (metabolic fatigue), resulting in inhibition of Ca²⁺ release from the SR or direct inhibition of the contractile apparatus (2). The results of the present study show that the [Ca²⁺]_i in the fibers of the study subjects did not remain elevated long enough to autolyze either µ-calpain or calpain-3 appreciably, at least not at the sites where the calpains are located. This seems entirely reasonable, given that force production after such types of fatiguing exercise normally fully recovers within 30 min (2, 13), which suggests that there could not have been large-scale activation of proteases and consequent muscle fiber damage. However, if the subjects were to perform a more prolonged exercise protocol sufficient to cause a long-lasting (>24 h) decrease in force production, often referred to as "low-frequency fatigue" (9, 11), there might well be substantial autolysis of µ-calpain or calpain-3 and consequent proteolytic damage (19, 32).

Such calpain activation and damage might also occur when muscles are subjected to eccentric contractions (1), particularly in dystrophic muscle (33), possibly due to the occurrence of a sustained influx of extracellular Ca²⁺ through stretch-sensitive sarcolemmal channels. As shown in Fig. 3, when the duration of Ca²⁺ exposure is increased to 15 or 60 min, there is appreciable autolysis of both calpains at comparatively low [Ca²⁺] (2.5 µM), particularly with regard to calpain-3. These results suggest that the longer intracellular [Ca²⁺]_i remains elevated in vivo, the greater the extent of autolysis of µ-calpain and calpain-3 and possibly the greater the extent of proteolytic damage.

Comparison of µ-calpain and calpain-3 autolysis in human and rat muscle. As mentioned previously, we found that µ-calpain and calpain-3 both seemed to demonstrate a slightly higher Ca²⁺ sensitivity in the human muscle than in the rat EDL muscle (Fig. 2). It is possible that this finding reflects differences in the types of muscle fibers, with the rat EDL muscle fibers likely being predominantly fast-twitch fibers, whereas those in the human muscle most likely a mixture of the various fiber types. It is also possible that the difference could be linked to the respective ranges over which [Ca²⁺]_i varies during normal activity in the human vs. rat muscle fibers. In addition, there are potentially multiple determinants for the autolysis rate constants of µ-calpain and calpain-3, such as time and [Ca²⁺]_i, and these may differ slightly between rats and humans.

In conclusion, our findings confirm that the autolysis of both µ-calpain and calpain-3 is tightly regulated by [Ca²⁺]_i in skeletal muscle across a range close to but evidently above that reached during normal activity.

	GRANTS

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This research was supported by National Health and Medical Research Council of Australia Grant 280623.

	ACKNOWLEDGMENTS

 
We thank Brendan Jackson and Jacinta Baldwin for help with human tissue sample collection.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. M. Murphy, Dept. of Zoology, La Trobe Univ., Victoria 3086, Australia (e-mail: r.murphy{at}latrobe.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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