Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany
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ABSTRACT |
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Fiber-type transitions in adult skeletal muscle induced by chronic low-frequency stimulation (CLFS) encompass coordinated exchanges of myofibrillar protein isoforms. CLFS-induced elevations in cytosolic Ca2+ could activate proteases, especially calpains, the major Ca2+-regulated cytosolic proteases. Calpain activity determined by a fluorogenic substrate in the presence of unaltered endogenous calpastatin activities increased twofold in low-frequency-stimulated extensor digitorum longus (EDL) muscle, reaching a level intermediate between normal fast- and slow-twitch muscles. µ- and m-calpains were delineated by a calpain-specific zymographical assay that assessed total activities independent of calpastatin and distinguished between native and processed calpains. Contrary to normal EDL, structure-bound, namely myofibrillar and microsomal calpains, were abundant in soleus muscle. However, the fast-to-slow conversion of EDL was accompanied by an early translocation of cytosolic µ-calpain, suggesting that myofibrillar and microsomal µ-calpain was responsible for the twofold increase in activity and thus involved in controlled proteolysis during fiber transformation. This is in contrast to muscle regeneration where m-calpain translocation predominated. Taken together, we suggest that translocation is an important step in the control of calpain activity in skeletal muscle in vivo.
calpain activation; calpastatin; chronic low-frequency stimulation; fast-to-slow transition; translocation
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INTRODUCTION |
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PROTEOLYSIS IN SKELETAL MUSCLE serves three major functions: 1) protein turnover at steady-state conditions, 2) muscle wasting under catabolic conditions, and 3) sarcomeric remodeling related to adaptive responses. Numerous studies have focused on protein catabolism, e.g., sepsis (35), immobilization- and denervation-induced atrophy (33, 37), muscle fiber injury (22), and necrosis (30). The impact of proteolysis as an essential step in the sarcomeric remodeling during muscle fiber conversion is increasingly recognized (10, 34). Thus adaptive responses of skeletal muscle to altered functional demands encompass, in addition to altered expression levels, multiple exchanges of protein isoforms, e.g., enzymes and myofibrillar and membrane proteins (18, 19).
As pointed out by Furuno and Goldberg (6), the basal
degradative process in muscle proteolysis results from the action of
cytosolic proteases and does not involve lysosomal proteases. The
cytosolic proteolytic apparatus encompasses the
Ca2+-regulated cysteine proteases, the
calpains, and the ATP-dependent, ubiquitin-related proteasome complex
(for reviews, see Refs. 2, 14, 32). The role of these proteases in
protein turnover, muscle wasting, or adaptive changes is still a matter
of debate. The observation that intact myofilaments cannot be degraded
directly by the proteasome (29) suggests that calpains may
be involved in the initiation of myofibrillar and cytoskeletal protein
breakdown (11). It has been shown that calpains release
-actinin from the Z disk without degradation (8). In
addition, calpains have been shown to cleave the ryanodine receptor
(RyR) of fast-twitch rabbit muscle into two major fragments
(28).
Our observation that the amount of RyR is drastically reduced when fast-twitch rabbit muscle undergoes fast-to-slow conversion by chronic low-frequency stimulation (CLFS) may, therefore, be due to enhanced calpain activity during the transformation process. Calpains may also be involved in the CLFS-induced remodeling of the Z disks (24). The possibility that calpains play a role in muscle fiber transformation is corroborated by observations that increases in resting sarcoplasmic Ca2+ are an early effect of enhanced contractile activity by CLFS (4, 31).
The importance of proteolysis during muscle fiber conversion has previously been suggested by results from chronically electrostimulated rat muscle. The exchange of the fastest myosin heavy chain (MHC) isoform, MHCIIb, with the less fast MHCIId(x) and MHCIIa isoforms, became detectable in low frequency-stimulated rat muscle after 8-10 days. However, we observed a markedly enhanced protein synthesis rate of MHCIId(x) and MHCIIa as soon as 3 days after the onset of CLFS. In other words, their elevated synthesis markedly preceded their accumulation. These findings suggest that the newly synthesized MHC isoforms were rapidly degraded and not incorporated into the myofibrillar apparatus because release and degradation of the existing MHCIIb, the isoform no longer synthesized, seems to be a prerequisite for the isoform exchange at the level of the thick filament (34).
The present study addresses the role of proteolysis in transforming rat muscle and specifically focuses on the role of calpains in this process. Rat muscle was chosen because CLFS does not elicit any signs of fiber injury or deterioration in rat muscle (21). This is in contrast to the rabbit in which CLFS has been shown to elicit processes of fiber degeneration and regeneration (15). Fiber necrosis and repair, as well as invasion of the stimulated muscle by mononucleated cells, e.g., macrophages, would be accompanied by enhanced proteolytic activities, thus obscuring the role of proteolysis during fiber-type transformation. Fast-twitch extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of the rat were exposed to CLFS for time periods up to 20 days and compared with their contralateral, untreated muscles as well as with slow-twitch soleus (SOL) muscle. For further comparison, we also studied calpain activities in regenerating muscle. Calpain activity was determined in whole muscle homogenates, i.e., in the presence of endogenous calpastatin, using a calpain-specific fluorogenic substrate. The assessment of total calpain activity, in this case in the absence of calpastatin, was conducted using a zymographical assay previously established for studies on purified calpains (23). Its specificity for measurements on whole muscle homogenates was validated by determining Ca2+ dependency, pH optimum, inhibitor profiles, and initiation as well as inhibition of the Ca2+-induced calpain processing. Finally, measurements were conducted to distinguish between cytosolic and structure-associated calpains, especially in microsomal and myofibrillar fractions.
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METHODS |
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Animals, CLFS, and Marcaine-induced degeneration/regeneration. Adult male Wistar rats (400-470 g body wt) were used. CLFS (10 Hz, 0.2-ms impulse width, 24 h/day) was performed via electrodes implanted laterally to the peroneal nerve of the left hindlimb as previously described (21). After various periods of stimulation (12 h and 2, 4, 8, and 20 days), the animals (n = 3-6 for each time point) were killed under anesthesia, and contralateral unstimulated (control) EDL, TA, and SOL and stimulated EDL and TA muscles were quickly removed, weighed, and frozen in liquid nitrogen. For regeneration studies, the EDL muscle of the left hindlimb was surgically exposed under anesthesia, and with the use of a hypodermic injection needle, a total dose of 2.5 mg bupivacaine (Marcaine; Astra, Sodertalje, Sweden) was injected into the muscle. The treated animals (n = 3 for each time point) were killed under anesthesia 2 or 5 days later, and both the injected and contralateral muscles were removed and treated as above.
Preparation of muscle extract. The frozen muscles were pulverized under liquid nitrogen in a steel mortar. The following procedures were performed at 4°C. Muscle powder was homogenized at intense cooling by an ice-salt mixture in a fivefold volume of 20 mM Tris · HCl buffer (pH 7.4) containing 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol (DTT; Sigma), 10 µg/ml Pefabloc (Roth, Karlsruhe, Germany), and 10 µg/ml pepstatin A (Calbiochem) using a polytron homogenizer (Kinematica, Luzern, Switzerland) at 10,000 rpm. Three homogenization steps, each lasting 60 s with intervals of 3 min, were used. Extracts were separated from debris by 10-min centrifugation at 1,000 g. For separation of soluble and total particulate muscle fractions, the supernatant was subjected to 60-min centrifugation at 100,000 g in a Beckman ultracentrifuge, model TL-100, yielding a cytosolic (S-100) and a total particulate P-100 (containing myofibrils, mitochondria, and microsomes) fraction. Protein concentration was determined by the Bradford method (Bio-Rad protein assay) with BSA as a standard.
Calpain assay in the presence of endogenous calpastatin.
Calpain was measured using a modification of the assay of Edelstein and
co-workers (5).
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (SLY-AMC) served as a substrate for calpain (25). A stock
solution of 50 mM SLY-AMC was prepared in dimethyl sulfoxide and stored at 20°C. The following procedure was used for measuring calpain activity in muscle extracts: 30 µl muscle extract was incubated for
10 min at 37°C in a buffer solution (pH 7.4) containing 20 mM
Tris · HCl, 5 mM Ca2+, 1 mM DTT, 10 µg/ml
Pefabloc, and 10 µg/ml pepstatin A. After addition of 5 µl of the
substrate solution, buffer was added to adjust the volume of the assay
to 2 ml. Fluorescence of the liberated AMC was monitored in a
Perkin-Elmer fluorometer for 15 min at 37°C (excitation 380 nm,
emission 460 nm). Control assays were performed without
CaCl2 in the presence of 10 mM EDTA and 10 mM EGTA. Calpain
activity was thus determined as the Ca2+-dependent cleavage
of SLY-AMC. Its activity was expressed as arbitrary units per minute of
incubation time per milligram of muscle protein.
Calpastatin assay. The method for assaying inhibitory activity of calpastatin in the homogenates was performed according to Ref. 3, using rat kidney as a positive control (17). Briefly, homogenates were centrifuged for 20 min at 10,000 g at 4°C. Supernatant fractions were heated at 100°C for 10 min to inactivate endogenous calpains and other proteases. After centrifugation, aliquots of the cleared supernatants were added to a stock extract obtained by centrifugation of skeletal muscle homogenates (see Preparation of muscle extract) at 100,000 g for 60 min. After 30-min incubation at 37°C, 5 µl of 50 mM SLY-AMC were added (final concentration 0.125 mM), and remaining calpain activity was determined after 30 min.
Casein zymography. Casein zymography was performed using the original assay (23) with minor modifications. Briefly, 0.02% (wt/vol) casein (Sigma) was copolymerized in a 10% (wt/vol) acrylamide gel (pH 8.8). The casein gel was subjected to 15 min of preelectrophoresis (100 V, 30-40 mA) with Tris-glycine buffer (pH 8.3) containing 1 mM EGTA and 1 mM DTT. After protein loading (60 µg), electrophoresis was started (2-3 h, 100 V, 30-40 mA, 4°C). The gel was washed twice for 30 min in a buffer containing 20 mM Tris · HCl (pH 7.4) and then incubated at room temperature overnight in activation buffer (pH 7.4) containing 20 mM Tris · HCl, 10 mM DTT, and 4 mM Ca2+. Finally, the casein gel was stained for 2 h with acid-based Coomassie blue, followed by intensification in distilled water.
The specificity of calpain activity assessed by zymography was studied by determining the pH optimum and Ca2+ dependency. Ca2+-induced processing was performed before electrophoresis. Similarly, the effects of reversible (500 µM leupeptin) and irreversible (1 mM E-64) cysteine protease inhibitors were studied by incubations of the homogenates before electrophoresis. The intensity of the caseinolytic bands was estimated by integrating densitometry (ScanPack software; Biometra, Göttingen, Germany). The degree of translocation was estimated by referring structure-bound activity to total activity in both control and stimulated muscles.Analysis of calpain activities in fractionated muscle
homogenates.
Instead of EDL, TA muscle was used because of its much larger size that
permitted reproducible fractionations. The homogenates (see
Preparation of muscle extract) were centrifuged for 15 min at 14,000 g to obtain a myofibrillar pellet (P-14) that was
further purified by three centrifugations at the same conditions.
Cytosolic S-100 and microsomal P-100 fractions were obtained by 60-min
centrifugation of the 14,000 g supernatant at 100,000 g. Calpain activity of 50 µg protein of these fractions
was assessed by the zymographical assay. Assignment of myofibrillar
proteins to the various fractions was attained by immunoblot analyses
with antibodies specific to fast and slow MHC (26) and
-actinin (Sigma, antibody A 7811).
Immunohistochemistry. Nine-micrometer-thick frozen sections were air-dried, washed twice in phosphate-buffered saline (PBS), and incubated for 15 min in 3% H2O2 in methanol. Sections were blocked for 2 h in PBS (pH 7.4) containing 2% BSA and 10% horse serum. For staining embryonic MHC (MHCemb) and desmin (clone DE-B-5, Boehringer Mannheim), the primary mouse monoclonal antibodies were diluted in blocking solution and incubated overnight. After the incubation with the primary antibodies, sections were washed and reacted for 30 min with biotinylated secondary antibodies. Thereafter, sections were washed, incubated for 30 min with a biotin-avidin-horseradish peroxidase complex (Vectastain ABC reagent; Vector Laboratories), washed, and reacted for 6 min with diaminobenzidine as substrate (Sigma D-4293).
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RESULTS |
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Measurements of calpain activity in the presence of calpastatin
were performed on homogenates of control and low-frequency-stimulated EDL muscles. The specificity of the assay was established by using a
calpain-specific substrate at neutral pH and by addressing the Ca2+-dependent cleavage of substrate. Referred to the
unstimulated contralateral EDL muscles, CLFS induced significant
(P < 0.04) increases in free calpain activity after 4 days, reaching a twofold elevated level at 8 days without further
increasing with CLFS up to 20 days (Fig.
1A). Calpain activity
of stimulated EDL muscle thus reached a level intermediate
between fast- and slow-twitch (soleus) muscles. The substrate
concentration used in the assay based on Ref. 5 was below saturation.
Measurements were also performed at a 10-fold higher concentration of
SLY-AMC (1.25 mM), but under these conditions, stimulated muscles
displayed the same increases in calpain activity as determined at low
substrate (results not shown).
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Because calpain activity was measured in an assay using whole muscle homogenates, i.e., in the presence of calpastatin, the observed increase in activity might have been due to changes in calpastatin activity. Therefore, measurements were performed to compare calpastatin activity between control and stimulated TA muscles. According to analyses of stimulated and control muscles of three animals, no changes in calpastatin activity were detected after 8 days of CLFS, when calpain activity reached its maximum (Fig. 1B).
A zymographical assay was used to assess total calpain activity in
muscle homogenates. This assay is based on an electrophoretic separation before the measurement of activity. Thus calpastatin-bound calpain was liberated to quantify total calpain activities and to
distinguish calpain isoforms. As shown in Fig.
2, crude muscle homogenates yielded two
electrophoretically separated caseinolytic bands. These bands were
detected only in activation buffer containing 4 mM Ca2+ and
10 mM DTT. The faster migrating band was identified by comparison with
a commercially available calpain preparation from skeletal muscle
(Sigma) as m-calpain (not shown). According to Zhao and co-workers
(38), the slower band was tentatively designated as
µ-calpain.
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To further characterize the properties of the two skeletal muscle calpains, effects of Ca2+ activation and processing, as well as inhibition by leupeptin and E-64, were studied. For this purpose, homogenates were preincubated with inhibitors (leupeptin, E-64) or activators (Ca2+) and their combinations before electrophoresis. Ca2+-induced processing before electrophoretic separation revealed the expected cleaved fragments of both isoforms (Fig. 2A). The conditions for Ca2+-induced processing proved to be the same for the two isoforms (data not shown). The caseinolytic activity of the two bands was not affected by the reversible inhibitor leupeptin. However, Ca2+-induced processing was completely blocked in the presence of leupeptin. On the other hand, E-64 did not affect the caseinolytic activity in the absence of Ca2+ but inhibited it under Ca2+-activating conditions (Fig. 2B). This inhibition was suppressed by preincubation with leupeptin (Fig. 2B), confirming the competition of leupeptin and E-64 for the target sequence.
Because to our knowledge calpains have not been assessed by
zymographical assays in skeletal muscle, we determined pH profiles (Fig. 3) and kinetics of casein cleavage
(Fig. 4). Both isoforms exhibited maximum
activities in the neutral range (pH ~7.0-7.6). However,
differences existed between the time course of casein cleavage by the
two isoforms. The rate of casein cleavage by m-calpain was practically
linear during the first 2 h but leveled off with longer incubation
times (Fig. 4). Conversely, casein cleavage by µ-calpain became
detectable in the zymographical assay only after 2 h and then
accelerated with longer incubation times.
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Measurements of total calpain activities by the zymographical assay,
i.e., in the absence of calpastatin, in control and
low-frequency-stimulated muscles, did not reveal significant changes
for both isoforms at various periods of CLFS (Fig.
5). To reconcile this result with the
data on elevated calpain activity in the presence of calpastatin (Fig.
1A), we investigated the intracellular distribution of the
two calpains in control and stimulated muscles. For this purpose, crude
muscle homogenates were centrifuged to yield the S-100 supernatant
(cytosol) and total P-100 particulate fractions for the assessment of
cytosolic and structure-bound calpain activities. Total calpain
activity in control fast-twitch EDL muscles thus proved to be cytosolic
(Fig. 6). Contractile activity as induced by CLFS changed the distribution such that 4-day-stimulated EDL contained, in addition to cytosolic, also structure-bound calpain activity. This redistribution, which signifies a translocation of
protease activity, was more pronounced for µ-calpain than for m-calpain. In contrast to fast-twitch EDL muscle, calpain activity of
slow-twitch SOL muscle was present both in the cytosolic S-100 and the
total P-100 particulate fractions (Fig. 6). The CLFS-induced calpain
translocation was further analyzed by comparing control, 2-day, and
8-day-stimulated EDL muscles, including SOL muscles for comparison.
Control EDL muscles displayed negligible amounts of structure-bound
calpains, whereas considerable structure-bound calpain activities
appeared in the 2-day-stimulated muscles (Fig. 7). Prolonged periods of CLFS did not
lead to further increases in structure-bound activities.
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Several independent experiments were performed to confirm the
redistribution of calpain in low-frequency-stimulated EDL and TA
muscles. The results of these studies were compared with the fluorometrically determined calpain activities (Fig. 1A).
Increases in structure-bound activities were assessed by referring the
densitometrically evaluated intensities of the caseinolytic lanes from
stimulated muscles to the contralateral controls. The results indicated
>2-fold increases of structure-bound µ-calpain in 4-day and
8-day-stimulated muscles (Fig. 8). The
elevation in calpain activity (Fig. 1A) could thus be
explained by the observed translocation of µ-calpain.
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Because calpain translocation might involve activation at the
sarcolemma and/or proteolysis at the site of myofibrillar proteins, we
were interested to study in more detail its redistribution. For this
purpose, structure-bound activities were studied in myofibrillar and
microsomal fractions (see METHODS) prepared by differential centrifugation. As verified by immunoblot analyses (myosin,
-actinin), the P-14 fraction was highly enriched with myofibrils,
whereas the microsomal P-100 fraction contained only traces of
myofibrillar proteins (Fig.
9B). Zymographical analyses of
these fractions in 20-day-stimulated TA muscle indicated that
redistribution encompassed translocation of calpain activity to
myofibrils as well as to membranes. This pattern was similar to that of
normal SOL muscle (Fig. 9A).
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To compare alterations in calpain activity and distribution during
muscle fiber transformation with changes related to myogenesis, we
studied calpain profiles in regenerating rat EDL muscle. For this
purpose, EDL muscle was injected with Marcaine and examined 2 and 5 days later. As shown by staining for desmin (Fig.
10), 2-day-treated muscle contained
intact and degenerated fibers. As demonstrated by MHCemb
immunohistochemistry, no MHCemb expression was detected at
this time. However, as judged by MHCemb, fiber regeneration
was prominent in 5-day-treated muscle. According to zymographical
studies on the two stages of degeneration/regeneration, the
2-day-treated muscle exhibited a pronounced although transitory decrease in cytosolic µ-calpain (Fig.
11). In contrast, cytosolic m-calpain
was unaltered. Both the 2-day- and 5-day-treated muscles, however,
contained considerable amounts of translocated m-calpain in their
particulate fractions. Translocation was also evident for µ-calpain
that became detectable in the particulate fraction of 5-day-treated
muscle.
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DISCUSSION |
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The purpose of this study was to elucidate changes in calpain activity during CLFS-induced fiber-type transitions. The use of low frequency-stimulated rat muscle as an experimental model largely excludes the possibility that the observed changes in calpain activity are due to fiber damage. The sarcomeric remodeling during fast-to-slow fiber-type transitions encompasses the exchange of fast-type myofibrillar and other protein isoforms with their slower or slow counterparts. It can be assumed that this process involves finely tuned interactions of proteolytic and protein synthetic activities. Moreover, excision and degradation of the protein isoforms no longer synthesized during the remodeling process should depend on the concerted action of several proteolytic systems.
The present study focuses on the calpain system. The regulation of calpains encompasses active and inactive forms of both calpains and calpastatin. In addition, interactions with activators and/or cellular structures play a role. Taking this into account, the present study assesses calpain activities both in the presence and absence of calpastatin. As shown by measurements on whole muscle homogenates, CLFS enhances calpain activity in the presence of calpastatin a few days after the onset of stimulation. Under the conditions of our assay, cleavage of the specific substrate in the presence of endogenous calpastatin can only result from active calpains. The increase in active calpain, without changes in its total amount (see below), could result by specific activators (16), by a reduced interaction with calpastatin, or by translocation. The possibility of calpastatin being phosphorylated and thus inactivated (20) is supported by CLFS-induced increases in cAMP (13) and resting free Ca2+ (4, 31). Because changes in calpastatin activity were not observed, the enhanced calpain activity might result either from activation due to autoprocessing or from altered intracellular distribution. The results obtained by the zymographical assay in the absence of endogenous calpastatin make the former possibility unlikely because neither conspicuous changes in total calpain activity nor autoprocessed forms were detected. These results support the hypothesis that nonprocessed calpains are the physiologically active forms (12).
A major result of the present study is the pronounced effect of CLFS on the intracellular distribution of calpain that is suggested to initiate enhanced myofibrillar turnover by dissociation of thin and thick filament components from the myofibrillar surface (9). Before the increase in calpain activity, we observed calpain activity in the particulate fraction. This redistribution appears to be more pronounced in the case of µ-calpain than of m-calpain. The translocation of µ-calpain, which is evident in 2-day-stimulated muscles, may be interpreted under three different aspects: 1) enhanced Ca2+ sensitivity due to interaction with membrane phospholipids (32) and/or 2) access to specific membrane-associated substrates (1, 38), or 3) initiation of a calpain cascade (36). An enhancement in Ca2+ sensitivity would amplify the effect of the CLFS-induced increase in cytosolic Ca2+. Access to specific substrates would be in line with the steep decay of the RyR previously observed in low frequency-stimulated rabbit muscle (10). The translocation of µ-calpain that precedes the overall increase in calpain activity might be an important step in a heterolytic activation of m-calpain, a possibility suggested by Tompa and co-workers (36) on the basis of in vitro studies.
A comparison of our results from normal EDL and low-frequency-stimulated EDL muscles with normal soleus muscle reveals pronounced differences between fast- and slow-twitch muscles; moreover, it demonstrates that transforming fast-twitch muscle acquires properties similar to those of a slow-twitch muscle. In contrast to the normal EDL with calpain activity detectable mainly in the cytosol, SOL muscle contains abundant calpain activity in the particulate fraction. The high fractions of structure-bound µ- and m-calpains in SOL muscle are in agreement with the higher protein turnover of slow-twitch muscle compared with fast-twitch muscle (7).
Translocation of m-calpain to the cell membrane has been suggested to represent a conditioning step in myoblast fusion (27). Therefore, the possibility exists that the translocation observed in the stimulated muscle results from a similar process. Fiber degeneration/regeneration processes were previously demonstrated in low frequency-stimulated fast-twitch muscle of rabbit (15), but not in rat (21). In this context, the Marcaine experiment proved to be informative. It was performed to delineate calpain translocation by CLFS from that during myoblast fusion. The finding that translocation is mainly confined to m-calpain in regenerating muscle suggests that the translocation of µ-calpain is specifically related to the CLFS-induced transformation process.
In summary, CLFS up to 20 days induces in fast-twitch EDL muscle an increase in calpain activity as determined in the presence of endogenous calpastatin. This increase reaches a level intermediate between normal fast- and slow-twitch muscles. With the use of a calpain-specific zymographical assay for analysis of total µ- and m-calpain activities in muscle homogenates, we provide evidence that enhanced calpain activity neither results from an increase in total calpain nor from autoprocessing-induced calpain activation. However, enhanced contractile activity by CLFS induces calpain translocation to membranes and the myofibrillar apparatus of the muscle fiber that might be an important step in the activation of calpain to initiate controlled proteolysis during muscle fiber transformation. Interestingly, the increases in calpain activity coincide with the early alterations in MHC isoform composition, namely, the exchange of MHCIIb with MHCIId and MHCIIa (34).
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ACKNOWLEDGEMENTS |
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We thank Elmi Leisner for excellent technical assistance.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft Grant Pe 62/27.
Address for reprint requests and other correspondence: D. Pette, Faculty of Biology, Univ. of Konstanz, D-78457 Konstanz, Germany (E-mail: dirk.pette{at}uni-konstanz.de).
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. §1734 solely to indicate this fact.
Received 9 August 1999; accepted in final form 21 March 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arthur, GD,
Booker TS,
and
Belcastro AN.
Exercise promotes a subcellular redistribution of calcium-stimulated protease activity in striated muscle.
Can J Physiol Pharmacol
77:
42-47,
1998[ISI].
2.
Belcastro, AN,
Shewchuk LD,
and
Raj DA.
Exercise-induced muscle injury: a calpain hypothesis.
Mol Cell Biochem
179:
135-145,
1998[ISI][Medline].
3.
Bor-Rung, O,
and
Forsberg NE.
Determination of skeletal muscle calpain and calpastatin activities during maturation.
Am J Physiol Endocrinol Metab
261:
E677-E683,
1991
4.
Carroll, S,
Nicotera P,
and
Pette D.
Calcium transients in single fibers of low frequency stimulated fast-twitch muscle of rat.
Am J Physiol Cell Physiol
277:
C1122-C1129,
1999
5.
Edelstein, CL,
Wieder ED,
Yaqoob MM,
Gengaro PE,
Burke TJ,
Nemenoff RA,
and
Schrier RW.
The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.
Proc Natl Acad Sci USA
92:
7662-7666,
1995[Abstract].
6.
Furuno, K,
and
Goldberg AL.
The activation of protein degradation in muscle by Ca2+ or muscle injury does not involve a lysosomal mechanism.
Biochem J
237:
859-864,
1986[ISI][Medline].
7.
Goldberg, AL.
Protein synthesis in tonic and phasic skeletal muscles.
Nature
216:
1219-1220,
1967[ISI][Medline].
8.
Goll, DE,
Dayton R,
Singh I,
and
Robson RM.
Studies of the -actinin/actin interaction in the Z-disk by using calpain.
J Biol Chem
266:
8501-8510,
1991
9.
Goll, DE,
Thompson VF,
Taylor RG,
and
Christiansen JA.
Role of the calpain system in muscle growth.
Biochimie
74:
225-237,
1992[ISI][Medline].
10.
Hicks, A,
Ohlendieck K,
Göpel SO,
and
Pette D.
Early functional and biochemical adaptations to low-frequency stimulation of rabbit fast-twitch muscle.
Am J Physiol Cell Physiol
273:
C297-C305,
1997
11.
Huang, J,
and
Forsberg NE.
Role of calpain in skeletal-muscle protein degradation.
Proc Natl Acad Sci USA
95:
12100-12105,
1998
12.
Johnson, GVW,
and
Guttmann RP.
Calpains: intact and active?
Bioessays
19:
1011-1018,
1997[ISI][Medline].
13.
Kraus, WE,
Longabaugh JP,
and
Liggett SB.
Electrical pacing induces adenylyl cyclase in skeletal muscle independent of the -adrenergic receptor.
Am J Physiol Endocrinol Metab
263:
E226-E230,
1992
14.
Lecker, SH,
Solomon V,
Mitch WE,
and
Goldberg AL.
Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states.
J Nutr
129:
227S-237S,
1999
15.
Maier, A,
Gambke B,
and
Pette D.
Degeneration-regeneration as a mechanism contributing to the fast to slow conversion of chronically stimulated fast-twitch rabbit muscle.
Cell Tissue Res
244:
635-643,
1986[ISI][Medline].
16.
Melloni, E,
Michetti M,
Salamino F,
and
Pontremoli S.
Molecular and functional properties of a calpain activator protein specific for µ-isoforms.
J Biol Chem
273:
12827-12831,
1998
17.
Nakamura, M,
Imahori K,
and
Kawashima S.
Tissue distribution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats.
Comp Biochem Physiol B Biochem Mol Biol
89:
381-384,
1988[ISI].
18.
Pette, D,
and
Staron RS.
Mammalian skeletal muscle fiber type transitions.
Int Rev Cytol
170:
143-223,
1997[Medline].
19.
Pette, D,
and
Vrbová G.
What does chronic electrical stimulation teach us about muscle plasticity?
Muscle Nerve
22:
666-677,
1999[ISI][Medline].
20.
Pontremoli, S,
Viotti PL,
Michetti M,
Salamino F,
Sparatore B,
and
Melloni E.
Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation.
Biochem Biophys Res Commun
187:
751-759,
1992[ISI][Medline].
21.
Putman, CT,
Düsterhöft S,
and
Pette D.
Changes in satellite cell content and myosin isoforms in low-frequency stimulated fast muscle of hypothyroid rat.
J Appl Physiol
86:
40-51,
1999
22.
Raj, DA,
Booker TS,
and
Belcastro AN.
Striated muscle calcium-stimulated cysteine protease (calpain-like) activity promotes myeloperoxidase activity with exercise.
Pflügers Arch
435:
804-809,
1998[ISI][Medline].
23.
Raser, KJ,
Posner A,
and
Wang KKW
Casein zymography: a method to study µ-calpain, m-calpain, and their inhibitory agents.
Arch Biochem Biophys
319:
211-216,
1995[ISI][Medline].
24.
Salmons, S,
Gale DR,
and
Sréter FA.
Ultrastructural aspects of the transformation of muscle fibre type by long term stimulation: changes in Z discs and mitochondria.
J Anat
127:
17-31,
1978[ISI][Medline].
25.
Sasaki, T,
Kikuchi T,
Yumoto N,
Yoshimura N,
and
Murachi T.
Comparative specificity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic fluorogenic substrates.
J Biol Chem
259:
12489-12494,
1984
26.
Schneider, AG,
Sultan KR,
and
Pette D.
Muscle LIM protein: expressed in slow muscle and induced in fast muscle by enhanced contractile activity.
Am J Physiol Cell Physiol
276:
C900-C906,
1999
27.
Schollmeyer, JE.
Role of Ca2+ and Ca2+-activated protease in myoblast fusion.
Exp Cell Res
162:
411-422,
1986[ISI][Medline].
28.
Shevchenko, S,
Feng W,
Varsanyi M,
and
Shoshan-Barmatz V.
Identification, characterization and partial purification of a thiol-protease which cleaves specifically the skeletal muscle ryanodine receptor Ca2+ release channel.
J Membr Biol
161:
33-43,
1998[ISI][Medline].
29.
Solomon, V,
and
Goldberg AL.
Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts.
J Biol Chem
271:
26690-26697,
1996
30.
Spencer, MJ,
Croall DE,
and
Tiball JG.
Calpains are activated in necrotic fibers from mdx dystrophic mice.
J Biol Chem
270:
10909-10914,
1995
31.
Sréter, FA,
Lopez JR,
Alamo L,
Mabuchi K,
and
Gergely J.
Changes in intracellular ionized Ca concentration associated with muscle fiber type transformation.
Am J Physiol Cell Physiol
253:
C296-C300,
1987
32.
Suzuki, K,
and
Sorimachi H.
A novel aspect of calpain activation.
FEBS Lett
433:
1-4,
1998[ISI][Medline].
33.
Taillandier, D,
Aurousseau E,
Meynial-Denis D,
Bechet D,
Ferrara M,
Cottin P,
Ducastaing A,
Bigard X,
Guezennec C-Y,
Schmidt H-P,
and
Attaix D.
Coordinate activation of lysosomal, Ca2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle.
Biochem J
316:
65-72,
1996[ISI][Medline].
34.
Termin, A,
and
Pette D.
Changes in myosin heavy-chain isoform synthesis of chronically stimulated rat fast-twitch muscle.
Eur J Biochem
204:
569-573,
1992[Abstract].
35.
Tiao, G,
Lieberman M,
Fischer JE,
and
Hasselgren P-O.
Intracellular regulation of protein degradation during sepsis is different in fast- and slow-twitch muscle.
Am J Physiol Regulatory Integrative Comp Physiol
272:
R849-R856,
1997
36.
Tompa, P,
Baki A,
Schád E,
and
Friedrich P.
The calpain cascade. µ-Calpain activates m-calpain.
J Biol Chem
271:
33161-33164,
1996
37.
Wing, SS,
Haas AL,
and
Goldberg AL.
Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation.
Biochem J
307:
639-645,
1995[ISI][Medline].
38.
Zhao, X,
Posmatur R,
Kampfl A,
Liu S-J,
Wang KKW,
Newcomb JK,
Pike BR,
Clifton GL,
and
Hayes RL.
Subcellular localization and duration of µ-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study.
J Cereb Blood Flow Metab
18:
161-167,
1998[ISI][Medline].