©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calpains Are Activated in Necrotic Fibers from mdx Dystrophic Mice (*)

Melissa J. Spencer (1)(§), Dorothy E. Croall (3), James G. Tidball (1) (2)

From the (1) Department of Physiological Science and the (2) Jerry Lewis Neuromuscular Research Center, University of California, Los Angeles, California 90095 and the (3) Department of Biochemistry, University of Maine, Orono, Maine 04469-5735

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Death of dystrophin-deficient muscle purportedly results from increases in [Ca] that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and µ-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor µ-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of µ-calpain, indicative of increased activation. The extent of autoproteolysis of µ-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.


INTRODUCTION

Mutations in the gene for dystrophin that lead to the protein's absence underlie the muscular degeneration that occurs in Duchenne muscular dystrophy (DMD)() and mdx mouse dystrophy. However, the mechanisms by which dystrophin's absence leads to muscle cell death are unknown. Both mdx and DMD pathologies are characterized by myofibrillar protein loss, Z-disc disorganization, plasma membrane defects, and dilation of the sarcoplasmic reticulum (1, 2) . In DMD, these pathological changes first appear at about age two, when muscle necrosis begins and continues until death. mdx mice display these pathological changes at approximately 28 days when their muscles undergo one bout of necrosis, but then spontaneously regenerate (3) . mdx muscles do not undergo cycles of necrosis regeneration subsequent to this first regenerative episode (4) .

Muscle from DMD humans and mdx mice contain abnormally high concentrations of intracellular calcium during necrosis, which is believed to contribute to muscle cell death through the activation of proteases. Evidence for the pathological increase in intracellular [Ca] has been obtained in several studies using a variety of technical approaches (5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . One hypothesis to explain the elevated intracellular calcium concentrations states that dystrophin's normal function is to stabilize a calcium channel, and its absence results in the deregulation of that channel (14) . Alternatively, the increase in [Ca] may result from mechanical damage to the cell membrane that permits unregulated entry of calcium into the cell (15) .

The relationship between the increase in [Ca] and the pathology of dystrophin-deficient muscle suggests that calpains, ubiquitous calcium-dependent cysteine proteases, play a key role in degeneration of dystrophic muscle. Calpains exist as two main isoforms designated µ-calpain (or calpain I) and m-calpain (or calpain II) (16) . The two forms are classified based on their calcium requirements, with µ-calpain requiring between 1 and 70 µM calcium and m-calpain requiring 10-800 µM calcium for half-maximal activity in vitro(17, 18, 19) . Recently, two tissue-specific calpain isoforms have been described at the cDNA level (20, 21) . One form specific to skeletal muscle is designated p94. It is reported to be rapidly turned over and present at low concentrations relative to the m- and µ-isoforms (22) .

Although there are no published data to show whether calpains are activated in dystrophic muscle, several studies provided indirect evidence consistent with a role for calpain activation in the pathology of dystrophin-deficient muscle (23, 24, 25, 26, 27) . First, data show that when [Ca] is experimentally elevated in normal, healthy muscle, cytosolic cysteine proteases are activated that initiate widespread proteolysis (28, 29, 30) . Thus, calpains, which are calcium-sensitive cysteine proteases, may feasibly play a similar proteolytic role in dystrophin-deficient muscle. Calpains are capable of proteolyzing a broad range of structural proteins found in muscle, which also supports their possible involvement in muscular dystrophy. Z-disc proteins appear to be particularly susceptible to calpains. Treatment of myofibrils with purified calpain removes Z-discs and releases -actinin (31, 32, 33, 34, 35, 36) ; similar Z-disc disruption or ``streaming'' is commonly cited as an early event in DMD and mdx (37) . In addition, many myofibrillar and other cytoskeletal proteins are substrates for calpain such as troponins I and C, tropomyosin, filamin, desmin, talin, vinculin, -actinin, and integrin (38) . Finally, current evidence has shown that the collective concentration of calpains is increased in extracts of DMD muscle (26, 39) , which also supports the possibility that calpains play a role in degeneration of dystrophin-deficient muscle. The previous studies, however, measured the concentration and activation of calpain in tissue homogenates using conditions under which all calpains present would be activated, so that the actual in vivo activity could not be assessed.

In vitro studies have demonstrated that autolysis of amino-terminal peptides from each calpain subunit occurs upon activation and results in increased calcium sensitivity (40, 41) . Thus, proteolytic modification of the enzyme can be used as a direct indication of calpain activation. Although autolysis of calpains has not been demonstrated previously in vivo, calpain autoproteolysis in platelets coincides with activation of platelets in situ. Calpain autolysis in situ has been shown to coincide with relocalization of the enzyme to the membrane and cleavage of the membrane skeletal proteins, talin, spectrin, and filamin (42, 43, 44, 45) .

In the present investigation, we measured changes in the concentration and autoproteolytic modification of m-calpain and µ-calpain over the course of mdx dystrophy. By performing these analyses on muscles from 4-week (peak necrosis) and 14-week (regenerated) mice, the autoproteolytic modification of calpain can be correlated with the stage of the disease process. The mRNA levels and protein concentrations of the specific inhibitor of calpains, calpastatin, were also measured to determine whether changes in calpain's autoproteolytic modification could be secondary to changes in the regulation of calpastatin.


MATERIALS AND METHODS

Reagents

The following materials were used in this investigation: cesium trifluoroacetate, oligolabeling kit, and restriction enzymes (Pharmacia Biotech Inc.), [-P]dCTP (ICN), purified m-calpain (Sigma), recombinant calpastatin (Takara Biochemicals). Calpain cDNA probes to m- and µ-calpain were generously donated by Dr. John S. Elce (46) . Calpastatin cDNA probe was kindly donated by Dr. Masatoshi Maki (Kyoto University) (47) . The -actin probe was generously donated by Dr. Adrian Casillas (Dept. of Medicine, UCLA) who transcribed it from the original plasmid. Other chemicals were the highest grade obtainable from Fisher Scientific.

Animals

C57 and mdx mice were obtained from a colony generated at UCLA using breeding pairs originating from The Jackson Laboratories. Animals were housed at the UCLA vivarium and were 2, 4, or 14 weeks old at the time of sacrifice.

Antibody Production and Antisera Purification

Antisera were raised against oligopeptides of two different calpain sequences. One antigen corresponding to chicken calpain sequence (residues 282-301) (48) represents a highly conserved catalytic domain sequence present in m-, µ-, and the muscle-specific p94 calpains. The antisera were raised and characterized as described earlier (49) ; however, the antibody used for these experiments was of a much higher titer than in previously performed studies (49) , enabling sensitive detection of all known calpain isoforms. In this report it is referred to as anti-CCD (Conserved Catalytic Domain sequence). Immunoreactivity with this serum is independent of the autolytic modification of the calpain isoform. The other peptide antigen corresponded to the amino-terminal sequence for mammalian m-calpain (residues 4-23 from human sequences). The antiserum raised against this peptide was generated and characterized as described earlier (50) , which demonstrated its isoform specificity and selective reactivity with calpain prior to autoproteolysis, thereby recognizing only the un-autoproteolyzed m-calpain catalytic subunit. In this report, we refer to this serum as anti-NTm calpain (amiNo-Terminal-m). Anti-calpastatin was produced by immunizing a rabbit with recombinant human calpastatin domain I. The recombinant protein was initially purified on a Mono Q ion exchange column (Pharmacia) with a linear NaCl gradient at pH 7.6. The antisera were affinity-purified with antigen coupled to cyanogen bromide (CNBr)-activated Sepharose beads, prepared according to the manufacturer's instructions (Pharmacia Biotech Inc.). Following purification, the anti-calpastatin recognized one band of crude whole muscle extracts on immunoblots at 107 kDa.

Immunoblots

Proteins fractionated by ion exchange chromatography and an m-calpain standard were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (51) . Samples were then electrophoretically transferred to nitrocellulose for antibody overlays (52) . Nitrocellulose strips were incubated with the primary antibody, and immunoblots were performed as described earlier (49) except the secondary antibody (goat anti-rabbit) was conjugated to alkaline phosphatase. Color development was performed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate.

For preparation of whole muscle extracts to be analyzed by SDS-PAGE, mouse hind limb muscles previously frozen in liquid nitrogen were homogenized in 30 µl of reducing sample buffer (80 mM Tris-HCl, pH 6.8, 0.1 M dithiothreitol, 70 mM SDS, glycerol) per mg wet weight using a Dounce homogenizer. The samples were boiled for 1 min, centrifuged at 4 °C for 3 min at 12,000 g, and the supernatant was collected. The protein concentration was determined, and 50-75 µg were loaded onto 8% polyacrylamide gels. For each immunoblot, three C57 and three mdx samples were electrophoresed, and at least three experiments were performed for each age group.

Ion Exchange Chromatography

mdx and C57 mice were sacrificed in pairs, by cervical dislocation, and the hind limb muscles were dissected and homogenized with a Dounce homogenizer for 2 min in 5 volumes of buffer A (20 mM Tris, pH 7.6, 3 mM 2-mercaptoethanol, 5 mM EGTA, 10 mM NaCl) and centrifuged at 10,000 g at 4 °C for 20 min. The supernatant was then filtered through a 0.22 µM filter, and the concentration of the samples was adjusted with buffer A so that they were equal.

Samples were loaded onto the Mono Q column in buffer A and washed to baseline in the same buffer. Samples were eluted with a linear gradient of buffers A and B (20 mM Tris, pH 7.6, 3 mM -mercaptoethanol, 5 mM EGTA, 1.0 M NaCl) increasing from 0% B to 70% B in 21 ml. One-ml fractions were collected, and 65 µl of each fraction were loaded onto SDS-PAGE gels and blotted onto nitrocellulose paper. Duplicate sets of column fractions were analyzed; one for anti-CCD analysis (total calpain concentration) and one for anti-NTm calpain analysis (unproteolyzed m-calpain). The relative concentrations of calpain isoforms separated by ion exchange chromatography were measured by densitometry of immunoblots, and mdx values were expressed relative to control values.

RNA Blot Analysis

Total RNA was prepared from hind limb muscles using cesium trifluoroacetate, prepared according to the manufacturer's instructions. Total RNA (25 µg) was fractionated on 1.2% agarose gels containing 2.2 M formaldehyde (53) . Samples were transferred to uncharged nylon membranes electrophoretically and cross-linked to the membrane in a UV cross-linker (Bio-Rad) with 125 mJ. DNA probes were labeled with P using the random priming method. Membranes were hybridized with 2 10 cpm/ml of probe in the pre-hybridization fluid for 15-18 h. Washing was done with 0.2 SSC, 0.1% SDS at 42 °C for 2 h with at least 6 changes of wash buffer. Following washes, blots were exposed to Kodak autoradiographic film for 2-6 days.

Statistical Analysis

All quantitative data were analyzed using statistical analysis. For all experiments normalized to controls, Mann-Whitney analysis was utilized. Results were given as p to indicate the level.


RESULTS

Assays for Calpain Concentration in Prenecrotic, Necrotic, and Regenerative mdx Muscle

Calpain concentration was measured in mdx mouse muscle to determine if its elevation corresponds to a particular stage of the disease process. The antibody utilized was generated using a synthetic polypeptide from a highly conserved domain of calpain (49) that is approximately 70% conserved between m-calpain, µ-calpain, and p94; thus, this antibody recognizes all three isoforms of the catalytic subunit of calpain. Immunoblots of whole muscle extracts with anti-CCD revealed three bands migrating at molecular sizes of 78/80 kDa, 84 kDa, and 94 kDa (Fig. 1). The immunoreactive proteins at 78/80 kDa represent a mixture of the catalytic subunits of m-calpain, both pre- and post-autoproteolysis of the amino-terminal peptide of m-calpain, and autoproteolyzed µ-calpain. These are not resolved under the electrophoretic conditions used (54) . The 84-kDa band represents the unproteolyzed µ-calpain catalytic subunit. The 94-kDa band is believed to represent the skeletal muscle-specific p94 since the immunogen utilized is highly conserved between the 3 known isoforms of calpain.


Figure 1: Immunoblot showing specificity of anti-conserved catalytic domain peptide (anti-CCD). SDS-PAGE separation of high molecular mass protein standard stained with Coomassie Blue ( lane a), whole muscle extract of C57 muscle stained with Coomassie Blue ( lane b), immunoblot with anti-CCD on 500 ng of purified m-calpain ( lane c), and immunoblot, with anti-CCD, of the whole muscle extract shown in lane b ( lane d). Double arrows point to p78/80 ( lower band) and p84 kDa ( upper band) doublet. Arrowhead points to p94. Numbers indicate molecular mass in kilodaltons.



Muscle extracts were prepared from mdx mice at three stages of dystrophy: 2-week prenecrotic, 4-week peak necrotic, and 14-week regenerated and their age-matched controls. Samples were analyzed by immunoblotting with the anti-CCD serum. Calpain concentration was expressed relative to myosin heavy chain concentration so that changes in the concentration of fibrous components would not influence measurements of calpain concentration. Previous investigators (55) have shown that there is more connective tissue in mdx muscle than in age-matched controls. There are also significant increases in connective tissue concentration of mdx mice as they mature. If data were not expressed relative to a muscle protein, the change in concentration of muscle cells in the tissue during the necrotic or aging processes would cause an apparent change in calpain concentration in the tissue that would not necessarily reflect a change in calpain concentration in the muscle cells. Therefore, data were normalized to myosin heavy chain. Quantitation of total immunoreactive protein at 78/80 and 84 kDa by this method revealed a 1.81-fold increase (* p < 0.05) in the concentration of calpain in 4-week mdx muscles and a 1.59-fold increase (* p < 0.05) in calpain in adult mdx mouse muscles relative to age-matched controls (Fig. 2). This increase was attributable primarily to a significant increase in the amount of p78/80 which corresponds to a mixture of calpain isoforms. In contrast, p94 showed a significant decrease at the 4-week time point in mdx (63% of controls, * p < 0.05) but returned to control values in the adult mdx animals (91% of controls, * p < 0.3) (Fig. 2). Prenecrotic (2-week) mdx muscles were also assayed to determine whether calpain was constitutively expressed at higher levels in mdx muscle than in controls. This analysis showed no difference in calpain concentration between 2-week mdx or C57 muscles (Fig. 2). The increased concentration of total calpain in 4-week and 14-week mdx mice but not in 2-week prenecrotic mice indicates that the increase in calpain coincides with the onset of mdx muscle pathology.


Figure 2: Total calpain is increased in 4- week and adult, but not 2-week mdx muscle extracts. Histogram of densitometric measurements of calpain bands on immunoblots. Whole muscle extracts of C57 and mdx mice were prepared by homogenization in SDS-PAGE sample buffer. Samples were electrophoresed on 8% gels and immunoblotted with anti-CCD. For each experiment, 3 control and 3 mdx samples were run on the same gel and immunoblotted. Immunoreactive calpain areas were normalized to myosin heavy chain within the same sample and expressed relative to control values with control equal to 1. Values shown are the mdx value expressed relative to controls and are the average of three experiments. Error bars represent standard deviation between experiments.



Characterization of Increased Calpain Concentration

Whole muscle extracts from the gastrocnemius, quadriceps, and hamstrings of 4-week- and 14-week-old mice were separated by ion exchange chromatography and then by SDS-PAGE to characterize further the increased 78/80-kDa and 84-kDa calpains. Separation of the two isoforms, by ion exchange chromatography, allowed for the quantitation of m- and µ-calpain concentrations separately. In this procedure, µ-calpain elutes at approximately 180 mM NaCl and m-calpain at approximately 480 mM NaCl. The fractions were then analyzed by immunoblot analysis (Fig. 3). At the 4-week time point, only m-calpain concentration was shown to be significantly elevated in mdx mice (1.81-fold, * p < 0.05). At the 14-week time point, both m- and µ-calpains were elevated in mdx (2.3-fold and 1.4-fold, respectively), although the increase in calpain concentration relative to age-matched controls was not significant (Fig. 3). The previously observed increase in total calpain observed on immunoblots was therefore attributable to a significant increase in m-calpain at 4 weeks and the combined increase in both m- and µ-calpains at 14 weeks.


Figure 3: The increase in total calpain is due to an increase in m-calpain at the 4-week time point. Histogram showing calpain concentrations obtained from immunoblots of ion exchange-fractionated whole muscle extracts. mdx values are expressed relative to control values with control equal to 1. Bars show standard error between experiments.



Messenger RNA Analysis of C57 and mdx Muscles

We then investigated whether the increase in calpain concentration resulted from the induction of transcription of the genes for m- or µ-calpain. mRNA concentrations were assessed using cDNA probes and Northern blotting. The same blots were stripped and reprobed for -actin mRNA to allow normalization of data. Analysis of total RNA by Northern blots revealed that mRNA levels for these proteins remained the same as control tissues at all time points in the disease process (2-week, 4-week, and 14-week mice) (Fig. 4, 2-week not shown). Therefore, it appears that an increased half-life of the calpain proteins, rather than an increase in the rate of transcription, is responsible for the increased concentration in mdx tissues.


Figure 4: Northern analysis of mdx and C57 muscles; calpain expression is not regulated at the transcriptional level in mdx muscles. Twenty five micrograms of total RNA were electrophoresed on formaldehyde gels and analyzed by cDNA probes. a, m-calpain, 3.5 kb; b, µ-calpain, 3.5 kb; c, -actin. Lane 1, 4-week C57; lane 2, 4-week mdx; lane 3, 14-week C57; lane 4, 14-week mdx.



Assay of Autoproteolytic Activation of µ- and m-Calpain in mdx Dystrophy

The findings reported above show that calpain concentration is modified during mdx dystrophy; however, the data do not address changes in activation of the individual isoforms of calpain in vivo. Therefore, direct analyses of the amino-terminal propeptides of m- and µ-calpain were performed in order to determine the extent of autoproteolysis of these enzymes. The amino-terminal propeptide of calpain is cleaved upon calpain activation; therefore, antibodies directed against the amino terminus recognize only the inactive form of the enzyme. This difference between inactive and active calpain was exploited in the following study and utilized to determine the in vivo autoproteolytic modification of the calpains.

The analyses of m- and µ-calpain were performed slightly differently. Because the autoproteolyzed, p78 (active) and non-autoproteolyzed, p84 (inactive) forms of µ-calpain are resolved by SDS-PAGE, the extent of autoproteolysis of µ-calpain can be assessed directly by densitometric analysis of immunoblots of µ-calpain ion exchange fractions (54) . For m-calpain, the autolyzed 80-kDa subunit is not resolved from the unmodified subunit, and autolysis was assessed with anti-NTm calpain which recognizes the peptide lost from the 80-kDa subunit through autolysis. Therefore, anti-NTm calpain recognizes only inactive m-calpain. Thus, the extent of autoproteolysis was measured by comparing m-calpain reactivity with anti-CCD (total m-calpain) (Fig. 5 a), and the reactivity of the same samples with anti-NTm calpain (preautolysis only) (Fig. 5 b). The activation index (extent of autoproteolysis) was derived using the following formula: [mdx total calpain/C57 total calpain] [C57 pro-calpain/mdx pro-calpain].


Figure 5: Fractionation of calpain isoforms and immunoblotting with anti-CCD and anti-NTmcalpain. Whole extracts of adult mouse muscle were fractionated using ion exchange chromatography, electrophoresed on 8% SDS-PAGE, and immunoblotted with anti-CCD or anti-NTm calpain. Duplicate blots are shown where samples in a are overlaid with anti-CCD and samples in b with anti-NTm calpain. Double arrowheads point to pro-µ-calpain (upper band, 84 kDa) and autolyzed µ-calpain (lower band, 78 kDa). Fraction numbers (11, 12 and 18, 19) off the ion exchange column are indicated at the bottom of b. µ = µ-calpain fractions, m = m-calpain fractions, C57 = control fractions; MDX = mdx fractions.



This analysis showed that the activation of µ-calpain was the highest during peak necrosis (4-week mdx mice) (2.9-fold increase, * p < 0.05) when compared to age-matched controls and returned to levels near controls in regenerated muscle of 14-week mdx mice (Fig. 6). The extent of autoproteolysis of m-calpain did not differ significantly between mdx and control muscle at either time point.


Figure 6: µ-Calpain has the highest autoproteolytic activation at the stage of peak necrosis. Histogram showing calpain activity obtained from propeptide analysis of pre- and postautolysis isoforms of calpain. Six experiments were performed for each time point. These data show that an increase in µ-calpain activity occurs in the 4-week mice but returns to control levels in adult, regenerated mice. mdx values are expressed relative to control values with controls equal to 1. Bars show standard error between experiments.



Calpastatin Concentration Is Not Elevated in Adult mdx Mouse Muscle

All cells containing calpains also contain their specific endogenous inhibitor known as calpastatin. Thus, changes observed in the extent of autoproteolysis observed for µ-calpain could result from changes in calpastatin concentration. To determine if changes in calpain activation might be secondary to alterations in calpastatin concentration, we used both Northern blotting and Western blotting. Hind limb muscle from 4-week and adult mdx mice and age-matched controls were subjected to immunoblot or Northern analysis for calpastatin. No difference in calpastatin protein concentration was observed between mdx and age-matched controls at either age (Fig. 7). Furthermore, no difference in calpastatin mRNA concentration was detectable in Northern blot analysis (Fig. 8). Hence, up-regulation of calpastatin concentration is not the mechanism by which mdx muscles regulate calpain during necrosis or regeneration.


Figure 7: Calpastatin protein concentrations in mdx muscles are similar to age-matched controls. Immunoblots of whole muscle extracts from C57 and mdx hind limb muscles overlaid with anti-calpastatin. a, immunoblot of 4-week whole muscle extracts from mdx and C57 hind limb with anti-calpastatin. b, immunoblot of adult whole muscle extracts from mdx and C57 hind limb with anti-calpastatin. Anti-calpastatin recognizes a band in crude muscle extracts at 107 kDa.




Figure 8: Calpastatin mRNA concentrations in mdx muscles are similar to age-matched controls. Autoradiograph of 25 µg of total RNA extracted from mdx and C57 hind limb and probed with calpastatin cDNA probe (3.8 kb) ( a) or -actin cDNA probe (1.7 kb) ( b). Lower stringency washes of the nylon filter probed with calpastatin cDNA also revealed a faint band at 5.1 kb (not shown). Lane 1, 4-week C57 RNA; lane 2, 4-week mdx RNA; lane 3, 14-week C57 RNA; lane 4, 14-week mdx RNA.




DISCUSSION

Previous investigations have relied upon indirect evidence and speculation to conclude that calpains play a role in the wasting of dystrophic muscle. Recent progress in understanding the biochemistry of the calpain family of proteases has provided the tools to finally address this issue more definitively. The present investigation provides the first direct evidence that calpains are active components in the pathology of dystrophin-deficient muscle in that their concentrations and/or levels of autoproteolytic modification vary over the course of the disease.

In this study, three isoforms of calpains were quantified. Immunoblots of whole muscle extracts document that each calpain isoform appears to be regulated independently throughout the progression of necrosis and regeneration. Thus, each isoform may make distinct contributions to the process of muscle degeneration in muscular dystrophy. Results from immunoblotting also demonstrated that the total concentration of calpains is increased in necrotic muscle from mdx mice relative to controls. The increase is largely attributable to an increase in the m-calpain isoform and correlated with the onset of necrosis. The increased m-calpain protein did not result from an accompanying increase in its mRNA suggesting that regulation was not at the level of transcription. The total concentration of the other major calpain isoform, µ-calpain, was not changed throughout the progress of muscle necrosis and regeneration.

This is the first study to document the presence of the skeletal muscle-specific calpain, p94 protein, in muscle tissues and to show that its concentration decreases at peak muscle necrosis. In a previous study, p94 was not detected in fractionated rat muscle extracts, which the authors interpreted to suggest that p94 was very short-lived (22) . The discrepancy between the two reports may lie in differences in sample preparation, antibody titer, and/or antibody specificity. The difference is apparently not due to species differences as the anti-CCD also detected p94 in rat muscle extracts (not shown). The decrease in p94 concentration accompanying necrosis may reflect a decreased stability and increased turnover.

The protein concentration of calpains, however, may not be directly related to enzyme activity in vivo. It is well established that µ- and m-calpain undergo autoproteolysis which precedes, or accompanies, substrate hydrolysis (16-18, 40, 50, 54, 56-58). Evidence also clearly demonstrates that calpains become more sensitive to calcium after autoproteolysis and that removal of an amino-terminal peptide of the µ-calpain catalytic subunit is prerequisite for cleavage of other proteins (59) . It is still controversial whether the autoproteolysis of m-calpain is a prerequisite for substrate hydrolysis (50, 57, 58, 60) although it is agreed that if m-calpain has been proteolytically modified it is, or has been, activated. Thus, the loss of the amino-terminal peptide provides a minimal estimate of activation of m-calpain in vivo. We therefore examined covalent changes to the calpain catalytic subunits to assess their activation status within dystrophic muscles in vivo. Despite no significant change in µ-calpain concentration at peak necrosis, the fraction of autoproteolyzed, active µ-calpain increased substantially. Importantly, the activation status returned to control values in regenerated muscle. These changes in µ-calpain activation do not result from any regulatory influence of calpastatin, because we found no change in the concentration of calpastatin or its mRNA at the ages sampled. Our findings also show that m-calpain concentration increases during necrosis, although the extent to which it is autoproteolyzed does not change. However, these findings indicate that the total mass of autoproteolyzed m-calpain increases during necrosis because autolyzed m-calpain is a constant proportion of an increased mass of total m-calpain.

The results of the present study provide strong evidence that calpains are active components in the pathology of dystrophin-deficient muscle necrosis in that their concentrations and/or levels of activation of each isoform vary independently with the progress of the disease state. Although the data show here that calpain activation increases only 2-3-fold above controls, these values were averaged over the whole muscle. Even at 4 weeks of age, the proportion of the fibers that undergo necrosis at any one time is small, so that a 2-3-fold increase in the entire muscle represents a much higher level of calpain activation in the subpopulation that is experiencing necrosis. For example, Carnwath and Shotten (61) have shown that at the peak of mdx muscle necrosis, only 7% of the muscle cross-section is occupied by degenerating fibers. If the majority of the increased calpain activation occurs in these fibers, the actual increase in calpain activation may be as high as 20-30-fold in fibers undergoing degeneration. These studies show directly for the first time in any whole animal system that calpain activation varies during a biological process and is pertinent to events occurring in vivo. It remains for future studies to establish whether calpain plays an initiating role in proteolysis encountered in mdx dystrophy.


FOOTNOTES

*
This work was supported by Grants AR-40343 from the National Institutes of Health (to J. G. T.), DCB90-96188 and MCB93-19602 from the National Science Foundation (to D. E. C.), a grant from the Muscular Dystrophy Association (to J. G. T.), and the UCLA dissertation year fellowship from the UCLA graduate division (to M. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Physiological Science, UCLA, Los Angeles, CA 90095-1527. Tel.: 310-206-8389; Fax: 310-206-9184.

The abbreviations used are: DMD, Duchenne muscular dystrophy; PAGE, polyacrylamide gel electophoresis; kb, kilobase(s).


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