EXPERIMENTAL

CELL

RESEARCH

203,

107-114

(19%)

Calpain Concentration Is Elevated Although Net Calcium-Dependent Proteolysis Is Suppressed in Dystrophin-Deficient Muscle Departmentof Physiological

Science

and

MELISSA

J. SPENCER’

Jerry

Neuromuscular

Lewis

AND JAMES Research

INTRODUCTION Duchenne muscular dystrophy (DMD) and m& mouse dystrophy are both X-linked recessive diseases that result in severe muscle wasting attributed to the absence of the protein dystrophin [ 1, 21. Muscle necrosis occurring in dystrophin deficiency is characterized by Z-disc streaming, plasma membrane defects, and dilation of the sarcoplasmic reticulum in early stages of the diseases. The relationship between the absence of dystrophin and the ensuing pathology is uncertain, but a leading hypothesis asserts that dystrophin plays a structural role at the cell membrane and may mediate To whom

reprint

requests

should

University

of California,

Los

Angeles,

California

90024

thin filament-membrane associations. This hypothesis is supported by the observation of membrane defects in DMD muscle [3], the lack of lateral attachments between thin filaments and the myotendinous junction membrane in m& mice [4], and similarities in amino acid sequence between dystrophin and the cytoskeletal proteins a-actinin and spectrin [5]. Furthermore, recent findings [6] show that dystrophin-associated glycoproteins bind laminin extracellularly, indicating that dystrophin may be part of a chain of structural molecules linking thin filaments to the extracellular matrix. A characteristic feature of both DMD and mdx muscle cells that may provide insight into the relationship between dystrophin deficiency and muscle necrosis is elevated intracellular calcium levels [7-lo]. Although the mechanism by which the lack of dystrophin leads to elevated intracellular calcium is not understood, studies using peroxidase loading and osmotic stability as indicators of membrane leakiness have confirmed that mdx and DMD plasma membranes are leakier than controls [3, 111. In addition, a relationship between the elevated [Ca”], and enhanced protein degradation in muscle has been demonstrated [7]. In these experiments, protein degradation was shown to be 80% greater in the calcium-enriched mdx muscle. These data support a relationship between a lack of dystrophin, enhanced Recent evi[Ca2+lin7and increased protein degradation. dence indicates that dystrophin may function in regulating calcium channel activity in such a way that its absence leads to increased intracellular calcium [S]. Proteolysis in dystrophin-deficient muscle is expected to result from the activation of calcium-dependent proteases called calpains. Although calpains have been well characterizedbiochemically [12], their physiological role has not been well defined. One hypothesis is that they are housekeeping proteins involved in normal myofibrillar turnover and that elevation of [Ca2+lin to pat,hological levels results in extensive muscle necrosis via their activation. Studies by Kar and Pearson [13] and Reddy et al. 1141 have demonstrated elevated calpain activity in DMD muscle. In addition, calpain activity has been shown to be elevated in muscle from other dystrophic animal models such as the chicken [15], dy/ dy mouse [16], and hamster [17].

The concentration, activity, and distribution of calcium-dependent proteases (calpains) are compared in dystrophin-deficient (n&) and control mouse muscle. Calpains have been implicated previously as the protease responsible for the observed necrosis in dystrophin-deficient human muscle. Although these mouse and human muscular dystrophies have been attributed to similar genetic defects, the mouse dystrophy shows a brief necrotic episode while the human deficiency results in progressive, lethal muscle necrosis. Findings of the present study show that control mouse muscle contains more calcium-dependent proteolytic activity than dystrophin-deficient muscle. Paradoxically, adult, dystrophin-deficient mouse muscle contains higher concentrations of calpain than found in controls. Furthermore, indirect immunofluorescence using antisera produced against an oligopeptide found in the proteolytic domain of calpain shows that calpain distribution in dystrophin-deficient muscle is dispersed throughout the cytoplasm while immunolabeling of control muscle shows calpain concentrated at Z-discs. This redistribution is consistent with calpain activation in dystrophic muscle. These findings indicate that mdx mice possess the capability of suppressing calpain-mediated proteolysis. We speculate that this suppression may enable dystrophin-deficient mouse muscle to arrest necrosis D 1992 Academic Press, Inc. and regenerate successfully.

1

Center,

G. TIDBALL

be addressed. 107

All

Copyright 0 1992 rights of reproduction

0014-4827/92 55.00 by Academic Press, Inc. in any form reserved.

108

SPENCER

AND

In view of these previous findings, the hypothesis that increased [Ca”‘], in dystrophin-deficient muscle leads to enhanced calpain activation and the characteristic necrosis of muscular dystrophy seems plausible. However, while the genetic basis and physiological causes of necrosis are similar in mdx and DMD, the outcomes of these myopathies differ dramatically. In DMD, necrosis is progressive and results in early death of the patient, while in mdx, necrosis is arrested, the muscle regenerates, and the mice live normal lifespans. This important difference indicates that either calpain-mediated necrosis is not adequate to explain the pathology of dystrophin-deficient muscle or mdx muscle is capable of controlling necrosis by a mechanism unavailable to DMD muscle. Two possible mechanisms to explain calpainmediated necrosis followed by successful regeneration in mdr muscle are (1) calpain expression is downregulated after the initial onset of necrosis or (2) calpain is present but inactivated, possibly by its endogenous inhibitor calpastatin. In the present investigation, we examine the role of calpain in dystrophin-deficient muscle by comparing calpain’s concentration with its activity and distribution in adult mdx mouse muscle. MATERIALS

AND

METHODS

Materials. Keyhole limpet hemocyanin was purchased from Calbiochem (San Diego), Protein A Superose column was from Pharmacia, O.C.T. compound was from Miles Inc., gel/mount mounting media were from Biomeda Corp., and all other chemicals were obtained from Sigma. Purified bovine m-calpain was generously donated by Dr. Dorothy Croall (University of Maine). Animals. C57 and mdx mice were obtained from Dr. Richard Entrikin (University of California, Davis) and housed at the UCLA vivarium. Animals were 5 months to 1 year old at the time of sacrifice. Assays for net calcium-dependent proteolysis. Net calcium-dependent proteolysis was assayed in hydrolysates of mdz and C57 hindlimb muscles so that the net effect of modifications in the concentration of calpains and their inhibitor calpastatin could be measured. Mice were sacrificed by cervical dislocation and gastrocnemius and quadriceps muscles were dissected and homogenized with a Dounce homogenizer for 1 min in 10 vol of buffer A (100 mM Tris, pH 7.5,100 mM KCl, 10 mM 2mercaptoethanol, 0.1 mM ethylenediaminetetraacetic acid, 1.0 mM sodium azide) with 1 .O mM phenylmethylsulfonyl fluoride (PMSF) and 100 mg/liter ovomucoid trypsin inhibitor and centrifuged at 17,000g at 4°C for 15 min. The muscle extract from mdn mice had a&a of 5.8, corresponding to a free calcium concentration of 2 PM. This level of free calcium is well below the levels demonstrated to cause autolysis [18, 191 or association of calpains with particulates [20]. The supernatant was mixed with casein (5 mg/ml in buffer A) and the reaction was started by the addition of calcium (5.0 mM) at 27°C. Non-calcium-dependent activity was assessed by the addition of the same volume of buffer A to duplicate tubes. Tubes were incubated at 27°C for 1 h and the reaction was stopped by the addition of 5% trichloroacetic acid (TCA) to a final concentration of 2.5%. Tubes were centrifuged at 17,000g at 4°C and the absorbance of the supernatant was measured at 278 nm. Calcium-dependent activity was determined by subtracting the absorbance of the non-calciumcontaining samples from the absorbance of the calcium-containing samples. Antibody production. Anti-calpain was produced against a 20. amino-acid peptide from the chicken calpain cDNA corresponding to

TIDBALL amino acids 2822301 [21]. The chosen sequence is highly conserved between both m and + forms of calpain and both chicken and rabbit calpains [21, 221. This oligopeptide is contained in the proteolytic domain of the protein (domain II). The synthesized peptide was coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde. The peptide was dissolved in 0.1 M phosphate-buffered saline, pH 7.5 (12 mg/ml), and 1 ml was mixed with 200 ~1 of KLH (86 mg/ml in 50% glycerol). Slowly (over 10 min) 500 ~1 of 0.2% glutaraldehyde was added with continual stirring. The mixture was allowed to stir for 30 min at room temperature and dialyzed at 4’C overnight against 4 liters of 0.1 M PBS, pH 7.5. The dialysate (600 ~1 for one inoculation) was thoroughly mixed 1:l with Freunds complete adjuvant and injected subcutaneously into rabbits. Following the first injection of antigen, each subsequent boost was mixed 1:l with Freunds incomplete adjuvant. The animals were bled bimonthly and boosted weekly. lmmunoblots and antisera purification. Mouse gastrocnemius, quadriceps, and tihialis anterior muscles previously frozen in liquid nitrogen or fresh chicken pectoralis muscle were homogenized in 30 ~1 reducing sample buffer (80 mM Tris-HCl, pH 6.8,O.l M DTT, 70 mM sodium dodecyl sulfate, 1.0 mM bromphenol blue, glycerol) per milligram wet weight using a Dounce homogenizer. Soluble proteins were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie blue, and destained with 7.5% acetic acid and 7.5% methanol [23]. Samples were then electrophoretically transferred to nitrocellulose for antibody overlays. Nitrocellulose strips were incubated with anti-calpain for 90 min; washed in buffer B (50 mM Tris, pH 7.6, 150 mM NaCl, 0.02% NaN,) with 0.5% Tween-20, 0.2% gelatin, and 0.3% non-fat dry milk (blocking buffer) for 1 h; and then incubated in iodinated goat antirabbit for 90 min. After 1 h of washing in blocking buffer, the nitrocellulose was dried at room temperature and exposed to film for autoradiography. Antisera were purified by protein A affinity chromatography. The antisera (2 ml) were centrifuged at 10,OOOg to remove large particles and the volume was increased to 16 ml with buffer C (3 M sodium chloride, 1.5 M glycine) to avoid loss of antibody during filtration. After filtration through glass wool and then a 0.22-pm filter, the antisera were loaded onto a protein A Superose column preequilibrated in buffer C. After washing to baseline with buffer C, the nonspecific immunoglobulins were eluted with buffer D (0.1 M citric acid), pH 6.5. Purified anti-calpain eluted with buffer D at pH 4.5. Following this purification, the antisera recognized one band at 80 kDa on immunoblots against whole mouse muscle extracts (Fig. 1). Densitometry. Relative protein concentration was quantitated by densitometry of immunoblots and Coomassie blue-stained gels. Although samples prepared for SDS-PAGE used a constant ratio of muscle mass to sample buffer in preparing homogenates, variability in mass of protein per lane was observed in SDS-PAGE samples following electrophoresis. We therefore chose to normalize measurements of calpain mass obtained by densitometry of autoradiographs to densitometric measures of both myosin heavy chain (MHC) and total protein, in each lane of identically loaded gels stained with Coomassie blue. Mass ratios of calpain to whole sample protein and calpain to MHC are both provided. In older adult mdx mice there is a slight increase in muscle fibrosis so that even if calpain concentration in muscle cells remained constant, the calpain:whole sample protein ratio would decrease over the course of fibrosis. Expression of data as calpain:MHC may therefore provide a more biologically significant measure of calpain concentration. Calpain inhibition assay. The assay to assess whether anti-calpain inhibits calcium-dependent activity was performed by incubating 0.091 unit of partially purified chicken calpain with anti-calpain or with preimmune sera (control) for 90 min on ice with frequent vortexing (1.0 unit of calpain activity is defined as the amount of enzyme that catalyzed an increase in absorbance of 1.0 OD at 278 nm in 1 h). Casein (5 mg/ml in buffer A) was added to all tubes, and 5 mM calcium chloride to one-half of the antibody tubes and one-half of the

CALPAINS TABLE

1

Calpain Activity in C57 and mdx C57

Gastrocnemius Quadriceps a Activities b n, sample * Significant

IN DYSTROPHIN-DEFICIENT

calcium-dependent activity or (2) the direct inhibition of a non-calcium-dependent proteolytic enzyme by calcium.

Mouse Muscle”

(n, SD)’

mdr -5.1 -2.3

5.3 (n = 5, 2.9) 4.5 (n = 6, 2.4)

(n,

109

MUSCLE

SD)

(n = 4, 2.3)* (n = 6, 4.1)*

are expressed in units/mg of protein. size; SD, standard deviation. (P = 0.0001).

control tubes. The other half of each group received an equal volume of buffer A. After incubation for 1 h at 27°C the reaction was stopped with 2.5% TCA and the absorbance of the supernatant measured at 278 nm. Zmmunolabeling. Freshly dissected tibialis anterior muscle for sectioning was embedded in O.C.T. compound on cork and frozen in isopentane cooled by liquid nitrogen. Cryosections (10 pm) were stored dry at -20°C prior to labeling. For labeling, sections were removed from the freezer and fixed in 2% paraformaldehyde for 3 min at room temperature. All subsequent steps were performed at room temperature. After fixation, slides were washed in buffer B for 5 min and placed in blocking buffer for 30 min. Sections were washed in buffer B for 5 min and overlayed with anti-calpain or preimmune sera for 3 h. After rinsing in buffer B for 5 min, sections were blocked for 1 h and rinsed again in buffer B for 5 min. The sections were overlayed with FITC goat anti-rabbit secondary antibody (1:50) for 1 h and finally rinsed in buffer B for 5 min, blocking buffer for 15 min, and buffer B for 5 min before mounting with water miscible mounting media and viewed by epifluorescence.

Synthesis

of a Calpain

Antibody

As a result of the observation that net calcium-dependent proteolysis in whole muscle extracts of mdx muscle was significantly lower than that in C57 muscle, we were interested in determining the concentration of both active and inactive calpains in those muscles. We therefore synthesized a 20-amino-acid oligopeptide of calpain and produced polyclonal antibodies against it. Anti-calpain was shown on immunoblots to recognize one band each from mouse and chicken whole muscle extracts after purification of the antisera on protein A Superose (Fig. 1). The apparent molecular weight of the band in mouse muscle is 80 kDa while the apparent molecular weight of the band in chicken muscle is 88 kDa. This mass is consistent with that previously described for chicken calpain [21]. The antibody also recognized purified bovine m-calpain with an apparent molecular weight of 80 kDa (Fig. 2). In a calpain assay with casein as a substrate, the antibody was shown to inhibit all calcium-dependent activity while control samples, using normal rabbit serum in place of the antibody, showed no inhibition of calciumdependent proteolysis (data not shown). This indicates that the antibody recognizes and inactivates both m-

RESULTS

Calpain

Activity

Is Lower

in Adult

mdx Mouse

ABC

Muscle

Calpain assays were performed on whole muscle extracts of the gastrocnemius and quadriceps muscles from mdx and C57 mice according to an established protocol [24]. These muscles were chosen because a large mass of muscle was necessary to obtain calpain absorbance values. Whole muscle extracts were used so that these assays would reflect the net calpain activity in the presence of endogenous calpastatin. Calcium-dependent proteolysis was barely detectable in mdx mice and was consistently lower than control values (Table 1). All assay values are expressed per milligram of total protein assayed. The total concentration of protein assayed did not correlate with the level of calcium-dependent activity measured. Interestingly, the addition of calcium to the assay mixture caused calcium-dependent proteolysis to decrease to a value less than the amount occurring in the tubes not containing calcium. This occurrence resulted in negative net calcium-dependent activity based on the definition we had previously designated. Since this observation was made approximately 80% of the time, we inferred that there must be other mechanisms influencing net, calcium-dependent proteolysis such as: (1) the activation by calcium of an inhibitor of

DE

200

116 95 68

42

FIG. 1. SDS-PAGE separation and immunoblots of mouse and chicken muscle extracts under reducing conditions. Lane A: molecular weight standards (numbers indicate mass in kilodaltons). Lane B: mouse muscle extract stained with Coomassie blue. Lane C: chicken muscle extract stained with Coomassie blue. Lane D: autoradiograph of mouse muscle labeled with anti-calpain. Lane E: autoradiograph of chicken muscle labeled with anti-calpain.

110

SPENCER

AND

or total protein. In addition, when total protein was quantitated prior to sample loading (50 pg per lane), mdx muscle also showed a higher concentration of calpain on immunoblots (Fig. 2, lanes C and D). Data shown in Table 2 were not pooled because we observed substantial variations in activity of the labeled second antibody and in the levels of background labeling between experiments. These variations affected the signal-to-noise ratio, hence the densitometric values obtained. Therefore, data are shown by individual experiment.

200

116 95 480

Calpain Distribution Muscle

68

a 480

b A

6

CDE

FIG. 2. SDS-PAGE separation and immunoblots of mouse and chicken muscle extracts under reducing condition. The corresponding autoradiograph is shown below the gel. Panel a: SDS-PAGE stained with Coomassie blue. Lane A: molecular weight standards (numbers indicate mass in kilodaltons). Lane B: chicken muscle extract. Lane C: mdx muscle extract (50 +g). Lane D: (2.57 muscle extract (50 pg). Lane E: purified bovine m-calpain (400 ng). Panel b: corresponding autoradiograph of SDSPAGE. Lane B: autoradiograph of chicken muscle labeled with anti-calpain. Lane C: autoradiograph of mdx muscle labeled with anti-calpain. Lane D: autoradiograph of C57 muscle extract labeled with anti-calpain. Lane E: autoradiograph of purified bovine m-calpain labeled with anti-calpain.

Calpain

ABCDEFG

in mdx and C57 Mouse

HIJKLM

200

116

95

68

as anticipated in view of the antigenic location in domain II of both m- and /.L-

Concentration

Is Different

Many investigators support the hypothesis that calpain changes its location in the cell following activation by calcium [26]. Immunolocalization studies have demonstrated a change in calpains’ location in the cell with different physiological states such as myoblast fusion [26], mitosis [27], and platelet aggregation [28]. Thus, we were interested in determining whether calpain might have a different localization in a pathological state such as muscular dystrophy. We examined the immunolocalization of calpain in C57 and mdx mouse muscle in frozen tissue sections. The mdx muscles used in this study were from adult animals and exhibited central nuclei, typical of regenerated fibers, both in cross sections and in longitudinal

42

and p-calpain, oligopeptide’s calpain.

TIDBALL

42

Is Elevated in Adult

mdx Mice

Qualitative comparisons of immunoblots of whole muscle extracts reveal calpain concentration to be consistently elevated in mdx mice (Figs. 2 and 3), and densitometry of gels and autoradiographs confirms this (Table 2). As stated under Materials and Methods, we felt the best method for normalizing protein loading values was to use a prominent muscle protein (MHC) whose concentration reflects the mass of muscle in the sample through the course of the disease. However, mdx calpain levels were higher whether normalized relative to MHC

-

FIG. 3. SDS-PAGE separation of C57 and mdx muscle under reducing conditions. The corresponding autoradiograph labeled with anti-calpain is shown below the gel. Lane A: molecular weight standards (numbers indicate mass in kilodaltons). Lanes B, C: C57 tibialis anterior. Lanes D, E: C57 gastrocnemius. Lanes F, G: C57 quadriceps. Lanes H, I: mdx tibialis anterior. Lanes J, K: mdx gastrocnemius. Lanes L, M: mdx quadriceps.

CALPAINS

IN

DYSTROPHIN-DEFICIENT

TABLE

111

MUSCLE

2

Calpain Concentration in C57 and mdx Mouse Muscle Increase C57 (n, SD)” Experiment Calpain: Calpain: Experiment Calpain: Calpain: Experiment Calpain: Calpain: a n, sample

1 MHC total 2 MHC total 3 MHC total

mdx

(n, SD)

protein

0.278 0.043

(n = 4, 0.09) (n = 4, 0.01)

0.459 0.056

(n = 4, 0.05) (n = 4, 0.02)

65% 32%

protein

0.259 0.042

(n = 3, 0.12) (n = 3, 0.02)

0.293 0.058

(n = 3, 0.05) (n = 3, 0.01)

13% 38%

protein

0.121 0.029

(n = 5, 0.04) (n = 5, 0.01)

0.189 0.036

(n = 5, 0.01) (n = 5, 0.01)

56% 25%

size; SD, standard

in

[calpain]

deviation.

sections (Fig. 4). There appeared to be a slight increase in the amount of connective tissue present, compared to C57, that was infiltrated with mononucleated cells. The mdx fibers appeared to be generally larger, more variable in diameter, and separated into regions containing either large or small fibers. Immunolabeling of longitudinal sections of C57 muscle showed calpain to be localized in sharp periodic striations which coincided with Z-discs identified by Nomarski optics (Fig. 5). In addition, labeling at the surface of muscle fibers was apparent. This type of localization confirms calpain localization previously documented by other researchers [29-321. However, in mdx muscle anti-calpain staining was more diffuse than in C57 muscle and appeared only slightly more concentrated at some Z-discs than elsewhere in the fiber. Hence, calpain distribution differs in mdx and C57 muscle. DISCUSSION DMD and mdx muscle cells are characterized by elevated intracellular calcium levels that precede degradation of muscle proteins [7]. Muscle necrosis in both DMD and mdx dystrophy are characterized by hypercontracted myofibrils, loss of Z-discs, and grouped fiber destruction. In mdx muscle, necrosis begins at about 2 weeks of age and progresses to 4 weeks of age, the stage of peak necrosis [33, 341. Although early stages of necrosis are similar in DMD and mdx dystrophy, necrosis is progressive in DMD but successfully reversed in mdx. The common characteristic of elevated [Ca2+], in dystrophic muscle has provided the basis for speculation that calpain may be involved in the necrotic processes in dystrophin-deficient and other dystrophic muscle. Calpain is a calcium-dependent protease that can remove Z-discs from purified myofibrils and degrade muscle-specific proteins such as troponins T and I, tropomyosin, desmin, filamin, titin, and nebulin [35].

Indirect evidence for calcium-mediated proteolysis in muscular dystrophies has been given by studies using protease inhibitors to treat dystrophic animal models. Leupeptin, an inhibitor of calpain and other proteases, delayed muscle degeneration in dystrophic chickens [36], increased the growth rate in dystrophic chick muscle cells [37], and improved muscle morphology of dy/dy mice [38]. Other studies have more directly implicated calpain as the source of the proteolysis. Kar and Pearson [l3] examined a panel of human muscular dystrophies and showed calpain activity of whole muscle homogenates to be elevated only in DMD and Becker dystrophy. Reddy et al. [14] showed calpain activity to be elevated 5-fold in DMD patients. Similar observations have been made in dystrophic muscles that are not dystrophin deficient. For example, in dystrophic hamster [I71 and dy/dy mice [16], purified calpain activity was increased 1.5-fold and 1.9-fold, respectively. In the chick model [ 151,whole muscle homogenates had significantly higher calpain activity and this increase coincided with the onset of muscular necrosis. The location of calpain in muscle has been variably reported in previous investigations. The majority of these studies have shown calpain located at I-Z-I complexes [30] and Z-discs [31, 321 of skeletal muscle. In tissue culture, the protein has been observed to be at the surface membrane of L6 cells [26]. In the present study, calpain staining of longitudinal sections was markedly different between mdx and control muscle. Z-disc and surface membrane labeling was apparent in both types of muscle but was much more localized in control muscle. We are unable to ascertain the amount of the calpain that is active vs inactive by immunolabeling techniques, although we speculate that the cytoplasmically located calpain was a previously activated form that is being inhibited by calpastatin. This hypothesis is consistent with the “membrane activation theory” proposed by Mellgren [25] in which calpain associates with the membrane in response to a rise in [Ca2+lin, auto-

SPENCER

AND

TIDBALL

MDX

FIG. 4. C57 and mdn muscle stained with hematoxylin and eosin. Panel A: longitudinal section of C57 muscle. Panel section of mdr muscle. Panel C: cross section of C57 muscle. Panel D: cross section of mdx muscle. Arrowheads denote Parentheses surround connective tissue infiltrated by mononucleated cells. Arrows point to a necrotic cell. Bar is 50 pm.

lyzes, and is released to the cytoplasm in an activated form. We chose to measure calpain activity from crude homogenates to enable us to estimate net calpain activity in ho. Thus, what is being measured is active calpain relative to its specific inhibitor, calpastatin, which is also present in the crude homogenate. The adult mdx mice in these studies exhibited an elevated calpain concentration with a corresponding decrease in calpain activity. There are two probable explanations for these results: (1) a decreased sensitivity of calpain to calcium in adult mdx mice or (2) an upregulation of the specific calpain inhibitor calpastatin. The former explanation is unlikely because the calcium concentration in the assays was 5 mM; too high for a change in sensitivity of the enzyme to be apparent. Therefore, upregulation of calpastatin and suppression of calpain activity is the most

B: longitudinal central nuclei.

likely mechanism by which the mdx mouse muscle is able to regenerate. A study on DMD muscle [I41 showing that calpain activity increased dramatically while calpastatin activity remained unchanged suggests that upregulation of calpastatin is indeed the mechanism by which mdr muscle regenerates. These investigators also noted that the calcium dependence of calpain from DMD muscle was the same as controls verifying that a change in the sensitivity of calpain probably does not occur. In contrast, Rabbani et al. [ 161 carried out a similar experiment in the dy/dy mouse model, one that is not dystrophin deficient, and observed no change in the activities of isolated calpain relative to calpastatin. Hence, it seems feasible that the mechanism by which the mdn mice are able to compensate for the enhanced calpain concentration is by an upregulation of calpastatin to a level that suppresses all calpain activity.

CALPAINS

IN

DYSTROPHIN-DEFICIENT

FIG. 5. Immunolabeling of longitudinal sections of mouse skeletal muscle with anti-calpain. Panel Panel B: corresponding fluorescent micrograph of C57 muscle labeled with anti-calpain. Panel C: phase corresponding fluorescent micrograph of mdx muscle labeled with anti-calpain. Bar is 46 pm.

To our knowledge, this is the first time calpain concentration has been studied in parallel with its distribution and net calcium-dependent protease activity. This approach provides the investigator a more extensive evaluation of active calpain present in the tissue. In the future, we intend to study other ages of mdx mice, follow-

113

MUSCLE

A: phase micrograph of C57 muscle. micrograph of mdx muscle. Panel D:

ing calpain’s concentration, activity, and distribution different stages in the progression of the disease.

at

The authors thank Dr. Dorothy Croall for donation of purified calpain and helpful discussions. This work was supported by a grant from the NIH (AR403431 and the Muscular Dystrophy Association.

114

SPENCER

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