The FASEB Journal Research Communication

Effect of calpain and proteasome inhibition on Ca2 -dependent proteolysis and muscle histopathology in the mdx mouse
Alexandre Briguet, Michael Erb, Isabelle Courdier-Fruh, Patrizia Barzaghi, Gesa Santos, Holger Herzner, Cyrille Lescop, Herve Siendt, Marco Henneboehle, Philipp Weyermann, Josef P. Magyar, Judith Dubach-Powell, Gunther Metz,1 and Thomas Meier
Santhera Pharmaceuticals, Liestal, Switzerland Dystrophin deciency is the underlying molecular cause of progressive muscle weakness observed in Duchenne muscular dystrophy (DMD). Loss of functional dystrophin leads to elevated levels of intracellular Ca2 , a key step in the cellular pathology of DMD. The cysteine protease calpain is activated in dystrophin-decient muscle, and its inhibition is regarded as a potential therapeutic approach. In addition, previous work has shown that the ubiquitin-proteasome system also contributes to muscle protein breakdown in dystrophic muscle and, therefore, also qualies as a potential target for therapeutic intervention in DMD. The relative contribution of calpain- and proteasome-mediated proteolysis induced by increased Ca2 levels was characterized in cultured muscle cells and revealed initial Ca2 inux-dependent calpain activity and subsequent Ca2 -independent activity of the ubiquitin-proteasome system. We then set out to optimize novel small-molecule inhibitors that inhibit both calpain as well as the 20S proteasome in a cellular system with impaired Ca2 homeostasis. On administration of such inhibitors to mdx mice, quantitative histological parameters improved signicantly, in particular with compounds strongly inhibiting the 20S proteasome. To investigate the role of calpain inhibition without interfering with the ubiquitin-proteasome system, we crossed mdx mice with transgenic mice, overexpressing the endogenous calpain inhibitor calpastatin. Although our data show that proteolysis by calpain is strongly inhibited in the transgenic mdx mouse, this calpain inhibition did not ameliorate muscle histology. Our results indicate that inhibition of the proteasome rather than calpain is required for histological improvement of dystrophin-decient muscle. In conclusion, we have identied novel proteasome inhibitors that qualify as potential candidates for pharmacological intervention in muscular dystrophy.Briguet, A., Erb, M., Courdier-Fruh, I., Barzaghi, P., Santos, G., Herzner, H., Lescop, C., Siendt, H., Henneboehle, M., Weyermann, P., Magyar, J. P., Dubach-Powell, J., Metz, G., Meier, T. Effect of calpain and proteasome inhibition on Ca2 -dependent proteolysis and muscle histopathology in the mdx mouse. FASEB J. 22, 4190 4200 (2008)
ABSTRACT 4190

Key Words: Duchenne muscular dystrophy dystrophin deciency small-molecule inhibitors calpastatin in vivo efcacy

Dystrophin deficiency in human muscle tissue leads to the degeneration of myobers, which, in conjunction with inammatory processes and incomplete regeneration of damaged myobers, results in a complex and fatal pathology called Duchenne muscular dystrophy (DMD) (1). Loss of functional dystrophin protein, which in healthy muscle cells links the cortical actin cytoskeleton to the extracellular matrix, causes membrane instability and uncontrolled Ca2 inux into muscle bers during cycles of muscle contraction and relaxation (25). Elevated subsarcolemmal Ca2 levels in dystrophic muscle have been associated with the overactivation of calpain, a cytosolic Ca2 -dependent cysteine protease, and a direct link to increased breakdown of intracellular proteins has been discussed (4, 6). For example, autolyzed forms of the 2 ubiquitously expressed calpain I and calpain II, also known as - and m-calpain, respectively, are detected in muscle extracts of mdx mice (7), indicating calpain overactivation. Although the relationship between changes in calpain expression, activation, and location suggests that calpain mediates proteolysis in dystrophic muscle, support of this hypothesis through in vivo data is limited (6). Initial genetic evidence favoring the role of calpain in muscle dystrophy was obtained by overexpressing calpastatin, a specic endogenous calpain inhibitor, in the mdx mouse (8). On the other hand, pharmacological experiments with calpain inhibitors administered to mdx mice did not conclusively support a pivotal role of calpain-mediated protein degradation in dystrophic muscle. For example, data obtained with leupeptin (9) are inconclusive because this tripeptide aldehyde inhibitor is not specic for calpain (10, 11). Likewise,
Correspondence: Santhera Pharmaceuticals (Switzerland) Ltd, Hammerstrasse 47, CH-4410 Liestal, Switzerland. E-mail: guenther.metz@santhera.com doi: 10.1096/fj.07-099036
0892-6638/08/0022-4190 FASEB
1

another calpain inhibitor, BN82270, ameliorated muscle pathology in the mdx mouse, but this inhibitor also acts as antioxidant (12), and therefore no clear conclusion can be drawn with respect to its predominant pharmacological mode of action. The experiments presented here show that Ca2 inux not only activates calpain-mediated but also ubiquitin-proteasome system-mediated proteolysis in muscle cells. We synthesized collections of 2 chemically distinct classes of inhibitors with comparable potencies against calpain I but variable potencies against the 20S proteasome. These inhibitors were tested for their ability to inhibit the degradation of calpain- and ubiquitin-proteasome system-dependent proteolysis in muscle cells subject to pharmacologically induced Ca2 dysregulation. Furthermore, over 50 of these inhibitors were then administered to mdx mice during their initial phase of muscle degeneration occurring between 3 and 7 wk of age to assess their ability to ameliorate diseasespecic histological parameters. The results obtained in these studies indicate that therapeutic efcacy in the mdx mouse can be achieved by combined inhibition of calpain- and ubiqutin-proteasome-mediated proteolysis. Next we tested to what extent calpain inhibition alone was able to mitigate muscle pathology in the dystrophin-decient mouse. For this we engineered transgenic mdx mice overexpressing calpastatin, the endogenous calpain inhibitor. Although reduced calpain activity was observed in muscle tissue of these transgenic mice, histological investigations on muscles revealed no improvement in parameters over mdx mice, which is in contradiction to a previous report (8). Together with data from our screening efforts for small-molecule inhibitors with different target proles, we come to the conclusion that inhibition of the ubiquitin-proteasome system would offer a possible therapeutic approach for dystrophinopathies, while calpain does not appear to qualify as a drug target for this pathology. Our ndings are in line with recent experiments demonstrating that known proteasome inhibitors ameliorated muscle histology in mdx mice (13, 14).

with Ac-nLPnLD-amc (100 M, Biomol, Plymouth Meeting, PA, USA), and of the trypsin-like sites with Ac-RLR-amc (100 M, Biomol). The cathepsin B activity was assayed in reaction buffer (50 mM Na-acetate buffer, pH 5.5; 5 mM l-cystein HCl; 2.5 mM EDTA) using Z-RR-amc (200 M, Bachem) and 3 ng of cathepsin B puried from human liver (Calbiochem). The cathepsin L activity was assayed in the same reaction buffer using Z-FR-amc (50 M, Bachem) and 10 ng of cathepsin L puried from human liver (Calbiochem). The caspase-3 activity was assayed in reaction buffer (100 mM NaCl; 50 mM HEPES, pH 7.4; 5 mM L-cysteine HCl) using Ac-DEVD-amc (200 M, Bachem) and 20 U of human recombinant caspase-3 (Calbiochem). The thrombin activity was assayed in reaction buffer (20 mM Tris HCl, pH 8.4; 150 mM NaCl; 2.5 mM CaCl2) using H-D-CHA-Ala-Arg-amc (100 M, Pentapharm, Basel, Switzerland) and 4.5 10 5 unit of thrombin puried from human plasma (Novagen, Madison, WI, USA). For all enzymatic assays, the increase in uorescence ( ex 360 nm; em 440 nm) was measured at 30C over 30 min at 30 s intervals using a Spectramax Gemini XS uorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). Fluorometric proteolytic activity assay in muscle cells C2C12 myoblasts were cultured on gelatin coated 96-well plates (Becton Dickinson, Meylan, France) for 2 days in growth medium containing 20% fetal calf serum. To initiate myoblast fusion, the cells were switched to differentiation medium containing 5% horse serum for 24 h before use. To determine inhibitory concentration (IC50) values for the inhibition of the Ca2 -induced proteolysis, the cells were preincubated for 2 h with different dilutions of inhibitor compounds. Cells were rinsed with a reaction buffer containing 135 mM NaCl, 5 mM KCl, 4 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.25, and then incubated with 50 l of reaction buffer supplemented with 400 M of the uorogenic substrate Suc-LY-amc for 20 min at room temperature in the presence of the test compounds. To initiate proteolytic activity 50 l of reaction buffer supplemented with 20 M of the Ca2 -ionophore Br-A-23187 (Molecular Probes, Eugene, OR, USA) was added. This time is referred to T 0 min in Fig. 1. The increase in uorescence ( ex 360 nm; em 440 nm) was recorded over 60 min at 37C at 1 min intervals as described above. Inhibitors were either given together with the ionophore (T 0 min) or after ionophore treatment (T 30 min). For IC50 determination, the rate of uorescence increase was calculated over 1 h and reported in relation to the control proteolytic rate measured in the absence of inhibitor. Spectrin breakdown assay in cultured myoblasts C2C12 cells were cultured in 96-well plates as for the uorometric assay and preincubated for 2 h of the test inhibitor compounds. The cells were then rinsed with reaction buffer (see uorometric assay) and incubated with 100 l of reaction buffer containing the test compounds and 10 M Ca2 ionophore Br-A-23187 for 1 h at 37C. The reaction buffer was then removed and the cells lysed with 80 l/well of extraction buffer (80 mM TrisHCl, pH 6.8; 2% SDS) supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and mixed 1:1 with 2 SDS-sample buffer containing 0.2 M dithiothreitol (DTT). The breakdown of -spectrin was analyzed by Western blot using MAB1622 antibody (Chemicon, Temecula, CA, USA) at a dilution of 1:5000 and anti-mouse immunoglobulin G (IgG)
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MATERIALS AND METHODS

Cell-free uorometric protease inhibition assays For the calpain inhibition assays, 50 l of reaction buffer (100 mM imidazole, pH 7.5; 5 mM L-cysteine HCl) containing 10 mM CaCl2 and 500 M Suc-LY-amc (Bachem, Bubendorf, Switzerland) as calpain substrate was dispensed in white 96-well plates. A dilution series of test compounds dissolved in dimethyl sulfoxide (DMSO) was added to the reaction buffer. The reaction was initiated by adding 50 l of reaction buffer containing 0.1 U of puried porcine -calpain (Calbiochem, San Diego, CA, USA). The 20 S proteasome inhibition assay was performed similarly using reaction buffer (20 mM TrisHCl, pH 8.0; 0.5 mM EDTA) and 35 ng of puried rabbit 20 S proteasome (BostonBiochem, Cambridge, MA, USA). The activity of the chymotrypsin-like sites was assayed using Suc-LLVY-amc (200 M, Bachem), of the caspase-like sites
PROTEASE INHIBITION IN THE mdx MOUSE

Figure 1. Sequential calpain- and ubiquitin-proteasome system-mediated proteolysis of the uorogenic substrate Suc-LY-amc in Ca2 -ionophore treated myoblasts. A, C ) Increase in relative uorescence units (RFU) measured in the absence of inhibitor (control), in the presence of 10 mM EGTA or 1 M bortezomib added just before (A) or 30 min after (C ) the Ca2 -ionophore Br-A-23187. The base level was below the limit of quantication and was set to 0 at the time of adding the ionophore. Each data point represents the mean of 3 replicates. B, D) Summary histograms show the velocity of substrate cleavage measured in experiments A and C, respectively, relative to muscle cells treated with ionophore only (control, 100%). Quantication of the relative uorescence increase measured 15 to 30 min (black bars) or 30 to 60 min (white bars) after the addition of ionophore. Bars represent means sd of 3 independent experiments. *P 0.001 vs. control; unpaired Students t test. Alexa Fluor 680 secondary antibody at a dilution of 1:10,000. Fluorescence intensity of the -spectrin bands was quantied on Odyssey Imaging System (Li-Cor Biotechnology, Lincoln, NE, USA). Accumulation of ubiquitinated protein in cultured muscle cells Conuent C2C12 cells were switched to differentiation medium containing a dilution series of the test inhibitor compounds and incubated for 36 h without addition of a Ca2 ionophore. Thereafter the amount of ubiquitinated proteins was determined using a modication of a previously published cell-based ELISA method (15). Briey, ubiquitinated protein levels were determined immunologically with the monoclonal anti-ubiquitin antibody (clone P4D1, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:3000 and with appropriate peroxidase-coupled secondary antibodies using QuantaBlu Fluorogenic Peroxidase Substrate kit (Pierce, Rockford, IL, USA) for detection. Human plasma coagulation assay Fifty microliters of a 7.5 mM CaCl2 solution containing the test compounds was dispensed in transparent 96-well plate. Next, 25 l of human plasma (Swiss Red Cross) was added, and the coagulation was monitored by measuring the absorbance at 405 nm every minute for 1 h using a Victor 1420 Multilabel Counter (Wallac, Perkin Elmer, Waltham, MA, USA). Treatment of mdx mice with calpain/proteasome inhibitors Three-week-old male C57BL/10ScSn-mdx mice (Jackson Laboratory, Bar Harbor, ME, USA) were treated with calpain/proteasome inhibitors as a suspension in 50% PEG200/50% saline administered i.p. every second day for 4 wk at doses of 2 or 20 mg/kg. Control animals were treated with vehicle only using the same regimen. At the end of the treatment, mice were sacriced by CO2 asphyxiation. All procedures were performed in accordance with the Swiss regulations for animal experimentation and under the required licenses. Method for the quantication of dual calpain/proteasome inhibitor levels in muscle tissue For homogenization, deionized water was added to preweighed lyophilized muscle tissue sample from mdx mice treated with the
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dual calpain/proteasome inhibitor SNT198438 in a dened amount (1:11:2, w/v). A 20 l aliquot of a 2 g/mL solution of reference compound (SNT199217) in acetonitrile (ACN) was added to the homogenate as internal standard. The analyte and internal standard were extracted following a liquid-liquid extraction procedure with 5 ml methyl-tertbutyl ether. The organic layer was evaporated to dryness under a heated (40C) stream of nitrogen. The dried extract was reconstituted in 150 l 8:2 (v/v) 50 mM ammonium carbonate/ACN solution. A 20 l aliquot was injected into a liquid chromatography-mass spectrometry (LC-MS)/MS system for analysis. For calibration, increasing amounts of SNT198438 were added to blank muscle tissue homogenate and processed as described. Liquid chromatography was carried out on a binary gradient LC pump (Rheos 2000; Spectronex, Thermo Fisher Scientic, Reinach, Switzerland) equipped with an HTC PAL autosampler. The LC pump was used to load an aliquot of the processed samples on a guard column SecurityGuard C18 (Phenomenex, Torrance, CA, USA) followed by a SunFire C18 reversed phase column (50 2.1 mm, 3.5 m particle size) (Waters, Rupperswil, Switzerland) using 0.05% formic acid in 5 mM aqueous ammonium formate solution and 0.05% ammonium formate in ACN (20:80, v/v) mixture as the mobile phase at a ow rate of 0.2 ml/min. A gradient elution was performed increasing the mixture ratio from the initial 20:80 to 5:95 (v/v) within 2 min. MS/MS detection was performed with a TSQ Quantum tandem MS detector (Finnigan MAT, San Jose, CA, USA) with an electrospray ionization source operated in the positive ion mode. The specic transition of m/z 649.2 3 121.1 was selected to quantify the analyte SNT198438, whereas the transition of m/z 711.2 3 300.0 was used for the internal standard. The peak areas of the analyte and internal standard were determined using the Xcalibur 1.3 software (Thermo Fisher). A reliable and accurate quantication of SNT198438 in muscle tissue in unknown samples was possible within a linear quantication range between 2.5 and 1000 ng SNT198438/ml homogenate. Histological analysis Muscle samples were prepared and analyzed histologically as described previously (16). Briey, muscle cryosections were stained using Alexa Fluor 488 conjugated wheat-germ agglutinin (WGA-Alexa, Molecular Probes) to stain membranebound and extracellular epitopes and 1 g/ml 4 ,6-diamidino-2-phenylindole (DAPI; Molecular Probes) to stain nuclei. The variance coefcient of the muscle ber size and the percentage muscle bers with centralized nuclei were
BRIGUET ET AL.

The FASEB Journal

determined as described (16) by evaluating at least 3000 bers per diaphragm muscle and covering 90% of the area of limb muscles. NCAM-positive myobers were identied on cryosections using the monoclonal anti-NCAM antibody (MAB310 Millipore AG, Volketswil, Switzerland) and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, Soham, UK). Determination of -dystroglycan levels For quantication of -dystroglycan, muscle extracts were prepared using a buffer containing 2% SDS; 80 mM TrisHCl, pH 6.8; and 10 mM EDTA. Protein concentration in the extract was determined using Bradford reagent (Sigma-Aldrich, Munich, Germany), and protein loading on SDS-PAGE was normalized. The levels of -dystroglycan were determined by Western blot using NCL- -DG at a dilution of 1:1000 and anti-mouse IgG Alexa Fluor 680 secondary antibody at a dilution of 1:10,000. Fluorescence intensity of the -dystroglycan band was quantied on an Odyssey Imaging System. Generation of calpastatin transgenic mice The full-length mouse calpastatin coding sequence was excised from the I.M.A.G.E. Consortium clone 3710078 using EcoRV/NotI and was cloned downstream of the mouse muscle creatine kinase (MCK) promoter. The MCKcalpastatin fragment was excised from the vector using AhdI/NaeI, gel-puried, and used for pronuclear injection at the Biozentrum, University of Basel, transgenic mouse core facility. Founders were identied by polymerase chain reaction of clipped nger DNA (17) using the following primers: 5 mck CCA AGC TCC TGT CAT CGA TA and 3 calpastatin CAG CCT TAG TCT CTG TGG TA and mated with mdx mice. Only males were analyzed. All comparisons were made with age-matched, male mdx litter mates that were nontransgenic. Calpastatin protein expression levels were measured by Western blot using the anti-calpastatin antibody sc-20779 (Santa Cruz). Measure of Ca2 -dependent proteolytic activity in muscle extracts Frozen muscles were pulverized on liquid N2-cooled metal plates. The muscle powder was resuspended at 250 mg/ml in freshly prepared homogenization buffer (50 mM Tris HCl, pH 8.0; 2 mM EDTA; 1 mM PMSF; 0.1% -mercaptoethanol; 3% DMSO) and homogenized by sonication. The homogenate was then centrifuged for 10 min at 4C on a tabletop centrifuge; the supernatant was centrifuged a second time for 10 min. The protein concentration in the supernatant was then determined using Bradford reagent. For each muscle sample the proteolytic activity was measured in triplicate in 100 l reactions containing 90 l of substrate solution (100 mM imidazole, pH 7.5; 5 mM l-cysteine HCl), 50 g of muscle homogenate, and 1 g of Bodipy FL casein uorogenic substrate (Molecular Probes). The increase in uorescence ( ex 485 nm; em 538 nm) was measured at 30 s intervals over 30 min at 30C using a Spectramax Gemini XS uorescence plate reader (Molecular Devices). The activity was measured once in the presence of 10 mM CaCl2 and once in the presence of 100 mM EDTA instead of CaCl2. The Ca2 -dependent activity was then calculated by subtracting the activity measured in the absence of Ca2 from the activity measured in the presence of Ca2 .
PROTEASE INHIBITION IN THE mdx MOUSE

RESULTS Ca2 inux triggers sequential calpain- and proteasome-mediated proteolytic activity in cultured muscle cells We set out to evaluate the relative contribution of calpain and of the chymotrypsin-like proteasome site in the increased proteolytic activity as a consequence of impaired Ca2 homeostasis. For this we recorded the hydrolysis of the uorogenic substrate Suc-LYamc, a known substrate for both calpain and the chymotrypsin-like proteasome activity (18, 19), in C2C12 myoblasts treated with Ca2 -ionophore. On addition of the Ca2 -ionophore to Suc-LY-amc preloaded muscle cells, the emitted relative uorescence increased steadily over 1 h (Fig. 1A). Addition of EGTA prior to the Ca2 ionophore strongly reduced the rate of proteolytic activity, particularly during the initial 30 min of the experiment (Fig. 1A, B). In contrast, addition of the proteasome-specic inhibitor bortezomib (formerly known as PS-341; ref. 20) did not affect the initial proteolytic activity (rst 30 min) but strongly inhibited the late activity (between 30 and 60 min). When added 30 min after the initiation of the ionophore-mediated Ca2 inux, EGTA no longer prevented the proteolytic activity (Fig. 1C, D). In contrast, bortezomib added at this time point still largely prevented the hydrolysis of the uorogenic substrate. This experiment shows that the early proteolytic activity initiated by Ca2 inux is mediated predominantly by calpain and is largely independent of the ubiquitin-proteasome system activity, whereas the second phase of proteolysis is predominantly proteasome-mediated and no longer requires Ca2 inux. Based on these results we conclude that both calpain- and proteasome-mediated proteolysis can be initiated by impaired Ca2 homeostasis as also seen in dystrophin-decient muscle. These experiments further qualify both calpain as well as the proteasome as possible drug targets for DMD. Protease specicity prole of novel dipeptidic and tripeptidic calpain/proteasome inhibitors We have synthesized over 800 novel, small-molecule inhibitors with a dipeptidic or tripeptidic backbone, thereafter called SNT inhibitors (21) (Fig. 2A). They are chemically derived from the reference calpain inhibitor MDL28170 (22). Novel inhibitors were tested initially for their ability to inhibit calpain I and the chymotrypsin-like activity of the 20 S proteasome, using appropriate cell-free enzymatic assays. The IC50 values for the inhibition of the 2 proteases by these inhibitors were compared to those obtained with the 2 reference calpain inhibitors MDL28170 and A-705253 (23) as well
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Figure 2. Inhibition of calpain- and proteasome-mediated proteolysis by novel dipeptidic and tripeptidic SNT inhibitors. A) General structure of novel dipeptidic and tripeptidic inhibitors. B) Scatter plot representing the IC50 ( M) of exemplied dipeptidic and tripeptidic SNT inhibitors compared to MDL28170, A-705253, and bortezomib for calpain I (x axis) and the 20 S proteasome (y axis) in cell-free assays. The highest inhibitor concentration tested against both proteases was 1 M. C) Dose response curves for the inhibition of the Ca2 -ionophore-induced Suc-LY-amc proteolysis in C2C12 myoblasts. Data points represent means sd of 3 measurements. D) Novel calpain/proteasome SNT inhibitors block early and late proteolytic processes in muscle cells initiated by Ca2 inux. Bar histogram represents the relative inhibition of Ca2 -ionophore-induced Suc-LY-amc proteolysis by test compounds added to cultured myoblasts. Either compounds were given together with the ionophore and inhibition was measured for 30 min (left), or they were given 30 min after the ionophore and inhibition was measured from this time point up to 60 min (right). Bars represent means sd of 3 independent experiments.

as with bortezomib, a selective proteasome inhibitor with no calpain inhibitory activity up to 1 M. As shown in Fig. 2B, most tripeptidic inhibitors with IC50 for calpain I in the range 10 100 nM were also potent proteasome inhibitors with IC50 values ranging from 10 100 nM. As an example, the tripeptidic inhibitor SNT198438 had an IC50 for proteasome inhibition in the same range as bortezomib (Table 1). In contrast, dipeptidic inhibitors such as SNT197958, while showing comparable calpain inhibitory activity, were generally less potent proteasome inhibitors. Unlike MDL28170 and A-705253, however, dipeptidic SNT inhibitors still reached half-maximal inhibition of the chymotrypsin-like 20 S proteasome activity at concentrations below 1 M. The prole of selected di- and tripeptidic inhibitors against a panel of cysteine and serine proteases is listed in Table 1.
TABLE 1.

Inhibition of Ca2 -induced proteolytic activity in muscle cells To test the potential of selected novel calpain/proteasome inhibitors to affect intracellular proteolytic activity as a consequence of impaired Ca2 homeostasis, we determined proteolysis in cultured muscle cells loaded with the uorogenic substrate Suc-LY-amc. In this assay the IC50 values for reference inhibitors MDL28170 and A-705253 were 10.0 5.8 and 3.8 3.4 M, respectively (Fig. 2C), while the IC50 for SNT197958 was 0.427 0.127 M. In comparison, tripeptidic inhibitors were generally more potent inhibitors as exemplied by SNT198438 with an IC50 value of 0.008 0.003 M. This greatly improved cellular activity compared to the reference calpain inhibitors can be explained both by a chemistry optimized for better uptake in muscle

Protease specicity prole of example SNT inhibitors compared to reference inhibitors

IC50 (nM) Dipeptide SNT197958 Tripeptide SNT198438

SNT inhibitor

MDL28170

A-705253

Bortezomib

Calpain I 20 S chymotrypsin-like 20 S caspase-like 20 S trypsin-like Cathepsin-B Cathepsin-L Caspase-3 Thrombin

Values are means sd; n

23 3 10% inhibition at 1000 nM 1000 1000 100 0 57 10 1000 1000

58 9 1000 1000 1000 30 4 1100 360 ND 1000

1000 36 14 543 12 50% activation at 1000 nM 1000 1000 1000 1000

33 7 615 49 1000 1000 120 20 63 18 1000 1000

16 50

1 24 1000 1000 19 1 94 16 1000 1000

3. ND, not determined; 20 S, 20 S proteasome.

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BRIGUET ET AL.

cells (21) and inhibition of the proteasome-mediated component of the Ca2 -induced proteolytic activity. To verify the latter hypothesis, we determined the relative contribution of intracellular inhibition of calpain- and proteasome-mediated proteolysis by recording the time course of substrate cleavage following Ca2 -ionophore treatment of cultured muscle cells as described above. The calpain inhibitors MDL28170 and A-705253 added to cultured muscle cells at 10 M inhibited more than 60% of the early calpain-mediated activity (Fig. 2D). However, when added 30 min after the ionophore, these inhibitors blocked only a small proportion of the late proteasome-mediated activity. The remaining inhibition seen in this latter phase by selective calpain inhibitors might be due to the fact that residual calpain activity is present that can be inuenced by active site inhibitors but not by calcium removal using EGTA (Fig. 1). Conversely, the proteasome inhibitor bortezomib at 1 M predominantly inhibited the late phase of the Ca2 -induced proteolysis. Interestingly, novel dipeptidic and tripeptidic SNT inhibitors added at a concentration of 1 M blocked more than 80% of both the early calpain-mediated and the late ubiquitin-proteasome system-mediated activities. These experiments clearly demonstrate that the improved potency of novel SNT inhibitors against overall Ca2 induced proteolysis is based on their improved uptake into muscle cells (21) as well as their potent inhibition of both calpain- and proteasome-mediated cellular proteolytic activities as exemplied in our ndings with the model peptide substrate. Inhibition of calpain- and proteasome-specic proteolysis of muscle cell substrates To determine the efcacy of SNT inhibitors at preventing the breakdown of endogenous calpain substrates in muscle cells, we quantied the amount of the calpaingenerated 150 and 145 kDa fragments of -spectrin in muscle cells challenged with Ca2 -ionophore relative to the amount of the intact 260 kDa protein (24, 25).

As shown on the representative Western blots in Fig. 3A, SNT inhibitors offered a stronger protection against Ca2 -induced spectrin breakdown than reference calpain inhibitors. Specically, quantitative analysis revealed SNT197958 and SNT198438 had IC50 values of 0.302 0.325 and 0.440 0.173, M, respectively while the reference calpain inhibitors MDL28170 and A-705253 had IC50 values of 0.610 0.308 and 1.045 0.295 M, respectively (Fig. 3B). In contrast, bortezomib did not inhibit spectrin breakdown at concentrations up to 10 M (data not shown). As a measure for proteasome inhibition we quantied the amount of ubiquitinated proteins accumulated in cultured muscle cells after 36 h exposure to the inhibitors (26). Using a cell-based ELISA method the amount of accumulated ubiquitinated proteins were determined relative to the amount of ubiquitinated protein obtained in DMSO solvent-treated cultures (set as 100%; Fig. 3C). As anticipated, the reference calpain inhibitors (MDL28170 and A-705253) did not inuence the level of ubiquitinated proteins at concentrations up to 10 M. The dipeptidic inhibitor SNT197958 did not induce the accumulation of ubiquitinated proteins even though this compound was a weak inhibitor of the chymotrypsin-like proteasome activity in the cell-free enzymatic assay. The tripeptidic inhibitor SNT198438 and bortezomib, however, induced halfmaximal accumulation of ubiquitinated proteins at comparable concentrations of 0.115 0.045 and 0.041 0.005 M, respectively. In contrast to bortezomib-treated cultures, which showed lower levels of ubiquitinated proteins at concentrations higher than 0.1 M due to cellular toxicity (see below), ubiquitinated protein levels in SNT198438-treated cultures remained at high levels up to the highest concentration tested and without having an effect on cell viability. This lower cell toxicity observed with SNT198438 could be explained by its specicity for the chymotrypsin-like sites of the proteasome (Table 1). Indeed, inhibition of only one of the three 20S proteasome subsite

Figure 3. Protection of endogenous calpain and proteasome protein substrates in muscle cells. A) Western blot of extracts from Ca2 ionophore-teated C2C12 muscle cells. Top band: the uncleaved spectrin protein migrating at 260 kDa. Bottom band: the cleaved 150 kDa spectrin fragment. Inhibitor concentration is 10 M. B) Dose response curves for the inhibition of the Ca2 -ionophore-induced cleavage of -spectrin by inhibitors. Data from one representative experiment. C ) Dose response curves for the accumulation of ubiquitinated proteins in inhibitor-treated C2C12 myoblasts. Data points represent means sd from 3 replicates.
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activities was shown to block only a small proportion of the overall protein degradation in mammalian cells (27). In addition to the chymotrypsin-like activity, bortezomib inhibits the caspase-like proteasome activity. The relative importance of the different proteasome sites for protein degradation varies widely with the substrate. To further differentiate the prole of SNT inhibitors compared to bortezomib, a cell survival assay was performed where C2C12 myoblasts were exposed for 48 h to test compounds. Bortezomib-induced cell 0.090 M death with an EC50 value of 0.093 (mean sd, n 3) in this assay. In comparison, the tripeptidic dual inhibitor SNT198438 appeared to be less toxic with an EC50 value of 8.8 6.6 M (n 3). The dipeptidic inhibitor SNT197958 showed cellular toxicity only at a high concentration (EC50: 35 21 M, n 3) and in the same range as the 2 reference calpain inhibitors MDL28170 (EC50: 100 M, n 3) and A-705253 (EC50: 54 39 M, n 3). SNT inhibitors did not inhibit either thrombin activity in an enzymatic assay at concentrations up to 1 M nor did they inhibit blood plasma coagulation. Interestingly, this is in clear contrast to leupeptin, a widely used calpain inhibitor that completely inhibited blood plasma coagulation (28). Tripeptidic calpain/proteasome inhibitor is detectable in muscle tissue and ameliorates quantitative histological parameters in mdx mice Selected SNT inhibitors were tested to show whether they would improve disease-specic histological parameters in the mdx mouse. To this end we administered the SNT inhibitors i.p. at a dose of 20 mg/kg and additionally 2 mg/kg for the most potent inhibitors. The treatment was performed every second day for a duration of 4 wk in mdx mice aged 3 wk at the beginning of the experiment. This treatment period was chosen because it encompasses the peak of muscle degeneration in mdx mice (29). In general, over the 4 wk treatment, body weight gain was similar in SNTand vehicle-treated animals, indicating good tolerability of the compounds The mdx mice treated with SNT198438 gained an average of 189 26% (n 6) of body weight, growing from 8.9 1.2 g at the start of treatment to 23.1 1.7 g after 4 wk. In the same period, vehicle-treated animals grew from 7.1 1.5 to 20.2 2.4 g, corresponding to 159 28% (n 16) weight gain. LC-MS/MS analysis of samples from treated animals taken 2 days after the last i.p. administration revealed dose-dependent amounts of nonmetabolized SNT198438 in the quadriceps and gastrocnemius muscle (Table 2). At the histological level, dystrophic muscle in mdx mice is characterized by a high variability in the cross-sectional muscle ber size and a high proportion of muscle bers with centrally located myonuclei (30). Both of these parameters, quantied as the
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TABLE 2. mdx mice

Quantication of SNT198438 in muscles of treated

Muscle

Dose (mg/kg)

SNT198438 (ng/ml)

SNT198438 (nM)

Quadriceps Gastrocnemius lateral

2 20 2 20

9.4 38.9 6.3 47.3

14 59 5 73

For each dose administered to mdx mice, concentration of SNT198438 was measured in muscles pooled from 3 4 animals treated for 4 wk i.p. every second day at the indicated dose. Muscle samples were taken 48 h after the last application.

variance coefcient of the muscle ber size and the percentage of muscle ber with centralized nuclei, were signicantly ameliorated in several muscles of treated mdx mice. In particular, the diaphragm muscle of 20 mg/kg SNT198438-treated mice showed an improved ber size distribution (Fig. 4A, B). Although the variance coefcient of the muscle ber size rises from 237 22 in the wild type to a value of 402 32 in the mdx mice, it is reduced to 329 25 on treatment. This corresponds to an improvement of 44% (Fig. 4C). Likewise, the variance coefcient of the muscle ber size was ameliorated in the quadriceps, gastrocnemius, and biceps muscle by 49%, 22% and 31%, respectively. Consistently, the percentage of muscle bers with centralized nuclei was reduced by 35% in the diaphragm over the 4-wk period studied (Fig. 4C). Again this amelioration could also be observed for the other muscles (data not shown). Dystrophic muscle is also characterized by reduced expression of -dystroglycan, a transmembrane component of the dystrophin-glycoprotein complex (DGC) (31). As determined by quantitative Western blot, the total amount of the mature 43 kDa -dystroglycan protein was partially normalized in the diaphragm muscle of SNT198438-treated mdx mice (Fig. 4D). This nding is in line with studies demonstrating that proteasome inhibitors restore elevated levels of membrane-associated DGC proteins, including -dystroglycan, in the mdx mouse (13, 14, 32). Requirement of proteasome inhibition for in vivo efcacy More than 50 SNT inhibitors were tested in the same way as SNT198438 for in vivo efcacy in the mdx mouse. These inhibitors included approximately equal numbers of di- and tripeptidic compounds, all with high potencies against calpain (IC50 100 nM) but variable potencies against the proteasome, ranging from IC50 10 nM to 1 M. Weak proteasome inhibitors produced only a small or no improvement of the muscle histopathological symptoms, while more potent proteasome inhibitors including SNT198438 showed the best efcacy in mice. Even though many factors including
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Figure 4. Amelioration of disease-specic parameters on application of SNT198438 in mdx mice (20 mg/kg i.p. every second day). A) Fluorescence micrographs of representative WGA-Alexa and DAPIstained diaphragm muscle cross sections taken from wild-type, vehicle-treated mdx, and SNT198438treated mdx mice. B) Histogram representing the ber size distribution in wild-type (n 10), controltreated mdx (n 20), and SNT198438-treated mdx (n 6) diaphragm muscle. For each diaphragm muscle, 3000 bers were analyzed. Data points represent mean values. C) Box plot representing the variance coefcient (16) of muscle ber size (left) and the percentage of muscle bers with centralized nuclei (right) measured in wild-type, vehicle-treated mdx, and SNT198438-treated mdx diaphragm muscle. *P 0.05, **P 0.001 vs. mdx ; Mann-Whitney U test. D) Restoration of -dystroglycan in the diaphragm muscle of SNT198438-treated mdx mice: levels of the 43 kDa -dystroglycan protein in SNT198438-treated mdx mice compared to levels found in wild-type and vehicle-treated mdx mice as determined by Western blot analysis (example shown). Data are means sd of 5 mice. *P 0.01, **P 0.001 vs. vehicle-treated mdx ; unpaired Students t test.

bioavailability and metabolic stability can inuence the in vivo efcacy of a chemical agent, this large series of in vivo tests pointed toward a dominant role of the proteasomal inhibition for efcacy. This observation led us to question to what extent calpain inhibition alone would improve the disease phenotype in the mdx mouse. To this end, we generated 2 lines of transgenic mice overexpressing the endogenous calpain-specic inhibitor calpastatin in skeletal muscle. These mice were then crossed with mdx mice to obtain calpastatin overexpression in the dystrophin-decient genetic background termed tg/mdx. As shown in Fig. 5A, both mouse lines expressed high levels of calpastatin compared to nontransgenic mice both in the wild type and in the mdx background. These levels of calpastatin were sufcient to inhibit more than 60% of the total Ca2 -dependent proteolytic activity in muscles of 3-wk-old mice and more than 90% of this activity at 7 wk of age (Fig. 5B). The effect of this calpain inhibition on disease-specic histological parameters was analyzed in 7-wk-old mdx mice in the same manner as for the SNT inhibitors. Neither the muscle ber diameter distribution nor the percentage of muscle ber with centralized nuclei was improved in either the diaphragm or quadriceps muscle of calpastatin-overexpressing mdx mice (Fig. 6A, B; Table 3). We further evaluated the effect of calpastatin overexpression on the percentage of necrotic area and on the cross-sectional area occupied by regenerating muscle bers using NCAM staining as a monitor (Fig. 6C). In quadriceps the necrotic area in tg/mdx mice covered 14 3% (mean sd; n 6) of the muscle cross sectional area compared to 13 5 (n 8) in control mdx mice. Muscle ber regeneration assessed by the NCAMpositive area was measured to be 8 5% in the qudriceps muscle of mdx mice compared to 7 2%
PROTEASE INHIBITION IN THE mdx MOUSE

in tg/mdx mice, indicating that calpastatin overexpression did not alter muscle ber regeneration in the mdx mouse muscle.

Figure 5. Overexpression of a calpastatin transgene in skeletal muscle of mdx mice. A) Western blot showing the expression levels of calpastatin in the quadriceps muscle of nontransgenic mdx mice (mdx) and 2 different lines of calpastatin transgenic mdx mice (tg/mdx) at the age of 3 wk (top panel) and 7 wk (bottom panel). For each transgenic line, calpastatin expression is shown for 23 samples. B) Bar histogram represents calpain activity measured as the Ca2 -dependent proteolysis of Bodipy-FL-labeled casein in muscle homogenates of nontransgenic (gray bars) and calpastatin-transgenic mice (black bars). Bars represent means sd of 4 8 mice.
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Figure 6. Histogram representing the ber size distribution in wild-type (n 4), mdx (n 10), and tg/mdx (n 10; pooled data from both tg lines, 5 animals each) diaphragm (A) and quadriceps muscle (B). For each diaphragm, 3000 muscle bers were analyzed; for each quadriceps, 2000. Data points represent means sd. C ) Staining of quadriceps of mdx and tg/mdx mice (red, NCAM; green, WGA; blue, DAPI). Scale bars 100 m.

DISCUSSION DMD is a genetic disease that leads to progressive muscle weakness and culminates in early morbidity and mortality of affected patients. There is currently no effective treatment available that can stop or even slow the progression of this disease for prolonged periods of time. As demonstrated previously, dystrophin deciency results in elevated Ca2 inux and impaired Ca2 homeostasis, which in turn activates calpain proteases that contribute to the degradation of muscle proteins (5, 6). Calpain enzymes are known to perform only limited proteolysis of target substrates and are therefore unlikely to be the predominant contributor of muscle protein breakdown (33). However, calpain may play a role in initiating the breakdown of myobrillar proteins by releasing protein fragments that become suitable substrates for the ubiquitin-proteasome system. One objective of this study was to determine to which extent calpain and the ubiquitin-proteasome system contribute to proteolysis induced by excessive Ca2 inux into muscle cells. We have shown that on Ca2 ionophore treatment cultured C2C12 myoblasts develop an initial Ca2 inux-dependent calpain activity and a subsequent activity of the ubiquitin-proteasome system, which no longer requires elevated Ca2 levels. Our nding is in line with a recent report that Ca2 ionophore-treated myotubes show an enhanced protea-

some activity that depends, at least in part, on the calpain system (34). These experiments are in agreement with the idea that both calpain and the ubiquitinproteasome system act synergistically in the Ca2 -dependent proteolysis observed in dystrophic muscle. The nature of the molecular link between these two proteolytic systems, however, remains unclear. Taking into consideration calpain as well as the proteasome as potential drug targets for DMD, we have developed two classes of novel, muscle-cell permeating dual calpain/proteasome inhibitors. Tripeptidic calpain/proteasome inhibitors showed superior proteasome inhibition compared to dipeptidic inhibitors as well as higher potency in inhibiting cellular Ca2 -induced proteolysis induced by Ca2 inux. Furthermore, selected tripeptidic inhibitors such as SNT198438 were toxic to muscle cells only at concentrations 2 orders of magnitude higher than the marketed proteasome inhibitor bortezomib. In addition, the EC50 for cellular toxicity of SNT198438 was 3 log-units higher than the corresponding IC50 for Ca2 induced intracellular proteolysis, thereby showing a clear separation of efcacy and toxicity. Further, we report that the amount of SNT198438, a potent tripeptidic inhibitor of Ca2 -induced proteolysis, increases with dose in muscle tissue and ameliorates the histological and biochemical manifestation of dystrophin deciency in muscle of mdx mice, a prerequisite for the development of this class of inhibitors as a potential treatment for DMD. To the best

TABLE 3. Variance coefcient of muscle ber size and percentage muscle bers with centralized nuclei in muscles of calpastatin-overexpressing tg/mdx mice compared to wild-type and mdx mice
Diaphragm tg/mdx 1203 tg/mdx 1204 Quadriceps tg/mdx 1203 tg/mdx 1204

Variable

WT

mdx

WT

mdx

Variance coefcient Centralized nuclei (%) n

236 3.3 4

14 2.1

359 18.8 9

17 3.3

375 15.9 5

25 6.0

354 15.7 5

17 2.4

270 2.1 4

11 0.9

362 19 58.7 3.6 10

356 59.9 6

47 7.1

350 53.4 6

16 10.9

Data are means sd. WT, wild type; tg/mdx, calpastatin-overexpressing transgenic mice crossed with mdx mice (two lines, 1203 and 1204, have been established).

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of our knowledge, this is the rst report on a protease inhibitor that was specically optimized to prevent proteolysis in muscle tissue. The pharmacological testing of a large series of SNT inhibitors in vivo allowed us to highlight the importance of inhibiting the chymotrypsinlike activity of the proteasome to achieve therapeutic efcacy in mdx mice. These experiments demonstrated the important role of proteasome inhibitory activity but did not allow one to establish to which extent calpain inhibition was required to ameliorate histopathology in the mdx mouse. To directly test the relative contribution of calpain activity to muscle cell damage, we created lines of transgenic mdx mice overexpressing calapstatin, the endogenous inhibitor of calpain. Surprisingly, we found that calpain inhibition by calpastatin overexpression does not improve the muscle histology of mdx mice, thereby questioning the potential of pharmacological calpain inhibition as an approach to attenuate the consequences of dystrophin deciency in muscle. We cannot fully explain the discrepancy between our ndings and published results (8) that reported histological improvements in mdx mice overexpressing calpastatin. Although the transgenic mice described previously expressed the full-length human calpastatin protein under the control of a human skeletal actin promoter fragment, transgenic mice from our laboratory expressed the full-length mouse calpastatin gene under the control of an established 1.3 kb muscle creatine kinase promoter. Spencer and Mellgren (8) reported calpastatin activity at 4 wk of age, whereas we analyzed calpastatin activity at 3 wk of age ( 60% inhibition) and 7 wk of age ( 90%inhibition), which is a critical period of the muscle regeneration in the mdx mouse. The potential mosaic expression pattern of calpastatin driven by the MCK promoter (35) may lead to calpastatin being expressed in only 50% of muscle bers. Consequently, the reduced calpain activity in individual bers could be higher or lower than the average of 60 90% seen in our study. If we assumed that such mosaic-like expression occurred, one would nevertheless anticipate that the proportion of bers in which calpain activity is reduced by at least 60 90% would proportionally contribute to an expected amelioration of muscle ber histology. However, in our experiments we did not detect any improvement in mdx muscle overexpressing calpastatin. Besides the histological analysis discussed above and as further evidence we performed the NCAM staining with the same protocol as in the previously published paper but did not nd signs of amelioration in the calpastatin transgenic mdx mice. Our studies provide direct evidence that inhibition of calpain does not prevent histopathological features in the mdx mouse and therefore may not offer a valid approach for pharmacological invention in DMD. In contrast, inhibition of the ubiquitin-proteasome system exhibits desirable effects encouraging the development of proteasome inhibitors. Given their inhibitory activity and lower cellular toxicity, selected novel SNT inhibitors as described here
PROTEASE INHIBITION IN THE mdx MOUSE

qualify as potential candidates for a pharmacological inhibition of Ca2 -induced proteolytic pathways in dystrophin-decient myobers. Finally, our studies reveal that it is the inhibition of the proteasome rather than calpain that renders these dual inhibitors efcacious in the mdx mouse model.
The MCK promotor was generously supplied by Prof. Markus Ruegg (Biozentrum, Basel, Switzerland).

REFERENCES
1. 2. 3. 4. Nowak, K. J., and Davies, K. E. (2004) Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment. EMBO Rep. 5, 872 876 Petrof, B. J. (1998) The molecular basis of activity-induced muscle injury in Duchenne muscular dystrophy. Mol. Cell. Biochem. 179, 111123 Berchtold, M. W., Brinkmeier, H., and Muntener, M. (2000) Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol. Rev. 80, 12151265 Mallouk, N., Jacquemond, V., and Allard, B. (2000) Elevated subsarcolemmal Ca2 in mdx mouse skeletal muscle bers detected with Ca2 -activated K channels. Proc. Natl. Acad. Sci. U. S. A. 97, 4950 4955 Alderton, J. M., and Steinhardt, R. A. (2000) Calcium inux through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J. Biol. Chem. 275, 94529460 Tidball, J. G., and Spencer, M. J. (2000) Calpains and muscular dystrophies. Int. J. Biochem. Cell Biol. 32, 15 Spencer, M. J., Croall, D. E., and Tidball, J. G. (1995) Calpains are activated in necrotic bers from mdx dystrophic mice. J. Biol. Chem. 270, 10909 10914 Spencer, M. J., and Mellgren, R. L. (2002) Overexpression of a calpastatin transgene in mdx muscle reduces dystrophic pathology. Hum. Mol. Genet. 11, 26452655 Badalamente, M. A., and Stracher, A. (2000) Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition. Muscle Nerve 23, 106 111 Donkor, I. O. (2000) A survey of calpain inhibitors. Curr. Med. Chem. 7, 11711188 Otto, H. H., and Schirmeister, T. (1997) Cysteine proteases and their inhibitors. Chem. Rev. 97, 133172 Burdi, R., Didonna, M. P., Pignol, B., Nico, B., Mangieri, D., Rolland, J. F., Camerino, C., Zallone, A., Ferro, P., Andreetta, F., Confalonieri, P., and De Luca, A. (2006) First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant. Neuromuscul. Disord. 16, 237248 Bonuccelli, G., Sotgia, F., Schubert, W., Park, D. S., Frank, P. G., Woodman, S. E., Insabato, L., Cammer, M., Minetti, C., and Lisanti, M. P. (2003) Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. Am. J. Pathol. 163, 16631675 Bonuccelli, G., Sotgia, F., Capozza, F., Gazzerro, E., Minetti, C., and Lisanti, M. P. (2007) Localized treatment with a novel FDA-approved proteasome inhibitor blocks the degradation of dystrophin and dystrophin-associated proteins in mdx mice. Cell Cycle 6, 12421248 Courdier-Fruh, I., Barman, L., Briguet, A., and Meier, T. (2002) Glucocorticoid-mediated regulation of utrophin levels in human muscle bers. Neuromuscul. Disord. 12(Suppl 1), S95S104 Briguet, A., Courdier-Fruh, I., Foster, M., Meier, T., and Magyar, J. P. (2004) Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse. Neuromuscul. Disord. 14, 675 682 Moll, J., Barzaghi, P., Lin, S., Bezakova, G., Lochmuller, H., Engvall, E., Muller, U., and Ruegg, M. A. (2001) An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy. Nature 413, 302307

5.

6. 7. 8. 9. 10. 11. 12.

13.

14.

15. 16.

17.

4199

18.

19.

20.

21.

22.

23.

24. 25.

26.

Sasaki, T., Kikuchi, T., Yumoto, N., Yoshimura, N., and Murachi, T. (1984) Comparative specicity and kinetic studies on porcine calpain I and calpain II with naturally occurring peptides and synthetic uorogenic substrates. J. Biol. Chem. 259, 12489 12494 Arribas, J., and Castano, J. G. (1993) A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli. J. Biol. Chem. 268, 2116521171 Adams, J., Palombella, V. J., Sausville, E. A., Johnson, J., Destree, A., Lazarus, D. D., Maas, J., Pien, C. S., Prakash, S., and Elliott, P. J. (1999) Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 59, 26152622 Lescop, C., Herzner, H., Siendt, H., Bolliger, R., Hennebohle, M., Weyermann, P., Briguet, A., Courdier-Fruh, I., Erb, M., Foster, M., Meier, T., Magyar, J. P., and von Sprecher, A. (2005) Novel cell-penetrating alpha-keto-amide calpain inhibitors as potential treatment for muscular dystrophy. Bioorg. Med. Chem. Lett. 15, 5176 5181 Mehdi, S., Angelastro, M. R., Wiseman, J. S., and Bey, P. (1988) Inhibition of the proteolysis of rat erythrocyte membrane proteins by a synthetic inhibitor of calpain. Biochem. Biophys. Res. Commun. 157, 11171123 Neuhof, C., Fabiunke, V., Deibele, K., Speth, M., Moller, A., Lubisch, W., Fritz, H., Tillmanns, H., and Neuhof, H. (2004) Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts. Biol. Chem. 385, 10771082 Harris, A. S., and Morrow, J. S. (1988) Proteolytic processing of human brain alpha spectrin (fodrin): identication of a hypersensitive site. J. Neurosci. 8, 2640 2651 Saido, T. C., Yokota, M., Nagao, S., Yamaura, I., Tani, E., Tsuchiya, T., Suzuki, K., and Kawashima, S. (1993) Spatial resolution of fodrin proteolysis in postischemic brain. J. Biol. Chem. 268, 25239 25243 Tanahashi-Hori, T., Tanahashi, N., Tanaka, K., and Chiba, T. (2003) Conditional knockdown of proteasomes results in cellcycle arrest and enhanced expression of molecular chaperones Hsp70 and Hsp40 in chicken DT40 cells. J. Biol. Chem. 278, 1623716243

27.

28. 29. 30.

31.

32.

33. 34.

35.

Kisselev, A. F., Callard, A., and Goldberg, A. L. (2006) Importance of the different proteolytic sites of the proteasome and the efcacy of inhibitors varies with the protein substrate. J. Biol. Chem. 281, 8582 8590 Aoyagi, T., Miyata, S., Nanbo, M., Kojima, F., and Matsuzaki, M. (1969) Biological activities of leupeptins. J. Antibiot. (Tokyo) 22, 558 568 Carnwath, J. W., and Shotton, D. M. (1987) Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J. Neurol. Sci. 80, 39 54 Coulton, G. R., Morgan, J. E., Partridge, T. A., and Sloper, J. C. (1988) The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation. Neuropathol. Appl. Neurobiol. 14, 5370 Durbeej, M., and Campbell, K. P. (2002) Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. Curr. Opin. Genet. Dev. 12, 349 361 Assereto, S., Stringara, S., Sotgia, F., Bonuccelli, G., Broccolini, A., Pedemonte, M., Traverso, M., Biancheri, R., Zara, F., Bruno, C., Lisanti, M. P., and Minetti, C. (2006) Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment. Am. J. Physiol. Cell Physiol. 290, C577582 Goll, D. E., Thompson, V. F., Li, H., Wei, W., and Cong, J. (2003) The calpain system. Physiol. Rev. 83, 731 801 Menconi, M. J., Wei, W., Yang, H., Wray, C. J., and Hasselgren, P. O. (2004) Treatment of cultured myotubes with the calcium ionophore A23187 increases proteasome activity via a CaMK II-caspase-calpain-dependent mechanism. Surgery 136, 135142 Dunant, P., Larochelle, N., Thirion, C., Stucka, R., Ursu, D., Petrof, B. J., Wolf, E., and Lochmuller, H. (2003) Expression of dystrophin driven by the 1.35-kb MCK promoter ameliorates muscular dystrophy in fast, but not in slow muscles of transgenic mdx mice. Mol. Ther. 8, 80 89 Received for publication November 20, 2007. Accepted for publication July 24, 2008.

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