Title: Lipid Peroxidation Inhibition Blunts Nuclear Factor-κB Activation, Reduces Skeletal Muscle Degeneration, and Enhances Muscle Function in mdx Mice
Abstract: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease resulting from lack of the sarcolemmal protein dystrophin. However, the mechanism leading to the final disease status is not fully understood. Several lines of evidence suggest a role for nuclear factor (NF)-κB in muscle degeneration as well as regeneration in DMD patients and mdx mice. We investigated the effects of blocking NF-κB by inhibition of oxidative stress/lipid peroxidation on the dystrophic process in mdx mice. Five-week-old mdx mice received three times a week for 5 weeks either IRFI-042 (20 mg/kg), a strong antioxidant and lipid peroxidation inhibitor, or its vehicle. IRFI-042 treatment increased forelimb strength (+22%, P < 0.05) and strength normalized to weight (+23%, P < 0.05) and decreased fatigue (−45%, P < 0.05). It also reduced serum creatine kinase levels (P < 0.01) and reduced muscle-conjugated diene content and augmented muscle-reduced glutathione (P < 0.01). IRFI-042 blunted NF-κB DNA-binding activity and tumor necrosis factor-α expression in the dystrophic muscles (P < 0.01), reducing muscle necrosis (P < 0.01) and enhancing regeneration (P < 0.05). Our data suggest that oxidative stress/lipid peroxidation represents one of the mechanisms activating NF-κB and the consequent pathogenetic cascade in mdx muscles. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease resulting from lack of the sarcolemmal protein dystrophin. However, the mechanism leading to the final disease status is not fully understood. Several lines of evidence suggest a role for nuclear factor (NF)-κB in muscle degeneration as well as regeneration in DMD patients and mdx mice. We investigated the effects of blocking NF-κB by inhibition of oxidative stress/lipid peroxidation on the dystrophic process in mdx mice. Five-week-old mdx mice received three times a week for 5 weeks either IRFI-042 (20 mg/kg), a strong antioxidant and lipid peroxidation inhibitor, or its vehicle. IRFI-042 treatment increased forelimb strength (+22%, P < 0.05) and strength normalized to weight (+23%, P < 0.05) and decreased fatigue (−45%, P < 0.05). It also reduced serum creatine kinase levels (P < 0.01) and reduced muscle-conjugated diene content and augmented muscle-reduced glutathione (P < 0.01). IRFI-042 blunted NF-κB DNA-binding activity and tumor necrosis factor-α expression in the dystrophic muscles (P < 0.01), reducing muscle necrosis (P < 0.01) and enhancing regeneration (P < 0.05). Our data suggest that oxidative stress/lipid peroxidation represents one of the mechanisms activating NF-κB and the consequent pathogenetic cascade in mdx muscles. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD. Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease leading to loss of ambulation by the 13th year and to death, usually in early adulthood.1Dubowitz V Muscle Disorders in Childhood. ed 2. W.B. Saunders, Philadelphia1995Google Scholar The disease results from absence of the protein dystrophin, which is an essential component of the dystrophin-glycoprotein complex that maintains membrane integrity of muscle fibers by linking cytoskeleton to extracellular matrix.2Ervasti JM Ohlendieck K Kahl SD Gaver MG Campbell KP Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle.Nature. 1990; 345: 315-319Crossref PubMed Scopus (824) Google Scholar, 3Ervasti JM Campbell KP Membrane organization of the dystrophin-glycoprotein complex.Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1121) Google Scholar, 4Yoshida M Ozawa E Glyoprotein complex anchoring dystrophin to sarcolemma.J Biochem. 1990; 108: 748-752Crossref PubMed Scopus (452) Google Scholar, 5Ibraghimov-Beskrovnaya O Ervasti JM Leveille CJ Slaughter CA Sernett SW Campbell KP Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1202) Google Scholar Although the primary genetic defect is known, how this mutation gives rise to the final disease status is not fully understood. The mechanisms responsible for the pathological hallmarks of the dystrophic process, such as necrosis, phagocytosis, infiltration of inflammatory cells, initial efficient regeneration followed by a decline and secondary fibrosis, have not been definitively identified. DMD pathogenesis is frequently studied in the genetically homologous animal, the mdx mouse, despite relevant clinical and pathological differences. The murine model exhibits late muscle weakness, a slow disease progression, similar extensive degeneration and regeneration occurring between 2 and 12 weeks of age, but no proliferation of connective tissue in limb muscles.6Connolly AM Keeling RM Mehta S Pestronk A Sanes JR Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin alpha2-deficient mice.Neuromuscul Disord. 2001; 11: 703-712Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 7Granchelli JA Pollina C Hudecki MS Pre-clinical screening of drugs using the mdx mouse.Neuromuscul Disord. 2000; 10: 235-239Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Several lines of evidence suggest that oxidative stress might be involved in the dystrophic process. 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31: 1405-1416Crossref PubMed Scopus (268) Google Scholar which is involved in the up-regulation of antioxidant enzymes such as glutathione peroxidase and catalase.28Zhou LZ Johnson AP Rando TA NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells.Free Radic Biol Med. 2001; 31: 1405-1416Crossref PubMed Scopus (268) Google Scholar An involvement of NF-κB in myogenesis has been suggested because its activity was shown to be required by human and rat myoblasts to fuse into myotubes and to express muscle-specific proteins such as myosin heavy chain and caveolin 3.29Kaliman P Canicio J Testar X Palacin M Zorzano A Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway.J Biol Chem. 1999; 274: 17437-17444Crossref PubMed Scopus (113) Google Scholar In addition, it has also been demonstrated that systemic administration of the NF-κB inhibitor curcumin stimulates muscle regeneration after traumatic injury, suggesting that modulation of NF-κB activity within muscle tissue could be beneficial for muscle repair.30Thaloor D Miller KJ Gephart J Mitchell PO Pavlath GK Sys-temic administration of the NF-κB inhibitor curcumin stimulates muscle regeneration after traumatic injury.Am J Physiol. 1999; 277: C320-C329PubMed Google Scholar Very recently, NF-κB activity has been demonstrated to be increased in muscles of either DMD patients21Monici MC Aguennouz M Mazzeo A Messina C Vita G Activation of nuclear factor-kappaB in inflammatory myopathies and Duchenne muscular dystrophy.Neurology. 2003; 60: 993-997Crossref PubMed Scopus (148) Google Scholar or mdx mice,16Kumar A Boriek AM Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy.FASEB J. 2003; 17: 386-396Crossref PubMed Scopus (231) Google Scholar, 17Kumar A Takada Y Boriek AM Aggarwal BB Nuclear factor-kappaB: its role in health and disease.J Mol Med. 2004; 82: 434-448Crossref PubMed Google Scholar but its effective role in DMD pathogenesis is not clear to date. Interestingly, we have reported the novel observation of increased immunoreactivity for NF-κB in the cytoplasm of all regenerating fibers and in 20 to 40% of necrotic fibers in DMD as well as in inflammatory myopathies.21Monici MC Aguennouz M Mazzeo A Messina C Vita G Activation of nuclear factor-kappaB in inflammatory myopathies and Duchenne muscular dystrophy.Neurology. 2003; 60: 993-997Crossref PubMed Scopus (148) Google Scholar Taken together, this evidence suggests that reactive oxygen intermediates might be involved in the dystrophic process, triggering an inflammatory cascade that leads to NF-κB activation and to the subsequent release of inflammatory mediators. This work hypothesis would also indicate that the interruption of this cascade might have a therapeutic potential. To confirm and clarify this issue, we used as a pharmacological tool (±)-5-emisuccinoyl-2-[2-(acetylthio)ethyl]-2,3-dihydro-4,6,7-trimethylbenzofuran (IRFI-042), a synthetic, vitamin E analogue. Vitamin E has been suggested to act as potential inhibitor of NF-κB activation31Campo GM Ceccarelli S Squadrito F Altavilla D Dorigotti L Caputi AP Raxofelast (IRFI 016): a new hydrophilic vitamin E-like antioxidant agent.Cardiovasc Drug Rev. 1997; 15: 157-173Crossref Scopus (18) Google Scholar; nevertheless the marked lipophilicity of this vitamin limits its therapeutic potential with low circulating levels and poor tissue distribution after somministration. IRFI-042 is a less lipophilic compound with powerful antioxidant properties due to the combination in the same molecule of a chain-breaking moiety (characteristic of phenols related to α-tocopherol) with the reducing ability of a thiol group (dual antioxidant). Moreover this compound shows no systemic toxicity even after high dosage (up to 1 g/kg).31Campo GM Ceccarelli S Squadrito F Altavilla D Dorigotti L Caputi AP Raxofelast (IRFI 016): a new hydrophilic vitamin E-like antioxidant agent.Cardiovasc Drug Rev. 1997; 15: 157-173Crossref Scopus (18) Google Scholar IRFI-042 possesses a strong inhibitory activity on both oxidative stress/lipid peroxidation and NF-κB activation demonstrated in different experimental models, such as endotoxin-induced shock,32Altavilla D Squadrito G Minutoli L Deodato B Bova A Sardella A Seminara P Passaniti M Urna G Venuti SF Caputi AP Squadrito F Inhibition of nuclear factor-kappaB activation by IRFI 042, pro-tects against endotoxin-induced shock.Cardiovasc Res. 2002; 54: 684-693Crossref PubMed Scopus (51) Google Scholar organ ischemia/reperfusion injury,33Altavilla D Deodato B Campo GM Arlotta M Miano M Squadrito G Saitta A Cucinotta D Ceccarelli S Ferlito M Tringali M Minutoli L Caputi AP Squadrito F IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia reperfusion injury.Cardiovasc Res. 2000; 47: 515-528Crossref PubMed Scopus (77) Google Scholar neurotoxicity,34Marini H Altavilla D Bellomo M Adamo EB Marini R Laureanti F Bonaccorso MC Seminara P Passaniti M Minutoli L Bitto A Calapai G Squadrito F Modulation of IL-1 beta gene expression by lipid peroxidation inhibition after kainic acid-induced rat brain injury.Exp Neurol. 2004; 188: 178-186Crossref PubMed Scopus (33) Google Scholar and impaired wound healing process.35Altavilla D Galeano MR Marini H Squadrito F Hydrophilic dual vitamin E-like antioxidants as modulators of inflammatory response in low-flow states and impaired wound healing.Curr Med Chem Anti-Inflamm Anti-Allergy Agents. 2003; 2: 265-273Crossref Google Scholar The aim of our study was to test the novel hypothesis that the modulation of NF-κB activity by oxidative stress/lipid peroxidation inhibition may influence the skeletal muscle pathology in mdx mice, with respect to the functional, morphological, and biochemical patterns. Male mdx and wild-type C57BJ/10 (WT) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facilities. Mice were housed in plastic cages in a temperature-controlled environment with a 12-hour light/dark cycle and free access to food and water. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85-23, revised 1996). Five-week-old mdx and WT mice have been treated for 5 weeks with intraperitoneal injections with either IRFI 042 (n:8; 20 mg/kg three times a week) or vehicle (n:8; dimethyl sulfoxide/NaCl 0.9%; 0.1:1 v/v; 0.2 mg/kg three times a week). At the end of the experiments, animals were anesthetized with an intraperitoneal administration of sodium pentobarbital (80 mg/kg). Then, blood, collected by intracardiac puncture, was drawn to analyze creatine kinase (CK) levels and the biceps, quadriceps, and extensor digitorum longus (EDL) muscles were removed bilaterally and immediately frozen in liquid nitrogen-cooled isopentane and stored at −80°C for morphological and biochemical evaluations. Mice were weighed and examined for forelimb strength at baseline and after 5 weeks of treatment. Strength testing consisted of five separate measurements using a grip meter attached to a force transducer that measures peak force generated (Stoelting Co., Wooddale, IL). The mouse grabs the trapeze bar as it is pulled backward and the peak pull force in grams is recorded on a display. The three highest measurements for each animal were averaged to give the strength score. We calculated also the degree of fatigue by comparing the first two pulls to the last two pulls. The decrement between pulls one and two and pulls four and five gives a measure of fatigue.6Connolly AM Keeling RM Mehta S Pestronk A Sanes JR Three mouse models of muscular dystrophy: the natural history of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin alpha2-deficient mice.Neuromuscul Disord. 2001; 11: 703-712Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Blood samples were centrifuged at 6000 rpm and the serum was stored at −80°C until the day of analysis. Serum CK was evaluated at 37°C using a commercially available kit (Randox Laboratories Ltd., Antrim, UK). The results were expressed as U/L. Ten-μm-thick transverse cryostat sections were obtained from the midpoint of the biceps and EDL muscle body. The whole muscle cross-sections (corresponding to a mean area of 2.15 mm2 in biceps and 1.69 mm2 in EDL), stained with hematoxylin and eosin (H&E), were examined by a blinded observer, using the AxioVision 2.05 image analysis system equipped with Axiocam camera scanner (Zeiss, Munchen, Germany). The following four areas were recognized with patchy distribution: 1) normal fibers, identified by the presence of peripheral nuclei; 2) centrally nucleated fibers, identified by normal size but with central nuclei; 3) regenerating fibers, identified by small size, basophilic cytoplasm, and central nuclei; 4) necrotic fibers, identified by pale cytoplasm and phagocytosis. The results were expressed as the ratio of the area occupied by normal fibers, centrally nucleated fibers, regenerating fibers, or necrotic fibers divided by the total surface area as a percentage. Seven-μm-thick transverse cryostat sections from biceps and EDL muscles were incubated for 120 minutes at 37°C in rabbit polyclonal antibody against phospho-NF-κB p65 subunit (Ser276) (1:50; Cell Signaling Technology, Beverly, MA). It selectively binds to the NF-κB p65 only when phosphorylated at serine 276, ie, it is activated and can then undergo nuclear translocation. Nonspecific binding of immunoglobulin was blocked with 5% normal horse serum. Immunodetection was performed using a biotin-avidin system (DAKO, Milan, Italy) followed by horseradish peroxidase staining with 3,3-diaminobenzidine tetrahydrochloride. Estimation of the tissue content of CDs was performed to evaluate the extent of lipid peroxidation in tissue as previously shown.31Campo GM Ceccarelli S Squadrito F Altavilla D Dorigotti L Caputi AP Raxofelast (IRFI 016): a new hydrophilic vitamin E-like antioxidant agent.Cardiovasc Drug Rev. 1997; 15: 157-173Crossref Scopus (18) Google Scholar Samples of biceps muscle were collected in polyethylene tubes and then washed with 1 ml of butylated hydroxytoluene (BHT) (1 mg/ml in phosphate buffer). The samples, after drying in absorbent paper, were frozen at 4°C until the analysis. The biochemical assay of CDs required previous lipid extraction from the tissue samples by chloroform/methanol (2:1). The lipid layer was dried under nitrogen atmosphere and then dissolved in cyclohexane. Muscle contents of CDs was measured at 232 nm by using a spectrophotometric technique. The amount of muscle CDs was expressed as ΔABS/mg protein. GSH activity was evaluated to estimate endogenous defenses against oxidative stress. The levels in biceps muscles were determined as previously described.31Campo GM Ceccarelli S Squadrito F Altavilla D Dorigotti L Caputi AP Raxofelast (IRFI 016): a new hydrophilic vitamin E-like antioxidant agent.Cardiovasc Drug Rev. 1997; 15: 157-173Crossref Scopus (18) Google Scholar Briefly, tissue samples were homogenized with a Ultra-turrax (IKA, Staufen, Germany) homogenizer in a solution containing 5% trichloroacetic acid and 5 mmol/L ethylenediamine tetraacetic acid at 4°C. Then each sample was centrifuged at 15,000 × g for 10 minutes at 4°C. Homogenate supernatant (0.4 ml) was added in polyethylene dark tubes containing 1.6 ml of Tris-ethylenediamine tetraacetic acid buffer 0.4 mol/L, pH 8.9. After vortexing, 40 μl of 10 mmol/L dithiobisnitrobenzoic acid were added. The samples were vortexed again and the absorbance was read after 5 minutes at 412 nm. The values of unknown samples were drawn from a standard curve plotted by assaying different known concentrations of GSH. The amount of muscle GSH was expressed as μmol/g protein. NF-κB binding activity in quadriceps muscle specimens was performed in a 15-μl binding reaction mixture containing 1% binding buffer [50 μg/ml of double-stranded poly (dI-dC), 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 0.5 mmol/L ethylenediamine tetraacetic acid, 0.5 mmol/L dithiothreitol, 1 mmol/L MgCl2, and 10% glycerol], 15 μg of nuclear proteins, and 35 fmol (50,000 cpm, Cherenkov counting) of double-stranded NF-κB consensus oligonucleotide (5′-AGT TGA GGG GAC TTT CCC AGG C-3′; Promega, Madison, WI) that was end-labeled with [γ-32P] ATP (3000 Ci/mmol at 10 mCi/ml; Amersham Life Sciences, Arlington Heights, IL) using T4 polynucleotide kinase. The binding reaction mixture was incubated at room temperature for 20 minutes and analyzed by electrophoresis on 5% nondenaturing polyacrylamide gels. After electrophoresis, the gels were dried using a gel-drier and exposed to Kodak X-ray films at −70°C. The binding bands were quantified by scanning densitometry of a bio-image analysis system (Bio-Profil; Celbio, Milan, Italy). The results were expressed as relative integrated intensity compared with normal controls, considering exposure time, background levels, and known protein concentration of an Epstein-Barr virus nuclear antigen-1 extract, which was used as electrophoretic mobility shift assay control. Samples from quadriceps muscles were homogenized in lysis buffer (1% Triton X-100, 20 mmol/L Tris/HCl, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 5 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L phenylmethyl sulfonyl fluoride, 1% aprotinin, 15 μg ml leupeptin). Protein samples (40 μg) were denatured in reducing buffer (62 mmol/L Tris, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.003% bromophenol blue) and separated by electrophoresis on sodium dodecyl sulfate (12%) polyacrylamide gel with prestained standard proteins (Bio-Rad, Milan, Italy) to achieve a more accurate molecular weight determination. The separated proteins were transferred onto a nitrocellulose membrane using the transfer buffer (39 mmol/L glycine, 48 mm Tris, pH 8.3, 20% methanol) at 200 mA for 1 hour. The membranes were stained with Ponceau S (0.005% in 1% acetic acid) to confirm equal amounts of protein and were blocked with 5% non-fat dry milk in Tris-buffered saline-0.1% Tween for 1 hour at room temperature, washed three times for 10 minutes each in Tris-buffered saline-0.015% Tween, and incubated with rabbit monoclonal antibody against TNF-α (Chemicon, Temecula, CA) in Tris-buffered saline-0.1% Tween overnight at 4°C. After washing three times for 10 minutes each in Tris-buffered saline-0.15% Tween, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Pierce, Milan, Italy) for 1 hour at room temperature. After washing, the membranes were analyzed by the enhanced chemiluminescence system according to the manufacturer's protocol (Amersham). The protein signals were quantified by scanning densitometry using a bio-image analysis system (Bio-Profil, Celbio). The results from each experimental group were expressed as relative integrated intensity compared with control muscle measured with the same batch. Equal loading of protein was assessed on stripped blots by immunodetection of β-actin with a rabbit monoclonal antibody (Cell Sig-naling, Celbio) diluted 1:500 and peroxidase-conjugated goat anti-rabbit IgG (Pierce) diluted 1:15,000. All anti-bodies are purified by protein A and peptide affinity chromatography. IRFI 042 was supplied by Biomedica Foscama Research Centre, Ferentino, Italy. All substances were prepared fresh daily and administered in a volume of 1 ml/kg. Results are expressed as mean ± SD. Statistical evaluation was performed by using one-way analysis of variance followed by Dunnett's post hoc tests and paired Student's t-test with the use of the InPlotPrism software version 3.0 (GraphPad Software, San Diego, CA). P values <0.05 were considered significant. Body weight was not significantly different among the animal groups at baseline as well as after treatment. Comparing the values longitudinally, at the end of the experiment all groups had an increased body weight (P < 0.01) (Figure 1A). At baseline, strength and strength normalized to weight were significantly lower in both mdx mice groups (assigned to IRFI 042 or to vehicle treatment) compared to WT groups (allocated to IRFI 042 or to vehicle treatment) (P < 0.05). At the end of treatment, IRFI 042-treated mdx mice had higher forelimb strength (+22%, P < 0.05) and strength normalized to weight (+23%, P < 0.05) compared to vehicle-treated mdx mice (Figure 1, B and C). In all groups the somatic growth paralleled with an increment of strength if compared with baseline values (P < 0.01 in mdx + IRFI 042, P < 0.05 in the other groups) (Figure 1B), but when normalized to weight only the IRFI 042-treated mdx mice showed a significant amelioration in strength (P < 0.05) (Figure 1C). At baseline, the percentage of fatigue was significantly higher in both mdx mice groups compared to WT groups (P < 0.01); furthermore, there was not any significant difference between the two mdx groups (Figure 1D). After treatment we found in both mdx groups a higher level of fatigue compared to WT groups (P < 0.001 in mdx+ vehicle, P < 0.01 in mdx+ IRFI 042), but the value was significantly lower in IRFI 042-treated compared to vehicle-treated mdx (−45%, P < 0.05). Comparing the data longitudinally, the percentage of fatigue increased in the vehicle-treated (P < 0.05) and remained stable in the IRFI 042-treated mdx mice (Figure 1D). IRFI 042 did not cause any significant change in the forelimb strength, strength normalized to body weight, and fatigue of WT animals. Low CK levels were observed in WT animals treated either with vehicle or IRFI 042 (WT+ vehicle = 221 ± 33 U/L, WT+ IRFI 042 = 145 ± 28 U/L). Mdx mice showed a significant increase in serum CK levels (mdx + vehicle = 2662 ± 79 U/L, P < 0.01 versus WT+ vehicle). IRFI 042 administration resulted in a marked reduction of the enzyme levels (mdx +