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Metabolic remodeling agents show beneficial effects in the dystrophin- deficient mdx mouse model

Metabolic remodeling agents show beneficial effects in the dystrophin- deficient mdx mouse model Background: Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods: Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects. Results: We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions: The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice. Keywords: Duchenne muscular dystrophy, Muscle, AICAR, GW501516, Metabolism Background receptor γ coactivator 1 α (PGC-1α), the master regula- Muscle is a plastic tissue that responds and adapts to en- tor of mitochondrial biogenesis [8], seems to control vironmental changes [1]. Energy balance is one of the muscle wasting [3]. PGC-1α overexpression increases checkpoints between muscle growth/hypertrophy and mitochondrial content [9] and resistance to fatigue [9] protein breakdown [2], and >10% of atrophy-related and reduces the rapid muscle atrophy associated with genes are directly involved in energy production [3-5]. denervation, fasting, and FoXO 3 activation [3]. Re- Mitochondrial dysfunction activates various proteolytic cently, mice overexpressing PGC-1α have been shown to systems [2] and is associated with muscle atrophy in sev- have an increased lifespan and to be protected from sar- eral myopathies [6,7]. Peroxisome proliferator-activated copenia [10]. Therefore, targeting mitochondrial biogen- esis and metabolism up-regulation may have beneficial * Correspondence: knagaraju@cnmcresearch.org effects in muscle diseases. Center for Genetic Medicine Research, Children’s National Medical Center, Evidence for the beneficial effects of submaximal aer- Washington, DC, USA obic activities in DMD patients is slowly emerging. A re- Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA cent review on the management and care of DMD Full list of author information is available at the end of the article © 2012 Jahnke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 2 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 patients recommend that ambulatory and early non- LB 940, Berthold Technologies). LDH activity was nor- ambulatory-stage boys participate in regular submaximal malized to protein concentration. functional activities [11]. The molecular mechanisms by which exercise provides beneficial effects are currently Measurement of contraction properties unclear. However, increased PPARδ and AMPK activities Mice were anesthetized with 100mg/kg ketamine and have been implicated in these beneficial effects [12]. We 10mg/kg xylazine. EDL muscle was isolated and placed hypothesized that exercise mimetics activating PPARδ in Ringer’s solution (137mM NaCl, 24mM NaHCO , and AMPK pathways are beneficial to dystrophin defi- 11mM glucose, 5mM KCl, 2mM CaCl , 1mM MgSO , 2 4 cient skeletal muscle. In the present study, we have used 1mM NaH PO , and 0.025mM tubocurarine chloride) 2 4 agonists of PPARδ (GW501516) and AMPK (AICAR) to maintained at 25°C and bubbled with 95% O -5% CO . 2 2 activate beneficial endurance exercise-induced signaling Contractile properties were measured according to pathways in mdx mice. We have demonstrated that en- Brooks et al. [13], using an in vitro test apparatus (model durance mimetics can improve muscle function by halt- 305B, Aurora Scientific). A fatigue protocol was per- ing the cycle of muscle regeneration/degeneration in formed (20 min, 100 Hz). EDL muscle was subjected to a dystrophin-deficient mice. series of 120 isometric tetanic contractions (400 ms). Methods Animal experiments Behavioral activity measurement and grip strength All mice were handled according to Washington DC testing Veterans Affairs Medical Center’s Institutional Animal All animals were weighed before and after drug treatment. Care and Use Committee guidelines under approved Grip strength and open-field activity were assessed using a mdx protocol # 01079. C57BL/10ScSn-Dmd /J (mdx) grip strength meter (Columbus Instruments, Columbus, 8-week-old male mice, 20-30g, were purchased from OH) and open-field Digiscan apparatus (Omnitech Elec- The Jackson Laboratory housed in an individually vented tronics, Columbus, Ohio), respectively, as described previ- cage system (with a 12-h light–dark cycle, standard ously [14]. Tissues were either embedded in OCT or mouse chow and water ad libitum). Mice were rested wrapped in foil and then frozen in isopentane chilled in li- for 10–14 days, acclimatized on the behavioral instru- quid nitrogen. Blood was collected by cardiac puncture, ment for 1 week and then baseline grip strength and be- and serum was collected by centrifuging blood for 10min havioral activity was performed. At 12 weeks of age, the at 10,000rpm and then stored at −80°C. mice were given AICAR (250mg/kg [80μl]; Alexis) by intraperitoneal injection and/or GW501516 (7.5mg/kg Muscle cell extraction [80μl]; Alexis) by oral gavage, 5 days/week for 4 weeks. Leg muscles that were not harvested as previously DMSO in PBS (1/2 vol/vol) was used as a vehicle control described were used for satellite cell extraction. Tendons and concentration of DMSO was the same in vehicle and aponeuroses were removed. Muscles were minced, and drug treatments. placed in digestion medium (2.4U/ml dispase II, 100mg/ ml collagenase A), vortexed, and incubated at 37°C. After Extensor digitorum longus (EDL) fiber isolation and digestion, tubes were placed on ice, and 25ml of DMEM- staining 1% PS-2% L-Glut were added. The mixture was filtered The EDL muscles of 12-week-old mice were harvested with a 100-μm cell strainer and centrifuged (800g, 4°C, and incubated in DMEM with 2mg/ml collagenase for 3min). The pellet was resuspended in 25ml DMEM-1% 2 h. EDL fibers were separated with Pasteur pipets. Fibers PS-2% L-Glut), filtered with a 70-μm cell strainer, and were rinsed, stained with 10-nonyl acridine orange (NAO; centrifuged (800g, 4°C, 3min). The same operation was Sigma) (15min), rinsed, fixed with 4% formalin (10min), repeated with a 40-μm cell strainer. Cell extracts were fro- and mounted. Pictures were taken. Fluorescence levels zen and stored at −80°C. were analyzed with ImageJ software (NIH). LDH activity Flow cytometry analyses Lactate dehydrogenase activity of muscle lysate was mea- Mitochondrial content and inner membrane potential (ΔΨ) sured using 2.5μl of protein extract (1:2 dilution), 225μl were assessed with NAO and 3, 3’-dihexyloxacarbocyanine assay buffer (2.5ml of 1 M Tris [pH 7.6], 500μlof iodide (DiOC6) (Invitrogen) as described [15]. Cell immu- 200mM EDTA, and 500μl of 5mM NADH,H , and 48ml noreactivity against MyoD (Dako) was assessed with water). Oxidation of NADH, H was recorded after pyru- Hoechst 33342 (Sigma) as described [15]. Cells were ana- vate addition (10μl, 100mM). NADH fluorescence was lyzed on a FACSCalibur (BD Biosciences, San Jose, CA, detected by luminescence/fluorescence analyzer (Mithras USA) with BD Cell Quest ProTM 4.0.2. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 3 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Mitochondrial DNA to nuclear DNA ratio analysis T. Stereology System, Olympus America Inc., Center Total DNA was extracted from muscle cells using DNeasy Valley, PA). Pictures were processed using ImageJ. Fi- blood and tissue kit (Qiagen). The content of mtDNA was brotic red areas were expressed as a percentage of the calculated using real-time quantitative PCR by measuring total tissue section. the threshold cycle ratio (ΔCt) of a mitochondrial- encoded gene (ND1, forward 5’- GGA CCT AAG CCC Western blotting AAT AAC GA-3’, reverse 5’-GCT TCA TTG GCT ACA Protein homogenates were extracted as previously CCT TG-3’) versus a nuclear-encoded gene (Beta-globu- described [16]. Proteins were separated on 4-12% Nupage lin, forward 5’-CTT CTG GCT ATG TTT CCC TT-3’, Bis/Tris gels. After electro transfer, membranes were satu- reverse 5’-GTT CTC AGG ATC CAC ATG CA-3’). rated with 5% non-fat dry milk (1h, 20°C) and incubated overnight with primary antibody against FoXO1 (1/1,000) NADH activity (cell signaling), utrophin A (1/1,000) (DSHB), or vinculin Frozen sections were incubated in working solution (8mg/ (1/10,000) (Sigma), then with the corresponding secondary 5ml NADH and 10mg/5ml NBT, 30 min, 37°C) Sections antibodies (1/5,000) (Dako) for 90 min. Immunoreactivity were rinsed thrice in water, with three exchanges each in was determined by chemiluminescence and quantified with 30, 60, and 90% acetone solution, then incubated in 90% Quantity One (Bio-Rad). acetone until a faint purplish cloud was seen over each section. Sections were then rinsed several times with water RNA extraction and MiRNA gene expression and mounted. All sections were stained at the same time RNA was extracted using an miRNeasy Mini Kit (Qiagen, to avoid experimental variation. Pictures were analyzed Valencia, CA). Reverse transcription (RT) was performed using ImageJ. with a TaqMan microRNA reverse transcription kit (Life Technologies Co., Applied Byosystems, Carlsbad, CA). Immunohistochemistry and cytokine analysis miRNA expression was calculated using real-time quanti- Isolated muscle cells and frozen sections were fixed in tative PCR by measuring the threshold cycle ratio (ΔCt) ethanol (except for developmental myosin heavy chain of miRNA31 (3’-AGGCAAGAUGCUGGCAUAGCUG-5’) [dMHC] staining), rinsed, and incubated (30 min, 20°C) and miRNA133a (3’-UUUGGUCCCCUUCAACCAGCU with blocking solution (PBS, 2% BSA, 0.5% Triton G-5’) versus endogenous control snoRNA202 (3’-GCUG X-100, 0.1% Tween 20, 20% sheep serum). Samples were UACUGACUUGAUGAAAGUACUUUUGA-5’). mRNA washed and incubated with dMHC (DSHB), MyoD expression was calculated using real-time quantitative (Dako), or IgM overnight at 4°C, then washed and incu- PCR by measuring the threshold cycle ratio (ΔCt) of PGC- bated for 60 min (20°C) with the appropriate secondary 1α mRNA (5′ CCT GGC CGA GTT CTT TGA A 3′,5′ antibody and Hoechst 33342 (9.0μM, 10 min) and ana- GCC AGA TTT GCT TGT TTG G 3′), cyt c mRNA (5' lyzed as described above. TGC CCA GTG CCA CAC TGT 3', 5' CTG TCT TCC Cytokine expression in EDL muscle lysate was GCC CGA ACA 3'), PDK-4 mRNA (5′ CCG CTG TCC assessed by flow cytometry with a Mouse Inflammation ATG AAG CA 3′,5′ GCA GAA AAG CAA AGG ACG Kit (BD Biosciences 552364), as described in the manu- TT 3′) versus endogenous control GAPDH mRNA (5’ facturer’s instructions. CCG TTC AGC TCT GGG ATG AC 3’,5’ TTC TCA GCA ATG CAT CCT GC 3’). Hematoxylin and eosin (H&E) staining and fibrosis measurement Statistical analyses EDL muscle sections were stained with H&E. The fol- The mean difference between treated and untreated lowing parameters were assessed: the number of total mice was determined by one-way analysis of variance. fibers present, total fibers with central nuclei, total per- Scheffé’s post hoc test was used to identify specific mean ipheral nuclei (dark-blue nuclei), total central nuclei, re- differences. generating fibers (purple), degenerating fibers (pale pink), and inflammation (an interstitial group of 10 Results smaller inflammatory cells with dark-blue nuclei in a Effect of dystrophin deficiency on mitochondrial field) in five non-overlapping fields in each EDL muscle metabolism section. Fibers intersecting the left and top borders of Evaluation of mitochondrial mass (NAO staining) in EDL the field were not counted, and nuclei farther than one fibers of mdx mice and WT control mice (Figure 1A) nuclear diameter from the fiber border were considered showed that dystrophin-deficient muscle fibers have central nuclei. Frozen sections were stained with Van decreased mitochondrial mass, indicating that these Gieson stain (Sigma-Aldrich, St. Louis, MO). Sections muscle have a lower capacity to use oxidative energy. were imaged (bright field, 4× objective, Olympus C.A.S. Furthermore, assessment of LDH activity (Figure 1B) Jahnke et al. Skeletal Muscle 2012, 2:16 Page 4 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 1 Effect of dystrophin deficiency on mitochondrial mass and activity. (A) Isolated EDL fibers were stained with NAO dye to assess mitochondrial mass (n = 3 animals and 30 fibers per muscles). Pictures of the dye fluorescence were taken at the same setting. Fluorescence was quantified with ImageJ. (B) LDH enzyme activity was measured in TA muscle extracts (n = 6). (C) Isometric tetanic maximal force on EDL muscle from mdx and WT mice (n = 6). (D) Fatigue force measurements of EDL muscles from WT and mdx mice (n = 6). Data are means ± SE. * P < 0.05 vs. WT mice. *** P < 0.001 vs. WT mice. demonstrated that the mdx mice had a greater capacity treated groups (statistically significant for the quadriceps to produce lactate. These muscles also showed lower spe- (GW, +12.7%; AICAR, +14.6% ; GW&AICAR, 13.7%) cific force than those of WT mice (Figure 1C), and fa- (Figure 2B) and soleus (GW, +14.3%; AICAR, +17.4%) tigue testing showed that dystrophin-deficient muscle (Figure 2C). Interestingly, abdominal fat was decreased was more fatigable than that of WT mice (Figure 1D), in response to all three treatments (Figure 2D), and the suggesting that dystrophin deficiency leads to significant decrease was statistically significant for both single- alterations in mitochondrial function and muscle metab- treatment groups. olism. Comparison of the ratio of mtDNA to nDNA in Grip strength and open-field animal activity tests were the gastrocnemius muscle of vehicle treated and drug performed before and after drug treatment. We found a treated groups also suggested a trend in an increase of significant increase in forelimb grip strength in the the mtDNA in the AICAR and combination groups (data GW501516-treated and combination-treatment groups not shown). (Figure 2E,F). The increase in hind limb grip strength was significant for all three treatments (Figure 2G). Effect of GW and AICAR on muscle weight and behavioral Since these drugs influenced body weight, we normal- activity measures ized data to body weight. Both forelimb and hind limb The average body mass of the treated mice was signifi- grip strength increased significantly with GW501516 cantly higher than that of vehicle-treated mice (Figure 2A). (+19%, +13%, respectively) and combination treatment Treatments increased the body mass by ~10% (GW, 9.2%; (+25%, +13%, respectively) (Figure 2F). Behavioral activ- AICAR, 11.3%; GW&AICAR, 10.63%). A general in- ity measures did not significantly change for the single crease in the weight of the EDL, gastrocnemius, quadri- treatments but the combination treatment group showed ceps, soleus, and TA muscles was found in the drug- significantly increased movement time (89%) (Figure 2I) Jahnke et al. Skeletal Muscle 2012, 2:16 Page 5 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 2 Effect of GW and AICAR on body mass, muscle mass, and behavioral activity. (A-D) Mass of vehicle control- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice: mass of the whole body (A), quadriceps (B), soleus (C), and abdominal fat (D) after euthanasia. Grip strength was measured using a grid at 12 and 16 weeks of age: (E) Maximal forelimb grip strength, (F) normalized maximal forelimb strength, (G) maximal hind-limb grip strength, (H) normalized maximal hind-limb strength of all the groups. The overall activity of the mice was measured using the open-field Digiscan apparatus at 12 and 16 weeks of age: (I) movement time and (J) rest time activity. Behavioral activity is presented as a percentage of the initial activity measured at 12 weeks of age before treatment. Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. and decreased rest time (Figure 2J), suggesting an overall (Figure 3H) (GW, +43%; AICAR, +29%; GW&AICAR, beneficial effect on these parameters. 26%) and SOL (Figure 3I) muscle (GW, +35%; AICAR, +26%; GW&AICAR, 13.7%) in response to drug treat- Effect of GW and AICAR on mitochondrial activity ment. Soleus muscles expressed more myosin heavy We evaluated the impact of these drugs on mitochondrial chain type I (Figure 3J), whereas only GW showed a sta- activity in muscle cells isolated from hind limb muscles of tistically significant increase in type IIA fibers dystrophin-deficient mdx mice. A significant increase in (Figure 3K). This increase in oxidative capacity was also mitochondrial mass, as indicated by NAO staining, was observed in EDL muscles, in which the ratio of the found in the GW501516-treated cells (Figure 3A,B). We height of the twitch force (P ) to the time to reach this saw no significant increase in either the AICAR- or maximal force (tpt) was increased in single-treated mice combination-treated groups. Mitochondrial ΔΨ,as assessed (GW, +18%; AICAR, +24% ) (Figure 3L). Finally, LDH by DiOC6 staining, was significantly increased in response activity in the TA was decreased in all three treatment to AICAR treatment (Figure 3C,D) but not GW501516 groups, but the decrease was only statistically significant or combination treatment. Gastrocnemius muscle from for GW-treated mice (−30%) (Figure 3M). treated mdx mice expressed significantly more PGC-1 α, cyt c mRNAs (Figure 3E,F) in comparison to vehicle Effect of GW and AICAR on satellite cell activation and treated group but increase in PDK4 mRNA did not muscle regeneration and degeneration reach statistical significance (Figure 3G). We also found We found that the number of dMHC-positive fibers was that NADH activity was significantly increased in EDL significantly decreased in the drug-treated groups Jahnke et al. Skeletal Muscle 2012, 2:16 Page 6 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 3 Effect of GW and AICAR on mitochondrial activity. Fluorescent-activated cell scanning (FACS) of primary myoblasts isolated from untreated and drug-treated mdx mice. Representative dot plot FACS overlay of mitochondrial mass with nonyl acridine orange (NAO) staining (A and B) and staining for mitochondrial activity with 3, 3’-dihexyloxacarbocyanine iodide (DiOC6) in myoblasts derived from the muscles of treated mice (C, D). Histograms show geometric mean fluorescence of NAO and DiOC6 in dystrophin-deficient myoblasts. Quantification of mRNA expression of PGC-1 α (E), Cyt c (F) and PDK-4 (G) relative to GAPDH mRNA expression by RTqPCR in gastrocnemius with n = 2 for each group. Quantification of NADH activity in histological sections of EDL (H) and soleus (I) muscles, with immunolabeling of the soleus muscle for Type I (J) and type IIA (K) fibers. (L) EDL twitch force parameters, ratio of the maximal twitch force (P ) to the time required to reach this force (tpt), and (M) lactate dehydrogenase activity of TA muscle. All experiments involved vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. (Figure 4A,B), and MyoD expression in isolated skeletal miRNA133 was also increased in the treated groups, but muscle cells was markedly decreased in the GW- and the differences did not reach statistical significance (data combination-treated groups (Figure 4C). Furthermore, not shown). Expression of FoXO1, which controls muscle the number of EDL fibers without central nucleation wasting, was decreased in AICAR (−34%) and combin- increased in the single-treated groups (Figure 4D). Im- ation drug-treated mice (−36%)(Figure 4F). Serum CK portantly, miRNA31a expression, known to be associated levels showed huge variations but no statistically signifi- with muscle regeneration/degeneration, was significantly cant changes (data not shown). Finally, IgM immunos- down-regulated in diaphragms of treated mice (GW, taining was significantly decreased in gastrocnemius -28%; AICAR, -63%; GW&AICAR, -67%) (Figure 4E). sections of treated muscle (GW, -48%; AICAR, -69%; Jahnke et al. Skeletal Muscle 2012, 2:16 Page 7 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 4 Effect of GW and AICAR on satellite cell activation, muscle regeneration, and degeneration. (A) Immunostaining of gastrocnemius muscle for developmental myosin (MyoD) heavy chain. (B) Histogram representing the area of regenerating fibers in gastrocnemius muscle. (C) Percentage of MyoD-positive cells isolated from muscles. (D) Fibers with no central nuclei. (E) MiRNA expression in diaphragm muscle (n = 4 for each group). (F) Western blotting for FoXO1 in EDL muscle lysate. Vinculin was used as an internal control for protein loading. The expression was normalized to that of vinculin and expressed as a percentage of the vehicle expression. (G) IgM-positive immunolabeling of muscle section to identify degenerated fibers. Data are means ± SE from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. GW&AICAR, -54%) (Figure 4G). Overall, these data sug- progression and response therapy. We found that the gested a strong reduction in muscle degeneration. red-positive area was significantly decreased in the treated groups (GW501516, -25.6%; AICAR, -27.5%; Effect of GW and AICAR on diaphragm fibrosis, utrophin GW&AICAR, -27.2%) (Figure 5B). Evaluation of cytokine A expression, muscle cytokines, and inflammation expression in TA muscle lysate revealed that mdx mice Utrophin expression in skeletal muscle was studied be- had significantly increased IL-6 and IL-10 levels. Drug cause some of these improvements may have been due to treatment did not significantly affect IL-6 expression utrophin expression [17]. The level of utrophin A was (Figure 5C), but IL-10 levels were significantly decreased significantly increased in the treated groups over that in in GW&AICAR-treated mice (−45%) (Figure 5D), and untreated mice (GW, +112.97%,; AICAR, +84.97%; not in individual drug-treated mice. EDL muscle demon- GW&AICAR, +94.19%) (Figure 5A). We also measured strated a statistically significant decrease in inflammatory fibrosis in the diaphragm which occurs early in the dis- infiltrates in the GW group but not the other two groups ease and serves as a useful marker for assessing disease (Figure 5E). Jahnke et al. Skeletal Muscle 2012, 2:16 Page 8 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 5 Effect of GW and AICAR on diaphragm fibrosis, utrophin A expression, muscle cytokines, and inflammation. (A) Western blotting for utrophin in EDL muscles from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. Vinculin was used as an internal control for protein loading. The expression was normalized to that of vinculin and expressed as a percentage of the vehicle expression. (B) Van Gilson staining of the diaphragms of mice treated with vehicle, GW501516, AICAR, or GW&AICAR. The percentage of fibrosis was then calculated by measuring the area of the fibrosis and the area of the whole section from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. IL-6 (C) and IL-10 (D) analyses were performed by flow cytometry on EDL muscle lysate (n = 2), with 30,000 events per tube. WT muscle was used as a control for cytokine expression. (E) Inflammation was also analyzed by Histologic examination after H&E staining (1 inflammation = a cluster of 10 nuclei). Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. Discussion in mice [23]. Conversely, oxidative capacity activation In this study, we have demonstrated that dystrophic decreases muscle wasting in most cases [10]. Recently, muscle displays mitochondrial dysfunction similar to PPARδ have been demonstrated to be involved in satel- that in golden retriever muscular dystrophy [18]. Meta- lite cells proliferation and muscle regeneration [24]. bolic impairment has previously been reported [19], and Moreover, PGC-1α overexpression inhibits muscle atro- dystrophin-deficient myoblasts have been described as phy during fasting and denervation [3] and significantly having a pronounced respiratory impairment [20]. This improves dystrophic muscle [25]. Grumati et al. have deficiency is not the primary cause of muscle weakness demonstrated that correcting mitochondrial impairment in dystrophin deficiency; however, it may play a signifi- in collagen VI deficiency significantly improves muscle cant additional role that can be important for the time function [26]. Therefore, strategies that target mitochon- course of the disease. Defects in fatty acid oxidation drial up-regulation may be beneficial to dystrophic leads to the accumulation of fatty acylCoA and diacylgly- muscle. cerol, inducing insulin signaling disruption and causing In the present study, we have used two known muscle atrophy [21]. Similarly, alterations in mitochon- endurance-mimetic drugs, AICAR and GW501516, to drial functions caused by mtDNA mutations are activate endurance exercise-induced signaling pathways. involved in muscle loss during aging [22]. Mitochondrial AICAR is a mimetic of endurance training that activates fission and remodeling also contribute to muscle atrophy AMPK activity, an energy status sensor in the cell [27]. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 9 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 In normal mice, intraperitoneal injection of AICAR suggested by a marked decrease in FoXO1 and IgM ex- raises the level of PGC-1α expression and increases pression in fibers. Together, these results clearly indicate mitochondrial biogenesis in muscle [28], reducing that muscle degeneration is decreased in treated mice. muscle fatigability and increasing muscle performance FoXO1 transcription is lower in high-oxidative mouse [12]. GW501516 is a PPARβ/δ agonist, a transcription soleus than in low-oxidative gastrocnemius, TA, and factor that is co-activated by PGC-1α. Like AICAR, this quadriceps muscles [40]. In vivo, FoXO1 inhibits high drug is known to increase the amount of mitochondria oxidative fiber-related gene expression and oxidative and promote mitochondrial metabolism [29,30] and fatty metabolism-enhancing factor activity [41]. Skeletal mus- acid oxidation [31] in vivo and in vitro and has been cles of FoXO1-over-expressing mice had fewer type I tested as a therapeutic for type II diabetes [29,31,32]. fibers, as well as smaller type I and type II fibers [41]. More interestingly, the increase in muscle performance The phenotype of our treated mice became more oxida- that is stimulated by PPARδ/β activity is independent of tive, consistent with this change. A decrease in muscle exercise training in mice [33] and combination treat- degradation could explain the diminution in satellite cell ments with these drugs have been shown to have syner- activation that we observed. gistic beneficial effects in vivo in WT mice [12]. We also found a decrease in inflammation and fat tis- Our study clearly demonstrates that endurance sue in the treated mice. Increased IL-6 levels are mimetics improve muscle function and overall activity in involved in metabolic and structural changes in muscle dystrophic mice. The stimulation of mitochondrial bio- and in muscle loss during cachexia [42]. However, IL-6 genesis by GW501516 and/or AICAR that we have inhibition has significantly reversed skeletal muscle wast- observed is consistent with previous studies [31,33-35]. ing in rodents [42]. Our data suggest that also Miura et al. reported the use of GW501516 to slow the GW501516 and AICAR improve muscle function myogenic program and increase utrophin A expression through inflammation down-regulation. Adipose tissue in 5 weeks old mdx mice. More recently, Ljubicic et al plays a crucial endocrine role through the production of have reported that AICAR supplementation accompan- adipokines. Aberrant intracellular signaling cascades that ied with bout of exercise also improve muscle function regulate both inflammatory and immune processes are in 5 weeks old MDX mice. In our study, we have further known to contribute substantially to degeneration shown a decrease in LDH activity in TA muscles and in- [43,44]. Therefore, fat reduction is very interesting, since crease in NADH activity, together with an increase in it can reduce inflammation and have an impact on both type I/IIA fibers in soleus muscle, an increase of mRNA degeneration and regeneration. GW501516 has been expression of PGC-1α, cyt c and a trend for PDK-4 sug- shown to be involved in inflammatory pathway regula- gesting that the phenotype of treated muscle shifts from tion [45]. However, further experiments are needed to glycolytic to oxidative type. Recently, Selsby et al. found delineate the link between proinflammatory fat tissue that enhancing PGC-1α expression rescues dystrophic and muscle inflammation. muscle and that a switch from fast - to slow -twitch muscle is involved [25]. Moreover, utrophin expression increased, as in Miura et al., and could be part of the Conclusions process of improving muscle function. Evidence suggests In summary, this study demonstrates that the use of en- that utrophin is likely to compensate for the lack of dys- durance mimetics in mdx mice induces an improvement trophin in DMD muscle [17,36] and to decrease muscle in the structural integrity and reduces the degeneration/ pathology [37,38]. This suggests the possibility that the regeneration of mdx mouse muscle, probably through an increase of Utrophin A might partly restore the dys- increase in oxidative metabolism in the fibers. Our study trophin associated glycoprotein and help to improve and other recent work underline the high potential of muscle function. Slow-twitch fibers have been reported pharmacological activators of AMPK and PPARδ as part to have higher utrophin expression than do fast-twitch of rational drug treatments for muscular dystrophies. fibers. Therefore, the increase in utrophin expression with treatment could be a result of the change in fiber Abbreviations metabolism. The presence of fibers with no central nu- AMPK: 5' adenosine monophosphate-activated protein kinase; AICAR: 5- clei and the increase in peripheral nuclei suggest that de- aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, AICA-riboside; DIOC6: 3,3′-dihexyloxacarbocyanine iodide; DMD: Duchenne muscular generation/regeneration has been halted by this dystrophy; dMHC: Developmental myosin heavy chain; DMSO: Dimethyl therapeutic intervention. This evidence is further corro- sulphoxide; EDL: Extensor digitorum longus; Glut: Glutamine; LDH: Lactate borated by the concomitant down-regulation of activated deshydrogenase; mtDNA: mitochondrial Deoxyribonucleic acid; NADH: Nicotinamide adenine dinucleotide; NAO: 10-nonyl acridine orange; satellite cells and dMHC-positive regenerated fibers and P/S: Penicillin / Streptomycin; PGC-1α: Peroxisome proliferator-activated a decrease in miRNA-31, which are involved in muscle receptor gamma coactivator 1-alpha; PPARδ: Peroxisome proliferator- degeneration [39]. Stabilization of myofiber structure is activated receptor delta. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 10 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Competing interests 9. Lin J, Wu H, Tarr PT, et al: Transcriptional co-activator PGC-1 alpha drives Dr. Nagaraju is one of the co-founders and member of the board of the formation of slow-twitch muscle fibers. Nature 2002, 418:797–801. directors of ReveraGen BioPharma Inc, a biopharmaceutical company 10. Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT: Increased engaged in the discovery and development of proprietary, small molecule muscle PGC-1alpha expression protects from sarcopenia and metabolic therapeutics for the treatment of neuromuscular diseases. This work was disease during aging. Proc Natl Acad Sci USA 2009, 106:20405–20410. funded by Department of Defense USAMRAA grant W81XWH-05-1-0616 11. Bushby K, Finkel R, Birnkrant DJ, et al: Diagnosis and management of (Mouse Drug Screening Core to K. Nagaraju), the Foundation to Eradicate Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological Duchenne, Inc., the Muscular Dystrophy Association, NIH grant R01- and psychosocial management. Lancet Neurol 2010, 9:77–93. AR050478 (K. Nagaraju) and Cristal Ball funding. 12. Narkar VA, Downes M, Yu RT, et al: AMPK and PPARdelta agonists are Dr. Hoffman is one of the co-founders and member of the board of directors exercise mimetics. Cell 2008, 134:405–415. of ReveraGen BioPharma Inc, a biopharmaceutical company engaged in the 13. Brooks SV, Faulkner JA: Contractile properties of skeletal muscles from discovery and development of proprietary, small molecule therapeutics for young, adult and aged mice. J Physiol 1988, 404:71–82. the treatment of neuromuscular diseases. This work was funded by NIH 14. Spurney CF, Gordish-Dressman H, Guerron AD, et al: Preclinical drug trials grant 1U54HD053177-01A1 (Wellstone Muscular Dystrophy Center to E.P. in the mdx mouse: assessment of reliable and sensitive outcome Hoffman). measures. Muscle Nerve 2009, 39:591–602. Dr JAHNKE, Dr Van Der Meulen, Mrs Johnston, Dr Ghimbovschi and Dr 15. Jahnke VE, Sabido O, Freyssenet D: Control of mitochondrial biogenesis, Partridge report no disclosures. ROS level, and cytosolic Ca2+ concentration during the cell cycle and the onset of differentiation in L6E9 myoblasts. Am J Physiol Cell Physiol 2009, 296:C1185–C1194. Authors’ contributions 16. Jahnke VE, Sabido O, Defour A, et al: Evidence for mitochondrial VEJ, PhD: designed research, conducted experiments, performed data respiratory deficiency in rat rhabdomyosarcoma cells. PLoS One 2010, analysis, and wrote the manuscript. JHM VD, PhD: conducted experiments, 5:e8637. performed data analysis. HKJ: conducted experiments. SG, PhD: conducted experiments, performed data analysis. TP, PhD: contributed to scientific 17. Perkins KJ, Davies KE: The role of utrophin in the potential therapy of discussion on the manuscript. EPH, PhD: provided reagents/lab facilities. KN, Duchenne muscular dystrophy. Neuromuscul Disord 2002, PhD: designed research, interpreted the data, provided reagents/lab facilities, 12(Suppl 1):S78–S89. wrote the manuscript. All authors read and approved the final manuscript. 18. Guevel L, Lavoie JR, Perez-Iratxeta C, et al: Quantitative proteomic analysis of dystrophic dog muscle. J Proteome Res 2011, 10:2465–2478. 19. Even PC, Decrouy A, Chinet A: Defective regulation of energy metabolism Acknowledgments in mdx-mouse skeletal muscles. Biochem J 1994, 304(Pt 2):649–654. The authors wish to acknowledge Dr. Deborah McClellan for editorial 20. Onopiuk M, Brutkowski W, Wierzbicka K, et al: Mutation in assistance. Funding to KN in part by the Department of Defense USAMRAA dystrophin-encoding gene affects energy metabolism in mouse grant W81XWH-05-1-0616 (Mouse Drug Screening Core); W81XWH-11-1- myoblasts. Biochem Biophys Res Commun 2009, 386:463–466. 0782; National Institutes of Health grants K26RR032082; R01-AR050478 21. Koves TR, Li P, An J, et al: PPARgamma coactivator-1alpha -mediated (KN);1U54HD053177-01A1 (Wellstone Muscular Dystrophy Center); metabolic remodeling of skeletal myocytes mimics exercise training and 2R24HD050846-06 (Integrated Molecular Core for Rehabilitation Medicine) reverses lipid-induced mitochondrial inefficiency. J Biol Chem 2005, 3:3. and Muscular dystrophy association. 22. Figueiredo PA, Mota MP, Appell HJ, Duarte JA: The role of mitochondria in aging of skeletal muscle. Biogerontology 2008, 9:67–84. Author details 23. Romanello V, Guadagnin E, Gomes L, et al: Mitochondrial fission and Center for Genetic Medicine Research, Children’s National Medical Center, remodeling contributes to muscle atrophy. EMBO J 2010, 29:1774–1785. Washington, DC, USA. Department of Integrative Systems Biology, George 24. Angione AR, Jiang C, Pan D, Wang YX, Kuang S: PPARdelta regulates Washington University School of Medicine and Health Sciences, Washington, satellite cell proliferation and skeletal muscle regeneration. Skelet Muscle DC, USA. Integrative Systems Biology and Pediatrics, Research Center for 2011, 1:33. Genetic Medicine Children's National Medical Center, 111 Michigan Avenue, NW Washington, DC 20010, USA. 25. Selsby JT, Morine KJ, Pendrak K, Barton ER, Sweeney HL: Rescue of dystrophic skeletal muscle by PGC-1alpha involves a fast to slow fiber Received: 25 April 2012 Accepted: 23 July 2012 type shift in the mdx mouse. PLoS One 2012, 7:e30063. Published: 21 August 2012 26. Grumati P, Coletto L, Sandri M, Bonaldo P: Autophagy induction rescues muscular dystrophy. Autophagy 2011, 7:426–428. 27. Hardie DG: Minireview: the AMP-activated protein kinase cascade: the References key sensor of cellular energy status. Endocrinology 2003, 144:5179–5183. 1. Freyssenet D: Energy sensing and regulation of gene expression in Epub 2003 Sep 5174. skeletal muscle. J Appl Physiol 2007, 102:529–540. 28. Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M, Holloszy JO: 2. Sandri M: Signaling in muscle atrophy and hypertrophy. Physiology Activation of AMP-activated protein kinase increases mitochondrial (Bethesda) 2008, 23:160–170. enzymes in skeletal muscle. J Appl Physiol 2000, 88:2219–2226. 3. Sandri M, Lin J, Handschin C, et al: PGC-1alpha protects skeletal muscle 29. Tanaka T, Yamamoto J, Iwasaki S, et al: Activation of peroxisome from atrophy by suppressing FoxO3 action and atrophy-specific gene proliferator-activated receptor delta induces fatty acid beta-oxidation in transcription. Proc Natl Acad Sci USA 2006, 103:16260–16265. skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci 4. Brault JJ, Jespersen JG, Goldberg AL: Peroxisome proliferator-activated USA 2003, 100:15924–15929. Epub 12003 Dec 15915. receptor gamma coactivator 1alpha or 1beta overexpression inhibits 30. Brunmair B, Staniek K, Dorig J, et al: Activation of PPAR-delta in isolated muscle protein degradation, induction of ubiquitin ligases, and disuse rat skeletal muscle switches fuel preference from glucose to fatty acids. atrophy. J Biol Chem 2010, 285:19460–19471. Diabetologia 2006, 49:2713–2722. 5. Lecker SH, Jagoe RT, Gilbert A, et al: Multiple types of skeletal muscle 31. Dressel U, Allen TL, Pippal JB, Rohde PR, Lau P, Muscat GE: The peroxisome atrophy involve a common program of changes in gene expression. proliferator-activated receptor beta/delta agonist, GW501516, regulates FASEB J 2004, 18:39–51. the expression of genes involved in lipid catabolism and energy 6. Muller FL, Song W, Liu Y, et al: Absence of CuZn superoxide dismutase uncoupling in skeletal muscle cells. Mol Endocrinol 2003, 17:2477–2493. leads to elevated oxidative stress and acceleration of age-dependent Epub 2003 Oct 2402. skeletal muscle atrophy. Free Radic Biol Med 2006, 40:1993–2004. 32. Kramer DK, Al-Khalili L, Guigas B, Leng Y, Garcia-Roves PM, Krook A: 7. Mansouri A, Muller FL, Liu Y, et al: Alterations in mitochondrial function, Role of AMP kinase and PPARdelta in the regulation of lipid and hydrogen peroxide release and oxidative damage in mouse hind-limb glucose metabolism in human skeletal muscle. J Biol Chem 2007, skeletal muscle during aging. Mech Ageing Dev 2006, 127:298–306. 282:19313–19320. 8. Puigserver P, Spiegelman BM: Peroxisome proliferator-activated receptor- gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator 33. Wang YX, Zhang CL, Yu RT, et al: Regulation of Muscle Fiber Type and and metabolic regulator. Endocr Rev 2003, 24:78–90. Running Endurance by PPARdelta. PLoS Biol 2004, 2:E294. Jahnke et al. 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Tinsley JM, Fairclough RJ, Storer R, et al: Daily Treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse. PLoS One 2011, 6:e19189. 39. Cacchiarelli D, Incitti T, Martone J, et al: miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy. EMBO Rep 2011, 12:136–141. 40. Allen DL, Unterman TG: Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors. Am J Physiol Cell Physiol 2007, 292:C188–C199. 41. Kamei Y, Miura S, Suzuki M, et al: Skeletal muscle FOXO1 (FKHR) transgenic mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red muscle) fiber genes, and impaired glycemic control. J Biol Chem 2004, 279:41114–41123. 42. Tisdale MJ: Molecular pathways leading to cancer cachexia. Physiology (Bethesda) 2005, 20:340–348. 43. Kumar 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, 17:386–396. 44. Porter JD, Guo W, Merriam AP, et al: Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle. Neuromuscul Disord 2003, 13:223–235. 45. Coll T, Alvarez-Guardia D, Barroso E, et al: Activation of peroxisome proliferator-activated receptor-{delta} by GW501516 prevents fatty acid-induced nuclear factor-{kappa}B activation and insulin resistance in skeletal muscle cells. Endocrinology 2010, 151:1560–1569. doi:10.1186/2044-5040-2-16 Cite this article as: Jahnke et al.: Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model. Skeletal Muscle 2012 2:16. 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Metabolic remodeling agents show beneficial effects in the dystrophin- deficient mdx mouse model

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Copyright © 2012 by Jahnke et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-2-16
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22908954
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Abstract

Background: Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods: Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects. Results: We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions: The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice. Keywords: Duchenne muscular dystrophy, Muscle, AICAR, GW501516, Metabolism Background receptor γ coactivator 1 α (PGC-1α), the master regula- Muscle is a plastic tissue that responds and adapts to en- tor of mitochondrial biogenesis [8], seems to control vironmental changes [1]. Energy balance is one of the muscle wasting [3]. PGC-1α overexpression increases checkpoints between muscle growth/hypertrophy and mitochondrial content [9] and resistance to fatigue [9] protein breakdown [2], and >10% of atrophy-related and reduces the rapid muscle atrophy associated with genes are directly involved in energy production [3-5]. denervation, fasting, and FoXO 3 activation [3]. Re- Mitochondrial dysfunction activates various proteolytic cently, mice overexpressing PGC-1α have been shown to systems [2] and is associated with muscle atrophy in sev- have an increased lifespan and to be protected from sar- eral myopathies [6,7]. Peroxisome proliferator-activated copenia [10]. Therefore, targeting mitochondrial biogen- esis and metabolism up-regulation may have beneficial * Correspondence: knagaraju@cnmcresearch.org effects in muscle diseases. Center for Genetic Medicine Research, Children’s National Medical Center, Evidence for the beneficial effects of submaximal aer- Washington, DC, USA obic activities in DMD patients is slowly emerging. A re- Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA cent review on the management and care of DMD Full list of author information is available at the end of the article © 2012 Jahnke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 2 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 patients recommend that ambulatory and early non- LB 940, Berthold Technologies). LDH activity was nor- ambulatory-stage boys participate in regular submaximal malized to protein concentration. functional activities [11]. The molecular mechanisms by which exercise provides beneficial effects are currently Measurement of contraction properties unclear. However, increased PPARδ and AMPK activities Mice were anesthetized with 100mg/kg ketamine and have been implicated in these beneficial effects [12]. We 10mg/kg xylazine. EDL muscle was isolated and placed hypothesized that exercise mimetics activating PPARδ in Ringer’s solution (137mM NaCl, 24mM NaHCO , and AMPK pathways are beneficial to dystrophin defi- 11mM glucose, 5mM KCl, 2mM CaCl , 1mM MgSO , 2 4 cient skeletal muscle. In the present study, we have used 1mM NaH PO , and 0.025mM tubocurarine chloride) 2 4 agonists of PPARδ (GW501516) and AMPK (AICAR) to maintained at 25°C and bubbled with 95% O -5% CO . 2 2 activate beneficial endurance exercise-induced signaling Contractile properties were measured according to pathways in mdx mice. We have demonstrated that en- Brooks et al. [13], using an in vitro test apparatus (model durance mimetics can improve muscle function by halt- 305B, Aurora Scientific). A fatigue protocol was per- ing the cycle of muscle regeneration/degeneration in formed (20 min, 100 Hz). EDL muscle was subjected to a dystrophin-deficient mice. series of 120 isometric tetanic contractions (400 ms). Methods Animal experiments Behavioral activity measurement and grip strength All mice were handled according to Washington DC testing Veterans Affairs Medical Center’s Institutional Animal All animals were weighed before and after drug treatment. Care and Use Committee guidelines under approved Grip strength and open-field activity were assessed using a mdx protocol # 01079. C57BL/10ScSn-Dmd /J (mdx) grip strength meter (Columbus Instruments, Columbus, 8-week-old male mice, 20-30g, were purchased from OH) and open-field Digiscan apparatus (Omnitech Elec- The Jackson Laboratory housed in an individually vented tronics, Columbus, Ohio), respectively, as described previ- cage system (with a 12-h light–dark cycle, standard ously [14]. Tissues were either embedded in OCT or mouse chow and water ad libitum). Mice were rested wrapped in foil and then frozen in isopentane chilled in li- for 10–14 days, acclimatized on the behavioral instru- quid nitrogen. Blood was collected by cardiac puncture, ment for 1 week and then baseline grip strength and be- and serum was collected by centrifuging blood for 10min havioral activity was performed. At 12 weeks of age, the at 10,000rpm and then stored at −80°C. mice were given AICAR (250mg/kg [80μl]; Alexis) by intraperitoneal injection and/or GW501516 (7.5mg/kg Muscle cell extraction [80μl]; Alexis) by oral gavage, 5 days/week for 4 weeks. Leg muscles that were not harvested as previously DMSO in PBS (1/2 vol/vol) was used as a vehicle control described were used for satellite cell extraction. Tendons and concentration of DMSO was the same in vehicle and aponeuroses were removed. Muscles were minced, and drug treatments. placed in digestion medium (2.4U/ml dispase II, 100mg/ ml collagenase A), vortexed, and incubated at 37°C. After Extensor digitorum longus (EDL) fiber isolation and digestion, tubes were placed on ice, and 25ml of DMEM- staining 1% PS-2% L-Glut were added. The mixture was filtered The EDL muscles of 12-week-old mice were harvested with a 100-μm cell strainer and centrifuged (800g, 4°C, and incubated in DMEM with 2mg/ml collagenase for 3min). The pellet was resuspended in 25ml DMEM-1% 2 h. EDL fibers were separated with Pasteur pipets. Fibers PS-2% L-Glut), filtered with a 70-μm cell strainer, and were rinsed, stained with 10-nonyl acridine orange (NAO; centrifuged (800g, 4°C, 3min). The same operation was Sigma) (15min), rinsed, fixed with 4% formalin (10min), repeated with a 40-μm cell strainer. Cell extracts were fro- and mounted. Pictures were taken. Fluorescence levels zen and stored at −80°C. were analyzed with ImageJ software (NIH). LDH activity Flow cytometry analyses Lactate dehydrogenase activity of muscle lysate was mea- Mitochondrial content and inner membrane potential (ΔΨ) sured using 2.5μl of protein extract (1:2 dilution), 225μl were assessed with NAO and 3, 3’-dihexyloxacarbocyanine assay buffer (2.5ml of 1 M Tris [pH 7.6], 500μlof iodide (DiOC6) (Invitrogen) as described [15]. Cell immu- 200mM EDTA, and 500μl of 5mM NADH,H , and 48ml noreactivity against MyoD (Dako) was assessed with water). Oxidation of NADH, H was recorded after pyru- Hoechst 33342 (Sigma) as described [15]. Cells were ana- vate addition (10μl, 100mM). NADH fluorescence was lyzed on a FACSCalibur (BD Biosciences, San Jose, CA, detected by luminescence/fluorescence analyzer (Mithras USA) with BD Cell Quest ProTM 4.0.2. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 3 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Mitochondrial DNA to nuclear DNA ratio analysis T. Stereology System, Olympus America Inc., Center Total DNA was extracted from muscle cells using DNeasy Valley, PA). Pictures were processed using ImageJ. Fi- blood and tissue kit (Qiagen). The content of mtDNA was brotic red areas were expressed as a percentage of the calculated using real-time quantitative PCR by measuring total tissue section. the threshold cycle ratio (ΔCt) of a mitochondrial- encoded gene (ND1, forward 5’- GGA CCT AAG CCC Western blotting AAT AAC GA-3’, reverse 5’-GCT TCA TTG GCT ACA Protein homogenates were extracted as previously CCT TG-3’) versus a nuclear-encoded gene (Beta-globu- described [16]. Proteins were separated on 4-12% Nupage lin, forward 5’-CTT CTG GCT ATG TTT CCC TT-3’, Bis/Tris gels. After electro transfer, membranes were satu- reverse 5’-GTT CTC AGG ATC CAC ATG CA-3’). rated with 5% non-fat dry milk (1h, 20°C) and incubated overnight with primary antibody against FoXO1 (1/1,000) NADH activity (cell signaling), utrophin A (1/1,000) (DSHB), or vinculin Frozen sections were incubated in working solution (8mg/ (1/10,000) (Sigma), then with the corresponding secondary 5ml NADH and 10mg/5ml NBT, 30 min, 37°C) Sections antibodies (1/5,000) (Dako) for 90 min. Immunoreactivity were rinsed thrice in water, with three exchanges each in was determined by chemiluminescence and quantified with 30, 60, and 90% acetone solution, then incubated in 90% Quantity One (Bio-Rad). acetone until a faint purplish cloud was seen over each section. Sections were then rinsed several times with water RNA extraction and MiRNA gene expression and mounted. All sections were stained at the same time RNA was extracted using an miRNeasy Mini Kit (Qiagen, to avoid experimental variation. Pictures were analyzed Valencia, CA). Reverse transcription (RT) was performed using ImageJ. with a TaqMan microRNA reverse transcription kit (Life Technologies Co., Applied Byosystems, Carlsbad, CA). Immunohistochemistry and cytokine analysis miRNA expression was calculated using real-time quanti- Isolated muscle cells and frozen sections were fixed in tative PCR by measuring the threshold cycle ratio (ΔCt) ethanol (except for developmental myosin heavy chain of miRNA31 (3’-AGGCAAGAUGCUGGCAUAGCUG-5’) [dMHC] staining), rinsed, and incubated (30 min, 20°C) and miRNA133a (3’-UUUGGUCCCCUUCAACCAGCU with blocking solution (PBS, 2% BSA, 0.5% Triton G-5’) versus endogenous control snoRNA202 (3’-GCUG X-100, 0.1% Tween 20, 20% sheep serum). Samples were UACUGACUUGAUGAAAGUACUUUUGA-5’). mRNA washed and incubated with dMHC (DSHB), MyoD expression was calculated using real-time quantitative (Dako), or IgM overnight at 4°C, then washed and incu- PCR by measuring the threshold cycle ratio (ΔCt) of PGC- bated for 60 min (20°C) with the appropriate secondary 1α mRNA (5′ CCT GGC CGA GTT CTT TGA A 3′,5′ antibody and Hoechst 33342 (9.0μM, 10 min) and ana- GCC AGA TTT GCT TGT TTG G 3′), cyt c mRNA (5' lyzed as described above. TGC CCA GTG CCA CAC TGT 3', 5' CTG TCT TCC Cytokine expression in EDL muscle lysate was GCC CGA ACA 3'), PDK-4 mRNA (5′ CCG CTG TCC assessed by flow cytometry with a Mouse Inflammation ATG AAG CA 3′,5′ GCA GAA AAG CAA AGG ACG Kit (BD Biosciences 552364), as described in the manu- TT 3′) versus endogenous control GAPDH mRNA (5’ facturer’s instructions. CCG TTC AGC TCT GGG ATG AC 3’,5’ TTC TCA GCA ATG CAT CCT GC 3’). Hematoxylin and eosin (H&E) staining and fibrosis measurement Statistical analyses EDL muscle sections were stained with H&E. The fol- The mean difference between treated and untreated lowing parameters were assessed: the number of total mice was determined by one-way analysis of variance. fibers present, total fibers with central nuclei, total per- Scheffé’s post hoc test was used to identify specific mean ipheral nuclei (dark-blue nuclei), total central nuclei, re- differences. generating fibers (purple), degenerating fibers (pale pink), and inflammation (an interstitial group of 10 Results smaller inflammatory cells with dark-blue nuclei in a Effect of dystrophin deficiency on mitochondrial field) in five non-overlapping fields in each EDL muscle metabolism section. Fibers intersecting the left and top borders of Evaluation of mitochondrial mass (NAO staining) in EDL the field were not counted, and nuclei farther than one fibers of mdx mice and WT control mice (Figure 1A) nuclear diameter from the fiber border were considered showed that dystrophin-deficient muscle fibers have central nuclei. Frozen sections were stained with Van decreased mitochondrial mass, indicating that these Gieson stain (Sigma-Aldrich, St. Louis, MO). Sections muscle have a lower capacity to use oxidative energy. were imaged (bright field, 4× objective, Olympus C.A.S. Furthermore, assessment of LDH activity (Figure 1B) Jahnke et al. Skeletal Muscle 2012, 2:16 Page 4 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 1 Effect of dystrophin deficiency on mitochondrial mass and activity. (A) Isolated EDL fibers were stained with NAO dye to assess mitochondrial mass (n = 3 animals and 30 fibers per muscles). Pictures of the dye fluorescence were taken at the same setting. Fluorescence was quantified with ImageJ. (B) LDH enzyme activity was measured in TA muscle extracts (n = 6). (C) Isometric tetanic maximal force on EDL muscle from mdx and WT mice (n = 6). (D) Fatigue force measurements of EDL muscles from WT and mdx mice (n = 6). Data are means ± SE. * P < 0.05 vs. WT mice. *** P < 0.001 vs. WT mice. demonstrated that the mdx mice had a greater capacity treated groups (statistically significant for the quadriceps to produce lactate. These muscles also showed lower spe- (GW, +12.7%; AICAR, +14.6% ; GW&AICAR, 13.7%) cific force than those of WT mice (Figure 1C), and fa- (Figure 2B) and soleus (GW, +14.3%; AICAR, +17.4%) tigue testing showed that dystrophin-deficient muscle (Figure 2C). Interestingly, abdominal fat was decreased was more fatigable than that of WT mice (Figure 1D), in response to all three treatments (Figure 2D), and the suggesting that dystrophin deficiency leads to significant decrease was statistically significant for both single- alterations in mitochondrial function and muscle metab- treatment groups. olism. Comparison of the ratio of mtDNA to nDNA in Grip strength and open-field animal activity tests were the gastrocnemius muscle of vehicle treated and drug performed before and after drug treatment. We found a treated groups also suggested a trend in an increase of significant increase in forelimb grip strength in the the mtDNA in the AICAR and combination groups (data GW501516-treated and combination-treatment groups not shown). (Figure 2E,F). The increase in hind limb grip strength was significant for all three treatments (Figure 2G). Effect of GW and AICAR on muscle weight and behavioral Since these drugs influenced body weight, we normal- activity measures ized data to body weight. Both forelimb and hind limb The average body mass of the treated mice was signifi- grip strength increased significantly with GW501516 cantly higher than that of vehicle-treated mice (Figure 2A). (+19%, +13%, respectively) and combination treatment Treatments increased the body mass by ~10% (GW, 9.2%; (+25%, +13%, respectively) (Figure 2F). Behavioral activ- AICAR, 11.3%; GW&AICAR, 10.63%). A general in- ity measures did not significantly change for the single crease in the weight of the EDL, gastrocnemius, quadri- treatments but the combination treatment group showed ceps, soleus, and TA muscles was found in the drug- significantly increased movement time (89%) (Figure 2I) Jahnke et al. Skeletal Muscle 2012, 2:16 Page 5 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 2 Effect of GW and AICAR on body mass, muscle mass, and behavioral activity. (A-D) Mass of vehicle control- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice: mass of the whole body (A), quadriceps (B), soleus (C), and abdominal fat (D) after euthanasia. Grip strength was measured using a grid at 12 and 16 weeks of age: (E) Maximal forelimb grip strength, (F) normalized maximal forelimb strength, (G) maximal hind-limb grip strength, (H) normalized maximal hind-limb strength of all the groups. The overall activity of the mice was measured using the open-field Digiscan apparatus at 12 and 16 weeks of age: (I) movement time and (J) rest time activity. Behavioral activity is presented as a percentage of the initial activity measured at 12 weeks of age before treatment. Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. and decreased rest time (Figure 2J), suggesting an overall (Figure 3H) (GW, +43%; AICAR, +29%; GW&AICAR, beneficial effect on these parameters. 26%) and SOL (Figure 3I) muscle (GW, +35%; AICAR, +26%; GW&AICAR, 13.7%) in response to drug treat- Effect of GW and AICAR on mitochondrial activity ment. Soleus muscles expressed more myosin heavy We evaluated the impact of these drugs on mitochondrial chain type I (Figure 3J), whereas only GW showed a sta- activity in muscle cells isolated from hind limb muscles of tistically significant increase in type IIA fibers dystrophin-deficient mdx mice. A significant increase in (Figure 3K). This increase in oxidative capacity was also mitochondrial mass, as indicated by NAO staining, was observed in EDL muscles, in which the ratio of the found in the GW501516-treated cells (Figure 3A,B). We height of the twitch force (P ) to the time to reach this saw no significant increase in either the AICAR- or maximal force (tpt) was increased in single-treated mice combination-treated groups. Mitochondrial ΔΨ,as assessed (GW, +18%; AICAR, +24% ) (Figure 3L). Finally, LDH by DiOC6 staining, was significantly increased in response activity in the TA was decreased in all three treatment to AICAR treatment (Figure 3C,D) but not GW501516 groups, but the decrease was only statistically significant or combination treatment. Gastrocnemius muscle from for GW-treated mice (−30%) (Figure 3M). treated mdx mice expressed significantly more PGC-1 α, cyt c mRNAs (Figure 3E,F) in comparison to vehicle Effect of GW and AICAR on satellite cell activation and treated group but increase in PDK4 mRNA did not muscle regeneration and degeneration reach statistical significance (Figure 3G). We also found We found that the number of dMHC-positive fibers was that NADH activity was significantly increased in EDL significantly decreased in the drug-treated groups Jahnke et al. Skeletal Muscle 2012, 2:16 Page 6 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 3 Effect of GW and AICAR on mitochondrial activity. Fluorescent-activated cell scanning (FACS) of primary myoblasts isolated from untreated and drug-treated mdx mice. Representative dot plot FACS overlay of mitochondrial mass with nonyl acridine orange (NAO) staining (A and B) and staining for mitochondrial activity with 3, 3’-dihexyloxacarbocyanine iodide (DiOC6) in myoblasts derived from the muscles of treated mice (C, D). Histograms show geometric mean fluorescence of NAO and DiOC6 in dystrophin-deficient myoblasts. Quantification of mRNA expression of PGC-1 α (E), Cyt c (F) and PDK-4 (G) relative to GAPDH mRNA expression by RTqPCR in gastrocnemius with n = 2 for each group. Quantification of NADH activity in histological sections of EDL (H) and soleus (I) muscles, with immunolabeling of the soleus muscle for Type I (J) and type IIA (K) fibers. (L) EDL twitch force parameters, ratio of the maximal twitch force (P ) to the time required to reach this force (tpt), and (M) lactate dehydrogenase activity of TA muscle. All experiments involved vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. (Figure 4A,B), and MyoD expression in isolated skeletal miRNA133 was also increased in the treated groups, but muscle cells was markedly decreased in the GW- and the differences did not reach statistical significance (data combination-treated groups (Figure 4C). Furthermore, not shown). Expression of FoXO1, which controls muscle the number of EDL fibers without central nucleation wasting, was decreased in AICAR (−34%) and combin- increased in the single-treated groups (Figure 4D). Im- ation drug-treated mice (−36%)(Figure 4F). Serum CK portantly, miRNA31a expression, known to be associated levels showed huge variations but no statistically signifi- with muscle regeneration/degeneration, was significantly cant changes (data not shown). Finally, IgM immunos- down-regulated in diaphragms of treated mice (GW, taining was significantly decreased in gastrocnemius -28%; AICAR, -63%; GW&AICAR, -67%) (Figure 4E). sections of treated muscle (GW, -48%; AICAR, -69%; Jahnke et al. Skeletal Muscle 2012, 2:16 Page 7 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 4 Effect of GW and AICAR on satellite cell activation, muscle regeneration, and degeneration. (A) Immunostaining of gastrocnemius muscle for developmental myosin (MyoD) heavy chain. (B) Histogram representing the area of regenerating fibers in gastrocnemius muscle. (C) Percentage of MyoD-positive cells isolated from muscles. (D) Fibers with no central nuclei. (E) MiRNA expression in diaphragm muscle (n = 4 for each group). (F) Western blotting for FoXO1 in EDL muscle lysate. Vinculin was used as an internal control for protein loading. The expression was normalized to that of vinculin and expressed as a percentage of the vehicle expression. (G) IgM-positive immunolabeling of muscle section to identify degenerated fibers. Data are means ± SE from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. *** P < 0.001 vs. vehicle-treated control mice. GW&AICAR, -54%) (Figure 4G). Overall, these data sug- progression and response therapy. We found that the gested a strong reduction in muscle degeneration. red-positive area was significantly decreased in the treated groups (GW501516, -25.6%; AICAR, -27.5%; Effect of GW and AICAR on diaphragm fibrosis, utrophin GW&AICAR, -27.2%) (Figure 5B). Evaluation of cytokine A expression, muscle cytokines, and inflammation expression in TA muscle lysate revealed that mdx mice Utrophin expression in skeletal muscle was studied be- had significantly increased IL-6 and IL-10 levels. Drug cause some of these improvements may have been due to treatment did not significantly affect IL-6 expression utrophin expression [17]. The level of utrophin A was (Figure 5C), but IL-10 levels were significantly decreased significantly increased in the treated groups over that in in GW&AICAR-treated mice (−45%) (Figure 5D), and untreated mice (GW, +112.97%,; AICAR, +84.97%; not in individual drug-treated mice. EDL muscle demon- GW&AICAR, +94.19%) (Figure 5A). We also measured strated a statistically significant decrease in inflammatory fibrosis in the diaphragm which occurs early in the dis- infiltrates in the GW group but not the other two groups ease and serves as a useful marker for assessing disease (Figure 5E). Jahnke et al. Skeletal Muscle 2012, 2:16 Page 8 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Figure 5 Effect of GW and AICAR on diaphragm fibrosis, utrophin A expression, muscle cytokines, and inflammation. (A) Western blotting for utrophin in EDL muscles from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. Vinculin was used as an internal control for protein loading. The expression was normalized to that of vinculin and expressed as a percentage of the vehicle expression. (B) Van Gilson staining of the diaphragms of mice treated with vehicle, GW501516, AICAR, or GW&AICAR. The percentage of fibrosis was then calculated by measuring the area of the fibrosis and the area of the whole section from vehicle- (n = 8), GW- (n = 6), AICAR- (n = 8), and GW&AICAR-treated (n = 6) mice. IL-6 (C) and IL-10 (D) analyses were performed by flow cytometry on EDL muscle lysate (n = 2), with 30,000 events per tube. WT muscle was used as a control for cytokine expression. (E) Inflammation was also analyzed by Histologic examination after H&E staining (1 inflammation = a cluster of 10 nuclei). Data are means ± SE. * P < 0.05 vs. vehicle-treated control mice. **P < 0.01 vs. vehicle-treated control mice. Discussion in mice [23]. Conversely, oxidative capacity activation In this study, we have demonstrated that dystrophic decreases muscle wasting in most cases [10]. Recently, muscle displays mitochondrial dysfunction similar to PPARδ have been demonstrated to be involved in satel- that in golden retriever muscular dystrophy [18]. Meta- lite cells proliferation and muscle regeneration [24]. bolic impairment has previously been reported [19], and Moreover, PGC-1α overexpression inhibits muscle atro- dystrophin-deficient myoblasts have been described as phy during fasting and denervation [3] and significantly having a pronounced respiratory impairment [20]. This improves dystrophic muscle [25]. Grumati et al. have deficiency is not the primary cause of muscle weakness demonstrated that correcting mitochondrial impairment in dystrophin deficiency; however, it may play a signifi- in collagen VI deficiency significantly improves muscle cant additional role that can be important for the time function [26]. Therefore, strategies that target mitochon- course of the disease. Defects in fatty acid oxidation drial up-regulation may be beneficial to dystrophic leads to the accumulation of fatty acylCoA and diacylgly- muscle. cerol, inducing insulin signaling disruption and causing In the present study, we have used two known muscle atrophy [21]. Similarly, alterations in mitochon- endurance-mimetic drugs, AICAR and GW501516, to drial functions caused by mtDNA mutations are activate endurance exercise-induced signaling pathways. involved in muscle loss during aging [22]. Mitochondrial AICAR is a mimetic of endurance training that activates fission and remodeling also contribute to muscle atrophy AMPK activity, an energy status sensor in the cell [27]. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 9 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 In normal mice, intraperitoneal injection of AICAR suggested by a marked decrease in FoXO1 and IgM ex- raises the level of PGC-1α expression and increases pression in fibers. Together, these results clearly indicate mitochondrial biogenesis in muscle [28], reducing that muscle degeneration is decreased in treated mice. muscle fatigability and increasing muscle performance FoXO1 transcription is lower in high-oxidative mouse [12]. GW501516 is a PPARβ/δ agonist, a transcription soleus than in low-oxidative gastrocnemius, TA, and factor that is co-activated by PGC-1α. Like AICAR, this quadriceps muscles [40]. In vivo, FoXO1 inhibits high drug is known to increase the amount of mitochondria oxidative fiber-related gene expression and oxidative and promote mitochondrial metabolism [29,30] and fatty metabolism-enhancing factor activity [41]. Skeletal mus- acid oxidation [31] in vivo and in vitro and has been cles of FoXO1-over-expressing mice had fewer type I tested as a therapeutic for type II diabetes [29,31,32]. fibers, as well as smaller type I and type II fibers [41]. More interestingly, the increase in muscle performance The phenotype of our treated mice became more oxida- that is stimulated by PPARδ/β activity is independent of tive, consistent with this change. A decrease in muscle exercise training in mice [33] and combination treat- degradation could explain the diminution in satellite cell ments with these drugs have been shown to have syner- activation that we observed. gistic beneficial effects in vivo in WT mice [12]. We also found a decrease in inflammation and fat tis- Our study clearly demonstrates that endurance sue in the treated mice. Increased IL-6 levels are mimetics improve muscle function and overall activity in involved in metabolic and structural changes in muscle dystrophic mice. The stimulation of mitochondrial bio- and in muscle loss during cachexia [42]. However, IL-6 genesis by GW501516 and/or AICAR that we have inhibition has significantly reversed skeletal muscle wast- observed is consistent with previous studies [31,33-35]. ing in rodents [42]. Our data suggest that also Miura et al. reported the use of GW501516 to slow the GW501516 and AICAR improve muscle function myogenic program and increase utrophin A expression through inflammation down-regulation. Adipose tissue in 5 weeks old mdx mice. More recently, Ljubicic et al plays a crucial endocrine role through the production of have reported that AICAR supplementation accompan- adipokines. Aberrant intracellular signaling cascades that ied with bout of exercise also improve muscle function regulate both inflammatory and immune processes are in 5 weeks old MDX mice. In our study, we have further known to contribute substantially to degeneration shown a decrease in LDH activity in TA muscles and in- [43,44]. Therefore, fat reduction is very interesting, since crease in NADH activity, together with an increase in it can reduce inflammation and have an impact on both type I/IIA fibers in soleus muscle, an increase of mRNA degeneration and regeneration. GW501516 has been expression of PGC-1α, cyt c and a trend for PDK-4 sug- shown to be involved in inflammatory pathway regula- gesting that the phenotype of treated muscle shifts from tion [45]. However, further experiments are needed to glycolytic to oxidative type. Recently, Selsby et al. found delineate the link between proinflammatory fat tissue that enhancing PGC-1α expression rescues dystrophic and muscle inflammation. muscle and that a switch from fast - to slow -twitch muscle is involved [25]. Moreover, utrophin expression increased, as in Miura et al., and could be part of the Conclusions process of improving muscle function. Evidence suggests In summary, this study demonstrates that the use of en- that utrophin is likely to compensate for the lack of dys- durance mimetics in mdx mice induces an improvement trophin in DMD muscle [17,36] and to decrease muscle in the structural integrity and reduces the degeneration/ pathology [37,38]. This suggests the possibility that the regeneration of mdx mouse muscle, probably through an increase of Utrophin A might partly restore the dys- increase in oxidative metabolism in the fibers. Our study trophin associated glycoprotein and help to improve and other recent work underline the high potential of muscle function. Slow-twitch fibers have been reported pharmacological activators of AMPK and PPARδ as part to have higher utrophin expression than do fast-twitch of rational drug treatments for muscular dystrophies. fibers. Therefore, the increase in utrophin expression with treatment could be a result of the change in fiber Abbreviations metabolism. The presence of fibers with no central nu- AMPK: 5' adenosine monophosphate-activated protein kinase; AICAR: 5- clei and the increase in peripheral nuclei suggest that de- aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, AICA-riboside; DIOC6: 3,3′-dihexyloxacarbocyanine iodide; DMD: Duchenne muscular generation/regeneration has been halted by this dystrophy; dMHC: Developmental myosin heavy chain; DMSO: Dimethyl therapeutic intervention. This evidence is further corro- sulphoxide; EDL: Extensor digitorum longus; Glut: Glutamine; LDH: Lactate borated by the concomitant down-regulation of activated deshydrogenase; mtDNA: mitochondrial Deoxyribonucleic acid; NADH: Nicotinamide adenine dinucleotide; NAO: 10-nonyl acridine orange; satellite cells and dMHC-positive regenerated fibers and P/S: Penicillin / Streptomycin; PGC-1α: Peroxisome proliferator-activated a decrease in miRNA-31, which are involved in muscle receptor gamma coactivator 1-alpha; PPARδ: Peroxisome proliferator- degeneration [39]. Stabilization of myofiber structure is activated receptor delta. Jahnke et al. Skeletal Muscle 2012, 2:16 Page 10 of 11 http://www.skeletalmusclejournal.com/content/2/1/16 Competing interests 9. Lin J, Wu H, Tarr PT, et al: Transcriptional co-activator PGC-1 alpha drives Dr. Nagaraju is one of the co-founders and member of the board of the formation of slow-twitch muscle fibers. Nature 2002, 418:797–801. directors of ReveraGen BioPharma Inc, a biopharmaceutical company 10. Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT: Increased engaged in the discovery and development of proprietary, small molecule muscle PGC-1alpha expression protects from sarcopenia and metabolic therapeutics for the treatment of neuromuscular diseases. This work was disease during aging. Proc Natl Acad Sci USA 2009, 106:20405–20410. funded by Department of Defense USAMRAA grant W81XWH-05-1-0616 11. Bushby K, Finkel R, Birnkrant DJ, et al: Diagnosis and management of (Mouse Drug Screening Core to K. Nagaraju), the Foundation to Eradicate Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological Duchenne, Inc., the Muscular Dystrophy Association, NIH grant R01- and psychosocial management. Lancet Neurol 2010, 9:77–93. AR050478 (K. Nagaraju) and Cristal Ball funding. 12. Narkar VA, Downes M, Yu RT, et al: AMPK and PPARdelta agonists are Dr. Hoffman is one of the co-founders and member of the board of directors exercise mimetics. Cell 2008, 134:405–415. of ReveraGen BioPharma Inc, a biopharmaceutical company engaged in the 13. Brooks SV, Faulkner JA: Contractile properties of skeletal muscles from discovery and development of proprietary, small molecule therapeutics for young, adult and aged mice. J Physiol 1988, 404:71–82. the treatment of neuromuscular diseases. This work was funded by NIH 14. Spurney CF, Gordish-Dressman H, Guerron AD, et al: Preclinical drug trials grant 1U54HD053177-01A1 (Wellstone Muscular Dystrophy Center to E.P. in the mdx mouse: assessment of reliable and sensitive outcome Hoffman). measures. Muscle Nerve 2009, 39:591–602. Dr JAHNKE, Dr Van Der Meulen, Mrs Johnston, Dr Ghimbovschi and Dr 15. Jahnke VE, Sabido O, Freyssenet D: Control of mitochondrial biogenesis, Partridge report no disclosures. ROS level, and cytosolic Ca2+ concentration during the cell cycle and the onset of differentiation in L6E9 myoblasts. Am J Physiol Cell Physiol 2009, 296:C1185–C1194. Authors’ contributions 16. Jahnke VE, Sabido O, Defour A, et al: Evidence for mitochondrial VEJ, PhD: designed research, conducted experiments, performed data respiratory deficiency in rat rhabdomyosarcoma cells. PLoS One 2010, analysis, and wrote the manuscript. JHM VD, PhD: conducted experiments, 5:e8637. performed data analysis. HKJ: conducted experiments. SG, PhD: conducted experiments, performed data analysis. TP, PhD: contributed to scientific 17. Perkins KJ, Davies KE: The role of utrophin in the potential therapy of discussion on the manuscript. EPH, PhD: provided reagents/lab facilities. KN, Duchenne muscular dystrophy. Neuromuscul Disord 2002, PhD: designed research, interpreted the data, provided reagents/lab facilities, 12(Suppl 1):S78–S89. wrote the manuscript. 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Skeletal MuscleSpringer Journals

Published: Aug 21, 2012

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