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TWEAK promotes exercise intolerance by decreasing skeletal muscle oxidative phosphorylation capacity

TWEAK promotes exercise intolerance by decreasing skeletal muscle oxidative phosphorylation capacity Background: Proinflammatory cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 are the major regulators of skeletal muscle mass in many catabolic conditions. However, their role in muscle metabolism remains largely unknown. In the present study, we investigated the role of TWEAK on exercise capacity and skeletal muscle mitochondrial content and oxidative metabolism. Methods: We employed wild-type and TWEAK-knockout (KO) mice and primary myotube cultures and performed biochemical, bioenergetics, and morphometric assays to evaluate the effects of TWEAK on exercise tolerance and muscle mitochondrial function and angiogenesis. Results: TWEAK-KO mice showed improved exercise tolerance compared to wild-type mice. Electron microscopy analysis showed that the abundance of subsarcolemmal and intermyofibrillar mitochondria is significantly increased in skeletal muscle of TWEAK-KO mice compared to wild-type mice. Furthermore, age-related loss in skeletal muscle oxidative capacity was rescued in TWEAK-KO mice. Expression of a key transcriptional regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and several other molecules involved in oxidative metabolism were significantly higher in skeletal muscle of TWEAK-KO mice. Moreover, treatment of primary myotubes with soluble TWEAK inhibited the expression of PGC-1α and mitochondrial genes and decreased mitochondrial respiratory capacity. Deletion of TWEAK also improved angiogenesis and transcript levels of vascular endothelial growth factor in skeletal muscle of mice. Conclusions: These results demonstrate that TWEAK decreases mitochondrial content and oxidative phosphorylation and inhibits angiogenesis in skeletal muscle. Neutralization of TWEAK is a potential approach for improving exercise capacity and oxidative metabolism in skeletal muscle. Keywords: Skeletal muscle, Exercise tolerance, TWEAK, Fn14, PGC-1α, PPARδ Background of new mitochondria and clearance of defunct mitochon- Skeletal muscle is the largest tissue of the human body dria are essential to meet cellular energy demand especially which ensures basic functions such as locomotion, me- during endurance exercise and to protect from many tabolism, and respiration. Skeletal muscle exhibits a high chronic conditions such as diabetes, heart failure, obesity, level of plasticity in response to physiological stressors. and aging [2,3]. Peroxisome proliferator-activated receptor For example, in response to exercise training, the pro- (PPAR)-γ coactivator 1α (PGC-1α) is a key player in portion of slow-type fibers and mitochondrial content regulating skeletal muscle fiber composition, mitochondrial within fibers is noticeably increased [1]. The biogenesis content, and oxidative metabolism, and maintenance of glucose, lipid, and energy homeostasis in response to physiological demands [4-6]. Transgenic mice overexpre- * Correspondence: ashok.kumar@louisville.edu ssing physiological levels of PGC-1α in skeletal muscle have Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, 500 South Preston Street, Louisville, KY 40202, USA increased mitochondrial biogenesis and oxidative capacity, Full list of author information is available at the end of the article © 2013 Sato 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. Sato et al. Skeletal Muscle 2013, 3:18 Page 2 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 are more resistant to fatigue and have improved aerobic tolerance and skeletal muscle bioenergetic function. performance [7-10]. Forced expression of PGC-1α also Our results show that, compared with wild-type mice, preserves skeletal muscle mass in various catabolic states TWEAK-KO mice have increased exercise tolerance, including denervation [11,12]. PGC-1α improves mito- which is associated with higher levels of skeletal muscle chondrial biogenesis by coactivating nuclear respiratory subsarcolemmal and intermyofibrillar mitochondria and factor (NRF)-1 [13], NRF-2 [14] and estrogen-related enhanced oxidative phosphorylation capacity. TWEAK receptor α [15-17] which, in turn, augments the expression represses the expression of PGC-1α and several other of hundreds of nuclear-coded mitochondrial genes [18]. molecules involved in mitochondrial biogenesis and Recent evidence also suggests a role for mitochondria in oxidative metabolism in vivo and in cultured myotubes. preventing muscle protein degradation in catabolic condi- Furthermore, vascularization and expression of vascular tions [19,20]. Thus, preserving mitochondria content and endothelial growth factor (VEGF) are increased in ske- function is essential to countering muscle wasting and to letal muscle of TWEAK-KO mice compared with wild- improving muscle homeostasis. type mice. These results support our hypothesis that Inflammatory cytokines such as tumor necrosis factor-α TWEAK causes exercise intolerance by suppressing (TNF-α) and interleukin-6 (IL-6) are some of the import- mitochondrial oxidative metabolism and angiogenesis. ant mediators of loss of skeletal muscle mass and function in many catabolic conditions [21-24]. TNF-α has been Methods reported to augment muscle protein degradation both Cell culture in vitro and in vivo [25-27]. Moreover, TNF receptor I Primary myoblasts were isolated from hind limb muscle knockout (KO) mice are protected from diet-induced of mice and cultured following the same protocol as pre- obesity due to increased thermogenesis [28]. Importantly, viously described [43]. Briefly, hind limb skeletal muscles TNF-α inhibits the expression of PGC-1α levels in from mice were aseptically isolated, minced into a coarse myotubes [29]. In animal models of cancer cachexia, mito- slurry, and enzymatically digested at 37°C for one hour chondrial content and biogenesis are reduced with in- by adding 200 IU/ml collagenase I (cat # LS004196; creased levels of IL-6 during the progression of the Worthington, Lakewood, NJ, USA) and 0.1% pronase condition whereas inhibition of IL-6 activity attenuates (EMD Chemicals, Billerica, MA, USA). The digested tumor-induced loss of skeletal muscle [30,31]. It is also slurry was filtered through a 70 μm filter and spun, and notable that while moderate exercise attenuates inflamma- isolated cells were resuspended and cultured initially in tion [32-34], acute exercise more robustly increases the F-10 medium (containing 20% fetal bovine serum (FBS) levels of inflammatory cytokines in obese compared with and supplemented with 10 ng/ml basic fibroblast growth lean subjects suggesting that rigorous exercise can further factor) and then in F-10 plus (Dulbecco’s) Modified aggravate the condition of overweight individuals [35]. Eagle’s Medium ((D)MEM) (1:1 ratio) based culture Despite these observations, cause-and-effect relationships medium supplemented with 15% FBS on culture dishes between different inflammatory mediators, exercise toler- coated with 10% matrigel (BD Biosciences, San Jose, CA, ance, and mitochondrial function in skeletal muscle have USA). Differentiation in primary myoblast cultures was not been clearly established using genetic mouse models. induced by replacing the growth medium with differenti- TNF-like weak inducer of apoptosis (TWEAK) is a major ation medium (2% horse serum in (D)MEM). proinflammatory cytokine of the TNF super family [36] that functions by binding to Fn14 receptors on target Animals cells [37-39]. TWEAK-Fn14 signaling mediates unique TWEAK-KO mice were provided by Dr. Avi Ashkenazi and context-dependent pleiotropic effects [40]. TWEAK (Genentech South, San Francisco, CA, USA) and have has been recently identified to be a key mediator that been previously described [40]. All the mice were in the causes skeletal muscle loss that occurs in response to C57BL/6 background, and their genotype was determined denervation, immobilization, and starvation [21,41,42]. by PCR from tail DNA. All animal procedures were Moreover, TWEAK-transgenic mice overexpressing phy- approved (protocol # 10129) by the Institutional Animal siological levels of TWEAK in skeletal muscle showed a Care and Use Committee and conformed to the American higher proportion of fast-type fibers (glycolytic) in soleus Physiological Society’s Guiding Principles in the Care and and extensor digitorum longus (EDL) muscle [41] indi- Use of Animals. cating that elevated levels of TWEAK cause a slow- to-fast type fiber switch. However, the role and mecha- Treadmill running protocol nisms by which TWEAK affects exercise capacity and Mice were subjected to treadmill running following bioenergetic function have not been studied. the same previously described protocol [44]. In brief, In the present study, we have used TWEAK-knockout 4.5-month-old wild-type and TWEAK-KO mice were (KO) mice to investigate the role of TWEAK in exercise matched for body weight and randomly assigned to Sato et al. Skeletal Muscle 2013, 3:18 Page 3 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 either a sedentary or exercise group. Mice were and 2.4 mM nitroblue tetrazolium (NBT, Sigma) at 37°C subjected to an acute bout of treadmill (Eco3/6 tread- in a humidity chamber for 45 minutes. The sections mill; Columbus Instruments, Columbus, OH, USA) run- were then washed in deionized water for three minutes, ning at 15 m/minute for 90 minutes. All mice in the dehydrated in 50% ethanol for two minutes, and mounted exercise group finished the 90-minute trial and were vis- for viewing with DPX mount medium (Electron Micros- ibly exhausted. Mice were sacrificed within 30 minutes copy Sciences, Hatfield, PA, USA). Digital photographs after exercise to study mitochondrial function. were taken from each section at 10X magnification under a Nikon Eclipse TE 2000-U microscope (Nikon, Melville, Exercise tolerance test NY, USA) with a Nikon digital camera (Digital Sight DS- The exercise tolerance test on mice was performed fol- Fi1), and fibers were quantified with imaging software lowing a method as previously described [45]. Briefly, all (Image J, NIH). At least 700 fibers were counted to deter- animals were run on a treadmill (Columbus Instru- mine SDH-positive fibers in each section in a blinded ments) at 10 m/minutes for five minutes at 0% degree fashion. The percentage of SDH stained fibers was then incline for acclimation for three days. On the exercise determined based on a criteria using integrated optical testing day, animals ran on the treadmill with a fixed density. slope of 10%. Mice first ran at 10 m/minute for five mi- nutes and the speed was increased by 2 m/minute every two minutes until they were exhausted or a maximal Measurement of mitochondrial bioenergetics speed of 46 m/minute was achieved. The criterion of Mitochondrial oxidative capacity was measured in isolated exhaustion was defined as the inability of the animal to mitochondria and in cultured myotubes using a Seahorse run on the treadmill for 10 seconds despite mechanical Bioscience XF24 extracellular flux analyzer (Billerica, MA, prodding. Running time and maximum speed achieved USA). For measurements in isolated mitochondria, tissue was measured whereas running distance, work and power from the limb muscle (approximately 50 mg) was isolated were calculated. within 30 minutes of exercise and homogenized in 1 ml of isolation buffer (220 mM mannitol, 70 mM sucrose, 5 mM Transmission electron microscopy 3-(N-morpholino)propanesulfonic acid (MOPS), 1 mM Soleus muscles from wild-type and TWEAK-KO mice ethylene glycol tetraacetic acid (EGTA), 0.3% fatty acid-free were fixed in 3% glutaraldehyde in 0.1 M cocodylate BSA, pH 7.2) using a Potter Elvehjem tube and a Teflon buffer overnight followed by fixing in 1% cocodylate- pestle. The homogenate was centrifuged at 500×g for five buffered osmium tetroxide. The tissue was dehydrated minutes at 4°C. The supernatant containing mitochondria through a series of graded alcohols, and embedded in was centrifuged at 10,000 × g for five minutes. After two LX-112 plastic (Ladd Research Industries, Williston, wash-centrifugation steps in BSA-free isolation buffer, VT, USA). Longitudinal sections (80 nm) were cut the mitochondria were suspended in respiration buffer using an ultramicrotome (LKB, Rockville, MD, USA)) (120 mM KCl, 25 mM sucrose, 10 mM 4-(2-hydroxyethyl)- and stained with uranium acetate and lead citrate. 1-piperazineethanesulfonic acid (HEPES), 1 mM MgCl ,5 Samples were analyzed using a transmission electron mM KH PO , pH to 7.2). Protein in the mitochondrial sus- 2 4 microscope (Philips CM 12; HZB, Berlin, Germany) pension was estimated using the Lowry DC assay (Biorad, operating at 60 kV. The pictures were captured at Hercules, CA, USA) and 5 to 12.5 μg of mitochondrial pro- 8,800x magnification using a 3.2 megapixel digital tein was sedimented in XF culture plates as described previ- camera (Sia-7C; Kodak, Rochester, NY, USA) at room ously [47]. Complex I-mediated, state 3 respiratory activity temperature. No imaging medium was used to was determined by measuring the oxygen consumption rate visualize the pictures, and images were stored as (OCR) after injection of pyruvate (5 mM), malate (2.5 mM) JPEG files. Image levels were equally adjusted using and ADP (1 mM). The OCR of mitochondria after exposure Photoshop CS2 software. to oligomycin (1 μg/ml) was used to estimate state 4 activ- ity; exposure to carbonyl cyanide p-trifluoromethoxyphe Succinate dehydrogenase (SDH) staining nylhydrazone (FCCP, 2 μM) was used to examine the SDH staining was performed as previously described uncoupled rate of respiration. Finally, succinate (10 mM) [46]. Briefly, transverse sections (8 μm) were cut from and rotenone (1 μM) were injected to assess maximal Com- the mid-belly of the TA muscles on a cryostat at −20°C plex II-mediated respiratory capacity. Data are expressed as and stored at −80°C until SDH staining was performed. pmol O /min/μg protein. For measurements in cultured The sections were dried at room temperature for 30 mi- cells, differentiated myoblasts were exposed to soluble nutes before incubation in a solution made up of 0.2 M TWEAK (R&D Systems, Minneapolis, MN, USA) for 72 phosphate buffer (pH 7.4), 0.1 M MgCl , 0.2 M succinic hours followed by the mitochondrial function assay acid (Sigma Chemical Company, St. Louis, MO, USA) outlined previously [48-51]. Sato et al. Skeletal Muscle 2013, 3:18 Page 4 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 RNA isolation and quantitative real time-PCR between control and treatment groups. A value P <0.05 RNA isolation and quantitative real time-PCR (qRT-PCR) was considered statistically significant. were performed as previously described [41]. Briefly, total RNA was isolated from homogenized mouse tissues using Results the TRIzol reagent (Invitrogen, Grand Island, NY, USA) Improvement in exercise tolerance in TWEAK-KO mice and an RNeasy Mini kit (QIAGEN, Valencia, CA, USA) We first studied the role of TWEAK in exercise tolerance according to the manufacturers’ protocols. First strand in mice. Male 4.5-month old wild-type and TWEAK-KO cDNA for PCR analyses were made using a reverse mice were acclimated for three days for treadmill running transcription system with 1 μgofpurified RNA usingoligo prior to the exercise tolerance test. After acclimation, the (dT) primer (Applied Biosystems, Grand Island, NY, USA) mice were run on a 10% slope until the mice were unable and the Omniscript reverse transcription kit (QIAGEN). to run on the treadmill for 10 seconds despite mechanical The quantification of mRNA expression was performed prodding. TWEAK-KO mice ran longer compared to using the SYBR green dye method on a sequence detec- wild-type mice (1,483 seconds for TWEAK-KOs versus tion system (Applied Biosystems, model 7300). Primers 1,170 seconds for the controls, Figure 1A). Since TWEAK- were designed using Vector NTI software (Invitrogen). KO mice could keep running on higher speed (30.6 m/ Primer sequences are available on request. The thermal minute for TWEAK-KOs versus 25.5 m/minute for the conditions consisted of an initial denaturation at 95°C for controls, Figure 1B), the difference in running distance 10 minutes, followed by 40 cycles of denaturation at 95°C became greater than 47% (463 meter for TWEAK-KOs for 15 seconds, annealing and extension at 60°C for one versus 314 meter for the controls, Figure 1C). Work and minute, and, for a final step, a melting curve of 95°C for power generated by TWEAK-KO mice were also higher 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds. compared to wild-type mice. TWEAK-KO mice generated All reactions were performed in duplicate to reduce vari- 22.2 J of work while control mice generated 15.2 J of work ation. Data normalization was accomplished using the (a difference of 46%) during the exercise test (Figure 1D). endogenous control (β-actin), and the normalized values In turn, TWEAK-KO mice exerted 14% more power than -ΔΔCt were subjected to a 2 formula to calculate the fold the controls (14.9 mW for TWEAK-KO versus 13.0 mW change between control and experimental groups. for the wild-type, Figure 1E) on the treadmill. These data established that TWEAK-KO mice have significantly im- proved exercise tolerance. Immunostaining for CD31 After cutting 8 μm thickness frozen sections of TA Ablation of TWEAK improves mitochondrial content in muscles, the sections were fixed by cold acetone for 10 skeletal muscle of mice minutes and dried in air for 30 minutes. The tissues We have previously shown that deletion of TWEAK in- were rinsed with phosphate buffered saline (PBS) twice, creases the proportion of type I fibers in soleus and EDL blocked in 2% BSA solution for one hour at room muscles of mice [41]. In this study, we investigated temperature followed by incubation with primary anti- whether TWEAK affects mitochondrial content in skel- bodies in 2% BSA solution (laminin, anti-rabbit 1:300, etal muscle of mice. The soleus muscle of 4.5-month old Sigma and CD31, anti-mouse 1:30, BD Biosciences, San TWEAK-KO and wild-type mice were isolated and used Jose, CA, USA) at 4°C in a humidity chamber overnight. to measure mitochondrial content by performing trans- The next day, the sections were rinsed with PBS for five mission electron microscopy. As shown in Figure 2A, minutes three times and incubated with secondary anti- the abundance and size of mitochondria was found to be body (Alexa Fluor-conjugated 488 anti-rabbit, 1:2500 increased in TWEAK-KO compared to wild-type mice. and 546 anti-mouse, 1:2500, Invitrogen) in 2% BSA solu- Quantitative analysis of mitochondria in electron micro- tion for one hour at room temperature. After washing graphs showed that the levels of subsarcolemmal and twice with PBS and once with deionized water, the intermyofibrillar mitochondria were increased by 42% sections were mounted with DPX mounting medium. and 32%, respectively, in TWEAK-KO mice compared to Digital photographs were taken from each section under wild-type mice (Figure 2B and Figure 2C). Because a Nikon Eclipse TE 2000-U microscope (Nikon) with a gastrocnemius (GA) muscle contains a mixture of slow and Nikon digital camera (Digital Sight DS-Fi1). Number of fast twitch fibers and greatly influences running capacity on CD31-postive vessels per myofiber was quantified using the treadmill, we measured mRNA levels of fiber type- NIS Elements BR 3.00 software (Nikon). specific myosin heavy chain (MyHC) isoforms in GA muscle of wild-type and TWEAK-KO mice. As shown in Statistical analysis Figure 2D, mRNA levels of MyHC I and MyHC IIA were The results are presented as means ± standard deviation increased by 2- and 2.7-fold, respectively in TWEAK-KO (SD). Student’s t-test was used to compare the difference mice compared to wild-type mice. These results further Sato et al. Skeletal Muscle 2013, 3:18 Page 5 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Wild-type; TWEAK-KO A. B. C. 600 200 D. E. Figure 1 Treadmill exercise tolerance test. After acclimatization, mice were run on a treadmill with a 10% slope and increasing speed to exhaustion. Maximum speed and running time were monitored, and distance, work, and power were calculated based on the individual performance. (A) Running time; (B) Speed; (C) Distance; (D) Work; and (E) Power. Data are represented as mean ± SD. N = 6 in each group. *P <0.05; values vary significantly between wild-type and TWEAK-KO mice. KO, knockout. suggest that genetic ablation of TWEAK increases the muscle, we investigated whether TWEAK modulates the slow-type fiber phenotype in skeletal muscle of mice. expression of PGC-1α, a critical regulator of the mito- SDH, also known as complex II in the mitochondrial chondrial biogenetic program in skeletal muscle [9,10]. respiratory chain, is a marker of oxidative capacity of skel- Interestingly, the mRNA levels of PGC-1α were found to be etal muscle at the fiber level. Therefore, we performed significantly higher in 4.5-month-old TWEAK-KO mice SDH staining on TA muscle of 4.5-month- and 12-month- compared to age-matched wild-type mice (Figure 4). old wild-type mice and TWEAK-KO mice and quantified Similarly, mRNA levels of PPARδ were significantly higher the percentage of SDH-positive fibers. Compared to wild- in skeletal muscle of TWEAK-KO mice compared to corre- type mice, fibers in TWEAK-KO mice stained more darkly sponding wild-type mice (Figure 4). In contrast, mRNA for SDH (Figure 3A). Furthermore, there was a significant levels of glycolytic enzymes such as hexokinase-2 (HK II) increase in the number of SDH-positive fibers in TWEAK- and phosphoglycerate mutase 2 (PGAM2) were reduced in KO mice compared to wild-type mice at the age of 4.5 skeletal muscle of TWEAK-KO mice compared to wild- months (Figure 3B). Likewise, 12-month-old TWEAK-KO type mice. No significant change was observed in the had more SDH-positive fibers compared to age-matched expression of pyruvate dehydrogenase kinase-4 (PDK4). wild-type mice (Figure 3A and Figure 3C). Taken together, Furthermore, mRNA levels of mitochondrial carnitine these results suggest that ablation of TWEAK in mice is palmitoyltransferase I (mCPT1), which is required for the sufficient to improve mitochondrial content and oxidative transport of long-chain fatty acyl-CoAs from the cyto- capacity of skeletal muscle. plasm into the mitochondrion, were significantly ele- vated in TWEAK-KO mice compared to wild-type mice (Figure 4). These results suggest that a deficiency of TWEAK-KO mice demonstrate increased expression of TWEAK reduces expression of glycolytic genes and PGC-1α and metabolic genes in skeletal muscle augments the expression of genes involved in oxidative To understand the mechanisms by which TWEAK affects metabolism in skeletal muscle. exercise capacity and mitochondrial content in skeletal Running time (seconds) Work (joule) -3 Power (watts X 10 ) Speed (meter/minute) Distance (meters) Sato et al. Skeletal Muscle 2013, 3:18 Page 6 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Figure 2 Transmission electron microscopy (TEM) analysis for mitochondria morphology and content and expression of muscle fiber type-specific genes in TWEAK-KO mice. Soleus muscle of six-month-old wild-type and TWEAK-KO mice were isolated and longitudinal sections were processed for TEM analysis. (A) Representative images of longitudinal soleus muscle are shown. The abundance and the size of mitochondria were increased in TWEAK-KO mice compared to wild-type mice. Arrows point to mitochondria in muscle sections. Circles are used to show representative enlarged mitochondria. Stars (‘*’) point to subsarcolemmal space in longitudinal sections. Scale bar: 1 μm. (B) Quantification of the number of subsarcolemmal mitochondria in soleus muscle of wild-type and TWEAK-KO mice. (C) Quantification of the number of intermyofibrillar mitochondria in soleus muscle sections of wild-type and TWEAK-KO mice. (D) Relative mRNA levels of MyHC type I, IIA and IIB in gastrocnemius muscle of 4.5-month-old wild-type and TWEAK-KO mice. Data are represented as mean ± SD. N = 3 in each group. *P <0.05; values vary significantly from wild-type mice. KO, knockout; TWEAK, TNF-like weak inducer of apoptosis. Ablation of TWEAK increases state 3 respiration in induced by addition of pyruvate, malate and ADP; state 4 skeletal muscle mitochondria respiration was induced by addition of oligomycin; FCCP We next sought to determine whether TWEAK regulates was added to determine maximal complex I-mediated mitochondrial function in skeletal muscle. After an acute respiratory activity; and, lastly, succinate and rotenone bout of treadmill running for 90 minutes, mitochondria were added to determine maximal complex II-mediated were isolated from hind limb muscles of wild type and respiratory activity. As shown in Figure 5A and 5B, mito- TWEAK-KO mice and mitochondrial function was exam- chondria from TWEAK-KO mice showed significantly in- ined by extracellular flux (XF) analysis. The sequential creased state 3 respiratory activities compared with wild addition of respiratory substrates and inhibitors of oxi- type mice. Mitochondrial coupling (as determined by dative phosphorylation were used to determine changes in calculating respiratory control ratios) and other indices of electron transport chain activity: state 3 respiration was mitochondrial function were not significantly different Sato et al. Skeletal Muscle 2013, 3:18 Page 7 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 A. Wild-type TWEAK-KO B. C. 50 50 4.5-month 1-year 40 40 Wild-type TWEAK-KO Wild-type TWEAK-KO Figure 3 Succinate dehydrogenase (SDH) staining analysis for oxidative capacity in TA muscle of TWEAK-KO mice. Frozen transverse TA muscle sections from 4.5- or 12-month old wild-type and TWEAK-KO mice were used to stain for SDH. (A) Representative SDH-stained images are presented here. TA muscle of TWEAK-KO showed relatively dark staining for SDH compared to wild-type mice. Scale bars: 50 μm. Quantification of SDH-positive fibers in TA muscle of (B) 4.5-month, and (C) 12-month old wild-type and TWEAK-KO mice. Data are presented as mean ± SD. *P <0.05; values vary significantly from wild-type mice. KO, knockout; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis. 2.0 Wild-type; TWEAK-KO * * 1.5 1.0 0.5 PGC-1α PPARδ HK II PGAM2 PDK4 mCPT1 Figure 4 qRT-PCR analysis of transcript levels of metabolic genes in skeletal muscle of wild-type and TWEAK-KO mice. TA muscle from 4.5-month-old wild-type and TWEAK-KO mice were isolated and processed to measure mRNA levels of PGC-1α, PPARδ, HK II, PGAM2, PDK4, and mCPT1. Data are represented as mean ± SD. N = 4 in each group. *P <0.05. values vary significantly from wild-type mice. HK II, hexokinase-2; KO, knockout; mCPT1, mitochondrial carnitine palmitoyltransferase 1; PDK4, pyruvate dehydrogenase kinase-4; PGAM2, phosphoglycerate mutase 2; PGC-1α, PPAR coactivator 1α; PPAR, peroxisome proliferator-activated receptor; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis. SDH-positive fibers Frequency (%) 1-year 4.5-month SDH-positive fibers Frequency (%) Relative mRNA levels Sato et al. Skeletal Muscle 2013, 3:18 Page 8 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 B. A. Wild-type; TWEAK-KO mice Wild-type; TWEAK-KO mice 60 60 Succ/Rot FCCP Oligo Pyr/Mal/ADP State 3 State 4 Complex I Complex II 05 10 15 20 Maximum Maximum Time (min) Figure 5 Mitochondrial respiration is enhanced in TWEAK-KO mice. Respiratory activity of mitochondria isolated from wild-type and TWEAK-KO mice subjected to an acute bout of treadmill running: (A) Extracellular flux assay of isolated mitochondria: mitochondrial activity was measured in the absence of substrate (state 1 respiration) or in the presence of pyruvate (5 mM), malate (2.5 mM) and ADP (1 mM) (state 3 respiration). State 4 respiration was estimated by measuring the oxygen consumption rate (OCR) after the addition of oligomycin (1 μg/ml). FCCP (4 μM) was added to assess maximal activity of mitochondria respiring on substrates providing electrons for complex I (that is, pyruvate and malate) or complex II (that is, succinate in the presence of rotenone; 10 mM and 1 μM, respectively). (B) Group data derived from panel A. N = 3 mice per group. *P <0.05 versus wild-type. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; KO, knockout; TWEAK, TNF-like weak inducer of apoptosis. between wild type and KO mice. These data suggest that assess the effects of TWEAK on mitochondrial function absence of TWEAK augments mitochondrial oxidative directly, we exposed differentiated myotubes to TWEAK capacity in skeletal muscle in exercised mice. and then assessed mitochondrial function as described previously [48-51]. As shown in Figure 7A and 7B, ex- posure of myotubes to TWEAK for 72 hours did not TWEAK represses the expression of PGC-1α and genes affect the basal mitochondrial OCR, nor did it affect regulating oxidative metabolism in cultured primary ATP-linked OCR or proton leak. However, the FCCP- myotubes stimulated OCR, indicative of maximal respiratory cap- We next studied whether TWEAK affects the expression acity, was remarkably diminished by TWEAK treatment. of PGC-1α and other genes in cultured primary myotubes. In addition, a modest, yet significant, increase in the Primary myoblasts prepared from wild-type mice were dif- non-mitochondrial OCR occurred in myotubes exposed ferentiated into myotubes by incubation in differentiation to100 ng/ml TWEAK, which may be due to increased medium for 96 hours followed by treatment with TWEAK production of reactive oxygen species. The diminishment for 72 hours. As shown in Figure 6, addition of soluble in mitochondrial respiratory capacity caused by TWEAK TWEAK dramatically reduced the expression of PGC-1α was associated with an increase in the extracellular acid- in myotubes. Treatment with TWEAK also significantly ification rate (ECAR), a surrogate measure of glycolysis reduced the mRNA levels of several mitochondrial genes (Figure 7C). Collectively, these results suggest that encoding proteins related to oxidative metabolism, such TWEAK directly modulates the bioenergetic capacity of as ATP synthase subunit beta (ATP5b), cytochrome c oxi- skeletal muscle. dase subunit I (Cox I), Cox subunit IV (COX IV), COX - 7b, cytochrome c (Cyt c), medium-chain acyl-coenzyme A Ablation of TWEAK improves angiogenesis in skeletal dehydrogenase (MCAD), PPARδ, and mCPT1 (Figure 6). muscle of mice These results further suggest that TWEAK inhibits the There is a causal relationship between PGC-1α and mitochondrial biogenetic program in skeletal muscle. angiogenesis. Skeletal muscle-specific overexpression of PGC-1α in mice results in increased angiogenesis, which TWEAK decreases maximal respiratory capacity and may contribute to enhanced exercise performance [52]. increases glycolysis in cultured primary myotubes Since TWEAK suppresses PGC-α in skeletal muscle, we Our studies in isolated mitochondria derived from ske- next sought to determine whether TWEAK also regu- letal muscle after exercise suggest that the absence of lates angiogenesis in skeletal muscle of mice. TA muscle TWEAK increases mitochondrial oxidative capacity. To of 4.5-month-old wild-type and TWEAK-KO mice OCR (pmol O /min/ug protein) OCR (pmol O /min/ug protein) 2 Sato et al. Skeletal Muscle 2013, 3:18 Page 9 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Control 10 ng/ml TWEAK 1.4 100 ng/ml TWEAK 1.2 1.0 * * * * 0.8 0.6 0.4 * 0.2 Figure 6 Effect of TWEAK on expression of PGC-1α and mitochondria oxidative metabolism genes in cultured primary myotubes. Primary myotubes prepared from wild-type mice were treated with soluble TWEAK protein (10 ng/ml or 100 ng/ml) for 72 hours followed by RNA isolation and performing qRT-PCR to study transcript levels of PGC-1α, Tfam, Tfam2m, Cyt-c, Cox I, Cox IV, Cox 7b, and MCAD. Data are presented as mean ± SD. *P <0.05; values vary significantly from untreated myotubes. Cox, cytochroms c oxidase; Cyt c, cytochrome c; MCAD, medium-chain acyl-coenzyme A dehydrogenase; PGC-1α, PPAR coactivator 1α; PPAR, peroxisome proliferator-activated receptor; Tfam, transcription factor A mitochondrial; TWEAK, TNF-like weak inducer of apoptosis. was isolated and immunostained with antibodies against intermyofibrillar mitochondria, increased SDH-positive CD31 (also known as platelet endothelial cell adhesion myofibers, and elevated expression of metabolic genes molecule-1), an endothelial-specific marker for capilla- such as PGC-1α,PPARδ, and mCPT-1 compared to wild- ries. Laminin staining was used to mark the periphery type mice (Figures 2, 3, and 4). Oxidative phosphorylation of fibers. As shown in Figure 8A, the number of CD31- is also increased in exercised TWEAK-KO mice compared positive capillaries was considerably increased in TWEAK- with wild-type mice (Figure 5), and treatment of myotubes KO mice compared with wild-type mice. The capillary- with TWEAK directly decreases mitochondrial biogenetic to-fiber ratio was increased by 29% in TWEAK-KO mice capacity and maximal respiratory activity (Figures 6 and (Figure 8B). Since VEGF positively regulates angiogenesis, 7). Moreover, vascularization and expression of VEGF in we also measured mRNA levels of VEGF in TA muscle of skeletal muscle are enhanced in TWEAK-KO mice com- wild-type and TWEAK-KO mice. As shown in Figure 8C, pared to their controls (Figure 8). These data suggest that mRNA levels of VEGF were significantly higher in TA TWEAK directly regulates the mitochondrial biogenetic muscle of TWEAK-KO mice compared to wild-type mice. program and that loss of TWEAK improves mitochondrial Furthermore, treatment with TWEAK also reduced the respiratory capacity and increases vascularization of ske- mRNA levels of VEGF in primary myotubes (Figure 8D). letal muscle tissue, which collectively lead to improved These results suggest that ablation of TWEAK stimulates exercise performance. angiogenesis in skeletal muscle of mice potentially through One of the potential mechanisms by which TWEAK increasing the expression of VEGF. might be attenuating exercise capacity is through diminishing the levels of PGC-1α in skeletal muscle. Discussion PGC-1α augments mitochondrial biogenesis, oxidative Exercise capacity in mammals is determined by multiple metabolism, and promotes fast-to-slow type fiber tran- factors including skeletal muscle oxidative metabolism sition [9,13,17,54-56]. Overexpression of PGC-1α in rat and vascularization. Our previous microarray study sug- primary culture cells leads to increased abundance of gested that TWEAK can modulate the expression of sev- slow oxidative-associated MyHC isoform [57]. Skeletal eral genes whose products are involved in mitochondrial muscle-specific PGC-1α KO mice demonstrate reduced dysfunction and fatty acid metabolism [53]. Our present endurance capacity with a shift from oxidative fiber type study demonstrates that compared to wild-type mice, to glycolytic fibers and increased levels of TNF-α in TWEAK-KO mice run longer and with higher speed skeletal muscle [45,58]. By contrast, muscle-specific during an exercise tolerance test (Figure 1). TWEAK- overexpression of PGC-1α enhances exercise perfor- KO mice show augmented levels of subsarcolemmal and mance with increased fatty acid oxidation and decreased Relative mRNA levels PGC-1α α ATP5b Tfam Tfb2m Cyt-c COX-I COX-IV COX-7b MCAD PPARδ mCPT1 Sato et al. Skeletal Muscle 2013, 3:18 Page 10 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 C. A. 70 O F Control Control 10 ng/ml TWEAK 10 ng/ml TWEAK 100 ng/ml TWEAK 100 ng/ml TWEAK 50 1.0 0.8 0.6 0.4 010 20 30 40 50 Time (min) 0.2 B. Control 10 ng/ml TWEAK 100 ng/ml TWEAK * * Basal ATP-linked Proton Maximum Reserve Non mito- mito OCR OCR Leak Capacity Capacity chondrial Figure 7 TWEAK regulates mitochondrial oxidative capacity and glycolytic flux. Extracellular flux analysis of primary differentiated myotubes treated with 0, 10, or 100 ng/ml TWEAK for 72 hours. (A) Mitochondrial function assay: after three baseline measurements, inhibitors or activators of electron transport were added sequentially to intact myotubes. Oxygen consumption rate (OCR) measurements were recorded after each exposure. O, oligomycin; F, FCCP; and A, antimycin A/rotenone. The OCR was normalized to total protein in each well. (B) Indices of mitochondrial function calculated from assay results in panel A. (C) Extracellular acidification rates (ECAR) of cells treated without or with TWEAK; ECAR is a surrogate measure of lactate and therefore is used as a measure of glycolytic flux. N = 3 per group; *P <0.05 versus control. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TWEAK, TNF-like weak inducer of apoptosis. glycogen usage during exercise [8,52]. These findings other molecules involved in mitochondrial oxidative suggest that PGC-1α is not only essential but sufficient phosphorylation in cardiomyocytes and forced expres- to determining skeletal muscle fiber composition. Our sion of PGC-1α attenuates TWEAK-induced cardiac experiments demonstrate that mRNA levels of PGC-1α dysfunction in mice [59]. are increased in skeletal muscle of TWEAK-KO mice The PPARs are ligand-modulated transcription factors with a concomitant increase in mitochondrial content in which three subtypes have been identified: α, β/δ, and and expression of genes whose products are involved in γ [60]. Previous studies have shown that their endogen- oxidative capacity (Figures 2 and 4). Furthermore, treat- ous ligands are composed of fatty acids and lipid me- ment of primary myotubes with TWEAK drastically re- tabolites and, therefore, certain PPARs mediate the duced levels of PGC-1α and other mitochondrial genes expression of genes whose products are involved in the (Figure 6) further suggesting that TWEAK represses the regulation of fatty acid metabolism in response to expression of PGC-1α leading to reduced mitochondrial changes in systemic fuel availability [60-62]. In skeletal content. These findings are consistent with functional muscle, levels of PPARδ are relatively higher compared analyses of mitochondrial function: ablation of TWEAK to PPARα or PPARγ [63]. Treatment with synthetic resulted in increased oxidative phosphorylation capacity PPARδ agonist or overexpression of PPARδ by retroviral in isolated mitochondria (Figure 5) and treatment of infection induces the levels of molecules which are in- myotubes with TWEAK decreased maximal respiratory volved in lipid metabolism and fatty acid oxidation, capacity (Figure 7). Although not tested in this study, it whereas overexpression of a dominant-negative PPARδ is possible that TWEAK also reduces the levels of PGC- mutant exerts opposite effects in C2C12 myotubes [64]. 1α in other organs which ultimately results in reduced Furthermore, it has been shown that muscle-specific exercise capacity. Indeed, Shi et al. recently reported overexpression of PPARδ in mice increases exercise that TWEAK represses the expression of PGC-1α and tolerance with a switch to increased number of type I OCR OCR (pmol O 2 /min/ug protein) (pmol O /min/ug protein) ECAR (mpH unit/min/μ μg protein) Sato et al. Skeletal Muscle 2013, 3:18 Page 11 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 A. B. D. C. 1.8 1.8 Control 1.8 1.6 1.6 1.6 10 ng/ml TWEAK 1.4 1.4 1.4 100 ng/ml TWEAK 1.2 1.2 1.2 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Wild-type Wild-type TWEAK-KO TWEAK-KO Figure 8 Analysis of capillary density in skeletal muscle of wild-type and TWEAK-KO mice. Transverse sections of TA muscle prepared from 4.5-month-old wild-type and TWEAK-KO mice were immunostained for CD31 (red) and counterstained for laminin (green). (A) Representative CD31- and laminin-immunostained and merged images are presented here. Scale bars: 50 μm. (B) Quantification of CD31-postive capillaries per myofiber in TA muscle. N = 8 in each group. (C) Relative mRNA levels of VEGF in TA muscle of wild-type and TWEAK-KO mice (N = 4 in each group) measured by qRT-PCR analysis. (D) Primary myotubes were treated with the indicated concentration of TWEAK for 24 hours followed by measurement of mRNA levels of VEGF by qRT-PCR. Relative mRNA levels of VEGF are shown here. Data are presented as mean ± SD. *P <0.05; values vary significantly from untreated myotubes. KO, knockout; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor. muscle fibers and up-regulation of molecules related to might be responsible for improvement in exercise capacity fatty acid metabolism. In contrast, PPARδ-null mice in TWEAK-KO mice. Data from cell culture experiments show decreased exercise performance compared to wild- further support this possibility, as treatment of myotubes type mice [65,66]. Although muscle relies mainly on fat with TWEAK resulted in diminished maximal respiratory and carbohydrate as energy resources, enhanced fatty capacity and increased glycolytic flux (Figure 7). acid utilization during exercise with glycogen sparing re- Although the mechanisms by which TWEAK reduces sults in improved exercise endurance capacity [67-69]. levels of PGC-1α in skeletal muscle remain unknown, a Haramizu et al. demonstrated that the expression levels recent study has shown that TWEAK increases membrane of gene products related to lipid metabolism in the translocation of adaptor protein TNF receptor associated muscle is correlated with the levels of fatty acid β- factor 2 (TRAF2), in an Fn14 dependent manner, in car- oxidation activity as well as exercise strength [70]. Our diomyocytes [59]. Furthermore, TWEAK treatment in- results demonstrate that transcript levels of PPARδ and creases the activation of canonical nuclear factor-kappaB mCPT1 are significantly increased while the expression (NF-κB) signaling in both skeletal muscle [41] and cardiac of molecules that are associated with glycolysis such as myocytes [59]. Knockdown of TRAF2 using small hair- HK II and PGAM2 is suppressed in skeletal muscle of pin RNA (shRNA) or selective blockade of IκBkinase-β TWEAK-KO mice compared to wild-type mice (Figure 6). (IKKβ, an upstream activator of canonical NF-κBsignal- These results suggest that enhanced fatty acid oxidation ing) prevented the TWEAK-mediated suppression of Merged Laminin CD31 Capillary to fiber ratio TWEAK-KO Wild-type Relative VEGF mRNA levels (Fold change) Relative VEGF mRNA levels (Fold change) Sato et al. Skeletal Muscle 2013, 3:18 Page 12 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 PGC-1α in cardiomyocytes suggesting that TWEAK re- inhibition of TWEAK activity using neutralizing antibodies pression of PGC-1α requires Fn14-TRAF2-IKKβ-NF-κB or pharmacological compounds could improve muscle signaling cascade [59]. Previously, the effects of TNF-α function and exercise capacity in patients with meta- on the mRNA expression of PPARδ and its target genes bolic disorders. have been investigated. Treatment with TNF-α reduces Abbreviations PPARδ-target genes, such as mCPT1 and PGC-1α, ATP5b: ATP synthase subunit beta; BSA: Bovine serum albumin; Cox: Cytochrome whereas addition of PPARδ agonist rescues this reduc- c oxidase; FCCP: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GA: Gastrocnemius; HK II: Hexokinase II; KO: Knockout; MCAD: Medium-chain tion in adipocytes [71,72]. Moreover, TNF-α impairs acyl-coenzyme A dehydrogenase; mCPT1: Mitochondrial carnitine mitochondrial biogenesis and function in skeletal palmitoyltransferase I; MyHC: Myosin heavy chain; NF-κB: Nuclear factor-kappaB; muscle [73]. While TWEAK can induce the expression OCR: Oxygen consumption rate; PCR: Polymerase chain reaction; PDK4: Pyruvate dehydrogenase kinase 4; PGAM2: Phosphoglycerate mutase 2; PGC-1α:PPAR of molecules associated with the autophagy-lysosomal coactivator 1α; PPAR: Peroxisome proliferator-activated receptor; qRT- system indicating it causes mitochondria dysfunction PCR: Quantitative real-time PCR; SDH: Succinate dehydrogenase; shRNA: Small [74], muscle-specific ablation of TRAF6, which is in- hairpin RNA; TA: Tibial anterior; TNF: Tumor necrosis factor; TRAF: TNF receptor- associated factor; TWEAK: TNF-like weak inducer of apoptosis; VEGF: Vascular volved in TWEAK signaling, suppresses the activation endothelial growth factor. of autophagy in response to denervation and cancer cachexia [75]. These results suggest that TWEAK is in- Competing interests The authors declare they have no competing interests. volved in reducing PPARδ and PGC-1α levels and their target genes leading to mitochondrial dysfunction and Authors’ contributions content. Since no change of PPARδ expression was AK and BGH conceived and designed the study. SS, YO, VM, JS, BGH and SB observed in skeletal muscle of PGC-1α KO mice or the performed experiments and analyzed the data. SS, BGH and AK wrote the manuscript. All authors read and approved the final manuscript. muscle-specific PGC-1α overexpressing transgenic mice [66], PGC-1α is not an upstream regulator for PPARδ. Acknowledgements Indeed, PGC-1α has a synergistic effect with PPARδ We are grateful to Dr. Avi Ashkenazi (Genentech South San Francisco, CA) for providing TWEAK-KO mice. This work was supported by NIH grants agonist to induce the expression of oxidative metabolic R01AR059810 and RO1AG029623 to AK. genes such as mCPT1 and PDK4 [76].Furthermore,it has been shown that PGC-1α directly coactivates the Author details Department of Anatomical Sciences and Neurobiology, University of Louisville mCPT1 and PDK4 promoter via PPARδ in a ligand- School of Medicine, 500 South Preston Street, Louisville, KY 40202, USA. dependent manner [76,77]. Therefore, it is likely that Diabetes and Obesity Center, Institute of Molecular Cardiology, and PGC-1α is a coactivator of PPARδ in terms of up- Department of Medicine, University of Louisville, Louisville, KY 40202, USA. Present address: Gastroenterology Division, University of Pittsburgh School of regulating these metabolic genes. Medicine, Pittsburgh, PA 15261, USA. 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TWEAK promotes exercise intolerance by decreasing skeletal muscle oxidative phosphorylation capacity

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Springer Journals
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Copyright © 2013 by Sato 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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10.1186/2044-5040-3-18
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23835416
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Abstract

Background: Proinflammatory cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 are the major regulators of skeletal muscle mass in many catabolic conditions. However, their role in muscle metabolism remains largely unknown. In the present study, we investigated the role of TWEAK on exercise capacity and skeletal muscle mitochondrial content and oxidative metabolism. Methods: We employed wild-type and TWEAK-knockout (KO) mice and primary myotube cultures and performed biochemical, bioenergetics, and morphometric assays to evaluate the effects of TWEAK on exercise tolerance and muscle mitochondrial function and angiogenesis. Results: TWEAK-KO mice showed improved exercise tolerance compared to wild-type mice. Electron microscopy analysis showed that the abundance of subsarcolemmal and intermyofibrillar mitochondria is significantly increased in skeletal muscle of TWEAK-KO mice compared to wild-type mice. Furthermore, age-related loss in skeletal muscle oxidative capacity was rescued in TWEAK-KO mice. Expression of a key transcriptional regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and several other molecules involved in oxidative metabolism were significantly higher in skeletal muscle of TWEAK-KO mice. Moreover, treatment of primary myotubes with soluble TWEAK inhibited the expression of PGC-1α and mitochondrial genes and decreased mitochondrial respiratory capacity. Deletion of TWEAK also improved angiogenesis and transcript levels of vascular endothelial growth factor in skeletal muscle of mice. Conclusions: These results demonstrate that TWEAK decreases mitochondrial content and oxidative phosphorylation and inhibits angiogenesis in skeletal muscle. Neutralization of TWEAK is a potential approach for improving exercise capacity and oxidative metabolism in skeletal muscle. Keywords: Skeletal muscle, Exercise tolerance, TWEAK, Fn14, PGC-1α, PPARδ Background of new mitochondria and clearance of defunct mitochon- Skeletal muscle is the largest tissue of the human body dria are essential to meet cellular energy demand especially which ensures basic functions such as locomotion, me- during endurance exercise and to protect from many tabolism, and respiration. Skeletal muscle exhibits a high chronic conditions such as diabetes, heart failure, obesity, level of plasticity in response to physiological stressors. and aging [2,3]. Peroxisome proliferator-activated receptor For example, in response to exercise training, the pro- (PPAR)-γ coactivator 1α (PGC-1α) is a key player in portion of slow-type fibers and mitochondrial content regulating skeletal muscle fiber composition, mitochondrial within fibers is noticeably increased [1]. The biogenesis content, and oxidative metabolism, and maintenance of glucose, lipid, and energy homeostasis in response to physiological demands [4-6]. Transgenic mice overexpre- * Correspondence: ashok.kumar@louisville.edu ssing physiological levels of PGC-1α in skeletal muscle have Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, 500 South Preston Street, Louisville, KY 40202, USA increased mitochondrial biogenesis and oxidative capacity, Full list of author information is available at the end of the article © 2013 Sato 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. Sato et al. Skeletal Muscle 2013, 3:18 Page 2 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 are more resistant to fatigue and have improved aerobic tolerance and skeletal muscle bioenergetic function. performance [7-10]. Forced expression of PGC-1α also Our results show that, compared with wild-type mice, preserves skeletal muscle mass in various catabolic states TWEAK-KO mice have increased exercise tolerance, including denervation [11,12]. PGC-1α improves mito- which is associated with higher levels of skeletal muscle chondrial biogenesis by coactivating nuclear respiratory subsarcolemmal and intermyofibrillar mitochondria and factor (NRF)-1 [13], NRF-2 [14] and estrogen-related enhanced oxidative phosphorylation capacity. TWEAK receptor α [15-17] which, in turn, augments the expression represses the expression of PGC-1α and several other of hundreds of nuclear-coded mitochondrial genes [18]. molecules involved in mitochondrial biogenesis and Recent evidence also suggests a role for mitochondria in oxidative metabolism in vivo and in cultured myotubes. preventing muscle protein degradation in catabolic condi- Furthermore, vascularization and expression of vascular tions [19,20]. Thus, preserving mitochondria content and endothelial growth factor (VEGF) are increased in ske- function is essential to countering muscle wasting and to letal muscle of TWEAK-KO mice compared with wild- improving muscle homeostasis. type mice. These results support our hypothesis that Inflammatory cytokines such as tumor necrosis factor-α TWEAK causes exercise intolerance by suppressing (TNF-α) and interleukin-6 (IL-6) are some of the import- mitochondrial oxidative metabolism and angiogenesis. ant mediators of loss of skeletal muscle mass and function in many catabolic conditions [21-24]. TNF-α has been Methods reported to augment muscle protein degradation both Cell culture in vitro and in vivo [25-27]. Moreover, TNF receptor I Primary myoblasts were isolated from hind limb muscle knockout (KO) mice are protected from diet-induced of mice and cultured following the same protocol as pre- obesity due to increased thermogenesis [28]. Importantly, viously described [43]. Briefly, hind limb skeletal muscles TNF-α inhibits the expression of PGC-1α levels in from mice were aseptically isolated, minced into a coarse myotubes [29]. In animal models of cancer cachexia, mito- slurry, and enzymatically digested at 37°C for one hour chondrial content and biogenesis are reduced with in- by adding 200 IU/ml collagenase I (cat # LS004196; creased levels of IL-6 during the progression of the Worthington, Lakewood, NJ, USA) and 0.1% pronase condition whereas inhibition of IL-6 activity attenuates (EMD Chemicals, Billerica, MA, USA). The digested tumor-induced loss of skeletal muscle [30,31]. It is also slurry was filtered through a 70 μm filter and spun, and notable that while moderate exercise attenuates inflamma- isolated cells were resuspended and cultured initially in tion [32-34], acute exercise more robustly increases the F-10 medium (containing 20% fetal bovine serum (FBS) levels of inflammatory cytokines in obese compared with and supplemented with 10 ng/ml basic fibroblast growth lean subjects suggesting that rigorous exercise can further factor) and then in F-10 plus (Dulbecco’s) Modified aggravate the condition of overweight individuals [35]. Eagle’s Medium ((D)MEM) (1:1 ratio) based culture Despite these observations, cause-and-effect relationships medium supplemented with 15% FBS on culture dishes between different inflammatory mediators, exercise toler- coated with 10% matrigel (BD Biosciences, San Jose, CA, ance, and mitochondrial function in skeletal muscle have USA). Differentiation in primary myoblast cultures was not been clearly established using genetic mouse models. induced by replacing the growth medium with differenti- TNF-like weak inducer of apoptosis (TWEAK) is a major ation medium (2% horse serum in (D)MEM). proinflammatory cytokine of the TNF super family [36] that functions by binding to Fn14 receptors on target Animals cells [37-39]. TWEAK-Fn14 signaling mediates unique TWEAK-KO mice were provided by Dr. Avi Ashkenazi and context-dependent pleiotropic effects [40]. TWEAK (Genentech South, San Francisco, CA, USA) and have has been recently identified to be a key mediator that been previously described [40]. All the mice were in the causes skeletal muscle loss that occurs in response to C57BL/6 background, and their genotype was determined denervation, immobilization, and starvation [21,41,42]. by PCR from tail DNA. All animal procedures were Moreover, TWEAK-transgenic mice overexpressing phy- approved (protocol # 10129) by the Institutional Animal siological levels of TWEAK in skeletal muscle showed a Care and Use Committee and conformed to the American higher proportion of fast-type fibers (glycolytic) in soleus Physiological Society’s Guiding Principles in the Care and and extensor digitorum longus (EDL) muscle [41] indi- Use of Animals. cating that elevated levels of TWEAK cause a slow- to-fast type fiber switch. However, the role and mecha- Treadmill running protocol nisms by which TWEAK affects exercise capacity and Mice were subjected to treadmill running following bioenergetic function have not been studied. the same previously described protocol [44]. In brief, In the present study, we have used TWEAK-knockout 4.5-month-old wild-type and TWEAK-KO mice were (KO) mice to investigate the role of TWEAK in exercise matched for body weight and randomly assigned to Sato et al. Skeletal Muscle 2013, 3:18 Page 3 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 either a sedentary or exercise group. Mice were and 2.4 mM nitroblue tetrazolium (NBT, Sigma) at 37°C subjected to an acute bout of treadmill (Eco3/6 tread- in a humidity chamber for 45 minutes. The sections mill; Columbus Instruments, Columbus, OH, USA) run- were then washed in deionized water for three minutes, ning at 15 m/minute for 90 minutes. All mice in the dehydrated in 50% ethanol for two minutes, and mounted exercise group finished the 90-minute trial and were vis- for viewing with DPX mount medium (Electron Micros- ibly exhausted. Mice were sacrificed within 30 minutes copy Sciences, Hatfield, PA, USA). Digital photographs after exercise to study mitochondrial function. were taken from each section at 10X magnification under a Nikon Eclipse TE 2000-U microscope (Nikon, Melville, Exercise tolerance test NY, USA) with a Nikon digital camera (Digital Sight DS- The exercise tolerance test on mice was performed fol- Fi1), and fibers were quantified with imaging software lowing a method as previously described [45]. Briefly, all (Image J, NIH). At least 700 fibers were counted to deter- animals were run on a treadmill (Columbus Instru- mine SDH-positive fibers in each section in a blinded ments) at 10 m/minutes for five minutes at 0% degree fashion. The percentage of SDH stained fibers was then incline for acclimation for three days. On the exercise determined based on a criteria using integrated optical testing day, animals ran on the treadmill with a fixed density. slope of 10%. Mice first ran at 10 m/minute for five mi- nutes and the speed was increased by 2 m/minute every two minutes until they were exhausted or a maximal Measurement of mitochondrial bioenergetics speed of 46 m/minute was achieved. The criterion of Mitochondrial oxidative capacity was measured in isolated exhaustion was defined as the inability of the animal to mitochondria and in cultured myotubes using a Seahorse run on the treadmill for 10 seconds despite mechanical Bioscience XF24 extracellular flux analyzer (Billerica, MA, prodding. Running time and maximum speed achieved USA). For measurements in isolated mitochondria, tissue was measured whereas running distance, work and power from the limb muscle (approximately 50 mg) was isolated were calculated. within 30 minutes of exercise and homogenized in 1 ml of isolation buffer (220 mM mannitol, 70 mM sucrose, 5 mM Transmission electron microscopy 3-(N-morpholino)propanesulfonic acid (MOPS), 1 mM Soleus muscles from wild-type and TWEAK-KO mice ethylene glycol tetraacetic acid (EGTA), 0.3% fatty acid-free were fixed in 3% glutaraldehyde in 0.1 M cocodylate BSA, pH 7.2) using a Potter Elvehjem tube and a Teflon buffer overnight followed by fixing in 1% cocodylate- pestle. The homogenate was centrifuged at 500×g for five buffered osmium tetroxide. The tissue was dehydrated minutes at 4°C. The supernatant containing mitochondria through a series of graded alcohols, and embedded in was centrifuged at 10,000 × g for five minutes. After two LX-112 plastic (Ladd Research Industries, Williston, wash-centrifugation steps in BSA-free isolation buffer, VT, USA). Longitudinal sections (80 nm) were cut the mitochondria were suspended in respiration buffer using an ultramicrotome (LKB, Rockville, MD, USA)) (120 mM KCl, 25 mM sucrose, 10 mM 4-(2-hydroxyethyl)- and stained with uranium acetate and lead citrate. 1-piperazineethanesulfonic acid (HEPES), 1 mM MgCl ,5 Samples were analyzed using a transmission electron mM KH PO , pH to 7.2). Protein in the mitochondrial sus- 2 4 microscope (Philips CM 12; HZB, Berlin, Germany) pension was estimated using the Lowry DC assay (Biorad, operating at 60 kV. The pictures were captured at Hercules, CA, USA) and 5 to 12.5 μg of mitochondrial pro- 8,800x magnification using a 3.2 megapixel digital tein was sedimented in XF culture plates as described previ- camera (Sia-7C; Kodak, Rochester, NY, USA) at room ously [47]. Complex I-mediated, state 3 respiratory activity temperature. No imaging medium was used to was determined by measuring the oxygen consumption rate visualize the pictures, and images were stored as (OCR) after injection of pyruvate (5 mM), malate (2.5 mM) JPEG files. Image levels were equally adjusted using and ADP (1 mM). The OCR of mitochondria after exposure Photoshop CS2 software. to oligomycin (1 μg/ml) was used to estimate state 4 activ- ity; exposure to carbonyl cyanide p-trifluoromethoxyphe Succinate dehydrogenase (SDH) staining nylhydrazone (FCCP, 2 μM) was used to examine the SDH staining was performed as previously described uncoupled rate of respiration. Finally, succinate (10 mM) [46]. Briefly, transverse sections (8 μm) were cut from and rotenone (1 μM) were injected to assess maximal Com- the mid-belly of the TA muscles on a cryostat at −20°C plex II-mediated respiratory capacity. Data are expressed as and stored at −80°C until SDH staining was performed. pmol O /min/μg protein. For measurements in cultured The sections were dried at room temperature for 30 mi- cells, differentiated myoblasts were exposed to soluble nutes before incubation in a solution made up of 0.2 M TWEAK (R&D Systems, Minneapolis, MN, USA) for 72 phosphate buffer (pH 7.4), 0.1 M MgCl , 0.2 M succinic hours followed by the mitochondrial function assay acid (Sigma Chemical Company, St. Louis, MO, USA) outlined previously [48-51]. Sato et al. Skeletal Muscle 2013, 3:18 Page 4 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 RNA isolation and quantitative real time-PCR between control and treatment groups. A value P <0.05 RNA isolation and quantitative real time-PCR (qRT-PCR) was considered statistically significant. were performed as previously described [41]. Briefly, total RNA was isolated from homogenized mouse tissues using Results the TRIzol reagent (Invitrogen, Grand Island, NY, USA) Improvement in exercise tolerance in TWEAK-KO mice and an RNeasy Mini kit (QIAGEN, Valencia, CA, USA) We first studied the role of TWEAK in exercise tolerance according to the manufacturers’ protocols. First strand in mice. Male 4.5-month old wild-type and TWEAK-KO cDNA for PCR analyses were made using a reverse mice were acclimated for three days for treadmill running transcription system with 1 μgofpurified RNA usingoligo prior to the exercise tolerance test. After acclimation, the (dT) primer (Applied Biosystems, Grand Island, NY, USA) mice were run on a 10% slope until the mice were unable and the Omniscript reverse transcription kit (QIAGEN). to run on the treadmill for 10 seconds despite mechanical The quantification of mRNA expression was performed prodding. TWEAK-KO mice ran longer compared to using the SYBR green dye method on a sequence detec- wild-type mice (1,483 seconds for TWEAK-KOs versus tion system (Applied Biosystems, model 7300). Primers 1,170 seconds for the controls, Figure 1A). Since TWEAK- were designed using Vector NTI software (Invitrogen). KO mice could keep running on higher speed (30.6 m/ Primer sequences are available on request. The thermal minute for TWEAK-KOs versus 25.5 m/minute for the conditions consisted of an initial denaturation at 95°C for controls, Figure 1B), the difference in running distance 10 minutes, followed by 40 cycles of denaturation at 95°C became greater than 47% (463 meter for TWEAK-KOs for 15 seconds, annealing and extension at 60°C for one versus 314 meter for the controls, Figure 1C). Work and minute, and, for a final step, a melting curve of 95°C for power generated by TWEAK-KO mice were also higher 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds. compared to wild-type mice. TWEAK-KO mice generated All reactions were performed in duplicate to reduce vari- 22.2 J of work while control mice generated 15.2 J of work ation. Data normalization was accomplished using the (a difference of 46%) during the exercise test (Figure 1D). endogenous control (β-actin), and the normalized values In turn, TWEAK-KO mice exerted 14% more power than -ΔΔCt were subjected to a 2 formula to calculate the fold the controls (14.9 mW for TWEAK-KO versus 13.0 mW change between control and experimental groups. for the wild-type, Figure 1E) on the treadmill. These data established that TWEAK-KO mice have significantly im- proved exercise tolerance. Immunostaining for CD31 After cutting 8 μm thickness frozen sections of TA Ablation of TWEAK improves mitochondrial content in muscles, the sections were fixed by cold acetone for 10 skeletal muscle of mice minutes and dried in air for 30 minutes. The tissues We have previously shown that deletion of TWEAK in- were rinsed with phosphate buffered saline (PBS) twice, creases the proportion of type I fibers in soleus and EDL blocked in 2% BSA solution for one hour at room muscles of mice [41]. In this study, we investigated temperature followed by incubation with primary anti- whether TWEAK affects mitochondrial content in skel- bodies in 2% BSA solution (laminin, anti-rabbit 1:300, etal muscle of mice. The soleus muscle of 4.5-month old Sigma and CD31, anti-mouse 1:30, BD Biosciences, San TWEAK-KO and wild-type mice were isolated and used Jose, CA, USA) at 4°C in a humidity chamber overnight. to measure mitochondrial content by performing trans- The next day, the sections were rinsed with PBS for five mission electron microscopy. As shown in Figure 2A, minutes three times and incubated with secondary anti- the abundance and size of mitochondria was found to be body (Alexa Fluor-conjugated 488 anti-rabbit, 1:2500 increased in TWEAK-KO compared to wild-type mice. and 546 anti-mouse, 1:2500, Invitrogen) in 2% BSA solu- Quantitative analysis of mitochondria in electron micro- tion for one hour at room temperature. After washing graphs showed that the levels of subsarcolemmal and twice with PBS and once with deionized water, the intermyofibrillar mitochondria were increased by 42% sections were mounted with DPX mounting medium. and 32%, respectively, in TWEAK-KO mice compared to Digital photographs were taken from each section under wild-type mice (Figure 2B and Figure 2C). Because a Nikon Eclipse TE 2000-U microscope (Nikon) with a gastrocnemius (GA) muscle contains a mixture of slow and Nikon digital camera (Digital Sight DS-Fi1). Number of fast twitch fibers and greatly influences running capacity on CD31-postive vessels per myofiber was quantified using the treadmill, we measured mRNA levels of fiber type- NIS Elements BR 3.00 software (Nikon). specific myosin heavy chain (MyHC) isoforms in GA muscle of wild-type and TWEAK-KO mice. As shown in Statistical analysis Figure 2D, mRNA levels of MyHC I and MyHC IIA were The results are presented as means ± standard deviation increased by 2- and 2.7-fold, respectively in TWEAK-KO (SD). Student’s t-test was used to compare the difference mice compared to wild-type mice. These results further Sato et al. Skeletal Muscle 2013, 3:18 Page 5 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Wild-type; TWEAK-KO A. B. C. 600 200 D. E. Figure 1 Treadmill exercise tolerance test. After acclimatization, mice were run on a treadmill with a 10% slope and increasing speed to exhaustion. Maximum speed and running time were monitored, and distance, work, and power were calculated based on the individual performance. (A) Running time; (B) Speed; (C) Distance; (D) Work; and (E) Power. Data are represented as mean ± SD. N = 6 in each group. *P <0.05; values vary significantly between wild-type and TWEAK-KO mice. KO, knockout. suggest that genetic ablation of TWEAK increases the muscle, we investigated whether TWEAK modulates the slow-type fiber phenotype in skeletal muscle of mice. expression of PGC-1α, a critical regulator of the mito- SDH, also known as complex II in the mitochondrial chondrial biogenetic program in skeletal muscle [9,10]. respiratory chain, is a marker of oxidative capacity of skel- Interestingly, the mRNA levels of PGC-1α were found to be etal muscle at the fiber level. Therefore, we performed significantly higher in 4.5-month-old TWEAK-KO mice SDH staining on TA muscle of 4.5-month- and 12-month- compared to age-matched wild-type mice (Figure 4). old wild-type mice and TWEAK-KO mice and quantified Similarly, mRNA levels of PPARδ were significantly higher the percentage of SDH-positive fibers. Compared to wild- in skeletal muscle of TWEAK-KO mice compared to corre- type mice, fibers in TWEAK-KO mice stained more darkly sponding wild-type mice (Figure 4). In contrast, mRNA for SDH (Figure 3A). Furthermore, there was a significant levels of glycolytic enzymes such as hexokinase-2 (HK II) increase in the number of SDH-positive fibers in TWEAK- and phosphoglycerate mutase 2 (PGAM2) were reduced in KO mice compared to wild-type mice at the age of 4.5 skeletal muscle of TWEAK-KO mice compared to wild- months (Figure 3B). Likewise, 12-month-old TWEAK-KO type mice. No significant change was observed in the had more SDH-positive fibers compared to age-matched expression of pyruvate dehydrogenase kinase-4 (PDK4). wild-type mice (Figure 3A and Figure 3C). Taken together, Furthermore, mRNA levels of mitochondrial carnitine these results suggest that ablation of TWEAK in mice is palmitoyltransferase I (mCPT1), which is required for the sufficient to improve mitochondrial content and oxidative transport of long-chain fatty acyl-CoAs from the cyto- capacity of skeletal muscle. plasm into the mitochondrion, were significantly ele- vated in TWEAK-KO mice compared to wild-type mice (Figure 4). These results suggest that a deficiency of TWEAK-KO mice demonstrate increased expression of TWEAK reduces expression of glycolytic genes and PGC-1α and metabolic genes in skeletal muscle augments the expression of genes involved in oxidative To understand the mechanisms by which TWEAK affects metabolism in skeletal muscle. exercise capacity and mitochondrial content in skeletal Running time (seconds) Work (joule) -3 Power (watts X 10 ) Speed (meter/minute) Distance (meters) Sato et al. Skeletal Muscle 2013, 3:18 Page 6 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Figure 2 Transmission electron microscopy (TEM) analysis for mitochondria morphology and content and expression of muscle fiber type-specific genes in TWEAK-KO mice. Soleus muscle of six-month-old wild-type and TWEAK-KO mice were isolated and longitudinal sections were processed for TEM analysis. (A) Representative images of longitudinal soleus muscle are shown. The abundance and the size of mitochondria were increased in TWEAK-KO mice compared to wild-type mice. Arrows point to mitochondria in muscle sections. Circles are used to show representative enlarged mitochondria. Stars (‘*’) point to subsarcolemmal space in longitudinal sections. Scale bar: 1 μm. (B) Quantification of the number of subsarcolemmal mitochondria in soleus muscle of wild-type and TWEAK-KO mice. (C) Quantification of the number of intermyofibrillar mitochondria in soleus muscle sections of wild-type and TWEAK-KO mice. (D) Relative mRNA levels of MyHC type I, IIA and IIB in gastrocnemius muscle of 4.5-month-old wild-type and TWEAK-KO mice. Data are represented as mean ± SD. N = 3 in each group. *P <0.05; values vary significantly from wild-type mice. KO, knockout; TWEAK, TNF-like weak inducer of apoptosis. Ablation of TWEAK increases state 3 respiration in induced by addition of pyruvate, malate and ADP; state 4 skeletal muscle mitochondria respiration was induced by addition of oligomycin; FCCP We next sought to determine whether TWEAK regulates was added to determine maximal complex I-mediated mitochondrial function in skeletal muscle. After an acute respiratory activity; and, lastly, succinate and rotenone bout of treadmill running for 90 minutes, mitochondria were added to determine maximal complex II-mediated were isolated from hind limb muscles of wild type and respiratory activity. As shown in Figure 5A and 5B, mito- TWEAK-KO mice and mitochondrial function was exam- chondria from TWEAK-KO mice showed significantly in- ined by extracellular flux (XF) analysis. The sequential creased state 3 respiratory activities compared with wild addition of respiratory substrates and inhibitors of oxi- type mice. Mitochondrial coupling (as determined by dative phosphorylation were used to determine changes in calculating respiratory control ratios) and other indices of electron transport chain activity: state 3 respiration was mitochondrial function were not significantly different Sato et al. Skeletal Muscle 2013, 3:18 Page 7 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 A. Wild-type TWEAK-KO B. C. 50 50 4.5-month 1-year 40 40 Wild-type TWEAK-KO Wild-type TWEAK-KO Figure 3 Succinate dehydrogenase (SDH) staining analysis for oxidative capacity in TA muscle of TWEAK-KO mice. Frozen transverse TA muscle sections from 4.5- or 12-month old wild-type and TWEAK-KO mice were used to stain for SDH. (A) Representative SDH-stained images are presented here. TA muscle of TWEAK-KO showed relatively dark staining for SDH compared to wild-type mice. Scale bars: 50 μm. Quantification of SDH-positive fibers in TA muscle of (B) 4.5-month, and (C) 12-month old wild-type and TWEAK-KO mice. Data are presented as mean ± SD. *P <0.05; values vary significantly from wild-type mice. KO, knockout; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis. 2.0 Wild-type; TWEAK-KO * * 1.5 1.0 0.5 PGC-1α PPARδ HK II PGAM2 PDK4 mCPT1 Figure 4 qRT-PCR analysis of transcript levels of metabolic genes in skeletal muscle of wild-type and TWEAK-KO mice. TA muscle from 4.5-month-old wild-type and TWEAK-KO mice were isolated and processed to measure mRNA levels of PGC-1α, PPARδ, HK II, PGAM2, PDK4, and mCPT1. Data are represented as mean ± SD. N = 4 in each group. *P <0.05. values vary significantly from wild-type mice. HK II, hexokinase-2; KO, knockout; mCPT1, mitochondrial carnitine palmitoyltransferase 1; PDK4, pyruvate dehydrogenase kinase-4; PGAM2, phosphoglycerate mutase 2; PGC-1α, PPAR coactivator 1α; PPAR, peroxisome proliferator-activated receptor; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis. SDH-positive fibers Frequency (%) 1-year 4.5-month SDH-positive fibers Frequency (%) Relative mRNA levels Sato et al. Skeletal Muscle 2013, 3:18 Page 8 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 B. A. Wild-type; TWEAK-KO mice Wild-type; TWEAK-KO mice 60 60 Succ/Rot FCCP Oligo Pyr/Mal/ADP State 3 State 4 Complex I Complex II 05 10 15 20 Maximum Maximum Time (min) Figure 5 Mitochondrial respiration is enhanced in TWEAK-KO mice. Respiratory activity of mitochondria isolated from wild-type and TWEAK-KO mice subjected to an acute bout of treadmill running: (A) Extracellular flux assay of isolated mitochondria: mitochondrial activity was measured in the absence of substrate (state 1 respiration) or in the presence of pyruvate (5 mM), malate (2.5 mM) and ADP (1 mM) (state 3 respiration). State 4 respiration was estimated by measuring the oxygen consumption rate (OCR) after the addition of oligomycin (1 μg/ml). FCCP (4 μM) was added to assess maximal activity of mitochondria respiring on substrates providing electrons for complex I (that is, pyruvate and malate) or complex II (that is, succinate in the presence of rotenone; 10 mM and 1 μM, respectively). (B) Group data derived from panel A. N = 3 mice per group. *P <0.05 versus wild-type. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; KO, knockout; TWEAK, TNF-like weak inducer of apoptosis. between wild type and KO mice. These data suggest that assess the effects of TWEAK on mitochondrial function absence of TWEAK augments mitochondrial oxidative directly, we exposed differentiated myotubes to TWEAK capacity in skeletal muscle in exercised mice. and then assessed mitochondrial function as described previously [48-51]. As shown in Figure 7A and 7B, ex- posure of myotubes to TWEAK for 72 hours did not TWEAK represses the expression of PGC-1α and genes affect the basal mitochondrial OCR, nor did it affect regulating oxidative metabolism in cultured primary ATP-linked OCR or proton leak. However, the FCCP- myotubes stimulated OCR, indicative of maximal respiratory cap- We next studied whether TWEAK affects the expression acity, was remarkably diminished by TWEAK treatment. of PGC-1α and other genes in cultured primary myotubes. In addition, a modest, yet significant, increase in the Primary myoblasts prepared from wild-type mice were dif- non-mitochondrial OCR occurred in myotubes exposed ferentiated into myotubes by incubation in differentiation to100 ng/ml TWEAK, which may be due to increased medium for 96 hours followed by treatment with TWEAK production of reactive oxygen species. The diminishment for 72 hours. As shown in Figure 6, addition of soluble in mitochondrial respiratory capacity caused by TWEAK TWEAK dramatically reduced the expression of PGC-1α was associated with an increase in the extracellular acid- in myotubes. Treatment with TWEAK also significantly ification rate (ECAR), a surrogate measure of glycolysis reduced the mRNA levels of several mitochondrial genes (Figure 7C). Collectively, these results suggest that encoding proteins related to oxidative metabolism, such TWEAK directly modulates the bioenergetic capacity of as ATP synthase subunit beta (ATP5b), cytochrome c oxi- skeletal muscle. dase subunit I (Cox I), Cox subunit IV (COX IV), COX - 7b, cytochrome c (Cyt c), medium-chain acyl-coenzyme A Ablation of TWEAK improves angiogenesis in skeletal dehydrogenase (MCAD), PPARδ, and mCPT1 (Figure 6). muscle of mice These results further suggest that TWEAK inhibits the There is a causal relationship between PGC-1α and mitochondrial biogenetic program in skeletal muscle. angiogenesis. Skeletal muscle-specific overexpression of PGC-1α in mice results in increased angiogenesis, which TWEAK decreases maximal respiratory capacity and may contribute to enhanced exercise performance [52]. increases glycolysis in cultured primary myotubes Since TWEAK suppresses PGC-α in skeletal muscle, we Our studies in isolated mitochondria derived from ske- next sought to determine whether TWEAK also regu- letal muscle after exercise suggest that the absence of lates angiogenesis in skeletal muscle of mice. TA muscle TWEAK increases mitochondrial oxidative capacity. To of 4.5-month-old wild-type and TWEAK-KO mice OCR (pmol O /min/ug protein) OCR (pmol O /min/ug protein) 2 Sato et al. Skeletal Muscle 2013, 3:18 Page 9 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 Control 10 ng/ml TWEAK 1.4 100 ng/ml TWEAK 1.2 1.0 * * * * 0.8 0.6 0.4 * 0.2 Figure 6 Effect of TWEAK on expression of PGC-1α and mitochondria oxidative metabolism genes in cultured primary myotubes. Primary myotubes prepared from wild-type mice were treated with soluble TWEAK protein (10 ng/ml or 100 ng/ml) for 72 hours followed by RNA isolation and performing qRT-PCR to study transcript levels of PGC-1α, Tfam, Tfam2m, Cyt-c, Cox I, Cox IV, Cox 7b, and MCAD. Data are presented as mean ± SD. *P <0.05; values vary significantly from untreated myotubes. Cox, cytochroms c oxidase; Cyt c, cytochrome c; MCAD, medium-chain acyl-coenzyme A dehydrogenase; PGC-1α, PPAR coactivator 1α; PPAR, peroxisome proliferator-activated receptor; Tfam, transcription factor A mitochondrial; TWEAK, TNF-like weak inducer of apoptosis. was isolated and immunostained with antibodies against intermyofibrillar mitochondria, increased SDH-positive CD31 (also known as platelet endothelial cell adhesion myofibers, and elevated expression of metabolic genes molecule-1), an endothelial-specific marker for capilla- such as PGC-1α,PPARδ, and mCPT-1 compared to wild- ries. Laminin staining was used to mark the periphery type mice (Figures 2, 3, and 4). Oxidative phosphorylation of fibers. As shown in Figure 8A, the number of CD31- is also increased in exercised TWEAK-KO mice compared positive capillaries was considerably increased in TWEAK- with wild-type mice (Figure 5), and treatment of myotubes KO mice compared with wild-type mice. The capillary- with TWEAK directly decreases mitochondrial biogenetic to-fiber ratio was increased by 29% in TWEAK-KO mice capacity and maximal respiratory activity (Figures 6 and (Figure 8B). Since VEGF positively regulates angiogenesis, 7). Moreover, vascularization and expression of VEGF in we also measured mRNA levels of VEGF in TA muscle of skeletal muscle are enhanced in TWEAK-KO mice com- wild-type and TWEAK-KO mice. As shown in Figure 8C, pared to their controls (Figure 8). These data suggest that mRNA levels of VEGF were significantly higher in TA TWEAK directly regulates the mitochondrial biogenetic muscle of TWEAK-KO mice compared to wild-type mice. program and that loss of TWEAK improves mitochondrial Furthermore, treatment with TWEAK also reduced the respiratory capacity and increases vascularization of ske- mRNA levels of VEGF in primary myotubes (Figure 8D). letal muscle tissue, which collectively lead to improved These results suggest that ablation of TWEAK stimulates exercise performance. angiogenesis in skeletal muscle of mice potentially through One of the potential mechanisms by which TWEAK increasing the expression of VEGF. might be attenuating exercise capacity is through diminishing the levels of PGC-1α in skeletal muscle. Discussion PGC-1α augments mitochondrial biogenesis, oxidative Exercise capacity in mammals is determined by multiple metabolism, and promotes fast-to-slow type fiber tran- factors including skeletal muscle oxidative metabolism sition [9,13,17,54-56]. Overexpression of PGC-1α in rat and vascularization. Our previous microarray study sug- primary culture cells leads to increased abundance of gested that TWEAK can modulate the expression of sev- slow oxidative-associated MyHC isoform [57]. Skeletal eral genes whose products are involved in mitochondrial muscle-specific PGC-1α KO mice demonstrate reduced dysfunction and fatty acid metabolism [53]. Our present endurance capacity with a shift from oxidative fiber type study demonstrates that compared to wild-type mice, to glycolytic fibers and increased levels of TNF-α in TWEAK-KO mice run longer and with higher speed skeletal muscle [45,58]. By contrast, muscle-specific during an exercise tolerance test (Figure 1). TWEAK- overexpression of PGC-1α enhances exercise perfor- KO mice show augmented levels of subsarcolemmal and mance with increased fatty acid oxidation and decreased Relative mRNA levels PGC-1α α ATP5b Tfam Tfb2m Cyt-c COX-I COX-IV COX-7b MCAD PPARδ mCPT1 Sato et al. Skeletal Muscle 2013, 3:18 Page 10 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 C. A. 70 O F Control Control 10 ng/ml TWEAK 10 ng/ml TWEAK 100 ng/ml TWEAK 100 ng/ml TWEAK 50 1.0 0.8 0.6 0.4 010 20 30 40 50 Time (min) 0.2 B. Control 10 ng/ml TWEAK 100 ng/ml TWEAK * * Basal ATP-linked Proton Maximum Reserve Non mito- mito OCR OCR Leak Capacity Capacity chondrial Figure 7 TWEAK regulates mitochondrial oxidative capacity and glycolytic flux. Extracellular flux analysis of primary differentiated myotubes treated with 0, 10, or 100 ng/ml TWEAK for 72 hours. (A) Mitochondrial function assay: after three baseline measurements, inhibitors or activators of electron transport were added sequentially to intact myotubes. Oxygen consumption rate (OCR) measurements were recorded after each exposure. O, oligomycin; F, FCCP; and A, antimycin A/rotenone. The OCR was normalized to total protein in each well. (B) Indices of mitochondrial function calculated from assay results in panel A. (C) Extracellular acidification rates (ECAR) of cells treated without or with TWEAK; ECAR is a surrogate measure of lactate and therefore is used as a measure of glycolytic flux. N = 3 per group; *P <0.05 versus control. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; TWEAK, TNF-like weak inducer of apoptosis. glycogen usage during exercise [8,52]. These findings other molecules involved in mitochondrial oxidative suggest that PGC-1α is not only essential but sufficient phosphorylation in cardiomyocytes and forced expres- to determining skeletal muscle fiber composition. Our sion of PGC-1α attenuates TWEAK-induced cardiac experiments demonstrate that mRNA levels of PGC-1α dysfunction in mice [59]. are increased in skeletal muscle of TWEAK-KO mice The PPARs are ligand-modulated transcription factors with a concomitant increase in mitochondrial content in which three subtypes have been identified: α, β/δ, and and expression of genes whose products are involved in γ [60]. Previous studies have shown that their endogen- oxidative capacity (Figures 2 and 4). Furthermore, treat- ous ligands are composed of fatty acids and lipid me- ment of primary myotubes with TWEAK drastically re- tabolites and, therefore, certain PPARs mediate the duced levels of PGC-1α and other mitochondrial genes expression of genes whose products are involved in the (Figure 6) further suggesting that TWEAK represses the regulation of fatty acid metabolism in response to expression of PGC-1α leading to reduced mitochondrial changes in systemic fuel availability [60-62]. In skeletal content. These findings are consistent with functional muscle, levels of PPARδ are relatively higher compared analyses of mitochondrial function: ablation of TWEAK to PPARα or PPARγ [63]. Treatment with synthetic resulted in increased oxidative phosphorylation capacity PPARδ agonist or overexpression of PPARδ by retroviral in isolated mitochondria (Figure 5) and treatment of infection induces the levels of molecules which are in- myotubes with TWEAK decreased maximal respiratory volved in lipid metabolism and fatty acid oxidation, capacity (Figure 7). Although not tested in this study, it whereas overexpression of a dominant-negative PPARδ is possible that TWEAK also reduces the levels of PGC- mutant exerts opposite effects in C2C12 myotubes [64]. 1α in other organs which ultimately results in reduced Furthermore, it has been shown that muscle-specific exercise capacity. Indeed, Shi et al. recently reported overexpression of PPARδ in mice increases exercise that TWEAK represses the expression of PGC-1α and tolerance with a switch to increased number of type I OCR OCR (pmol O 2 /min/ug protein) (pmol O /min/ug protein) ECAR (mpH unit/min/μ μg protein) Sato et al. Skeletal Muscle 2013, 3:18 Page 11 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 A. B. D. C. 1.8 1.8 Control 1.8 1.6 1.6 1.6 10 ng/ml TWEAK 1.4 1.4 1.4 100 ng/ml TWEAK 1.2 1.2 1.2 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 Wild-type Wild-type TWEAK-KO TWEAK-KO Figure 8 Analysis of capillary density in skeletal muscle of wild-type and TWEAK-KO mice. Transverse sections of TA muscle prepared from 4.5-month-old wild-type and TWEAK-KO mice were immunostained for CD31 (red) and counterstained for laminin (green). (A) Representative CD31- and laminin-immunostained and merged images are presented here. Scale bars: 50 μm. (B) Quantification of CD31-postive capillaries per myofiber in TA muscle. N = 8 in each group. (C) Relative mRNA levels of VEGF in TA muscle of wild-type and TWEAK-KO mice (N = 4 in each group) measured by qRT-PCR analysis. (D) Primary myotubes were treated with the indicated concentration of TWEAK for 24 hours followed by measurement of mRNA levels of VEGF by qRT-PCR. Relative mRNA levels of VEGF are shown here. Data are presented as mean ± SD. *P <0.05; values vary significantly from untreated myotubes. KO, knockout; TA, tibial anterior; TWEAK, TNF-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor. muscle fibers and up-regulation of molecules related to might be responsible for improvement in exercise capacity fatty acid metabolism. In contrast, PPARδ-null mice in TWEAK-KO mice. Data from cell culture experiments show decreased exercise performance compared to wild- further support this possibility, as treatment of myotubes type mice [65,66]. Although muscle relies mainly on fat with TWEAK resulted in diminished maximal respiratory and carbohydrate as energy resources, enhanced fatty capacity and increased glycolytic flux (Figure 7). acid utilization during exercise with glycogen sparing re- Although the mechanisms by which TWEAK reduces sults in improved exercise endurance capacity [67-69]. levels of PGC-1α in skeletal muscle remain unknown, a Haramizu et al. demonstrated that the expression levels recent study has shown that TWEAK increases membrane of gene products related to lipid metabolism in the translocation of adaptor protein TNF receptor associated muscle is correlated with the levels of fatty acid β- factor 2 (TRAF2), in an Fn14 dependent manner, in car- oxidation activity as well as exercise strength [70]. Our diomyocytes [59]. Furthermore, TWEAK treatment in- results demonstrate that transcript levels of PPARδ and creases the activation of canonical nuclear factor-kappaB mCPT1 are significantly increased while the expression (NF-κB) signaling in both skeletal muscle [41] and cardiac of molecules that are associated with glycolysis such as myocytes [59]. Knockdown of TRAF2 using small hair- HK II and PGAM2 is suppressed in skeletal muscle of pin RNA (shRNA) or selective blockade of IκBkinase-β TWEAK-KO mice compared to wild-type mice (Figure 6). (IKKβ, an upstream activator of canonical NF-κBsignal- These results suggest that enhanced fatty acid oxidation ing) prevented the TWEAK-mediated suppression of Merged Laminin CD31 Capillary to fiber ratio TWEAK-KO Wild-type Relative VEGF mRNA levels (Fold change) Relative VEGF mRNA levels (Fold change) Sato et al. Skeletal Muscle 2013, 3:18 Page 12 of 14 http://www.skeletalmusclejournal.com/content/3/1/18 PGC-1α in cardiomyocytes suggesting that TWEAK re- inhibition of TWEAK activity using neutralizing antibodies pression of PGC-1α requires Fn14-TRAF2-IKKβ-NF-κB or pharmacological compounds could improve muscle signaling cascade [59]. Previously, the effects of TNF-α function and exercise capacity in patients with meta- on the mRNA expression of PPARδ and its target genes bolic disorders. have been investigated. Treatment with TNF-α reduces Abbreviations PPARδ-target genes, such as mCPT1 and PGC-1α, ATP5b: ATP synthase subunit beta; BSA: Bovine serum albumin; Cox: Cytochrome whereas addition of PPARδ agonist rescues this reduc- c oxidase; FCCP: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GA: Gastrocnemius; HK II: Hexokinase II; KO: Knockout; MCAD: Medium-chain tion in adipocytes [71,72]. Moreover, TNF-α impairs acyl-coenzyme A dehydrogenase; mCPT1: Mitochondrial carnitine mitochondrial biogenesis and function in skeletal palmitoyltransferase I; MyHC: Myosin heavy chain; NF-κB: Nuclear factor-kappaB; muscle [73]. While TWEAK can induce the expression OCR: Oxygen consumption rate; PCR: Polymerase chain reaction; PDK4: Pyruvate dehydrogenase kinase 4; PGAM2: Phosphoglycerate mutase 2; PGC-1α:PPAR of molecules associated with the autophagy-lysosomal coactivator 1α; PPAR: Peroxisome proliferator-activated receptor; qRT- system indicating it causes mitochondria dysfunction PCR: Quantitative real-time PCR; SDH: Succinate dehydrogenase; shRNA: Small [74], muscle-specific ablation of TRAF6, which is in- hairpin RNA; TA: Tibial anterior; TNF: Tumor necrosis factor; TRAF: TNF receptor- associated factor; TWEAK: TNF-like weak inducer of apoptosis; VEGF: Vascular volved in TWEAK signaling, suppresses the activation endothelial growth factor. of autophagy in response to denervation and cancer cachexia [75]. These results suggest that TWEAK is in- Competing interests The authors declare they have no competing interests. volved in reducing PPARδ and PGC-1α levels and their target genes leading to mitochondrial dysfunction and Authors’ contributions content. Since no change of PPARδ expression was AK and BGH conceived and designed the study. SS, YO, VM, JS, BGH and SB observed in skeletal muscle of PGC-1α KO mice or the performed experiments and analyzed the data. SS, BGH and AK wrote the manuscript. All authors read and approved the final manuscript. muscle-specific PGC-1α overexpressing transgenic mice [66], PGC-1α is not an upstream regulator for PPARδ. Acknowledgements Indeed, PGC-1α has a synergistic effect with PPARδ We are grateful to Dr. Avi Ashkenazi (Genentech South San Francisco, CA) for providing TWEAK-KO mice. This work was supported by NIH grants agonist to induce the expression of oxidative metabolic R01AR059810 and RO1AG029623 to AK. genes such as mCPT1 and PDK4 [76].Furthermore,it has been shown that PGC-1α directly coactivates the Author details Department of Anatomical Sciences and Neurobiology, University of Louisville mCPT1 and PDK4 promoter via PPARδ in a ligand- School of Medicine, 500 South Preston Street, Louisville, KY 40202, USA. dependent manner [76,77]. Therefore, it is likely that Diabetes and Obesity Center, Institute of Molecular Cardiology, and PGC-1α is a coactivator of PPARδ in terms of up- Department of Medicine, University of Louisville, Louisville, KY 40202, USA. Present address: Gastroenterology Division, University of Pittsburgh School of regulating these metabolic genes. Medicine, Pittsburgh, PA 15261, USA. 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Skeletal MuscleSpringer Journals

Published: Jul 8, 2013

References