THE MUSCLE-HYPERTROPHIC EFFECT OF IS ADDITIVE TO THE HYPERTROPHIC EFFECT OF MYOSTATIN SUPPRESSION KYUNG HO KIM, MS,1 YONG SOO KIM, PhD,2 and JINZENG YANG, PhD2 1 Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii, USA 2 Department of Human Nutrition Food and Animal Sciences, University of Hawaii, 1955 East–West Road, Honolulu, Hawaii 96822, USA Accepted 18 October 2010 ABSTRACT: Introduction: In this study we investigated the terminal prodomain (MSTN-pro) and a C-terminal b combined effect of myostatin (MSTN) suppression and -agonist active form. The cleaved MSTN-pro remains non- (clenbuterol) administration on muscle hypertrophy and the phos- phorylation of muscle 4E-BP1 and p70S6k, two downstream covalently associated with the active MSTN and effectors of the Akt/mTOR anabolic pathway. Methods: Female inhibits its biological activity by preventing binding heterozygous MSTN-prodomain transgenic mice (an MSTN to its receptor.2,14,15 The important role of MSTN- suppression model) and wild-type littermates were given 0 or 20 ppm of clenbuterol (CL) in their drinking water, and muscle pro in regulating MSTN activity was demonstrated samples were collected at 1 and 2 weeks after treatment. by a dramatic increase in skeletal muscle mass in Results: CL increased body and muscle mass in both geno- transgenic mice that overexpress MSTN-pro.16,17 types. Levels of phosphorylated muscle 4E-BP1 and p70S6k were higher in MSTN-prodomain transgenic mice than in wild- MSTN binds to activin receptor type IIB type mice. CL increased the phosphorylation of 4E-BP1 and (ActRIIB) to exert its biological activity.15,18,19 The Conclusions: p70S6k in both genotypes. The muscle-hyper- binding of MSTN to its receptor leads to phospho- trophic effect of CL is additive to the effect of MSTN suppres- sion. The combination of MSTN suppression and treatment with rylation of transcription factors Smad2 and Smad3 b-agonists may be an effective therapeutic approach to combat and complex formation with Smad4, resulting in muscle-wasting conditions. nuclear translocation of the Smad complex and Muscle Nerve 43: 700–707, 2011 consequent regulation of transcription of down- stream target genes.20–22 Although the activation Loss of muscle mass can adversely affect human of Smad signaling by MSTN has been well charac- health and viability, and molecules involved in the terized, very little is known about the molecular regulation of skeletal muscle growth are valuable mechanisms that connect Smads activation to mus- targets in developing therapeutic strategies for cle protein metabolism. Recent studies, however, muscle-wasting conditions. Myostatin (MSTN) and indicate there is cross-talk between Smads signaling some b- agonists, such as clenbuterol and the Akt/mammalian target-of-rapamycin (CL), have emerged as powerful regulators of skel- (mTOR) anabolic pathway. For example, muscle etal muscle growth and mass.1,2 Both CL adminis- fiber atrophy induced by Smad2/3 activation was tration and MSTN inhibition have individually prevented by the presence of constitutively active demonstrated their therapeutic potential for pre- Akt.23 It was also reported that Akt phosphoryla- venting or reversing muscle loss in various condi- tion was inhibited by MSTN in human myotubes, tions that lead to muscle atrophy or wasting.3–13 and the effect was dependent upon the presence MSTN, a member of the transforming growth of Smad2 and 3.24 The mTOR, a downstream tar- factor-beta (TGF-b) superfamily of growth and dif- get of Akt, is an integral control point for various ferentiation factors, is a potent negative regulator signals involved in muscle hypertrophy and atro- of myogenesis during development, and it also phy.25–27 Activation of mTOR promotes the phos- inhibits postnatal skeletal muscle growth.2 MSTN is phorylaion of two key downstream effectors that synthesized mainly in skeletal muscle as a precur- are closely associated with translational regulation sor form that is proteolytically cleaved into an N- of protein synthesis: p70S6 kinase (p70S6k) and eukaryotic initiation factor 4E binding protein 1 28 Abbreviations: 4E-BP1, eukaryotic initiation factor 4E binding protein 1; (4E-BP1). Recent results have indicated that myo- ActRIIB, activin receptor type IIB; AC, adenyl cyclase; ANOVA, analysis of variance; CL, clenbuterol; DTT, dithiothreitol; EDL, extensor digitorum lon- statin inhibits muscle hypertrophy in part through gus; EDTA, ethylene-diamine tetraacetic acid; EGTA, ethylene-glycol tetra- inhibition of protein synthesis mediated by the acetic acid; eIF4E, elongation initiation factor 4E; MSTN, myostatin; 29–31 MSTN-pro, myostatin prodomain; mTOR, mammalian target of rapamycin; Akt/mTOR pathway. PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reac- It is well documented that administration of b- tion; PMSF, phenylmethylsulfonylfluoride; PVDF, polyvinylidene difluoride; rpS6, ribosomal protein S6; SDS, sodium dodecylsulfate; TBS, Tris-buf- adrenergic agonists such as CL induces a dramatic fered saline; TBST, Tris-buffered saline plus Tween 20; TGF-b, transform- increase in skeletal muscle growth in various mam- ing growth factor-beta; TSC, tuberous sclerosis complex 5,32,33 Key words: b-agonist, clenbuterol, mTOR pathway, muscle growth, malian species. The growth- and muscle-pro- myostatin, myostatin prodomain moting action of b-adrenergic agonists is short-lived, Correspondence to: Y. S. Kim; e-mail: [email protected] as the effect disappears during prolonged treat- VC 2010 Wiley Periodicals, Inc. ment.5,32,34,35 The temporal nature of the growth- Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10. 1002/mus.21950 promoting responsiveness to b-adrenergic agonists is

700 MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 caused by receptor desensitization operating at both Previous studies have demonstrated the effectiveness the receptor level and downstream from receptors.36 of the 20-ppm dose (about 5 mg/kg body weight/ b b Studies with transgenic mice lacking the 1-, 2-, or day) of CL via drinking water on promoting body b b 40 both 1-and 2-adrenergic receptors have indicated and muscle growth. At 7 and 14 days after admin- that b-adrenergic agonists mediate the hypertrophic istration, animals were euthanized (about half in b effect through binding to the 2-adrenergic recep- each killing) by CO2 asphyxiation, and gastrocne- 37 b tor. The 2- is a member of mius, plantaris, soleus, and extensor digitorum lon- the G-protein–coupled receptor superfamily, and gus muscles of the hind leg were rapidly dissected ligand binding induces GDP-to-GTP exchange on out, weighed, snap-frozen in liquid nitrogen, and the Ga subunit and subsequent activation of Ga. stored at 80C until analysis. At the time of killing, b Thus, the classic signaling pathway of 2-adrenergic 7 days after administration, mice were randomized receptor involves activation of adenyl cyclase (AC) to euthanasia groups so the mean starting body and subsequent formation of cAMP.36 The link weights of each euthanasia group within the geno- b a between the classic 2-adrenergic receptor/G /AC/ type were almost equal. cAMP signaling pathway and the change in protein metabolism induced by the administration of b-adre- Genotyping. DNA was extracted from tail samples nergic agonists has remained elusive, but recent by phenol/chloroform extraction after solubiliza- results indicate that, like MSTN, b-adrenergic ago- tion of the tissue in a Tris buffer (50 mM, pH 8.0) nists may induce muscle hypertrophy via the Akt/ containing 0.5% sodium dodecylsulfate (SDS), 0.1 mTOR signaling pathway.38,39 M ethylene-diamine tetraacetic acid (EDTA), and Although there have been numerous demon- proteinase K (0.7 mg/ml). The extracted DNA was strations of the muscle growth–promoting effect of subjected to polymerase chain reaction (PCR) CL administration or MSTN suppression, the effect amplification with a primer set unique to the trans- of a combination of CL administration and MSTN genic mice. The forward and reverse primers were 50-GACAGCAGTGATGGCTCT-30 and 50-CTTGTCA inhibition on skeletal muscle growth has not been 0 investigated. Therefore, we designed an experi- TCGTCGTCCTTGTAATCGGTAC-3 , respectively. PCR conditions were the same as those described ment in which CL was fed to wild-type and MSTN- 17 suppressed mice to assess the combined effect of previously. The PCR products were subjected to CL administration and MSTN suppression on body electrophoresis in a 1.2% agarose gel and stained and muscle growth. At the same time, we exam- with ethidium bromide to examine for the pres- ined the phosphorylation of 4E-BP1 and p70S6k, ence of the transgenic PCR product. two key downstream effectors of the mTOR path- Measurement of Skeletal Muscle DNA and RNA Con- way, during treatment. centration. Plantaris muscle samples were homog- enized in 20 volumes of ice-cold distilled water, then duplicate aliquots were added to 0.5 volume METHODS of 0.6N ice-cold perchloric acid. The aforemen- Animals and Sample Collection. All procedures tioned mixtures were used to separate DNA and using experimental animals were approved by the RNA following the procedure of Munro and institutional animal care and use committee at the Fleck.41 RNA concentration was measured by University of Hawaii. All mice were maintained in absorption at 260 nm, and DNA concentration was temperature- and humidity-controlled conditions measured by a fluorescence spectrophotometry with a 12-h light–dark cycle and had free access to method using Hoechst H33258 dye, as described food and water. For MSTN suppression, we used a previously.42 Protein concentrations in muscle ho- transgenic mouse strain that overexpresses the mogenates were determined using the modified MSTN-prodomain (MSTN-pro) and exhibits a signif- Lowry protein assay kit (Pierce, Rockford, Illinois) icant increase in skeletal muscle mass, regardless of to estimate muscle protein content. age and gender.17 To produce the heterozygous MSTN-pro transgenic and wild-type littermate geno- Western Blot Analysis of Phosphorylation of 4E-BP1 typesusedinthisstudy,femaleB6SJLF1micewere and p70S6k. The procedure described by Dreyer mated to heterozygous MSTN-pro transgenic male et al.43 was used for Western blot analysis. Briefly, mice. Pups were weaned at 28 days of age, and tail extensor digitorum longus muscle samples were ho- tissue samples were collected for genotyping at the mogenized at 4C in 9 volumes of ice-cold lysis time of weaning. At 35 days of age, female mice buffer [50 mM Tris-HCl (pH 7.4), 250 mM manni- were separated by their genotypes, and randomly tol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 assigned to one of the two groups: 0 or 20 ppm CL. mM EDTA, 1 mM ethylene-glycol teraacetic acid CL was administered in the drinking water for 14 (EGTA), 1% Triton X-100, 1 mM dithiothreitol days, and body weights were monitored periodically. (DTT), 1 mM benzamidine, 0.1 mM

MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 701 phenylmethylsulfonylfluoride (PMSF), and 5 lg/ml soybean trypsin inhibitor]. The homogenates were centrifuged at 6000g for 10 min at 4C, and aliquots of the supernatants were saved for Western blot detection of p70S6k. For Western blot detection of 4E-BP1, 50 ll of the aforementioned supernatants were heated at 100C for 10 min, centrifuged for 30 min at 10,000g and 4C, and the supernatants were saved. Protein concentrations in supernatants were determined using the modified Lowry protein assay kit (Pierce) to estimate muscle protein content. The protein concentration of supernatants was not signif- icantly (P < 0.01) affected by the genotype and CL administration, and thus equal volumes of superna- FIGURE 1. tants were loaded per lane with sodium dodecylsul- Effect of CL administration on body weight gain in wild-type and MSTN-pro mice. Solid and dotted lines indicate 0- fate–polyacrylamide gel electrophoresis (SDS-PAGE). and 20-ppm CL administration, respectively, to wild-type (W, Twenty microliters of the protein extracts for open circles) and MSTN-pro mice (M, filled circles). Values are p70S6k and 4E-BP1 were subjected to 7.5% and expressed as mean 6 SEM. (A) Genotype effect (***P < 15% SDS-PAGE, respectively, and then proteins 0.001). (B) CL effect (**P < 0.01; ***P < 0.001; NS, not were transferred to polyvinylidene difluoride significant). (PVDF) membranes. The blots were blocked for 3 h at room temperature in Tris-buffered saline (20 mM tion (P < 0.001; Fig. 1), and they grew significantly Tris-HCl, 150 mM NaCI at pH 7.5) containing 3% faster than wild-type mice over the 14-day period bovine serum albumin (BSA), followed by incuba- (P < 0.001, Fig. 1). The growth-promoting effect tion with primary antibody in Tris-buffered saline of CL was evident in both wild-type and MSTN-pro plus 0.01% Tween 20 (TBST, 20 mM Tris-HCl, 150 mice (Fig. 1), demonstrating that the growth-pro- mM NaCl at pH 7.5) at 4C overnight. The primary moting effect of CL is additive to the growth-pro- antibodies were rabbit anti–phospho-p70S6k moting effect of MSTN suppression. The temporal (Thr389; 1:1000; Cell Signaling, Beverly, Massachu- effect of CL on body weight gain was evident in setts) and rabbit anti–phospho-4E-BP1 (Thr37/46; both genotypes (Fig. 1): the increase in body 1:1000; Cell Signaling). The blots were washed twice weight gain occurred at up to 7–10 days of CL with TBST and incubated with horseradish peroxi- administration, and no further increase was dase–conjugated anti-rabbit antibody (1:2500; Invi- observed thereafter. trogen, Carlsbad, California) in TBST for 2 h at Skeletal muscle weights of wild-type and MSTN- room temperature. After washing, the membrane pro mice as affected by CL administration for 7 was developed with an enhanced chemiluminescent and 14 days are summarized in Table 1. As reagent (ECL Plus Western Detection System; GE expected, gastrocnemius, plantaris, extensor digito- Healthcare Biosciences, Pittsburgh, Pennsylvania), rum longus (EDL), and soleus muscles of MSTN- followed by apposition of the membrane to autora- pro mice were significantly (P < 0.001) heavier diographic films (Hyperfilm ECL; Kodak). After than those of wild-type mice during the experi- obtaining an appropriate image, band intensity was mental period. The protein concentration of plan- quantitatively analyzed using Quantity One analysis taris muscle of MSTN-pro mice was greater than software (Bio-Rad, Hercules, California). that of wild-type mice (P < 0.001). CL administra- tion significantly increased the four muscle weights Statistical Analysis. All values are expressed as of both MSTN-pro and wild-type mice after both mean 6 SEM. Two-way analysis of variance the 7- and 14-day administration periods. CL (ANOVA) was performed using Prism5 software administration also increased the protein concen- (GraphPad, San Diego, California) to examine the tration of plantaris muscle in both MSTN mice effects of genotype, CL, and interaction on body and wild-type mice (P < 0.001), leading to the and muscle weights; muscle DNA and RNA con- highest muscle protein concentration in CL-fed centrations; and the levels of phosphorylation of MSTN-pro mice. When concentrations of soluble 4E-BP1 and p70S6k. proteins in lysis buffer were measured from EDL RESULTS muscle during Western blot analysis, genotype and Effects of Clenbuterol on Body and Muscle Weights of CL administration had no effect on soluble pro- Wild-Type and MSTN-Pro Mice. Consistent with our tein concentration. Thus, it is postulated that the previous report,17 MSTN-pro mice were heavier increase in total muscle protein concentration in than wild-type mice at the time of CL administra- MSTN-pro mice or by CL administration is

702 MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 Table 1. Muscle weights of wild-type and MSTN-pro mice as affected by CL administration. Wild-type MSTN-pro Significance 0 ppm 20 ppm 0 ppm 20 ppm GT CL GT CL 7 days after administration Number of animals 12 10 15 15 Initial body wt (g) 17.7 (0.47) 17.7 (0.37) 18.5 (0.49) 18.7 (0.37) P < 0.05 NS NS Final body wt (g) 18.5 (0.44) 19.3 (0.40) 19.7 (0.47) 21.8 (0.40) P < 0.001 P < 0.01 NS Gastrocnemius wt (mg) 87.6 (3.73) 95.0 (3.41) 110.8 (4.30) 136.6 (3.51) P < 0.001 P < 0.001 P < 0.05 Plantaris wt (mg) 9.78 (0.517) 10.41 (0.676) 12.58 (0.451) 15.92 (0.549) P < 0.001 P < 0.001 P < 0.05 EDL wt (mg) 6.87 (0.511) 7.51 (0.367) 9.39 (0.397) 11.22 (0.229) P < 0.001 P < 0.01 NS Soleus wt (mg) 4.15 (0.381) 5.93 (0.377) 4.85 (0.295) 6.13 (0.362) NS P < 0.001 NS Muscle protein (%)* 21.1 (0.14) 25.9 (0.20) 24.7 (0.17) 28.5 (0.20) P < 0.001 P < 0.001 NS 14 days after administration Number of animals 12 14 11 14 Initial body wt (g) 17.7 (0.36) 17.6 (0.30) 18.8 (0.56) 18.6 (0.37) P < 0.01 NS NS Final body wt (g) 19.1 (0.34) 20.6 (0.24) 21.6 (0.60) 22.8 (0.37) P < 0.001 P < 0.01 NS Gastrocnemius wt (mg) 84.9 (2.75) 106.6 (2.49) 119.1 (4.10) 151.1 (3.99) P < 0.001 P < 0.001 NS Plantaris wt (mg) 9.53 (0.340) 13.24 (0.498) 13.85 (0.429) 18.68 (0.722) P < 0.001 P < 0.001 NS EDL wt (mg) 6.33 (0.339) 9.07 (0.307) 8.59 (0.605) 11.16 (0.338) P < 0.001 P < 0.001 NS Soleus wt (mg) 4.46 (0.287) 5.97 (0.229) 5.13 (0.192) 6.96 (0.311) P < 0.01 P < 0.001 NS Muscle protein (%)* 20.1 (0.12) 25.2 (0.17) 24.1 (0.14) 27.4 (0.15) P < 0.001 P < 0.001 NS

Values are expressed as mean (SEM). CL, clenbuterol; GT, genotype; wt, weight; EDL, extensor digitorum longus; NS, not significant. *Muscle protein concentration was measured in plantaris muscle using the modified Lowry method. probably due to an increase in insoluble myofibril- effect disappeared at 14 days after administration. lar proteins. The increases in weight gain of the Likewise, CL administration significantly (P < gastrocnemius (23%) and plantaris (27%) at 7-day 0.001) decreased muscle DNA concentrations of CL administration in MSTN-pro mice were signifi- both wild-type and MSTN-pro mice, leading to a cantly (P < 0.05) greater than those in wild-type lowest DNA concentration in MSTN-pro mice mice (8.5% for gastrocnemius and 6.4% for planta- treated with CL. The extent of decrease in muscle ris). However, at 14 days after CL administration, DNA concentration was greater at 14 days after CL the increases in weight gain of the gastrocnemius administration (13.9% in wild-type and 9.5% in (26.9%) and plantaris (34.9%) by CL in MSTN-pro MSTN-pro) than at 7 days of CL administration mice were not different from those in the wild-type (5.3% in wild-type and 5.9% in MSTN-pro). mice (25.6% for gastrocnemius and 38.9% for Because CL administration lowered DNA concen- plantaris), suggesting that a synergistic effect of tration, it significantly (P < 0.05) increased the MSTN suppression and CL administration on the muscle RNA/DNA ratio in both wild-type and growth of these muscles may occur only during the MSTN-pro mice, resulting in the highest RNA/ early period of CL administration. DNA ratio in the MSTN-pro mice treated with CL.

Effects of CL on Muscle DNA and RNA Concentrations Effect of CL Administration on Phosphorylation of in Wild-Type and MSTN-Pro Mice. DNA and RNA Muscle 4E-BP1 and p70S6k in Wild-Type and MSTN- concentrations in plantaris muscle were measured to Pro Mice. The phosphorylation of 4E-BP1 and/or determine indirectly the myonuclei domain size and p70S6k has been shown to play an important role protein synthetic capacity, respectively (Table 2). No in regulating protein synthesis, and thus the levels significant difference in muscle RNA concentration of phosphorylation of these proteins in EDL was observed between wild-type and MSTN-pro mice muscles were examined using Western blot analysis during the experimental period. Muscle DNA con- (Figs. 2 and 3). In our Western blot analysis of the centration of the MSTN-pro mice was significantly phosphorylation of 4E-BP1 and p70S6k, we did not (P < 0.001) lower than that of wild-type mice during perform immunoblot against the total amount of the experimental period (5.9% at day 7 and 7.6% at these proteins. Accordingly, the levels of phospho- day 14). As a result of the lower muscle DNA con- rylation expressed in this study represent the phos- centration, the muscle RNA/DNA ratio of MSTN- phorylated amount of those proteins per unit tis- pro mice was significantly (P < 0.05) greater than sue volume, not the phosphorylated proportion of that of wild-type mice. these proteins. Thus, these results cannot reveal CL administration significantly (P < 0.05) whether the changes in phosphorylated 4E-BP1 or increased muscle RNA concentrations of both the p70S6k observed in this study occurred in the ab- wild-type and MSTN-pro mice at 7 days, but the sence of changes in the total amount of these

MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 703 Table 2. Muscle DNA and RNA concentrations in wild-type and MSTN-pro mice as affected by CL administration. Wild-type MSTN-pro Significance 0 ppm 20 ppm 0 ppm 20 ppm GT CL GT CL 7 days after administration Number of animals 12 10 15 15 DNA (mg/g wet muscle) 1.22 (0.010) 1.15 (0.015) 1.14 (0.30) 1.08 (0.012) P < 0.001 P < 0.001 NS RNA (mg/g wet muscle) 1.13 (0.044) 1.27 (0.030) 1.24 (0.031) 1.36 (0.031) NS P < 0.05 NS RNA/DNA ratio 1.01 (0.035) 1.11 (0.032) 1.09 (0.037) 1.27 (0.036) P < 0.01 P < 0.001 NS 14 days after administration Number of animals 12 14 11 14 DNA (mg/g wet muscle) 0.95 (0.031) 0.87 (0.026) 0.88 (0.030) 0.79 (0.012) P < 0.01 P < 0.01 NS RNA (mg/g wet muscle) 1.13 (0.032) 1.15 (0.027) 1.15 (0.022) 1.15 (0.019) NS NS NS RNA/DNA ratio 1.22 (0.072) 1.35 (0.055) 1.32 (0.035) 1.45 (0.034) P < 0.05 P < 0.05 NS

Values are expressed as mean (SEM). CL, clenbuterol; NS, not significant; GT, genotype. proteins. However, a recent study showed that The increase in phosphorylated 4E-BP1 by CL myostatin blockage did not significantly affect mus- administration was greater at 7 days of CL adminis- cle concentrations of p70S6k or 4E-BP1,31 indicat- tration (38.2% for wild-type and 69.2% for MSTN- ing that modulation of MSTN activity does not pro) than at 14 days of CL administration (24.0% affect the muscle concentration of these proteins. for wild-type and 13.9% for MSTN-pro). Likewise, The levels of both phosphorylated 4E-BP1 and the increase in phosphorylated p70S6k by CL p70S6k were significantly (P < 0.001) greater in administration was greater at 7 days of CL adminis- MSTN-pro mice than in wild-type mice during the tration (70.1% for wild-type and 41.7% for MSTN- experimental period. CL administration significantly pro) than at 14 days of CL administration (22.2% (P < 0.001) increased the levels of phosphorylated for wild-type and 18.5% for MSTN-pro). This result 4E-BP1 and p70S6k regardless of genotype, leading suggests that, like other cellular responses, the effect to the highest levels of phosphorylation of these pro- of CL on phosphorylation of 4E-BP1 and p70S6k is teins in MSTN-pro mice treated with CL. This result diminished during long-term administration of CL. suggests that both MSTN suppression and CL administration stimulate the phosphorylation of 4E- DISCUSSION BP1 and p70S6k, two downstream effectors of the The results from this study show that the muscle mTOR pathway, and the phosphorylations of 4E- hypertrophic effect of CL is additive to the muscle BP1 and p70S6k are summed when the MSTN sup- hypertophic effect of MSTN suppression, an pression and CL administration are combined. important implication for future therapeutic

FIGURE 2. Effect of CL administration on 4E-BP1 phosphorylation at Thr37/46 in EDL muscles of wild-type and MSTN-pro mice. Rep- resentative immunoblot images are shown above the bar graph. Each lane sequentially represents the group in the bar graph. Bar graph shows image intensity values, expressed as mean 6 SEM. Sample numbers correspond to those shown in Tables 1 and 2. ***P < 0.001.

704 MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 FIGURE 3. Effect of CL administration on p70S6k phosphorylation at Thr389 in EDL muscles of wild-type and MSTN-pro mice. Repre- sentative immunoblot images are shown above the bar graph. Each lane sequentially represents the group in the bar graph. Bar graph shows image intensity values expressed as mean 6 SEM. Sample numbers correspond to those shown in Tables 1 and 2. *P < 0.05; ***P < 0.001 approaches aimed to reverse muscle-wasting condi- mius muscle of MSTN-pro mice was not different tions. Our study, however, did not examine the from that of wild-type mice,17 indicating that the changes in muscle function during the hypertro- increase in muscle mass in the MSTN-pro group phy induced by the combination of CL administra- was mostly due to an increase in fiber size (hyper- tion and MSTN suppression, and therefore future trophy) and not an increase in fiber number studies will need to investigate muscle functionality (hyperplasia). Taken together, these results suggest as affected by the combination of b-agonist treat- that the myonuclear domain size of MSTN-pro ment and MSTN suppression. mice probably was not maintained during the The temporal nature of the growth-promoting enhanced muscle hypertrophy induced by MSTN effect of CL was observed in both wild-type and suppression. b-Adrenergic agonists have been MSTN-pro mice, suggesting that the desensitization shown to increase muscle RNA concentration dur- process of muscle b-adrenoceptors appears not be ing the early period of administration and influenced by chronic suppression of MSTN activ- decrease muscle DNA concentration.32 This is con- ity. It also appears that, during the early period of sistent with our results that show CL administra- CL administration, CL may act synergistically with tion for 7 days increased muscle RNA concentra- the hypertrophic effect of MSTN suppression, tions and decreased muscle DNA concentrations of because the increase in body and muscle weight both the wild-type and MSTN-pro mice, resulting gains of MSTN-pro mice by CL was significantly in an increased RNA/DNA ratio by CL administra- greater than that of wild-type mice at 7 days after tion. This suggests that, like the MSTN suppres- CL administration, whereas the increase at 14 days sion, myonuclear domain size was not maintained of administration was similar between the two during the CL-induced muscle hypertrophy. genotypes. The mTOR pathway has emerged as a critical These results show that the muscle DNA con- mediator of cell growth and proliferation by inte- centration in MSTN-pro mice was lower than that grating signals from growth factors, nutrients, and of wild-type mice but without a difference in RNA the energy status of cells.44 The serine/threonine concentration, resulting in higher RNA/DNA kinase mTOR interacts with multiple protein part- ratios in MSTN-pro mice. These results are consist- ners, forming two distinctive multiprotein com- ent with a previous study that showed muscle DNA plexes, mTORC1 and mTORC2. The mTORC1 concentration and RNA/DNA ratio of MSTN-defi- complex mediates functions that are sensitive to cient transgenic mice are lower and higher, respec- rapamycin, such as protein translation, whereas tively, than those of wild-type mice, without differ- mTORC2 mediates functions not sensitive to rapa- ences in muscle RNA concentration between the mycin. mTORC1 activation by Akt induces the two groups.30 The total fiber number in gastrocne- phosphorylation of two downstream effectors,

MSTN and CL Interaction and Muscle Growth MUSCLE & NERVE May 2011 705 ribosomal protein p70S6k and 4E-BP1, a suppres- p70S6k phosphorylation, CL treatment also pro- sor of the cap binding protein eIF4E, resulting in moted the phosphorylation of Akt.38 Furthermore, increased protein translation.44 Muscle growth also the CL effect was suppressed by rapamycin.38 In appears to be regulated by the mTOR pathway, as agreement with the results of previous studies, demonstrated by studies showing that the hyper- increased levels of phosphorylated 4E-BP1 and trophic response induced by overload or regenerat- p70S6k were observed by CL administration in our ing muscle growth was blocked by rapamycin, an study, supporting the role of the Akt/mTOR path- inhibitor of mTOR.25,45 Anabolic stimuli, such as way in b-–induced skeletal muscle overloading, resistance exercise, and essential hypertrophy. Notably, our result demonstrated that amino acid ingestion, have been shown to increase CL administration increased the phosphorylation of the phosphorylation of p70S6k and 4E-BP1 along 4E-BP1 and p70s6k not only in wild-type mice but with a corresponding increase in protein synthe- also in MSTN-pro mice, resulting in a summation of sis.25,43,46–48 Conversely, atrophying muscles have the levels of phosphorylation of the two molecules. decreased levels of phosphorylation of the down- The findings further show that skeletal muscle stream targets of mTOR.25,49 masses of treatment groups were closely related to In our study we observed that levels of phosphoryl- the levels of phosphorylation of 4E-BP1 and p70S6k ated 4E-BP1 (Thr37/46) and p70S6k (Thr389)were and the RNA/DNA ratio of treatment groups. It is greater in MSTN-pro transgenic mice compared with thus suggested that the post-receptor signaling of wild-type mice, suggesting that MSTN also regulates both MSTN suppression and b-receptor activation muscle mass probably through the mTOR pathway. by CL induces the phosphorylation of 4E-BP1 and This result is in agreement with a recent report that p70S6k to enhance protein translation, and the showed MSTN overexpression in mice skeletal muscle combination of MSTN suppression and b-receptor by electrotransfer of MSTN-expression vector attenu- activation induces the summation of 4E-BP1 and ated the phosphorylation of 4E-BP1 (Thr37/46).29 In p70S6k phosphorylation and subsequent protein addition to the decrease in 4E-BP1 phosphorylation, translation. In this study we did not extensively the study also observed a decrease in the phosphoryla- examine signaling intermediates of the Akt/mTOR tion of TSC2 (Thr1462), ribosomal protein S6 (Ser235/ pathway, and more studies are needed to under- 236), and Akt (Thr308) in response to MSTN overex- stand the role of the Akt/mTOR pathway in the con- pression, supporting the role of the Akt/mTOR sig- vergence of MSTN and b-adrenergic agonist signal- naling pathway in MSTN regulation of skeletal muscle ing to 4E-BP1 and p70S6k phosphorylation. mass. Interestingly, in another study using a different In conclusion, the results of this study demon- model, where MSTN activity was suppressed by the strate that the skeletal muscle hypertrophic effect of administration of anti-MSTN antibody in mice,31 CL is additive to the hypertrophic effect of MSTN sup- MSTN suppression did not affect the phosphorylation pression. Although CL is not approved for use in of Akt (Thr308 and Ser473) and 4E-BP1 (Thr36/45, humans, a few trials with , a b-adrenergic Ser64,andThr69), but it increased the phosphoryla- agonist approved for human use, have shown a lim- tion of p70S6k (Thr389) as well as rpS6, a substrate for ited positive effect in muscular dystrophy patients.4,5,13 p70S6k. In that study it was also noted that rapamy- A new generation of adrenergic agonists, including cin, a pharmacological blocker of mTOR, eliminated and , appear to have more the phosphorylation of p70S6k and rpS6, but it did powerful skeletal muscle anabolic effects, with fewer not block the increase in muscle protein synthetic side effects.50–52 Our findings thus indicate the poten- rate induced by MSTN suppression.31 These results tial of the combination of MSTN suppression and suggest that MSTN regulation of muscle protein syn- treatment with b-agonists in the prevention or reversal thesis can be independent of Akt/mTOR signaling of muscle-wasting conditions. The results on 4E-BP1 even though we cannot rule out the possibility that and p70S6k phosphorylation also show that the path- the discrepancy resulted from differences in MSTN ways of MSTN and CL signaling converge to the phos- modulation in conjunction with sampling factors. phorylation of 4E-BP1 and p70S6k, two downstream The mTOR pathway is known to take diverse signals effectors of the Akt/mTOR anabolic pathway, to regu- and produce a myriad of responses,44 and thus fur- late skeletal muscle hypertrophy. ther studies are needed to clearly define the details of The authors thank Dr. Michael Dunn for his critical review of this the involvement of the Akt/mTOR pathway in MSTN manuscript. This study was supported by a USDA–TSTAR grant signaling. (2004-34135-15196). Like MSTN, b-adrenergic agonists also induce muscle hypertrophy through the Akt/mTOR signal- REFERENCES ing pathway.38,39 For example, CL treatment in 70 1. Lynch GS,Ryall JG. 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