THE MUSCLE-HYPERTROPHIC EFFECT OF CLENBUTEROL 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-adrenergic 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-adrenergic receptor 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 À80C 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