Functional redundancy of type I and type II receptors in INAUGURAL ARTICLE the regulation of skeletal muscle growth by myostatin and activin A Se-Jin Leea,b,1, Adam Lehara, Yewei Liua, Chi Hai Lyc,d, Quynh-Mai Phama, Michael Michauda, Renata Rydzike, Daniel W. Youngstrome, Michael M. Shenf, Vesa Kaartineng, Emily L. Germain-Leeh,i, and Thomas A. Randoc,d,j aThe Jackson Laboratory for Genomic Medicine, Farmington, CT 06032; bDepartment of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT 06030; cPaul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA 94305; dDepartment of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305; eDepartment of Orthopaedic Surgery, University of Connecticut School of Medicine, Farmington, CT 06030; fDepartment of Genetics and Development, Columbia University, New York, NY 10032; gDepartment of Biologic and Materials Sciences and Prosthodontics, University of Michigan School of Dentistry, Ann Arbor, MI 48109; hDepartment of Pediatrics, University of Connecticut School of Medicine, Farmington, CT 06030; iConnecticut Children’s Center for Rare Bone Disorders, Farmington, CT 06032; and jNeurology Service, VA Palo Alto Health Care System, Palo Alto, CA 94304 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2012. Contributed by Se-Jin Lee, October 13, 2020 (sent for review September 14, 2020; reviewed by Chen-Ming Fan and S. Paul Oh) Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family atrophy following falls and hip fracture surgery, age-related member that normally acts to limit muscle growth. The function of sarcopenia, Charcot–Marie–Tooth disease, and cachexia due to MSTN is partially redundant with that of another TGF-β family chronic obstructive pulmonary disease, end-stage kidney disease, member, activin A. MSTN and activin A are capable of signaling and cancer. through a complex of type II and type I receptors. Here, we inves- The finding that certain inhibitors of MSTN signaling can − − tigated the roles of two type II receptors (ACVR2 and ACVR2B) and increase muscle mass even in Mstn / mice revealed that the two type I receptors (ALK4 and ALK5) in the regulation of muscle function of MSTN as a negative regulator of muscle mass is mass by these ligands by genetically targeting these receptors ei- partially redundant with at least one other TGF-β family member ther alone or in combination specifically in myofibers in mice. We (13, 14), and subsequent studies have identified activin A as one PHYSIOLOGY show that targeting signaling in myofibers is sufficient to cause of these cooperating ligands (15, 16). MSTN and activin A share significant increases in muscle mass, showing that myofibers are many key regulatory and signaling components. For example, the the direct target for signaling by these ligands in the regulation of activities of both MSTN and activin A can be modulated extra- muscle growth. Moreover, we show that there is functional redun- cellularly by naturally occurring inhibitory binding proteins, in- dancy between the two type II receptors as well as between the cluding follistatin (17, 18) and the follistatin-related protein, two type I receptors and that all four type II/type I receptor com- FSTL-3 or FLRG (19, 20). Moreover, MSTN and activin A also binations are utilized in vivo. Targeting signaling specifically in appear to share receptor components. Based on in vitro studies, myofibers also led to reductions in overall body fat content and MSTN is capable of binding initially to the activin type II re- improved glucose metabolism in mice fed either regular chow or a ceptors, ACVR2 and ACVR2B (also called ActRIIA and high-fat diet, demonstrating that these metabolic effects are the ActRIIB) (18) followed by engagement of the type I receptors, result of enhanced muscling. We observed no effect, however, on ALK4 and ALK5 (21). In previous studies, we presented genetic either bone density or muscle regeneration in mice in which sig- evidence supporting a role for both ACVR2 and ACVR2B in naling was targeted in myofibers. The latter finding implies that mediating MSTN signaling and regulating muscle mass in vivo. MSTN likely signals to other cells, such as satellite cells, in addition Specifically, we showed that mice expressing a truncated, to myofibers to regulate muscle homeostasis. dominant-negative form of ACVR2B in skeletal muscle (18) or myostatin | activin | receptors | skeletal muscle Significance yostatin (MSTN) is a secreted signaling molecule that Myostatin and activin A are secreted signaling molecules that Mnormally acts to limit skeletal muscle growth (for review, act to limit skeletal muscle growth. Here, we show that myo- see ref. 1). Mice lacking MSTN exhibit dramatic increases in statin and activin A utilize four receptor components to signal muscle mass throughout the body, with individual muscles directly to muscle fibers. These findings have implications for growing to about twice the normal size (2). MSTN appears to strategies to target this signaling pathway for clinical applica- play two distinct roles in regulating muscle size, one to regulate tions to treat patients with muscle loss. the number of muscle fibers that are formed during development and a second to regulate the growth of those fibers postnatally. Author contributions: S.-J.L. and T.A.R. designed research; S.-J.L., A.L., Y.L., C.H.L., Q.-M.P., The sequence of MSTN has been highly conserved through M.M., R.R., and E.L.G.-L. performed research; S.-J.L., M.M.S., and V.K. contributed new evolution, with the mature MSTN peptide being identical in reagents/analytic tools; S.-J.L., C.H.L., D.W.Y., and T.A.R. analyzed data; S.-J.L. and T.A.R. wrote the paper; and S.-J.L., D.W.Y., E.L.G.-L., and T.A.R. provided project administration. species as divergent as humans and turkeys (3). The function of Reviewers: C.-M.F., Carnegie Institution for Science; and S.P.O., Barrow Neurological MSTN has also been conserved, and targeted or naturally oc- Institute. curring mutations in MSTN have been shown to cause increased Competing interest statement: The authors are in the process of filing a patent applica- muscling in numerous species, including cattle (3–5), sheep (6), tion based on some of the findings reported in this paper. dogs (7), rabbits (8), rats (9), swine (10), goats (11), and humans This open access article is distributed under Creative Commons Attribution-NonCommercial- (12). Numerous pharmaceutical and biotechnology companies NoDerivatives License 4.0 (CC BY-NC-ND). have developed biologic agents capable of blocking MSTN ac- 1To whom correspondence may be addressed. Email: [email protected]. tivity, and these have been tested in clinical trials for a wide This article contains supporting information online at https://www.pnas.org/lookup/suppl/ range of indications, including Duchenne and facioscapulo- doi:10.1073/pnas.2019263117/-/DCSupplemental. humeral muscular dystrophy, inclusion body myositis, muscle www.pnas.org/cgi/doi/10.1073/pnas.2019263117 PNAS Latest Articles | 1of11 Downloaded by guest on September 25, 2021 carrying deletion mutations in Acvr2 and/or Acvr2b (13) have receptor messenger RNA (mRNA) levels in various muscles significantly increased muscle mass. One limitation of the latter showed that we were able to reduce expression significantly in study, however, was that we could not examine the consequence each of the floxed lines, with the residual expression likely rep- of complete loss of both receptors using the deletion alleles, as resenting RNA made either by type I fibers or by nonmuscle cells double homozygous mutants die early during embryogenesis (SI Appendix, Fig. S1). Significantly, in mice in which a given (22). Moreover, the roles that the two type I receptors, ALK4 receptor was targeted in muscle, expression levels of each of the and ALK5, play in regulating MSTN and activin A signaling in other receptors were relatively unchanged, indicating that there muscle in vivo have not yet been documented using genetic ap- was not a compensatory up-regulation of expression of any of the proaches. Here, we present the results of studies in which we other receptors. used floxed alleles for each of the type II and type I receptor As we reported previously (23), targeting Acvr2b alone in genes in order to target these receptors alone and in combination muscle fibers resulted in increases in muscle mass of about 8 to in muscle fibers. We show that these receptors are functionally 12% in females and about 4 to 6% in males depending on the redundant and that signaling through each of these receptors specific muscle examined (Fig. 1A and Table 1). Targeting Acvr2 contributes to the overall control of muscle mass. alone resulted in similar, statistically significant increases in the quadriceps and gastrocnemius but not in the pectoralis or tri- Results ceps. Targeting both receptors simultaneously resulted in much To determine the effect of targeting the two known type II re- more substantial increases in all muscles examined, with the ceptors for MSTN specifically in skeletal muscle, we generated greatest effect being seen in the quadriceps (58 and 50% in fe- mice carrying floxed alleles for both Acvr2 and Acvr2b (23, 24) males and males, respectively) and gastrocnemius (72 and 62% and then targeted these alleles using a transgene expressing cre in females and males, respectively). These results show defini- recombinase from a myosin light chain promoter/enhancer tively that Acvr2 and Acvr2b are functionally redundant with one (Myl1-cre) (25), which we showed to be expressed by skeletal another with respect to limiting muscle mass and that targeting muscle fibers but not by satellite cells (23).