Regulation of muscle mass by follistatin and activins

Abstract

Myostatin is a TGF-β family member that normally acts to limit skeletal muscle mass. Follistatin is a myostatin-binding protein that can inhibit myostatin activity in vitro and promote muscle growth in vivo. Mice homozygous for a mutation in the Fst gene have been shown to die immediately after birth but have a reduced amount of muscle tissue, consistent with a role for follistatin in regulating myogenesis. Here, we show that Fst mutant mice exhibit haploinsufficiency, with muscles of Fst heterozygotes having significantly reduced size, a shift toward more oxidative fiber types, an impairment of muscle remodeling in response to cardiotoxin-induced injury, and a reduction in tetanic force production yet a maintenance of specific force. We show that the effect of heterozygous loss of Fst is at least partially retained in a Mstn-null background, implying that follistatin normally acts to inhibit other TGF-β family members in addition to myostatin to regulate muscle size. Finally, we present genetic evidence suggesting that activin A may be one of the ligands that is regulated by follistatin and that functions with myostatin to limit muscle mass. These findings potentially have important implications with respect to the development of therapeutics targeting this signaling pathway to preserve muscle mass and prevent muscle atrophy in a variety of inherited and acquired forms of muscle degeneration.

Figure 1

Effect of heterozygous loss of Fst on muscle mass. Bottom panel shows percent decrease in muscle weights in Fst^+/− mice compared with wild-type mice. Middle panel shows percent decrease in muscle weights in Fst^+/−, Mstn^+/− mice compared with Fst^+/+, Mstn^+/− mice. Top panel shows percent decrease in muscle weights in Fst^+/−, Mstn^−/− mice compared with Fst^+/+, Mstn^−/− mice. All calculations were made from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).

Figure 2

Fiber type analysis. A, Sections of gastrocnemius muscles either stained with hematoxylin and eosin or incubated with antibodies against type I (red), type IIa (green), or type IIb (green) MHC isoforms. Note that muscles of Fst^+/− mice had increased numbers of small, darkly stained fibers, which corresponded to type IIa fibers, as well as increased numbers of type I fibers. B, Fiber type distributions in the EDL and soleus muscles. Note the appearance of type I fibers and the decrease in proportion of type IIa fibers in Fst^+/− EDL muscle. C, Distribution of type I, IIa, and IIb fiber diameters in the gastrocnemius muscle. Solid gray bars represent muscle fibers from wild-type mice, and open black bars represent muscle fibers from Fst^+/− mice. Note the shift in the distributions toward fibers with smaller diameters in muscles of Fst^+/− mice. H&E, hematoxylin and eosin; wt, wild type.

Figure 3

Force measurements in the soleus and EDL muscles of wild-type and Fst^+/− mice. Note the decreased twitch and tetanic force with no change in specific force in muscles of Fst^+/− mice. n.s., Nonsignificant; P > 0.20 wt, wild type.

Figure 4

Impaired muscle regeneration in Fst^+/− mice. A, Sections of gastrocnemius muscles of wild-type and Fst^+/− mice after cardiotoxin-induced injury. At 21 d after injury, note the centrally located nuclei characteristic of regenerating fibers in the wild-type injured muscle (see inset) and the significantly increased extent of fibrosis in the Fst^+/− muscle. At 4 d after injury, note the presence of neonatal myosin in both wild type and Fst^+/− muscle. B, Quantification of amount of fibrosis at 21 d after cardiotoxin-induced injury as assessed by measurement of percent fibrotic area relative to total injured area. H&E, Hematoxylin and eosin; wt, wild type.

Figure 5

Effect of a mutation in Fstl3 and in genes encoding inhibin β-subunits on muscle mass. A, Diagram of Fstl3-targeting strategy. Mice carrying the targeted allele were crossed to EIIa-cre transgenic mice (33) to generate mice in which recombination had occurred between the outside LoxP sites (denoted by triangles), thereby resulting in a mutant allele in which exons 3–5 were completely deleted in the germline. B, Effects of the deletion mutation in Fstl3 either alone or in combination with heterozygous loss of Fst. C, Diagram of InhβC/InhβE-targeting strategy. D, Effect of Inhβ mutations in male mice. In the bar graphs shown in Panels b and d, numbers represent percent increase or decrease in muscle mass relative to wild-type mice and were calculated from the data shown in Table 1. Data from Mstn^+/− mice (21) are shown for comparison. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green). TK, Thymidine kinase.