Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

Abstract

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Keywords: activin; myostatin; receptors; skeletal muscle.

Fig. 1.

Effect of targeting type II and type I receptors in myofibers on muscle weights. (A and B) Relative weights of pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius/plantaris (green) muscles in mice in which Acvr2 and/or Acvr2b (A) or Alk4 and/or Alk5 (B) were targeted. Numbers are expressed as percent increase/decrease relative to the same receptor genotypes but in the absence of Myl1-cre. (C) Gastrocnemius/plantaris muscle weights of individual wild-type C57BL/6 and Mstn^−/− mice or individual mice in which Acvr2/Acvr2b or Alk4/Alk5 were targeted in myofibers. Bars indicates mean values. (D) Relative muscle weights of mice in which an individual type II receptor (Acvr2 or Acvr2b) was targeted along with an individual type I receptor (Alk4 or Alk5). (E and F) Relative muscle weights of mice in which an individual type II or type I receptor was targeted along with Cfc1b (E) or Mstn (F). Numbers are expressed as percent increase/decrease relative to the same receptor genotypes but in the absence of Myl1-cre, and the color code is the same as in A and B. The numbers shown in A, B, D, and F were calculations based on muscle weights shown in Tables 1–3, which also contain the numbers of mice in each group. ^aP < 0.001 vs. cre⁻; ^bP < 0.01 vs. cre⁻; ^cP < 0.05 vs. cre⁻; ^dP < 0.001 vs. Mstn fl/fl, cre⁺; ^eP < 0.01 vs. Mstn fl/fl, cre⁺; ^fP < 0.05 vs. Mstn fl/fl, cre⁺.

Fig. 2.

Lack of effect of targeting Acvr2 and Acvr2b in myofibers on muscle regeneration following chemical injury. (A–C) Distribution of myofiber CSAs (A), mean CSAs (B), and number of Pax7⁺ cells (C) in Acvr2 fl/fl, Acvr2b fl/fl mice with or without Myl1-cre either uninjured or 5 or 21 DPI.

Fig. 3.

Total body fat content by DXA analysis, plasma leptin levels, fasting blood glucose levels, and fasting plasma insulin levels in 1-y-old mice lacking MSTN and mice in which both type II receptors were targeted in myofibers. Numbers of mice in each group are shown underneath the bars.

Fig. 4.

Effect of a high-fat diet on Mstn^−/− mice and mice in which Acvr2 and Acvr2b have been targeted in myofibers. (A) Weight gain in male mice placed on a high-fat diet starting at 12 wk of age. (B and C) Fasting blood glucose levels (B) and GTTs (C) in 12 wk-old male mice on standard diets or after placement on a high-fat diet for 4 wk. In C, the numbers of mice in each group are the same as shown in A.

Fig. 5.

Lack of bone effects of targeting Acvr2 and Acvr2b in myofibers. (A, Top) DXA analysis of wild-type C57BL/6 mice either uninjected (n = 9) or injected weekly with the ACVR2B/Fc decoy receptor at a dose of 10 mg/kg (n = 8) starting at 10 wk of age. (A, Bottom) DXA analysis of Acvr2 flox/flox, Acvr2b flox/flox mice with (n = 8) and without (n = 12) the Myl1-cre transgene at the same ages as in A, Top. *P < 0.05, **P < 0.001. (B) MicroCT images of femurs taken from these same mice at 16 wk of age. (C) Bone volume/total volume fraction, trabecular thickness, trabecular number, apparent density, and cortical thickness of femurs and L4 and L5 vertebrae determined by microCT analysis in these mice at 16 wk of age. Numbers of mice in each group are shown underneath the bars.