Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway

Abstract

Myostatin and activin A are structurally related secreted proteins that act to limit skeletal muscle growth. The cellular targets for myostatin and activin A in muscle and the role of satellite cells in mediating muscle hypertrophy induced by inhibition of this signaling pathway have not been fully elucidated. Here we show that myostatin/activin A inhibition can cause muscle hypertrophy in mice lacking either syndecan4 or Pax7, both of which are important for satellite cell function and development. Moreover, we show that muscle hypertrophy after pharmacological blockade of this pathway occurs without significant satellite cell proliferation and fusion to myofibers and without an increase in the number of myonuclei per myofiber. Finally, we show that genetic ablation of Acvr2b, which encodes a high-affinity receptor for myostatin and activin A specifically in myofibers is sufficient to induce muscle hypertrophy. All of these findings are consistent with satellite cells playing little or no role in myostatin/activin A signaling in vivo and render support that inhibition of this signaling pathway can be an effective therapeutic approach for increasing muscle growth even in disease settings characterized by satellite cell dysfunction.

Fig. 1.

Effect of blocking the myostatin/activin A pathway in Sdc4^−/− mice. (A) Percentage increase in weights of the pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius/plantaris (green) muscles of WT and Sdc4^−/− mice resulting either from the presence of the F66 transgene or from administration of ACVR2B/Fc (10 mg/kg for 4 wk). Actual weights, SEs, and P values are shown in Table 1. (B) Hematoxylin and eosin-stained sections prepared from gastrocnemius muscles showing the hypertrophy induced by the F66 transgene and by ACVR2B/Fc. (C) Distribution of fiber sizes in gastrocnemius muscles of mice carrying the F66 transgene (blue bars) or injected with ACVR2B/Fc (red bars) compared with control mice (gray bars) either lacking the transgene or injected with vehicle. (D) Mean fiber diameters in the gastrocnemius muscles.

Fig. 2.

Effect of the F66 transgene in Pax7^−/− mice. (A) Percentage increase in weights of the pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius/plantaris (green) muscles of WT, Pax7^+/−, and Pax7^−/− mice resulting from the presence of the F66 transgene. Actual weights, SEs, and P values are shown in Table 2. (B) Hematoxylin and eosin-stained sections prepared from gastrocnemius muscles showing the hypertrophy induced by the F66 transgene in Pax7^−/− mice. (C) Distribution of fiber sizes in gastrocnemius muscles of WT mice (blue bars) and Pax7^−/− mice without (gray bars) or with (red bars) the F66 transgene.

Fig. 3.

No substantial myofiber incorporation from satellite cells after ACVR2B/Fc-induced muscle hypertrophy. (A) Schema of experimental time line. Bullet-arrows indicate the time of vehicle or ACVR2B/Fc administration; d, day; tmx, tamoxifen. Vehicle and ACVR2B/Fc-treated samples are indicated at top. (B–E) X-gal stained (blue) TA muscle sections from lineage-labeled Pax7^CE/+, R26R^LacZ mice treated with vehicle (B and D) and ACVR2B/Fc (C and E), counterstained with Nuclear Fast Red; B and C, low magnification; D and E, high magnification; asterisks, spindle myofibers; arrows, β-gal⁺ satellite cells. (F and G) Immunostaining of dystrophin (green) to delineate myofiber boundaries for identifying myonuclei (arrowheads) stained by DAPI (red). (H–M) Immunostaining of PAX7 (green), BrdU (red), and Laminin (white), counterstained with DAPI (blue); H and I, overlaid images of PAX7 and BrdU; J and K, same images further overlaid with Laminin and DAPI; L and M, rare examples of a PAX7⁺BrdU⁺ satellite cell and a PAX7⁻BrdU⁺ myonucleus, respectively. For H–M, arrows, filled arrowheads, and open triangles indicate satellite cells, myonuclei, and interstitial cells, respectively. Color scheme for antigens is the same as in H–K.

Fig. 4.

Effect of targeting Acvr2b in myofibers. (A) Gene targeting strategy. Locations of exons 2–10 (E2–E10) are shown as black boxes, FRT (Flippase recognition target) sites are denoted by open triangles, and LoxP sites are denoted by filled triangles. The neo cassette was removed by crossing mice carrying the targeted allele with transgenic mice expressing FLP (Flippase) recombinase in the germline, resulting in an Acvr2b^flox allele. Recombination between the LoxP sites results in a deletion allele (Acvr2b^Δ) lacking exons 2–4. Southern blot analysis of genomic DNA digested with ScaI (S) and hybridized using the probe indicated is predicted to give 6.5-kb and 5.5-kb fragments for the Acvr2b^flox and Acvr2b^Δ alleles, respectively. (B) Southern blot analysis of genomic DNA isolated from various tissues or cell preparations (as indicated) using either the probe shown in A or a probe for the MLC-cre transgene. (C) Northern blot analysis of total RNA (25 μg) isolated from the gastrocnemius muscle using probes corresponding either to the entire coding sequence or to the extracellular and transmembrane domains of ACVR2B. Blots were reprobed with S26 (ribosomal protein) as a loading control. (D) Percentage increase or decrease in weights of the pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius/plantaris (green) muscles of Acvr2b^+/flox or Acvr2b^flox/flox mice resulting from the presence of the MLC-cre transgene. Actual weights, SEs, and P values are shown in Table 5.

Fig. P1.

Blockade of the myostatin/activin pathway leads to dramatic growth of muscles throughout the body, as illustrated by the transgenic mouse expressing a potent myostatin/activin inhibitor, follistatin (Upper Right), compared with a normal mouse (Upper Left) [Images reprinted from ref. (Copyright 2001, National Academy of Sciences, USA).]. The data presented in this paper demonstrate that muscle fiber hypertrophy induced by myostatin/activin inhibition does not require the activation of satellite cells or their fusion to myofibers (diagram). The plasmalemmal surface of the muscle fiber is denoted by the green line, and the basement membrane surrounding the muscle fiber is shown by the gray line. Myonuclei are denoted by red ovals, a satellite cell is denoted by a small blue oval, and new myonuclei derived from fusion of satellite cells to the myofiber are shown by larger blue ovals.