Figure 4.

Local administration of bivalent FSTL3-Fc increases muscle mass in healthy mice

(A) Schematic presentation of intramuscular injection (i.m.) of bi-FSTL3-Fc into the right calf.

(B) Distribution of trypan blue 10 min after i.m. injection into the right calf.

(C) Schematic presentation of the protocol. bi-FSTL3-Fc, ActRIIB-Fc, or control Fc was injected intramuscularly into the right calf (hindlimb) of 6-week-old male mice, twice weekly for 2 weeks. All mice were sacrificed 2 days after final injection (n = 10 for each group).

(D) Representative macroscopic images of GC muscles excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Muscles on the right hindlimb were injected, while muscles on the left side were used as contralateral counterparts. Images are representative of different experiments (n = 10 independent samples). Scale bar: 1 cm.

(E) Normalized weight of muscles excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Muscle weight was normalized to the body weight. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by analysis of variance (ANOVA) with Tukey-Kramer post hoc test for multiple comparison; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. GC, gastrocnemius; QF, quadriceps femoris; TA, tibialis anterior; and Ham, hamstrings; uninjected, left hindlimb; injected, right hindlimb.

(F) Representative cross-sectional images of myofibers in the GC muscle excised from mice with 2-week local administration of bi-FSTL3-Fc, ActRIIB-Fc, or control Fc. Top: immunohistochemistry (IHC) for laminin, middle: quantification using Hybrid Cell Count software, bottom: hematoxylin and eosin staining (H&E). Images are representative of different experiments (n = 5 independent samples). Scale bar: 100 μm.

(G) Quantification and distribution of muscle fiber cross-sectional area of (F). The differences in muscle fiber cross-sectional area were analyzed using Wilcoxon rank-sum test, and p values were adjusted with Benjamini-Hochberg correction for multiple comparisons; ∗∗∗∗p < 0.0001.

(H) Normalized weight of the pancreas of mice after a 2-week treatment. Weight of the pancreas was normalized to the body weight. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by ANOVA with Tukey-Kramer post hoc test; n.s., not significant, ∗∗p < 0.01.

(I) The blood glucose concentration of mice after a 2-week-treatment. Mice were fasted for 2.5 hr. Data are means ± SD from n = 10 independent experiments. Differences between the conditions were analyzed by ANOVA with Tukey-Kramer post hoc test; n.s., not significant, ∗p < 0.05.