Age-dependent effect of myostatin blockade on disease severity in a murine model of limb-girdle muscular dystrophy

Abstract

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.

Figure 1

Comparison of relative muscle weights [muscle weight (mg)/body weight (g)] for gastrocnemius (gastroc), plantaris, tibialis anterior (TA), and quadriceps (quad) muscles and heart for scgd^−/− mice treated with control or MSTN antibody beginning at either 4 weeks (A) or 20 weeks of age (B). Mice treated with MSTN antibody (Ab) beginning at 4 weeks showed gains in all skeletal muscles examined 12 weeks later (gastroc, n = 11; plantaris, n = 12; TA, n = 12; quad, n = 12; *t-test, P < 0.01), but no change was detected in the heart (n = 6). Mice treated with MSTN Ab beginning at 20 weeks showed gains in muscle weight for TA (n = 8) and quad (n = 8) only (^#P < 0.001). Plantaris muscles showed no change (n = 8 control, n = 7 MSTN Ab), while gastrocnemius showed a significant loss of muscle weight (n = 7, ^†P < 0.0006). Again, no change in heart weight was detected (n = 4).

Figure 2

Analysis of Masson’s trichrome-stained gastrocnemius and diaphragm sections for scgd^−/− antibody-treated mice. Representative trichrome-stained sections of gastrocnemius muscle from control antibody (Ab) and MSTN Ab-treated mice beginning at 4 weeks (A) and 20 weeks (B) of age. MetaMorph 6.1 software quantification of area of fibrosis (blue staining) in gastrocnemius from animals Ab-treated beginning at 4 weeks (C) and 20 weeks (D) (*t-test, P = 0.03). Representative trichrome-stained sections of diaphragm muscle from control Ab and MSTN Ab-treated mice beginning at 4 weeks (E) and 20 weeks (F) of age. MetaMorph quantification of fibrosis in diaphragm from animals Ab-treated at 4 weeks (G) and 20 weeks (H) (*P = 0.01).

Figure 3

Muscle fiber areas and percent centrally nucleated fibers in scgd^−/− mice treated with control antibody or MSTN antibody beginning at 4 weeks or 20 weeks of age. Diaphragm muscle fiber areas (μm²) of mice treated beginning at 4 weeks (A) with control (n = 6) or MSTN antibody (Ab) (n = 6) (*t-test, P = 0.01) and at 20 weeks (B) with control (n = 6) or MSTN Ab (n = 4) (*P = 0.03). For all Ab-treated groups ∼200 to 300 fibers were measured per field (×10 magnification), with two fields counted. Myofiber areas from WT (n = 3, >220 fibers/field, two fields) diaphragm, 31 weeks of age, were also measured to include as a nondiseased control. Percentage of fibers containing central nuclei in diaphragm muscle from mice treated with Ab beginning at 4 weeks (C) (n = 5, average 1100 fibers examined/mouse, *P = 0.04) or at 20 weeks (D) (n = 4, average 975 fibers examined/mouse).

Figure 4

MSTN expression decreases in dystrophic muscle and loss of expression enhances relative muscle weight and improves cardiac function in scgd^−/− mstn^−/− mice. A: Western blot analysis of MSTN propeptide expression or the indicated control proteins in WT and scgd^−/− quadriceps muscle (80 μg protein loaded). mstn^−/− muscle was included as a negative control. Quantitation is shown in the graph on the right. B: Comparison of relative muscle weights for gastrocnemius, quadriceps, and soleus (WT, n = 20; scgd^−/−, n = 28; mstn^−/−, n = 16; scgd^−/− mstn^−/−, n = 28) and heart (WT, n = 10; scgd^−/−, n = 14; mstn^−/−, n = 8; scgd^−/− mstn^−/−, n = 14) (*t-test, P < 0.000001 scgd^−/− relative to WT, ^¶P < 0.01 mstn^−/− relative to WT, ^†P < 0.01 scgd^−/− mstn^−/− relative to mstn^−/−, except heart, P = 0.04). C: Comparison of cardiac fractional shortening by echocardiography (*P = 0.00001 relative to WT, ^§P = 0.02 relative to scgd^−/−).

Figure 5

Analysis of Masson’s trichrome-stained gastrocnemius and diaphragm sections for WT, scgd^−/−, mstn^−/−, and scgd^−/− mstn^−/− mice. A: Representative trichrome-stained sections of diaphragm muscle. B: MetaMorph quantification of area of fibrosis in diaphragm muscle (WT, n = 3; mstn^−/−, n = 3; scgd^−/−, n = 12; scgd^−/− mstn^−/−, n = 11; *t-test, P = 0.005). C: Hydroxyproline assay data obtained from diaphragm muscle (WT, n = 3; mstn^−/−, n = 3; scgd^−/−, n = 4; scgd^−/− mstn^−/−, n = 5; *P = 0.002 relative to scgd^−/−). D: Percentage of fibers containing central nuclei in diaphragm muscle (WT, n = 4; mstn^−/−, n = 3; scgd^−/−, n = 3; scgd^−/− mstn^−/−, n = 3; average 640 fibers examined/mouse, *P = 0.03 relative to scgd^−/−). E: Representative trichrome-stained sections of gastrocnemius muscle.

Figure 6

Representative semiquantitative RT-PCR data. Expression of satellite cell markers Pax7 and M-cadherin in diaphragm tissue of WT, scgd^−/−, mstn^−/−, and scgd^−/− mstn^−/− mice of ∼36 weeks of age. Data are representative of that seen from analysis of three to four mice per genotype per marker, although data from only two mice in each group are shown (mouse 1 and mouse 2). L7 is a housekeeping control mRNA.