Treatment with ActRIIB-mFc Produces Myofiber Growth and Improves Lifespan in the Acta1 H40Y Murine Model of Nemaline Myopathy

Abstract

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)-mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice.

Supplemental Figure S1

Additional pathologic studies of skeletal muscle in wild-type (WT) and Acta1 H40Y mice. A: Gomori trichrome staining of the diaphragm and gastrocnemius muscles reveal the degree of myofiber hypertrophy and pathologic inclusion burden seen after vehicle (VEH) or ActRIIB-mFc (ActRIIB) treatment of WT or Acta1 H40Y mice. B: Electron microscopy (EM) reveals several pathologic abnormalities in Acta1 H40Y quadriceps muscle, including nemaline rods, and aggregates of mitochondria (yellow arrows), sometimes surrounded by myofibrillar disarray. Other areas (right) have nemaline rods/bodies without evidence of mitochondrial abnormalities or large-scale myofibrillar disorganization. Scale bars: 40 μm (A); 2 μm (B).

Figure 1

Phenotypic findings in VEH- and ActRIIB-mFc–treated mice. A: Total body weight of VEH- and ActRIIB-mFc –treated mice. B: Forelimb grip force of VEH- and ActRIIB-mFc–treated mice. C: Open field activity (expressed in meters traveled in 10 minutes) of VEH- and ActRIIB-mFc–treated mice. D: Kaplan-Meier survival curves for VEH- and ActRIIB-mFc–treated mice. Bars and asterisks denote significant differences between treatment groups with relevant comparisons. Note that the bar and asterisk in A illustrate a significant difference between the H40Y/VEH and H40Y/ActRIIB groups only between 6.5 and 9 weeks of age. The survival comparison in D between WT/ActRIIB and H40Y/ActRIIB did not reach statistical significance (P = 0.062). Data are expressed as means ± SEM (A–C). n = 25 for the WT/VEH group, 19 for the WT/ActRIIB group, 32 for the H40Y/VEH group, and 24 for the H40Y/ActRIIB group (A–C) and n = 28 for the WT/VEH group, 25 for the WT/ActRIIB group, 37 for the H40Y/VEH group, and 29 for the H40Y/ActRIIB group (D). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. ActRIIB, ActRIIB-mFc treated; VEH, vehicle; WT, wild type.

Figure 2

Skeletal muscle histopathologic findings in VEH- and ActRIIB-mFc–treated mice. A: Individual muscle weights from VEH- and ActRIIB-mFc–treated mice. Bars and asterisks denote significant (P < 0.05) differences between treatment groups with relevant comparisons. B: Representative areas of transversely sectioned quadriceps muscle after sectioning and staining with Gomori trichrome. Acta1 H40Y mice have pathologic aggregates of purple/red material that corresponds to aggregates of nemaline rod material. C: Frequency histogram illustrating myofiber size distribution in the quadriceps muscles of VEH- and ActRIIB-treated mice. Asterisks reflect significant differences between treated and untreated animal groups, both in WT and Acta1 H40Y mice. ^∗P < 0.05. Scale bar = 40 μm (B). ActRIIB, ActRIIB-mFc treated; VEH, vehicle; WT, wild type.

Figure 3

Genitourinary (GU) tract histopathologic findings in VEH- and ActRIIB-mFc–treated mice. A and B: Gross and histologic evidence of urethral obstruction leading to bladder distension, inflammation (asterisk) and necrosis in a subset of Acta1 H40Y mice that died spontaneously during the trial. C and D: Assessment of copulatory plugs. C: Axial section of the GU tract of an ActRIIB-mFc–treated Acta1 H40Y mouse at 16 weeks of age. A copulatory plug is evident, as is a large amount of urethral striated muscle surrounding the prostate. There is no evidence of inflammation or tissue necrosis in this specimen. D: Quantification of mice exhibiting intraurethral copulatory plugs out of (n) mice per group. E: Quantification of mice exhibiting GU tissue inflammation out of (n) mice per group. F and G: Immunostaining of urethral striated muscles for skeletal muscle actin reveals pathologic aggregates of actin (corresponding to nemaline rod aggregates) in numerous fibers in Acta1 H40Y mice (G), whereas these aggregates are absent in WT littermates (F). H: Frequency histogram illustrating myofiber size distribution in urethral striated muscles of VEH- and ActRIIB-mFc–treated mice. ^∗P < 0.05, ^∗∗∗P < 0.001. ActRIIB, ActRIIB-mFc treated; VEH, vehicle; WT, wild type.

Figure 4

Expression of proteins related to myofiber growth in VEH- and ActRIIB-mFc–treated mice. A: Western blots reveal protein expression of growth-associated proteins in the quadriceps muscles of individual WT or Acta1 H40Y mice. B: Quantified protein expression (normalized to GAPDH expression) for each growth-associated protein shown in A. Protein extracted from the quadriceps muscle of four 16-week-old mice per treatment group were tested. ^∗P < 0.05, ^∗∗P < 0.01. ActRIIB, activin type IIB receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VEH, vehicle; WT, wild type.