Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice

Abstract

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.

Figure 1

Myostatin expression in wild-type and Mtm1δ4 mice. A: Western blot analysis performed on the triceps muscle of 43 day-old animals demonstrates similar levels of myostatin expression in Mtm1δ4 mice and their wild-type littermates. These are nonadjacent lanes from the same immunoblot and using the same exposure time, as denoted by the vertical line. B: Expression of myostatin normalized to GAPDH expression in vehicle-treated wild-type and Mtm1δ4 mice at 43 days of life. Numbers are shown in reference to the mean value for the wild-type animals, and reflect the mean (SEM) value of 3 animals for each genotype.

Figure 2

Behavioral findings in vehicle- and ActRIIB-mFC–treated mice. A: Body weight of vehicle- and ActRIIB-mFC–treated mice. Measurements are the running mean (SEM) of (n) animals in each treatment group. B: Antigravity hanging performance of vehicle- and ActRIIB-mFC–treated mice. C: Forelimb grip force of vehicle- and ActRIIB-mFC–treated mice. D: Measurements are mean (SEM) values of (n) animals in each treatment group. E: Kaplan-Meier survival curves for vehicle- (Veh) and ActRIIB-mFC–treated Mtm1δ4 mice. *P < 0.05.

Figure 3

Gross evaluation of vehicle- and ActRIIB-mFC–treated mice. A: Gross examination of skinned animals at 43 days of life (DOL) or at end stage (∼9 weeks) reveals increased muscle bulk in wild-type animals as a result of treatment with ActRIIB-mFC. Treatment-related increase in muscle bulk was apparent in Mtm1δ4 mice at 43 days of life; however, muscle atrophy in the terminal phase of disease prevents differentiation of vehicle- and ActRIIB-mFC–treated Mtm1δ4 animals at end stage. B: Weights of individual muscles in treated and untreated animals at 43 days of life. *P < 0.05.

Figure 4

Histologic evaluation of vehicle- and ActRIIB-mFC–treated mice. A: H&E-stained transverse sections from the quadriceps muscles of vehicle- and ActRIIB-mFC–treated wild-type and Mtm1δ4 mice. Histologic findings in wild-type mice were similar at all three time points. B: Counts of centrally nucleated fibers in quadriceps muscles from mice at 43 days of life (DOL) and at end stage. C: H&E-stained transverse sections from the diaphragm muscles of vehicle- and ActRIIB-mFC–treated wild-type and Mtm1δ4 mice. Scale bars: 100 μm (A); 200 μm (C). *P < 0.05.

Figure 5

Histologic analysis according to fiber type. A: Immunostaining of transverse sections from the quadriceps muscles of an ActRIIB-mFC–treated wild-type or Mtm1δ4 mouse at 43 days of life for dystrophin (red) and either myosin heavy chain type 1, type 2a, or type 2b fibers (green). Scale bar = 100 μm. Staining was performed in all treatment groups at 35 and 43 days of life and at end stage (determined by complete hindlimb paralysis or a >20% loss in body weight), and myofiber size was quantitated according to fiber type. B: Percentages of type 1, 2a, and 2b fibers were counted. C–E: Muscles from animals at 35 days of life, 43 days of life, and end stage were analyzed. Mean MinFeret diameter measurements for type 1 (C), type 2a (D), and type 2b (E) fibers are shown. The legend for panel C also applies to panels D and E. F and G: Frequency histograms demonstrate the distribution of fiber sizes in the quadriceps muscles of vehicle- and ActRIIB-mFC–treated wild-type (F) and Mtm1δ4 (G) mice. *P < 0.01. **P < 0.0001.

Figure 6

Ultrastructural evaluation of vehicle- and ActRIIB-mFC–treated mice. A–D: Ultrastructural examination of gastrocnemius muscle from vehicle- and ActRIIB-mFC–treated wild-type animals reveals no differences in the organization of the contractile apparatus between treated and untreated mice. Similarly, vehicle- and ActRIIB-mFC–treated Mtm1δ4 mice exhibit appropriate organization of contractile elements, but also exhibit abnormalities of T-tubule and sarcoplasmic reticulum architecture (insets), as previously reported.E: Myofiber degeneration in Mtm1δ4 mice is characterized by disorganization of organelles and abnormalities in the contractile apparatus, which are unaffected by treatment with ActRIIB-mFC. F: Atrophic fibers, identifiable by folding and redundancy of the basal lamina (inset), are observed in Mtm1δ4 animals treated with ActRIIB-mFC. Scale bars: 500 nm (A–D); 2 μm (E and F).