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. 2014 Mar;124(3):1350-63.
doi: 10.1172/JCI71206. Epub 2014 Feb 24.

Reducing Dynamin 2 Expression Rescues X-linked Centronuclear Myopathy

Free PMC article

Reducing Dynamin 2 Expression Rescues X-linked Centronuclear Myopathy

Belinda S Cowling et al. J Clin Invest. .
Free PMC article

Abstract

Centronuclear myopathies (CNM) are congenital disorders associated with muscle weakness and abnormally located nuclei in skeletal muscle. An autosomal dominant form of CNM results from mutations in the gene encoding dynamin 2 (DNM2), and loss-of-function mutations in the gene encoding myotubularin (MTM1) result in X-linked CNM (XLCNM, also called myotubular myopathy), which promotes severe neonatal hypotonia and early death. Currently, no effective treatments exist for XLCNM. Here, we found increased DNM2 levels in XLCNM patients and a mouse model of XLCNM (Mtm1(-/y)). Generation of Mtm1(-/y) mice that were heterozygous for Dnm2 revealed that reduction of DNM2 in XLCNM mice restored life span, whole-body strength, and diaphragm function and increased muscle strength. Additionally, classic CNM-associated histological features, including fiber atrophy and nuclei mispositioning, were absent or reduced. Ultrastructural analysis revealed improvement of sarcomere organization and triad structures. Skeletal muscle-specific decrease of Dnm2 during embryogenesis or in young mice after disease onset revealed that the rescue associated with downregulation of Dnm2 is cell autonomous and is able to stop and potentially revert XLCNM progression. These data indicate that MTM1 and DNM2 regulate muscle organization and force through a common pathway. Furthermore, despite DNM2 being a key mechanoenzyme, its reduction is beneficial for XLCNM and represents a potential therapeutic approach for patients.

Figures

Figure 1
Figure 1. DNM2 levels in XLCNM.
(A) Representative Western blot of XLCNM patient muscle lysates for DNM2, MTM1, and GAPDH loading control. (B) Relative level of DNM2 protein expression determined by densitometry, standardized to GAPDH. n = 5 patients. TA (C) and diaphragm (E) skeletal muscle lysates from 5-week-old WT and Mtm1–/y mice were immunoblotted for DNM2 and GAPDH. Relative levels of DNM2 protein determined by densitometry of DNM2 immunoreactive polypeptides, standardized to GAPDH, for TA (D) and diaphragm (F). DNM2 expression is represented as fold difference from WT lysate. n = 4 mice. (G) Diaphragm muscle sections stained for H&E. Scale bars: 100 μm. All graphs depict mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2. Reduced DNM2 expression greatly rescues the life span of Mtm1–/y mice.
(A) Life span of all mice, represented as percentage survival. All mice in groups WT, Dnm2+/–, and Mtm1–/yDnm2+/– survived to at least 12 months. (B) Mouse whole-body weight. (CF) Relative level of DNM2 protein was determined by densitometry of DNM2 standardized to GAPDH (blots on Supplemental Figure 5). DNM2 level is represented as fold difference from WT lysate. Expression was determined in 8-week-old, 16-week-old, and 6-month-old mice from diaphragm (DIA) (C), gastrocnemius (GAS) (D), TA (E), and soleus (SOL) (F) muscles. n = 2–8 mice. (G) mRNA levels were quantified by qRT-PCR analysis, with DNM2 levels expressed relative to GAPDH. Graph represents 3 independent experiments. Gastrocnemius (H), TA (I), and soleus (J) muscle weights (n = 5–13 mice). All graphs depict mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. w, weeks of age; m, months of age.
Figure 3
Figure 3. CNM histological features are greatly rescued in Mtm1–/y mice with reduced DNM2 expression.
Transverse TA sections from 8-week-old (A) or 16-week-old (C) mice were stained with H&E (upper panel) or SDH (lower panel). Scale bars: 300 μm; 25 μm (high magnification). (B) Transverse muscle sections from 8-week-old mice viewed by TEM. Arrow indicates membrane accumulation around nucleus. Scale bar: 0.5 μm. Transverse TA muscle sections from 8-week-old mice (D) and 16-week-old (E) mice were analyzed for fiber area. Fiber size is grouped into 500-μmβ intervals, and represented as the percentage of total fibers. n = 5–7 mice. (F) The frequency of fibers with internal or central nuclei was scored. n = 5 mice. Internal nuclei are defined as not subsarcolemmal or central. Images were not analyzed in Mtm1–/y mice at 16 weeks, as they usually die before this age. All graphs depict mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. Improved muscle strength and endurance of Mtm1–/y mice with reduced DNM2 expression.
(A) The string test was performed on mice weekly from 3 to 8 weeks. A fall was considered equal to 20 seconds. (B) The absolute maximal force of the TA muscle was measured in 8-week-old and 16-week-old mice. (C) Specific maximal force of the TA. Mtm1–/y mice usually die before 16 weeks and were therefore not measured at that age. (D) Fatigue in TA muscle, measured as the time taken to reach 50% of maximum muscle force. Muscle fatigue was unable to be measured in Mtm1–/y mice at 8 weeks due to extreme muscle weakness. All graphs depict mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. n = minimum 5 mice per group.
Figure 5
Figure 5. Improved muscle ultrastructure of Mtm1–/y mice with reduced DNM2 expression.
TA muscles from 8-week-old (A) and 16-week-old (B) mice were imaged by TEM. Scale bars: 0.5 μm (A); 1 μm (B).
Figure 6
Figure 6. Integrity of triads in TA muscle from 8-week-old mice.
(A) Transverse and longitudinal muscle sections were stained with RyR1 or costained with RyR1 (green) and α-actinin (red) antibodies and imaged by confocal microscopy. Scale bars: 20 μm (transverse); 5 μm (longitudinal). (B) Muscles were imaged by TEM. Arrows point to normally localized triads, shown in high magnification insert. Scale bars: 200 nm; 100 nm (high magnification). (C) Percentage of triads visualized per sarcomere. Graph depicts mean ± SEM. *P < 0.05. (D) Transverse muscle sections were stained with caveolin 3 and imaged by confocal microscopy. Scale bars: 50 μm.
Figure 7
Figure 7. Long-term phenotype of Mtm1–/y mice with reduced DNM2 expression.
(A) 12-month-old WT (left) and Mtm1–/yDnm2+/– (right) mice. (B) Footprint test indicating the angle of hind foot position. Raw data in Supplemental Figure 7. (C) Four-paw grip test. (D) Rotarod test performed under acceleration mode (4–40 rpm in 5 minutes). n = 3 trials/mouse/d. (E) The hanging test requires mice to be suspended from a cage lid for up to 60 seconds. n = 3 trials/mouse (F) A plethysmograph test for resting breathing measurements was performed on 6-month-old mice. Inspiratory/expiratory/relaxation time, and breathing frequency are shown; complementary results in Supplemental Figure 8. (G) Maximal muscle force was measured in diaphragm from 6-month-old mice. Force-frequency relationship and specific maximal force under twitch and tetanus (100 Hz) are depicted. (H) Longitudinal diaphragm sections were stained with H&E. Scale bars: 100 μm. All graphs depict mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. n = minimum 5 mice.
Figure 8
Figure 8. Reducing DNM2 in skeletal muscle alone ameliorates the life span and pathology of Mtm1–/y mice.
(A) Life span of all mice represented as a percentage of survival. Mtm1–/y mice with reduced DNM2 in muscle depicted as Mtm1–/yDnm2skm+/–. (B) Body weight. (C) Weight of gastrocnemius, TA, and soleus muscles as a percentage of total body weight. (D) Transverse TA sections were stained with H&E (top panel) or SDH (lower panel). Scale bars: 100 μm. (E) Transverse sections were analyzed for fiber area. Fiber size is grouped into 500 μmβ intervals, and represented as a percentage of total fibers. (F) The frequency of fibers with internal or central nuclei was counted in TA muscle. (G) Relative level of DNM2 protein from TA muscles, standardized to GAPDH. DNM2 level is represented as a fold difference from WT lysate. All graphs depict mean + SEM. *P < 0.05; ***P < 0.001. n = 4–12 mice, aged 16 weeks.
Figure 9
Figure 9. Reducing DNM2 in skeletal muscle after the onset of symptoms ameliorates the life span and pathology of Mtm1–/y mice.
(A) Life span of all mice represented as a percentage of survival. (B) Body weight. (C) Weight of gastrocnemius, TA, and soleus muscles represented as a percentage of total body weight. (D) Transverse TA sections were stained with H&E (top panel) or SDH (lower panel). Scale bars: 100 μm. (E) Transverse sections were analyzed for fiber area. Fiber size is grouped into 500 μmβ intervals. (F) The frequency of fibers with internal or central nuclei were counted. (G) Relative level of DNM2 protein from TA muscles, standardized to GAPDH. DNM2 level is represented as a fold difference from WT control lysate. All graphs depict mean + SEM. *P < 0.05; **P < 0.01; ***P < 0.001. n = 4–12 mice, aged 16 weeks.

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