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. 2011 Jan;121(1):70-85.
doi: 10.1172/JCI44021. Epub 2010 Dec 6.

Myotubularin Controls Desmin Intermediate Filament Architecture and Mitochondrial Dynamics in Human and Mouse Skeletal Muscle

Free PMC article

Myotubularin Controls Desmin Intermediate Filament Architecture and Mitochondrial Dynamics in Human and Mouse Skeletal Muscle

Karim Hnia et al. J Clin Invest. .
Free PMC article


Muscle contraction relies on a highly organized intracellular network of membrane organelles and cytoskeleton proteins. Among the latter are the intermediate filaments (IFs), a large family of proteins mutated in more than 30 human diseases. For example, mutations in the DES gene, which encodes the IF desmin, lead to desmin-related myopathy and cardiomyopathy. Here, we demonstrate that myotubularin (MTM1), which is mutated in individuals with X-linked centronuclear myopathy (XLCNM; also known as myotubular myopathy), is a desmin-binding protein and provide evidence for direct regulation of desmin by MTM1 in vitro and in vivo. XLCNM-causing mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal IF assembly and architecture in muscle cells and both mouse and human skeletal muscles. Adeno-associated virus-mediated ectopic expression of WT MTM1 in Mtm1-KO muscle reestablished normal desmin expression and localization. In addition, decreased MTM1 expression and XLCNM-causing mutations induced abnormal mitochondrial positioning, shape, dynamics, and function. We therefore conclude that MTM1 is a major regulator of both the desmin cytoskeleton and mitochondria homeostasis, specifically in skeletal muscle. Defects in IF stabilization and mitochondrial dynamics appear as common physiopathological features of centronuclear myopathies and desmin-related myopathies.


Figure 1
Figure 1. MTM1 interacts with desmin in vitro and in muscle.
(A) GST–MTM1–full-length (MTM1-FL), GST-MTM1-GRAM PH domain (GRAM), or GST recombinant proteins were incubated with mouse myotube or muscle homogenates. Endogenous desmin interacted with MTM1-FL, but not GST or GRAM. CB, Coomassie blue staining of purified recombinant GST fusions. (B) Far-Western blot using recombinant desmin protein overlaid with GST-MTM1-FL and revealed with anti-GST (left) or anti-desmin (right) antibodies. (C) Top: co-IP assays with lysates from COS-1 cells (cotransfected with MTM1 and desmin constructs), myotubes, and skeletal muscle using anti-MTM1–specific antibody or a frataxin antibody as a control (CTR). Antibodies used for IB are indicated. Bottom: co-IP of MTM1 with desmin in skeletal (Sk) muscle using the anti-desmin antibody. (D) Localization of MTM1 and desmin in WT and Mtm1-KO mouse skeletal muscles. Longitudinal and transversal sections showed colocalization of MTM1 and desmin at the z-disc structure and at the sarcolemma. Desmin localization was abnormal in Mtm1-KO muscle. Scale bar: 20 μm. (E) Desmin did not precipitate with MTM1 in extracts from cardiac muscle. Longitudinal sections of cardiac muscle were stained for MTM1 and desmin. Scale bars: 10 μm.
Figure 2
Figure 2. Dissection of the MTM1-desmin interaction.
(A) Schematic of the MTM1 protein domains. Pulldown of GST-fusion domains of MTM1 with extracts from COS-1 cells overexpressing desmin. Desmin interacted with the ΔGRAM domain. (B) Prediction model for MTM1 based on the MTMR2 crystallographic model using PyMol software. The outlined region (shown in detail at right) represents the potential domain for interaction with desmin, composed of 4 independent loops with indicated residues exposed to outside space. aas labeled in red are implicated in desmin binding; histograms represent the relative binding of listed MTM1 constructs (deletion and aa mutation). (C) Prediction model for MTM1, showing loops implicated in desmin interaction based on peptide mapping and competition experiments (Supplemental Figure 3, D and E). (D) Desmin binding domain. Schematic representation of desmin domains. Dashed lines outline the common region between Y2H clones. The region of desmin implicated in MTM1 binding was determined by peptide mapping experiments. Quantitation of desmin peptides’ affinity for MTM1 compared with a non-desmin peptide (CTR). Data were correlated from 3 independent experiments, and statistical analysis of the difference in intensities between positive peptides was set at *P ≤ 0.05.
Figure 3
Figure 3. Effect of MTM1 depletion and mutation on desmin expression and localization.
(A) Desmin was overexpressed in an XLCNM patient’s myoblast (R474X, F238 frameshift, and R241C mutations) compared with controls (CTR1–CTR3); corresponding histograms show desmin protein and mRNA levels over 3 independent experiments (*P ≤ 0.05). (B) Desmin aggregation in XLCNM patient myotubes and muscle biopsies. XLCNM mutations are denoted; arrowheads indicate desmin aggregates. Scale bars: 50 μm. (C) Effect of Mtm1-KD (in C2C12) or -KO background (primary myoblasts from Mtm1-KO muscle) on desmin expression and solubility. (D and E) Quantification of C. (F) Effect of WT and XLCNM-linked or artificial (asterisks) MTM1 mutations on desmin interaction. Histograms show that several XLCNM mutations located in the interacting domain interfered with desmin binding. Quantification of the level of immunoprecipitated desmin/input relative to control (MTM1-FL) was correlated from 3 independent experiments. *P ≤ 0.05. (G) Effect of DRM-linked desmin mutations on MTM1 binding. Far-Western blot of recombinant WT and mutated desmin using recombinant MTM1 protein for overlay suggested that MTM1 only bound to DES-L370P and WT desmin. Lanes were run on the same gel but were noncontiguous (white line).
Figure 4
Figure 4. Ectopic expression of MTM1 in Mtm1-KO muscle restores normal desmin expression and localization.
(A) H&E-stained transversal section of tibialis anterior from Mtm1-KO muscles injected with AAV-MTM1, AAV, or PBS. AAV-MTM1 rescued muscle fiber atrophy and muscle weight (B). Data correlated from 2 independent experiments (n = 6 mice per group). *P ≤ 0.05. Scale bars: 100 μm. (C) Ectopic expression of MTM1 transgene in Mtm1-KO muscle restored normal desmin localization in muscle. Arrowheads indicate aggregates of desmin in Mtm1-KO muscle injected with AAV or PBS. Scale bars: 50 μm. (D) Partial localization (along the z-disc) between MTM1 and desmin in AAV-MTM1–injected muscle. Scale bars: 50 μm. (E) Ectopic expression of MTM1 transgene in Mtm1-KO muscle restored normal desmin expression level in injected muscles. Data correlated from 2 independent experiments (n = 3 mice per group). *P ≤ 0.05.
Figure 5
Figure 5. Effect of MTM1 on desmin filament polymerization.
(A) Effect of MTM1 on in vitro assembly of desmin filaments. Assembly of recombinant desmin alone (scale bars: 200 nm) or in the presence of MTM1 WT (scale bars: 100 nm) or the control protein Sumo (scale bars: 200 nm) was monitored by electron microscopy at the indicated times. Addition of WT MTM1 led to irregular and ribbon-like filaments. (B) Filament parameters (width and length at 5 minutes of assembly) in the presence or absence of MTM1. Scale bar: 200 nm. (C) MTM1 cosedimented with desmin and interfered with polymerization. SDS-PAGE stained with Coomassie blue showed desmin exclusively in the pellet fraction (P; polymerized) in the presence of GST or GST-MTM1S209A, while it was also present in the soluble fraction (S; unpolymerized) in the presence of GST-MTM1. (D) Increasing amounts of recombinant GST-MTM1 caused a decrease in desmin polymerization. Lanes were run on the same gel but were noncontiguous (white line). Quantification of desmin assembly (calculated as the ratio of band intensities in pellet/supernatant) in the presence of increasing amounts (4, 8, and 16 μM) of GST-MTM1, GST-MTM1S209A, or GST. Data were correlated from 2 independent experiments. *P ≤ 0.05. (E) Model of MTM1’s effect on desmin assembly in vitro. In phase 1 of desmin assembly, 8 tetrameric subunits, made from 2 antiparallel, half-staggered coiled-coil dimers, associate laterally to form ULFs after initiation of assembly. In phase 2, ULFs and short filaments longitudinally anneal to other ULFs. In phase 3, filaments evolve to radial compacted structures. MTM1 addition (yellow) interferes with filament assembly in vitro, leading to a branched-like phenotype at the squiggles step and a ribbon-like structure.
Figure 6
Figure 6. Effect of desmin on MTM1 PI phosphatase activity.
(A) GST-MTM1 recombinant proteins were incubated with PtdIns(3,5)P2 alone or in the presence of full-length desmin or specified desmin peptides (versus Sumo as a control protein and control peptide, respectively). No significant variation in the PtdIns5P/PtdIns(3,5)P2 ratio per mg protein was observed after quantification. Coomassie blue gel of purified GST-MTM1 and desmin are also shown. (B) Immunoprecipitated MTM1-containing complexes from cells cotransfected with MTM1-B10 and desmin-myc or with MTM1-B10 and NFL-myc (neurofilament light chain) were incubated with PtdIns(3,5)P2. Immunoprecipitated complexes were confirmed by Western blot analysis with anti-B10 and anti-myc antibodies. No significant changes in MTM1 phosphatase activity were found by quantification of the PtdIns5P/PtdIns(3,5)P2 ratio. (C) Immunolabeling of MTM1 substrate in Mtm1-KO and -KD cells showed accumulation of PtdIns(3,5)P2 compared with control cells. Single cells are outlined (original magnification, ×63). (D) MTM1 mutations within the desmin-binding domain (e.g., MTM1K255A and MTM1S209A) did not interfere with MTM1 PI phosphatase activity in cells, but XLCNM mutations (e.g., MTM1R241C and MTM1R421Q) or the catalytic inactive mutation (MTM1C375S) significantly impaired this activity. Single cells are outlined (original magnification, ×63). Histograms represent fluorescent intensities of PtdIns(3,5)P2 over 2 independent experiments. *P ≤ 0.05.
Figure 7
Figure 7. MTM1 has a role in mitochondrial dynamics in muscle cells.
(A) Effect of MTM1 mutations on mitochondrial morphology in C2C12 cells. Single cells are outlined. Scale bar: 10 μm. (B) Quantitation of mitochondrial phenotypes observed as normal or collapsed. More than 100 cells per transfection were counted for 2 independent experiments. Nontransfected (NT) cells served as a control. *P ≤ 0.05. (C) Confocal microscopy images after MitoTracker Red staining showed accumulation/collapse of mitochondria at the perinuclear region in Mtm1-KO and -KD myoblasts. Scale bar: 20 μm. Also shown is quantitation of mitochondrial phenotypes observed over 2 independent experiments, as well as position of mitochondria with respect to nuclei (0 μm) in control and Mtm1-KD C2C12 cells. *P ≤ 0.05. (D) Overexpression of MTM1-WT, but not MTM1-S209A, in Mtm1-KD cells restored mitochondrial morphology. Single cells are outlined (original magnification, ×63). Boxed regions are shown at higher magnification at right (scale bar: 10 μm). (E) Ultrastructural observations of Mtm1-KD cells by electron microscopy revealed the presence of swollen mitochondria (arrowheads). Boxed regions are shown at higher magnification below (original magnification, ×20,000). Scale bars: 1 μm.
Figure 8
Figure 8. The MTM1-desmin complex is associated with mitochondria and is involved in mitochondrial dynamics/motility.
(A) Subcellular fractionation of muscles from WT or Mtm1-KO mice (lanes labeled 1 and 2, respectively) showed accumulation of desmin in the mitochondrial fraction in Mtm1-depleted muscle along with the mitochondrial markers prohibitin and cytochrome c. The cytosolic protein HSP70 served as a control. (B) MTM1, desmin, and cytochrome c levels in mitochondrial fractions in WT and Mtm1-KO muscle relative to the mitochondrial protein (prohibitin). Data correlated from 2 independent experiments (3 mice per group; 2 tibialis anterior muscles per mouse). *P ≤ 0.05. (C) Number of motile mitochondria in the peripheral and perinuclear region of scramble and Mtm1-KD myoblasts. *P ≤ 0.05. (D) Mean velocity of mitochondria in scramble versus Mtm1-KD myoblasts. (C and D) N, number of total cells monitored; n, number of mitochondria scored. (E) Spot plot depicting velocity of individual mitochondria and their cellular position in relation to nucleus (0 μm) in scramble and Mtm1-KD myoblasts. Data correlated from 2 independent experiments. (F) Depletion of MTM1 in muscle did not affect the plectin-desmin interaction. Mitochondrial and microsomal fractions from WT and Mtm1-KO muscles were subjected to IP with an anti-plectin antibody and revealed by anti-desmin antibody. Prohibitin and β-DG were used as mitochondrial and microsomal markers, respectively. Data were correlated from 2 independent experiments. *P ≤ 0.05.
Figure 9
Figure 9. Involvement of MTM1 in mitochondrial function and homeostasis in muscle.
(A) Decreased cytochrome oxidase activity in Mtm1-KD C2C12 and XLCNM patient myoblasts and myotubes. Cell lines from 3 XLCNM patients were assayed. 3 independent experiments were analyzed. *P ≤ 0.05. (B) ATP content of total muscle homogenates from WT and Mtm1-KO mice. *P ≤ 0.05. (C) Mitochondria freshly isolated from mouse muscle were used to measure Ca2+-induced MPT. Changes observed during the 18-minute period are shown for WT and Mtm1-KO mitochondria in the presence and absence of Ca2+. (D) Cytochrome c release was monitored from freshly isolated mitochondria from WT and Mtm1-KO muscles. Mitochondria were incubated with or without Ca2+, followed by analysis of cytochrome c released to the supernatant. (BD) Data were correlated from 2 independent experiments (3 mice per group; 2 tibialis anterior muscles per mouse). (E) Decreased MTM1 levels did not enhance apoptotic events. TUNEL assay was used to determine the apoptotic rate of control and Mtm1-KD cells. FACS analysis showed no significant difference in the profile of control and KD samples. Data are representative of 3 independent experiments. (F) Diagram illustrating IF-binding proteins and their associated diseases together with potential connection to mitochondrial dynamics and function. EB-MD, epidermolysis bullosa with muscular dystrophy; Mb, membrane.

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