AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Anna Buj-Bello, Françoise Fougerousse, Yannick Schwab, Nadia Messaddeq, Danièle Spehner, Christopher R Pierson, Muriel Durand, Christine Kretz, Olivier Danos, Anne-Marie Douar, Alan H Beggs, Patrick Schultz, Marie Montus, Patrice Denèfle, Jean-Louis Mandel, Anna Buj-Bello, Françoise Fougerousse, Yannick Schwab, Nadia Messaddeq, Danièle Spehner, Christopher R Pierson, Muriel Durand, Christine Kretz, Olivier Danos, Anne-Marie Douar, Alan H Beggs, Patrick Schultz, Marie Montus, Patrice Denèfle, Jean-Louis Mandel

Abstract

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Figures

Figure 1.
Figure 1.
Pathology of tibialis anterior (TA) muscle of 4 week-old Mtm1 mutant mice. (A) Weight of wild-type (WT) and Mtm1/HSA [mKO (muscle-specific knockout)] TA muscles (n = 4 and 8 muscles for WT and mKO, respectively). Note the important reduction of muscle mass in mutant mice (*P < 0.001). (B) Area of TA myofibers. The curve represents the percentage of muscle fibers per area group (myofiber areas were divided into 20 groups, n = 496 and 771 for WT and mKO fibers, respectively, P < 0.001). The curve is shifted to the left in mKO animals indicating a general decrease of myofiber areas. (C) Hematoxylin and eosin (left panels, HE, magnification ×400) and nicotimanide adenine dinucleotide tetrazolium reductase (NADH-TR) (right panels, magnification ×200) staining of TA cross-sections from WT (top) and mKO (bottom) mice at 4 weeks of age. Note the presence of very small myofibers (arrow) and nuclei beneath the sarcolemma. Mitochondrial oxidative staining is often distributed as a ring at the periphery of the muscle fibers.
Figure 2.
Figure 2.
Myotubularin replacement leads to increased muscle volume. (A) Expression of myotubularin in muscle-specific knockout (mKO) muscle after rAAV2/1 transduction. phosphate-buffered saline (PBS) and adeno-associated virus (AAV)-treated mutant tibialis anterior (TA) cross-sections (mKO-PBS and mKO-AAV) were immuno-labeled with a rabbit polyclonal antibody against myotubularin. The antibody labels aspecifically nuclei in mutant muscle, whereas it detects myotubularin throughout the fiber with reinforcement in the sarcolemmal region 4 weeks after rAAV2/1-CMV-Mtm1 vector transfer (magnification ×400). (B) Weight of mutant TA muscle after myotubularin re-expression. The weight of isolated TA was measured 4 weeks after viral transduction (mKO-AAV) and compared with PBS-injected wild-type (WT) and mKO muscles (n = 6 for WT-PBS and mKO-AAV, n = 7 for mKO-PBS). The weight is lower by about four times in mutant animals (mKO-PBS, *P = 2 × 10−6), but significantly increased after rAAV (recombinant AAV) transfer (*P = 2 × 10−5). (C) Evaluation of mean myofiber area 4 weeks after intramuscular injection of rAAV2/1-CMV-Mtm1 vector. The size of Mtm1-deficient TA muscle fibers is smaller than WT animals (n = 473 from five mice and n = 1555 from four mice for WT-PBS and mKO-PBS, respectively, *P < 0.001). Viral transduction leads to a strong increase in myofiber area (n = 636 from four mice for mKO-AAV, *P < 0,001). (D) Distribution of muscle fibers from TA according to their size. The areas were subdivided into 20 subgroups ranging from the smallest to the biggest area, the interval between each subgroup corresponds to 82 µm2. The curve represents the percentage of fibers that belongs to each of the subgroups according to genotype and treatment. Note the strong reduction of very small fibers in mKO-AAV muscle.
Figure 3.
Figure 3.
Correction of XLMTM (X-linked myotubular myopathy) pathology by viral-mediated myotubularin treatment. (A) Percentage of muscle fibers with internalized nuclei after adeno-associated virus (AAV)-mediated myotubularin expression. Myonuclei were considered as internalized if detached from the sarcolemma. The number of fibers with internal nuclei is increased in mutant tibialis anterior (TA) muscle (n = 311 for WT-PBS (wild-type-phosphate-buffered saline) and n = 1181 for mKO(muscle-specific knockout)-PBS, *P < 0,001) and significantly reduced 4 weeks after viral injection (n = 487 for mKO-AAV, *P < 0,001). (B) Histological aspect of myotubularin-deficient skeletal muscle 4 weeks after rAAV2/1-CMV-Mtm1 transfer. Hematoxylin and eosin, HE (upper panels, magnification ×400) and nicotimanide adenine dinucleotide tetrazolium reductase (NADH-TR) (lower panels, ×630) stainings of TA cross-sections from WT (left), mutant (middle) and AAV-treated (right) mice. Note the recovery of oxidative reactivity pattern in myotubularin-expressing mKO muscle. (C) Immunostaining of mKO muscle cross-sections 4 weeks after rAAV-Mtm1 injection with myotubularin antibodies (left) and Hoechst (middle). Right panel is the merge of MTM1 labeling and Hoechst. Right panel shows an inflammatory infiltrate in a region where myotubularin is highly overexpressed (×400).
Figure 4.
Figure 4.
Recovery of the contractile force in Mtm1-deficient skeletal muscle. (A) Extensor digitorum longus (EDL) and tibialis anterior (TA) muscle cross-sections 4 weeks after viral injection. The photographs illustrate the important increase in the volume of both mutant muscles. The bars represent 1 mm. (B) The histogram represents the isometric contractile force (mean ± SD) of isolated EDL and TA normalized by muscle length. Strength was also measured 4 weeks after injection. Results show a significant decrease in muscle force between wild-type and mKO animals at 8 weeks of age (*P < 0.001), whereas an important recovery of strength is observed after myotubularin treatment (force is about five times higher in treated versus untreated muscles, P-value between mKO-PBS (muscle-specific knockout-phosphate-buffered saline) and mKO-AAV (muscle-specific knockout-adeno-associated virus) is P = 0.0006 for both muscles).
Figure 5.
Figure 5.
Subcellular localization of myotubularin in skeletal muscle. (A) Detection of endogenous myotubularin by western blot. Left panel shows the presence of myotubularin in wild-type (WT) but not KO skeletal muscle and heart. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoreactivity was used as an internal control. Right panel illustrates the level of expression of myotubularin 6 weeks after adeno-associated virus (AAV) injection; 75 and 7.5 µg of proteins were loaded for phosphate-buffered saline (PBS) (LTA) and AAV (RTA)-injected WT muscles, respectively. A lower exposure of myotubularin band in RTA (RTA*) is shown to illustrate the doublet. (B) Localization of endogenous myotubularin in skeletal muscle. Semithin cryosections (0.5 µm) of WT muscle were stained for MTM1 (myotubularin), α-actinin (Z-lines), ryanodine receptor (RYR) and triadin (both in triads). Occasional α-actinin-positive Z-lines appear yellow when oblique orientation of the sarcomeres in sections superimpose the Z-lines with the adjacent triadic regions. The bar represents 5 µm. (C) Localization of overexpressed myotubularin in skeletal muscle. Semithin Mtm1-deficient (upper panels) and WT-AAV (middle and lower panels) muscle cryosections were stained for MTM1, SERCA1 (sarco-endoplasmic reticulum calcium ATPase 1) and α-actinin. Myotubularin antibody binds aspecifically to a region between I-bands in WT and KO myofibers, which probably corresponds to the M-band. Note myotubularin location at the sarcolemma (arrowheads) and around the Z-line (arrows) in WT-AAV muscle. The Z-line and I-band (endoplasmic reticulum) were labeled with antibodies against α-actinin and SERCA1, respectively. The bar represents 5 µm. (D) Subcellular localization of overexpressed myotubularin by immunoelectron microscopy. Triads (arrows) are labeled by r1947-coupled gold particles. Note the position of mitochondria (m, bar represents 200 nm). (E) Subcellular fractionation of skeletal muscle overexpressing myotubularin (upper panel) and wild-type muscle (lower). Proteins from total homogenate (HT), S1 (supernatant at 1000g), S2 (10 000g), S3 (100 000g) and the corresponding pellet fractions (P1, P2 and P3) were analyzed by immunoblotting. Myotubularin is distributed along all fractions. We used myosin heavy chain (MHC) and GAPDH as internal controls for cytosolic fractions. GRP78/BiP and caveolin 3 (CAV3) immunoreactivities are indicative of membrane fractions. Antibody r1947 was used in all illustrated experiments.
Figure 6.
Figure 6.
Effect of overexpression of myotubularin in skeletal muscle. (A) Presence of needle-like structures (left panel, arrows) and vacuoles (right panel) in wild-type muscle overexpressing myotubularin. Semithin cross-sections of tibialis anterior were stained with toluidine blue (×630, left and ×1000, right). (B) Electron micrographs illustrate the nature of myotubularin-induced needle-like structures (left and middle panels). They resemble myelin-like structures and are occasionally associated with honey combs-like structures (arrow, middle panel). Note that the basal lamina (arrowhead) is not included. Vacuoles often contain degenerative membrane aggregates (arrow, right panel). Occasional authophagic vacuoles are also present (arrowhead). Bars represent 2 µm in the left and right panels and 400 nm in the middle panel. (C) Immunogold detection with an anti-myotubularin antibody shows labeling of the sarcolemma (arrowheads) and an intense signal on the membrane aggregates. Bars indicate 200 nm. (D) Left panel: central section of an electron tomogram from a cellular region containing such a membrane assembly. Membrane sheets clearly appear as being composed of two bilayers. Vesicular structures can be found attached to the membrane sheets (arrow). Central panel: section through the membrane assembly normal to the section plane as indicated by the dotted line in the left panel. This cross-section demonstrates that the lipid assemblies are flat layers and not tubular structures. Right panel: surface representation of the lipid layers which evidences the twisted nature of the close to parallel lipid assemblies. The bar represents 200 nm in the left and central panels and 125 nm in the right panel.

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