Muriel Durand

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

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.

Phenotypic Correction of α-Sarcoglycan Deficiency by Intra-arterial Injection of a Muscle-specific Serotype 1 rAAV Vector

α-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in α-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human α-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, α-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated α-sarcoglycan gene transfer as a therapeutic approach.

Six and Eya expression during human somitogenesis and MyoD gene family activation.

This report describes the characterisation of the expression profile of several myogenic determination genes during human embryogenesis. The data were obtained from axial structures and limb buds of human embryos aged between 3 and 8 weeks of development. Using in situ hybridisation to detect Pax3 and MyoD gene family mRNAs, and immunochemistry to follow Six and Eya protein accumulation, we have been able to establish the chronology of accumulation of these gene products. As in mouse, the first transcripts detected in myotomes of 3 week-old embryos are Pax3 and Myf5, followed by the expression of myogenin. MyoD appears to be activated well after Myf5, myogenin and MRF4 in the early myotome, whereas, in limb bud muscles, the presence of all four of these mRNAs is concomitant from 6 weeks. Six1, Six4 and Six5 homeoproteins are detected later than Myf5 activation. These Six homeoproteins are first observed in the cytoplasm of myogenin expressing cells. At later stages of development, Six1 and Six5, but not Six4, are translocated into the nuclei of myogenic cells, concomitantly with MyHCemb expression. Eya1 and Eya2 proteins, potential Six cofactors, were also detected in myogenin positive cells, but their accumulation was delayed and was mainly cytoplasmic. These results preclude that early activation of Myf5, myogenin and MRF4 is under the control of Six and Eya proteins, while Six and Eya proteins would be involved in later steps of myogenic differentiation.

Force impairment in calpain 3–deficient mice is not correlated with mechanical disruption

Defects in human calpain 3 are responsible for limb‐girdle muscular dystrophy type 2A, an autosomal‐recessive disorder characterized mainly by late‐onset proximal muscular atrophy. A corresponding murine model has previously been generated by gene targeting. In this report, muscular activity of calpain 3–deficient (capn3−/−) mice was evaluated at different ages. Growth curves showed a progressive global muscular atrophy. Histological examination throughout the lifespan of mice confirmed the dystrophic lesions. Whole animal tests showed only a mild significant impairment of the forelimbs. Studies of the mechanical properties of selected isolated fast‐ and slow‐twitch muscles demonstrated that slow‐twitch muscles were significantly weaker in capn3−/− mice than in wild‐type mice. Three different tests showed that there was no membrane disruption, suggesting a nonmechanical etiology of capn3−/− mice dystrophy. These findings are consistent with a mechanism involving signaling systems. Muscle Nerve 27: 616–623, 2003

The muscle-specific enolase is an early marker of human myogenesis.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) is active as a dimer formed from three different subunits, α, β and γ, encoded by separate genes. The expression of these genes is developmentally regulated in a tissue-specific manner. A shift occurs during development, from the unique embryonic isoform αα, towards specific isoforms in two tissues with high energy demands: αβ and γγ in the nervous system, αβ and ββ in striated muscles. The αα remains widely distributed in adult tissues. Here we report the results of the first extensive study of β enolase expression during human development. Indeed, the β subunit is specifically expressed at early stages of human myogenesis. Immunocytochemical analyses demonstrated that it is first detected in the heart of 3-week-old embryos and in the myotomal compartment of somites from 4-week-old embryos. At this stage, the muscle-specific sarcomeric protein titin is expressed in this structure, which will give rise to all body skeletal muscles, but embryonic myosin heavy chain is not yet present. Analyses at the protein level show that, during human ontogenesis, myogenesis is accompanied by an increase in β enolase expression and by a decrease in the expression of the two other α and γ subunits. Furthermore, β enolase subunit is expressed in proliferating myoblasts from both embryonic and post-natal muscles. In addition, clonal analysis of primary cell cultures, obtained from the leg muscle of a 7-week-old human embryo, revealed that the β subunit is present in the dividing myoblasts of all four types, according to the classification of Edom-Vovard et al. [(1999) J Cell Sci 112: 191–199], but not in cells of the non-myogenic lineage. Myoblast fusion is accompanied by a large increase in β enolase expression. Our results demonstrate that this muscle-specific isoform of a glycolytic enzyme (β enolase) is among the earliest markers of myogenic differentiation in humans.

Calpain3 expression during human cardiogenesis.

Transcripts of calpain3, the gene involved in limb girdle muscular dystrophy type 2A, appear in organs other than the skeletal muscle during human development, the first of which being the early embryonic heart. We examined more precisely the spatio-temporal transcription pattern of calpain3 during human cardiogenesis and the appearance of its protein in fetal tissues, and correlated it to titin expression. Different events of the hearts maturation can be recognized: (i) the presence of titin RNA or protein constitute very precocious developmental cardiac markers appearing before the fusion of the two lateral endocardial tubes; (ii) the disappearance of calpain3 RNA from the ventricular compartment later in the embryonic heart. Finally, although calpain3 transcripts are present in the heart, the corresponding protein is not detected elsewhere than in skeletal muscle.