Karine Charton

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

A human skeletal muscle interactome centered on proteins involved in muscular dystrophies: LGMD interactome

BackgroundThe complexity of the skeletal muscle and the identification of numerous human disease-causing mutations in its constitutive proteins make it an interesting tissue for proteomic studies aimed at understanding functional relationships of interacting proteins in both health and diseases.MethodWe undertook a large-scale study using two-hybrid screens and a human skeletal-muscle cDNA library to establish a proteome-scale map of protein-protein interactions centered on proteins involved in limb-girdle muscular dystrophies (LGMD). LGMD is a group of more than 20 different neuromuscular disorders that principally affect the proximal pelvic and shoulder girdle muscles.Results and conclusionThe interaction network we unraveled incorporates 1018 proteins connected by 1492 direct binary interactions and includes 1420 novel protein-protein interactions. Computational, experimental and literature-based analyses were performed to assess the overall quality of this network. Interestingly, LGMD proteins were shown to be highly interconnected, in particular indirectly through sarcomeric proteins. In-depth mining of the LGMD-centered interactome identified new candidate genes for orphan LGMDs and other neuromuscular disorders. The data also suggest the existence of functional links between LGMD2B/dysferlin and gene regulation, between LGMD2C/γ-sarcoglycan and energy control and between LGMD2G/telethonin and maintenance of genome integrity. This dataset represents a valuable resource for future functional investigations.

Removal of the calpain 3 protease reverses the myopathology in a mouse model for titinopathies

The dominant tibial muscular dystrophy (TMD) and recessive limb-girdle muscular dystrophy 2J are allelic disorders caused by mutations in the C-terminus of titin, a giant sarcomeric protein. Both clinical presentations were initially identified in a large Finnish family and linked to a founder mutation (FINmaj). To further understand the physiopathology of these two diseases, we generated a mouse model carrying the FINmaj mutation. In heterozygous mice, dystrophic myopathology appears late at 9 months of age in few distal muscles. In homozygous (HO) mice, the first signs appear in the Soleus at 1 month of age and extend to most muscles at 6 months of age. Interestingly, the heart is also severely affected in HO mice. The mutation leads to the loss of the very C-terminal end of titin and to a secondary deficiency of calpain 3, a partner of titin. By crossing the FINmaj model with a calpain 3-deficient model, the TMD phenotype was corrected, demonstrating a participation of calpain 3 in the pathogenesis of this disease.

RNA-targeting approaches for neuromuscular diseases

Although most molecular therapy strategies for genetic diseases are based on gene replacement, interesting alternative approaches target RNA. These strategies rely on the modification of the mutated genes expression in vivo by modulating pre-mRNA splicing, mRNA stability or mRNA translation. Here, we review recent progress using these RNA-based approaches in the field of muscle and muscle-related genetic diseases. Different molecular tools, including modified antisense oligonucleotides, pre-mRNA trans-splicing molecules, ribozymes or chemical compounds have been used successfully on patient cells or animal models of disease. These diverse strategies show tremendous therapeutic potential and several clinical trials have been initiated with Duchenne muscular dystrophy patients with promising results.

A new titinopathy: Childhood-juvenile onset Emery-Dreifuss–like phenotype without cardiomyopathy

Objective: To identify the genetic defects present in 3 families with muscular dystrophy, contractures, and calpain 3 deficiency. Methods: We performed targeted exome sequencing on one patient presenting a deficiency in calpain 3 on Western blot but for which mutations in the gene had been excluded. The identification of a homozygous truncating mutation in the M-line part of titin prompted us to sequence this region in 2 additional patients presenting similar clinical and biochemical characteristics. Results: The 3 patients shared similar features: coexistence of limb-girdle weakness and early-onset diffuse joint contractures without cardiomyopathy. The biopsies showed rimmed vacuoles, a dystrophic pattern, and secondary reduction in calpain 3. We identified a novel homozygous mutation in the exon Mex3 of the TTN gene in the first patient. At protein level, this mutation introduces a stop codon at the level of Mex3. Interestingly, we identified truncating mutations in both alleles in the same region of the TTN gene in patients from 2 additional families. Molecular protein analyses confirm loss of the C-ter part of titin. Conclusions: Our study broadens the phenotype of titinopathies with the report of a new clinical entity with prominent contractures and no cardiac abnormality and where the recessive mutations lead to truncation of the M-line titin and secondary calpain 3 deficiency.

Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy

Background— Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus–mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results— Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle–restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions— The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.

CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.

Circulating miRNAs are generic and versatile therapeutic monitoring biomarkers in muscular dystrophies

The development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD.

AAV-mediated transfer of FKRP shows therapeutic efficacy in a murine model but requires control of gene expression

&NA; Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol‐5‐phosphate transferase, is a participant in &agr;‐dystroglycan (&agr;DG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock‐in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of &agr;DG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of &agr;DG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV‐Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock‐out indicated a dose‐dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles.

The Phenotype of Dysferlin-Deficient Mice Is Not Rescued by Adeno-Associated Virus–Mediated Transfer of Anoctamin 5

Mutations in dysferlin and anoctamin 5 are the cause of muscular disorders, with the main presentations as limb-girdle muscular dystrophy or Miyoshi type of distal myopathy. Both these proteins have been implicated in sarcolemmal resealing. On the basis of similarities in associated phenotypes and protein functions, we tested the hypothesis that ANO5 protein could compensate for dysferlin absence. We first defined that the main transcript of ANO5 expressed in skeletal muscle is the 22-exon full-length isoform, and we demonstrated that dysferlin-deficient (Dysf (prmd)) mice have lower Ano5 expression levels, an observation that further enhanced the rational of the tested hypothesis. We then showed that AAV-mediated transfer of human ANO5 (hANO5) did not lead to apparent toxicity in wild-type mice. Finally, we demonstrated that AAV-hANO5 injection was not able to compensate for dysferlin deficiency in the Dysf (prmd) mouse model or improve the membrane repair defect seen in the absence of dysferlin. Consequently, overexpressing hANO5 does not seem to provide a valuable therapeutic strategy for dysferlin deficiency.