Laurence Suel

NF-κB-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A

Limb‐girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF‐NF‐κBB/INF‐κBBα survival pathway. In this study, the consequences of altered NF‐BNF‐κBB/IBNF‐κBBBα pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular‐FLICE inhibitory protein (C‐FLIP), which is dependent on the NF‐BNF‐κBB pathway in normal muscle cells, is down‐regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF‐BNF‐κBB is readily acti vated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF‐BNF‐κBB target genes. IBNF‐κBBBα is expressed following NF‐BNF‐κBB activation independent of the CAPN3 status, whereas expression of C‐FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF‐BNF‐κBB‐dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF‐BNF‐κBB pathway could be part of the mecha nism responsible for the muscle wasting resulting from CAPN3 deficiency.—Benayoun, B., Baghdiguian, S., Lajmanovich, A., Bartoli, M., Daniele, N., Gicquel, E., Bourg, N., Raynaud, F., Pasquier, M.‐A., Suel, L., Lochmuller, H., Lefranc, G., Richard, I. NF‐BNF‐κBB‐dependent expression of the antiapoptotic factor C‐FLIP is regulated by calpain 3, the protein involved in limb‐girdle muscular dystrophy type 2A. FASEB J. 22, 1521–1529 (2008)

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.

Cardiac ankyrin repeat protein is a marker of skeletal muscle pathological remodelling

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, α‐sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin–proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21WAF1/CIP1, is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast‐twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21WAF1/CIP1 is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

Lack of correlation between outcomes of membrane repair assay and correction of dystrophic changes in experimental therapeutic strategy in dysferlinopathy.

Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.

A new pathway encompassing calpain 3 and its newly identified substrate cardiac ankyrin repeat protein is involved in the regulation of the nuclear factor‐κB pathway in skeletal muscle

A multiprotein complex encompassing a transcription regulator, cardiac ankyrin repeat protein (CARP), and the calpain 3 protease was identified in the N2A elastic region of the giant sarcomeric protein titin. The present study aimed to investigate the function(s) of this complex in the skeletal muscle. We demonstrate that CARP subcellular localization is controlled by the activity of calpain 3: the higher the calpain 3, the more important the sarcomeric retention of CARP. This regulation would occur through cleavage of the N‐terminal end of CARP by the protease. We show that, upon CARP over‐expression, the transcription factor nuclear factor NF‐κB p65 DNA‐binding activity decreases. Taken as a whole, CARP and its regulator calpain 3 appear to occupy a central position in the important cell fate‐governing NF‐κB pathway. Interestingly, the expression of the atrophying protein MURF1, one of NF‐κB main targets, remains unchanged in presence of CARP, suggesting that the pathway encompassing calpain3/CARP/NF‐κB does not play a role in muscle atrophy. With NF‐κB also having anti‐apoptotic effects, the inability of calpain 3 to lower CARP‐driven inhibition of NF‐κB could reduce muscle cell survival, hence partly accounting for the dystrophic pattern observed in limb girdle muscular dystrophy 2A, a pathology resulting from the protease deficiency.

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.

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.

G.P.229

Several published studies strongly implied the existence of functional relationship between calpain 3 and titin proteins. Calpain 3, the cysteine protease mutated in Limb Girdle Muscular Dystrophy type 2A (LGMD2A), has at least 2 different binding sites on titin, which is also one of calpain 3 substrates. Moreover, Tibial Muscular Dystrophy (TMD) and LGMD2J, both caused by the mutations in the last exons of the titin gene, are characterised by secondary reduction of calpain 3 expression. We also showed that the last domains of titin are not present in the TMD/LGMD2J muscle. Here, we tested several calpain 3 properties in vivo and in vitro conditions to better clarify the relationship between calpain 3 and titin in the M-band of the sarcomere, as well as its possible implication in the pathogenesis of LGMD2A and LGMD2J. An in vitro analysis validated the C-terminal part of the titin protein as a proteolytic substrate for calpain 3. Moreover, we demonstrated that TMD/LGMD2J mutations induce disruption of these cleavages in vitro while calpain 3 is still able to interact with titin. Interestingly and unexpectedly, we showed that the disappearance of the M-band titin seen in vivo as a consequence of TMD/LGMD2J mutations is still observed in the absence of calpain 3, indicating that this event is not caused by a calpain 3 proteolytic cleavage.