Marc Bartoli

Calpain 3 Is Activated through Autolysis within the Active Site and Lyses Sarcomeric and Sarcolemmal Components

ABSTRACT Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer

Deficiency of the dysferlin protein presents as two major clinical phenotypes: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin is known to participate in membrane repair, providing a potential hypothesis to the underlying pathophysiology of these diseases. The size of the dysferlin cDNA prevents its direct incorporation into an adeno-associated virus (AAV) vector for therapeutic gene transfer into muscle. To bypass this limitation, we split the dysferlin cDNA at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Intramuscular injection of the corresponding vectors into a dysferlin-deficient mouse model led to the expression of full-length dysferlin for at least 1 year. Importantly, systemic injection in the tail vein of the two vectors led to a widespread although weak expression of the full-length protein. Injections were associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.

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.

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)

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.

Calcium-dependent plasma membrane repair requires m- or μ-calpain, but not calpain-3, the proteasome, or caspases

Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the non-catalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive E1 ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or mu-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury.

Down-regulation of striatin, a neuronal calmodulin-binding protein, impairs rat locomotor activity.

Striatin, an intraneuronal, calmodulin-binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein-protein interactions, suggesting a role within a dendritic Ca(2+)-signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A-ODN) or mismatch ODN (M-ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A-ODN-treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A-ODN-treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down-regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A-ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines.

A third of LGMD2A biopsies have normal calpain 3 proteolytic activity as determined by an in vitro assay

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependent protease. We developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 as a substrate for active calpain 3 molecules. A total of 79 human biopsies have been analysed using an unbiased single blind method. Results were confronted with the molecular diagnosis for confirmation. Proteolytic activity was either reduced or absent in 68% of LGMD2A biopsies. In the remaining 32%, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties might be impaired to give rise to the LGMD2A phenotype. Our assay is easily adaptable to routine and appears to be more sensitive than common analysis by immunodetection.

Mannosidase I inhibition rescues the human α-sarcoglycan R77C recurrent mutation

Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.

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.