Muscle‐specific calpain‐3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction

CAPN3 (also called p94/calpain‐3) is a skeletal muscle‐specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb‐girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril‐based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.     

Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.     

Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3

Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.     

Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway

Mutations in the non-lysosomal cysteine protease calpain 3 cause limb-girdle muscular dystrophy type 2A (LGMD2A). Our previous studies of the calpain 3 knockout mouse (C3KO) suggested a role for calpain 3 in sarcomere formation and remodeling. Calpain 3 may mediate remodeling by cleavage and release of myofibrillar proteins, targeting them for ubiquitination and proteasomal degradation. Loss of proper protein turnover may be the basis for this muscle disease. To test this hypothesis in vivo, we used an experimental model of hindlimb unloading and reloading that has been shown to induce sarcomere remodeling. We showed that the rate of atrophy and especially the rate of growth are decreased in C3KO muscles under conditions promoting sarcomere remodeling. In wild-type mice, an elevated level of ubiquitinated proteins was observed during muscle reloading, which is presumably necessary to remove atrophy-specific and damaged proteins. This increase in ubiquitination correlated with an increase in calpain 3 expression. C3KO muscles did not show any increase in ubiquitination at the reloading stage, suggesting that calpain 3 is necessary for ubiquitination and that it acts upstream of the ubiquitination machinery. We found upregulation of heat shock proteins in C3KO muscles following challenge with a physiological condition that requires highly increased protein degradation. Furthermore, old C3KO mice show evidence of insoluble protein aggregate formation in skeletal muscles. These studies suggest that accumulation of aged and damaged proteins can lead to cellular toxicity and a cell stress response in C3KO muscles, and that these characteristics are pathological features of LGMD2A.     

A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity

Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43‐year‐old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild‐type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.     

Mdm muscular dystrophy: interactions with calpain 3 and a novel functional role for titin's N2A domain

Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.     

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.     

cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were suspected to have LGMD2A, based on western blot results. Four of these patients were shown to have LGMD2I upon molecular analysis, whereas 16 of the remaining 42 patients harbored mutations in CAPN3 by both direct genomic sequencing and cDNA analyses. In 10 patients, we identified both mutant alleles. In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish origin, indicating a five- to sixfold lower prevalence in Denmark compared to other European countries. A total of 16 different CAPN3 mutations were identified, of which 5 were novel. The present study demonstrates the value of cDNA analysis for CAPN3 in LGMD2A patients and indicates that calpainopathy is an uncommon cause of LGMD in the Denmark.     

Characterization of lobulated fibers in limb girdle muscular dystrophy type 2A by gene expression profiling

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in CAPN3, which encodes an intracellular cysteine protease. To elucidate the fundamental molecular changes that may be responsible for the pathological features of LGMD2A, we employed cDNA microarray analysis. We divided LGMD2A muscles into two groups according to specific pathological features: an early-stage group characterized by the presence of active necrosis and a regeneration process and a later-stage group characterized by the presence of lobulated fibers. After comparing the gene expression profiles of the two groups of LGMD2A muscles with control muscles, we identified 29 genes whose mRNA expression profiles were specifically altered in muscles with lobulated fibers. Interestingly, this group included genes that encode actin filament binding and regulatory proteins, such as gelsolin, PDZ and LIM domain 3 (PDLIM3) and troponin I1. Western blot analysis confirmed the upregulation of these proteins. From these results, we propose that abnormal increased expression of actin filament binding proteins may contribute to the changes of the intra-myofiber structures, observed in lobulated fibers in LGMD2A.     

Molecular diagnosis in LGMD2A: Mutation analysis or protein testing?

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain-3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS-PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain-3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain-3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype-protein-phenotype correlations and provide insights into a critical protein domain.     

Ryanodine receptors in human bladder smooth muscle

The role of intracellular Ca2+ release in the activation of human bladder smooth muscle is controversial. We have measured the expression of mRNA encoding for the ryanodine receptor (RyR) isoforms (RyR1, RyR2 and RyR3) in isolated human detrusor smooth muscle. mRNA for RyR2 was detected in all samples but no mRNA for RyR1 or RyR3 could be found. Human bladder smooth muscle cells in culture are unresponsive to caffeine, suggesting the absence of a functional RyR system. However, mRNA encoding for RyR2 was detected in these cells. Using saponin-permeabilized cells, a Ruthenium Red-sensitive Ca(2+)-dependent 45Ca2+ release could be demonstrated from the sarcoplasmic reticulum (SR). These data confirm the functional presence of Ca(2+)-induced Ca2+ release (CICR) in cells and suggest that the properties of the RyR2 isoform in human detrusor may change when the cells are maintained in culture. The implications of these observations to detrusor smooth muscle function are discussed.     

High incidence of 550delA mutation of CAPN3 in LGMD2 patients from Russia

Autosomal recessive limb gird muscular dystrophy (LGMD2) is a clinically and genetically heterogeneous group of diseases that are characterized by progressive atrophy and weakness of the proximal limb muscles. At least eight genetic loci leading to LGMD2 are recognized. The proportion of particular gene involved in producing different forms of LGMD2 shows a marked geographical variation. We studied 19 LGMD2 patients from Russia (15 families) and found calpain 3 (CAPN3) gene mutations in most of the patients studied. Sequence analysis of the fourth exons revealed two sibs - heterozygous compound for a 15-bp deletion (nt598-612) and 550 adenine deletion, and two sibs homozygous for a 550delA. We developed assay based on allele specific amplification (ASA) for rapid screening of the 550delA. The ASA assay of the LGMD2 patients under study showed that 7 patients from 6 families were homozygous for 550delA and 7 patients from 4 families were heterozygous for 550delA. A linkage analysis employing four microsatellites flanking the LGMD2A locus was performed. We found complete haplotype identity in most cases what favors the possibility of a common founder. Heterozygous carriers of 550delA were found in general population. The crude estimate of the mutation frequency is 1/150. Hum Mutat 15:295, 2000.     

Seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: from LGMD2A to LGMD2G

The autosomal recessive limb-girdle muscular dystrophies (AR-LGMDs) are a heterogeneous group of disorders of progressive weakness of the pelvic and shoulder girdle musculature. The clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression resembling clinically Xp21 Duchenne muscular dystrophy (DMD) to milder forms with later onset and slower course. Eight genes are mapped for the AR-LGMDs; they are: LGMD2A (CAPN3) at 15q, LGMD2B (dysferlin) at 2p, LGMD2C (gamma-SG) at 13q, LGMD2D (alpha-SG) at 17q, LGMD2E (beta-SG) at 4q, LGMD2F (6-SG) at 5q, LGMD2G at 17q, and more recently LGMD2H at 9q. The LGMD2F (delta-SG) and LGMD2G genes were mapped in Brazilian AR-LGMD families. Linkage analysis in two unlinked families excluded the eight AR-LGMD genes, indicating that there is at least one more gene responsible for AR-LGMD. We have analyzed 140 patients (from 40 families) affected with one of seven autosomal recessive LGMD loci, that is, from LGMD2A to LGMD2G. The main observations were: 1) all LGMD2E and LGMD2F patients had a severe condition, but considerable inter- and intra-familial clinical variability was observed among patients from all other groups; 2) serum CK activities showed the highest values in LGMD2D (alpha-SG) patients among sarcoglycanopathies and LGMD2B (dysferlin) patients among nonsarcoglycanopathies; 3) comparison between LGMD2A (CAPN3) and LGMD2B (dysferlin) showed that the first have on average a more severe course and have calf hypertrophy more frequently (86% versus 13%); and 4) inability to walk on toes was observed in approximately 70% of LGMD2B patients.     

The distribution and characterization of skeletal muscle lesions in dysferlin-deficient SJL and A/J mice

The pathogenesis of limb-girdle muscular dystrophy type 2B (LGMD2B) dysferlinopathy remains to be investigated. The distribution and characterization of skeletal muscle lesions were examined in two different LGMD2B mouse models, SJL and A/J mice (at 10 and 35 weeks old), in association with the endoplasmic reticulum (ER) stress. SJL mice showed an earlier age of onset and a faster progression of skeletal muscle lesions as compared with those of A/J mice; the sensitivity difference to muscular dystrophic lesions between SJL and A/J mice was observed in the lumbar muscles (particularly, lumbar longissimus and sublumbar muscles); the lesions seen mainly in SJL mice at 35 weeks old consisted of degeneration, necrosis, fatty infiltration, variation in muscle fiber size and atrophy in muscle fibers. Enzyme-histochemically, the fast-twitch muscle fiber was predominant for the degenerative changes seen in the rectus femoris and lateral longissimus muscles of SJL mice. Immunohistochemically, the main reactive cell type observed in and around degenerative and/or necrotic muscle fibers was macrophages, demonstrable with an anti-F4/80 antibody. Because the analyses of spliced XBP1 mRNA, a marker of ER stress, did not show the increased expression, it was considered that ER stress did not affect the progression of skeletal muscle lesions in SJL mice with the advanced stage of dysferlinopathy.