The effect of calpain 3 deficiency on the pattern of muscle degeneration in the earliest stages of LGMD2A

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 gene. In a large family affected by LGMD2A with four severely affected members, three additional asymptomatic relatives had very high serum creatine kinase concentrations. All were homozygous for the R110X mutation and showed a total absence of calpain 3 in the muscle. Histological analysis of muscle in these three rare preclinical cases showed a consistent but unusual pattern, with isolated fascicles of degenerating fibres in an almost normal muscle. This pattern was also seen in one patient with early stage LGMD2A who had a P82L missense mutation and a partial deficiency of calpain 3 in the muscle, but was not seen in early stage patients affected by other forms of LGMD. These findings suggest that a peculiar pattern of focal degeneration occurs in calpainopathy, independently of the type of mutation or the amount of calpain 3 in the muscle.     

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150?nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.     

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.     

Regulation of NF-kappaB and ICAM-1 expression in human airway epithelial cells.

The aim of this study was to elucidate the redox regulation of cytokine-induced NF-kappaB activation and NF-kappaB mediated gene induction in A549 cells and primary cultures of human airway epithelial cells. In A549 cells, Western blot analysis showed transient depletion of IkappaBalpha after 15 min IL-1beta treatment followed by its reappearance after 60 min, indicating efficient NF-kappaB-driven gene induction. A similar pattern was observed in primary epithelial cells however, the kinetics were slower and depletion was less. In primary airway epithelial cells IkappaBalpha levels were 59.8+/-8.5% of control following 30 min treatment with IL-1beta and in A549 cells 29.1+/-8.5% of control following 15 min IL-1beta treatment. Cytokine-induced IkappaBalpha depletion was associated with NF-kappaB nuclear accumulation and subsequent resynthesis of IkappaBalpha and upregulation of ICAM-1 in both cell types. The antioxidant, NAC (20 mM) had no effect on the kinetics of cytokine-induced IkappaBalpha depletion or NF-kappaB p65 nuclear translocation in either cell type and failed to influence kappaB dependent IkappaBalpha resynthesis. H2O2 treatment alone or in combination with cytokines had no significant effects on IkappaBalpha depletion, NF-kappaB p65 nuclear translocation or ICAM-1 expression in either cell type but did cause significant activation of p38 MAPK. These results suggest that cytokine-induced NF-kappaB activation in cultured human airway epithelial cells does not involve an NAC-sensitive oxidant stress and that H2O2-induced oxidant stress does not result in effective NF-kappaB activation and NF-kappaB mediated gene induction.     

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.     

Biochemical and ultrastructural evidence of endoplasmic reticulum stress in LGMD2I

Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.     

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.     

Clinical variability in calpainopathy: what makes the difference?

Limb girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders characterised by progressive weakness of the pelvic and shoulder girdle muscles and a great variability in clinical course. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the calpain-3 gene (CAPN-3). More than 100 pathogenic mutations have been identified to date, however few genotype : phenotype correlation studies, including both DNA and protein analysis, have been reported. In this study we screened 26 unrelated LGMD2A Brazilian families (75 patients) through Single-Stranded Conformation Polymorphism (SSCP), Denaturing high-performance liquid chromatography (DHPLC) and sequencing of abnormal fragments which allowed the identification of 47 mutated alleles (approximately 90%). We identified two recurrent mutations (R110X and 2362-2363AG > TCATCT) and seven novel pathogenic mutations. Interestingly, 41 of the identified mutations (approximately 80%) were concentrated in only 6 exons (1, 2, 4, 5, 11 and 22), which has important implications for diagnostic purposes. Protein analysis, performed in 28 patients from 25 unrelated families showed that with exception of one patient (with normal/slight borderline reduction of calpain) all others had total or partial calpain deficiency. The effects of type of mutation, amount of calpain in the muscle, gender and ethnicity of affected patients on clinical course (age of onset and ascertainment) were analysed. Interestingly, it was observed that, on average, African-Brazilian calpainopathy patients are more severely affected than Caucasians.     

Characterization of NF-kappaB expression in Hodgkin's disease: inhibition of constitutively expressed NF-kappaB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells.

Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-kappaB, the precise physiologic and clinical significance of NF-kappaB expression is currently undefined. Expression of active NF-kappaB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-kappaB inhibitors gliotoxin and MG132 and the effect of NF-kappaB inhibition utilizing an adenovirus vector carrying a dominant-negative IkappaBalpha mutant (Ad5IkappaB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-kappaB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-kappaB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-kappaB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IkappaB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-kappaB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-kappaB in HRS cell survival and resistance to apoptosis.     

IkappaBalpha independent induction of NF-kappaB and its inhibition by DHMEQ in Hodgkin/Reed-Sternberg cells

Constitutive nuclear factor kappaB (NF-kappaB) activation characterizes Hodgkin/Reed-Sternberg (H-RS) cells. Blocking constitutive NF-kappaB has been shown to be a potential strategy to treat Hodgkin lymphoma (HL). Here, for the first time we show that although constitutive NF-kappaB level of H-RS cell lines is very high, topoisomerase inhibitors further enhance NF-kappaB activation through IkappaB kinase activation in not only H-RS cell lines with wild-type IkappaBalpha, but also in those with IkappaBalpha mutations and lacking wild-type IkappaBalpha. Thus, both constitutive and inducible NF-kappaB are potential targets to treat HL. We also present the data that indicate the involvement of IkappaBbeta in NF-kappaB induction by topoisomerase inhibitors. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) inhibited constitutive NF-kappaB activity and induced apoptosis of H-RS cell lines. DHMEQ also inhibited the growth of H-RS cells without significant systemic toxicity in a NOD/SCID/gammac(null) (NOG) mice model. DHMEQ and topoisomerase inhibitors revealed enhancement of apoptosis of H-RS cells by blocking inducible NF-kappaB. Results of this study suggest that both constitutive and inducible NF-kappaB are molecular targets of DHMEQ in the treatment of HL. The results also indicate that IkappaBbeta is involved in NF-kappaB activation in H-RS cells and IkappaBbeta substitutes for IkappaBalpha in H-RS cells lacking wild-type IkappaBalpha.     

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.     

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.     

A novel pathogenic variant in TNPO3 in a Hungarian family with limb-girdle muscular dystrophy 1F

Limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous muscular diseases that predominantly affect the proximal muscles. Pathogenic variants in TNPO3 have been associated with a rare, autosomal dominant limb-girdle muscular dystrophy 1F (LGMD1F) in a large Italian-Spanish family and an isolated LGMD1F case. Here we present two individuals from a Hungarian family with an early-onset, slowly progressive muscular dystrophy. Both the female proband and her affected son had delayed early motor milestones including first walking at 14 months and 18 months, respectively. Both present with progressive weakness of facial, bulbar, axial, and distal muscles especially of the lower extremities. Electromyography indicated myogenic damage and muscle biopsy from the proband showed myopathic alterations with sarcoplasmic masses and signs of mitochondrial dysfunction. Exome sequencing of the female proband identified a novel c.2767delC p.(Arg923AspfsTer17) variant in TNPO3. Sanger sequencing confirmed the presence of the TNPO3 variant in the affected son; the unaffected son did not have the variant. The identification of the c.2767delC variant further supports the clinical significance of TNPO3 and expands the clinical spectrum of TNPO3-associated LGMD1F.     

Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A

Background

Limb girdle muscular dystrophy type 2A (LGMD2A) is characterised by wide variability in clinical features and rate of progression. Patients with two null mutations usually have a rapid course, but in the remaining cases (two missense mutations or compound heterozygote mutations) prognosis is uncertain.

Methods

We conducted what is to our knowledge the first systematic histopathological, biochemical and molecular investigation of 24 LGMD2A patients, subdivided according to rapid or slow disease progression, to determine if some parameters could correlate with disease progression.

Results

We found that muscle histopathology score and the extent of regenerating and degenerating fibres could be correlated with the rate of disease course when the biochemical and molecular data do not offer sufficient information. Comparison of clinical and muscle histopathological data between LGMD2A and four other types of LGMD (LGMD2B–E) also gave another important and novel result. We found that LGMD2A has significantly lower levels of dystrophic features (ie degenerating and regenerating fibres) and higher levels of chronic changes (ie lobulated fibres) compared with other LGMDs, particularly LGMD2B. These results might explain the observation that atrophic muscle involvement seems to be a clinical feature peculiar to LGMD2A patients.

Conclusions

Distinguishing patterns of muscle histopathological changes in LGMD2A might reflect the effects of a disease‐specific pathogenetic mechanism and provide clues complementary to genetic data.     

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.     

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype-phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.