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ORIGINAL ARTICLE
Year : 2012  |  Volume : 135  |  Issue : 6  |  Page : 878-886

Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India


1 Department of Neurochemistry, National Institute of Mental Health & Neuro Sciences, Bangalore, India
2 Department of Neuropathology, National Institute of Mental Health & Neuro Sciences, Bangalore, India
3 Department of Neurology, National Institute of Mental Health & Neuro Sciences, Bangalore, India

Date of Acceptance12-Oct-2010
Date of Web Publication23-Jul-2012

Correspondence Address:
M M Srinivas Bharath
National Institute of Mental Health & Neuro Sciences, P.B # 2900, Hosur Road, Bangalore 560 029
India
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Source of Support: None, Conflict of Interest: None


PMID: 22825607

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   Abstract 

Background & objectives: Calpain-3, a Ca [2]+ -dependent protease has been implicated in the pathology of neuromuscular disorders (NMDs). The current study aimed to analyze calpain-3 expression in cases diagnosed as muscular dystrophy from the Indian population.
Methods: Calpain-3 Western blot analysis in muscle biopsies of immunohistochemically confirmed cases of Duchenne muscular dystrophy (DMD) (n=10), dysferlinopathy (n=30) and sarcoglycanopathy (n=8) was carried out. Calpain-3 Western blotting was also used in a blinded study to identify cases of calpain-3 deficiency in 28 NMD patients with potential muscular dystrophy.
Results: Calpain-3 appeared as a full length 94 kDa band with an autolytic product (~60 kDa) on Western blots with antibody NCL-CALP-12A2 (Ab-2). Eight of the 10 DMD samples showed absence of 94 kDa band but presence of 60 kDa band while one case of sarcoglycanopathy showed absence of both. Twenty one of the 30 dysferlinopathy samples showed both bands while six showed only the 60 kDa band and three showed absence of both. In the blinded study, five NMD cases with potential muscular dystrophy that showed complete absence of both bands in retrospect exhibited clinical features of limb girdle muscular dystrophy 2A (LGMD2A).
Interpretation & conclusions: While the study revealed a consistent pattern of calpain-3 in DMD, one sarcoglycanopathy and three dysferlinopathy samples exhibited secondary reduction in calpain-3. It was recognized that both calpain-3 bands should be considered to confirm calpain deficiency. Further, western blot offers an economical and fast preliminary screening method for LGMD2A especially in cases of complete absence of calpain-3 prior to conclusive diagnosis by genetic testing.

Keywords: Calpain - limb girdle muscular dystrophy - muscular dystrophy - neuromuscular disorders - western blot


How to cite this article:
Renjini R, Gayathri N, Nalini A, Srinivas Bharath M M. Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India. Indian J Med Res 2012;135:878-86

How to cite this URL:
Renjini R, Gayathri N, Nalini A, Srinivas Bharath M M. Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India. Indian J Med Res [serial online] 2012 [cited 2021 May 8];135:878-86. Available from: https://www.ijmr.org.in/text.asp?2012/135/6/878/98878

Calpains are Ca 2+ -dependent cysteine proteases that modulate the activities of various intracellular proteins and enzymes [1] . Among the various isoforms, calpain-3 (p94) isoform is specifically expressed in the skeletal muscle [2] . It has been established that a functional calpain-3 is mandatory for the structural organization, maintenance and turnover of sarcomere [3] . Calpain-3 interacts with several cytoskeletal proteins, regulates intracellular signal transduction and is critical for the functioning of skeletal muscle [4],[5] . Loss of calpain-3 is linked to pathological conditions such as muscular atrophy and dystrophy [6],[7] . Recessive mutations in calpain-3 causing defective protease activity results in limb girdle muscular dystrophy-2A (LGMD2A) or calpainopathy [8] . Interestingly, secondary calpain-3 deficiency is seen in other muscular dystrophies independent of genetic mutations [9],[10] . Conversely, a normal calpain-3 profile is observed in a few genetically confirmed LGMD2A cases [11],[12] . Hence, the role of calpain-3 in muscular dystrophy is not fully understood.

Due to the absence of defined mutational hot spots and presence of mutations in more than one gene, genetic analysis of calpain-3 is not straightforward [8],[13] . Although histology and immunohistochemistry are dependable [13],[14] , Western blot is considered as the easiest method for calpain-3 analysis [15] . Although autolytic activity of calpain-3 could also be used to analyze its status, the absence or reduction of the autocatalytic product on Western blots is the main indicator of calpain-3 deficiency [11],[13],[16] .

In this study, calpain-3 profile was compared in muscle biopsies from immunohistochemically confirmed cases of Duchenne muscular dystrophy (DMD), sarcoglycanopathy and dysferlinopathy from a tertiary referral centre for neuromuscular disorders in India. Calpain-3 profile was also analyzed in a blinded study to screen for potential LGMD2A samples from cases of neuromuscular disorders (NMDs) with potential muscular dystrophy.


   Material & Methods Top


Bulk chemicals were obtained from Merck & Co. Inc (Whitehouse Station, NJ, USA); anti-calpain-3 antibodies from Novocastra Laboratories (Newcastle upon Tyne, UK); nitrocellulose membrane from Millipore (Billerica, MA, USA); secondary antibodies from Bangalore Genei (Bangalore, Karnataka, India) were obtained.

Patients (n=82; M: F= 55: 27; age ranging from 13 months to 63 yr) with muscle diseases were evaluated at the Neuromuscular Disorders clinic of the National Institute of Mental Health and Neurosciences, Bangalore, India, during 2006-2009 and selected non-consecutively, subject to availability following preliminary diagnostic procedures. Skeletal muscle biopsies performed after obtaining informed consent were submitted for routine diagnosis to the department of Neuropathology, NIMHANS. Eighty three muscle samples including spinal muscular atrophy (SMA) (n=2), mitochondrial myopathy (n=2), suspected progressive myoclonic epilepsy (PME) (n=1), vasculitis (n=1), normal human autopsy sample (n=1) and immunohistochemically confirmed cases of DMD (n=10), dysferlinopathy (n=30), sarcoglycanopathies (n=8) were analyzed. The remaining 28 were blinded NMD samples with potential muscular dystrophy, which were found to be positive for the muscle proteins dystrophin, dysferlin, merosin, β dystroglycan and sarcoglycans (a, b, g and d) as confirmed by routine immunohistochemistry (IHC). The study protocol was approved by the Institutional Scientific Ethics Committee. Detailed clinical histories of all patients were recorded. The biopsy was conducted by a neurologist, under local anaesthesia for adults and under general anaesthesia for infants/young children. A moderately weak muscle (biceps or vastus lateralis) free from previous trauma was selected for uniformity. Fresh biopsy samples thus obtained were snap-frozen in isopentane precooled in liquid nitrogen and stored at -80 0 C. Autopsy muscle sample of one healthy subject who died due to non-neuromuscular disease was obtained and stored at -80 0 C until analysis.

The diagnosis of LGMD was based on clinical profile, family history, muscle biopsy studies, elevated serum creatine kinase (CK) levels and electromyography, according to the criteria described earlier [17] .

Preparation of protein extracts, SDS PAGE and Western blot: Total muscle proteins were extracted as described earlier [18] . Frozen muscle tissue (20 mg) was minced in 20 volumes of extraction buffer (75 mM Tris-HCl, pH 6.8, 15% SDS, 20% glycerol, 5% dithiothreitol and 0.001% bromophenol blue), sonicated for 30 sec (6 X 5 sec), boiled at 95 o C for 5 min and centrifuged at 15000 g (5 min); 10 ml of the solubilized proteins in the supernatant was subjected to 10 per cent SDS-PAGE and the gels were subjected to calpain-3 Western blotting using monoclonal antibodies (NCL-CALP-2C4 or Ab-1, NCL-CALP-12A2 or Ab-2 and NCL-CALP-11B3 or Ab-3) [18] . For myosin profile (loading control), protein extracts were run on 6 per cent SDS-PAGE and stained with coomassie brilliant blue. To avoid artifactual protein degradation which might reduce calpain-3 signal, precaution was taken to preserve the integrity of the biopsies and to maintain optimal protein loading and gel transfer conditions.

Semi-quantitative analysis of calpain-3 Western blots: Semi-quantitative analysis of calpain-3 expression in the blinded samples of NMD with potential dystrophy was carried out as described earlier [13] . The calpain-3 bands (94 and 60 kDa) on Western blots were scored as stronger than normal (5), normal (4), slightly reduced (3), reduced (2), markedly reduced (1) or absent (0) compared with positive control as calpain-3/ myosin ratio using the myosin band from the same patient (internal control). Samples with score "0" for both calpain-3 bands were repeated and considered as calpain-3 negative.

Histological and immunohistochemical analysis: A portion of the skeletal muscle biopsy oriented transversely was flash frozen in isopentane pre-cooled in liquid nitrogen; 8 m thick serial cryosections were subjected to routine staining [haematoxilin-eosin (HE), modified gomori trichrome (MGT), succinic dehydrogenase (SDH), nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), adenosine tri phosphate (ATPase) pH 9.5, 4.6 and acid phosphatase]. Immunostaining to monoclonal antibodies against dystrophin (1, 2, 3), sarcoglycans (α, β, γ, δ), dysferlin, β-dystroglycan and merosin as primary and HRP tagged goat anti-mouse as secondary antibodies were carried out.


   Results Top


Three monoclonal calpain-3 antibodies were used on muscle biopsies from PME (non-dystrophic control). Ab-1 detected only the 94 kDa band (full-length calpain-3) with a faint 30 kDa autocatalytic product [Figure 1]A. Ab-2 detected both the 94 kDa band and the autocatalytic band appearing as a doublet at ~60 kDa [Figure 1]B. Ab-3 detected not only the 94 kDa and the 60 kDa autocatalytic doublet bands but also the m and m-calpains around 85 kDa [Figure 1]C. Since the presence of autocatalytic 60 kDa band (irrespective of the 94 and 30 kDa bands) is considered as a marker of active calpain-3 [13] , Ab-2 was selected to analyze all the biopsies. In the normal sample, only 60 kDa bands were visible possibly due to the long post-mortem interval and consequent artifactual degradation [Figure 1]D.
Figure 1: Characterization of the monoclonal antibodies used in the study. Western blot using 3 monoclonal antibodies against calpain-3 in muscle biopsies are shown. (A ) Western blot with Ab-1 [lanes 1 & 2 correspond respectively to 10 & 20 ml of muscle extract from the positive control (PME)]. (B) western blot with Ab-2 [lanes 1 & 2 correspond respectively to 10 & 20 ml of muscle extract from PME]. (C) western blot with Ab-3 (Lanes 1-3 correspond 10 ml each of the total extract from 3 dystrophic muscle samples, respectively). (D) Western blot with Ab-2 (lanes 1 & 2 correspond to 10 ml each of the total extract from PME and normal autopsy muscle respectively). The protein molecular weights (MW) in kDa in each blot are shown.

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Calpain-3 pattern in muscular dystrophies: Calpain-3 Western blots using Ab-2 in biopsies of vasculitis, SMA and mitochondrial myopathy displayed both the 94 and 60 kDa bands [Figure 2]A. However, SMA (2 cases; lanes 3 and 5) comparatively showed low levels of 94 kDa band indicating higher autolytic activity of calpain-3.
Figure 2: Western blot analysis with Ab-2 to investigate the status of calpain-3 in different muscle biopsies including muscular dystrophies. (A) calpain-3 profile in PME (lane 1), vasculitis (lane 2), SMA (lanes 3 & 5), and mitochondrial myopathy (lanes 4 & 6) are shown. (B) calpain-3 profile in DMD muscle biopsies (lanes 1-5 & 6-10) compared with positive control (PME) (+) is shown. (C) calpain-3 profile in dysferlinopathy muscle biopsies (lanes 1-4, 5-8 & 9-13) compared with positive control (PME) (+) is shown. (D) calpain-3 profile in sarcoglycanopathy muscle biopsies (lanes 1-4 & 5-7) compared with positive control (PME) (+) is shown. The corresponding myosin band in each sample is indicated below the blot.

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Eight of the 10 DMD samples showed marked reduction or absence of the 94 kDa band but positive 60 kDa band ([Figure 2]B, lanes 1, 4, 5 and 6-10) indicating higher calpain-3 activity. Twenty one of the 30 dysferlinopathy samples showed both calpain-3 bands while six showed only the 60 kDa product and three showed complete absence of both bands. [Figure 2]C shows 13 such samples of which 9 samples exhibited both species (lanes 1, 2 and 5-8, 9, 11 and 13), two exhibited only the 60 kDa product (lanes 3 and 4). However, two exhibited absence of both bands (lanes 10 and 12, [Figure 2]C) which might represent secondary reduction in calpain-3 downstream of dysferlin loss. Among the sarcoglycanopathies, one case (No. 4) showed complete absence of calpain-3 bands ([Figure 2]D, lane 4) while other samples exhibited normal pattern.

Western based detection of calpain-3 deficiency in blinded samples: Calpain-3 Western blotting was carried out on the 28 cases of NMDs with potential muscular dystrophy where the researcher was blinded to the identity of the samples [Figure 3]. Twenty eight cases (Male : Female = 14:14), biopsied at age ranging from 2-63 yr had age at onset ranging from 6 months to 14 yr. Their clinical diagnoses were LGMD (n=18), congenital muscular dystrophy (CMD, n=2), distal myopathy with rimmed vacuoles (DMRV, n=3), DMD (n=1) and spinal muscular atrophy (SMA, n=5). Histologically, majority (n=21) were diagnosed to have LGMD, while CMD (n=4), DMRV (n=2) and SMA (n=1) constituted the rest. Immunohistochemically, preserved labelling to dystrophin, sarcoglycans (α, β, γ, δ), β dystroglycan, merosin and dysferlin was noted in all cases.
Figure 3: Western blot analysis of calpain-3 using Ab-2 in muscle biopsies from unclassified muscular dystrophies (blinded cases). Calpain-3 Western blot in 13 out of 28 unclassified NMD muscle biopsies compared with positive control (PME) (indicated by +) were carried out. While A shows 5 unclassified cases (lanes 1 to 5), B shows 4 cases (lanes 1 to 4) and C shows 4 cases (lanes 1 to 4). Among the 13 samples, lanes 1 and 3 in C corresponding to samples 3 and 4 (as shown in Table) are calpain-3 negative.

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The Western data of the 28 cases revealed 23 cases with preserved 60 kDa product and 20 of these 23 cases exhibited the 94 kDa band. We found heterogeneity in the calpain-3 pattern and the relative abundance of 94 and 60 kDa bands suggesting that direct association with the disease condition and severity would be difficult. However, based on the semi-quantitative analysis of the calpain-3/ myosin ratio, complete absence of all calpain-3 bands (score 0,0) was noted in five cases (case nos. 1, 2, 3, 4 and 5 in the Table), which could be potential cases of LGMD2A. There were four samples with score (0,1) with markedly reduced calpain-3 expression. Interestingly, none of the samples showed presence of only 94 kDa with the absence of the 60 kDa band. On the contrary, there were samples (lanes 1& 5 in [Figure 3]A) which showed marked reduction or absence of the 94 kDa and reduced 60 kDa band.

Histopathology and clinical evaluation of selected calpain-3 deficient cases: Following Western blotting, only the cases with (0,0) score as analysed by semiquantitative analysis of calpain-3 were analyzed further. Accordingly, five cases with primary calpain-3 deficiency were selected from the blinded study (cases 1-5 in the Table) for review of the stained sections of muscle biopsies. Dystrophic features were noted in all cases with presence of atrophic angulated fibers with clumped nuclei and a few muscle giant cells in one of the cases. In addition, inflammatory infiltrates in the interstitium and perivascular region comprising lymphocytes and eosinophils were noted in all (representative [Figure 4]A and B, case 3 in the [Table 1], corresponding to lane 1 of [Figure 3]C). There was no evidence of lobulated fibers on SDH and NADH-Tr. Mosaic pattern was seen on ATPase reaction. Immunostaining with monoclonal antibodies against dystrophin (DYS 1, 2 3), sarcoglycans (α, β, γ, δ), dysferlin, β dystroglycan and merosin showed uniform intense labelling along the membrane in all the fibers suggesting that these five biopsies did not represent DMD/BMD or sarcoglycanopathy or dysferlinopathy [Figure 4] C-K.
Figure 4: Histological and immunohistochemical analysis of muscle biopsy from a representative case (case no. 3 in Table) of calpain-3 deficiency. Transverse section of skeletal muscle stained for haematoxilin-eosin shows variation in diameter, myophagocytosis and dense interstitial inflammation (A) X200. Note the presence of eosinophils (↑q) (B) X400. Immunohistochemical staining to antibodies against dystrophin ( C: DYS 1; D: DYS 2; E: DYS 3), Merosin (F), sarcoglycans ( G: α; H: β; I: δ; J: γ) and dysferlin (K) shows uniform labelling along the membrane in all the fibers X200.

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Table 1: Salient features of cases (n=9) with calpain-3 deficiency as per the Western blot analysis

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A detailed follow up of the patients confirmed that these cases indeed exhibited all the classical features of LGMD2A. Among the five cases (four men and one woman), the mean age at presentation was 21.5 ± 6.47 yr (range: 14-31 yr), mean age at onset was 16.83 ± 6.68 yr (11-27 yr) and mean duration of illness was 4.5 ± 2.6 yr (2-9 yr). Three patients (with ages at onset 11, 11 and 12 yr, respectively) presented with classical scapulo-humeral phenotype, multiple contractures and tip-toe walking. The remaining three patients presented with pelvo-femoral phenotype with weakness in limb girdle distribution with age at onset as 18, 22 and 27 yr. Contractures were not prominent. This group had a later age at onset as compared to scapulo-humeral form. The mean CK value was 3574.33 ± 2924.1 IU/l (range 511-8392 IU/l).


   Discussion Top


In this study, we compared the calpain-3 protein profile across muscular dystrophy samples from India. In calpain-3 Western blots, all the bands must be considered in the context of other muscle proteins and optimal tissue preservation instead of determining the abnormal levels of the full length protein only [13] . We found a typical pattern for calpain-3 with a full length protein (94 kDa) and an autolytic product (60 kDa). But the identity of the proteolytic product is unknown. We did not find a prominent 30 kDa band in our blots probably due to limited sensitivity or degradation specific to our samples. We considered only the complete absence of the 60 and 94 kDa bands rather than reduced expression as an indicator of calpain-3 deficiency (LGMD2A).

In DMD, eight of the 10 biopsies showed absence of the 94 kDa band indicating increased autolytic activity [26] . Increased calpain activity has been localized to atrophic fibers in DMD and might be important but not specific to DMD [19],[20] . Hence, calpain inhibition could be a therapeutic option in DMD [21] . The intracellular location of calpain is directly associated with its cellular role under normal physiological conditions and in DMD [22] . Interestingly, dystrophin is an important substrate for calpain-3 [23] .

The interaction between calpain-3 and dysferlin is not completely defined [17] . Calpain-3 probably interacts physically with dysferlin [24] which might reduce dysferlin membrane labelling in LGMD2A biopsies [13] . A secondary reduction in calpain-3 expression is observed in dysferlinopathy [9] . In our study, although we did not observe a significant change in calpain-3 bands in 70 per cent dysferlinopathy biopsies, consistent with the gene expression data published earlier [25] , six cases showed absence of 94 kDa band and three showed complete absence of both bands.

If the clinical evaluation is suggestive of calpainopathy, the first step is to rule out other LGMDs by immunohistochemistry of other muscle proteins that might cause secondary reduction of calpain-3. Although this is an indirect method, it is still the best preliminary option. In this study, samples which were positive for dystrophin, dysferlin, sarcoglycans and merosin immunolabelling were considered for calpain-3 Western blotting. We found one case of calpain-3 deficiency secondary to deficiency of b-sarcoglycan and three cases secondary to deficiency of dysferlin. However, none of the 28 blinded cases of NMDs showed any significant reduction in other muscle proteins.

Recently Pathak et al[26] reported a study based on semi-quantitative protein analysis with clinical and histopathological correlation in LGMD2A in India. According to their data based on 171 cases of clinically suspected LGMD, unclassified muscular dystrophy or myopathy, based on histopathology, immunohistochemistry and Western blots, 75 of the biopsies showed complete calpain-3 deficiency of which 45 per cent had complete loss and 39 per cent had partial loss of calpain-3 protein. In that study, the calpain-3 status was based exclusively on the full length calpain-3 band at 94 kDa. However, approaches such as ours and others [26] are applicable only in cases showing complete absence of calpain-3. There might be LGMD2A cases in the remaining 23 samples that went undetected due to normal calpain-3 pattern. While some mutations cause calpain-3 reduction, others may not affect protein levels but rather cause functional inactivation [11],[16],[27],[28],[29],[30] .

Hence, biopsies with normal calpain-3 on Western blot should be confirmed by a functional in vitro autolytic assay followed by genetic analysis [27],[28],[29],[30] . Although genetic testing provides significant information about the disease, it is not the first method of choice for diagnosis of LGMD2A, as it is expensive, labour intensive and might be ambiguous due to lack of definite mutational hotspots [9] . Therefore, Western blot based preliminary screening method can be used as the first step to quickly screen LGMD2A in a high-throughput setting in clinically suggestive cases, before conclusive diagnosis based on immunohistochemistry and molecular genetics.


   Acknowledgment Top


This work was financially supported by the Department of Science and Technology, New Delhi, India. The first author (RR) acknowledges the Council for Scientific and Industrial Research, India, for providing the junior research fellowship. The authors thank all the patients and their families.

 
   References Top

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