Calpain 3 Is Important for Muscle Regeneration

Methods

Patients

Twenty-two patients with genetically verified LGMD2A (age 37 ± 14 years) participated in this study described in (Table 1) and also in two previous studies.^[24,25] All investigations were performed with informed consent and in accordance with the Declaration of Helsinki, and the study was approved by the Danish Committee System on Biomedical Research Ethics. To be able to determine if mutations in specific domains of CAPN3 affect regeneration more than others we included as many homozygous patients with LGMD2A as possible. Thirteen patients were homozygous for mutations in CAPN3, and six were functionally homozygous, because one allele was a null allele confirmed in previous studies.^[26–28] They found calpain 3 was not detected by western blotting, which is probably due to its degradation by nonsense-mediated mRNA decay. Three patients harbored two null alleles, and were all severely affected. For comparison of the three patients harboring two null alleles (age 48 ± 17 years), we included five clinically matched patients with Becker muscular dystrophy (BMD) (age 31 ± 16 years) and five patients with limb-girdle muscular dystrophy type 2I (LGMD2I) (age 27 ± 12 years) who were compound heterozygous for the p.L276I mutation in the fukutin-related protein gene FKRP (Table 1). Diagnoses for the patients with Becker muscular dystrophy were based on deletions in the dystrophin gene were identified in all five BMD patients.

The modified Gardner-Medwin and Walton clinical severity score,^[29] was used for our patients: grade 0 = hyperCKaemia, all activities normal; grade 1 = normal gait, unable to run freely, myalgia, atrophy; grade 2 = unable to walk on tiptoes, waddling gait; grade 3 = evident muscle weakness, stepping gait, climbing stairs with banister; grade 4 = difficulty in rising from the floor, presence of Gowers' sign; grade 5 = unable to rise from the floor; grade 6 = unable to climb stairs; grade 7 = unable to rise from a chair; grade 8 = unable to walk without assistance; grade 9 = unable to eat, drink or sit unassisted.

Muscle Force

The ankle dorsal flexion and knee extension forces, corresponding to the muscle that was biopsied, were measured by dynamometry.^[30,31] Muscle force measurements were compared to the ankle dorsal flexion (196 ± 52 N) and knee extension (269 ± 77 N) forces found in healthy subjects (14 males and 13 females, age 32 ± 12 years). For eleven patients with LGMD2A muscle force measurements corresponding to the muscle that was biopsied at the time of biopsy were not obtainable. However five of them had a clinical evaluation of the biopsied muscle on the MRC scale whereas the remaining six did not have any functional assessment at the time of biopsy (Table 1).

Muscle Biopsy and Histochemistry

Percutaneous needle muscle biopsies were obtained as part of the diagnostic procedure and snap-frozen in liquid nitrogen-cooled isopentane, and stored at -80°C. The quadriceps femoris was biopsied in sixteen patients, the tibialis anterior in thirteen and the biceps/triceps brachii in four patients. Ten μm serial cryo-sections were used for all stains. Sections were stained with hematoxylin and eosin for general histopathological evaluation, and assessment of internally nucleated fibers (INF). A myofiber was considered INF if at least one nucleus was non-peripheral. Internal nuclei originate from activated satellite cells, and reside internal for a period of several months after regeneration initiation in skeletal muscle (Figure 1).^[19,32,33] Two investigators (SH and TOK) independently assessed all biopsies blinded for patient data. Complete sections were evaluated for the number of INF and necrotic fibers. In addition we determined number of whorled fibers, representing an irregular regeneration of mature myofibers where the sarcomere alignment in the outer layers is perpendicular to the core.^[34]

Pathological Severity

Biopsies were graded on a 3-point pathological severity scale based on the dystrophic changes as previously described:^[2] 1 = active dystrophic process (marked increase of fiber size variability, active degeneration and regeneration, marked increase of connective tissue); 2 = moderate dystrophic process (marked increase of fiber size variability, increased central nuclei, few degenerating and regenerating fibers, slight increase of connective tissue); 3 = mild myopathic picture (moderate increase of fiber size variability, increased central nuclei).

Immunohistochemistry

For immunohistochemistry, all sections were, fixed in acetone and methanol (1:1) or 4% paraformaldehyde (for MyoD), and subsequently blocked in buffer (3% fetal calf serum in PBS) prior to staining. Primary antibodies were diluted 1:100. To assess the number of myofibres presently undergoing regeneration, sections were stained with neonatal myosin heavy chain (nMHC) (Vector Laboratories, Burlingame, CA, USA). Muscle fibers showing a faint nMHC reaction (intermediate stain) and nuclear clumps (with positive nMHC staining) were excluded from the calculation, in order to consider only active regenerating fibers. To confirm that fibers expressing nMHC were actually regenerating the adjacent section were stained with the intermediate filament vimentin (clone V9, Novocastra, UK). Both nMHC and vimentin are usually expressed 1–3 weeks after initiation of regeneration (Figure 1).^[20,22] Co-expression of nMHC and vimentin during regeneration has previously been described in patients with muscular dystrophy.^[35] Satellite cells were visualized with antibodies against myogenic transcription factors MyoD (Vector Laboratories) targeting activated cells and differentiating cells with myogenin (clone F5D, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). These myogenic markers are expressed within 24 hours and 1–7 days respectively of satellite cell activation (Figure 1).^[18,21] Positive nuclei were confirmed by DAPI nuclear stain (Invitrogen, Carlsbad, CA, USA) and to be in a satellite cell position under the basal lamina by using an antibody against laminin (L9393; Sigma, St Louis, Missouri, USA). Alexa 488 and 594 (Invitrogen, Carlsbad, CA) secondary anti- mouse and anti-goat antibodies were used at a 1:500 dilution in PBS buffer. For detection of apoptosis in sub-sarcolemmal myonuclei, sections were TUNEL-stained using the manufacturer's protocol (Roche Diagnostics, Hvidovre, Denmark), and subsequently stained with cleaved PARP fragment antibody (Biosource, Camarillo, CA, USA), Laminin alpha-2 (clone 22B2, Novocastra, UK) and DAPI nuclear stain. Fibers that were nuclei-positive for all three stains were considered apoptotic.^[36–38] The sections were observed under a Nikon 80i microscope with epi-fluorescence.

Western Blot and Densitometric Analysis

Calpain 3 was analyzed by western blot (Figure 2) using mouse anti-human calpain 3, clone 12A2 (Novocastra) recognizing the full size and 60 kDa bands and clone 2C4 recognizing full size and 30 kDa band as previously described.^[2] Lanes of healthy control muscles were loaded on each gel. Dried gels and blots were scanned at 600 dpi on an Epson GT8000 flatbed scanner using white light for gels and blue for blots. Each image was stored as a 16-bit TIFF grayscale and ImageJ v1.41 software was used for the densitometric analysis. The presence of calpain 3 (94 + 60 bands) of each sample was normalized to the amount of skeletal myosin bands in the post-transfer Coomassie blue-stained gels and quantity of calpain 3 is expressed as percentage of control rounded off to nearest 5 percent.

Genetic Analyses

Genetic analysis was performed as described elsewhere.^[24,25] Primers and conditions for testing mutations in FKRP were performed as described before.^[39]

Statistics

Statistical significance among groups was determined using Student's unpaired T-test. A significance level of p < 0.05 was considered significant. All numbers provided are mean ± SD. Logarithmic and linear regression analysis was used, and p-value was determined from a Pearson correlation coefficient table of critical values. For pathological severity comparison a Freeman-Halton extension of the Fisher exact probability test for a two-row by three-column contingency table was used.

BMC Musculoskelet Disord. 2012;13(43) © 2012  BioMed Central, Ltd.

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