Calpain 3 is important for muscle regeneration: evidence from patients with limb girdle muscular dystrophies

Abstract

Background: Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in the CAPN3 gene and complete lack of functional calpain 3 leads to the most severe muscle wasting. Calpain 3 is suggested to be involved in maturation of contractile elements after muscle degeneration. The aim of this study was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration.

Methods: We studied muscle regeneration in 22 patients with LGMD2A with calpain 3 deficiency, in five patients with LGMD2I, with a secondary reduction in calpain 3, and in five patients with Becker muscular dystrophy (BMD) with normal calpain 3 levels. Regeneration was assessed by using the developmental markers neonatal myosin heavy chain (nMHC), vimentin, MyoD and myogenin and counting internally nucleated fibers.

Results: We found that the recent regeneration as determined by the number of nMHC/vimentin-positive fibers was greatly diminished in severely affected LGMD2A patients compared to similarly affected patients with LGMD2I and BMD. Whorled fibers, a sign of aberrant regeneration, was highly elevated in patients with a complete lack of calpain 3 compared to patients with residual calpain 3. Regeneration is not affected by location of the mutation in the CAPN3 gene.

Conclusions: Our findings suggest that calpain 3 is needed for the regenerative process probably during sarcomere remodeling as the complete lack of functional calpain 3 leads to the most severe phenotypes.

Figure 1

Sequential presence of regeneration markers MyoD, myogenin, neonatal myosin heavy chain (nMHC), vimentin and internally nucleated fibers (INF) based on previous reports [18,20-22,32,33].

Figure 2

Western blot used for densitometric analysis of calpain 3 amount using mouse anti-human calpain 3, clone 12A2 (Novocastra) and for patient 9M mouse anti-human calpain 3, clone 2C4 (Novocastra). Homogenate from a healthy control muscle (Con) and from patients were loaded and 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.

Figure 3

Hematoxylin & eosin and neonatal myosin heavy chain stains are shown for patients with Limb-Girdle muscular dystrophy type 2A, patients with two CAPN3 null alleles (2NA), patients with Becker muscular dystrophy and patients with LGMD2I (A), (B). Asterisks denote whorled fibers. Bar is 100 μm. Comparison of internally nucleated fibers (INF) in patients with LGMD2A, those with two CAPN3 null alleles (2NA), BMD, and LGMD2I shows no difference among groups (C). In the same patient groups, the relationship between both nMHC and vimentin-positive fibers (black bars) and corresponding muscle strength of biopsied muscle, expressed as percentage of normal, (grey bars) is shown (D).

Figure 4

Distribution of patients with LGMD2A ordered by mutation site and grouped into domains I-IV of CAPN3. Level of internally nucleated fibers and fibers expressing both neonatal myosin heavy chain and vimentin are displayed with Gardner-Medwin-Walton scale and pathological severity.

Figure 5

Regeneration markers, internally nucleated fibers (INF) (A) and fibers expressing the regeneration markers neonatal myosin heavy chain and vimentin (B) showing no correlation with level of calpain 3 in patients with (• LGMD2A) or (Δ LGMD2I). Each symbol represents a patient. Correlation between the fibers expressing the regeneration markers neonatal myosin heavy chain and vimentin and age at biopsy (C).

Figure 6

Whorled fibers in patients with LGMD2A with missense mutations (MM) and those with two null-alleles (2NA). Findings are shown as the mean and range (p < 0.06) (A). Necrotic fibers and level of calpain 3 in patients with LGMD2A (p < 0.05) (B).