This study suggests that functional calpain 3 is a prerequisite for adequate muscle regeneration, and indicate that part of the pathogenesis in LGMD2A may relate to a deficiency in regenerating damaged muscle fibers. This is based on: Severely affected patients with CAPN3 null-alleles, i.e. no functional calpain 3, had almost no recent regeneration and since the clinical and pathological severity of LGMD2A, LGMD2I and BMD is comparable, we can assume that it is the lack of calpain 3 in patients with LGMD2A, and not the severity of the disease, that affects the regenerative process. Our findings are consistent with studies of calpain 3 knockout mice which have decreased levels of embryonic myosin heavy chain and abnormal sarcomere organization .
We found that patients with LGMD2A whom did not harbor null-alleles, while having no detectable level of calpain 3, were still able to regenerate their muscles. It is possible that minute quantities of calpain 3, not detectable by Western blotting, still have remaining function shown for a LGMD2A patient homozygous for p.R748Q . This could be sufficient to maintain sarcomere remodeling at a very basic level of muscle regeneration. In addition, function of calpain 3 may not always correspond to the level of calpain 3 protein detected by Western blot, so the mutant protein may be inactive even if it is present . Studies of inactive calpain 3 knock-in mice suggests that calpain 3 may have other functions than acting as a protease and it is possible that calpain 3-dependent muscle regeneration, in particular sarcomere remodeling and incorporation of developmental myosins is based on non-proteolytic functions of calpain 3 rather than the obvious protease . Consistent with this, we have found that severely affected patients with LGMD2I having a secondary loss of calpain 3 protein, while having a high level of nMHC/vimentin positive fibers. While the loss of calpain 3 can be considerable, the residual albeit normal calpain 3 in LGMD2I patients, is apparently sufficient to maintain the regenerative process.
The mechanism by which calpain 3 acts in the regeneration process is unknown but it has been suggested that calpain 3 aids the shift of non-muscle myosin heavy chain in pre-myofibrils to muscle myosin heavy chain during myofibrillogenesis . This implies that calpain 3 deficiency would lead to a prominent decrease in post-fusion incorporation of nMHC, hence the regeneration of the myofiber after damage is attenuated. This is also supported by a study of patients with LGMD2A showing that patients with near normal levels of calpain 3 had slow disease progression, whereas patients with absent or very low quantities of calpain 3 had rapid progression and early onset of weakness . This is consistent with the findings for the CAPN3 null-alleles patients, where the absence of calpain 3 as seen in patients with two CAPN3 null-alleles has a profound negative impact on the regenerative response, so severely affected patients with LGMD2A with the highest need for regeneration have the most impeded response.
We noted that regeneration among the patients with LGMD2A whom did not harbor null-alleles, had a tendency of age-dependency, consistent with other findings of age negatively affecting muscle regeneration [42, 43]. In fact, one common feature among the four patients with the highest level of recent regeneration is that they were among the youngest of all included LGMD2A patients when they were examined and the biopsy was taken. In other words, it appears that in this study the ability to maintain a higher level of recent regeneration is better at a younger age. However, on a grander scale, with young patients having other calpain 3 mutations than what we include in this study, this may change. The three severely affected patients with CAPN3 null-alleles in the present study were older than the severely affected patients with LGMD2I and BMD, so it is possible that the regenerative capacity was reduced even further by the increased age.
A key aspect of this study is the fact that muscle regeneration varies tremendously among the patients. One may ask why some mildly affected patients display as much recent regeneration as severely affected patients or why regeneration in terms of INF of mildly affected patients spans from 2.5% to 23.2%. There is no simple answer to these questions, however, we propose that ultimately muscle regeneration is a matter of degeneration and capability. Since the effect of the various mutations on the function of calpain 3 are not well understood, two different patients can experience the same clinical course, even though the mutations may affect different functions of calpain 3. The level and frequency of muscle degeneration may vary to an extent, if a mutation e.g. affects the processing of AHNAK, thus the dysferlin-mediated membrane repair, or a different mutation impedes the sarcomere remodeling. For this reason we have focused on the difference between complete lack of calpain 3 in the calpain 3 null patients and patients having some calpain 3 left affected by various mutations.
The patients with LGMD2A with two CAPN3 null-alleles have a high number of INF. Thus, the initial steps of regeneration such as fusion of myoblasts to the damaged myofiber and migration of the nuclei to the center of the fiber, is not affected by loss of functional calpain 3, unlike the subsequent part of myofibrillogenesis . Calpain 3 has been reported to modulate the myogenic factor MyoD in fully differentiated C2C12, thus promoting the formation of reserve cells in these myotubes, which would correspond to satellite cells in vivo [5, 44]. In calpain 3 null patients, the repeated regeneration/regeneration cycle could ultimately cause exhaustion of the satellite cell pool. However, we do not have any evidence this is taking place as it would require an in-depth analysis of satellite cell activation in a larger cohort of calpain 3 null patients, due to the fact that activated satellite cells in vivo are only detectable within a few days.
Calpain 3 null patients had a very clear tendency of whorled fibers (p < 0.06), compared to the patients with LGMD2A having missense mutations. Whorled fibers are an obvious indication of sarcomeres in disarray, as part of the fiber contains sarcomeres perpendicular to the fiber direction. While whorled fibers have been described earlier as a feature of LGMD , we are not aware of any previous study, which demonstrates this difference in LGMD2A. As whorled fibers are seen in other LGMD and BMD, predominantly severely affected patients, it is clear that whorled fibers is not caused by calpain 3 deficiency, but some yet unknown mechanism likely related to massive degeneration. A study of rabbit skeletal muscle immobilized in the most stretched position found that the whorled fibers represent an irregular regeneration, where the sarcomere alignment in the outer layers is perpendicular to the core .
Necrotic fibers, a sign of degeneration, are dependent on the level of calpain 3 in the patients with LGMD2A in our study. While it is difficult to point to one specific reason calpain 3 deficiency causes necrotic fibers, it is possible that in some myofibers the combination of a severed link between calpain 3 and dysferlin-mediated membrane repair and dysfunctional sarcomere remodeling ultimately leads to the demise of the myofiber.
We did not find a significant number of apoptotic myonuclei in patients with LGMD2A, which suggests that myonuclear apoptosis is not a primary feature of calpain 3 deficiency in the patients included in this study. This seems consistent with observations in calpain 3 knockout mouse muscle . However, it has previously been reported that lack of calpain 3 leads to apoptosis . This discrepancy can be attributed to either choice of apoptotic markers or difference in genotype of the patients.
More information from in vitro studies of enzyme activity of mutated calpain 3 is necessary before any correlation between function and phenotype can be made. It is often assumed that all of these mutations reduce/abolish the function of calpain 3, but this is not always the case [6, 46]. The null alleles clearly abolish protein function, but missense mutations in CAPN3 have unpredictable consequences at protein level [46, 47]. This may result in rapid autolysis, thus reducing the function of calpain 3 dramatically.