Vínculos Interorganizacionales

From the above described models and experiments reported in the thesis, multiple experimental research lines can be suggested.

Study of specific pathogenic mutant protein species

Disease causing mutations that support a link between different LGMD genes provide interesting study material. As an example a recent LGMD case was reported with a clear Miyoshi Myopathy phenotype and diagnosis [225]. Surprisingly, no mutation could be detected in the DYSF gene. On the contrary, the disease was linked to a non-synonymous SNP in the CAPN3 gene [225]. This suggests that CAPN3 protein is important for correct Dysferlin function. It will be interesting to see how this protein variant behaves especially in respect to Dysferlin protein complex. Will it bind to Dysferlin and AHNAK with equal affinity, and retain its cleavage potential towards AHNAK?

Satellite cell activation and myogenenesis

A common hypothesis for the cause of LGMD lies in a gradual loss in the ability to activate satellite cells to participate in muscle regeneration. As muscle ages or sustains increasing levels of damage, the regenerative potential of the tissue diminishes, which ultimately results in fibrosis and inflammation. It is commonly assumed that enhanced regeneration, caused by impaired muscle maintenance causes LGMD. This regeneration might be caused by factors intrinsic to muscle, but also to systemic factors [58]. However, a direct role for LGMD protein in myogenesis has not been considered, given the adult onset of the disease. There is little information in literature on satellite cell count in LGMD tissues, or expression

of LGMD proteins in satellite cells. In fact only Dysferlin expression has been reported in the muscle stem cell population [67].

With our CAPN3 cleavage motif we surprisingly identified many putative CAPN3 substrates that link to mitosis and transcriptional regulation. During mitosis the cytoskeleton is effectively remodelled, which could potentially benefit from a protease that enables focal cytoskeleton remodeling. In addition we observed that CAPN3 regulates protein sumoylation through proteolysis of PIAS E3 SUMO ligases. SUMO (Small Ubiquitin Like Modifier) is an 8 kDa protein that is reversibly conjugated to lysine residues to influence protein function and localization [88]. Described targets include cytoskeletal proteins [116], which is in agreement with the idea that CAPN3 regulates the cytoskeleton. In addition, SUMO has been well characterized in cell cycle progression and mitosis [97], in agreement with a potential role for CAPN3 in myogenic reserve cells. However, little is known about SUMO in muscle.

CAPN3 was long considered to be expressed only upon myoblast fusion, when the storage protein Titin is produced in excess. Although CAPN3 deficient primary myoblasts show no apparent defect in cell cycle withdrawal [156], the recent observation of CAPN3 expression in a population of non-differentiating myogenic reserve cells in cultured C2C12 cells [238] opens interesting new avenues towards a role for CAPN3 in cell proliferation. In these cells CAPN3 regulates MyoD protein levels [238]. Based on these data it is interesting to speculate that also in skeletal muscle tissue CAPN3 is involved in myogenic differentiation.

Indeed, CAPN3 deficient myoblasts have enhanced fusion capacity and mature myofibers contain an increased number of myonuclei compared to controls [152,156]. This is in agreement with a defect in reserve cell maintenance. Additionally, in the absence of CAPN3 proteins Integrins are deregulated and the levels of β-Catenin at the cortical cytoskeleton are strongly reduced [156]. Finally, we observed an increase in the protein levels of Brother of CDO precursor (BOC) in LGMD2A skeletal muscle protein homogenates. BOC is important for myoblast fusion.

To substantiate the evidence for a role of CAPN3 at the pre-myofiber stage in muscle, it is paramount to show its expression in muscle satellite cells. To this end wild-type mouse myofibers can be isolated, which harbour satellite cells in the surface. These cells can be differentiated in vitro, allowing for the staining of CAPN3 protein in subsequent stages of myogenesis. Costaining for different cell markers will allow for the definition of a distinct CAPN3 expression myogenic cell pool. It would be interesting to define SUMO expression in a similar series of experiments to further define the cross-talk between CAPN3 and SUMO. Subsequently, similar studies should be performed in CAPN3 deficient myofibers, to uncover whether

CAPN3 plays an active role in myogenesis.

Dysferlin, CAPN3 and AHNAK at focal adhesions

The observation that Dysferlin copurifies with Focal Complex proteins suggests it is in close vicinity to these adhesion sites. It will be interesting to see whether Dysferlin can be observed at focal adhesions in living cells. Focal adhesion proteins are difficult to stain due to the high turnover, and the density of the complex. A potential solution to this would be to construct GFP-Dysferlin expressing cells to visualize its trafficking during cell movement and myoblast fusion. This experiment can also be performed in THP1 cells, which acquire adherence potential as a result of differentiation. Also in these cells, we obtained biochemical support for the physical interaction between Dysferlin and Focal Complexes.

AHNAK stabilizes CAPN3 in inactive form

CAPN3 likely requires a docking protein to maintain it in its inactive form. At the sarcomere Titin acts as a CAPN3 stabilizer and storage facility. At the cell periphery, AHNAK could serve a similar role. Both proteins can directly interact and AHNAK contains at least one CAPN3 binding site [118]. Moreover, CAPN3 can proteolyse AHNAK [118], extending the analogy with Titin [107]. In the absence of Dysferlin both AHNAK and CAPN3 are often seen as secondarily reduced [7,119]. It would therefore be interesting to investigate whether AHNAK can truly stabilize CAPN3. To test for such a role, cells expressing either complete recombinant miniAHNAK, containing the CAPN3 binding site could be used to measure CAPN3 RNA and protein levels. At increasing expression of AHNAK, storage potential for CAPN3 would similarly increase, possibly driving increased protein expression. In addition, homology or motif searches in Titin and AHNAK, might give clues for a potential interaction motif.

Does CAPN3 regulate the interaction between Dysferlin and Focal Complex proteins?

We could show that CAPN3 proteolytically regulates the interaction between AHNAK and Dysferlin at the cell periphery ([118], Chapter 3). Dysferlin interacts with Focal Complex proteins, such as PARVB and Vinculin (Chapters 2 and 7). Moreover, Dysferlin is required to anchor PARVB to the sarcolemma [174], similar to AHNAK [118]. CAPN3 can directly interact with PARVB [248] and was shown to proteolyse Talin and Vinculin [242]. This suggests parallels in Dysferlin’s interactions with structural proteins. It would therefore be interesting to test whether the interaction between Vinculin or PARVB and Dysferlin has a similar dependence on CAPN3 proteolysis.

Mitochondria and LGMD

The importance of Mitochondria in muscle physiology is imminent. Moreover, mitochondrial deficits have been observed in many, if not all, myopathies, but it is often unclear whether these are primary or secondary to the disease. CAPN3 was additionally shown to exist at the triads [155]. Triads are distinct structures in the skeletal muscle fiber, where Rough Endoplasmic Reticulum membrane is in close contact with the T-tubules [155]. There it regulates RYR1 and Aldolase-A [155]. Moreover, mitochondrial deficits are a feature of CAPN3 deficient mice [154]. These observations indicate a role for CAPN3 in energy metabolism and excitation-contraction coupling. The latter is interesting because of its calcium- sensor, which is sensitive to fluctuations in buffered calcium environment [191].

It was recently shown that removal of mitochondria through regulated autophagy underlies muscle atrophy [216]. Moreover, it was suggested that the fibre type switch from oxidative, mitochondria-rich type II fibers to glycolytic, mitochondria- poor type I fibers is in part achieved through mitochondrial autophagy [216]. In Dysferlinopathy, proteomic analysis revealed an decrease in fast Type II fiber marker proteins and a concommittant increase in slow type I markers, suggesting a different fiber type composition [70]. In this regard it is interesting that we observed that a large number of mitochondrial proteins co-immunoprecipitates with Dysferlin from protein homogenates of myogenic origin (Chapter 2).

The DYSF gene expresses two Dysferlin isoforms with alternative first exons [202]. Interestingly, the low-abundant form contains a putative N-terminal mitochondrial targeting signal. This suggests that Dysferlin might localize to mitochondria. Given that Dysferlin also interacts with components of the autophagy system (Chapter 2), and be degraded through this route [83] it is intriguing to speculate that Dysferlin might be involved in autophagy of mitochondria. To test for a function of Dysferlin in mitochondria it is important to show its physical presence at these organelles. This can be achieved through cell fractionation to obtain relatively pure mitochondria. In addition, microscopy studies will give insight in its localization. Definitive proof can only be obtained through electron microscopy. To gain insight into Dysferlin function at mitochondria it is imperative to compare mitochondria form Dysferlin deficient and wild-type myogenic cells. Connecting the remaining LGMD genes

Considering the high amount of mechanical stress and the enormous differences in force output that the skeletal muscle tissue is subject to, it needs preset molecular systems to sense damage and achieve rapid and efficient repair of damage. We propose that the LGMD genes encode for muscle sensors that allow for effective damage sensing and subsequent repair in light of proper myofiber maintenance. It

will therefore be important to insert the remaining LGMD genes into this schematic of structural and maintenance defects that appear to underlie LGMD. This might yield a truly unifying theory of LGMD pathogenicity, and provide directions for a LGMD therapy.

Myoblast

Myotube

Tissue