dystrophy
Morrée, A. de
Citation
Morrée, A. de. (2011, January 12). Functional protein networks unifying limb girdle muscular dystrophy. Retrieved from https://hdl.handle.net/1887/16329
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/16329
Note: To cite this publication please use the final published version (if
applicable).
Maintenance and remodeling are central to skeletal muscle physiology and disturbed
in LGMD
Introduction
Limb Girdle Muscular Dystrophy (LGMD) is a heterogeneous group of muscle disorders characterized by a late onset and progressive muscle weakness and wasting [30]. Mutations in at least 21 different genes result in a similar clinical presentation, but it is largely unclear whether or not the causative gene products are mechanistically related [160]. Given the adult onset it has been suggested that LGMD is the result of impaired muscle regeneration due to exhaustion of the regenerative potential. A high level of myofiber damage would result in exhaustion of the pool of satellite cells and therefore eventually impair replacement of myofibers. Here we discuss the recent advances in our understanding of the pathophysiology of LGMD including those described in this thesis and the possibility that impaired membrane maintenance and disturbed regeneration are a common denominator of LGMD.
The most common form of LGMD in many populations is LGMD2A, and is caused by mutations in the gene encoding Calpain 3 (CAPN3) [211]. CAPN3 is a calcium-sensitive cysteine protease, and a member of the non-lysosomal non- denaturing Calpain proteases [17]. CAPN3 is often secondarily reduced in LGMD2B [7], which is caused by mutations in the gene encoding Dysferlin (DYSF) [15].
Dysferlin is a calcium-sensitive C2 domain containing single-pass transmembrane domain protein involved in membrane repair [12]. It was shown that CAPN3 can be a component of the Dysferlin protein complex [120] and both proteins were proposed to be important for repair of skeletal muscle damage [13,17]. During muscle contractions the myofibers sustain continuous damage to the internal sarcomere, the anchoring complexes that link the sarcomere to the extracellular matrix and the sarcolemma. Such damage requires rapid and efficient repair to prevent myofiber degeneration, and defects to such repair systems have been suggested as a pathogenic mechanism common to these forms of LGMD (Chapter 1). In the following sections we will discuss the myofiber’s response to damage at the level of the sarcomere, the sarcolemma and the cytoskeleton anchors, with focus on the proteins CAPN3 and Dysferlin. Finally, the remaining LGMD proteins are shortly discussed in light of the findings on CAPN3 and Dysferlin.
Sarcomere damage
During contraction the contractile apparatus sustains mechanical damage that requires efficient and rapid repair [17]. The sarcomere consists of a number of giant proteins that interact to form a dense structure that can generate mechanical force (Chapter 1, Figure 1). Apart from contraction, there is little space for movement of proteins, and the diffusion rate is estimated to be low [17]. However, when damage to the sarcomere occurs, potentially toxic protein fragments need
to be efficiently removed. Moreover, the damaged proteins must be replaced.
Therefore, the muscle requires a preset system to remove and replace proteins from the dense sarcomere in response to damage. CAPN3 is one such potential mechanical sensor and actor [17].
CAPN3 is a muscle specific protease that is a member of the Calpain cysteine protease family [234]. It is highly similar to the well-studied ubiquitously expressed Calpains 1 and 2, sharing ~50% amino acid identity with these proteins [138,235]. In addition it contains three insertion sequences (IS) without any homology to other Calpains, which are used to regulate its enzymatic activity [236]. Full-length CAPN3 is proteolytically inactive. The IS1 sequence blocks the proteolytic cavity ([72], Chapter 4). Upon activation it proteolytically removes the IS1 sequence by autolysis (Figure 1). Hereupon the protease continues as a proteolytically active intramolecular heterodimer, and gains access to its potential full substrate spectrum. At the same time, CAPN3 will intermolecularly cleave itself at the remaining two insertion sequences. This feature enables the rapid deactivation of CAPN3. In vitro studies have indicated that CAPN3 is activated and deactivated within a time-span of 10 minutes [138].
In intact skeletal muscle fibers CAPN3 appears to be much more stable compared to in vitro [191]. CAPN3 contains a functional calcium sensor, consisting of five consecutive EF hands in its C-terminus, similar to Calpains 1 and 2 [138,139]. Its calcium sensitivity however, lies in the nanomolar range, thereby greatly exceeding that of Calpain 1 (micromolar) and Calpain 2 (millimolar) [191]. Moreover, in skeletal muscle most of the CAPN3 protein localizes to the sarcomere [191]. There it can directly interact with Titin [232], and it has been suggested that this interaction stabilizes CAPN3 in its inactive form [233], proving a possible explanation for the apparent discrepancy between the in vivo and in vitro measurements.
Figure 1: Schematic overview of the model for CAPN3 autolytic activation and deactivation. The IS1 sequence is highlighted in red.
Inactive Activated Proteolytic Deactivated
Inactive Activated Proteolytic Deactivated
A recent model for CAPN3 function suggests that upon sarcomere damage CAPN3 is released by Titin, whereupon it would rapidly activate, and deactivate itself [17]. Due to this short half-life, CAPN3 activity would be spatially restricted [17]. Thereby proteolytic processing of the local sarcomere unit is achieved, thus allowing for the relaxation of the dense sarcomere structure and subsequent replacement of damaged proteins, while at the same time safeguarding a temporally restricted enzymatic activity. CAPN3 would therefore be involved in controlled degeneration of the myofiber, and indeed it was shown to function upstream of the ubiquitin-proteasome pathway [153]. This model suggests that the sarcomere has a build-in highly reactive protease to control sarcomere remodeling.
In support of this model it was shown that CAPN3 deficient mice cannot undergo extensive sarcomere remodeling in response to muscle atrophy and degeneration in hind-limb suspension experiments [153]. Transgenic mice that overexpress CAPN3 show no phenotype, but the myofibers appear immature [237]. In fact western blot analysis showed that most of the CAPN3 protein is still present in its inactive form, suggesting that the muscle contains excess docking sites to store the excess CAPN3 protein [237]. As most of the CAPN3 molecules are stored at the sarcomere the best candidate docking protein is Titin. Indeed it has been estimated that the number of CAPN3 binding sites on available Titin molecules greatly exceeds the number of expressed CAPN3 molecules [17].
Interestingly, miss-sense mutations in the CAPN3-binding site in Titin also cause LGMD [104]. Elegant proof for the stabilizing role of Titin comes from experiments with transgenic mice. Mice that carry these same Titin mutations in the CAPN3- binding site show a mild progressive myopathy reminiscent of LGMD in humans.
When the a-phenotypic CAPN3 overexpressing mice were crossed onto the Titin transgenic mice, this severely aggravated that muscle phenotype, suggesting that the muscle had lost its buffering capacity for the excess CAPN3 [122]. Crossing CAPN3 null mice onto this Titin mutant background had no aggravating effect.
It is hypothesized that upon sarcomere damage, the locally stored CAPN3 is released by Titin [17]. Dependent on the free calcium concentration it rapidly activates itself through autoproteolysis [17,191]. The dense structure of the sarcomere restricts its activity to its direct vicinity [17,191]. It will rapidly break down all giant proteins in its direct neighbourhood, and through continuing autoproteolysis, quickly deactivate itself, before its activity can spread beyond where it is needed [17]. The question that remains is therefore: what are the in vivo substrates of CAPN3?
Due to its instability only few in vivo substrates of CAPN3 are known. Using a combination of bioinformatics, biochemistry and cell biology we could show that these few substrates share a common sequence motif in the vicinity of the
CAPN3 cleavage site (Chapter 4). We could subsequently show that this motif can transform non-substrates into substrates, and that this motif is shared by
>300 other proteins. Based on bioinformatics analyses, the majority of the CAPN3 target proteins are involved in cytoskeleton organization. This confirms that indeed CAPN3 will target local structural proteins upon activation.
In conclusion, CAPN3 is stored locally in inactive form to enable rapid remodeling of local cytoskeleton architecture when needed. It is therefore a highly plausible candidate for a muscle repair agent.
Dysferlin and membrane maintenance
Dysferlin is critical for calcium-dependent sarcolemmal repair [13]. Dysferlin localizes to the sarcolemma and to intracellular vesicles [13]. Upon membrane damage and calcium entry it rapidly accumulates at the site of the lesion, and is thought to deliver excess membrane for patch-fusion repair of the sarcolemma [12,13]. Indeed Dysferlin deficient myofibers cannot efficiently repair laser- inflicted membrane wounds, and show sub-sarcolemmal vesicle accumulation [13]. Moreover, this repair function appears to be conserved in other Dysferlin expressing cell types, such as macrophage-like THP1 cells (Chapter 7). Dysferlin contains seven C2 domains, which are believed to function in calcium-sensitive interactions with proteins and phospholipids [244]. Such interactions would aid Dysferlin in performing its maintenance function.
Among the identified interaction partners are the membrane fusogens Annexin A1 and Annexin A2, of which the latter binds in a calcium-dependent manner [162]. These proteins are involved in membrane repair in non-muscle cells [179]
and are predicted to aid in Dysferlin vesicle docking to the cell membrane. Trim72 (MG53) is a redox sensor that functions upstream of Dysferlin and is important for vesicle nucleation [34–36]. Lastly, Caveolin 3, an essential component of caveolae and mutated in LGMD1C, interacts with Dysferlin [172], and is involved in Dysferlin trafficking [110,111].
By immunoprecipitation followed by mass spectrometry analysis we have shown that Dysferlin forms a complex with many other proteins, in a context-dependent manner during myogenic differentiation (Chapter 2). Among its complex partners are vesicle-related proteins including those that are involved in endocytosis (Chapter 2). This is consistent with the observation that in adult muscle most of Dysferlin staining is found at the sarcolemma. Moreover, experiments in caveolin negative cells strongly suggested that Dysferlin needs to be anchored at the sarcolemma to prevent its rapid endocytosis [111]. Dysferlin is often secondarily reduced in LGMD1C, or caveolin 3 deficiency [172]. This suggests that Dysferlin also has a function while being localized to the sarcolemma.
Cortical cytoskeleton restructuring
For membrane repair to occur two separate processes require tight coordination [180]. First the local cortical cytoskeleton needs to be reshaped to allow for subsequent restructuring of the damaged membrane. In a second phase excess membrane from intracellular stores is recruited to enable patch fusion repair.
In the absence of Dysferlin this second phase is blocked as evidenced by the accumulation of subsarcolemmal vesicles [13]. A possible role for Dysferlin in the first phase has not previously been considered.
We showed that Dysferlin forms a protein complex with focal adhesion components such as Vinculin, Talin and α-Actinin, both in cells of myogenic origin (myoblasts, myotubes, mature muscle tissue) and in differentiated macrophages (Chapters 2 and 7). Focal Complexes are based on the transmembrane Integrin receptors, which can interact with extracellular matrix molecules such as fibronectin and collagen. Upon direct interaction between Integrin heterodimers (α- and β-subunit) and the extracellular matrix the cell responds by utilizing this attachment site as a transient scaffold for the intracellular filamentous Actin network. Proteins such as Vinculin, Talin, paxilin and β-Parvin (PARVB) regulate this scaffolding function [40,41].
A possible explanation for this observation is that Dysferlin links the two steps of repair. Recent elegant biochemical experiments showed that at the site of membrane damage, focal adhesion proteins such as Talin, and Vimentin, are proteolytically modified by Calpain proteases [183]. This is rapidly followed by the arrival of new vesicles, which are enriched for Actin and Integrin β1 (ITGB1) [183]. Presumably, these proteins will allow for restoration of the resting situation. We propose a model (Figure 2) in which Dysferlin can target vesicular membrane stores, and through its interaction with focal adhesion components it can coordinate cytoskeletal remodeling. Previous work showed that Dysferlin
Figure 2: A schematic model for Dysferlin function in membrane maintenance. A) In resting conditions Dysferlin is at the cell membrane, in a macromolecular complex with AHNAK, PARVB and Vinculin, and thereby attached to focal adhesions. This occurs nearby caveolea, which prevent Dysferlin endocytosis. CAPN3 is docked to AHNAK. B) Upon membrane wounding and Calcium entry, the Dysferlin/AHNAK interaction undergoes a conformational change and CAPN3 is released. S-AHNAK is released from L-AHNAK. C) CAPN3 autolytically activates and cleaves all structural proteins in its close vicinity, including AHNAK, Vinculin and Talin, to ensure focal cytoskeleton remodeling. Dysferlin is endocytosed and S-AHNAK shifts to the nucleus.
D) Dysferlin containing repair vesicles nucleated by Trim72 allow for patch-fusion repair and ATP release, and restoration of the resting situation. The membrane source of these vesicles is unclear.
recruits PARVB to the sarcolemma [174]. PARVB can directly interact with Integrin linked kinase (ILK), and is important for stabilizing focal adhesions [84,173,261].
In the absence of Dysferlin PARVB does not localize to the sarcolemma [174],
Ca2+
[Ca2+]
Ca2+
[Ca2+]
Endosome Nucleus
Ca2+
Endosome? ER/Golgi? Mitochondrion?
A B
C D
[ATP]
VINC VINC
VINC
TLN
PARVB ILK
ACTN ITGAITGB
L-AHNAK S-AHNAK
CAV3
Cell Membrane Cell Membrane
Cell Membrane Cell Membrane
CAV3
CAV3
TLN
TLN
PARVB
PARVB
ITGAITGB
ITGAITGB
F-ACTIN F-ACTIN
L-AHNAK
S-AHNAK
L-AHNAK S-AHNAK
ILK
ACTN
ACTN Dysferlin
CAPN3
but the functional consequence of this is unknown. Upon membrane damage and calcium entry, Dysferlin might change confirmation, allowing rapid disintegration of the cytoskeleton anchors and subsequent repair processes. It will be interesting to see with which other focal adhesion components Dysferlin directly interacts and if this occurs in a calcium-sensitive manner. In addition, localization of these proteins in Dysferlin deficient muscle might give clear indications for the validity of this model. It is intriguing to speculate that Dysferlin acts as a sensor to coordinate the remodeling of structural proteins in addition to aiding patch-fusion of membranes.
Calpain proteolysis and the cortical cytoskeleton
Calpain proteolysis is important for cytoskeleton remodeling in response to membrane damage. It is therefore interesting that we could identify the skeletal muscle specific Calpain family member CAPN3 as a component of the Dysferlin protein complex. As described above, the majority of CAPN3 protein localizes to the sarcomere, where it most likely functions as a build-in proteolytic switch to enable local sarcomere remodeling. It is less clear what the function of CAPN3 outside the sarcomere is. We hypothesized that CAPN3 is involved in Dysferlin dependent membrane repair, by focal remodeling of the cortical cytoskeleton, to clear the way for repair vesicles.
When ectopically expressed CAPN3 increases the turnover of focal adhesions by proteolytic cleavage of the focal adhesion components Vinculin and Talin [242], two proteins also identified in the Dysferlin protein complex (Chapters 2 and 7).
Subsequently cells become round and lose their normally tight adherence [242].
This is in agreement with the model of CAPN3 proteolysis regulating the cortical Actin cytoskeleton. Moreover, in skeletal muscle cryosections CAPN3 staining was observed at costameres [118] (Chapter 3), which are important adhesion sites of the mature myofibers, and are strongly enriched for Integrin complexes. This suggests that in mature muscle CAPN3 localizes to Integrin-based adherence complexes, possibly, in analogy to sarcomere remodeling, to regulate cytoskeletal remodeling.
Recent experiments with CAPN3 deficient myoblasts showed that membrane repair processes are not severely impaired in the absence of functional CAPN3 protein [182]. Therefore, CAPN3 is dispensable for membrane repair, contrary to Calpain 2, which is essential [182,183]. However, it cannot be excluded that CAPN3 is involved in sarcolemmal repair. Possibly, there is functional redundancy between the Calpains, as their substrate targets largely overlap (including Vinculin, Talin and Vimentin). It would therefore be interesting to develop a chemical or molecular sensor to monitor subcellular CAPN3 activity, with the objective to
acquire insight into where and when this protease is active. We could show that a ten amino acid motif is sufficient for CAPN3 proteolysis, which would theoretically allow for the development of a fluorescent sensor based on this short peptide sequence, fused to a fluorescent group, similar to what has been achieved for Calpain 1 and 2.
Due to the instability of CAPN3 the question remains how the myofiber retains a local inactive pool of CAPN3 molecules at these peripheral sites. A candidate for this function might be the giant protein AHNAK, which has overlapping localization in skeletal muscle and can directly interact with recombinant proteolytically inactive CAPN3 (Chapter 3).
The AHNAK connection
AHNAK is a 700 kDa protein that is strongly expressed in cells with barrier properties and cells with a high susceptibility to mechanical stress [220]. In the cytosol two distinct major AHNAK pools exist, being at the cytoskeleton [19], and on vesicles [53]. It is found on the luminal side of enlargeosome vesicles, and can shift from the cytoplasm to the cell membrane upon calcium entry or cell contact formation [52,53]. We observed that the giant protein AHNAK is directly interacting with Dysferlin C2A through its most C-terminal 500 amino acids [119]. In addition the same AHNAK domain can directly interact with inactive CAPN3 [118].
AHNAK has been hypothesized to function as molecular scaffold to integrate multiple functions [176]. It can interact with filamentous Actin and is important for laminin-based cell-matrix adhesion in myelating Schwann cells [220]. A C-terminal 72 kDa fragment promotes Actin polymerization [101], and loss of peripheral AHNAK destabilizes the cortical Actin cytoskeleton [19,220]. In skeletal muscle cryosections we observed that AHNAK localizes to costameres and the sarcomere [118,119]. Interestingly, we observed that in skeletal muscle myoblasts recombinant miniAHNAK redistributes in response to Integrin inhibition (Chapter 6). This suggests that it is in close vicinity to and possibly part of Focal Complexes.
AHNAK is part of the Dysferlin protein complex [119]. Moreover, AHNAK is secondarily reduced in LGMD2B muscle [119], suggesting that Dysferlin is important for localizing or stabilizing AHNAK to the sarcolemma. The direct interaction between Dysferlin and AHNAK is calcium insensitive [119], but regulated by CAPN3 proteolysis ([118], Chapter 3). CAPN3 can directly interact with AHNAK and cleave at its N- and C-terminus [118]. Cleaved AHNAK fragments have lost their affinity for Dysferlin, indicating that CAPN3 mediated proteolysis regulates the interaction between Dysferlin and AHNAK [118]. This is consistent with our hypothesis that CAPN3 regulates structural components to aid Dysferlin function. From these data we hypothesized that AHNAK is important in connecting the cortical cytoskeleton
with the sarcolemma, and that its interaction with Dysferlin is important for the integration of membrane and cytoskeletal architecture.
We propose that CAPN3 is docked in inactive form on peripheral AHNAK. AHNAK is localized to the sarcolemma through its direct interaction with Dysferlin [119].
In addition it binds the cortical Actin cytoskeleton [19] and stabilizes adhesion sites. Upon damage to the sarcolemma, a conformational change in the Dysferlin- AHNAK complex results in the release and subsequent activation of CAPN3.
CAPN3 subsequently proteolyzes AHNAK and other cytoskeletal components, thereby allowing for Dysferlin trafficking and the turnover of cell membrane and cytoskeletal components.
We observed that the AHNAK gene generates a small and a large protein isoform, which can directly interact (Chapter 5). The large isoform localizes to the peripheral Actin cytoskeleton. The small isoform is also seen in nuclear speckles enriched for spliceosomal proteins, and there it affects mRNA splicing. It would be interesting to speculate that the small isoform functions as a mRNA modifying factor in response to cortical cytoskeleton remodeling.
Commonalities beyond skeletal muscle
During muscle maintenance communication with infiltrating immune cells coordinates tissue repair and regeneration. Upon muscle damage, immune cells are recruited to remove necrotic myofibers through phagocytosis. To arrive at the site of damage the infiltrating immune cells will need to remove the extracellular matrix. This is mediated by pro-inflammatory M1 macrophages. Concurrent cytokine signalling will inhibit myofibers to secrete additional extracellular matrix constituents until the clearance of dead cells is completed. Then the immune response gradually switches to a contra-inflammatory M2 profile, where signalling results in reconstitution of the extracellular matrix. In addition, satellite cells are activated to participate in myofiber regeneration. It was shown that the communication between the muscle and immune system is essential for degeneration-regeneration cycles.
Dysferlin plays an important role in this communication, as it is also expressed by infiltrating monocytes and macrophages [66]. In muscle cells Dysferlin is involved in the secretion of chemokines [68] and cytokines [45] that attract immune cells. In Dysferlin deficient muscle the inflammasome is deregulated and cytokine secretion is modified [210]. As a result, the recruited neutrophils and monocytes arrive later and linger longer, thereby prolonging the inflammatory response [45]. It has been suggested that this improper immune response aggravates the muscle pathology.
Dysferlin is also strongly expressed in monocytes and increased upon
differentiation (Chapter 7). Phagocytosis assays indicated that Dysferlin deficient LGMD2B monocytes behave more aggressively than matched control cells and phagocytose more particles [193], suggesting that not only immune cell recruitment, but also immune cell function is impaired in Dysferlinopathy. Recent experiments showed that muscle specific restoration of Dysferlin expression in Dysferlin deficient mice recued contraction-induced skeletal muscle pathology [168,185], suggesting that the misbalanced immune response is secondary to the muscle dysfunction. However, at later ages, these mice might still develop a mild dystrophic pathology [168], indicating that the modified immune response might be pathologically relevant during disease progression.
We observed that Dysferlin-depleted THP1 cells show a differentiation defect (Chapter 7). They adhere less efficiently than cells treated with a non-target shRNA, which provides an alternative explanation for the decreased recruitment efficiency of Dysferlin deficient monocytes in damaged mouse skeletal muscle tissue. Like in myogenic cells, immunoprecipitation of Dysferlin resulted in co-purification of Focal Complex components, such as Vinculin. Interestingly, in the absence of Dysferlin, Integrin β3 (ITGB3) RNA levels were strongly and specifically deregulated, as was its intracellular trafficking in response to an Integrin inhibitory peptide or ITGB3 specific antibody. This strongly suggests that deregulation of Integrin expression, specifically ITGB3, is a feature of Dysferlin deficient monocytes and macrophages.
We observed that in differentiated THP1 cells, Dysferlin is rapidly endocytosed upon Integrin inhibition, together with ITGB3. This suggests that its trafficking is linked to Integrin-based cell adhesion. In support of this we observed that ITGB3 trafficking is disturbed in the absence of Dysferlin. ITGB3 is involved in macrophage phagocytosis of extracellular matrix and apoptotic cells, by forming cell contacts.
The process of phagocytosis is analogous to that of endocytosis, and also requires coordinated restructuring of cell membrane and cytoskeleton. Therefore, the reported increase of phagocytotic capacity in Dysferlin deficient monocytes might be explained by our findings that ITGB3 trafficking is deregulated in the absence of Dysferlin protein. This opens the possibility of a potential immunomodulatory treatment of Dysferlinopathies.
In myogenic cells, and differentiated THP1 cells, Integrin complexes copurify with Dysferlin, while DGC complex partners do not. This strongly suggests a specific role for Dysferlin at Integrin based cell adhesion. In migrating cells, the Integrin-based Focal Complexes are essential for cellular movement [40]. The Focal Complexes form tight anchoring structures, but show a high turnover [40,41]. Therefore, they allow for simultaneous tight adherence to the extracellular matrix and rapid cellular migration. Skeletal muscle myofibers are non-migratory. However, during contraction the myofiber strongly changes in
size, while maintaining a tight connection to the extracellular matrix to translate the mechanical force. To prevent excessive membrane damage due to protein anchorage, it might be beneficial for the muscle fiber to maintain a subset of tight anchoring complexes that can be rapidly adapted to accommodate the relative movement of the myofiber membrane to the surrounding extracellular matrix.
In summary
Both muscle and monocyte cells require proper maintenance of cell-cell contacts and timely restructuring of membrane/cytoskeleton architecture. We propose that the triad of Dysferlin, AHNAK and CAPN3 is important for maintenance of Integrin based cell-cell contacts. Dysfunction of any of the three proteins results in impaired maintenance at the level of molecular cell contact points.
Future perspectives in LGMD studies
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.
