On the role of dysferlin in striated muscle: membrane repair, t‐tubules and Ca2+ handling

Dysferlin is a 237 kDa membrane‐associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)‐tubule network and the tight coordination of excitation–contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t‐tubules, and it is suspected that dysferlin regulates t‐tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.


Introduction
The healthy function of skeletal muscle is dependent on invaginations of the sarcolemma known as transverse (t)-tubules, which facilitate excitation-contraction (EC) coupling.During EC coupling, the action potential depolarises the sarcolemma and t-tubule membranes, which causes a conformational change in the associated L-type Ca 2+ channel (LTCC), also known as the dihydropyridine receptor (DHPR).In turn, this initiates the release of Ca 2+ from the sarcoplasmic reticulum (SR) via its Ca 2+ -release channel, the ryanodine receptor type 1 (RYR1), causing a transient increase in intracellular Ca 2+ concentration.These Ca 2+ ions bind to myofilaments causing the cell to contract (for review, see Calderón et al., 2014).
Skeletal muscle damage is associated with changes in t-tubule structure (Takekura et al., 2001), and data from human and animal studies suggest that forceful muscle contraction leads to sarcolemmal damage (Cooper & Head, 2015;Hamer et al., 2002;McNeil & Khakee, 1992;Newham et al., 1986;Roche et al., 2010).Given these considerations, an efficient damage repair mechanism is likely to be important in maintaining muscle health.Experiments using in vitro damage assays suggest that many proteins cooperate to regulate muscle membrane repair in a Ca 2+ -dependent manner.These are known collectively as the membrane repair complex (for review, see Cardenas et al., 2016;Demonbreun et al., 2015;Wallace & McNally, 2009).In the late 1990s, dysferlin mutations were implicated in the development of human muscular dystrophies, known as dysferlinopathies, such as Miyoshi myopathy (MM) and limb-girdle muscular dystrophy (LGMD) R2 dysferlin-related (Bushby et al., 1998;Liu et al., 1998;Straub et al., 2018).Cell death, inflammation and muscle wastage, which are characteristic of dysferlinopathy, have been linked to the failure of muscle membrane repair mechanisms and more recently to the dysregulation of intracellular Ca 2+ handling (Baek et al., 2017;Bansal et al., 2003;Cenacchi et al., 2005;Lukyanenko et al., 2022;Rosales et al., 2010).Bansal et al. (2003) used a laser membrane damage assay to show that dysferlin is a key regulator of the muscle repair mechanism.Data from rodent models indicate that dysferlin regulates the structure of the skeletal muscle t-tubule system (Ampong et al., 2005;Demonbreun et al., 2014;Kerr et al., 2013;Klinge et al., 2007Klinge et al., , 2010;;Roche et al., 2011;Waddell et al., 2011) and further studies indicated dysferlin may regulate Ca 2+ signalling by modulating the activity and expression of key EC coupling proteins such as the LTCC and RYR1 (Kerr et al., 2013;Lloyd et al., 2023;Lukyanenko et al., 2017Lukyanenko et al., , 2022;;Muriel et al., 2022).
Therefore, given that dysferlin has been implicated in multiple mechanisms known to support skeletal muscle physiology, it is clear why dysferlin mutations primarily manifest in skeletal myopathy (Bushby et al., 1998;Liu et al., 1998).However, clinical observations showed that some dysferlinopathy patients presented with a cardiac phenotype (Moore et al., 2022;Nishikawa et al., 2016;Wenzel et al., 2007).Like skeletal muscle, cardiac muscle contains extensive t-tubule networks with associated EC coupling proteins, which facilitate the fast and synchronous rise in intracellular Ca 2+ necessary for a healthy heartbeat (Fig. 1B and C; for reviews, see Bers, 2001;Smith et al., 2018).It is well established that remodelling of the cardiac t-tubule network is associated with cardiac pathologies such as heart failure (for review, see Dibb et al., 2022) and atrial fibrillation (Lenaerts et al., 2009).The mechanisms that drive cardiac t-tubule remodelling are not properly understood.However, recent evidence indicates that dysferlin associates with cardiac t-tubules (Hofhuis et al., 2020), suggesting a similar relationship between dysferlin and the t-tubule network may exist in both striated muscle types.Whilst there is a lack of evidence for cardiac sarcolemmal damage under physiological conditions, damage to the sarcolemma has been observed in specific cardiomyopathies (Clarke et al., 1995;Kostin et al., 2003).Membrane repair is, therefore, likely to be important in the heart but the existence or function of a membrane repair mechanism comparable to that observed in skeletal muscle has not been fully investigated.Han et al. (2007) were the first to show evidence for Ca 2+ -dependent dysferlin-mediated membrane repair in the heart using a laser damage assay.Recent findings show that dysferlin also regulates aspects of cardiac EC coupling (Hofhuis et al., 2020;Wei et al., 2015;Wenzel et al., 2007).
The function of dysferlin is diverse and so it is unsurprising that its role in health and disease remains to be elucidated in full.Here we discuss the current literature on the function of dysferlin in striated muscle.We will examine the mechanics of dysferlin and its proposed function in the membrane repair mechanism, the t-tubule network and intracellular Ca 2+ handling.We will also consider some of these mechanisms in light of dysferlin's relationship with other major membrane-associated proteins such as mitsugumin 53 (MG53; Cai et al., 2009b,c;Lek et al., 2013;Matsuda et al., 2012), caveolin-3 (cav-3;Cai et al., 2009c;Hernández-Deviez et al., 2006, 2008;Matsuda et al., 2001) and the annexin family (Bittel et al., 2020;Croissant et al., 2020;Demonbreun, Allen, et al., 2016;Lennon et al., 2003).Although dysferlin regulates and, in turn, is regulated by a wider range of proteins, they remain outside of the scope of this review.
More information regarding these proteins has been provided elegantly by Cooper and McNeil (2015).

The function of dysferlin in skeletal muscle
Dysferlin is a 237 kDa membrane-associated protein that was named due to its homology with Fer-1, a protein involved in vesicle-membrane fusion in spermatids from Caenorhabditis elegans (Bushby et al., 1998).Whilst dysferlin is present at varying levels in a range of tissue types, it is highly expressed in striated muscle (Redpath et al., 2014;Uhlén et al., 2015).Dysferlin is a member of the ferlin family together with myoferlin and otoferlin.The family is characterised by multiple C2 domains (Fig. 1A; Lek et al., 2012), and many C2 domains have roles in Ca 2+ -dependent membrane binding (for review, see Cho & Stahelin, 2006).While dysferlin's role in membrane repair was suggested over two decades ago (Bansal et al., 2003), it is now recognised as a fundamental player within a wider complex of repair proteins (Cardenas et al., 2016;Demonbreun et al., 2015;Wallace & McNally, 2009).

How does dysferlin regulate skeletal muscle membrane repair?
Dysferlin's role in the regulation of vesicle fusion.In vitro damage assays have yielded substantial information regarding the membrane repair process in skeletal muscle, and dysferlin's proposed role in this process is summarised in Fig. 2. In uninjured skeletal muscle, dysferlin is predominantly localised to the sarcolemma and t-tubule membrane (Ampong et al., 2005;Klinge et al., 2007;McDade et al., 2014;Roche et al., 2011).However, in response to membrane damage in vitro, some dysferlin translocates to the cytoplasm via endocytosis (Mcdade et al., 2021).Dysferlin is thought to facilitate the aggregation of exocytotic repair vesicles into a larger repair structure called the repair patch (Fig. 2B; Davenport et al., 2016;Klinge et al., 2010;Lek et al., 2013;McDade and Michele, 2014).Davenport et al. (2016) clearly visualised this patch-mediated repair process in Xenopus oocytes and showed that dysferlin preferentially localises to the damage site in a similar pattern to that seen in muscle cells (Lek et al., 2013;McDade et al., 2014).This pathway is one of the multiple proposed mechanisms by which cell membranes are thought to be repaired, as summarised by Blazek et al., 2015. Bittel et al. (2020) used total internal reflection fluorescence microscopy (TIRFM) to show dysferlin-containing vesicles fusing with the plasma membrane of skeletal myoblasts in a damage-dependent manner.Notably, vesicles appear to accumulate beneath the sarcolemma in skeletal muscle cells in dysferlin-deficient mouse models, which may indicate vesicles failing to fuse with Figure 1.Dysferlin's structure and its relationship with the striated muscle t-tubule and EC coupling system A, dysferlin contains seven C2 domains with the first, C2A, being located closest to the N-terminus whilst the last, C2G, is located towards the C-terminus.Calpain-cleavable dysferlin isoforms are characterized by the alternatively spliced exon 40a situated between the C2E and C2F domains.Cleavage at the exon 40a site produces a functional C-terminal fragment, mini-dysferlin c 72, which consists of the C2F and C2G domains together with the transmembrane (TM) domain.B, dysferlin is expressed at the t-tubule membrane and the sarcolemma in healthy skeletal myocytes and it is thought to physically interact with the L-type Ca 2+ channel (LTCC) and ryanodine receptor type 1 (RyR1) at the triad, possibly via its C2C and C2D domains.Dysferlin is also thought to buffer Ca 2+ in the triadic cleft via its C2A domain (Lukyanenko et al., 2022).Each skeletal myocyte is innervated via a neuromuscular junction (NMJ), but in the absence of a neuronal signal, Ca 2+ ions are stored in the sarcoplasmic reticulum (SR) and the cell remains in a relaxed state.C, dysferlin is also found at the sarcolemma and t-tubule membrane in cardiac myocytes and at high concentrations at the intercalated disc (ICD).It is unclear whether dysferlin also maintains LTCC/RyR2 proximity in cardiac myocytes.Cardiac myocytes are not individually innervated by neurones but are, instead, arranged in an electrical syncytium via the ICD.The cardiac EC coupling process is summarized as follows.
(1) The arrival of the cardiac action potential leads to the depolarisation of the cardiac myocyte.(2) A small amount of Ca 2+ then moves through the LTCC on the t-tubule membrane and binds to RyR2 on the SR causing them to open.
(3) This leads to a greater efflux of Ca 2+ from the SR known as the systolic Ca 2+ transient (systole).(4) This sharp increase in cytosolic Ca 2+ causes the sarcomeric machinery to contract, thus rapidly ejecting blood from the heart.For the heart to refill, cardiac myocytes must relax (diastole).( 5) This is initiated by the removal of the majority of Ca 2+ from the cytoplasm into the SR via the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) and (6) the remainder is largely removed from the cell via the Na + /Ca 2+ exchanger (NCX).
the membrane (Bansal et al., 2003;Cenacchi et al., 2005;Hornsey et al., 2013).These findings suggest that dysferlin likely facilitates vesicle-vesicle and vesicle-membrane fusion and that these events may contribute significantly to the dysferlin-mediated muscle membrane repair mechanism.
Dysferlin's C2 domains have diverse functions in regulating membrane repair and intracellular Ca 2+ handling.Ca 2+ is thought to be essential for dysferlin-mediated membrane repair following in vitro wounding as described above.Dysferlin's seven C2 domains are thought to have distinct functions during membrane repair, and while all interact with Ca 2+ , the Ca 2+ dependence of repair relies primarily on five of these domains (C2C to C2G; Abdullah et al., 2014;Muriel et al., 2022).The C2A and C2B domains are arguably most important for membrane repair since their deletion negatively impacts in vitro membrane repair efficiency the most; however, the activity of the C2A and C2B domains during repair appears to be less affected by Ca 2+ concentration when compared to dysferlin's other C2 domains (Muriel et al., 2022).This shows that the Ca 2+ binding affinities of dysferlin's C2 domains do not directly correlate with their membrane repair capacity, and therefore further investigation is required to understand how Ca 2+ regulates dysferlin-mediated membrane repair.

Figure 2. Dysferlin and the skeletal muscle membrane repair mechanism
A, tensile forces cause damage to the sarcolemma and t-tubule network, which leads to an influx of Ca 2+ ions and oxidative products from the extracellular space thus initiating the Ca 2+ -dependent membrane repair mechanism.B, the oxidative environment stimulates MG53 oligomerisation, and a local increase in Ca 2+ leads to the cleavage of dysferlin by calpain enzymes to produce mini-dysferlin c 72. Dysferlin is endocytosed and incorporated into repair vesicles.Dysferlin mediates the fusion of repair vesicles with each other to form larger vesicular structures and it is suspected that these repair vesicles may also contribute to t-tubule regeneration.C, the formation of a larger repair patch facilitates the sealing of the membrane wound, and the lateral recruitment of sarcolemma-derived dysferlin is thought to facilitate the integration of repair vesicles and the repair patch to the damage site.D, in the final stages of membrane repair, two distinct repair structures are formed, namely the repair cap and shoulder, each with a specific protein localisation signature.The annexin-rich repair cap is supported by the phosphatidylserine (PS)-rich shoulder, which is characterised by other repair proteins such as dysferlin, MG53, Esp homology domain proteins (EHDs) and BIN-1.Cap, repair cap; S, repair shoulder.
J Physiol 602.9 Abdullah et al. (2014) described that all of dysferlin's C2 domains bind lipids but the lipid binding characteristics of the C2A domain have been studied in most detail.The C2A domain is thought to bind various species of phospholipids, such as phosphatidylserine, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, in a damage-dependent manner (Cai et al., 2009a;Davis et al., 2002;Therrien et al., 2009).A naturally occurring mutation (V76D) within dysferlin's C2A domain impacts its membrane-binding properties in vitro (Hofhuis et al., 2017) and other C2A mutations such as W52R inhibit the binding between dysferlin C2A and MG53, thus disrupting the assembly of the membrane repair complex (Matsuda et al., 2012).This suggests that dysferlin C2A supports membrane repair through both phospholipid binding and protein assembly and goes some way to explaining the mechanisms through which patients carrying C2A mutations may develop dysferlinopathy (Illarioshkin et al., 2000).
Furthermore, each of dysferlin's C2 domains except the C2B domain also regulates specific aspects of intracellular Ca 2+ handling at baseline or in response to stress due to osmotic shock injury in vitro (Muriel et al., 2022).This could be partially attributed to the heterogeneity between the Ca 2+ binding affinities of the C2 domains, but further study is required.Disease-causing mutations have been observed in all of dysferlin's C2 domains, which shows that each C2 domain plays an essential role in supporting in vivo muscle function (Krahn et al., 2009).However, it is clear from the literature that our understanding of the functions of dysferlin's C2 domains is severely lacking.
Dysferlin interacts with MG53 and caveolin-3.Whilst there is strong evidence that dysferlin is a major regulator of skeletal muscle membrane repair, there are other key proteins that function in tandem with dysferlin and form part of the wider membrane repair complex.
MG53, also known as TRIM72, is a muscle-specific vesicle-trafficking protein that facilitates the assembly of the membrane repair complex in skeletal muscle.MG53 is a key early regulator of skeletal muscle membrane repair such that MG53-null mice present with skeletal myopathy (Cai et al., 2009a,c;Weisleder et al., 2009).Membrane damage and an influx of oxidative products are thought to cause MG53 oligomerisation (Cai et al., 2009;Fig. 2B).This is important for both MG53-mediated recruitment of repair vesicles and the binding of MG53 to the damaged membrane via an interaction with phosphatidylserine.Whilst the initial activation of MG53 may occur in a Ca 2+ -independent manner, Ca 2+ is still required for efficient membrane repair (Cai et al., 2009a).In no or low Ca 2+ conditions, dysferlin-positive vesicle pools remain spatially separated from MG53-positive regions of damaged plasma membrane in cultured human myotubes.However, in higher Ca 2+ conditions, MG53 and dysferlin-positive vesicles interdigitate, and the lesion is successfully repaired (Lek et al., 2013).This suggests that MG53 is primarily responsible for the rapid accumulation of repair vesicles and the repair patch to the damage site (Cai et al., 2009a).In contrast, dysferlin may primarily regulate Ca 2+ -dependent vesicle-membrane fusion events during repair, as vesicles aggregate near to, but fail to merge with the damaged sarcolemma in dysferlin-deficient muscle (Bansal et al., 2003;Cenacchi et al., 2005;Hornsey et al., 2013) or in the absence of Ca 2+ (Lek et al., 2013).Therefore, the Ca 2+ -dependent interaction between dysferlin and MG53 appears essential for membrane repair in skeletal muscle cells following experimental damage.Dysferlin and MG53 likely act synergistically to recruit and fuse reparative vesicles to regions of damaged membrane at least in an in vitro setting (Fig. 2B and C).
MG53 and dysferlin are known to form a protein complex with caveolin-3 (cav-3; Cai et al., 2009c;Matsuda et al., 2001), a muscle-specific member of the caveolin family that regulates the formation of caveolae (Lisanti et al., 1994;Tang et al., 1996).Mutations in cav-3 are known to cause LGMD1C (rippling muscle disease) in humans, as well as other major conditions such as distal myopathy and hyperCKaemia (Gazzerro et al., 2010;Minetti et al., 1998;Straub et al., 2018).Cav-3 is a major regulator of dysferlin localisation in skeletal muscle cells as it transports dysferlin to the cell membrane and supports dysferlin's position by inhibiting dysferlin endocytosis (Hernández-Deviez et al., 2006, 2008).This may explain why dysferlin localizes differently in skeletal muscle cells from rippling muscle disease patients that harbour cav-3 mutations (Matsuda et al., 2001).However, whilst both dysferlin and cav-3 interact directly with MG53, they do not interact directly with each other (Flix et al., 2013), suggesting cav-3's influence over dysferlin's localisation is likely mediated via other proteins such as MG53.Similarly, MG53 mislocalisation as a consequence of cav-3 mutations correlates with defective membrane repair in cultured muscle cells (Cai et al., 2009c).It is evident, therefore, that dysferlin, MG53 and cav-3 possess an important interdependent relationship during skeletal muscle membrane repair and tightly regulate each other's localisation.Targeting this molecular complex may be therapeutically useful in specific disease settings.For example, overexpression of MG53 improved skeletal and cardiac muscle function in an animal model of delta-sarcoglycanopathy by upregulating dysferlin expression and improving cav-3-mediated dysferlin trafficking (He et al., 2012).
The membrane repair cap and shoulder form during membrane resealing.In the later stages of the laser-induced membrane repair process, the proteins of the membrane repair complex can be categorised into two subdomains due to their proposed association with distinct structures.These are known as the repair cap and shoulder (Demonbreun, Quattrocelli, et al., 2016).Dysferlin and MG53 were observed to localise to the phosphatidylserine-enriched shoulder region along with other repair proteins such as Esp 15 homology domain protein (EHD) 1 and 2 and Bridging-integrator 1 (BIN-1, also known as Amph-II).The shoulder region is distinct from but supports the annexin-rich repair cap, which is characterised by the expression of multiple annexin proteins (Fig. 2D; Demonbreun, Quattrocelli, et al., 2016).McDade et al. (2014) hypothesised that sarcolemma-derived dysferlin is trafficked to the periphery of the membrane tear to create an 'active zone' for Ca 2+ -dependent dysferlin-mediated vesicle fusion for the effective resealing of the sarcolemma (Fig. 2C).Whilst Demonbreun, Quattrocelli, et al. (2016) also suggest that dysferlin may be laterally recruited to the repair shoulder from the adjacent sarcolemma, they did not observe any evidence of vesicle recruitment to the repair apparatus.This contradicts the current hypothesis that vesicle recruitment is a major contributor to dysferlin-mediated membrane repair and warrants further investigation.
As discussed by Blazek et al. (2015), vesicle-mediated repair is just one of multiple mechanisms that may contribute to membrane repair, and it is possible that different in vitro membrane repair models evoke similar yet distinct repair pathways.These pathways may also differ from those that are activated in vivo.Furthermore, species-specific differences and differences in phenotype or maturity of cells used to model the muscle repair process in vitro may also account for disparate experimental findings.Therefore, the use of more physiological techniques and appropriate species to model the muscle membrane repair pathway will be an important component of future dysferlin research.
Dysferlin and the annexins share a bidirectional relationship during membrane repair.The mechanisms by which dysferlin regulates intracellular membrane dynamics are largely unknown.However, increasing evidence suggests that annexin proteins are essential for regulating dysferlin's intracellular localisation and its activity during membrane repair.The annexin proteins are involved in Ca 2+ -dependent membrane binding and vesicle trafficking (for review, see Gerke et al., 2005) and they have well-established roles in the membrane repair pathway (Koerdt et al., 2019).A very early report showed that dysferlin binds annexin A1 and A2 in skeletal muscle in a Ca 2+ -dependent manner, and annexin A1 and A2 also localised differently in dysferlin-deficient muscle cells with a reduced membrane repair capacity (Lennon et al., 2003).Whilst disruption of annexin A1 has been shown to inhibit membrane repair in some non-cardiac cell lines (McNeil et al., 2006), some data suggest that annexin A1 is not required for skeletal muscle membrane repair but is more important for muscular regeneration by mediating myoblast fusion (Leikina et al., 2015).Despite these findings, a separate study found that annexin A1 does localise to the repair cap during the repair process in muscle cells (Demonbreun, Quattrocelli, et al., 2016).This observation is similar to observations suggesting annexin A1 contributes to vesicle trafficking and repair patch formation in Xenopus oocyte membrane repair (Davenport et al., 2016).
Annexin A2 rapidly localises to the damaged sarcolemma and subsarcolemmal dysferlin-positive vesicles in response to Ca 2+ .Annexin A2 is also thought to regulate dysferlin localisation, which may partially explain why membrane repair efficiency is reduced when annexin A2 is ablated or is prevented from moving to the sarcolemma (Bittel et al., 2020).Other members of the annexin family, like annexins A5 and A6, have also been implicated in muscle membrane repair.Whilst both annexin A5 and A6 appear to localise to the damage site in a dysferlin-independent manner, annexin A6 is thought to recruit dysferlin to the membrane damage site (Carmeille et al., 2016;Croissant et al., 2020;Demonbreun, Allen, et al., 2016, 2019;Swaggart et al., 2014).Annexin A6 is also one of the first proteins to be recruited to the annexin-rich repair cap (Demonbreun, Quattrocelli, et al., 2016), and repair cap formation is thought to be dependent on Ca 2+ , which is consistent with the idea that Ca 2+ binding is an important property of the annexin family (Gerke et al., 2005;Grieve et al., 2012;Hayes et al., 2004).
These data show that dysferlin and various members of the annexin family share a functional co-dependency during muscle membrane repair in vitro.The idea that the annexin-mediated transport of dysferlin to membrane damage sites requires Ca 2+ may further indicate why low Ca 2+ is correlated with a reduced membrane repair efficiency even when dysferlin is expressed (Han et al., 2007;Lek et al., 2013).Furthermore, because the annexins are characterised as vesicle-trafficking proteins, their relationship with dysferlin lends support to the idea that repair vesicles are a major contributor to dysferlin-mediated membrane repair.However, we know very little about how dysferlin and annexins might cooperate during vesicle trafficking and so further investigation is clearly necessary.
What is mini-dysferlin c 72?As is the case with most biological processes, our understanding of the membrane repair process has developed slowly and incrementally.
J Physiol 602.9 However, the discovery of mini-dysferlin c 72 is a significant development in membrane repair and dysferlin research.Native dysferlin can be cleaved by Ca 2+ -dependent proteolytic calpain enzymes in a damage-dependent manner to generate a functional 72 kDa C-terminal protein fragment named mini-dysferlin c 72, which consists of only two C2 domains (C2F and C2G) and the transmembrane domain (Fig. 1A).The mini-dysferlin c 72 fragment has been observed to localise independently at the damage site in in vitro muscle membrane damage assays (Ballouhey et al., 2021;Lek et al., 2013;Redpath et al., 2014).
Mini-dysferlin c 72 regulates membrane repair in vitro.Krahn et al. (2010) described a patient who presented with a less severe case of dysferlinopathy whilst expressing a truncated form of dysferlin, which was coincidentally very similar in structure to mini-dysferlin c 72.The truncated mini-dysferlin was cloned and the exogenous expression of this recombinant mini-dysferlin was able to restore membrane repair capacity in an in vitro laser wounding assay in skeletal muscle cells (Krahn et al., 2010).However, this mini-dysferlin construct was unable to fully ameliorate muscle dysfunction when expressed in dysferlin-deficient mice (Lostal et al., 2012).
Many human organs such as skeletal muscle, brain, liver, pancreas, lungs and heart all express two isoforms of dysferlin.Only one of these contains the exon 40a calpain cleavage site (Redpath et al., 2014).The exogenous expression of a dysferlin isoform containing exon 40a, and thus able to produce mini-dysferlin c 72, was shown to rescue the perturbed membrane repair mechanism in cultured human dysferlin-null myoblasts.However, the expression of an uncleavable dysferlin variant lacking exon 40a was unable to improve repair to the same extent (Ballouhey et al., 2021).This strongly suggests that mini-dysferlin c 72 is the primary regulator of sarcolemmal repair under these conditions, but further investigation is required.
Mini-dysferlin c 72 is produced by calpain cleavage and is similar in structure to vesicle-trafficking synaptotagmin proteins.While dysferlin's calpain cleavage site is known to be encoded by exon 40a (Fig. 1A; Ballouhey et al., 2021;Redpath et al., 2014), there is still debate as to which calpain enzymes cleave dysferlin (Ballouhey et al., 2021;Huang et al., 2008;Mellgren et al., 2009).However, calpain-3 is thought to interact with dysferlin in skeletal muscle (Goll et al., 2003;Huang et al., 2005), and mutations in calpain-3 cause a type of muscular dystrophy known as LGMD R1 calpain-3-related (Richard et al., 1995;Straub et al., 2018).This suggests dysferlin cleavage may be essential for muscle health.Interestingly, both otoferlin and myoferlin are also cleaved by calpains to generate similar 'mini-ferlin' proteins (Piper et al., 2017;Redpath et al., 2014) indicating calpain cleavage may be a common and important driver of ferlin protein function.
Mini-dysferlin c 72 is very similar in structure to the synaptotagmin proteins, which are also characterised by possessing two C2 domains.Notably, synaptotagmins are thought to regulate Ca 2+ -dependent vesicle trafficking (Chapman, 2002), and synaptotagmin VII-deficient mice show signs of skeletal muscle pathology, which may result from compromised membrane integrity (Chakrabarti et al., 2003).This is yet further evidence to support the idea that vesicle-membrane fusion drives dysferlin-mediated membrane repair, and importantly, it may be mini-dysferlin c 72 that is the primary driver of this process as suggested by Lek et al. (2013).Mini-dysferlin c 72 may be of some clinical significance for the treatment of dysferlin-deficiency-related muscle disease.This is because the shorter mini-dysferlin C 72 RNA sequence is less restrictive when considering viral-based therapeutic strategies.For example, mini-dysferlin c 72 can be inserted into an adeno-associated viral (rAAV) vector used for gene transfer therapies much like the 'Nano-dysferlin' construct described in the literature (Krahn et al., 2010;Llanga et al., 2017;Naso et al., 2017).However, mini-dysferlin could not rescue muscle pathology in dysferlin-deficient mice (Lostal et al., 2012) suggesting factors other than membrane repair contribute to dysferlinopathy (Kerr et al., 2013;Lukyanenko et al., 2017Lukyanenko et al., , 2022;;Muriel et al., 2022).Almost all dysferlin research has been conducted in ignorance of the fact that dysferlin can be cleaved by calpain enzymes to produce mini-dysferlin c 72.At present mini-dysferlin research is in its infancy, but the current literature suggests an important role for mini-dysferlin c 72 in membrane repair possibly through repair vesicle dynamics.Therefore, future investigations must consider the cleavage potential of dysferlin to properly understand its function in striated muscle.
An overview of dysferlin and the membrane repair complex.Further investigation is required to better understand muscle membrane repair, especially in an in vivo setting, but a general outline can be drawn from the literature (Fig. 2).Dysferlin resides at the sarcolemma and t-tubule membrane in uninjured mature muscle cells (Ampong et al., 2005;Klinge et al., 2010;Roche et al., 2011) but partially redistributes to the cytoplasm following damage (Klinge et al., 2010).Mini-dysferlin c 72 is also produced in a damage-dependent manner (Ballouhey et al., 2021;Lek et al., 2013;Redpath et al., 2014).Dysferlin appears to be a primary regulator of Ca 2+ -dependent vesicle trafficking and fusion, which is thought to contribute to the resealing of membranes (Davis et al., 2002;McDade & Michele, 2014;Therrien et al., 2009).Dysferlin's localisation is dynamic and it is regulated by multiple proteins such as cav-3 (Hernández-Deviez et al., 2006, 2008), annexin A2 and A6 (Bittel et al., 2020;Demonbreun, Allen, et al., 2016), and MG53 (Cai et al., 2009a).
MG53 is recruited very rapidly to damage sites (Cai et al., 2009a) and binds with dysferlin's C2A domain in the presence of Ca 2+ (Matsuda et al., 2012), facilitating the recruitment of dysferlin-positive vesicles to the membrane (Lek et al., 2013).It is thought that MG53 helps to accumulate repair vesicles to the membrane lesion (Cai et al., 2009a), whereas dysferlin facilitates the fusion of those vesicles with each other to form the repair patch (Bansal et al., 2003;Cenacchi et al., 2005;Davenport et al., 2016;Hornsey et al., 2013;Selcen et al., 2001).Data suggest that mini-dysferlin c 72 plays a major role in vesicle-membrane fusion but this is still under investigation (Lek et al., 2013).
The localisation of MG53 and dysferlin at the repair shoulder together with other suspected repair proteins such as BIN-1 and EHDs is regulated by phospholipid signalling (Demonbreun, Quattrocelli, et al., 2016).The repair shoulder is distinct from the repair cap, which is rich in annexin proteins, and its formation is dependent on Ca 2+ and remodelling of the actin cytoskeleton (Demonbreun, Quattrocelli, et al., 2016).Although dysferlin does not appear to localise with the annexin-rich cap at this stage, dysferlin's function during membrane repair is regulated by a complex and intricate relationship with various annexin proteins (for reviews, see Gerke et al., 2005;Koerdt et al., 2019).
The majority of research has focused on the role of dysferlin at the sarcolemma, but t-tubule membranes are simply extensions of the cell membrane that are also subjected to damage following muscle injury (Takekura et al., 2001).The mechanisms that regulate sarcolemma resealing may also regulate t-tubule growth and repair, and dysferlin is likely to be a key player in both processes (Fig. 2A-D).

How does dysferlin regulate skeletal muscle t-tubule dynamics?
A common feature of striated muscle is the t-tubule network, which is essential for regulating contractility (for reviews, see Al-Qusairi & Laporte, 2011;Eisner et al., 2017).Dysferlin and other major membrane repair proteins, such as MG53 and annexin A1, are enriched at longitudinal branches of the t-tubule system in over-stretched skeletal muscle, which are particularly susceptible to damage (Takekura et al., 2001;Waddell et al., 2011).This shows that the membrane repair complex may also regulate t-tubules.Multiple lines of evidence point to a relationship between dysferlin and t-tubules in both mature and developing skeletal muscle (Demonbreun et al., 2014;Klinge et al., 2007Klinge et al., , 2010)).During myotube development in vitro, dysferlin localises to developing t-tubules with BIN-1, another well-established t-tubule regulatory protein (Klinge et al., 2007;Lee et al., 2002).Dysferlin can also repair membrane lesions in cultured immature muscle cells (Klinge et al., 2007), which indicates that the function of dysferlin is multifaceted from early in the life cycle.

Dysferlin localises to the uninjured and regenerating t-tubule network.
There is uncertainty as to what proportion of dysferlin localises to the sarcolemma at rest, but its association with the t-tubule membrane is well established (Ampong et al., 2005;Kerr et al., 2013;Klinge et al., 2010;Roche et al., 2011;Waddell et al., 2011).The localisation of dysferlin at the t-tubule is comparable to that of other proteins that regulate t-tubule biogenesis and maintenance in skeletal muscle, such as BIN-1 (Lee et al., 2002;Prokic et al., 2020) and cav-3 (Fig. 1B; Parton et al., 1997;Ralston & Ploug, 1999).When skeletal muscle t-tubules are damaged pharmacologically, dysferlin redistributes from the t-tubule to the cytoplasm (Huang et al., 2007;Klinge et al., 2010).During cellular regeneration, dysferlin re-localises from the cytoplasm to the regenerating t-tubule network, which implicates dysferlin in mature t-tubule turnover (Fig. 2; Klinge et al., 2010).Furthermore, in response to osmotic shock injury, the disruption of t-tubule morphology is more severe in dysferlin-deficient muscle fibres than in wild-type fibres (Kerr et al., 2013).This shows that dysferlin helps to maintain t-tubule integrity in response to stress.
Dysferlin may act synergistically with BIN-1 to regulate t-tubule growth and morphology.In addition to BIN-1, which is known to generate tubule-like structures in both skeletal (Lee et al., 2002) and cardiac muscle cells (Caldwell et al., 2014;Lawless et al., 2019), dysferlin can generate t-tubule-like networks in non-muscle cell lines that do not normally contain t-tubules (Hofhuis et al., 2017).Much like mature t-tubules, these dysferlin-generated tubules have direct connections to the cell membrane and thus the extracellular space, an essential feature for t-tubule function (Al-Qusairi & Laporte, 2011;Brette & Orchard, 2003).Dysferlin is distinct within the ferlin family, as neither myoferlin nor otoferlin can promote tubule growth (Hofhuis et al., 2017).Tubules generated by dysferlin expression alone are shorter and less branched when compared to those that formed in response to BIN-1 expression only.Co-transfection with BIN-1 did not inhibit the tubulation activity of dysferlin, but instead BIN-1 co-localised with dysferlin at the tubular membrane.These data indicate J Physiol 602.9 that the functionality of dysferlin and BIN-1 during tubule formation is not identical but they appear to act synergistically when co-expressed (Hofhuis et al., 2017).Whilst dysferlin alone can drive tubule formation in non-tubule-containing cells (Hofhuis et al., 2017), the role of dysferlin in vivo is unknown, but it may be to fine-tune the morphology of the t-tubule network.This is because disordered t-tubules are still present in adult mouse skeletal muscle that lacks dysferlin, but still expresses other t-tubule-associated proteins such as BIN-1 and cav-3 (Demonbreun et al., 2014;Klinge et al., 2010).Taken together, these data strongly suggest that dysferlin regulates t-tubule maintenance and regeneration in skeletal muscle but the mechanisms by which dysferlin does so are mostly unknown.However, we would postulate that t-tubule dynamics are at least in part regulated by dysferlin-mediated vesicle trafficking and fusion.

How does dysferlin modulate EC coupling in skeletal
muscle?Evidence suggests that inefficiency within the membrane repair mechanism is not the only driver of muscle pathology in dysferlinopathy (Krahn et al., 2010;Lostal et al., 2012;Roche et al., 2010), and recent investigations have suggested that dysferlin also regulates intracellular Ca 2+ handling in skeletal muscle (Kerr et al., 2013(Kerr et al., , 2014;;Lukyanenko et al., 2017;Muriel et al., 2022).Other major repair proteins such as MG53 and cav-3 are also thought to regulate intracellular Ca 2+ handling in skeletal muscle by modulating the activity of EC coupling proteins such as LTCC and RyR1 (Ahn et al., 2016;Weiss et al., 2008).This suggests that there is a crossover between the Ca 2+ -dependent membrane repair complex and EC coupling regulatory mechanisms.
Dysferlin-knockout skeletal muscle is more susceptible to pathological Ca 2+ handling.In vitro osmotic shock injury brings about more pronounced pathological changes in dysferlin-knockout (KO) skeletal muscle cells compared to wild-type control cells, which include t-tubule damage, an increase in basal intracellular Ca 2+ concentration, a reduction in Ca 2+ transient amplitude and LTCC aggregation.These effects are mostly ameliorated by the expression of recombinant Venus-dysferlin, removal of extracellular Ca 2+ , or inhibition of the LTCC or RyR1 (Kerr et al., 2013;Lukyanenko et al., 2017).Systemic treatment with diltiazem also promotes muscle recovery in dysferlin-null muscle fibres after stretch-induced muscle damage in vivo (Kerr et al., 2013).These findings suggest that dysferlin protects against pathological Ca 2+ handling via the regulation of the LTCC and RyR1 and that the dysregulation of Ca 2+ likely contributes to dysferlinopathy.
Osmotic shock injury also causes a larger increase in Ca 2+ sparks and Ca 2+ waves in skeletal myocytes lacking dysferlin (Kerr et al., 2013;Lukyanenko et al., 2017).This is consistent with data showing dysferlin may protect against pathological Ca 2+ sparks and Ca 2+ waves by buffering Ca 2+ within the triadic cleft via its C2A domain, thus limiting Ca 2+ -induced Ca 2+ release (Lukyanenko et al., 2022).This may explain the importance of C2A's high Ca 2+ binding affinity and indicates an important dual role for C2A in regulating both EC coupling and membrane repair (Abdullah et al., 2014;Cai et al., 2009c;Lukyanenko et al., 2022;Muriel et al., 2022).It has also been suggested that dysferlin binds the SR luminal protein calsequestrin-1, suggesting dysferlin could also modulate SR Ca 2+ release (Flix et al., 2013).These data provide strong evidence that dysferlin is a key regulator of healthy EC coupling at the triadic junction.
Dysferlin regulates EC coupling protein expression and may support LTCC/RyR1 coupling.Dysferlin's C2C and C2D domains have been shown to regulate Ca 2+ transient amplitude (Muriel et al., 2022).As there is evidence to suggest dysferlin binds both the LTCC and RyR1 (Ampong et al., 2005;Flix et al., 2013), dysferlin C2C and C2D may support a physical coupling between the LTCC and RyR1, but further investigation is required (Fig. 1B inset).In support of a structural role for dysferlin at the triad, a lack of dysferlin has been shown to increase RyR1 cluster size in skeletal muscle cells (Barefield et al., 2021).
It should also be noted that the loss of dysferlin is associated with an increase in calsequestrin-1 and LTCC expression and a decrease in RyR1 expression coupled with an increase in RyR1 cluster size (Barefield et al., 2021;Lloyd et al., 2023).However, this effect is myofibre-type specific, which may explain why some muscles are preferentially affected in dysferlinopathy patients (for review, see Cardenas et al., 2016).Careful consideration should, therefore, be given to the muscles used to study dysferlin, especially when considering intracellular Ca 2+ handling.
Taken together, these data suggest that in addition to its established role in Ca 2+ -dependent membrane repair, dysferlin is also important for Ca 2+ signalling in skeletal myocytes, especially in response to stress.This supports the emerging hypothesis that aberrations in intracellular Ca 2+ handling are a driver of dysferlinopathy and other muscular dystrophies (for review, see Vallejo-Illarramendi et al., 2014) and emphasizes the need for further study into dysferlin's role in EC coupling.

How does dysferlin regulate cardiac biology?
Comparable to skeletal muscle, cardiac myocytes also have a well-developed t-tubule network.T-tubules provide a larger surface area for the formation of Ca 2+ -release units known as cardiac dyads between the LTCC on the t-tubule membrane and the cardiac ryanodine receptor isoform, RYR2, located on the SR (for review, see Bers, 2001).The tight control of cardiac t-tubule structure is essential for efficient EC coupling and strong contractility and therefore regulates the rhythmic beat of the myocardium (for review, see Smith et al., 2018).The cardiac EC coupling pathway is summarised in Fig. 1C.T-tubule networks are disrupted or lost completely in cardiac disease states, such as atrial fibrillation (Lenaerts et al., 2009) and heart failure (Cannell et al., 2006;Dibb et al., 2009).T-tubule degradation and aberrant intracellular Ca 2+ handling due to mechanical stretching of papillary muscles is thought to be indicative of t-tubule loss in response to elevated wall stress during heart failure (Frisk et al., 2016).The pathological and physiological characteristics of skeletal and cardiac muscle in EC coupling and t-tubule function are similar.Therefore, insights into the functionality of one type of striated muscle are likely to shed light on the parallel, less understood mechanisms of the other.It is evident from the information discussed here that the last two decades have provided a wealth of literature that suggests a role for dysferlin in membrane repair, t-tubule dynamics and Ca 2+ homeostasis in skeletal muscle.In comparison, the literature on the role of dysferlin in the heart is severely limited, yet multiple studies have yielded results that demonstrate dysferlin is important for various aspects of cardiac biology.
Dysferlin regulates cardiac membrane repair and function.Due to the very limited regenerative capacity of cardiac myocytes (Pasumarthi & Field, 2002), maintenance of the cell population is essential to prevent cell death as this can drive cardiac disease (Kostin et al., 2003).Compromised cardiac sarcolemmal integrity has been correlated with some cardiomyopathies (Clarke et al., 1995;Kostin et al., 2003), but it is unclear whether compromised membrane integrity is a major contributor to cardiac dysfunction.Early investigations showed that dysferlin is enriched at the cell membrane and in intracellular vesicles in cardiac myocytes and that dysferlin is a primary regulator of Ca 2+ -dependent sarcolemmal repair following laser injury in cultured cardiac tissue slices (Han et al., 2007).This shows that laser-induced membrane damage evokes a similar dysferlin-mediated response in both striated muscle types.However, much more is known regarding this proposed dysferlin-mediated repair pathway in skeletal muscle compared to the heart.
Cav-3, annexin A6 and MG53 are also thought to be involved in cardiac membrane repair in vitro, mirroring observations from skeletal muscle (Wang et al., 2010;Wenzel et al., 2007).It should be noted, however, that whilst MG53 is a major regulator of cardiac membrane repair in mice, it is not thought to be expressed in the human heart (Lemckert et al., 2016).These clear species-specific differences in the proposed cardiac membrane repair system emphasize the importance of selecting an appropriate model for the study of the role of dysferlin in the human heart.Cardiac dysferlin is primarily localised to the intercalated disc (ICD; Fig. 1C), a specialised feature of cardiac muscle, not found in skeletal muscle, which is subject to high tensile forces (Chase et al., 2009;Goossens et al., 2007;Shadrin et al., 2016).Whilst the function of dysferlin at the ICD is not known, it has been suggested that high tensile force may necessitate increased dysferlin-mediated membrane repair (Chase et al., 2009).
Cardiomyopathy is usually absent or mild in dysferlin-null animals, at least in young individuals under laboratory conditions (Chase et al., 2009;Hofhuis et al., 2020;Wei et al., 2015;Wenzel et al., 2007).Most reports show little to no evidence of cardiac hypertrophy or contractility in hearts or isolated cardiac myocytes from young dysferlin-deficient mice compared to controls (Chase et al., 2009;Hofhuis et al., 2020;Wei et al., 2015;Wenzel et al., 2007).Similarly, signs of skeletal muscle pathology are largely absent in young dysferlin-deficient mice and tend to develop later in a muscle-specific manner, partly reminiscent of the disease progression in human patients (Bansal et al., 2003;Hornsey et al., 2013;Lostal et al., 2010;van Putten et al., 2020).Wenzel et al. (2007) described that only two of seven human LGMD R2 dysferlin-related patients presented with signs of mild cardiomyopathy, including LV dilatation and a reduced ejection fraction.This suggests that the consequence of dysferlin dysfunction may be more variable or less severe in the myocardium than in the skeletal musculature, but the reasons for this are not clear.
Dysferlin deficiency negatively impacts cardiac physiology in response to stress.Stress exercise in the form of running causes a reduction in cardiac function and accelerated cell damage in dysferlin-deficient hearts (Han et al., 2007).Similar effects were observed after isoprenaline treatment in two further mouse models of dysferlin deficiency (Wenzel et al., 2007).The increase in contractile force brought about by exercise or isoprenaline treatment is thought to bring about damage to cardiac myocytes (Clarke et al., 1995), which may partially explain why dysferlin-deficient hearts are less resistant to such stressors.However, it was not investigated whether the stress-induced cardiac dysfunction in dysferlin-deficient hearts was caused by elevated membrane damage and/or perturbed intracellular Ca 2+ handling.
Dysferlin plays a role in cardiac ageing.Multiple lines of investigation have shown that a loss of dysferlin exacerbates the ageing process.Whilst the ultrastructure of young dysferlin-deficient myocytes appears more or less normal (Chase et al., 2009;Wenzel et al., 2007), J Physiol 602.9 pathological changes begin to manifest from 6 months of age, including delamination of the fascia adherens and vacuolation throughout the cytoplasm, often near the ICD (Chase et al., 2009).This suggests dysferlin is important for ICD stability and function.Broader histological analysis revealed an increase in fibrosis and serum troponin-T levels in aged dysferlin-deficient mice compared to age-matched controls (Han et al., 2007).Similarly, in a cohort of dysferlin-deficient A/J mice, approximately half were identified as showing signs of mild age-related cardiomyopathy (Chase et al., 2009).
Dysferlin deficiency does not bring about gross changes in the mammalian heart under normal healthy conditions, and Han et al. (2007) suggest this could be due to the absence of excessive mechanical stress in the heart during this time.However, dysferlin-mediated support of membrane integrity and structure is likely to be important for the maintenance of heart health in response to acute or chronic stressors such as exercise, β-adrenergic stimulation, cardiac disease or ageing.

Dysferlin regulates the cardiac t-tubule network.
Dysferlin associates with the adult t-tubule network in mice and rats (Chase et al., 2009;Hofhuis et al., 2020), which is likely to represent dysferlin's spatial localisation in the human heart.Various changes are observed within the dysferlin-deficient cardiac t-tubule network including a reduction in density and branching together with a shift towards a more axial orientation in moderately aged mice (Hofhuis et al., 2020).The close relationship between dysferlin and the cardiac t-tubule network appears to begin during early development.In the developing mouse heart, dysferlin expression increases at around postnatal day 6 or 7 (Hofhuis et al., 2020;Wei et al., 2015), correlating with the approximate start of murine t-tubule development (Reynolds et al., 2013).These data suggest a potential role for dysferlin in cardiac t-tubule development, but it is possible that dysferlin simply associates with t-tubule membranes constructed by other proteins such as BIN-1 (Caldwell et al., 2014;Lawless et al., 2019).
In MG53-KO mice, cardiac t-tubules develop normally but the efficiency of t-tubule repair is reduced after damage (Zhang et al., 2017), which would suggest that the mechanisms and proteins which drive the initial growth versus the repair or regrowth of pre-existing cardiac t-tubules are not identical.MG53 is thought to work with dysferlin to regulate the mouse cardiac t-tubule network but MG53 is absent in the human heart (Cai et al., 2009a;Lemckert et al., 2016;Zhang et al., 2017).This suggests fundamental species-specific differences in the way dysferlin regulates cardiac t-tubules.Cardiac t-tubule development and remodelling are poorly understood.Therefore, if future investigations provide strong evidence that dysferlin and the membrane repair complex are key regulators of cardiac t-tubules, this will significantly progress the cardiac t-tubule field.However, the lack of MG53 in the human heart points to major differences in the way the sarcolemma and t-tubule membranes are regulated between striated muscle types.This shows that additional models will be required to properly understand the function of dysferlin and associated proteins in regulating the human cardiac t-tubule network.
Dysferlin modulates Ca 2+ handling in the heart.Due to dysferlin's relationship with the cardiac t-tubule network and its proposed interaction with the LTCC and RyR1 in skeletal muscle, it was hypothesised that dysferlin also regulates intracellular Ca 2+ handling in the heart (Ampong et al., 2005;Hofhuis et al., 2020;Kerr et al., 2013;Lukyanenko et al., 2017).Data from mice suggest that the absence of cardiac dysferlin slows the decay of the Ca 2+ transient (Wei et al., 2015), which parallels human data showing that some LGMD R2 dysferlin-related patients presented with slowed repolarisation (Hofhuis et al., 2020;Wenzel et al., 2007).Hofhuis et al. (2020) observed a decrease in the L-type Ca 2+ current (I Ca,L ) and SR Ca 2+ content in mouse dysferlin-deficient myocytes, but the Ca 2+ transient amplitude in these cells was comparable to control, which they suggest is due to an increase in fractional SR Ca 2+ release in myocytes lacking dysferlin.They also found increased Ca 2+ spark frequency in dysferlin-deficient cells; this may facilitate cardiac arrhythmias (Landstrom et al., 2017).The current literature, albeit sparse, suggests that dysferlin is an essential regulatory protein for cardiac EC coupling, especially under conditions of stress.Thus, we would postulate that dysferlin may be protective against cardiac arrhythmias and the progression of other cardiac diseases.

Concluding remarks and future directions
The importance of dysferlin in skeletal muscle health was first realised over 20 years ago when dysferlin mutations were implicated in muscular dystrophy.Subsequent investigations, using mostly in vitro damage assays, identified dysferlin as a membrane-associated protein and a key regulator of Ca 2+ -dependent sarcolemma repair.However, more physiological studies cast some doubt on whether a reduced membrane repair capacity is a major driver of muscle pathology, at least in animal models of injury.Future experiments should focus on better understanding the most important mechanisms underpinning the progression of dysferlinopathy as this will be essential for improving clinical outcomes.Dysferlin also regulates t-tubule dynamics and intracellular Ca 2+ handling, two factors that underpin healthy muscle physiology.Although there is substantial literature on dysferlin in skeletal muscle, there are many questions still to be addressed.For example, it is still unclear how each of dysferlin's C2 domains contributes to membrane repair and there is debate as to how Ca 2+ regulates dysferlin's complex role within muscle cells.Moreover, it is still uncertain how the wider complex of membrane repair proteins interact with and regulate dysferlin.Also, the idea that mini-dysferlin c 72 is functionally distinct from the full-length dysferlin protein is an exciting, yet poorly understood concept.
Indirect evidence suggests that dysferlin regulates the repair and maintenance of t-tubules, but a clear role for dysferlin in initial t-tubule development has not yet been established.Although t-tubules are simply extensions of the sarcolemma, we cannot say whether dysferlin regulates these membrane subdivisions via a shared or distinct mechanism, but some findings suggest there are at least some commonalities between the two pathways.Increasing evidence to show that dysferlin regulates skeletal muscle EC coupling hints that the dysregulation of intracellular Ca 2+ is an unappreciated, if not a primary contributing factor to the muscular dysfunction observed in dysferlinopathy.Some membrane repair proteins are known to regulate EC coupling, which suggests there may be some crossover between the membrane repair and intracellular Ca 2+ handling mechanism.However, our current understanding of how these pathways are linked is very poor.The more we understand the behaviour of dysferlin in healthy skeletal muscle, the better equipped we will be to treat the muscular dysfunction that arises due to mutations within the dysferlin gene.
The relative lack of cardiac dysferlin research in the last two decades is surprising, but dysferlin is now earning its identity as an important protein in cardiac physiology.The similarities between skeletal muscle cells and cardiac myocytes are extensive.It is not surprising, therefore, that dysferlin may play a similar role in the heart as it does in skeletal muscle.Specifically, dysferlin has been shown to drive Ca 2+ -mediated sarcolemma repair in cardiac tissue slices in vitro, and regulate t-tubule structure together with some aspects of cardiac EC coupling, especially in response to stress.At present, it is suspected that a comparable collection of membrane repair proteins to that described in skeletal muscle facilitates wound repair in cardiac myocytes.However, further investigation is needed to examine how susceptible cardiac myocytes are to membrane damage, and how this contributes to cardiomyopathy.The increasing evidence for a role for dysferlin in the regeneration of t-tubules and the tight regulation of EC coupling suggests we should endeavour to properly elucidate the function of dysferlin within the heart.In the process, we will better understand basic cardiac function and find new protein targets for cardiac disease therapies.