Sarcolemmal Repair Research Articles

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

Dysferlin is a 237kDa 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.

Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.

Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction–induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non–muscle-targeted gene therapy for LGMD2B.

Mitochondrial dysfunction and consequences in calpain-3-deficient muscle

BackgroundNonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.MethodsCell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.ResultsWe find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.ConclusionOur results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.

Autoantibodies targeting TRIM72 compromise membrane repair and contribute to inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM) involve chronic inflammation of skeletal muscle and subsequent muscle degeneration due to an uncontrolled autoimmune response; however, the mechanisms leading to pathogenesis are not well understood. A compromised sarcolemmal repair process could promote an aberrant exposure of intramuscular antigens with the subsequent initiation of an inflammatory response that contributes to IIM. Using an adoptive transfer mouse model of IIM, we show that sarcolemmal repair is significantly compromised in distal skeletal muscle in the absence of inflammation. We identified autoantibodies against TRIM72 (also known as MG53), a muscle-enriched membrane repair protein, in IIM patient sera and in our mouse model of IIM by ELISA. We found that patient sera with elevated levels of TRIM72 autoantibodies suppress sarcolemmal resealing in healthy skeletal muscle, and depletion of TRIM72 antibodies from these same serum samples rescues sarcolemmal repair capacity. Autoantibodies targeting TRIM72 lead to skeletal muscle fibers with compromised membrane barrier function, providing a continuous source of autoantigens to promote autoimmunity and further amplifying humoral responses. These findings reveal a potential pathogenic mechanism that acts as a feedback loop contributing to the progression of IIM.

Dysregulation of Mitochondrial Ca2+ Uptake and Sarcolemma Repair Underlie Muscle Weakness and Wasting in Patients and Mice Lacking MICU1

SUMMARYMuscle function is regulated by Ca2+, which mediates excitation-contraction coupling, energy metabolism, adaptation to exercise, and sarcolemmal repair. Several of these actions rely on Ca2+ delivery to the mitochondrial matrix via the mitochondrial Ca2+ uniporter, the pore of which is formed by mitochondrial calcium uniporter (MCU). MCU’s gatekeeping and cooperative activation are controlled by MICU1. Loss-of-protein mutation in MICU1 causes a neuromuscular disease. To determine the mechanisms underlying the muscle impairments, we used MICU1 patient cells and skeletal muscle-specific MICU1 knockout mice. Both these models show a lower threshold for MCU-mediated Ca2+ uptake. Lack of MICU1 is associated with impaired mitochondrial Ca2+ uptake during excitation-contraction, aerobic metabolism impairment, muscle weakness, fatigue, and myofiber damage during physical activity. MICU1 deficit compromises mitochondrial Ca2+ uptake during sarcolemmal injury, which causes ineffective repair of the damaged myofibers. Thus, dysregulation of mitochondrial Ca2+ uptake hampers myofiber contractile function, likely through energy metabolism and membrane repair.

Dysferlin Mutants: Defects in Trafficking and Association with Proteins of the Transverse Tubule

Dysferlinopathies are a class of muscular dystrophies caused by a loss of function or expression of dysferlin, a 237 kDa single-pass transmembrane protein expressed in many tissues, including skeletal muscle. Dysferlin has been implicated in sarcolemmal repair and in the regulation of calcium signaling following mechanical stress. Dysferlin binds to several proteins associated with the transverse tubules, including the dihydropyridine receptor (DHPR) and Caveolin 3, to stabilize the triad junction and perhaps to serve as a scaffold for repair machinery. Mutations disrupting these interactions may lead to disruption of Ca signaling after mechanical stress, which in turn leads to fiber loss, inflammation and muscle atrophy associated with muscular dystrophy. Mutations to dysferlin may also to lead to aberrant trafficking, causing accumulation in compartments other than the T-tubules, therefore removing or reducing dysferlin's function. We are studying several point mutations found in dysferlinopathy patients, as well as domain deletion mutants, to determine their effects on trafficking, association with other membrane proteins, and dimerization. Our results to date indicate that mutations or deletions that are thought to be pathogenic are at least partially associated with altered trafficking to the T-tubules and, for some, disruption of binding to Cav3, but not to changes in its ability to dimerize. Supported by the Jain Foundation and NIH RO1 AR064268.

Exon Skipping in a Dysf-Missense Mutant Mouse Model

Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.

Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy

The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.

Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair.

Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an ‘eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions.

Abstract 86: Compromised Sarcolemmal Membrane Repair Contributes to the Progression of Heart Failure

A conserved sarcolemmal membrane repair response exist to counteract membrane damage and restore membrane barrier function in order to maintain normal cellular homeostasis. This response can involve various mechanisms including activation of signaling pathways that trigger vesicular trafficking to the site of injury followed by vesicular fusion with the damaged portion of the membrane to patch the membrane disruption. Previous studies indicate that compromised repair capacity can exacerbate cardiac injury while increasing membrane repair capacity can reduce cardiac pathology. In our studies, membrane repair assays on cardiac and non-cardiac cell lines demonstrated that this process is dependent on activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis through the downstream target Akt1. One mechanism found to increase membrane repair following PI3K/Akt1 activation is elevated exocytotic and endocytotic activity. Further studies indicate that the PI3K/Akt1 pathway is relevant to membrane repair in native hearts. Thick slices of myocardium from explanted human and mouse hearts were probed using multi-photon microscopy to determine the membrane repair capacity. These studies indicate decreased repair capacity in failing human myocardium as well as in mouse hearts following transaortic constriction (TAC). This membrane repair response requires PI3K/Akt1 signaling as genetically modified mice null for Akt1 show compromised sarcolemmal membrane repair. Additionally, PI3K or Akt1 inhibition prevents membrane resealing in non-failing human or mouse myocardium. The compromised membrane repair observed in failing human myocardium can be ameliorated by PI3K or Akt1 agonists. Treatment of TAC mice with multiple therapeutic compounds known to increase membrane repair capacity can minimize the development of structural and functional hallmarks of heart failure. Our results indicate that failing cardiomyocytes exhibit compromised membrane repair and that increased PI3K/Akt1 signaling can increase repair capacity thereby demonstrating potential as a heart failure treatment.

Plasma Membrane Repair inHealth and Disease.

Since an intact membrane is required for normal cellular homeostasis, membrane repair is essential for cell survival. Human genetic studies, combined with the development of novel animal models and refinement of techniques to study cellular injury, have now uncovered series of repair proteins highly relevant for human health. Many of the deficient repair pathways manifest in skeletal muscle, where defective repair processes result in myopathies or other forms of muscle disease. Dysferlin is a membrane-associated protein implicated in sarcolemmal repair and also linked to other membrane functions including the maintenance of transverse tubules in muscle. MG53, annexins, and Eps15 homology domain-containing proteins interact with dysferlin to form a membrane repair complex and similarly have roles in membrane trafficking in muscle. These molecular features of membrane repair are not unique to skeletal muscle, but rather skeletal muscle, due to its high demands, is more dependent on an efficient repair process. Phosphatidylserine and phosphatidylinositol 4,5-bisphosphate, as well as Ca(2+), are central regulators of membrane organization during repair. Given the importance of muscle health in disease and in aging, these pathways are targets to enhance muscle function and recovery from injury.

Abstract 359: Tripartite Motif Protein 58 (trim58) is a Novel Regulator of Membrane Repair With Increased Expression in Failing Cardiac Muscle

In recent years, members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to be both positively and negatively regulated in various disease pathologies. TRIM72 (MG53) has been directly linked with regulation of sarcolemmal membrane repair in striated muscle cells, including cardiomyocytes. Recently, we were first to identify that a novel tripartite motif family member, TRIM58, is a negative regulator of the cell membrane repair process in striated muscle cells. Overexpression of TRIM58 decreases the membrane repair capacity of cultured myoblasts as measured by dye occlusion following laser-mediated disruption of the sarcolemmal membrane. We also find that TRIM58 can directly interact with TRIM72/MG53 and alter signaling through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Protein profiling experiments show that TRIM58 expression increases during the development of heart failure suggesting that TRIM58 may be relevant in the development of cardiac failure. Western blot and histological examination in both human (Fig.1) and mouse transverse aortic constriction (TAC) heart failure samples clearly show an increased expression of TRIM58 in cardiac tissue. Our results suggest that TRIM58 levels might serve as a potential prognostic marker and that TRIM58 may be a therapeutic target for the management of cardiovascular disease.

Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair.

Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy.Objectives: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy.Methods: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts.Results: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation.Conclusion: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.

Ion-Mobility Mass Spectrometry Assay for Incorporation of Phytanic Acid into Muscle Phospholipids

Human dysferlinopathies (Limb-Girdle Muscular Dystrophy 2b, Myoshi Myopathy) are muscle-wasting syndromes caused by mutations in the dysferlin protein. Dysferlin loss impairs sarcolemmal repair that may contribute to disease progression. One therapeutic approach is to treat subjects with compounds that fortify the natural membrane tendency to reseal after damage, and minimize the inflammation that impedes regeneration and amplifies tissue destruction. Phytanic acid is a saturated branched chain fatty acid comprised of a 16 carbon aliphatic chain with 4 methyl groups (4ME 16:0); it is incorporated into phospholipids and triglycerides. Model phospholipid bilayers containing phytanic acid exhibit greater electrical stability than bilayers composed of straight-chain phospholipids. Phytanic acid-containing phospholipids may improve the resistance of the muscle fiber sarcolemma to stretch-induced damage. To measure phytanic acid incorporation into muscle phospholipids, dysferlin-deficient A/J mice were maintained on a defined diet supplemented with 2% phytol for three weeks; control animals received the defined diet without phytol. Muscle tissue lipids were analyzed by mass spectrometry. The muscle phosphatidylcholine species profiles were similar between phytol and control diet, except that some additional species were detected in the phytol diet muscle. The two most abundant novel species (m/z 790.7 and 862.7) are tentatively identified as PC 20:0-16:0 and PC 20:0-22:6. To confirm the presence of phytanic acid, the ion mobility of these species was compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids. The ion mobility is intermediate between PC species containing either zero or two phytanic acid chains, consistent with having one phytanic acid chain. We estimate that these species represent 5% of PC. Using this methodology, we will identify phytanic acid containing species in the other classes, in order to determine the total amount of phytanic acid-containing phospholipid incorporated in the muscle.

G.P.286: Validation of in silico variation effect prediction tools in missense mutations of dysferlinopathy

In silico prediction tools for genetic variation have now become the commodity in genetic analysis. Each tool utilizes their own algorithm to give us the results. Dysferlin (DYSF) is a transmembrane protein, crucial for sarcolemmal repair, and its recessive genetic defects bring about progressive muscular weakness either as Miyoshi distal myopathy or limb girdle muscular dystrophy 2B. The Leiden database lists one of the most corroborating data set on the pathogenicity of each genotype in the field of muscular dystrophy. In this study, we compared the in silico analysis results and the published pathogenicity of the dysferlin mutations listed on Leiden database. We suggest a new cut-off value that can be optimally used to test a new mutation with in silico tools. We used PROVEAN and SIFT online software to predict a possibly damaging mutation. This is a prediction tool used mainly to test the impact on the biological function of a protein by a missense mutation or amino acid substitution. We used the Leiden open variation database on dysferlin mutations and utilized the complete sequence variants data provided by the website. PROVEAN tool showed a remarkably better result to test dysferlin nonsense mutations than SIFT tool. On using PROVEAN tool for dysferlin missense mutation, we propose a new cut-off value of −5.365. Individualized cut-off values for other genes will expand the usefulness of in silico prediction tools.

P.5.1 Sarcolemmal repair and reorganization of microtubule

Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B, and is involved in sarcolemmal repair. Proteomics analyses have identified multiple dysferlin-binding partners including cytoskeletal proteins. Affixin, a binding partner of dysferlin, regulates cytoskeletal actin. We reported that affixin, β- and γ-actin accumulated at the sarcolemmal injury site of wild-type mice in response to membrane rapture. α-tubulin and microtubule-associated proteins have been reported as dysferlin binding partners, however, their involvement in sarcolemmal repair remains unclear. The purpose of this study is to examine involvement of microtubule in sarcolemmal repair. To clarify molecular behavior of α-tubulin in sarcolemmal repair, GFP2-tagged human α-tubulin was expressed in mouse FDB muscle (C57BL/6J and dysferlin-deficient SJL) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. GFP2-α-tubulin expressed in wild-type mice was localized at sarcolemma and T-tubule, and observed as a striated pattern. This striation pattern of GFP2-α-tubulin was slightly disorganized in SJL mice. After sarcolemmal injury, GFP2-α-tubulin in wild-type mice was disassembled and accumulates slower compared to GFP-β- and γ -actin. In SJL mice, GFP2-α-tubulin around the injury site was depolymerized, however, accumulation of GFP2-α-tubulin was not observed. α-Tubulin may also have a role in sarcolemmal repair.

Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca2+dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.