Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy.

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Lack of MG53 in human heart precludes utility as a biomarker of myocardial injury or endogenous cardioprotective factor.

AIMS: Mitsugumin-53 (MG53/TRIM72) is an E3-ubiquitin ligase that rapidly accumulates at sites of membrane injury and plays an important role in membrane repair of skeletal and cardiac muscle. MG53 has been implicated in cardiac ischaemia-reperfusion injury, and serum MG53 provides a biomarker of skeletal muscle injury in the mdx mouse model of Duchenne muscular dystrophy. We evaluated the clinical utility of MG53 as a biomarker of myocardial injury. METHODS AND RESULTS: We performed Langendorff ischaemia-reperfusion injury on wild-type and dysferlin-null murine hearts, using dysferlin deficiency to effectively model more severe outcomes from cardiac ischaemia-reperfusion injury. MG53 released into the coronary effluent correlated strongly and significantly (r = 0.79-0.85, P < 0.0001) with functional impairment after ischaemic injury. We initiated a clinical trial in paediatric patients undergoing corrective heart surgery, the first study of MG53 release with myocardial injury in humans. Unexpectedly, we reveal although MG53 is robustly expressed in rat and mouse hearts, MG53 is scant to absent in human, ovine, or porcine hearts. Absence of MG53 in 11 human heart specimens was confirmed using three separate antibodies to MG53, each subject to epitope mapping and confirmed immunospecificity using MG53-deficient muscle cells. CONCLUSION: MG53 is an effective biomarker of myocardial injury and dysfunction in murine hearts. However, MG53 is not expressed in human heart and therefore does not hold utility as a clinical biomarker of myocardial injury. Although cardioprotective roles for endogenous myocardial MG53 cannot be extrapolated from rodents to humans, potential therapeutic application of recombinant MG53 for myocardial membrane injury prevails.

Calpains, cleaved mini-dysferlinC72, and L-type channels underpin calcium-dependent muscle membrane repair.

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice.

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.

Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

The murine stanniocalcin 2 gene is a negative regulator of postnatal growth.

Stanniocalcin (STC), a secreted glycoprotein, was first studied in fish as a classical hormone with a role in regulating serum calcium levels. There are two closely related proteins in mammals, STC1 and STC2, with functions that are currently unclear. Both proteins are expressed in numerous mammalian tissues rather than being secreted from a specific endocrine gland. No phenotype has been detected yet in Stc1-null mice, and to investigate whether Stc2 could have compensated for the loss of Stc1, we have now generated Stc2(-/-) and Stc1(-/-) Stc2(-/-) mice. Although Stc1 is expressed in the ovary and lactating mouse mammary glands, like the Stc1(-/-) mice, the Stc1(-/-) Stc2(-/-) mice had no detected decrease in fertility, fecundity, or weight gain up until weaning. Serum calcium and phosphate levels were normal in Stc1(-/-) Stc2(-/-) mice, indicating it is unlikely that the mammalian stanniocalcins have a major physiological role in mineral homeostasis. Mice with Stc2 deleted were 10-15% larger and grew at a faster rate than wild-type mice from 4 wk onward, and the Stc1(-/-) Stc2(-/-) mice had a similar growth phenotype. This effect was not mediated through the GH/IGF-I axis. The results are consistent with STC2 being a negative regulator of postnatal growth.

Loss of ACTN3 gene function alters mouse muscle metabolism and shows evidence of positive selection in humans.

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein alpha-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associated with elite athlete status and human muscle performance, suggesting that alpha-actinin-3 deficiency influences the function of fast muscle fibers. Here we show that loss of alpha-actinin-3 expression in a knockout mouse model results in a shift in muscle metabolism toward the more efficient aerobic pathway and an increase in intrinsic endurance performance. In addition, we demonstrate that the genomic region surrounding the 577X null allele shows low levels of genetic variation and recombination in individuals of European and East Asian descent, consistent with strong, recent positive selection. We propose that the 577X allele has been positively selected in some human populations owing to its effect on skeletal muscle metabolism.

Expression of Gtf2ird1, the Williams syndrome-associated gene, during mouse development.

The gene GTF2IRD1 is localized within the critical region on chromosome 7 that is deleted in Williams syndrome patients. Genotype-phenotype comparisons of patients carrying variable deletions within this region have implicated GTF2IRD1 and a closely related homolog, GTF2I, as prime candidates for the causation of the principal symptoms of Williams syndrome. We have generated mice with an nls-LacZ knockin mutation of the Gtf2ird1 allele to study its functional role and examine its expression profile. In adults, expression is most prominent in neurons of the central and peripheral nervous system, the retina of the eye, the olfactory epithelium, the spiral ganglion of the cochlea, brown fat adipocytes and to a lesser degree myocytes of the heart and smooth muscle. During development, a dynamic pattern of expression is found predominantly in musculoskeletal tissues, the pituitary, craniofacial tissues, the eyes and tooth buds. Expression of Gtf2ird1 in these tissues correlates with the manifestation of some of the clinical features of Williams syndrome.

Characterisation of a P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow.

BACKGROUND: Gene transfer of the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) provides a mechanism for drug resistance and the selective expansion of gene-modified cells in vivo. Possible clinical applications for this strategy include chemoprotection to allow dose escalation of alkylating chemotherapy, or combining MGMT(P140K) expression with a therapeutic gene in the treatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivo selection to develop allogeneic micro-transplantation protocols that rely on post-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels of donor/recipient chimerism. METHODS: Using stably transfected murine embryonic stem (ES) cells, we have generated a C57BL/6 transgenic mouse line with expression of MGMT(P140K) within the hematopoietic compartment for use as a standard source of donor HSC in such models. Functional characterisation of transgene expression was carried out in chemotherapy-treated transgenic mice and in allogeneic recipients of transgenic HSC. RESULTS: Expression of the transgene provided chemoprotection and allowed in vivo selection of MGMT(P140K)-expressing cells in transgenic mice after exposure to O6-benzylguanine (BG) and N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). In an allogeneic transplant experiment in which transgenic HSC were engrafted into 129 strain recipients following low intensity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy. CONCLUSIONS: This MGMT(P140K) transgenic mouse line provides a useful source of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplant conditioning elements can be investigated independently of gene transfer efficiency.

Membrane lymphotoxin contributes to anti-leishmanial immunity by controlling structural integrity of lymphoid organs.

Lymphotoxin (LT)alpha in combination with LTbeta forms membrane-bound heterotrimeric complexes with a crucial function in lymph node (LN) organogenesis and correct morphogenesis of secondary lymphoid tissue. To study the role of membrane LT (mLT) in lymphoid tissue organogenesis we generated an LTbeta-deficient mouse strain on a pure genetic C57BL/6 background (B6.LTbeta-/-) and compared it to a unique series of LTalpha-, TNF- and TNF/LTalpha-gene-targeted mice on an identical genetic background (B6.LTalpha-/-, B6.TNF-/- and B6 TNF/LTalpha-/-). B6.LTbeta-/- mice lacked peripheral LN with the exception of mesenteric LN, and displayed a disturbed micro-architecture of the spleen, although less profoundly than LTalpha- or TNF/LTalpha-deficient mice. Radiation bone marrow chimeras (B6.WT-->B6.LTbeta-/- developed Peyer's patch (PP)-like lymphoid aggregates in the intestinal wall indicating a possible role for soluble LTalpha(3) in the formation of the PP anlage. After infection with Leishmania major, B6.LTbeta-/- mice developed a fatal disseminating leishmaniasis resulting in death after 8 to 14 weeks, despite the natural resistance of the C57BL/6 genetic background (B6.WT) mice to the parasite. Both, the cellular and the humoral anti-L. major immune responses were delayed and ineffective. However, the expression pattern of the key cytokines IFN-gamma and IL-12 were comparable in B6.WT and B6.LTbeta-/- mice. Infection of radiation bone marrow chimeras showed that it is the LTbeta-dependent presence of lymphoid tissue and not the expression of mLT itself that renders mice resistant to leishmaniasis.