Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system.

AIMS: The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart. METHODS AND RESULTS: We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to ß-adrenergic receptor stimulation. CONCLUSIONS: Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.

Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy.

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system.

Treatment with human immunoglobulin G improves the early disease course in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe hereditary myopathy. Standard treatment by glucocorticosteroids is limited because of numerous side effects. The aim of this study was to test immunomodulation by human immunoglobulin G (IgG) as treatment in the experimental mouse model (mdx) of DMD. 2 g/kg human IgG compared to human albumin was injected intraperitoneally in mdx mice at the age of 3 and 7 weeks. Advanced voluntary wheel running parameters were recorded continuously. At the age of 11 weeks, animals were killed so that blood, diaphragm, and lower limb muscles could be removed for quantitative PCR, histological analysis and ex vivo muscle contraction tests. IgG compared to albumin significantly improved the voluntary running performance and reduced muscle fatigability in an ex vivo muscle contraction test. Upon IgG treatment, serum creatine kinase values were diminished and mRNA expression levels of relevant inflammatory markers were reduced in the diaphragm and limb muscles. Macrophage infiltration and myopathic damage were significantly ameliorated in the quadriceps muscle. Collectively, this study demonstrates that, in the early disease course of mdx mice, human IgG improves the running performance and diminishes myopathic damage and inflammation in the muscle. Therefore, IgG may be a promising approach for treatment of DMD. Two monthly intraperitoneal injections of human immunoglobulin G (IgG) improved the early 11-week disease phase of mdx mice. Voluntary running was improved and serum levels of creatine kinase were diminished. In the skeletal muscle, myopathic damage was ameliorated and key inflammatory markers such as mRNA expression of SPP1 and infiltration by macrophages were reduced. The study suggests that IgG could be explored as a potential treatment option for Duchenne muscular dystrophy and that pre-clinical long-term studies should be helpful.

Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres.

Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition.

From ventriculomegaly to severe muscular atrophy: expansion of the clinical spectrum related to mutations in AIFM1.

The apoptosis-inducing factor (AIF) functions as a FAD-dependent NADH oxidase in mitochondria. Upon apoptotic stimulation it is released from mitochondria and migrates to the nucleus where it induces chromatin condensation and DNA fragmentation. So far mutations in AIFM1, a X-chromosomal gene coding for AIF, have been described in three families with 11 affected males. We report here on a further patient thereby expanding the clinical and mutation spectrum. In addition, we review the known phenotypes related to AIFM1 mutations. The clinical course in the male patient described here was characterized by phases with rapid deterioration and long phases without obvious progression of disease. At age 2.5 years he developed hearing loss and severe ataxia and at age 10 years muscle wasting, swallowing difficulties, respiratory insufficiency and external opthamoplegia. By next generation sequencing of whole exome we identified a hemizygous missense mutation in the AIFM1 gene, c.727G>T (p.Val243Leu) affecting a highly conserved residue in the FAD-binding domain. Summarizing what is known today, mutations in AIFM1 are associated with a progressive disorder with myopathy, ataxia and neuropathy. Severity varies greatly even within one family with onset of symptoms between birth and adolescence. 3 of 12 patients died before age 5 years while others were still able to walk during young adulthood. Less frequent symptoms were hearing loss, seizures and psychomotor regression. Results from clinical chemistry, brain imaging and muscle biopsy were unspecific and inconsistent.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.

A de novo interstitial deletion of 2p23.3-24.3 in a boy presenting with intellectual disability, overgrowth, dysmorphic features, skeletal myopathy, dilated cardiomyopathy.

Interstitial deletions of the distal part of chromosome 2p are rare, with only six reported cases involving regions from 2p23 to 2pter. Most of these were cytogenetic investigations. We describe a 14-year-old boy with an 8.97 Mb deletion of 2p23.3-24.3 detected by array comparative genomic hybridization (array CGH) who had intellectual disability (ID), unusual facial features, cryptorchidism, skeletal myopathy, dilated cardiomyopathy (DCM), and postnatal overgrowth (macrocephaly and tall stature). We compared the clinical features of the present case to previously described patients with an interstitial deletion within this chromosomal region and conclude that our patient exhibits a markedly different phenotype. Additional patients are needed to further delineate phenotype-genotype correlations.

Motor performance of young dystrophic mdx mice treated with long-circulating prednisolone liposomes.

For Duchenne muscular dystrophy (DMD), a common myopathy that leads to severe disability, no causal therapy is available. Glucocorticosteroids improve patients' muscle strength, but their long-term use is limited by negative side effects. Thus, pharmacological modifications of glucocorticosteroids are required to increase the efficacy by drug targeting. Liposomal encapsulation augments systemic half-life and local tissue concentrations of glucocorticosteroids and, at the same time, reduces systemic side effects. In this study, the efficacy of novel, long-circulating, polyethylene-glycol-coated liposomes encapsulating prednisolone was compared with free prednisolone in the treatment of mdx mice, a well-established animal model for DMD. Using an objective and sensitive computerized 24-hr detection system of voluntary wheel-running in single cages, we demonstrate a significant impairment of the running performance in mdx compared with black/10 control mice aged 3-6 weeks. Treatment with liposomal or free prednisolone did not improve running performance compared with saline control or empty liposomes. Histopathological parameters, including the rate of internalized nuclei and fiber size variation, and mRNA and protein expression levels of transforming growth factor (TGF)-ß and monocytes chemotactic protein (MCP)-1 also remained unchanged. Bioactivity in skeletal muscle of liposomal and free prednisolone was demonstrated by elevated mRNA expression of muscle ring finger protein 1 (MuRF1), a mediator of muscle atrophy, and its forkhead box transcription factors (Foxo1/3). Our data support the assessment of voluntary running to be a robust and reproducible outcome measure of skeletal muscle performance during the early disease course of mdx mice and suggest that liposomal encapsulation is not superior in treatment efficacy compared with conventional prednisolone. Our study helps to improve the future design of experimental treatment in animal models of neuromuscular diseases.

Effects of bioturbation on the fate of oil in coastal sandy sediments--an in situ experiment.

Effects of bioturbation by the common lugworm Arenicola marina on the fate of oil hydrocarbons (alkanes and PAHs) were studied in situ during a simulated oil spill in a shallow coastal area of Roskilde fjord, Denmark. The fate of selected oil compounds was monitored during 120 d using GC-MS and bioturbation activity (feces production and irrigation) was measured regularly during the experiment and used as input parameters in a mechanistic model describing the effects of A. marina on the transport and degradation of oil compounds in the sediment. The chemical analytical data and model results indicated that A. marina had profound and predictable effects on the distribution, degradation and preservation of oil and that the net effect depended on the initial distribution of oil. In sediment with an oil contaminated subsurface-layer A. marina buried the layer deeper in the sediment which clearly enhanced oil persistence. Conversely, A. marina stimulated both the physical removal and microbial degradation of oil compounds in uniformly oil contaminated sediments especially in deeper sediment layers (10-20 cm below the surface), whereas the fate of oil compounds deposited in surface layers (0-5 cm) mainly was affected by removal processes induced by wave actions and other bioturbating infauna such as Nereis diversicolor, Corophium volutator and Hydrobia spp. present in the experimental plots.

Dysferlin associates with the developing T-tubule system in rodent and human skeletal muscle.

Mutations in the dysferlin gene cause limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. Dysferlin mainly localizes to the sarcolemma in mature skeletal muscle where it is implicated in membrane fusion and repair. In different forms of muscular dystrophy, a predominantly cytoplasmic localization of dysferlin can be observed in regenerating myofibers, but the subcellular compartment responsible for this labeling pattern is not yet known. We have previously demonstrated an association of dysferlin with the developing T-tubule system in vitro. To investigate the role of dysferlin in adult skeletal muscle regeneration, we studied dysferlin localization at high resolution in a rat model of regeneration and found that the subcellular labeling of dysferlin colocalizes with the developing T-tubule system. Furthermore, ultrastructural analysis of dysferlin-deficient muscle revealed primary T-tubule anomalies similar to those seen in caveolin-3-deficient muscle. These findings indicate that dysferlin is necessary for correct T-tubule formation, and dysferlin-deficient skeletal muscle is characterized by abnormally configured T-tubules.

A common MLP (muscle LIM protein) variant is associated with cardiomyopathy.

RATIONALE: We previously discovered the human 10T-->C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. OBJECTIVE: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. METHODS AND RESULTS: We now report the generation and detailed analysis of the corresponding Mlp(W4R/+) and Mlp(W4R/W4R) knock-in animals, which develop an age- and gene dosage-dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. CONCLUSIONS: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that (W4R-MLP) might contribute significantly to human cardiovascular disease.

New aspects on patients affected by dysferlin deficient muscular dystrophy.

Mutations in the dysferlin gene lead to limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. A cohort of 36 patients affected by dysferlinopathy is described, in the first UK study of clinical, genetic, pathological and biochemical data. The diagnosis was established by reduction of dysferlin in the muscle biopsy and subsequent mutational analysis of the dysferlin gene. Seventeen mutations were novel; the majority of mutations were small deletions/insertions, and no mutational hotspots were identified. Sixty-one per cent of patients (22 patients) initially presented with limb girdle muscular dystrophy 2B, 31% (11 patients) with a Miyoshi phenotype, one patient with proximodistal mode of onset, one patient with muscle stiffness after exercise and one patient as a symptomatic carrier. A wider range of age of onset was noted than previously reported, with 25% of patients having first symptoms before the age of 13 years. Independent of the initial mode of presentation, in our cohort of patients the gastrocnemius muscle was the most severely affected muscle leading to an inability to stand on tiptoes, and lower limbs were affected more severely than upper limbs. As previous anecdotal evidence on patients affected by dysferlinopathy suggests good muscle prowess before onset of symptoms, we also investigated pre-symptomatic fitness levels of the patients. Fifty-three per cent of the patients were very active and sporty before the onset of symptoms which makes the clinical course of dysferlinopathy unusual within the different forms of muscular dystrophy and provides a challenge to understanding the underlying pathomechanisms in this disease.

Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy.

Skeletal muscle requires an efficient and active membrane repair system to overcome the rigours of frequent contraction. Dysferlin is a component of that system and absence of dysferlin causes muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels and a prominent inflammatory infiltrate. We have observed that dysferlinopathy patient biopsies show an excess of immature fibres and therefore investigated the role of dysferlin in muscle regeneration. Using notexin-induced muscle damage, we have shown that regeneration is attenuated in a mouse model of dysferlinopathy, with delayed removal of necrotic fibres, an extended inflammatory phase and delayed functional recovery. Satellite cell activation and myoblast fusion appear normal, but there is a reduction in early neutrophil recruitment in regenerating and also needle wounded muscle in dysferlin-deficient mice. Primary mouse dysferlinopathy myoblast cultures show reduced cytokine release upon stimulation, indicating that the secretion of chemotactic molecules is impaired. We suggest an extension to the muscle membrane repair model, where in addition to fusing patch repair vesicles with the sarcolemma dysferlin is also involved in the release of chemotactic agents. Reduced neutrophil recruitment results in incomplete cycles of regeneration in dysferlinopathy which combines with the membrane repair deficit to ultimately trigger dystrophic pathology. This study reveals a novel pathomechanism affecting muscle regeneration and maintenance in dysferlinopathy and highlights enhancement of the neutrophil response as a potential therapeutic avenue in these disorders.

Sarcoglycanopathies: can muscle immunoanalysis predict the genotype?

Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (alpha-, beta-, gamma-, delta-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed alpha-sarcoglycan deficient LGMD2D, 7 patients beta-sarcoglycan deficient LGMD2E, 3 patients gamma-sarcoglycan deficient LGDM2C, and one patient delta-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and beta-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD.

From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis.

The dysferlin gene is mutated in limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T-tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+-dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full-length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.

Severe phenotype in infantile facioscapulohumeral muscular dystrophy.

While much is known about the clinical course of adult FSHD, the third most common inherited muscular dystrophy, data on the "infantile phenotype" and especially on the progression of the disease in children are limited. We have followed a cohort of 7 patients with infantile FSHD for 9-25 years and here report the clinical and genetic findings in this group. Infantile FSHD is relatively rare, amounting to 4% of all of our FSHD patients. Despite some variability in the progression, infantile FSHD has a more consistent phenotype than adult FSHD. Although they had normal motor milestones, all patients showed facial weakness from early childhood, and subsequently were severely affected with rapid progression of the disease, marked muscular wasting, weakness, and hyperlordosis. None of the patients have shown signs of nocturnal hypoventilation or cardiomyopathy so far. No correlation was found between sex and the severity of phenotype whereas all but one patient had very short fragment sizes of the D4Z4 repeat. Only two patients had a de novo mutation: 3 patients inherited the mutation from a parent with somatic mosaicism, and one was inherited from a parent with classical adult FSHD. One patient was unusual in having one allele inherited from his father who showed somatic mosaicism and one allele with an additional de novo mutation. We conclude that infantile FSHD is a severe and rapidly progressive disease, and this needs to be taken into account in the advice given to patients diagnosed in early childhood. However, our data also suggest that the risk to an individual with classical FSHD of having a child with the infantile form is low.

Reversible infantile hypoglycorrhachia: possible transient disturbance in glucose transport?

Facilitated glucose transporter isoform 1 deficiency syndrome (GLUT1 DS), caused by impaired GLUT1-mediated glucose transport into the brain, is characterized by hypoglycorrhachia. The defect in the facilitative glucose transporter isoform 1 (GLUT1) can be confirmed by functional, quantitative, and molecular analyses. Diagnostic difficulties arise when these analyses are normal and hypoglycorrhachia remains unexplained. Three infants presenting with seizures and hypoglycorrhachia at 2, 4, and 6 weeks of age, which suggests GLUT1 deficiency syndrome, are reported. The seizures responded to a ketogenic diet in Patients 1 and 3 and phenobarbitone in Patient 2. Repeated GLUT1 analyses were normal. When treatment was discontinued, all patients remained seizure-free and developed normally. Subsequent lumbar punctures showed the return to normoglycorrhachia. We conclude that these cases might represent a transient disturbance in GLUT1-mediated glucose transport. The biomolecular basis for this clinical observation remains unknown. Though no treatment is required, clinical follow-up and repeated lumbar punctures are necessary to distinguish this benign condition from the original GLUT1 deficiency syndrome.

Severe Fusobacteria infections (Lemierre syndrome) in two boys.

UNLABELLED: Abscess formation is a rare cause of febrile illness in childhood but always has to be considered in such clinical presentations. Belonging to the resident flora of the oropharyngeal region, Fusobacteria are known to cause local infections; from here they may extend to other sites via the bloodstream or are aspirated into the lung (Lemierre disease). We report on two boys with Lemierre disease due to infection by Fusobacteria in monoculture causing two different clinical phenotypes. Case 1 presented with a large subphrenic abscess and pneumonic infiltration of the right middle lobe. Primary focus of infection was periodontal disease. Case 2 presented with a life-threatening septicaemia due to a retropharyngeal abscess and perforated otitis media followed by osteomyelitis of the atlas and thrombosis of the left sigmoid sinus and internal jugular vein. CONCLUSION: Fusobacteria should be considered in any abscess formation in children. A thorough examination of the oropharyngeal region as a possible site of primary manifestation is mandatory.