dysferlin in a hyperckaemic patient with caveolin 3 mutation and in c2c12 cells after p38 map kinase inhibition

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription

Extracellular matrix and nuclear abnormalities in skeletal muscle of a patient with Walker–Warburg syndrome caused by POMT1 mutation

AbstractWalker–Warburg syndrome (WWS) is an autosomal recessive disorder characterized by congenital muscular dystrophy, structural eye abnormalities and severe brain malformations. We performed an immunohistochemical and electron microscopy study of a muscle biopsy from a patient affected by WWS carrying a homozygous frameshift mutation in O-mannosyltransferase 1 gene (POMT1). α-Dystroglycan glycosylated epitope was not detected in muscle fibers and intramuscular peripheral nerves. Laminin α2 chain and perlecan were reduced in muscle fibers and well preserved in intramuscular peripheral nerves. The basal lamina in several muscle fibers showed discontinuities and detachment from the plasmalemma. Most nuclei, including myonuclei and satellite cell nuclei, showed detachment or complete absence of peripheral heterochromatin from the nuclear envelope. Apoptotic changes were detected in 3% of muscle fibers. The particular combination of basal lamina and nuclear changes may suggest that a complex pathogenetic mechanism, affecting several subcellular compartments, underlies the degenerative process in WWS muscle

Collagen VI–NG2 axis in human tendon fibroblasts under conditions mimicking injury response

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI–NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI–NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor β1 (TGFβ1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFβ1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair

Cyclosporine A in Ullrich Congenital Muscular Dystrophy: Long-Term Results

Six individuals with Ullrich congenital muscular dystrophy (UCMD) and mutations in the genes-encoding collagen VI, aging 5–9, received 3–5 mg/kg of cyclosporine A (CsA) daily for 1 to 3.2 years. The primary outcome measure was the muscle strength evaluated with a myometer and expressed as megalimbs. The megalimbs score showed significant improvement (P = 0.01) in 5 of the 6 patients. Motor function did not change. Respiratory function deteriorated in all. CsA treatment corrected mitochondrial dysfunction, increased muscle regeneration, and decreased the number of apoptotic nuclei. Results from this study demonstrate that long-term treatment with CsA ameliorates performance in the limbs, but not in the respiratory muscles of UCMD patients, and that it is well tolerated. These results suggest considering a trial of CsA or nonimmunosuppressive cyclosporins, that retains the PTP-desensitizing properties of CsA, as early as possible in UCMD patients when diaphragm is less compromised

Exon Skipping-Mediated Dystrophin Reading Frame Restoration for Small Mutations

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Collagen VI myopathies: pathogenic mechanism and therapeutic strategies

Collagen (Col) VI is a major component of the extracellular matrix which, in skeletal muscle, is localized just outside the basement membrane. Deficiency of ColVI in humans due to mutations of COL6 genes gives rise to Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD), and Myosclerosis Myopathy. About 70 different COL6 gene  mutations have been associated to ColVI myopathies which, although present  a wide range of clinical features, share a common pathogenesis. This mechanism, initially identified in the Col6a1-/- model (Irwin et al., 2003), and then in cultures from BM and UCMD patients (Angelin et al., 2007), involves a mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). The pat hogenic role of PTP opening, causing the release of proapoptotic factors, has been confirmed by the normalizing effect of cyclosporine A (CsA) on  the mitochondrial defect and on the increased apoptotic rate in both the mouse model and in a selected group of patients (Merlini et al., 2008). We have recently demonstrated that the persistence of abnormal mitochondria and apoptosis are amplified by defective autophagy (Grumati et al., 2010). In fact, forced activation of autophagy by genetic, dietary and pharmacological approaches restore myofiber survival and ameliorate the dystrophic phenotype in mice. Since also muscle cells of BM and UCMD patients  present a defective activation of the autophagic machinery, it will be possible to restore this activity by using  a low protein diet or drugs capable to reactivate autophagy. To monitor the effects of therapies on ColVI-related myopathies highly invasive muscle/skin biopsies have been so far utilized. We have recently obtained evidence that ColVI expression in blood macrophages from BM and UCMD patients can be detected at levels comparable to those observed in muscle biopsies and cultured skin fibroblasts (Gualandi et al., 2011). These data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing highly invasive biopsies in the diagnosis and monitoring of ColVI myopathies. 1. Irwin WA et al., Nat Genet 2003;35:267-71 2. Angelin A et al., PNAS USA 2007;104:991-6 3. Merlini L et al., PNAS USA 2008;105:5225-9 4. Gualandi F et al., Muscle Nerve 2011;44:80-

Autophagy is defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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Ultrastructural changes in muscle cells of patients with collagen VI-related myopathies

Collagen VI is an extracellular matrix protein expressed in several tissues including skeletal muscle. Mutations in COL6A genes cause Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD) and Myosclerosis Myopathy (MM). Collagen VI deficiency causes increased opening of the mitochondrial permeability transition pore (mPTP), leading to ultrastructural and functional alterations of mitochondria, amplified by impairment of autophagy. Here we report for the first time ultrastructural studies on muscle biopsies from BM and UCMD patients, showing swollen mitochondria with hypodense matrix, disorganized cristae and paracrystalline inclusions, associated with dilated sarcoplasmic reticulum and apoptotic changes. These data were supported by scanning electron microscopy analysis on BM and UCMD cultured cells, showing alterations of the mitochondrial network. Morphometric analysis also revealed a reduced short axis and depicted swelling in about 3% of mitochondria. These data demonstrate that mitochondrial defects underlie the pathogenetic mechanism in muscle tissue of patients affected by collagen VI myopathie