Macrophages: A minimally invasive tool for monitoring collagen VI myopathies

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Early Morphological Changes of the Rectus Femoris Muscle and Deep Fascia in Ullrich Congenital Muscular Dystrophy

Ullrich congenital muscular dystrophy (UCMD) is a severe form of muscular dystrophy caused by the loss of function of collagen VI, a critical component of the muscle-tendon matrix. Magnetic resonance imaging of UCMD patients’ muscles shows a peculiar rim of abnormal signal at the periphery of each muscle, and a relative sparing of the internal part. The mechanism/s involved in the early fat substitution of muscle fiber at the periphery of muscles remain elusive. We studied a muscle biopsy of the rectus femoris/deep fascia (DF) of a 3-year-old UCMD patient, with a homozygous mutation in the COL6A2 gene. By immunohistochemical and ultrastructural analysis, we found a marked fatty infiltration at the interface of the muscle with the epimysium/DF and an atrophic phenotype, primarily in fast-twitch fibers, which has never been reported before. An unexpected finding was the widespread increase of interstitial cells with long cytoplasmic processes, consistent with the telocyte phenotype. Our study documents for the first time in a muscle biopsy the peculiar pattern of outside-in muscle degeneration followed by fat substitution as already shown by muscle imaging, and an increase of telocytes in the interstitium of the deep fascia, which highlights a potential involvement of this structure in the pathogenesis of UCMD

Effects on Collagen VI mRNA Stability and Microfibrillar Assembly of Three COL6A2 Mutations in Two Families with Ullrich Congenital Muscular Dystrophy

We recently reported a severe deficiency in collagen type VI, resulting from recessive mutations of the COL6A2 gene, in patients with Ullrich congenital muscular dystrophy. Their parents, who are all carriers of one mutant allele, are unaffected, although heterozygous mutations in collagen VI caused Bethlem myopathy. Here we investigated the consequences of three COL6A2 mutations in fibroblasts from patients and their parents in two Ullrich families. All three mutations lead to nonsense-mediated mRNA decay. However, very low levels of undegraded mutant mRNA remained in patient B with compound heterozygous mutations at the distal part of the triple-helical domain, resulting in deposition of abnormal microfibrils that cannot form extensive networks. This observation suggests that the C-terminal globular domain is not essential for triple-helix formation but is critical for microfibrillar assembly. In all parents, the COL6A2 mRNA levels are reduced to 57-73% of the control, but long term collagen VI matrix depositions are comparable with that of the control. The almost complete absence of abnormal protein and near-normal accumulation of microfibrils in the parents may account for their lack of myopathic symptoms

Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation

Background: Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. Case presentation: A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. Conclusions: This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology

Integrin binding site within the gC1q domain orchestrates EMILIN-1-induced lymphangiogenesis.

Lymphatic vessels (LVs) play a pivotal role in the control of tissue homeostasis and also have emerged as important regulators of immunity, inflammation and tumor metastasis. EMILIN-1 is the first ECM protein identified as a structural modulator of the growth and maintenance of LV; accordingly, Emilin1-/- mice display lymphatic morphological alterations leading to functional defects as mild lymphedema, leakage and compromised lymph drainage. Many EMILIN-1 functions are exerted by the binding of its gC1q domain with the E933 residue of α4 and α9β1 integrins. To investigate the specific regulatory role of this domain on lymphangiogenesis, we generated a transgenic mouse model expressing an E933A-mutated EMILIN-1 (E1-E933A), unable to interact with α4 or α9 integrin. The mutant resulted in abnormal LV architecture with dense, tortuous and irregular networks; moreover, the number of anchoring filaments was reduced and collector valves had aberrant narrowed structures. E933A mutation also affected lymphatic function in lymphangiography assays and made the transgenic mice more prone to lymph node metastases. The finding that the gC1q/integrin interaction is crucial for a correct lymphangiogenesis response was confirmed and reinforced by functional in vitro tubulogenesis assays. In addition, ex vivo thoracic-duct ring assays revealed that E1-E933A-derived lymphatic endothelial cells had a severe reduction in sprouting capacity and were unable to organize into capillary-like structures. All these data provide evidence that the novel "regulatory structural" role of EMILIN-1 in the lymphangiogenic process is played by the integrin binding site within its gC1q domain

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