Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.

Background: Camptocormia, as an isolated sign, could be a diagnostic challenge since it might be caused by several central and peripheral nervous system diseases, including Parkinson disease, dystonia, MSA, Alzheimer disease, ALS, CIDP, myasthenia and myopathies.

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full‐length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013. © 2012 Wiley Periodicals, Inc.

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1(-/-) mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established.

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery–Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.

Autophagy is crucial in the turnover of cell components both in constitutive conditions and in response to starvation. Defects of this degradative system play a role in various diseases, but little is known about autophagy in muscular dystrophies. Mutations in any of the three genes coding for collagen VI cause several muscle diseases in humans, including Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and Congenital myosclerosis. Collagen VI null (Col6a1−/−) mice display an early onset myopathic phenotype characterized by organelle defects, mitochondrial dysfunction and spontaneous apoptosis, leading to myofiber degeneration. We found that persistence of abnormal organelles and apoptosis are caused by defective autophagy. Indeed, skeletal muscles of Col6a1−/− mice displayed an impairment of autophagic flux, which matched the lower induction of Beclin 1 and Bnip3 and the lack of autophagosomes after starvation. Notably, reactivation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1−/− mice. Furthermore, muscle biopsies from patients affected by UCMD and BM showed reduced levels of Beclin 1 and Bnip3. These findings indicate that defective activation of the autophagic machinery plays a pathogenic role in congenital muscular dystrophies.

We have defined the expression and localization of the two novel collagen type VI chains, alpha5 and alpha6, in normal muscle tissue and cultures. In muscle sections, the alpha6 chain was detected in the endomysium and perimysium, while the alpha5 chain was restricted to the myotendinous junctions. In muscle cultures, the alpha5 chain was absent while the alpha6 was present in traces in the ECM; the treatment with pro-fibrotic factor TGF-beta1 did not affect the expression of alpha5 chain, while, the alpha6 chain was markedly increased. In order to define the involvement of alpha6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD) and Congenital Muscular Dystrophy 1A (MDC1A). We found that alpha6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the alpha5 chain was undetectable. Our data indicate a differential distribution of alpha5 and alpha6 chains in skeletal muscle; while the alpha5 chain may have a specialized function in areas of tissues subjected to tensile stress, the alpha6 chain appears to be involved in (and/or contributes to) ECM remodeling upon fibrosis.

We recently demonstrated that lower doses of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed to cationic core–shell nanoparticles (NPs) induce widespread dystrophin restoration, even 45 days after intra-peritoneal (I.P) treatment, in mdx mice. Here we describe persistent, albeit low, levels of AON-induced skipped transcript in muscles from mdx mice sacrificed three months after NP-AON treatment, associated with well-maintained dystrophin expression in skeletal muscles, detectable by immunostaining and western blotting. We also administered by oral route NP-AON in mdx mice: rescued dystrophin protein is detectable in intestinal smooth muscle by immunofluorescence and western blot analysis. Nanoparticles labeled with IR-Dye (Li-COR Biosciences) were used to evaluate biodistribution in mdx mice injected I.P or orally, by using Odyssey Imager–Li-COR Biosciences. Twenty four hours after I.P injection the NPs are still in the peritoneal cavity. After longer time the peritoneal cavity becomes less fluorescent, while the fluorescence diffuses widely in all the body, especially in lymphatic tissues (spleen) suggesting a body distribution via lymphatic vessels. Fluorescence is detectable up to 22 days. The time course of NP-IR-Dye after oral administration demonstrates the persistence of NPs in intestinal lumen for at least 48 h. During this time some fluorescence is also visible outside from the intestine, as in the spleen. The Telethon Italy Grant GGPO9093 (to AF, PB, ML, MNM) is acknowledged.

Dystrophin is a subsarcolemmal protein critical for the integrity of muscle fibers by linking the actin cytoskeleton to the extracellular matrix via the dystroglycan complex (DGC). DGC also occurs at dermal–epidermal junction in skin. Here we report for the first time that epidermal melanocytes express dystrophin. Dystrophin full-length muscle isoform (mDp427) is clearly detectable in skin sections at RNA analysis. By immohistochemistry, dystrophin is selectively expressed at the basal layer of melanocytes where it co-localizes with basement membrane (BM) components. Dystrophin is absent in the epidermis of DMD patients, while dystroglycans and BM components were normally expressed. Interestingly, cultured DMD melanocytes display decreased adhesive capacity with respect to controls. Moreover, ultrastructural analysis of skin from DMD patients reveals melanocytes miss-localization, morphological nuclear heterogeneity and degenerative changes. Our study suggest that epidermal dystrophin acts in stabilizing melanocytes adhesion to the BM and that this function is impaired in DMD patients. Considering that melanocytes cultures can be easily obtained by conventional skin biopsy, they may represent a feasible and reliable cellular model for studying and monitoring dystrophinopathies.

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy are inherited muscle disorders caused by mutations of genes encoding the extracellular matrix protein collagen VI (ColVI). Mice lacking ColVI (Col6a1(-/-)) display a myopathic phenotype associated with ultrastructural alterations of mitochondria and sarcoplasmic reticulum, mitochondrial dysfunction with abnormal opening of the permeability transition pore (PTP) and increased apoptosis of muscle fibers. Treatment with cyclosporin (Cs) A, a drug that desensitizes the PTP by binding to cyclophilin (Cyp)-D, was shown to rescue myofiber alterations in Col6a1(-/-) mice and in UCMD patients, suggesting a correlation between PTP opening and pathogenesis of ColVI muscular dystrophies. Here, we show that inactivation of the gene encoding for Cyp-D rescues the disease phenotype of ColVI deficiency. In the absence of Cyp-D, Col6a1(-/-) mice show negligible myofiber degeneration, rescue from mitochondrial dysfunction and ultrastructural defects, and normalized incidence of apoptosis. These findings (i) demonstrate that lack of Cyp-D is equivalent to its inhibition with CsA at curing the mouse dystrophic phenotype; (ii) establish a cause-effect relationship between Cyp-D-dependent PTP regulation and pathogenesis of the ColVI muscular dystrophy and (iii) validate Cyp-D and the PTP as pharmacological targets for the therapy of human ColVI myopathies.

Ullrich congenital muscular dystrophy and Bethlem myopathy are skeletal muscle diseases due to mutations in the genes encoding collagen VI. Myoblasts from patients affected by Ullrich congenital muscular dystrophy display functional and ultrastructural mitochondrial alterations and increased apoptosis due to inappropriate opening of the permeability transition pore, a mitochondrial inner membrane channel. These alterations could be normalized by treatment with cyclosporin A, a widely used immunosuppressant that desensitizes the permeability transition pore independently of calcineurin inhibition. Here we report the results of an open pilot trial with cyclosporin A in five patients with collagen VI myopathies. Prior to treatment, all patients displayed mitochondrial dysfunction and increased frequency of apoptosis, as determined in muscle biopsies. Both these pathological signs were largely normalized after 1 month of oral cyclosporin A administration, which also increased muscle regeneration. We measured muscle strength with an hand-held myometer before and after treatment (hand grip, elbow flexion, knee flexion and extension bilaterally). The sum of the eight scores was recorded as megascore. In the three wheelchair bound patients with UCMD, aged 8.9–9.5 years, the megascore increased from 140 N to 192 N after 2 years of treatment. One of the two patients with BM was followed for 4 months only and her megascore moved from 652 N to 695 N. The megascore of the other BM patient changed from 863 N to 961 N after 2 years. In five untreated patients with BM the mean megascore during 3.4 years changed from 727 N to 623 N (−29 N per year). Treatment with CsA beyond the study period has shown in all the patients a favourable effect on muscle strength as measured with myometry.

Mutations of COL6A1-A3 genes, encoding the extracellular matrix protein collagen VI, cause muscular diseases, among which Bethlem Myopathy (BM) and Ullrich Congenital Muscular Dystrophy (UCMD) are the mostly described. Studies on the mouse model of these disorders (Col6a1−/−) and the patients affected by UCMD have shown an increased rate of spontaneous apoptosis, ultrastructural alterations in mitochondria and sarcoplasmic reticulum and a latent mitochondrial dysfunction in skeletal muscle. We demonstrated that these dysfunctions were caused by inappropriate opening of the permeability transition pore (PTP), a mitochondrial inner membrane channel that plays a role in several forms of cell death and can be desensitized by cyclosporin (Cs) A after binding to cyclophilin (Cyp) D. Indeed, the mitochondrial phenotype could be rescued both by treatment with CsA or its non-immunosuppressive derivative, Debio 025, and by genetic inactivation of CypD in the Col6a1−/− mice. Moreover, the treatment with CsA of UCMD patients led to rescue from mitochondrial dysfunction, normalization of apoptotic rates and increased muscle regeneration. Recent studies have shown that mitochondrial dysfunction is also present in mouse models of DMD and MDC1A, and in muscle-derived cells isolated from patients with LGMD2B. On the basis of all these data, we decided to investigate if mitochondria play a key pathogenic role in BM and in other muscular dystrophies, and to evaluate the role of the different muscle cell types (myoblasts versus fibroblasts) in the pathogenesis and the progression of the COL6 related diseases.

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2–AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2–AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as α-, β-, γ- and δ-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

Osteoporose, uma doença metabólica caracterizada por baixa massa óssea, deterioração da microarquitetura do tecido ósseo e aumento da suscetibilidade a fraturas, é comumente vista como um problema de saúde feminino. Essa visão tem fundamentos: em comparação com os homens as mulheres têm densidade mineral óssea menor, têm vida mais longa e perdem massa óssea mais rapidamente, principalmente após a menopausa, devido à diminuição acentuada dos níveis séricos de estrógeno. Entretanto, nos últimos 20 anos a osteoporose no homem tem sido reconhecida como um problema de saúde pública devido à ocorrência cada vez maior de fraturas por fragilidade. Cerca de 30% de todas as fraturas de quadril ocorrem em homens. Estudos recentes mostram que a probabilidade de fratura por fragilidade do quadril, vértebra ou punho em homens brancos após os 50 anos, pelo resto de suas vidas, situa‐se em torno de 13%, 40% nas mulheres. Os homens apresentam perda de massa óssea e fraturas mais tardiamente do que as mulheres. Embora os homens mais idosos tenham maior risco de fratura, cerca de metade das fraturas de quadril ocorre antes dos 80 anos. A expectativa de vida tem aumentado para ambos os sexos no Brasil e em todo o mundo, porém em uma velocidade maior para homens do que para mulheres. Esta Diretriz foi baseada em uma revisão sistemática da literatura com relação a prevalência, etiologia, diagnóstico e tratamento da osteoporose em homens.Osteoporosis, a metabolic disease characterized by low bone mass, deterioration of the bone tissue microarchitecture and increased susceptibility to fractures, is commonly regarded as a women's health problem. This point of view is based on the fact that compared with men, women have lower bone mineral density and longer lifespans and lose bone mass faster, especially after menopause, due to a marked decrease in serum estrogen levels. However, in the last 20 years, osteoporosis in men has become recognized as a public health problem due to the occurrence of an increasingly higher number of fragility fractures. Approximately 30% of all hip fractures occur in men. Recent studies show that the probability of fracture due to hip, vertebral or wrist fragility in Caucasian men older than fifty years, for the rest of their lives, is approximately 13% versus a 40% probability of fragility fractures in women. Men show bone mass loss and fractures later than women. Although older men have a higher risk of fracture, approximately half of all hip fractures occur before the age of 80. Life expectancy is increasing for both sexes in Brazil and worldwide, albeit at a higher rate for men than for women. This Guideline was based on a systematic review of the literature on the prevalence, etiology, diagnosis and treatment of osteoporosis in men.

Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects.We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.

The collagen VI related myopathies include Bethlem (BM) and Ullrich (UCMD) muscular dystrophies, a pure limb girdle presentation (LGMD) and the Myosclerosis Myopathy.