Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration following peripheral nerve injury.

Following peripheral nerve injury, successful axonal growth and functional recovery require Schwann cell (SC) reprogramming into a reparative phenotype, a process dependent upon c-Jun transcription factor activation. Unfortunately, axonal regeneration is greatly impaired in aged organisms and following chronic denervation, which can lead to poor clinical outcomes. While diminished c-Jun expression in SCs has been associated with regenerative failure, it is unclear whether the inability to maintain a repair state is associated with the transition into an axonal growth inhibition phenotype. We here find that reparative SCs transition into a senescent phenotype, characterized by diminished c-Jun expression and secretion of inhibitory factors for axonal regeneration in aging and chronic denervation. In both conditions, the elimination of senescent SCs by systemic senolytic drug treatment or genetic targeting improved nerve regeneration and functional recovery, increased c-Jun expression and decreased nerve inflammation. This work provides the first characterization of senescent SCs and their influence on axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration and functional recovery after peripheral nerve injuries.

Denervation Drives YAP/TAZ Activation in Muscular Fibro/Adipogenic Progenitors.

Loss of motoneuron innervation (denervation) is a hallmark of neurodegeneration and aging of the skeletal muscle. Denervation induces fibrosis, a response attributed to the activation and expansion of resident fibro/adipogenic progenitors (FAPs), i.e., multipotent stromal cells with myofibroblast potential. Using in vivo and in silico approaches, we revealed FAPs as a novel cell population that activates the transcriptional coregulators YAP/TAZ in response to skeletal muscle denervation. Here, we found that denervation induces the expression and transcriptional activity of YAP/TAZ in whole muscle lysates. Using the PdgfraH2B:EGFP/+ transgenic reporter mice to trace FAPs, we demonstrated that denervation leads to increased YAP expression that accumulates within FAPs nuclei. Consistently, re-analysis of published single-nucleus RNA sequencing (snRNA-seq) data indicates that FAPs from denervated muscles have a higher YAP/TAZ signature level than control FAPs. Thus, our work provides the foundations to address the functional role of YAP/TAZ in FAPs in a neurogenic pathological context, which could be applied to develop novel therapeutic approaches for the treatment of muscle disorders triggered by motoneuron degeneration.

Episodic Binge-like Ethanol Reduces Skeletal Muscle Strength Associated with Atrophy, Fibrosis, and Inflammation in Young Rats.

Binge Drinking (BD) corresponds to episodes of ingestion of large amounts of ethanol in a short time, typically ≤2 h. BD occurs across all populations, but young and sports-related people are especially vulnerable. However, the short- and long-term effects of episodic BD on skeletal muscle function have been poorly explored. Young rats were randomized into two groups: control and episodic Binge-Like ethanol protocol (BEP) (ethanol 3 g/kg IP, 4 episodes of 2-days ON-2-days OFF paradigm). Muscle function was evaluated two weeks after the last BEP episode. We found that rats exposed to BEP presented decreased muscle strength and increased fatigability, compared with control animals. Furthermore, we observed that skeletal muscle from rats exposed to BEP presented muscle atrophy, evidenced by reduced fiber size and increased expression of atrophic genes. We also observed that BEP induced fibrotic and inflammation markers, accompanied by mislocalization of nNOSµ and high levels of protein nitration. Our findings suggest that episodic binge-like ethanol exposure alters contractile capacity and increases fatigue by mechanisms involving atrophy, fibrosis, and inflammation, which remain for at least two weeks after ethanol clearance. These pathological features are common to several neuromuscular diseases and might affect muscle performance and health in the long term.

The functional and molecular effects of problematic alcohol consumption on skeletal muscle: a focus on athletic performance.

Background: Chronic alcohol misuse is associated with alcoholic myopathy, characterized by skeletal muscle weakness and atrophy. Moreover, there is evidence that sports-related people seem to exhibit a greater prevalence of problematic alcohol consumption, especially binge drinking (BD), which might not cause alcoholic myopathy but can negatively impact muscle function and amateur and professional athletic performance.Objective: To review the literature concerning the effects of alcohol consumption on skeletal muscle function and structure that can affect muscle performance.Methodology: We examined the currently available literature (PubMed, Google Scholars) to develop a narrative review summarizing the knowledge about the effects of alcohol on skeletal muscle function and exercise performance, obtained from studies in human beings and animal models for problematic alcohol consumption.Results: Exercise- and sport-based studies indicate that alcohol consumption can negatively affect muscle recovery after vigorous exercise, especially in men, while women seem less affected. Clinical studies and pre-clinical laboratory research have led to the knowledge of some of the mechanisms involved in alcohol-related muscle dysfunction, including an imbalance between anabolic and catabolic pathways, reduced regeneration, increased inflammation and fibrosis, and deficiencies in energetic balance and mitochondrial function. These pathological features can appear not only under chronic alcohol misuse but also in other alcohol consumption patterns.Conclusions: Most laboratory-based studies use chronic or acute alcohol exposure, while episodic BD, the most common drinking pattern in amateur and professional athletes, is underrepresented. Nevertheless, alcohol consumption negatively affects skeletal muscle health through different mechanisms, which collectively might contribute to reduced sports performance.

Driving fibrosis in neuromuscular diseases: Role and regulation of Connective tissue growth factor (CCN2/CTGF).

Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.

Role of Matricellular CCN Proteins in Skeletal Muscle: Focus on CCN2/CTGF and Its Regulation by Vasoactive Peptides.

The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins' role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides.

Nervio occipital mayor: trayecto, relaciones anatómicas e implicancias clínicas de sus posibles sitios de atrapamiento / Great occipital nerve: course, anatomical relations and clinical implications of their potential entrapment sites

RESUMEN: El nervio occipital mayor (NOM) se forma del ramo dorsal del nervio espinal C2 y asciende entre la musculatura cervical posterior para inervar la piel del cuero cabelludo. Diversos autores han descrito su recorrido, sin embargo, es escasa la información referente a la relación que presenta este nervio con el músculo oblicuo inferior de la cabeza (OIC) y su trayecto intramuscular. El objetivo de este estudio fue determinar el recorrido y relaciones que el NOM estableció en el intervalo existente entre los músculos OIC y músculo trapecio (T). Para ello, se midieron las distancias verticales y horizontales a la altura de la protuberancia occipital externa y línea mediana, y se dividió al músculo OIC en tercios para observar variaciones del recorrido de este nervio. Junto con medir el diámetro del NOM, se midieron las distancias vertical y horizontal de este nervio a través de cinco puntos de referencia muscular y un punto de referencia vascular. Estos puntos musculares fueron: a) sobre el vientre del músculo OIC (punto 1); b) en la cara profunda del músculo semiespinoso de la cabeza (SEC) (punto 2); c) en la cara superficial del músculo SEC (punto 3); d) en la cara profunda del músculo T (punto 4); y e) en la cara superficial del músculo T (punto 5). A este se sumó el punto 6, en el cual se establecieron las distancias vertical y horizontal con la arteria occipital a la altura de la cara superficial del músculo T. Para ello se disecaron 18 cabezas (36 triángulos suboccipitales) de cadáveres adultos brasileños pertenecientes al laboratorio de Anatomía de la Universidade Federal de Alagoas (UFAL), Maceió, Brasil. Las distancias verticales y horizontales obtenidas respecto de los seis puntos fueron: 63,67 y 27,15 mm (punto 1); 53,89 y 21,44 mm (punto 2); 30,61 y 14,49 mm (punto 3); 20,39 y 22,8 mm (punto 4); 5,86 y 33,46 mm (punto 5); 5,99 y 35,56 mm (punto 6), respectivamente. En relación al músculo OIC, el NOM se ubicó en un 72,22 % de las muestras en el tercio medio de este músculo, 19,44% en su tercio lateral y un 8,33 % en su tercio medial. Todos estos hallazgos deben ser considerados al momento de diagnosticar correctamente posibles atrapamientos del NOM en la región cervical profunda, siendo además, una contribución para el éxito de procedimientos quirúrgicos de esta región.

SUMMARY: The great occipital nerve (GON) is formed from the dorsal branch of the C2 spinal nerve and ascends between the posterior cervical musculature to innervate the skin of the scalp. Various authors have described its course, however, there is little information regarding the relationship that this nerve presents with the obliquus capitis inferior (OCI) and its intramuscular path. The objective of this study was to determine the route and relationships that the GON established in the interval between the OCI muscles and the trapezius muscle (T). For this, the vertical and horizontal distances were measured at the height of the external occipital protuberance and median line, and the OCI muscle was divided into thirds to observe variations in the path of this nerve. Along with measuring the diameter of the GON, the vertical and horizontal distances of this nerve were measured through five muscle reference points and one vascular reference point. These muscle points were: a) on the belly of the OCI muscle (point 1); b) in the deep face of the semispinalis capitis muscle (SCM) (point 2); c) on the surface of the SCM (point 3); d) on the deep face of the T (point 4); and e) on the surface face of the T (point 5). To this was added point 6, in which the vertical and horizontal distances were established with the occipital artery at the height of the superficial face of the T. For this, 18 heads (36 suboccipital triangles) of Brazilian adult corpses belonging to the Anatomy laboratory of the Universidade Federal de Alagoas (UFAL), Maceió, Brazil, were dissected. The vertical and horizontal distances obtained with respect to the six points were: 63.67 and 27.15 mm (point 1); 53.89 and 21.44 mm (point 2); 30.61 and 14.49 mm (point 3); 20.39 and 22.8 mm (point 4); 5.86 and 33.46 mm (point 5); 5.99 and 35.56 mm (point 6), respectively. In relation to the OCI, the GON was located in 72.22 % of the samples in the middle third of this muscle, 19.44 % in its lateral third and 8.33 % in its medial third. All these findings should be considered when correctly diagnosing possible entrapments of GON in the deep cervical region, being a contribution to the success of surgical procedures in this region.

HIF-hypoxia signaling in skeletal muscle physiology and fibrosis.

Hypoxia refers to the decrease in oxygen tension in the tissues, and the central effector of the hypoxic response is the transcription factor Hypoxia-Inducible Factor α (HIF1-α). Transient hypoxia in acute events, such as exercising or regeneration after damage, play an important role in skeletal muscle physiology and homeostasis. However, sustained activation of hypoxic signaling is a feature of skeletal muscle injury and disease, which can be a consequence of chronic damage but can also increase the severity of the pathology and worsen its outcome. Here, we review evidence that supports the idea that hypoxia and HIF-1α can contribute to the establishment of fibrosis in skeletal muscle through its crosstalk with other profibrotic factors, such as Transforming growth factor ß (TGF-ß), the induction of profibrotic cytokines expression, as is the case of Connective Tissue Growth Factor (CTGF/CCN2), or being the target of the Renin-angiotensin system (RAS).

Role of hypoxia in skeletal muscle fibrosis: Synergism between hypoxia and TGF-ß signaling upregulates CCN2/CTGF expression specifically in muscle fibers.

Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-ß on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-ß1 under hypoxic conditions. Hypoxia and TGF-ß1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-ß signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-ß1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-ß signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.

Denervation-induced skeletal muscle fibrosis is mediated by CTGF/CCN2 independently of TGF-ß.

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type ß (TGF-ß) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-ß signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-ß signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-ß signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.

Effect of Alcohol on Hippocampal-Dependent Plasticity and Behavior: Role of Glutamatergic Synaptic Transmission.

Problematic alcohol drinking and alcohol dependence are an increasing health problem worldwide. Alcohol abuse is responsible for approximately 5% of the total deaths in the world, but addictive consumption of it has a substantial impact on neurological and memory disabilities throughout the population. One of the better-studied brain areas involved in cognitive functions is the hippocampus, which is also an essential brain region targeted by ethanol. Accumulated evidence in several rodent models has shown that ethanol treatment produces cognitive impairment in hippocampal-dependent tasks. These adverse effects may be related to the fact that ethanol impairs the cellular and synaptic plasticity mechanisms, including adverse changes in neuronal morphology, spine architecture, neuronal communication, and finally an increase in neuronal death. There is evidence that the damage that occurs in the different brain structures is varied according to the stage of development during which the subjects are exposed to ethanol, and even much earlier exposure to it would cause damage in the adult stage. Studies on the cellular and cognitive deficiencies produced by alcohol in the brain are needed in order to search for new strategies to reduce alcohol neuronal toxicity and to understand its consequences on memory and cognitive performance with emphasis on the crucial stages of development, including prenatal events to adulthood.

The inhibition of CTGF/CCN2 activity improves muscle and locomotor function in a murine ALS model.

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset progressive neurodegenerative disease characterized by upper and lower motoneuron degeneration. A total of 20% of familial ALS (fALS) cases are explained by mutations in the superoxide dismutase 1 (SOD1) enzyme. Although more than 20 years have passed since the generation of the first ALS mouse model, the precise molecular mechanisms of ALS pathogenesis remain unknown. CTGF/CCN2 is a matricellular protein with associated fibrotic activity that is up-regulated in several chronic diseases. The inhibition of CTGF/CCN2 with the monoclonal neutralizing antibody FG-3019 reduces fibrosis in several chronic disorders including the mdx mice, a murine model for Duchenne muscular dystrophy (DMD). In this work, we show that there are increased levels of CTGF/CCN2 in skeletal muscle and spinal cord of hSOD1G93A mice. In this scenario, we show evidence that FG-3019 not only reduces fibrosis in skeletal muscle of hSOD1G93A mice, but also improves muscle and locomotor performance. We demonstrate that treatment with FG-3019 reduces muscle atrophy in hSOD1G93A mice. We also found improvement of neuromuscular junction (NMJ) innervation together with a reduction in myelin degeneration in the sciatic nerve, suggesting that alterations in nerve-muscle communication are partially improved in FG-3019-treated hSOD1G93A mice. Moreover, we also found that CTGF/CCN2 is expressed in astrocytes and neurons, predominantly in dorsal areas of spinal cord from symptomatic hSOD1G93A mice. Together, these results reveal that CTGF/CCN2 might be a novel therapeutic target to ameliorate symptoms and improve the quality of life of ALS patients.

Fatiga postparto: revisión de la literatura / Postpartum fatigue: review of the literature

RESUMEN En el postparto surgen una serie de cambios físicos y psicosociales los que convierten esta etapa en un momento de mayor vulnerabilidad a problemas de salud para las mujeres. Uno de estos cambios es en la calidad y cantidad de sueño nocturno, el que trae consigo la fatiga postparto. En todos los estudios, las características propias de la fatiga en el postparto son asociadas a la enfermedad mental, dejando invisibilizada su relación con el bienestar de la mujer durante este periodo. El objetivo de este estudio es realizar una actualización en fatiga relacionada a la salud mental de la mujer durante el periodo postparto. Método: Se revisaron 34 estudios en inglés, publicados en los últimos cinco años, en PubMed y Web of Sciencie. Resultados: Los estudios demuestran que entre 60% a 65% de las mujeres durante el postparto tiene un sueño de corta duración, durmiendo menos de 6 horas, refiriendo la fatiga como uno de los síntomas más serios después del parto. Diversos estudios han confirmado la relación entre fatiga y depresión postparto, incluso algunos destacan la relación bidireccional entre ambas, donde la fatiga predice síntomas depresivos y viceversa. La fatiga, además, se ha relacionado a alteraciones en el funcionamiento diurno de la mujer, peso materno y construcción del vínculo madre-hijo. Conclusión: La presente revisión reafirma la necesidad de profundizar en los efectos de la fatiga postparto en el bienestar materno y de esta forma, realizar intervenciones que potencien la salud mental positiva durante el periodo postparto.

ABSTRACT A series of physical and psychosocial changes arise in the postpartum. This make a moment of great vulnerability for different problems for women. One of these changes is in the quality and quantity of nocturnal sleep, which brings a postpartum fatigue. In all studies, the characteristics of fatigue in the postpartum period are associated with mental illness, and not with women wellbeing during this period. The objective of this study is to have an update on fatigue related to the mental health of women during the postpartum period. Method: We reviewed 34 studies in English, published in the last five years, in PubMed and Web of Science. Results: Studies show that between 60% to 65% of women during postpartum have a short sleep time, they slept less than 6 hours. This show us fatigue as one of the most serious symptoms after childbirth. Several studies have confirmed the relationship between fatigue and postpartum depression, where even some highlight the bidirectional relationship between them, where fatigue predicts depressive symptoms and vice versa. Moreover, fatigue has been related to alterations in the diurnal functioning of the woman, maternal weight and construction of the mother-child bond. Conclusion: This review reaffirms the need to deepen the effects of postpartum fatigue on maternal wellbeing and, in this way, to carry out interventions that promote positive mental health during the postpartum period.

ALS skeletal muscle shows enhanced TGF-ß signaling, fibrosis and induction of fibro/adipogenic progenitor markers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motoneurons degenerate leading to muscle wasting, paralysis and eventually death from respiratory failure. Several studies indicate that skeletal muscle contributes to disease progression; however the molecular mechanisms remain elusive. Fibrosis is a common feature in skeletal muscle under chronic damage conditions such as those caused by muscular dystrophies or denervation. However, the exact mechanisms of fibrosis induction and the cellular bases of this pathological response are unknown. We show that extracellular matrix (ECM) components are augmented in skeletal muscles of symptomatic hSOD1G93A mice, a widely used murine model of ALS. These mice also show increased TGF-ß1 mRNA levels, total Smad3 protein levels and p-Smad3 positive nuclei. Furthermore, platelet-derived growth factor receptor-α (PDGFRα), Tcf4 and α-smooth muscle actin (α-SMA) levels are augmented in the skeletal muscle of symptomatic hSOD1G93A mice. Additionally, the fibro/adipogenic progenitors (FAPs), which are the main producers of ECM constituents, are also increased in these pathogenic conditions. Therefore, FAPs and ECM components are more abundant in symptomatic stages of the disease than in pre-symptomatic stages. We present evidence that fibrosis observed in skeletal muscle of symptomatic hSOD1G93A mice is accompanied with an induction of TGF-ß signaling, and also that FAPs might be involved in triggering a fibrotic response. Co-localization of p-Smad3 positive cells together with PDGFRα was observed in the interstitial cells of skeletal muscles from symptomatic hSOD1G93A mice. Finally, the targeting of pro-fibrotic factors such as TGF-ß, CTGF/CCN2 and platelet-derived growth factor (PDGF) signaling pathway might be a suitable therapeutic approach to improve muscle function in several degenerative diseases.

Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis.

Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.

Sarcolemmal targeting of nNOSµ improves contractile function of mdx muscle.

Nitric oxide (NO) is a key regulator of skeletal muscle function and metabolism, including vasoregulation, mitochondrial function, glucose uptake, fatigue and excitation-contraction coupling. The main generator of NO in skeletal muscle is the muscle-specific form of neuronal nitric oxide synthase (nNOSµ) produced by the NOS1 gene. Skeletal muscle nNOSµ is predominantly localized at the sarcolemma by interaction with the dystrophin protein complex (DPC). In Duchenne muscular dystrophy (DMD), loss of dystrophin leads to the mislocalization of nNOSµ from the sarcolemma to the cytosol. This perturbation has been shown to impair contractile function and cause muscle fatigue in dystrophic (mdx) mice. Here, we investigated the effect of restoring sarcolemmal nNOSµ on muscle contractile function in mdx mice. To achieve this, we designed a modified form of nNOSµ (NOS-M) that is targeted to the sarcolemma by palmitoylation, even in the absence of the DPC. When expressed specifically in mdx skeletal muscle, NOS-M significantly attenuates force loss owing to damaging eccentric contractions and repetitive isometric contractions (fatigue), while also improving force recovery after fatigue. Expression of unmodified nNOSµ at similar levels does not lead to sarcolemmal association and fails to improve muscle function. Aside from the benefits of sarcolemmal-localized NO production, NOS-M also increased the surface membrane levels of utrophin and other DPC proteins, including ß-dystroglycan, α-syntrophin and α-dystrobrevin in mdx muscle. These results suggest that the expression of NOS-M in skeletal muscle may be therapeutically beneficial in DMD and other muscle diseases characterized by the loss of nNOSµ from the sarcolemma.

ACE2 is augmented in dystrophic skeletal muscle and plays a role in decreasing associated fibrosis.

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.

Copper reduces Aß oligomeric species and ameliorates neuromuscular synaptic defects in a C. elegans model of inclusion body myositis.

Alzheimer's disease and inclusion body myositis (IBM) are disorders frequently found in the elderly and characterized by the presence of amyloid-ß peptide (Aß) aggregates. We used Caenorhabditis elegans that express Aß in muscle cells as a model of IBM, with the aim of analyzing Aß-induced muscle pathology and evaluating the consequences of modulating Aß aggregation. First, we tested whether the altered motility we observed in the Aß transgenic strain could be the result of a compromised neuromuscular synapse. Our pharmacological analyses show that synaptic transmission is defective in our model and suggest a specific defect on nicotine-sensitive acetylcholine receptors (AChRs). Through GFP-coupled protein visualization, we found that synaptic dysfunction correlates with mislocalization of ACR-16, the AChR subunit essential for nicotine-triggered currents. Histological and biochemical analysis allowed us to determine that copper treatment increases the amyloid deposits and decreases Aß oligomers in this model. Furthermore, copper treatment improves motility, ACR-16 localization, and synaptic function and delays Aß-induced paralysis. Our results indicate that copper modulates Aß-induced pathology and suggest that Aß oligomers are triggering neuromuscular dysfunction. Our findings emphasize the importance of neuromuscular synaptic dysfunction and the relevance of modulating the amyloidogenic component as an alternative therapeutic approach for this debilitating disease.

Intracellular amyloid formation in muscle cells of Abeta-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification.

BACKGROUND: The amyloid beta-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Abeta aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Abeta is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Abeta is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. RESULTS: In the present work, we found that intracellular Abeta aggregation in muscle cells of Caenorhabditis elegans overexpressing Abeta peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type Abeta. We show that intracellular amyloid aggregation of wild type Abeta is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Abeta-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. CONCLUSION: Our data show that intracellular Abeta amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Abeta aggregation may be part of a cell protective mechanism.

Inclusion body myositis: a view from the Caenorhabditis elegans muscle.

Inclusion body myositis (IBM) is the most common myopathy in people over 50 years of age. It involves an inflammatory process that, paradoxically, does not respond to anti-inflammatory drugs. A key feature of IBM is the presence of amyloid-beta-peptide aggregates called amyloid deposits, which are also characteristic of Alzheimer's disease. The use of animals that mimic at least some characteristics of a disease has become very important in the quest to elucidate the molecular mechanisms underlying this and other pathogeneses. Although there are some transgenic mouse strains that recreate some aspects of IBM, in this review, we hypothesize that the great degree of similarity between nematode and human genes known to be involved in IBM as well as the considerable conservation of biological mechanisms across species is an important feature that must be taken into consideration when deciding on the use of this nematode as a model. Straightforward laboratory techniques (culture, transformation, gene knockdown, genetic screenings, etc.) as well as anatomical, physiological, and behavioral characteristics add to the value of this model. In the present work, we review evidence that supports the use of Caenorhabditis elegans as a biological model for IBM.