RESUMEN
The skeletal muscle has a very remarkable ability to regenerate upon injury under physiological conditions; however, this regenerative capacity is strongly diminished in physio-pathological conditions, such as those present in diseased or aged muscles. Many muscular dystrophies (MDs) are characterized by aberrant inflammation due to the deregulation of both the lymphoid and myeloid cell populations and the production of pro-inflammatory cytokines. Pathological inflammation is also observed in old muscles due to a systemic change in the immune system, known as "inflammaging". Immunomodulation represents, therefore, a promising therapeutic opportunity for different skeletal muscle conditions. However, the use of immunomodulatory drugs in the clinics presents several caveats, including their low stability in vivo, the need for high doses to obtain therapeutically relevant effects, and the presence of strong side effects. Within this context, the emerging field of nanomedicine provides the powerful tools needed to control the immune response. Nano-scale materials are currently being explored as biocarriers to release immunomodulatory agents in the damaged tissues, allowing therapeutic doses with limited off-target effects. In addition, the intrinsic immunomodulatory properties of some nanomaterials offer further opportunities for intervention that still need to be systematically explored. Here we exhaustively review the state-of-the-art regarding the use of nano-sized materials to modulate the aberrant immune response that characterizes some physio-pathological muscle conditions, such as MDs or sarcopenia (the age-dependent loss of muscle mass). Based on our learnings from cancer and immune tolerance induction, we also discuss further opportunities, challenges, and limitations of the emerging field of nano-immunomodulation.
Asunto(s)
Envejecimiento , Sarcopenia , Humanos , Anciano , Músculo Esquelético/patología , Sarcopenia/patología , Inflamación/patología , InmunidadRESUMEN
Only in recent years, thanks to a precision medicine-based approach, have treatments tailored to the sex of each patient emerged in clinical trials. In this regard, both striated muscle tissues present significant differences between the two sexes, which may have important consequences for diagnosis and therapy in aging and chronic illness. In fact, preservation of muscle mass in disease conditions correlates with survival; however, sex should be considered when protocols for the maintenance of muscle mass are designed. One obvious difference is that men have more muscle than women. Moreover, the two sexes differ in inflammation parameters, particularly in response to infection and disease. Therefore, unsurprisingly, men and women respond differently to therapies. In this review, we present an up-to-date overview on what is known about sex differences in skeletal muscle physiology and disfunction, such as disuse atrophy, age-related sarcopenia, and cachexia. In addition, we summarize sex differences in inflammation which may underly the aforementioned conditions because pro-inflammatory cytokines deeply affect muscle homeostasis. The comparison of these three conditions and their sex-related bases is interesting because different forms of muscle atrophy share common mechanisms; for instance, those responsible for protein dismantling are similar although differing in terms of kinetics, severity, and regulatory mechanisms. In pre-clinical research, exploring sexual dimorphism in disease conditions could highlight new efficacious treatments or recommend implementation of an existing one. Any protective factors discovered in one sex could be exploited to achieve lower morbidity, reduce the severity of the disease, or avoid mortality in the opposite sex. Thus, the understanding of sex-dependent responses to different forms of muscle atrophy and inflammation is of pivotal importance to design innovative, tailored, and efficient interventions.
Asunto(s)
Sarcopenia , Caracteres Sexuales , Femenino , Humanos , Masculino , Atrofia Muscular/metabolismo , Envejecimiento/metabolismo , Músculo Esquelético/metabolismo , Caquexia/metabolismo , Inflamación/metabolismo , Sarcopenia/metabolismoRESUMEN
We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion ex vivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, in vivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Animales , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , MicroARNs/genética , Músculo EsqueléticoRESUMEN
Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.
Asunto(s)
Diferenciación Celular/genética , Macrófagos/metabolismo , Distrofia Muscular de Duchenne/genética , Regeneración/genética , Animales , Linaje de la Célula/genética , Modelos Animales de Enfermedad , Distrofina/genética , Humanos , Macrófagos/patología , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Mioblastos/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/patologíaRESUMEN
Chronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described exercised mdx (ex mdx) mouse model characterised by accelerated heart pathology, and the specific PKCθ inhibitor Compound 20 (C20), to show that inhibition of this kinase leads to a significant reduction in the number of immune cells infiltrating the heart, as well as necrosis and fibrosis. Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathy.
Asunto(s)
Cardiomiopatías/tratamiento farmacológico , Corazón/efectos de los fármacos , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Proteína Quinasa C-theta/antagonistas & inhibidores , Animales , Cardiomiopatías/metabolismo , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Distrofina/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miocardio/metabolismo , FenotipoRESUMEN
Skeletal muscle, the most abundant tissue in the body, is heterogeneous. This heterogeneity forms the basis of muscle diversity, which is reflected in the specialized functions of muscles in different parts of the body. However, these different parts are not always clearly delimitated, and this often gives rise to gradients within the same muscle and even across the body. During the last decade, several studies on muscular disorders both in mice and in humans have observed particular distribution patterns of muscle weakness during disease, indicating that the same mutation can affect muscles differently. Moreover, these phenotypical differences reveal gradients of severity, existing alongside other architectural gradients. These two factors are especially prominent in sarcoglycanopathies. Nevertheless, very little is known about the mechanism(s) driving the phenotypic diversity of the muscles affected by these diseases. Here, we will review the available literature on sarcoglycanopathies, focusing on phenotypic differences among affected muscles and gradients, characterization techniques, molecular signatures, and cell population heterogeneity, highlighting the possibilities opened up by new technologies. This review aims to revive research interest in the diverse disease phenotype affecting different muscles, in order to pave the way for new therapeutic interventions.
Asunto(s)
Mutación , Sarcoglicanopatías/clasificación , Sarcoglicanopatías/patología , Sarcoglicanos/metabolismo , Animales , Humanos , Sarcoglicanopatías/metabolismo , Sarcoglicanos/genéticaRESUMEN
Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions.
Asunto(s)
Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Animales , Diferenciación Celular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mioblastos/metabolismo , Regeneración/fisiología , TranscriptomaRESUMEN
Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mdx mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in mdxθ-/- mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in mdxθ-/- muscle compared with mdx muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from mdxθ-/- muscle compared with mdx derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in mdxθ-/- muscle compared with mdx. The expression level of Delta1 and Jagged1 was also higher in PKCθ-/- muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.
Asunto(s)
Cardiotoxinas/efectos adversos , Músculo Esquelético/lesiones , Distrofia Muscular de Duchenne/genética , Proteína Quinasa C-theta/genética , Células Madre/citología , Animales , Diferenciación Celular , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Factor de Transcripción PAX7/metabolismo , Receptor Notch1/metabolismo , Regeneración , Transducción de Señal , Trasplante de Células Madre , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq-/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Diafragma/efectos de los fármacos , Distrofia Muscular Animal/prevención & control , Proteína Quinasa C-theta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T/efectos de los fármacos , Animales , Diafragma/enzimología , Diafragma/inmunología , Diafragma/patología , Modelos Animales de Enfermedad , Fibrosis , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Distrofia Muscular Animal/enzimología , Distrofia Muscular Animal/inmunología , Distrofia Muscular Animal/patología , Necrosis , Proteína Quinasa C-theta/deficiencia , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Linfocitos T/enzimología , Linfocitos T/inmunología , Factores de TiempoRESUMEN
Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.
Asunto(s)
Antígenos Ly/genética , Epigénesis Genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de la Membrana/genética , Células Musculares/efectos de los fármacos , Mioblastos Esqueléticos/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Compuestos de Vanadio/farmacología , Animales , Western Blotting , Línea Celular , Células Cultivadas , Expresión Génica/efectos de los fármacos , Ratones Desnudos , Ratones Transgénicos , Microscopía Fluorescente , Células Musculares/citología , Células Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/lesiones , Músculo Esquelético/fisiopatología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Regeneración/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.
Asunto(s)
Caveolina 1/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo , Rabdomiosarcoma/metabolismo , Animales , Caveolina 1/genética , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas de Unión al ARN/genética , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process.
Asunto(s)
Potenciales de Acción , Canales de Cloruro/metabolismo , Isoenzimas/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Fenotipo , Proteína Quinasa C/metabolismo , Animales , Calcineurina/genética , Calcineurina/metabolismo , Cloruros/metabolismo , Isoenzimas/genética , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Proteína Quinasa C/genética , Proteína Quinasa C-thetaRESUMEN
Protein kinase Cθ (PKCθ) is a member of the novel calcium-independent PKC family, with a relatively selective tissue distribution. Most studies have focused on its unique role in T-lymphocyte activation and suggest that inhibition of PKCθ could represent a novel therapeutic approach in the treatment of chronic inflammation, autoimmunity and allograft rejection. However, considering that PKCθ is also expressed in other cell types, including skeletal muscle cells, it is important to understand its function in different tissues before proposing it as a molecular target for the treatment of immune-mediated diseases. A number of studies have highlighted the role of PKCθ in mediating several intracellular pathways, regulating muscle cell development, homoeostasis and remodelling, although a comprehensive picture is still lacking. Moreover, we recently showed that lack of PKCθ in a mouse model of Duchenne muscular dystrophy (DMD) ameliorates the progression of the disease. In the present article, we review new developments in our understanding of the involvement of PKCθ in intracellular mechanisms regulating skeletal muscle development, growth and maintenance under physiological conditions and recent advances showing a hitherto unrecognized role of PKCθ in promoting muscular dystrophy.
Asunto(s)
Isoenzimas/metabolismo , Músculo Esquelético/enzimología , Enfermedades Musculares/enzimología , Proteína Quinasa C/metabolismo , Homeostasis , Humanos , Proteína Quinasa C-thetaRESUMEN
Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3ß activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.
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Estrés del Retículo Endoplásmico/fisiología , Transducción de Señal/fisiología , Animales , Autofagia/fisiología , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Privación de Alimentos/fisiología , Isoenzimas/metabolismo , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/fisiopatología , Mioblastos/metabolismo , Mioblastos/patología , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Proteínas Proto-Oncogénicas c-akt/fisiología , Tapsigargina/farmacologíaRESUMEN
Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.
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Caveolina 1/biosíntesis , Movimiento Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Línea Celular , Colagenasas/metabolismo , Fase G1/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase S/fisiología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Inflammatory memory or trained immunity is a recently described process in immune and non-immune tissue resident cells, whereby previous exposure to inflammation mediators leads to a faster and stronger responses upon secondary challenge. Whether previous muscle injury is associated with altered responses to subsequent injury by satellite cells (SCs), the muscle stem cells, is not known. METHODS: We used a mouse model of repeated muscle injury, in which intramuscular cardiotoxin (CTX) injections were administered 50 days apart in order to allow for full recovery of the injured muscle before the second injury. The effect of prior injury on the phenotype, proliferation and regenerative potential of satellite cells following a second injury was examined in vitro and in vivo by immunohistochemistry, RT-qPCR and histological analysis. RESULTS: We show that SCs isolated from muscle at 50 days post-injury (injury-experienced SCs (ieSCs)) enter the cell cycle faster and form bigger myotubes when cultured in vitro, compared to control SCs isolated from uninjured contralateral muscle. Injury-experienced SCs were characterized by the activation of the mTORC 1 signaling pathway, suggesting they are poised to activate sooner following a second injury. Consequently, upon second injury, SCs accumulate in greater numbers in muscle at 3 and 10 days after injury. These changes in SC phenotype and behavior were associated with accelerated muscle regeneration, as evidenced by an earlier appearance of bigger fibers and increased number of myonuclei per fiber at day 10 after the second injury. CONCLUSIONS: Overall, we show that skeletal muscle injury has a lasting effect on SC function priming them to respond faster to a subsequent injury. The ieSCs have long-term enhanced regenerative properties that contribute to accelerated regeneration following a secondary challenge.
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Lesiones de Repetición , Animales , Ratones , Fibras Musculares Esqueléticas , Músculo Esquelético , Ciclo Celular , División CelularRESUMEN
Sergio Adamo prematurely left us on January 7th 2022, just one year after his retirement, leaving his family, friends and colleagues deeply sad and grieving. Sergio was a full Professor of Histology and Embryology at the Sapienza University of Rome. Since the foundation of the Institute of Histology and Embryology more than 50 years ago, he dedicated himself to the institution, research, and teaching with integrity, generosity, and a great sense of teamwork. Sergio's main research interests have been the mechanisms of myogenesis, muscle homeostasis and regeneration under normal and pathological conditions. Most relevant results obtained by Sergio and his collaborators indicate novel functions for the neurohypophyseal hormones, vasopressin and oxytocin, upon striated muscle differentiation, trophism, and homeostasis. Here we like to give the proper tribute to a mentor, a colleague and a sincere friend. He left an indelible mark on the professional and personal lives of all of us and his absence provokes a profound sense of emptiness.
RESUMEN
Insights into the molecular and cellular biology of embryonal rhabdomyosarcoma (ERMS), an aggressive paediatric tumour, are required in order to identify new targets for novel treatments that may benefit patients with this disease. The present study examined the functional effects of MKK3 and MKK6, two upstream kinases of p38, and found that the ectopic expression of MKK6 led to rapid p38 activation and the myogenic differentiation of ERMS cells, whereas MKK3 failed to induce differentiation, while maintaining the proliferation state. Myogenin and myosin heavy chain were induced in MKK6overexpressing ERMS cells and were inhibited by the p38 inhibitor, SB203580. The expression of Myc and ERKPO4 increased under the effect of SB203580, whereas it decreased in MKK6overexpressing cells. AKT activation was part of the myogenic program triggered by MKK6 overexpression alone. To the best of our knowledge, the present study demonstrates, for the first time, that the endogenous MKK6 pathway may be recovered by MEK/ERK inhibition (U0126 and trametinib) and that it concomitantly induces the reversal of the oncogenic pattern and the induction of the myogenic differentiation of ERMS cell lines. The effects of MEK/ERK inhibitors markedly increase the potential clinical applications in ERMS, particularly on account of the MEK inhibitorinduced early MKK6/p38 axis activation and of their antioncogenic effects. The findings presented herein lend further support to the antitumour effects of MKK6; MKK6 may thus represent a novel target for advanced personalised treatments against ERMS.
Asunto(s)
Rabdomiosarcoma Embrionario , Diferenciación Celular , Línea Celular Tumoral , Niño , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Rabdomiosarcoma Embrionario/genética , Rabdomiosarcoma Embrionario/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a, and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC-derived cardiac differentiation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hidrazonas/farmacología , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/biosíntesis , Miocitos Cardíacos/metabolismo , Sulfonas/farmacología , Animales , Antígenos de Diferenciación/biosíntesis , Caspasa 3/metabolismo , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Regulación de la Expresión Génica/fisiología , Hidrazonas/síntesis química , Hipoglucemiantes/farmacología , Células Madre Pluripotentes Inducidas/citología , Ratones , Miocitos Cardíacos/citología , Sulfonas/síntesis química , Vanadatos/farmacologíaRESUMEN
Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient. Graphic abstract: Figure 1.Satellite cell isolation using the ice-cold treatment method.