Testosterone induces up-regulation of mitochondrial gene expression in murine C2C12 skeletal muscle cells accompanied by an increase of nuclear respiratory factor-1 and its downstream effectors

The reduction in muscle mass and strength with age, sarcopenia, is a prevalent condition among the elderly, linked to skeletal muscle dysfunction and cell apoptosis. We demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells. Here, we analyzed the effect of testosterone on mitochondrial gene expression in C2C12 skeletal muscle cells. We found that testosterone increases mRNA expression of genes encoded by mitochondrial DNA, such as NADPH dehydrogenase subunit 1 (ND1), subunit 4 (ND4), cytochrome b (CytB), cytochrome c oxidase subunit 1 (Cox1) and subunit 2 (Cox2) in C2C12. Additionally, the hormone induced the expression of the nuclear respiratory factors 1 and 2 (Nrf-1 and Nrf-2), the mitochondrial transcription factors A (Tfam) and B2 (TFB2M), and the optic atrophy 1 (OPA1). The simultaneous treatment with testosterone and the androgen receptor antagonist, Flutamide, reduced these effects. H2O2-oxidative stress induced treatment, significantly decreased mitochondrial gene expression. Computational analysis revealed that mitochondrial DNA contains specific sequences, which the androgen receptor could recognize and bind, probably taking place a direct regulation of mitochondrial transcription by the receptor. These findings indicate that androgen plays an important role in the regulation of mitochondrial transcription and biogenesis in skeletal muscle.Fil: Pronsato, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Estradiol exerts antiapoptotic effects in skeletal myoblasts via mitochondrial PTP and MnSOD.

17beta-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. We have shown that E2 at physiological concentrations prevented apoptosis induced by H2O2 in C2C12 skeletal myoblasts. As we also demonstrated the presence of estrogen receptors in mitochondria, the present work was focused on the effects of E2 on this organelle. Specifically, we evaluated the actions of E2 on the mitochondrial permeability transition pore (MPTP) by the calcein-acetoxymethylester/cobalt method using fluorescence microscopy and flow cytometry. Pretreatment with E2 prevented MPTP opening induced by H2O2, which preceded loss of mitochondrial membrane potential. In addition, it was observed that H2O2 induced translocation of Bax to mitochondria; however, in the presence of the steroid this effect was abrogated suggesting that members of the Bcl-2 family may be regulated by E2 to exert an antiapoptotic effect. Moreover, E2 increased mitochondrial manganese superoxide dismutase protein expression and activity, as part of a mechanism activated by E2 that improved mitochondrial performance. Our results suggest a role of E2 in the regulation of apoptosis with a clear action at the mitochondrial level in C2C12 skeletal myoblast cells.Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Oxidative Stress in Muscle Diseases: Current and Future Therapy 2019

Increased oxidative stress has important molecular, structural, and functional muscle implications. In pathological conditions, reactive oxygen species (ROS) burst contributes to cellular dysfunction and the progression of muscle diseases. This special issue was designed to advance knowledge in the role of oxidative stress on muscle remodeling, in turn leading to innovative therapeutic approaches in a wide range of muscle diseases. Therefore, this special issue provides recent scientific advancements with researchers and practitioners who work in muscle scope. Articles included in this special issue address the molecular and cellular mechanisms involved in these processes as well as current therapies.Fil: Serra, Andrey. Universidade de Sao Paulo. Departamento de Fisiología; BrasilFil: Pinto, José Renato. Florida State University; Estados UnidosFil: Prokić, Marko. University of Belgrade, Belgrade; SerbiaFil: Arsa, Gisela. Universidade Federal do Mato Grosso do Sul; BrasilFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Non-polar extracts of Nicotiana glauca (Solanaceae) induce apoptosis in human rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common soft-tissue tumour in children andadolescents. It originates in normal skeletal muscle from myogenic cells that have failed tofully differentiate, and it usually has a poor prognosis. Current RMS therapy has manyadverse effects. Hence, new treatments are needed. Various pharmacological properties,such as analgesic, antineoplastic, antimicrobial, and antiparasitic properties, have beendemonstrated in species of the Solanaceae family. We performed ethanolic extraction fromleaves of Nicotiana glauca (Solanaceae), and the extract was successively partitioned withn-hexane, chloroform, and ethyl acetate. We evaluated the effects of extracts on RMScells, and we found that the extracts trigger apoptosis. By bio-guided fractionation assays,we identified the apoptotic agents. Morphological assessment after apoptotic cell inductionof cultured cells, mitochondrial and nuclear morphology by Mitotracker, and 4,6-diamidino-2-phenylindole (DAPI) staining, respectively, were analysed in fluorescentmicroscopy. The capacity of the cells to migrate or proliferate was analysed by the Petitassay, followed by methylene blue staining. NMR and GC-MS spectrometry were used toidentify palmitic acid and scopoletin as the phytochemicals responsible for the observedeffects. These results indicate that these compounds are apoptotic inducers and they couldbe useful as chemotherapeutic agents against muscle tumours.Rabdomiossarcoma (RMS) é o tumor de tecidos moles mais comum em crianças e adolescentes. Ele se origina no músculo esquelético normal a partir de las células miogênicas que no conseguiram-se diferenciar completamente e pelo general tem um prognóstico ruim. A terapia atual com RMS tem muitos efeitos adversos e portanto, novos tratamentos são necessários. Várias propriedades farmacológicas, como propriedades analgésica, antineoplásica, antimicrobiana e antiparasitária, foram demostradas em espécies da família Solanaceae. Neste trabalho, foi realizada a extração etanólica das folhas de Nicotiana glauca (Solanaceae), eo extrato foi particionado sucessivamente com n-hexano, clorofórmio y acetato de etila. Avaliamos os efeitos dos extratos nas células RMS e descobrimos que os extratos desencadeiam apoptose. Pelos ensaios de fracionamento bio-guiado, identificamos os agentes apoptóticos. Avaliação morfológica após indução da apoptose das células cultivadas ea morfologia mitocondrial e nuclear por coloração com Mitotracker e 4,6-diamidino-2-fenilindol (DAPI), respectivamente, foram analisadas com microscopia fluorescente. A capacidade das células para migrar ou proliferar foi analisada hair ensaio Petit, seguido pelacoloração com azul de metileno. Una espectrometría de RMN y GC-MS para uso utilizado para una identificación de ácido palmítico y una escopoletina como fitoquímicos responsáveis ​​pelos efeitos observados.Fil: Musso, Florencia Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Pronsato, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Milanesi, Lorena Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Faraoni, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell Morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.Fil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Ronda, Ana Carolina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Cátedra de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina

El 17β-Estradiol y la Testosterona protegen a las mitocondrias contra el estrés oxidativo en Células del Músculo Esquelético

En trabajos previos demostramos que la testosterona (T) y el 17β-estradiol (E2) protegen a las células musculares C2C12 de la apoptosis inducida por peróxido de hidrógeno (H2O2). Conjuntamente evidenciamos la existencia de receptores de estrógenos y andrógenos en las mitocondrias. El presente trabajo se ha centrado en caracterizar los efectos de ambos esteroides en esta organela, que conducen a la supervivencia celular. Específicamente, se evaluaron las acciones de T y E2 sobre el potencial de membrana mitocondrial con el colorante JC-1 y sobre el poro de permeabilidad transitoria mitocondrial (MPTP) mediante el método de calcein-acetoxymethylester/cobalt, utilizando microscopía de fluorescencia y citometría de flujo. Demostramos que T y E2 previenen la apertura del MPTP y la pérdida de potencial de membrana mitocondrial inducidas por H2O2. Además, observamos que el H2O2 aumenta los niveles de expresión proteica del canal aniónico dependiente de voltaje (VDAC) e induce la translocación de Bax a mitocondria. Sin embargo, en presencia de las hormonas la translocación de Bax fue inhibida lo cual sugiere que los miembros de la familia Bcl -2 pueden ser regulados por E2 y T. Los eventos moleculares desencadenados por E2 y T a nivel mitocondrial se reflejaron en la morfología de las organelas. El análisis microscópico de las células C2C12 y cultivos primarios de músculo esquelético de ratón, mediante tinciones con verde de Jano y Mitotracker reveló un efecto protector de los esteroides contra el daño por estrés oxidativo inhibiendo la redistribución y picnosis mitocondrial.We have previously shown that testosterone (T) and 17β-estradiol (E2) protect C2C12 muscle cells against apoptosis induced by hydrogen peroxide (H2O2). Since we also showed the presence of estrogen and androgen Receptors in mitochondria, this work was focused on the effects of both steroids on this organelle, which result in cellular survival. Specifically, we evaluated the actions of T and E2 on the mitochondrial membrane potential with JC-1 dye and on the mitochondrial permeability transition pore (MPTP) by the calceinacetoxymethylester (AM)/cobalt method, using fluorescence microscopy and flow cytometry. We demonstrated that T and E2 prevent MPTP opening and the loss of mitochondrial membrane potential induced by H2O2. In addition, it was observed that H2O2 increase voltage-dependent anion channel (VDAC) protein expression levels and induce translocation of Bax to mitochondria. However, in the presence of the steroids Bax translocation was abrogated suggesting that members of the Bcl-2 family may be regulated by E2 and T. The observed effects triggered by E2 and T were reflected on mitochondrial morphology. Microscopic analysis of C2C12 cells and primary cultures of mouse skeletal muscle, with Janus Green and Mitotracker staining revealed a protective effect of the steroids against oxidative stress damage which included mitochondrial redistribution and pyknosis of the organelle.Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Ronda, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto Argentino de Oceanografía (i); Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentin

Apoptosis As Cause Of Sarcopenia: Hormonal Regulation

The muscle mass declines with age, affecting the independence in theelderly. This process known as sarcopenia and the acceleration of myocytes lossthrough apoptosis might signify the main causative.Skeletal muscle tissue increases its size and shows a notable capacity to adapt to injury,due the existence of an undifferentiated group of myogenic-specific precursor cells,called satellite cells. The knowledge of mechanism that driven the apoptosis in satellitecells represents an important base to understand the etiology of sarcopenia.This chapter centers on the potential impact that the estrogen- and testosteroneregulationof satellite cell function has in elderly skeletal muscle, highlighting in therole that both steroids have on apoptotic signaling in myoblasts.Fil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Role of Sex Hormones in Cytoskeletal Structure: Implications in Cellular Lifespan

The cytoskeleton is composed of intracellular structures that maintain cellshape, interconnect organelles to each other, often attached to the cell membrane and isinvolved in signaling pathways. Sex steroids are effective regulators of cellmorphology and recent evidence indicates that it is obtained through the regulation ofthe actin cytoskeleton. Intriguingly, evidence implicates the actin cytoskeleton as botha sensor and mediator of apoptosis. Since it has been shown that sex hormonesaffect/regulate the cellular cytoskeleton, the aim of the present chapter is to discuss themolecular mechanism and targets, related to cytoskeleton, where sex steroids act andmodulate the apoptotic process and in consequence the cellular lifespan.Fil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

General Concepts of Skeletal Muscle and Apoptosis: Molecular Mechanisms and Regulation by Sex Steroids

Apoptosis is a physiologic process that take place during development andin the progression of specific diseases. In skeletal muscle, this process is poorly explored. Skeletal muscle represents an exceptional tissue regards apoptosis, by its multinucleated structure and its variable mitochondrial content. On the other hand, apoptosis of skeletal muscle tissue could have a wide spectrum of effects on organism since it is now well established that skeletal muscle not only generates force and movement; other functions are associated to this tissue. Skeletal muscle contributes to basal energy metabolism, in the storing for substrates such as amino acids and carbohydrates, in the keep of core temperature and blood glucose levels, and in the useof oxygen and energy during movement. Also, skeletal muscle acts as an endocrineorgan, thus could be regulated by owning or no own hormones. This chaptersummarizes the generalities of skeletal muscle at the molecular/structural and functional level and basic concepts of apoptosis, for a better understanding of the following chapters of this ebook. Which it focuses specifically, at a molecular level involving genomic regulation, on the actions of 17β-Estradiol and Testosterone in the homeostasis of skeletal muscle tissue in physiological and pathological conditions, converging in the relationship of muscle apoptosis and sexual hormones.Fil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Pronsato, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Molecular aspects of the early stages of elicitation of secondary metabolites in plants

Plants are a source of commercially important secondary metabolites. Elicitation of plant cells in culture represents a useful biotechnological tool to improve the production of these valuable metabolites. Greater knowledge on the mechanism of elicitation has basic as well as practical implications. This review summarizes molecular information available about the early stages of the elicitation process and the mode of action of elicitors. A description is first provided on the importance of plant secondary metabolism, its induction by elicitation, the elicitor concept and classification, factors affecting elicitation and the mechanisms involved, with a major emphasis on the intracellular transduction systems which mediate the actions of elicitors, namely, elicitor receptors, GTP binding proteins, the Ca2+ messenger system and the PI3K, PLC/IP3-DAG/PKC and adenylyl cyclase/cAMP/PKA pathways, which finally act through mitogen-activated protein kinases (MAPKs) affecting the expression of genes related to the biosynthesis of secondary metabolites. Relevant experimental approaches used to study these topics are also discussed.Fil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin