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.

Adverse Effects in Skeletal Muscle Following the Medicinal Use of Nicotiana glauca.

Nicotiana glauca is a cosmopolitan shrub, used in medicine to treat swellings, wounds, sores and cancer. However, its users lack of knowledge of the adverse effects. We seek to evaluate the effects of lipid extracts from N. glauca on myoblasts, identifying the compounds which cause undesirable effects. Myoblasts are important in muscle homeostasis, thus a high death rate of them cause myopathies. We performed an ethanolic extraction from leaves of N. glauca and the extract was successively partitioned with hexane, chloroform and ethyl acetate. The effects of extracts in C2C12 cells were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), Mitotracker and 4',6-diamidino-2-phenylindole (DAPI) staining, Western blotting, real-time PCR and immunofluorescence assays. Caspase activity was studied. The fraction with the highest apoptotic effects was analysed by chromatography, NMR and GC-MS spectrometry were used to identify the apoptotic agent, after which its biological activity was evaluated. The extracts from N. glauca induced apoptosis in C2C12 cells involving caspase-3/7. We found that the extracts trigger a defence response in muscle through Akt and heat shock protein 27 (HSP27). We identified an apoptotic agent as palmitic acid. These data suggest that the use of N. glauca in hormone replacement therapy, or in other therapies affects skeletal muscle homeostasis, worsening the negative effects of the menopause. Thus, the relevance of this work lies in the fact that it is the first time that a report about the molecular mechanism responsible for the side effects of medicinal use of N. glauca, has been shown. Moreover the compound responsible for these effects has been identified.

Effects of Photobiomodulation Therapy on Oxidative Stress in Muscle Injury Animal Models: A Systematic Review.

This systematic review was performed to identify the role of photobiomodulation therapy on experimental muscle injury models linked to induce oxidative stress. EMBASE, PubMed, and CINAHL were searched for studies published from January 2006 to January 2016 in the areas of laser and oxidative stress. Any animal model using photobiomodulation therapy to modulate oxidative stress was included in analysis. Eight studies were selected from 68 original articles targeted on laser irradiation and oxidative stress. Articles were critically assessed by two independent raters with a structured tool for rating the research quality. Although the small number of studies limits conclusions, the current literature indicates that photobiomodulation therapy can be an effective short-term approach to reduce oxidative stress markers (e.g., thiobarbituric acid-reactive) and to increase antioxidant substances (e.g., catalase, glutathione peroxidase, and superoxide dismutase). However, there is a nonuniformity in the terminology used to describe the parameters and dose for low-level laser treatment.

Testosterone modulates FoxO3a and p53-related genes to protect C2C12 skeletal muscle cells against apoptosis.

The loss of muscle mass and strength with aging, sarcopenia, is a prevalent condition among the elderly, associated with skeletal muscle dysfunction and enhanced muscle cell apoptosis. We have previously demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells, at different levels: morphological, biochemical and molecular. Since we have observed that testosterone reduces p-p53 and maintains the inactive state of FoxO3a transcription factor, induced by H2O2, we analyzed if the hormone was exerting its antiapoptotic effect at transcriptional level, by modulating pro and antiapoptotic genes associated to them. We detected the upregulation of the proapoptotic genes Puma, PERP and Bim, and MDM2 in response to H2O2 at different periods of the apoptotic process, and the downregulation of the antiapoptotic gene Bcl-2, whereas testosterone was able to modulate and counteract H2O2 effects. Furthermore, ERK and JNK kinases have been demonstrated to be linked to FoxO3a phosphorylation and thus its subcellular distribution. This work show some transcription level components, upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents.

17ß-Estradiol Protects Skeletal Myoblasts From Apoptosis Through p53, Bcl-2, and FoxO Families.

17ß-Estradiol (E2 ) protects several nonreproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2 O2 in skeletal myoblasts, reverting PKCδ, JNK, and p66Shc activation and exerting a beneficial action over mitochondria. Since genomic actions underlying the regulation of nuclear gene transcription are a common property of this steroid, the present work characterizes the transcriptional activity modulated by E2 to exert its antiapoptotic effect. We report that E2 protects skeletal myoblasts against apoptosis induced by H2 O2 modulating p53 and FoxO transcription factors and then their target genes Bcl-2, Bim, Puma, PERP, and MDM2, without affecting Noxa gene. The results presented in this work support the notion that the transcription factors FoxO and p53 coordinate apoptosis in C2C12 cells, and deepens our knowledge about a putative molecular mechanism by which E2 exerts beneficial effects against oxidative stress in skeletal myoblasts. J. Cell. Biochem. 118: 104-115, 2017. © 2016 Wiley Periodicals, Inc.

17ß-Estradiol and testosterone in sarcopenia: Role of satellite cells.

The loss of muscle mass and strength with aging, referred to as sarcopenia, is a prevalent condition among the elderly. Although the molecular mechanisms underlying sarcopenia are unclear, evidence suggests that an age-related acceleration of myocyte loss via apoptosis might be responsible for muscle perfomance decline. Interestingly, sarcopenia has been associated to a deficit of sex hormones which decrease upon aging. The skeletal muscle ability to repair and regenerate itself would not be possible without satellite cells, a subpopulation of cells that remain quiescent throughout life. They are activated in response to stress, enabling them to guide skeletal muscle regeneration. Thus, these cells could be a key factor to overcome sarcopenia. Of importance, satellite cells are 17ß-estradiol (E2) and testosterone (T) targets. In this review, we summarize potential mechanisms through which these hormones regulate satellite cells activation during skeletal muscle regeneration in the elderly. The advance in its understanding will help to the development of potential therapeutic agents to alleviate and treat sarcopenia and other related myophaties.

17ß-Estradiol Abrogates Apoptosis Inhibiting PKCδ, JNK, and p66Shc Activation in C2C12 Cells.

17ß-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2O2 in skeletal myoblasts. As we have also demonstrated a clear beneficial action of this hormone on skeletal muscle mitochondria, the present work further characterizes the signaling mechanisms modulated by E2 that are involved in mitochondria protection, which ultimately result in antiapoptosis. Here, we report that E2 through estrogen receptors (ERs) inhibited the H2O2-induced PKCδ and JNK activation, which results in the inhibition of phosphorylation and translocation to mitochondria of the adaptor protein p66Shc. In conjunction, the inhibition by the hormone of this H2O2-triggered signaling pathway results in protection of mitochondrial potential membrane. Our results provide basis for a putative mechanism by which E2 exerts beneficial effects on mitochondria, against oxidative stress, in skeletal muscle cells.

17ß-estradiol protects mitochondrial functions through extracellular-signal-regulated kinase in C2C12 muscle cells.

BACKGROUND/AIMS: We have previously shown that exposure to 17ß-estradiol (E2) prior to induction of apoptosis with H2O2 protects skeletal muscle cells against oxidative damage. However, the mechanism involved in the protective action of the hormone is poorly understood. In the present study, we focused on the mechanism by which ERK mediates this survival effect in connection with COXIV activity and mitochondrial membrane potential. METHODS: Immunocytochemistry, Western blot, cytochrome c oxidase complex IV (COXIV) activity, coimmunoprecipitation and JC-1 dye by flow cytometry were carried out using C2C12 myoblasts as experimental model. RESULTS: E2 is able to activate ERK and then induces its translocation to mitochondria. Using the pharmacological inhibitor of ERK activation U0126 we show that E2, through ERK activation, is able to enhance COXIV activity. Moreover, the hormone increases the interaction between COXIV and ERK. Also, we found that hydrogen peroxide decreases COXIV activity and that preincubation of the cells with E2 prior to induction of apoptosis prevents this effect. In addition, we observe that the estrogen inhibits the collapse of mitochondrial membrane potential induced by H2O2, involving ERK and COXIV. CONCLUSION: Our data demonstrate that E2 promotes ERK activation and translocation to mitochondria preventing the decline in COXIV activity and in turn, alteration of mitochondrial membrane potential by oxidative stress, in C2C12 myoblasts.

Actions of 17ß-estradiol and testosterone in the mitochondria and their implications in aging.

A decline in the mitochondrial functions and aging are two closely related processes. The presence of estrogen and androgen receptors and hormone-responsive elements in the mitochondria represents the starting point for the investigation of the effects of 17ß-estradiol and testosterone on the mitochondrial functions and their relationships with aging. Both steroids trigger a complex molecular mechanism that involves crosstalk between the mitochondria, nucleus, and plasma membrane, and the cytoskeleton plays a key role in these interactions. The result of this signaling is mitochondrial protection. Therefore, the molecular components of the pathways activated by the sexual steroids could represent targets for anti-aging therapies. In this review, we discuss previous studies that describe the estrogen- and testosterone-dependent actions on the mitochondrial processes implicated in aging.

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 (<20) or high (>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.

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 (<20) or high (>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.(AU)

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 (< 20) or high (> 60) passage numbers (identified as 1-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 C2Cl2 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.

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

17ß-Estradiol (E(2)) protects several non-reproductive tissues from apoptosis, including skeletal muscle. We have shown that E(2) at physiological concentrations prevented apoptosis induced by H(2)O(2) in C2C12 skeletal myoblasts. As we also demonstrated the presence of estrogen receptors in mitochondria, the present work was focused on the effects of E(2) on this organelle. Specifically, we evaluated the actions of E(2) on the mitochondrial permeability transition pore (MPTP) by the calcein-acetoxymethylester/cobalt method using fluorescence microscopy and flow cytometry. Pretreatment with E(2) prevented MPTP opening induced by H(2)O(2), which preceded loss of mitochondrial membrane potential. In addition, it was observed that H(2)O(2) 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 E(2) to exert an antiapoptotic effect. Moreover, E(2) increased mitochondrial manganese superoxide dismutase protein expression and activity, as part of a mechanism activated by E(2) that improved mitochondrial performance. Our results suggest a role of E(2) in the regulation of apoptosis with a clear action at the mitochondrial level in C2C12 skeletal myoblast cells.

High passage numbers induce resistance to apoptosis in C2C12 muscle cells. / 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 (< 20) or high (> 60) passage numbers (identified as 1-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 C2Cl2 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.

Role of 17ß-estradiol and testosterone in apoptosis.

17ß-Estradiol (E2) and Testosterone (T) exert actions in most animal tissues, in addition to the reproductive system. Thus, both sex steroid hormones affect growth and different cell functions in several organs. Accordingly, the nuclear estrogen (ER) and androgen (AR) receptors are ubiquitously expressed. Moreover, ER and AR may have non-classical intracellular localizations, e.g. plasma membrane, mitochondria and endoplasmic reticulum, raising additional complexity to the functional roles of E2 and T. In addition to the modulation of gene transcription by direct interaction with their cognate nuclear receptors, the steroids can rapidly activate signaling pathways by a non-genomic mechanism mediated by receptors identical to or different from known steroid receptors. Among various functions, E2 and T can regulate apoptosis through those pathways. In mitochondria, the presence of ER and AR and actions of estrogen and androgen have been shown, in keeping with the organelle being a control point of apoptosis. The most recurrent action for each steroid hormone is the protection of mitochondria against different insults, resulting in antiapoptosis. This review summarizes the molecular basis of the modulation of programmed cell death by E2 and T in several tissues.

Rol protector del 17ß-Estradiol y la testosterona en la apoptosis del músculo esquelético / Protective role of 17ß-Estradiol and testosterone in apoptosis of skeletal muscle

La sarcopenia, pérdida de masa y fuerza del músculo esqueléico, es una condición frecuente durante el envejecimiento. Conduce a incapacidad motora resultando en internación y mortalidad. Puesto que los niveles de estrógenos y/o testosterona disminuyen con la edad, la sarcopenia se ha asociado al déficit de estas hormonas. Aunque los mecanismos moleculares involucrados en esta patología no están totalmente dilucidados, existen evidencias indicando que la apoptosis es en parte responsable de la pérdida de miocitos en la adultez. Previamente demostramos que el 17ß-estradiol (E2) inhibe la apoptosis en la línea celular C2C12 de músculo esquelético a través de PI3K/Akt, MAPKs, HSP27 y receptores estrogénicos (ERs) con localización no clásica. Usando siRNAs específicos para silenciar las isoformas del ER, comprobamos que el E2 activa ERK involucrando a ERa, mientras que la activación de p38 MAPK es independiente de ERs. Confirmamos que el E2 puede inhibir la apoptosis a través de las MAPKs en cultivos primarios de músculo esquelético de ratón. Al igual que la E2, la testosterona bloquea la apoptosis. Las alteraciones morfológicas típicas de la apoptosis como fragmentación nuclear, desorganización del citoesqueleto, reorganización/disfunción mitocondrial y liberación de citocromo c, inducidos por H2O2 fueron suprimidas al preincubar las células con testosterona. Se requieren investigaciones adicionales para establecer un paralelismo entre los mecanismos de acción de ambas hormonas, que podrían estar implicados en patologías musculares asociadas a apoptosis. Los datos presentados en este estudio profundizan el conocimiento de las bases moleculares de la sarcopenia relacionada con estados de déficit de hormonas sexuales.

Extracellular-regulated kinase and p38 mitogen-activated protein kinases are involved in the antiapoptotic action of 17beta-estradiol in skeletal muscle cells.

17beta-Estradiol (E(2)) stimulates the mitogen-activated protein kinases (MAPKs) in various cellular types. We have shown that the hormone activates extracellular-regulated kinase (ERK) and p38 MAPK in skeletal muscle cells. However, the functions of MAPK modulation by the estrogen in muscle cells have not been studied yet. We have recently reported antiapoptotic actions of E(2) in C2C12 cells. Here, the role of MAPKs mediating the hormone effect in muscle cells was investigated. The results showed that cells exposed to 0.5 mM hydrogen peroxide (H(2)O(2)) presented cytoskeleton disorganization, mitochondrial redistribution, and picnotic/fragmented nuclei. Pretreatment with 10(-8) M E(2) prevented these morphological apoptotic characteristics, except in the presence of ERK or p38 MAPK inhibitors, U0126 and SB203580 respectively. Mitochondrial membrane integrity was also studied. Preincubation of cultures with 10(-8) M E(2) abrogated H(2)O(2) effects such as Janus Green oxidation, presence of cytochrome c oxidase activity in the cytoplasm, and SMAC/DIABLO release from mitochondria. When MAPKs were inhibited, the hormone could not prevent mitochondrial membrane damage exerted by oxidative stress. Blocking experiments with small interfering RNAs confirmed that both ERK and p38 MAPKs mediate the antiapoptotic effects of the hormone at the mitochondrial level. Further, some of the molecular mechanisms involved were also investigated. Thus, E(2) was able to induce AKT (Ser473) and BAD (Ser112) phosphorylation in C2C12 cells in the absence or in the presence of H(2)O(2) but not when the cultures were incubated with H(2)O(2) and MAPK inhibitors. Altogether, these results show that E(2) exerts a survival action in skeletal muscle cells involving ERK and p38 MAPK activation.

Participation of HSP27 in the antiapoptotic action of 17beta-estradiol in skeletal muscle cells.

Exposure to 17beta-estradiol prior to induction of apoptosis protects skeletal muscle cells against damage. The mechanism involved in this protective action of the hormone is poorly understood. In the present study, using the murine muscle cell line C2C12, evidence was obtained that inhibition of H(2)O(2)-induced apoptosis by the estrogen requires the participation of heat shock protein 27 (HSP27). Reverse transcriptase polymerase chain reaction, Western blot, and immunocytochemistry assays showed that 17beta-estradiol induces a time-dependent (5-60 min) increase in the expression of HSP27. In addition, in presence of quercetin, an inhibitor of HSPs, the antiapoptotic effect of the hormone was diminished. More specifically, blockage experiments with short interference RNA targeting HSP27 confirmed the role of this chaperone in the protective effect of the steroid. 17beta-Estradiol abolished caspase-3 cleavage elicited by H(2)O(2). Coimmunoprecipitation assays suggested physical interaction of HSP27 with caspase-3 in presence of estradiol. Furthermore, we observed that this chaperone interacts with estrogen receptors (ER) beta in mitochondria. Then, this study suggests that HSP27 plays a new role in the antiapoptotic action triggered by 17beta-estradiol by modulating caspase-3 activity and stabilizing ERbeta in skeletal muscle cells.