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.

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.

Effect of testosterone on the regulation of p53 and p66Shc during oxidative stress damage in C2C12 cells.

Accumulating evidence indicates that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells, at different levels: morphological, physiological, biochemical and molecular. In the present study we observed that H2O2 induces the mitochondrial permeability transition pore (mPTP) opening and exerts p53 activation in a time-dependent way, with a maximum response after 1-2h of treatment. Testosterone treatment, prior to H2O2, reduces not only p53 phosphorylation but also p66Shc expression, activation and its mitochondrial localization, at the same time that it prevents the mPTP opening. Furthermore, testosterone diminishes JNK and PKCßI phosphorylation induced by H2O2 and probably contributing thus, to reduce the activation of p66Shc. Thus, the mPTP opening, p53, JNK and PKCßI activation, as well as p66Shc mRNA increase, induced by oxidative stress, were reduced by testosterone pretreatment. The data presented in this work show some of the 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 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.

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.

Non-classical localization of androgen receptor in the C2C12 skeletal muscle cell line.

The classical model of testosterone action has been traditionally described as being mediated by the androgen receptor (AR) localized exclusively in the nucleus. However, there is increasing functional evidence for extranuclear localization of AR. We present biochemical and immunological data supporting mitochondrial and microsomal localization of AR in the C2C12 skeletal muscle cell line. As a first approach AR was detected by immunoblotting, using specific antibodies after subcellular fractionation, not only in nucleus and cytosol, but also in mitochondria and microsomes. We then established [(3)H] testosterone binding characteristics in total homogenates and subcellular fractions. Specific and saturable [(3)H] testosterone binding sites were detected in mitochondria and microsomes. Immunolocalization of the non-classical AR was also confirmed using confocal microscopy. Sucrose gradient fractionation demonstrated the presence of the AR in lipid rafts and caveolae. Besides, the AR interacts physically with Caveolin-1, association that is lost after testosterone treatment. Accordingly, Western blot analysis revealed a decrease of AR expression in the microsomal fraction after testosterone treatment, suggesting translocation of the membrane AR to another subcellular compartment. The non-classical distribution of native pools of AR in skeletal muscle cells suggests an alternative mode of AR localization/function.

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.

Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway.

Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H(2)O(2))-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H(2)O(2) cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H(2)O(2) induces BAD inactivation, inhibition of poly(ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H(2)O(2), and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.

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.