Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. in the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. in addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Dept Biochem & Immunol, Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 2006/58629-8Web of Scienc

Effects of short-term zidovudine exposure on mitochondrial DNA content and succinate dehydrogenase activity of rat skeletal muscle cells

Long-term use of zidovudine (AZT) may cause mitochondrial abnormalities in various tissues, including a toxic myopathy in AIDS patients associated with mitochondrial DNA (mtDNA) depletion. in the present study, we examine the short-term (48 h) effect of AZT (10, 30 and 100 mu g/ml) on the mitochondrial succinate dehydrogenase (SDH) and mtDNA content of rat cultured skeletal muscle. the effect of AZT on cytochrome c oxidase (COX) enzyme was also analyzed. the histochemical quantitative analysis of SDH showed that AZT 10, 30 and 100 mu g/ml increased by 7%, 9% and 13% the mitochondrial content. Conversely, treatment of rat cultures with 10 to 100 mu g/ml AZT reduced the mtDNA content by 23% to 66%, when compared to control values. the spontaneous contraction and the COX activity were not modified by up to 100 mu g/ml AZT. Taken together, these results show that short-term treatment with AZT can induce severe myotoxicity that involves mitochondrial proliferation and mtDNA depletion in the rat cultured myotubes. Our results also indicate that rat cultured skeletal muscle might be a valuable in vitro assay to evaluate the effect of drugs on mitochondria to predict their potential to induce mitochondrial toxicity. (C) 2007 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol INFAR, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol INFAR, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilWeb of Scienc

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05/59006-1]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)FAPESP[2006/58629-8

INCREASED EXPRESSION of ACETYLCHOLINE RECEPTORS in the DIAPHRAGM MUSCLE of MDX MICE

The absence of dystrophin in Duchenne muscular dystrophy (DMD) and in the mutant mdx mouse causes muscle degeneration and disruption of the neuromuscular junction. Based on evidence from the denervation-like properties of these muscles, we assessed the ligand-binding constants of nicotinic acetylcholine receptors (nAChRs) and the mRNA expression of individual subunits in membrane preparations of diaphragm muscles from adult (4-month-old) and aged (20-month-old) control and mdx mice. the concentration of nAChRs as determined by the maximal specific [(125)I]-alpha-bungarotoxin binding (Bmax) in the muscle membranes did not change with aging in both animal strains. When compared to age-matched control groups, the Bmax in mdx muscles was increased by 65% in adults, and by 103% in aged mice with no alteration of toxin affinity for nAChRs. Reverse-transcription polymerase chain reaction assays showed that mRNA transcripts for the nAChR alpha 1, gamma, alpha 7, and beta 2, but not the epsilon subunits, were more abundant in mdx than in control muscles. the results indicate increased expression of extrajunctional nAChRs in the mdx diaphragm and reflect impairment of nAChR regulation in dystrophin-deficient muscles. These observations may be related to the resistance to nondepolarizing muscle relaxants and the high sensitivity to depolarizing agents reported in DMD patients.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Escola Paulista Med, Nat Prod Sect, Dept Pharmacol, BR-04044020 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Expt Endocrinol Sect, BR-04044020 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Nat Prod Sect, Dept Pharmacol, BR-04044020 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Expt Endocrinol Sect, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

In vivo effects of the association of the psychoactive phenotiazine thioridazine on antitumor activity and hind limb paralysis induced by the native polypeptide crotamine

Crotamine is a cationic polypeptide composed by 42 amino acid residues with several pharmacological and biological properties, including the selective ability to enter and kill actively proliferating tumour cells, which led us to propose its use as a theranostic agent for cancer therapy. At the moment, the improvement of crotamine antitumoral efficacy by association with chemotherapeutic adjuvants is envisioned. In the present work, we evaluated the association of crotamine with the antitumoral adjuvant phenotiazine thioridazine (THD). In spite of the clear efficacy of these both compounds as anticancer agents in long-term in vivo treatment of animal model bearing implanted xenograph melanoma tumor, the expected mutual potentiation of the antitumor effects was not observed here. Moreover, this association revealed for the first time the influence of THD on crotamine ability to trigger the hind limb paralysis in mice, and this discovery may represent the first report suggesting the potential involvement of the CNS in the action of this snake polypeptide on the skeletal muscle paralysis, which was classically believed to be essentially limited to a direct action in peripheral tissues as the skeletal muscle. This is also supported by the observed ability of crotamine to potentiate the sedative effects of THD which action was consistently demonstrated to be based on its central action. The better characterization of crotamine properties in CNS may certainly bring important insights for the knowledge needed to pave the way toward the use of this molecule as a theranostic compound in human diseases as cancer

The kinin B-1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the-control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. the KKS (kallikrein kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B-1 and B-2, which are expressed in a variety of tissues. the local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-kappa B (nuclear factor kappa B) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B-1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-kappa B, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). in vivo, we observed an increase in kinin B-1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. in the same model, inhibition of the kinin B-1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and I kappa B (inhibitor of NF-kappa B) phosphorylation. Moreover, knockout of the kinin B-1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. in conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B-1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Fac Med Ribeirao Preto, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04039032 São Paulo, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Ctr Integrat Syst Biol CISBi, BR-14049900 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Pharmacol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04039032 São Paulo, BrazilWeb of Scienc