RESUMO
The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.
Assuntos
Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Animais , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Condicionamento Físico Animal , Resistência Física/genética , Dobramento de Proteína , Transporte Proteico , Transdução de Sinais , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Skeletal muscle undergoes remarkable adaptations in response to chronic decreases in contractile activity, such as a loss of muscle mass, decreases in both mitochondrial content and function, as well as the activation of apoptosis. Although these adaptations are well known, questions remain regarding the signaling pathways that mediated these changes. Autophagy is an organelle turnover pathway that could contribute to these adaptations. The purpose of this study was to determine whether denervation-induced muscle disuse would result in the activation of autophagy gene expression in both wild-type (WT) and Bax/Bak double knockout (DKO) animals, which display an attenuated apoptotic response. Denervation caused a reduction in muscle mass for WT and DKO animals; however, there was a 40% attenuation in muscle atrophy in DKO animals. Mitochondrial state 3 respiration was significantly reduced, and reactive oxygen species production was increased by two- to threefold in both WT and DKO animals. Apoptotic markers, including cytosolic AIF and DNA fragmentation, were elevated in WT, but not in DKO animals following denervation. Autophagy proteins including LC3II, ULK1, ATG7, p62, and Beclin1 were increased similarly following denervation for both WT and DKO. Interestingly, denervation markedly increased the localization of LC3II to subsarcolemmal mitochondria, and this was more pronounced in the DKO animals. Thus denervation-induced muscle disuse activates both apoptotic and autophagic signaling pathways in muscle, and autophagic protein expression does not exhibit a compensatory increase in the presence of attenuated apoptosis. However, the absence of Bax and Bak may represent a potential signal to trigger mitophagy in muscle.
Assuntos
Autofagia/fisiologia , Denervação Muscular , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/patologia , Transtornos Musculares Atróficos/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Skeletal muscle is highly adaptable in response to increases and decreases in contractile activity. The purpose of this study was to determine whether the preconditioning of skeletal muscle has a protective effect against subsequent denervation-induced apoptotic protein expression. To investigate this, we chronically stimulated the tibialis anterior and extensor digitorum longus muscles for 7 days (10 Hz, 3 h/day) before 7 days of denervation. Denervation reduced total cytochrome-c oxidase activity by 39%, which was likely a consequence of a decrease in subsarcolemmal (SS) mitochondria. This decrease in the SS subfraction was prevented by prior chronic stimulation and, as a result, maintained total mitochondrial content at control levels. The expression of Bax was elevated 2.2-fold by denervation, and prior chronic stimulation did not attenuate this increase. This produced a increase in the Bax-to-Bcl-2 ratio, indicating greater muscle apoptotic susceptibility. Denervation also decreased state 3 respiration in SS and intermyofibrillar mitochondria and elevated state 4 reactive oxygen species production within both mitochondrial subfractions. These changes were not prevented by prior chronic stimulation. Furthermore, the antioxidant protein MnSOD was also reduced by denervation, whereas Beclin-1 was markedly elevated. This suggests that autophagic cell death could also play a significant part in denervation-induced muscle atrophy. Thus, despite prior chronic stimulation, denervation increases the apoptotic susceptibility of skeletal muscle by altering the Bax-to-Bcl-2 ratio, by increasing reactive oxygen species production, and by reducing the expression of MnSOD. Whether a more extensive stimulation paradigm would be more effective in attenuating apoptosis before muscle disuse remains to be determined.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/fisiologia , Animais , Antioxidantes/metabolismo , Western Blotting , Peso Corporal/fisiologia , Estimulação Elétrica , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Técnicas In Vitro , Masculino , Contração Muscular/fisiologia , Denervação Muscular , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Tamanho do Órgão/fisiologia , Consumo de Oxigênio/fisiologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Apoptosis is an essential process that plays a critical role in both tissue development and maintenance. Apoptosis has been shown to be involved in skeletal muscle atrophy resulting from chronic muscular disuse, sarcopenia, and mitochondrial myopathies. Exercise may attenuate some of the proapoptotic adaptations that occur during these conditions. This review will focus on the factors influencing mitochondrially mediated apoptosis in skeletal muscle.
Assuntos
Apoptose/fisiologia , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/metabolismo , Humanos , Atrofia Muscular/patologiaRESUMO
Chronic muscle disuse induced by denervation reduces mitochondrial content and produces muscle atrophy. To investigate the molecular mechanisms responsible for these adaptations, we assessed 1) mitochondrial biogenesis- and apoptosis-related proteins and 2) apoptotic susceptibility and cell death following denervation. Rats were subjected to 5, 7, 14, 21, or 42 days of unilateral denervation of the sciatic or peroneal nerve. Muscle mass and mitochondrial content were reduced by 40-65% after 21 and 42 days of denervation. Denervation-induced decrements in mitochondrial content occurred along with 60% and 70% reductions in transcription factor A (Tfam) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, respectively. After 42 days of denervation, Bax was elevated by 115% and Bcl-2 was decreased by 89%, producing a 16-fold increase in the Bax-to-Bcl-2 ratio. Mitochondrial reactive oxygen species production was markedly elevated by 5- to 7.5-fold in subsarcolemmal mitochondria after 7, 14, and 21 days of denervation, whereas reactive oxygen species production in intermyofibrillar (IMF) mitochondria was reduced by 40-50%. Subsarcolemmal and IMF mitochondrial levels of MnSOD were also reduced by 40-50% after 14-21 days of denervation. The maximal rate of IMF mitochondrial pore opening (V(max)) was elevated by 25-35%, and time to V(max) was reduced by 20-25% after 14 and 21 days, indicating increased apoptotic susceptibility. Myonuclear decay, assessed by DNA fragmentation, was elevated at 7-21 days of denervation. Our data indicate that PGC-1alpha and Tfam are important factors that likely contribute to the reduced mitochondrial content after chronic disuse. In addition, our results illustrate that, despite the reduced mitochondrial content, denervated muscle has greater mitochondrial apoptotic susceptibility, which coincided with elevated apoptosis, and these processes may contribute to denervation-induced muscle atrophy.
Assuntos
Apoptose/fisiologia , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/inervação , Transtornos Musculares Atróficos/fisiopatologia , Adaptação Fisiológica , Animais , Núcleo Celular/metabolismo , Respiração Celular/fisiologia , Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Masculino , Denervação Muscular , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/patologia , Transtornos Musculares Atróficos/patologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Cardiolipin (CL) is a phospholipid that maintains the integrity of mitochondrial membranes. We previously demonstrated that CL content increases with chronic muscle use, and decreases with denervation-induced disuse. To investigate the underlying mechanisms, we measured the mRNA expression of 1) CL synthesis enzymes cardiolipin synthase (CLS) and CTP:PA-cytidylyltransferase-1 (CDS-1); 2) remodeling enzymes tafazzin and acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1); and 3) outer membrane CL enzymes, mitochondrial phospholipase D and phospholipid scramblase 3 (Plscr3), during chronic contractile activity (CCA)-induced mitochondrial biogenesis and denervation. With CCA, CDS-1 expression increased by 128%, parelleling CL levels. Surprisingly, denervation also led to large increases in CDS-1 and CLS, despite a decrease in mitochondria, possibly due to a compensatory mechanism to restore lost CL. ALCAT1 decreased by 32% with CCA, but increased by 290% following denervation, indicating that both CCA and denervation alter CL remodeling. CCA and denervation also elicited 60-90% increases in Plscr3, likely to facilitate CL movement to the outer membrane. The expression of these genes was not affected by aging, but changes due to CCA and denervation were attenuated compared with young animals. The absence of PPARγ coactivator-1α in knockout animals led to a decrease in CDS-1 and an increase in ALCAT1 mRNA levels, implicating PGC-1α in regulating both CL synthesis and remodeling. These data suggest that chronic muscle use and disuse modify the expression of mRNAs encoding CL metabolism enzymes. Our data also illustrate, for the first time, that PPARγ coactivator-1α regulates the CL metabolism pathway in muscle.
Assuntos
Cardiolipinas/metabolismo , Mitocôndrias Musculares/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Fatores Etários , Animais , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Denervação Muscular , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Atrofia Muscular/etiologia , Atrofia Muscular/genética , Atrofia Muscular/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Fatores de Tempo , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Alterations in contractile activity influence the intracellular homeostasis of muscle, which results in adaptations in the performance and the phenotype of this tissue. Denervation is an effective disuse model that functions to change the intracellular environment of muscle leading to a rapid loss in mass, a decrease in mitochondrial content, and an elevation in both proapoptotic protein expression and myonuclear apoptosis. Recent investigations have shown that alternative degradation pathways such as autophagy are activated in conjunction with apoptosis during chronic muscle disuse. We have previously shown that seven days of muscle disuse increases the expression of Beclin 1. Furthermore, we have also detected a significant increase in the expression of LC3-II, a known component of autophagy. In addition to its upregulation, denervation appears to induce the translocation of LC3-II to mitochondrial membranes. Collectively, these increases in protein expression suggest that autophagy signaling is upregulated in response to denervation, and that these pathways may preferentially target mitochondria for degradation in skeletal muscle.
Assuntos
Autofagia , Músculos/inervação , Músculos/patologia , Estresse Oxidativo , Transdução de Sinais , Animais , Proteínas Reguladoras de Apoptose , Proteína Beclina-1 , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Denervação Muscular , Proteínas/metabolismoRESUMO
Sirt1 is a NAD(+)-dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1alpha and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1alpha existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1alpha levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30-50% compared with the control muscle, whereas Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1alpha expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.