RESUMEN
The muscular dystrophy with myositis (mdm) mouse model results in a severe muscular dystrophy due to an 83-amino-acid deletion in the N2A region of titin, an expanded sarcomeric protein that functions as a molecular spring which senses and modulates the response to mechanical forces in cardiac and skeletal muscles. ANKRD1 is one of the muscle ankyrin repeat domain proteins (MARPs) a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. The aberrant over-activation of Nuclear factor Kappa B (NF-κB) and the Ankyrin-repeat domain containing protein 1 (ANKRD1) occurs in several models of progressive muscle disease including Duchenne muscular dystrophy. We hypothesized that mechanical regulation of ANKRD1 is mediated by NF-κB activation in skeletal muscles and that this mechanism is perturbed by small deletion of the stretch-sensing titin N2A region in the mdm mouse. We applied static mechanical stretch of the mdm mouse diaphragm and cyclic mechanical stretch of C2C12 myotubes to examine the interaction between NF-κΒ and ANKRD1 expression utilizing Western blot and qRTPCR. As seen in skeletal muscles of other severe muscular dystrophies, an aberrant increased basal expression of NF-κB and ANKRD1 were observed in the diaphragm muscles of the mdm mice. Our data show that in the mdm diaphragm, basal levels of NF-κB are increased, and pharmacological inhibition of NF-κB does not alter basal levels of ANKRD1. Alternatively, NF-κB inhibition did alter stretch-induced ANKRD1 upregulation. These data show that NF-κB activity is at least partially responsible for the stretch-induced expression of ANKRD1.
RESUMEN
The diaphragm is the "respiratory pump;" the muscle that generates pressure to allow ventilation. Diaphragm muscles play a vital function and thus are subjected to continuous mechanical loading. One of its peculiarities is the ability to generate distinct mechanical and biochemical responses depending on the direction through which the mechanical forces applied to it. Contractile forces originated from its contractile components are transmitted to other structural components of its muscle fibers and the surrounding connective tissue. The anisotropic mechanical properties of the diaphragm are translated into biochemical signals that are directionally mechanosensitive by mechanisms that appear to be unique to this muscle. Here, we reviewed the current state of knowledge on the biochemical pathways regulated by mechanical signals emphasizing their anisotropic behavior in the normal diaphragm and analyzed how they are affected in muscular dystrophies.
Asunto(s)
Diafragma , Contracción Muscular , Fuerza Muscular , Distrofias Musculares , Animales , Diafragma/metabolismo , Diafragma/patología , Diafragma/fisiopatología , Humanos , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofias Musculares/fisiopatologíaRESUMEN
Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species. The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription. In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.
Asunto(s)
Antioxidantes/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Sirtuina 1/biosíntesis , Superóxido Dismutasa/biosíntesis , Animales , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Humanos , Ratones , Células Musculares/metabolismo , Proteínas Musculares/genética , Ejercicios de Estiramiento Muscular , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Superóxido Dismutasa/genética , Transcripción Genética/fisiologíaRESUMEN
Ageing and obesity have common hallmarks: altered glucose and lipid metabolism, chronic inflammation and oxidative stress are some examples. The downstream effects of SIRT1 activity have been thoroughly explored, and their research is still in expanse. SIRT1 activation has been shown to regulate pathways with beneficiary effects on 1) ageing and obesity-associated metabolic disorders such as metabolic syndrome, insulin resistance and type-II diabetes with, 2) chronic inflammatory processes such as arthritis, atherosclerosis and emphysema, 3) DNA damage and oxidative stress with impact on neurodegenerative diseases, cardiovascular health and some cancers. This knowledge intensified the interest in uncovering the mechanisms regulating the expression and activity of SIRT1. This review focuses on the upstream regulatory mechanisms controlling SIRT1, and how this knowledge could potentially contribute to the development of therapeutic interventions.
Asunto(s)
Envejecimiento/metabolismo , Regulación de la Expresión Génica , Obesidad/metabolismo , Sirtuina 1/metabolismo , Animales , Daño del ADN , Diabetes Mellitus Tipo 2/metabolismo , Ejercicio Físico , Humanos , Hipoxia , Inflamación , Resistencia a la Insulina , Síndrome Metabólico , Ratones , NAD/metabolismo , Estrés Oxidativo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Riesgo , Estrés MecánicoRESUMEN
Increased activation of catabolic pathways, including apoptosis causes sarcopenia. However, the precise molecular mechanism that initiates apoptosis during aging is not well understood. Here, we report that aging alters miRNA expression profile in mouse skeletal muscle as evidenced by miRNA microarray and real-time PCR. We identified miR-434-3p as a highly downregulated miRNA in the skeletal muscle of aging mice. Myocytes transfected with miR-434-3p mimic prevents apoptosis induced by various apoptotic stimuli, and co-transfection of miR-434-3p antagomir abolishes the inhibitory role of miR-434-3p. We found that miR-434-3p inhibits apoptosis by targeting the eukaryotic translation initiation factor 5A1 (eIF5A1). Overexpression of miR-434-3p in myocytes reduces the loss of mitochondrial transmembrane potential, and activation of caspases-3, -8 and -9 by suppressing eIF5A1 in response to various apoptotic stimuli whereas inhibition of miR-434-3p reversed this scenario. Skeletal muscles from aging mice exhibit low levels of miR-434-3p and high levels of eIF5A1, suggesting a possible role for miR-434-3p in the initiation of apoptosis in aging muscle. Overall, our data identified for the first time that miR-434-3p is an anti-apoptotic miRNA that may be therapeutically useful for treating muscle atrophy in various pathophysiological conditions, including sarcopenia.
Asunto(s)
Envejecimiento/genética , Apoptosis/genética , MicroARNs/genética , Músculo Esquelético/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Envejecimiento/metabolismo , Animales , Caspasas/metabolismo , Regulación de la Expresión Génica , Potencial de la Membrana Mitocondrial/fisiología , Ratones , MicroARNs/metabolismo , Células Musculares/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
High-fat diet (HFD) plays a central role in the initiation of mitochondrial dysfunction that significantly contributes to skeletal muscle metabolic disorders in obesity. However, the mechanism by which HFD weakens skeletal muscle metabolism by altering mitochondrial function and biogenesis is unknown. Given the emerging roles of microRNAs (miRNAs) in the regulation of skeletal muscle metabolism, we sought to determine whether activation of a specific miRNA pathway would rescue the HFD-induced mitochondrial dysfunction via the sirtuin-1 (SIRT-1)/ peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) pathway, a pathway that governs genes necessary for mitochondrial function. We here report that miR-149 strongly controls SIRT-1 expression and activity. Interestingly, miR-149 inhibits poly(ADP-ribose) polymerase-2 (PARP-2) and so increased cellular NAD(+) levels and SIRT-1 activity that subsequently increases mitochondrial function and biogenesis via PGC-1α activation. In addition, skeletal muscles from HFD-fed obese mice exhibit low levels of miR-149 and high levels of PARP-2, and they show reduced mitochondrial function and biogenesis due to a decreased activation of the SIRT-1/PGC-1α pathway, suggesting that mitochondrial dysfunction in the skeletal muscle of obese mice may be because of, at least in part, miR-149 dysregulation. Overall, miR-149 may be therapeutically useful for treating HFD-induced skeletal muscle metabolic disorders in such pathophysiological conditions as obesity and type 2 diabetes.
Asunto(s)
MicroARNs/metabolismo , Recambio Mitocondrial/fisiología , Músculo Esquelético/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sirtuina 1/metabolismo , Factores de Transcripción/metabolismo , Animales , Dieta Alta en Grasa , Ratones , Ratones Obesos , MicroARNs/genética , Mitocondrias Musculares/metabolismo , Obesidad/genética , Obesidad/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Poli(ADP-Ribosa) Polimerasas/genética , Transducción de Señal/fisiología , Sirtuina 1/genética , Factores de Transcripción/genéticaRESUMEN
Muscle contraction is associated with the production of reactive oxygen species (ROS). Mechanisms of ROS scavenging are fundamental to avoid muscle damage. We had previously discovered a stretch-induced genetic program in myotubes that triggers an antioxidant defense. At the core of this mechanism, transcriptional activation of SIRT1 by the early growth response protein EGR1 results in increased MnSOD activity through the activation of Sod2 by SIRT1/FOXO pathway. In this report, we show experimental evidence that; a) EGR1 and SIRT1 proteins physically interact at the time of maximal Sirt1 induction, b) SIRT1 has a negative effect on the activation of the Sirt1 promoter by EGR1. Thus, the interaction between EGR1 and SIRT1 describes an autoregulatory loop that shuts down the stretch-induced Sirt1 expression.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Homeostasis , Mecanotransducción Celular , Músculo Esquelético/metabolismo , Sirtuina 1/metabolismo , Animales , Línea Celular , Ratones , Fibras Musculares Esqueléticas/metabolismo , Estrés Oxidativo , Regiones Promotoras Genéticas , Sirtuina 1/genética , Estrés MecánicoRESUMEN
Skeletal muscle aging is associated with increased inflammation and oxidative stress, a decrease in the ability to rebuild muscle after injury and in response to exercise. In this perspective, we discuss the mechanisms regulating Sirt1 activity and expression in skeletal muscles, emphasizing their implications in muscle physiology and the impairment of muscle function with age.
Asunto(s)
Músculo Esquelético/fisiología , Sirtuina 1/fisiología , Adaptación Fisiológica , Envejecimiento/fisiología , Animales , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ejercicio Físico/fisiología , Humanos , Inflamación/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Estrés Oxidativo/fisiología , Estrés MecánicoRESUMEN
The mechanical regulation of the forkhead box O (FOXO) subclass of transcription factors in the respiratory pump and its implication in aging are completely unknown. We investigated the effects of diaphragm stretch on three FOXO isoforms, Foxo1, Foxo3a, and Foxo4, in normal mice at different ages. We tested the hypotheses that 1) FOXO activities are regulated in response to diaphragm stretch and 2) mechanical properties of aging diaphragm are altered, leading to altered regulation of FOXO with aging. Our results showed that stretch downregulated FOXO DNA-binding activity by a mechanism that required Akt and IKK activation in young mice but that these pathways lost their mechanosensitivity with age. This aberrant regulation of FOXO with aging was associated with altered viscoelasticity, compliance, and extensibility of the aged diaphragm. Curiously, the dramatic decrease of the nuclear content of Foxo1 and Foxo3a, the two isoforms associated with muscle atrophy, with aging correlated with higher basal activation of Akt and IKK signaling in diaphragms of old mice. In contrast, the stability of Foxo4 in the nucleus became dependent on JNK, which is strongly activated in aged diaphragm. This finding suggests that Foxo4 was responsible for the FOXO-dependent transcriptional activity in aging diaphragm. Our data support the hypothesis that aging alters the mechanical properties of the respiratory pump, leading to altered mechanical regulation of the stretch-induced signaling pathways controlling FOXO activities. Our study supports a mechanosensitive signaling mechanism that is responsible for regulation of the FOXO transcription factors by aging.
Asunto(s)
Envejecimiento/fisiología , Factores de Transcripción Forkhead/metabolismo , Ventilación Pulmonar/fisiología , Animales , Adaptabilidad , Diafragma/metabolismo , Elasticidad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Isoformas de ProteínasRESUMEN
A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn(mdm/mdm)) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.
Asunto(s)
Diafragma/fisiopatología , Contracción Muscular , Proteínas Musculares/genética , Fuerza Muscular , Distrofia Muscular Animal/fisiopatología , Mutación , Miositis/fisiopatología , Proteínas Quinasas/genética , Animales , Western Blotting , Conectina , Diafragma/diagnóstico por imagen , Diafragma/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Mutantes , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Miofibrillas/metabolismo , Miofibrillas/patología , Cadenas Pesadas de Miosina/metabolismo , Miositis/genética , Miositis/metabolismo , Factores de Tiempo , Tomografía Computarizada por Rayos XRESUMEN
MRG15 is a novel chromodomain protein that is a member of a family of genes related to MORF4. MORF4 (mortality factor on chromosome 4) induces senescence in a subset of human tumor cell lines. Our previous results indicated that MRG15 (MORF-related gene on chromosome 15) could derepress the B-myb promoter by association with Rb. In this study, sucrose gradient analysis demonstrated that MRG15 was present in two distinct nuclear protein complexes, MAF1 (MRG15-associated factor 1) and MAF2. Rb was associated with MRG15 and PAM14 (a novel coil-coil protein) in MAF1, and a histone acetyl transferase, hMOF, was an MRG15 partner in MAF2. Analysis of deletion mutants of MRG15 indicated that the leucine zipper at the C-terminal region of MRG15 was important for the protein associations in MAF1 and that the N-terminal chromodomain was required for the assembly of the MAF2 protein complex. Consistent with these data was the fact that a histone acetyltransferase activity associated with MRG15 was lost when the chromodomain was deleted and that both mutant MRG15 proteins failed to activate the B-myb promoter. The various mechanisms by which MRG15 could activate gene transcription are discussed.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Genes myb , Nucleoproteínas/genética , Transactivadores/genética , Activación Transcripcional , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , Núcleo Celular/patología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 15 , Clonación Molecular , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Mapeo Restrictivo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Células Tumorales CultivadasRESUMEN
In this paper we demonstrate that the Candida albicans 20S proteasome is in vivo phosphorylated and is a good in vitro substrate (S(0.5) 14nM) of homologous protein kinase CK2 (CK2). We identify alpha6/C2, alpha3/C9, and alpha5/Pup2 proteasome subunits as the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. In vitro phosphorylation by homologous CK2 holoenzyme occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasomes from mammalian proteasomes which are phosphorylated by CK2 in the absence of polycations. The major in vivo phosphate acceptor is the alpha3/C9 subunit, being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by endoproteinase Glu-C digestion from in vivo labeled alpha3/C9 subunit, from in vitro phosphorylation by homologous CK2 holoenzyme, and from the recombinant alpha3/C9 subunit phosphorylated by recombinant human CK2-alpha subunit are identical, suggesting that CK2 is likely responsible for in vivo phosphorylation of this subunit. Direct mutational analysis shows that serine 248 is the residue of the alpha3/C9 subunit phosphorylated by CK2. The in vitro stoichiometry of phosphorylation of the alpha6/C2 and alpha3/C9 proteasome subunits by CK2 can be estimated as 0.7-0.8 and 0.4-0.5 mol of phosphate per mole of subunit, respectively. These results are consistent with the relative abundance of the unphosphorylated and phosphorylated isoforms of these subunits present in the purified 20S proteasome preparation. Our demonstration of phosphorylation of C. albicans proteasome suggests that phosphorylation might be a general mechanism of regulation of proteasome activity.