RESUMO
Maintenance of skeletal muscle structure and function requires efficient and precise metabolic control. Autophagy plays a key role in metabolic homeostasis of diverse tissues by recycling cellular constituents, particularly under conditions of caloric restriction, thereby normalizing cellular metabolism. Here we show that histone deacetylases (HDACs) 1 and 2 control skeletal muscle homeostasis and autophagy flux in mice. Skeletal muscle-specific deletion of both HDAC1 and HDAC2 results in perinatal lethality of a subset of mice, accompanied by mitochondrial abnormalities and sarcomere degeneration. Mutant mice that survive the first day of life develop a progressive myopathy characterized by muscle degeneration and regeneration, and abnormal metabolism resulting from a blockade to autophagy. HDAC1 and HDAC2 regulate skeletal muscle autophagy by mediating the induction of autophagic gene expression and the formation of autophagosomes, such that myofibers of mice lacking these HDACs accumulate toxic autophagic intermediates. Strikingly, feeding HDAC1/2 mutant mice a high-fat diet from the weaning age releases the block in autophagy and prevents myopathy in adult mice. These findings reveal an unprecedented and essential role for HDAC1 and HDAC2 in maintenance of skeletal muscle structure and function and show that, at least in some pathological conditions, myopathy may be mitigated by dietary modifications.
Assuntos
Autofagia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Homeostase , Músculo Esquelético/metabolismo , Animais , Eletroporação , Camundongos , Camundongos Mutantes , Músculo Esquelético/enzimologia , Reação em Cadeia da PolimeraseRESUMO
Recent work has demonstrated that autophagy, a phylogenetically conserved, lysosome-mediated pathway of protein degradation, is a key participant in pathological cardiac remodeling. One common feature of cell growth and autophagy is membrane biogenesis and processing. The exocyst, an octomeric protein complex involved in vesicle trafficking, is implicated in numerous cellular processes, yet its role in cardiomyocyte plasticity is unknown. Here, we set out to explore the role of small G protein-dependent control of exocyst function and membrane trafficking in stress-induced cardiomyocyte remodeling and autophagy. First, we tested in cultured neonatal rat cardiomyocytes (NRCMs) two isoforms of Ral (RalA, RalB) whose actions are mediated by the exocyst. In these experiments, mTOR inhibition in response to starvation or Torin1 was preserved despite RalA or RalB knockdown; however, activation of autophagy was suppressed only in NRCMs depleted of RalB, implicating RalB as being required for mTOR-dependent cardiomyocyte autophagy. To define further the role of RalB in cardiomyocyte autophagy, we analyzed hearts from mice lacking RalGDS (Ralgds(-/-)), a guanine exchange factor (GEF) for the Ral family of small GTPases. RalGDS-null hearts were similar to wild-type (WT) littermates in terms of ventricular structure, contractile performance, and gene expression. However, Ralgds(-/-) hearts manifested a blunted growth response (p<0.05) to TAC-mediated pressure-overload stress. Ventricular chamber size and contractile performance were preserved in response to TAC in Ralgds(-/-) mice, and load-induced cardiomyocyte autophagy was suppressed. Interestingly, TAC-induced activation of the fetal gene program was similar in both genotypes despite the relative lack of hypertrophic growth in mutant hearts. Together, these data implicate RalGDS-mediated induction of autophagy and exocyst function as a critical feature of load-induced cardiac hypertrophy.
Assuntos
Autofagia/fisiologia , Cardiomegalia/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Animais , Autofagia/genética , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fator ral de Troca do Nucleotídeo Guanina/genéticaRESUMO
Stress-induced hypertrophic growth of the myocardium is a pathogenetic milestone in the progression of heart failure. Some evidence suggests that suppression of pathological cardiac hypertrophy per se is a viable target for therapeutic intervention, and cardiomyocyte autophagy is an attractive mechanism for consideration as a means of controlling the hypertrophic response. However, although considerable insights have been gleaned in the molecular mechanisms governing cardiomyocyte autophagy, many details critical to rational targeting of the response remain unknown. Among them, mechanisms underlying the adaptive and maladaptive features of autophagy are obscure. With time and further study, it is possible that this near-ubiquitous cardiac response to stress will emerge as a target for therapeutic manipulation.
Assuntos
Autofagia/fisiologia , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Animais , Autofagia/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Coração/efeitos dos fármacos , Coração/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologiaRESUMO
BACKGROUND: The antiepileptic drug carbamazepine (CBZ) is a potent inducer of human drug metabolism, resulting in serious interactions with many commonly prescribed drugs. The molecular mechanisms underlying this response are not well understood, however, and the spectrum of CBZ-inducible genes in human liver has not been thoroughly investigated. METHODS: The availability of liver ribonucleic acid from 2 epileptic patients treated with CBZ and from 7 control subjects enabled us to study the global induction response of drug-metabolizing enzymes, drug transporters, and nuclear receptors in vivo. RESULTS: Using expression profiling, we identified 64 significantly up-regulated transcripts but only 1 significantly down-regulated transcript (SLC22A5). We confirmed the induction of several genes that previously have been shown to be inducible by drugs in vitro, including multiple cytochrome P450 (CYP) genes in the CYP1A, CYP2A, CYP2B, CYP2C, and CYP3A subfamilies, as well as glutathione S-transferase A1, uridine diphosphate-glucuronosyltransferase 1As, the drug transporter ABCC2, and the nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor). Moreover, we identified a number of additional genes not previously known to be induced by CBZ, including CYP39A1, sulfotransferase 1A1, glutathione S-transferase Z1, and the drug transporters SLCO1A2, ABCG2, and ABCB7, as well as the glucocorticoid and aldosterone receptors. In transactivation studies in CV-1 cells, we demonstrated that both CBZ and its major metabolite, CBZ-10,11-epoxide, activate the nuclear receptor PXR in a concentration-dependent fashion and at therapeutic concentrations with 50% inhibitory concentration values of approximately 50 micromol/L. CONCLUSIONS: CBZ is a potent inducer of a broad spectrum of drug-metabolizing enzymes and drug transporters in the human liver, and these effects are mediated at least in part by activation of PXR.
Assuntos
Carbamazepina/farmacologia , Perfilação da Expressão Gênica/métodos , Fígado/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Carbamazepina/uso terapêutico , Sistema Enzimático do Citocromo P-450/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Fígado/metabolismo , Masculino , Desintoxicação Metabólica Fase I/genética , Desintoxicação Metabólica Fase II/genética , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica/genéticaRESUMO
Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.