RESUMEN
The deleterious effects of statins on skeletal muscle are well known, but the mechanism associated with these effects remains unresolved. Statins are associated with mitochondrial damage, which may contribute to muscle myopathy. Here we demonstrate that simvastatin induces mitophagy in skeletal muscle cells and hypothesized that attenuating this process by silencing the mitophagy adapter p62/sequestosome-1 (SQSTM1) might mitigate myotoxicity. Surprisingly, silencing p62/SQSTM1 in differentiated C2C12 muscle cells exacerbated rather than attenuated myotoxicity. This inhibition of mitophagy in the face of statin challenge correlated with increased release of cytochrome c to the cytosol, activation of caspase-3, and lactate dehydrogenase (LDH) release. Correspondingly, targeted knockdown of Parkin, a canonical E3 ubiquitin ligase important for mitophagy, mirrored the effects of p62/SQSTM1 silencing. To corroborate these findings in vivo, we treated Parkin knockout mice with simvastatin for 2 wk. In line with our findings in vitro, these mitophagy-compromised mice displayed reduced spontaneous activity, loss of grip strength, and increased circulating levels of muscle damage marker LDH. Our findings demonstrate that mitophagy is an important mechanism to resist statin-induced skeletal muscle damage.-Ramesh, M., Campos, J. C., Lee, P., Song, Y., Hernandez, G., Sin, J., Tucker, K. C., Saadaeijahromi, H., Gurney, M., Ferreira, J. C. B., Andres, A. M. Mitophagy protects against statin-mediated skeletal muscle toxicity.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Mitofagia/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Simvastatina/farmacología , Animales , Caspasa 3/metabolismo , Línea Celular , Citocromos c/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Ratones Noqueados , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Interferencia de ARN , Proteína Sequestosoma-1/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Autophagy is regulated by nutrient and energy status and plays an adaptive role during nutrient deprivation and ischemic stress. Metabolic syndrome (MetS) is a hypernutritive state characterized by obesity, dyslipidemia, elevated fasting blood glucose levels, and insulin resistance. It has also been associated with impaired autophagic flux and larger-sized infarcts. We hypothesized that diet-induced obesity (DIO) affects nutrient sensing, explaining the observed cardiac impaired autophagy. We subjected male friend virus B NIH (FVBN) mice to a high-fat diet, which resulted in increased weight gain, fat deposition, hyperglycemia, insulin resistance, and larger infarcts after myocardial ischemia-reperfusion. Autophagic flux was impaired after 4 wk on a high-fat diet. To interrogate nutrient-sensing pathways, DIO mice were subjected to overnight fasting, and hearts were processed for biochemical and proteomic analysis. Obese mice failed to upregulate LC3-II or to clear p62/SQSTM1 after fasting, although mRNA for LC3B and p62/SQSTM1 were appropriately upregulated in both groups, demonstrating an intact transcriptional response to fasting. Energy- and nutrient-sensing signal transduction pathways [AMPK and mammalian target of rapamycin (mTOR)] also responded appropriately to fasting, although mTOR was more profoundly suppressed in obese mice. Proteomic quantitative analysis of the hearts under fed and fasted conditions revealed broad changes in protein networks involved in oxidative phosphorylation, autophagy, oxidative stress, protein homeostasis, and contractile machinery. In many instances, the fasting response was quite discordant between lean and DIO mice. Network analysis implicated the peroxisome proliferator-activated receptor and mTOR regulatory nodes. Hearts of obese mice exhibited impaired autophagy, altered proteome, and discordant response to nutrient deprivation.
Asunto(s)
Autofagia , Ayuno/metabolismo , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Obesidad/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Metabolismo Energético , Masculino , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Infarto del Miocardio/etiología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Obesidad/complicaciones , Obesidad/patología , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Mapas de Interacción de Proteínas , Proteolisis , Proteómica/métodos , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
Previously we reported that Myd88 contributed to tumor progression. To begin to decipher what may be inducing Myd88 dependent signaling we focused on proteins that could function as damage associated molecular pattern molecules (DAMPs) since DAMPs have been reported to be secreted by tumors, and certain DAMPs mediate effects through toll-like receptors. A screen of mammary carcinoma for DAMP expression showed HMGB1 and HSP60 were significantly elevated relative to normal mammary epithelium, and targeting these DAMPs, or receptors for these DAMPs influenced growth of tumor cells. Moreover, analysis using a Myd88 inhibitory peptide suggested that HMGB1 mediated its effects in a Myd88 dependent manner, and inhibiting Myd88 function decreased HMGB1 and HSP60 gene expression. Collectively, these data suggest that HMGB1 and HSP60 contribute to growth of mammary carcinoma cells, HMGB1 accomplishes this, at least in part, through Myd88 dependent signaling, and these DAMPs are expressed in a Myd88 dependent manner.
Asunto(s)
Proliferación Celular , Chaperonina 60/genética , Proteína HMGB1/genética , Factor 88 de Diferenciación Mieloide/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Chaperonina 60/inmunología , Chaperonina 60/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteína HMGB1/inmunología , Proteína HMGB1/metabolismo , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/antagonistas & inhibidores , Factor 88 de Diferenciación Mieloide/metabolismo , Péptidos/farmacología , Interferencia de ARN , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
Given that adverse remodeling is the leading cause of heart failure and death in the USA, there is an urgent unmet need to develop new methods in dealing with this devastating disease. Here we evaluated the efficacy of a short-course glucagon-like peptide-1 receptor agonist therapy-specifically 2-quinoxalinamine, 6,7-dichloro-N-(1,1-dimethylethyl)-3-(methylsulfonyl)-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB; aka Compound 2) - in attenuating adverse LV remodeling. We also examined the role, if any, of mitochondrial turnover in this process. Wild-type, Parkin knockout and MitoTimer-expressing mice were subjected to permanent coronary artery ligation, then treated briefly with DMB. LV remodeling and cardiac function were assessed by histology and echocardiography. Autophagy and mitophagy markers were examined by western blot and mitochondrial biogenesis was inferred from MitoTimer protein fluorescence and qPCR. We found that DMB given post-infarction significantly reduced adverse LV remodeling and the decline of cardiac function. This paralleled an increase in autophagy, mitophagy and mitochondrial biogenesis. The salutary effects of the drug were lost in Parkin knockout mice, implicating Parkin-mediated mitophagy as part of its mechanism of action. Our findings suggest that enhancing Parkin-associated mitophagy and mitochondrial biogenesis after infarction is a viable target for therapeutic mitigation of adverse remodeling.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Mitocondrias Cardíacas/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Quinoxalinas/administración & dosificación , Ubiquitina-Proteína Ligasas/genética , Remodelación Ventricular/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Pruebas de Función Cardíaca , Masculino , Ratones , Ratones Noqueados , Mitofagia , Infarto del Miocardio/etiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Quinoxalinas/farmacología , RatasRESUMEN
Mitophagy plays a major role in heart physiology. Impairment of Parkin-dependent mitophagy in heart is known to be deleterious. Obesity is a known cardiovascular risk factor. Impaired autophagy has been reported in models of obesity or hyperlipidemia/hypercholesterolemia; however less is known regarding obesity and mitophagy. The aim of this study was to evaluate the regulation of Parkin expression in hearts of mice fed a high fat diet. Interestingly, we found a significant decrease in Parkin protein in hearts of HFD mice compared those fed a low-fat diet. This was associated with mitochondrial dysfunction in the context of ischemia/reperfusion (I/R). This downregulation was not associated with a decrease in Parkin mRNA expression. We did not detect any change in the degradation rate of Parkin and only a slight decrease in its translation. The reduction of Parkin protein abundance in HFD hearts remains a mystery and will need further studies. However, Parkin depletion in the setting of obesity may contribute to cardiovascular risk.
RESUMEN
Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.
Asunto(s)
Apéndice Atrial/metabolismo , Procedimientos Quirúrgicos Cardíacos , Puente Cardiopulmonar , ADN Mitocondrial/metabolismo , Mitofagia , Daño por Reperfusión Miocárdica/metabolismo , Biogénesis de Organelos , ARN Mensajero/metabolismo , Anciano , Proteínas de Ciclo Celular , Puente de Arteria Coronaria , ADN/metabolismo , Daño del ADN , Femenino , GTP Fosfohidrolasas/metabolismo , Implantación de Prótesis de Válvulas Cardíacas , Humanos , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Polirribosomas , Factor de Transcripción TFIIIA/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Previous data obtained in our laboratory suggested that there may be constitutive signaling through the myeloid differentiation primary response gene 88 (Myd88)-dependent signaling cascade in murine mammary carcinoma. Here, we extended these findings by showing that, in the absence of an added Toll-like receptor (TLR) agonist, the myddosome complex was preformed in 4T1 tumor cells, and that Myd88 influenced cytoplasmic extracellular signal-regulated kinase (Erk)1/Erk2 levels, nuclear levels of nuclear factor-kappaB (NFκB) and signal transducer and activator of transcription 5 (STAT5), tumor-derived chemokine (C-C motif) ligand 2 (CCL2) expression, and in vitro and in vivo tumor growth. In addition, RNA-sequencing revealed that Myd88-dependent signaling enhanced the expression of genes that could contribute to breast cancer progression and genes previously associated with poor outcome for patients with breast cancer, in addition to suppressing the expression of genes capable of inhibiting breast cancer progression. Yet, Myd88-dependent signaling in tumor cells also suppressed expression of genes that could contribute to tumor progression. Collectively, these data revealed a multifaceted role for Myd88-dependent signaling in murine mammary carcinoma.