RESUMO
In cardiac ischemia-reperfusion injury, reactive oxygen species (ROS) generation and upregulation of the hypoxia-inducible protein BNIP3 result in mitochondrial permeabilization, but impairment in autophagic removal of damaged mitochondria provokes programmed cardiomyocyte death. BNIP3 expression and ROS generation result in upregulation of beclin-1, a protein associated with transcriptional suppression of autophagy-lysosome proteins and reduced activation of transcription factor EB (TFEB), a master regulator of the autophagy-lysosome machinery. Partial beclin-1 knockdown transcriptionally stimulates lysosome biogenesis and autophagy via mTOR inhibition and activation of TFEB, enhancing removal of depolarized mitochondria. TFEB activation concomitantly stimulates mitochondrial biogenesis via PGC1α induction to restore normally polarized mitochondria and attenuate BNIP3- and hypoxia-reoxygenation-induced cell death. Conversely, overexpression of beclin-1 activates mTOR to inhibit TFEB, resulting in declines in lysosome numbers and suppression of PGC1α transcription. Importantly, knockdown of endogenous TFEB or PGC1α results in a complete or partial loss, respectively, of the cytoprotective effects of partial beclin-1 knockdown, indicating a critical role for both mitochondrial autophagy and biogenesis in ensuring cellular viability. These studies uncover a transcriptional feedback loop for beclin-1-mediated regulation of TFEB activation and implicate a central role for TFEB in coordinating mitochondrial autophagy with biogenesis to restore normally polarized mitochondria and prevent ischemia-reperfusion-induced cardiomyocyte death.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Morte Celular/genética , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Accumulating evidence attests to a prosurvival role for autophagy under stress, by facilitating removal of damaged proteins and organelles and recycling basic building blocks, which can be utilized for energy generation and targeted macromolecular synthesis to shore up cellular defenses. These observations are difficult to reconcile with the dichotomous prosurvival and death-inducing roles ascribed to macroautophagy in cardiac ischemia and reperfusion injury, respectively. A careful reexamination of 'flux' through the macroautophagy pathway reveals that autophagosome clearance is markedly impaired with reperfusion (reoxygenation) in cardiomyocytes following an ischemic (hypoxic) insult, resulting from reactive oxygen species (ROS)-mediated decline in LAMP2 and increase in BECN1 abundance. This results in impaired autophagy that is 'ineffective' in protecting against cell death with ischemia-reperfusion injury. Restoration of autophagosome clearance and by inference, 'adequate' autophagy, attenuates reoxygenation-induced cell death.
Assuntos
Autofagia , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteína Beclina-1 , Redes Reguladoras de Genes/genética , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation and is implicated in causing cell death. We examined flux through the macroautophagy pathway as a determinant of the discrepant outcomes in cardiomyocyte cell death in this setting. METHODS AND RESULTS: Reversible left anterior descending coronary artery ligation was performed in mice with cardiomyocyte-restricted expression of green fluorescent protein-tagged microtubule-associated protein light chain-3 to induce ischemia (120 minutes) or ischemia/reperfusion (30-90 minutes) with saline or chloroquine pretreatment (n=4 per group). Autophagosome clearance, assessed as the ratio of punctate light chain-3 abundance in saline to chloroquine-treated samples, was markedly impaired with ischemia/reperfusion compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes compared with hypoxia alone, markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-green fluorescent protein tandem-tagged light chain-3 expression), and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species and mitochondrial permeabilization, leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by reactive oxygen species-mediated BECLIN-1 upregulation and a reduction in lysosome-associated membrane protein-2, a critical determinant of autophagosome-lysosome fusion. Restoration of lysosome-associated membrane protein-2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and to attenuate cell death after hypoxia-reoxygenation. CONCLUSION: Ischemia/reperfusion injury impairs autophagosome clearance mediated in part by reactive oxygen species-induced decline in lysosome-associated membrane protein-2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.
