Regulation of the Transcription Factor EB-PGC1α Axis by Beclin-1 Controls Mitochondrial Quality and Cardiomyocyte Death under Stress

Abstract

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 PGC1␣ 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 PGC1␣ transcription. Importantly, knockdown of endogenous TFEB or PGC1␣ 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. P reservation of healthy mitochondria is essential for energy generation and maintenance of contractile function in cardiac myocytes (1). In cardiac ischemia-reperfusion (IR) injury, mitochondrial permeabilization results in activation of programmed cell death pathways and cardiomyocyte loss (2). Removal of damaged mitochondria by macroautophagy, a lysosomal degradative pathway, is essential to prevent cardiomyocyte death and limit myocardial infarct size (3, 4). Cardiomyocyte autophagy is upregulated with IR injury (5), but autophagosome processing is impaired early after reperfusion, which prevents autophagic removal of damaged mitochondria (6). The hypoxic insult also provokes transcriptional induction of BNIP3 (Bcl2 and nineteen-kilodalton interacting protein 3), a prodeath Bcl2 family protein (7, 8) which is targeted to and permeabilizes mitochondria (9-11) and triggers cardiomyocyte death in IR injury (12). While BNIP3 has been suggested to facilitate mitochondrial autophagy by functioning as an adaptor to sequester damaged mitochondria within autophagosomes (13,14), increased BNIP3 expression provokes declines in lysosome numbers, with impaired autophagic flux, resulting in accumulation of damaged mitochondria and cardiomyocyte death (15). These observations implicate a failure of the autophagy-lysosome machinery to clear damaged mitochondria as a cause of cell death with IR inju...

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

“…Indeed, we have previously found that transcriptional suppression of the autophagy machinery with reperfusion-induced accumulation of BECN1 explains the lack of a protective role of the observed extent of autophagy induction in cardiac ischemia-reperfusion injury. 36,68 Further insights into this process could be gleaned via careful examination of the effects of intermittent fasting on the transcriptional program of the injured myocardium, in future studies.…”

“…Adenoviral particles for shRNA targeting Becn1 (shBecn1), 68 shLacZ and exogenous expression of human TFEB have been previously described. 77 Generation of conditional cardiac Tfeb transgenic mice The coding sequence of mouse Tfeb (translated from transcript variant 2, NP_001155194.1, that encodes a protein similar in size to human TFEB) with a N-terminal FLAG tag was cloned into the a-Myosin Heavy Chain promoter -TetOMin construct generously provided by Jeffery Robbins, Cincinnati Children's Hospital.…”

“…68,78 Transmission electron microscopy was performed on NRCMs fixed in a modified Karnovsky's fixative, as previously described. 68,78 Hypoxia was induced by culturing cells in an oxygen control cabinet (Coy Laboratories, Grass Lake, MI) mounted within an incubator and equipped with oxygen sensor for continuous oxygen level monitoring. A mixture of 95% nitrogen and 5% CO2 was infused and oxygen levels in the chamber were monitored and maintained at <1%, as described.…”

“…For assessment of mitochondrial polarization, NRCMs were incubated with JC-1 (10 mg/mL for 10 min) at 37 C in 5% CO 2 , the cells were trypsinized and subjected to flow cytometry on FACScan instrument (Becton-Dickinson, NJ), as previously described. 68,78 Cyflogic software (CyFlo) was employed to analyze 20,000 events per run. Mitochondrial DNA content was assessed with quantitative PCR analysis as previously described.…”

Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size

“…Myocardial Ischemia Reperfusion Studies-The methods of Weinheimer et al (60) were used. Mice were subjected to reversible left anterior descending (LAD) coronary artery occlusion to induce ischemia for 30 min, followed by 24 h of reperfusion.…”