Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death.
Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.
Center for Cardiovascular Research, Division of Cardiology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
Xiucui
X
Ma
Rebecca J
RJ
Godar
Haiyan
H
Liu
Abhinav
A
Diwan
D000143:Acids / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001343:Autophagy / Q000187:drug effects; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020123:Sirolimus / Q000494:pharmacology
D000143:Acids; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D020123:Sirolimus; C505650:BNIP3 protein, rat; C526109:Anxa3 protein, rat; C574124:TFEB protein, rat