Abstract 9176: Enhancing Lysosome Biogenesis Attenuates Bnip3-induced Cardiomyocyte Death
Rationale: Hypoxia-inducible pro-death protein, BNIP3 (Bcl2 and nineteen-kilodalton interacting protein-3) provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of cardiomyocyte autophagy accelerates Bnip3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating cardiomyocyte autophagy will attenuate Bnip3-induced cell death.
Methods and Results: Neonatal rat ventricular myocytes were adenovirally transduced with Bnip3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100nM) added at baseline and 24 hours; and cell death was assessed at 48 hours. Bnip3 expression increased autophagosome abundance 15-fold and caused a 3.5-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 increase in autophagosomes but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression); indicating autophagosome accumulation with impaired flux through the macroautophagy pathway. Bnip3 did not provoke lysosome permeabilization or alter lysosome pH; and BNIP3 protein did not localize to lysosomes by subcellular fractionation. Rather, Bnip3 expression caused a decline in lysosome numbers with decreased expression of lysosomal proteins-LAMP1 and LAMP2, indicating lysosome consumption and consequent autophagosome accumulation with Bnip3-induced autophagy. Forced expression of TFEB, a transcription factor that promotes lysosome biogenesis, increased lysosome numbers; reduced LC3-II/LC3-I ratio and p62 abundance, increased autolysosome to autophagosome ratio (indicating restored autophagic flux); and attenuated death in cells expressing Bnip3.
Conclusion: Bnip3 expression causes cardiomyocyte death with impaired autophagic flux secondary to reduced lysosome numbers indicating consumption in the autophagy process. Forced expression of TFEB, a lysosomal biogenesis factor, restores autophagic flux and attenuates Bnip3-induced cell death.
- © 2011 by American Heart Association, Inc.