Abstract 9176: Enhancing Lysosome Biogenesis Attenuates Bnip3-induced Cardiomyocyte Death
Abstract 9176: Enhancing Lysosome Biogenesis Attenuates Bnip3-induced Cardiomyocyte Death
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DOI：
10.1161/circ.124.suppl_21.a9176
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发表时间：
2011-11
期刊：
影响因子：
37.8
通讯作者：
Xiucui Ma;Rebecca J. Godar;A. Diwan
中科院分区：
文献类型：
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作者：
Xiucui Ma;Rebecca J. Godar;A. Diwan
基本原理：缺氧诱导的促死亡蛋白 BNIP3（Bcl2 和十九道尔顿相互作用蛋白 3）会引发线粒体通透，导致缺血再灌注损伤中的心肌细胞死亡。抑制心肌细胞自噬会阻止受损线粒体的去除，从而加速 Bnip3 诱导的细胞死亡。我们测试了刺激心肌细胞自噬会减弱 Bnip3 诱导的细胞死亡的假设。方法和结果：用 Bnip3（或 LacZ 作为对照；感染复数 = 100）腺病毒转导新生大鼠心室肌细胞；在基线和 24 小时添加雷帕霉素 (100nM) 刺激自噬；并在48小时评估细胞死亡。与对照相比，BNip3 表达使自噬体丰度增加 15 倍，并导致心肌细胞死亡增加 3.5 倍。雷帕霉素处理 Bnip3 表达细胞导致自噬体数量进一步增加，但不影响细胞死亡。 Bnip3 表达导致自噬体结合 LC3-II 和 p62 的积累；自噬体增加，但自溶酶体不增加（通过双荧光 mCherry-GFP-LC3 表达进行评估）；表明自噬体积累，通过巨自噬途径的通量受损。 Bnip3 不会引起溶酶体通透或改变溶酶体 pH 值；并且 BNIP3 蛋白没有通过亚细胞分级定位到溶酶体。相反，Bnip3 表达导致溶酶体数量下降，溶酶体蛋白 LAMP1 和 LAMP2 表达减少，表明 Bnip3 诱导的自噬导致溶酶体消耗和随后的自噬体积累。强制表达TFEB（一种促进溶酶体生物发生的转录因子），增加溶酶体数量； LC3-II/LC3-I 比率和 p62 丰度降低，自噬溶酶体与自噬体比率增加（表明自噬通量恢复）；并减少表达 Bnip3 的细胞的死亡。结论：BNip3 表达导致心肌细胞死亡，自噬通量受损，继发于溶酶体数量减少，表明自噬过程中的消耗。 TFEB（一种溶酶体生物发生因子）的强制表达可恢复自噬通量并减弱 Bnip3 诱导的细胞死亡。
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.