CAS Key Laboratory of Tissue Microenvironment and Tumor, Laboratory of Molecular Cardiology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences (CAS), CAS (S.G., J.T., Q.L., S.L., Y.Z., J.L., W.B., P.S., X.L., H.-T.Y.).
Program of Stem Cells and Regenerative Medicine, Zhongshan School of Medicine and The Fifth Affiliated Hospital, Sun Yat-Sen University, Guangdong, P.R. China (S.G., N.C.).
Circ Res. 2020 Sep 11;127(7):e148-e165. doi: 10.1161/CIRCRESAHA.119.316388. Epub 2020 Jul 22.
Impaired autophagic flux contributes to ischemia/reperfusion (I/R)-induced cardiomyocyte death, but the underlying molecular mechanisms remain largely unexplored.
To determine the role of LAPTM4B (lysosomal-associated transmembrane protein 4B) in the regulation of autophagic flux and myocardial I/R injury.
LAPTM4B was expressed in murine hearts but downregulated in hearts with I/R (30 minutes/2 hours) injury and neonatal rat cardiomyocytes with hypoxia/reoxygenation (6 hours/2 hours) injury. During myocardial reperfusion, LAPTM4B-knockout (LAPTM4B) mice had a significantly increased infarct size and lactate dehydrogenase release, whereas adenovirus-mediated LAPTM4B-overexpression was cardioprotective. Concomitantly, LAPTM4B mice showed higher accumulation of the autophagy markers LC3-II (microtubule-associated protein 1A/1B-light chain 3), but not P62, in the I/R heart, whereas they did not alter chloroquine-induced further increases of LC3-II and P62 in both sham and I/R hearts. Conversely, LAPTM4B-overexpression had opposite effects. The hypoxia/reoxygenation-reduced viability of neonatal rat cardiomyocytes, ratio of autolysosomes/autophagosomes, and function of lysosomes were further decreased by LAPTM4B-knockdown but reversed by LAPTM4B-overexpression. Moreover, the LAPTM4B-overexpression-mediated benefits were abolished by knockdown of lysosome-associated membrane protein-2 (an autophagosome-lysosome fusion protein) in vivo and by the autophagy inhibitor bafilomycin A1 in vivo. In contrast, rapamycin (Rapa) successfully restored the impaired autophagic flux in LAPTM4B mice and the subsequent myocardial I/R injury. Mechanistically, LAPTM4B regulated the activity of mTORC1 (mammalian target of rapamycin complex 1) via interacting with mTOR through its EC3 (extracelluar) domain. Thus, mTORC1 was overactivated in LAPTM4B mice, leading to the repression of TFEB (transcription factor EB), a master regulator of lysosomal and autophagic genes, during myocardial I/R. The mTORC1 inhibition or TFEB-overexpression rescued the LAPTM4B-induced impairment in autophagic flux and I/R injury, whereas TFEB-knockdown abolished the LAPTM4B-overexpression-mediated recovery of autophagic flux and cardioprotection.
The downregulation of LAPTM4B contributes to myocardial I/R-induced impairment of autophagic flux via modulation of the mTORC1/TFEB pathway. Graphic Abstract: A graphic abstract is available for this article.
自噬通量的损伤会导致缺血/再灌注（I/R）引起的心肌细胞死亡，但其中的分子机制仍很大程度上未被探索。
确定 LAPTM4B（溶酶体相关跨膜蛋白 4B）在调节自噬通量和心肌 I/R 损伤中的作用。
LAPTM4B 在鼠心中表达，但在 I/R（30 分钟/2 小时）损伤的心脏和缺氧/复氧（6 小时/2 小时）损伤的新生大鼠心肌细胞中下调。在心肌再灌注期间，LAPTM4B 敲除（LAPTM4B）小鼠的梗死面积和乳酸脱氢酶释放明显增加，而腺病毒介导的 LAPTM4B 过表达具有心脏保护作用。同时，LAPTM4B 小鼠在 I/R 心脏中显示出更高的自噬标记物 LC3-II（微管相关蛋白 1A/1B-轻链 3）的积累，但 P62 没有增加，而在 sham 和 I/R 心脏中，氯喹进一步增加 LC3-II 和 P62 没有改变。相反，LAPTM4B 过表达则产生相反的效果。LAPTM4B 敲低进一步降低了缺氧/复氧减少的新生大鼠心肌细胞活力、自噬溶酶体/自噬体的比值和溶酶体的功能，但 LAPTM4B 过表达则逆转了这一结果。此外，体内 LAPTM4B 敲低和体内自噬抑制剂巴弗洛霉素 A1 均消除了 LAPTM4B 过表达介导的益处。相反，雷帕霉素（Rapa）成功恢复了 LAPTM4B 小鼠受损的自噬通量和随后的心肌 I/R 损伤。在机制上，LAPTM4B 通过其 EC3（细胞外）结构域与 mTOR 相互作用，调节 mTORC1（雷帕霉素靶蛋白复合物 1）的活性。因此，在心肌 I/R 期间，LAPTM4B 小鼠中 mTORC1 过度激活，导致转录因子 EB（TFEB）的抑制，TFEB 是溶酶体和自噬基因的主要调节因子。mTORC1 抑制或 TFEB 过表达挽救了 LAPTM4B 引起的自噬通量损伤和 I/R 损伤，而 TFEB 敲低则消除了 LAPTM4B 过表达介导的自噬通量恢复和心脏保护作用。
LAPTM4B 的下调通过调节 mTORC1/TFEB 通路导致心肌 I/R 诱导的自噬通量损伤。
