Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury

Circ Res. 2020 Sep 11;127(7):e148-e165. doi: 10.1161/CIRCRESAHA.119.316388. Epub 2020 Jul 22.

Abstract

Rationale: Impaired autophagic flux contributes to ischemia/reperfusion (I/R)-induced cardiomyocyte death, but the underlying molecular mechanisms remain largely unexplored.

Objective: To determine the role of LAPTM4B (lysosomal-associated transmembrane protein 4B) in the regulation of autophagic flux and myocardial I/R injury.

Methods and results: 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.

Conclusions: 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.

Keywords: autophagosomes; autophagy; heart; lysosomes; mice.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagosomes / genetics
  • Autophagosomes / metabolism*
  • Autophagosomes / pathology
  • Autophagy*
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / metabolism
  • Cells, Cultured
  • Disease Models, Animal
  • Down-Regulation
  • Lysosomes / genetics
  • Lysosomes / metabolism*
  • Lysosomes / pathology
  • Male
  • Mechanistic Target of Rapamycin Complex 1 / metabolism*
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microtubule-Associated Proteins
  • Myocardial Infarction / genetics
  • Myocardial Infarction / metabolism*
  • Myocardial Infarction / pathology
  • Myocardial Infarction / prevention & control
  • Myocardial Reperfusion Injury / genetics
  • Myocardial Reperfusion Injury / metabolism*
  • Myocardial Reperfusion Injury / pathology
  • Myocardial Reperfusion Injury / prevention & control
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Rats, Sprague-Dawley
  • Sequestosome-1 Protein / metabolism
  • Signal Transduction

Substances

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • LC3 protein, rat
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Sequestosome-1 Protein
  • Sqstm1 protein, mouse
  • Sqstm1 protein, rat
  • TFEB protein, rat
  • Tcfeb protein, mouse
  • Mechanistic Target of Rapamycin Complex 1