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

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 lysosomal-associated transmembrane protein 4B (LAPTM4B) 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 (NRCMs) 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, while adenovirus-mediated LAPTM4B-overexpression was cardioprotective. Concomitantly, LAPTM4B-/- mice showed higher accumulation of the autophagy markers LC3-II, but not P62, in the I/R heart, while 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 NRCMs, 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 vitro and by the autophagy inhibitor bafilomycin A1 in vivo. In contrast, rapamycin successfully restored the impaired autophagic flux in LAPTM4B-/- mice and the subsequent myocardial I/R injury. Mechanistically, LAPTM4B regulated the activity of mammalian target of rapamycin complex 1 (mTORC1) via interacting with mTOR through its EC3 domain. Thus, mTORC1 was overactivated in LAPTM4B-/- mice, leading to the repression of transcription factor EB (TFEB), 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.

Neurotrophin-3 contributes to benefits of human embryonic stem cell-derived cardiovascular progenitor cells against reperfused myocardial infarction

“…The myocardial I/R model was induced as previously described 30 . Briefly, male C57BL/6 mice at 10‐12 weeks‐old were anesthetized via intraperitoneal injection (i.p.)…”

“…Recombinant adenoviruses (Ad) expressing short hairpin RNA were prepared as reported previously 30,33,34 . Detailed experimental methods are available in the Supplemental Materials section.…”

“…Surgical procedures and adenoviral delivery were carried out as previously described 30,34 . Briefly, the surgery process was the same as described above and a 30‐gauge needle containing 30 μL of diluted the scramble (sh‐scramble/negative control, shNC) or the short hairpin RNA against mouse NT‐3 (shNT‐3) (3 × 10 10 pfu/mL) was injected into the myocardium of LV at 3 sites.…”

“…Infarct size was measured by using 1% w/v of 2, 3, 5‐triphenyltetra‐zolium chloride (TTC, T8877, Sigma‐Aldrich) as previously described 30 . Detailed experimental methods are available in the Supplemental Materials section.…”

Guidelines for Regulated Cell Death Assays: A Systematic Summary, A Categorical Comparison, A Prospective

“…The membrane expands and bends, and then a spherical autophagosome is generated. Using electron microscopic examination of the obverse, it has been found that the diameter of autophagosomes ranges from 0.5 to 1.5 μm in mammals and ∼0.4 to 0.9 μm in yeast ( Takeshige et al, 1992 ; Gu et al, 2020 ). Besides autophagosomes, autophagy precursors (which include the cytoplasm, organelles, or bacteria) and autophagolysosomes (autophagosomes that bind to the lysosome) are also vital subcellular structures.…”