Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.
Pretreatment of berbamine protects the heart from ischemia/reperfusion (I/R) injury. However it is unknown whether it has cardioprotection when given at the onset of reperfusion (postconditioning (PoC)), a protocol with more clinical impact. Autophagy is upregulated in I/R myocardium and exacerbates cardiomyocyte death during reperfusion. However, it is unknown whether the autophagy during reperfusion is regulated by berbamine. Here we investigated whether berbamine PoC (BMPoC) protects the heart through regulation of autophagy by analyzing the effects of BMPoC on infarct size and/or cell death, functional recovery and autophagy in perfused rat hearts and isolated cardiomyocytes subjected to I/R. Berbamine from 10 to 100 nM given during the first 5 min of reperfusion concentration-dependently improved post-ischemic myocardial function and attenuated cell death. Similar protections were observed in cardiomyocytes subjected to simulated I/R. Meanwhile, BMPoC prevented I/R-induced impairment of autophagosome processing in cardiomyocytes, characterized by increased LC3-II level and GFP-LC3 puncta, and decreased p62 degradation. Besides, lysosomal inhibitor chloroquine did not induce additional increase of LC3-II and P62 abundance after I/R but it reversed the effects of BMPoC in those parameters in cardiomyocytes, suggesting that I/R-impaired autophagic flux is restored by BMPoC. Moreover, I/R injury was accompanied by enhanced expression of Beclin 1, which was significantly inhibited by BMPoC. In vitro and in vivo adenovirus-mediated knockdown of Beclin 1 in myocardium and cardiomyocytes restored I/R-impaired autophagosome processing, associated with an improvement of post-ischemic recovery of myocardial contractile function and a reduction of cell death, but it did not have additive effects to BMPoC. Conversely, overexpression of Beclin 1 abolished the cardioprotection of BMPoC as did by overexpression of an essential autophagy gene Atg5. Furthermore, BMPoC-mediated cardioprotection was abolished by a specific Akt1/2 inhibitor A6730. Our results demonstrate that BMPoC confers cardioprotection by modulating autophagy during reperfusion through the activation of PI3K/Akt signaling pathway.
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.
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