Bone marrow mesenchymal stem cell-derived exosomes carrying E3 ubiquitin ligase ITCH attenuated cardiomyocyte apoptosis by mediating apoptosis signal-regulated kinase-1.

Abstract

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been verified to perform an effective role in treating acute myocardial infarction (MI). Herein, we aimed to investigate the role of BMSC-derived exosomes carrying itchy E3 ubiquitin ligase (ITCH) in MI and the underlying mechanism involved. BMSCs were isolated from rat bone marrow and exosomes were extracted using ultra-high speed centrifugation. Exosomes uptake by cardiomyoblasts was determined by PKH-67 staining. Rat cardiomyoblast cell line H9C2 was stimulated by hypoxia, as in vitro model. H9C2 cell apoptosis was determined by flow cytometry. Cell viability was examined by cell counting kit-8 assay. Western blotting was performed to determine the expression of ITCH, apoptosis signal-regulated kinase-1 (ASK1), and apoptotic-related protein cleaved-caspase 3 and Bcl-2. Ubiquitination assay was employed to measure the levels of ASK1 ubiquitination. Exosomes derived from BMSCs were endocytosed by H9C2 cardiomyoblasts. BMSC-Exo downregulated cleaved-caspase 3 expression, upregulated Bcl-2 expression, further suppressed H9C2 cell apoptosis under hypoxia treatment, meanwhile the expression of ASK1 was downregulated, and similar effects were observed in BMSC-cultured supernatant (BMSC-S). However, these effects were reversed by exosome inhibitor GW4869. BMSC-derived exosomes enhanced ASK1 ubiquitination and degradation. Mechanically, exosomes of ITCH-knockdown BMSCs promoted H9C2 cell apoptosis and upregulated ASK1 expression. Overexpression of ITCH enhanced ASK1 ubiquitination and degradation. Further, the protein expression of ASK1 and cleaved-caspase 3 was upregulated and Bcl-2 protein expression was downregulated. ITCH-knockdown BMSC exosomes increased cardiomyoblast apoptosis. BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.