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

Abstract

Background 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. Methods 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. Results 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. Conclusion BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.

Unraveling the Signaling Dynamics of Small Extracellular Vesicles in Cardiac Diseases

“…Other studies use this strategy to produce exosomes derived from cardiosphere-derived cells (CDCs) or MSCs which carry miR-181, regulating macrophage polarization and reducing inflammation when they are administered after MI [131,132]. There is also evidence that the therapeutic use of MSC-derived exosomes carrying specific proteins such as Itchy E3 Ubiquitin Protein Ligase (ITCH), Fibronectin Type III Domain Containing 5 (FNDC5) or Stromal Cell-Derived Factor 1 (SDF1) promote cardiac repair after their administration [133][134][135] (Figure 2A) (Table 2).…”