Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: Initiated by hypoxia inducible factor 1α, presented by exosomes

Excessive tumor necrosis factor-alpha (TNF-alpha) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-alpha is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-alpha release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-alpha expression of cardiomyocytes, the role of hypoxia inducible factor-1 alpha (HIF-1 alpha) in TNF-alpha regulation and potential secretory pathway of TNF-alpha. Elevated TNF-alpha expression and HIF-1 alpha activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofiuorescence. TNF-alpha mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1 alpha small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1 alpha protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-alpha gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-alpha promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-alpha within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1 alpha initiates expression of TNF-alpha, mediated by exosomes in cardiomyocytes. (C) 2012 Elsevier Ltd. All rights reserved.