Excessive tumor necrosis factor-α (TNF-α) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-α 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-α release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-α expression of cardiomyocytes, the role of hypoxia inducible factor-1α (HIF-1α) in TNF-α regulation and potential secretory pathway of TNF-α. Elevated TNF-α expression and HIF-1α activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-α mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1α small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1α protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-α 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-α 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-α within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1α initiates expression of TNF-α, mediated by exosomes in cardiomyocytes.
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