Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase

An elevated level of tumor necrosis factor (TNF)-alpha is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-alpha activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-alpha and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-alpha-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-alpha with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-alpha-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-alpha-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-alpha-induced upregulation of interleukin (IL)-1 beta and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-alpha-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-alpha/beta and p65, degradation of IkappaB alpha, binding of NF-kappaB to its binding motif, and upregulation of IL-1 beta and VCAM-1 induced by TNF-alpha were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-alpha-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1 beta and VCAM-1, in human cardiomyocytes.