Glycated proteins stimulate reactive oxygen species production in cardiac myocytes - Involvement of Nox2 (gp91phox)-containing NADPH oxidase

Background - Nonenzymatic glycation that results in the production of early-glycation Amadori-modified proteins and advanced-glycation end products may be important in the pathogenesis of diabetic complications. However, the effects of early-glycated proteins, such as glycated serum albumin (Gly-BSA), are poorly defined. In this study, we investigated the effects of Gly-BSA on reactive oxygen species (ROS) production by cardiomyocytes. Methods and Results - Cultured neonatal rat cardiomyocytes were incubated with Gly-BSA or vehicle ( bovine serum albumin [BSA]) for up to 48 hours. Gly-BSA dose-dependently increased in situ ROS production (whole-cell dichlorodihydrofluorescein fluorescence), with an optimum effect at 400 mu g/mL after 24-hour incubation (152 +/- 10% versus BSA 100%; P < 0.01). Treatment with the NADPH oxidase inhibitor apocynin, a Nox2 (gp91phox) antisense oligonucleotide ( Nox2 AS), or the peptide gp91ds-tat significantly reduced Gly-BSA - induced ROS production at 24 hours (68.5 +/- 2.2%, 61.4 +/- 8.3%, and 53.2 +/- 5.4% reduction, respectively). NADPH-dependent activity in cell homogenates was also significantly increased by Gly-BSA at 24 hours (161 +/- 8% versus BSA) and was inhibited by diphenyleneiodonium, apocynin, NOX2AS, and the protein kinase C inhibitor bisindolylmaleimide I but not by a nitric oxide synthase inhibitor or mitochondrial inhibitors. Furthermore, bisindolylmaleimide I prevented Gly-BSA stimulated Rac1 translocation, an essential step for NADPH oxidase activation. Gly-BSA - induced increases in ROS were associated with apocynin-inhibitable nuclear translocation of nuclear factor-kappa B and an increase in atrial natriuretic factor mRNA expression. Conclusions - Gly-BSA stimulates cardiomyocyte ROS production through a protein kinase C - dependent activation of a Nox2-containing NADPH oxidase, which results in nuclear factor-kappa B activation and upregulation of atrial natriuretic factor mRNA. These findings suggest that early-glycated Amadori products may play a role in the development of diabetic heart disease.