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(Circulation. 2006;113:1235-1243.)
© 2006 American Heart Association, Inc.

Molecular Cardiology

Glycated Proteins Stimulate Reactive Oxygen Species Production in Cardiac Myocytes

Involvement of Nox2 (gp91phox)-Containing NADPH Oxidase

Min Zhang, MD, PhD; Ay Lin Kho, BSc; Narayana Anilkumar, PhD; Rakesh Chibber, PhD; Patrick J. Pagano, PhD; Ajay M. Shah, MD, FRCP; Alison C. Cave, PhD

From King’s College London (M.Z., A.L.K., N.A., R.C., A.M.S., A.C.C.), Cardiovascular Division, London, United Kingdom, and Hypertension and Vascular Research Division (P.J.P.), Henry Ford Hospital, Detroit, Mich.

Correspondence to Dr Alison C. Cave, Department of Cardiology, GKT School of Medicine, Bessemer Rd, London SE5 9PJ, UK. E-mail alison.cave{at}kcl.ac.uk

Received December 9, 2004; de novo received August 22, 2005; revision received December 6, 2005; accepted December 28, 2005.

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 µg/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-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-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.

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