Abstract 3940: Direct Myocardial Actions of Advanced Glycation Endproducts (AGEs) on Cytosolic Calcium Transients Mediated Through Reactive Oxygen Species
Advanced Glycation End products (AGEs) are formed through non enzymatic glycation of proteins in conditions such as diabetes and may influence cellular function through actions on cell surface receptors, the most common of which (RAGE) belonging to the immunoglobulin superfamily of receptors. AGEs are implicated in the development of diabetic micro and macrovascular disease and are thought to be important in the development of heart failure in diabetic patients. It is unclear whether AGEs contribute to myocardial abnormalities observed in diabetes through direct myocardial actions. We have therefore investigated the effects of AGEs on neonatal rat cardiomyocytes (NRCM). Western blot analysis demonstrated that AGE receptors (RAGE) were strongly expressed in NRCM. Incubation of NRCM with glycated albumin (AGE) over 24 hrs was associated with a dose dependant reduction in calcium transient amplitude. At 1g/L AGE (a typical concentration in diabetics), a 52±12% reduction in cytosolic Ca2+ transient amplitude (n=12, P<0.01) was observed, associated with a 68% reduction in the mRNA expression levels of SERCA2a. Using immunohistochemistry, we observed internalisation of the RAGE receptor following exposure of external AGE and translocation of the p47phox subunit of NADPH oxidase from the cell membrane to the perinuclear area representing activation of NADPH oxidase. This was associated with a 24% (n=12, P<0.001) increase in reactive oxygen species (ROS) production measured through DCF fluorescence. Furthermore the decrease in Ca2+ transient amplitude observed following exposure to AGEs was completely abated in the presence of the NAPDH oxidase inhibitor apocyanin (10uM), an inhibitor of reactive oxygen species (ROS) formation. This data demonstrate the presence and functionality of AGE receptors in myocardium. RAGE activation through a NADPH-dependent ROS pathway suppresses cytosolic Ca2+ transients, with SERCA as a potential target in this context. This data provides insight into the mechanisms of myocardial damage in diabetes that occur independent of vascular disease through advanced glycation endproducts.