Hydrogen peroxide and differential activation of nitric oxide synthases in cardiac myocytes

have important health benefits, recent clinical trials suggested that supplemental intake of Se above the adequate level may raise the risk of type 2 diabetes mellitus. However, the molecular mechanisms for the effect of dietary Se on the development of this disease are not understood. We examined the contribution of selenoproteins to increased risk of developing diabetes using animal models. Our data show that mice maintained on a Se-supplemented diet develop signs of hyperinsulinemia and have decreased insulin sensitivity. These effects are accompanied by elevated expression of a select group of selenoproteins. We also observed that reduced synthesis of selenoproteins caused by overexpression of a mutant selenocysteine tRNA promotes glucose intolerance and leads to a diabeteslike phenotype. The role of Se in the development of diabetes may be mediated, in part, by ER-resident selenoproteins. However, functions of these proteins are mostly not known. We will present data on functional characterization of these proteins. Overall, our findings suggest that changes in selenoprotein expression may dysregulate glucose homeostasis, consistent with the role of selenoproteins in the development of diabetes. doi:10.1016/j.freeradbiomed.2012.08.114

Symposium 10: Reactive Nitrogen Species and Reactive Oxygen Species in Cardiac Myocyte Signal Transduction Symp. 10.1

Hydrogen peroxide and differential activation of nitric oxide synthases in cardiac myocytes J. Sartoretto, H. Kalwa, T. Michel* Harvard Medical School, USA Nitric oxide and hydrogen peroxide are synthesized within cardiac myocytes and play key roles in the modulation of cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase. We have used biosensors and chemical sensors to detect hydrogen peroxide and nitric oxide in cardiac myocytes, and have discovered differential regulation of eNOS and nNOS via receptor-modulated signaling pathways involving hydrogen peroxide. Activation of the L-type calcium channel by hydrogen peroxide plays a critical role in activation of eNOS. This seminar will present new data exploring the interplay of reactive nitrogen and reactive oxygen species in the modulation of cardiac myocyte function and cellular signaling responses. Keywords: cardiac myocyte, nitric oxide synthase, hydrogen peroxide, biosensor

Symp. 10.2

In vivo dissection of redox-regulated protein kinase activation in the heart P. Eaton* King's College London, UK Antioxidant intervention trials aimed at limiting injury during human diseases, including those of the cardiovascular system, have generally failed. This may be because these reducing compounds interfere with everyday homeostatic cellular responses that actually require regulated oxidant production. Instead of simply causing injury, oxidants are now known to serve as signalling molecules transmitting information. An integral component of oxidant-induced signalling is the posttranslational oxidative modification of proteins. Proteins that respond in this way are ‘redox-sensors', and are crucial to the transduction pathways linking increased oxidant to an appropriate cellular response. Oxidative structural modifications can alter protein function, operating in much the same way as established posttranslational control mechanisms, especially phosphorylation. Our laboratory has investigated various forms of cardiovascular protein thiol oxidation. For example, proteomic studies assessing interprotein disulfide bond formation showed PKA RI subunits oxidise in response to H2O2 or nitrocysteine. RI oxidation is associated with kinase activation by a mechanism under continued investigation. PKG1alpha also formed an interprotein disulfide during oxidative stress, directly activating it independently of the classical NO-cGMP pathway. This has implications for vasoregulation, and explains at least in part how some oxidants may operate as an endothelium derived hyperpolarising factor (EDHF) to lower blood pressure without activating guanylate cyclase. Indeed when we generated ‘redoxdead' Cys42Ser PKG1alpha knock-in mice they were hypertense (measured in vivo using radio-telemetry) and their isolated resistance blood vessels were deficient in their vasodilatory response to hydrogen peroxide, acetylcholine or an EDHF protocol compared to wildtype littermates. This confirms the importance of oxidantinduced activation of PKG in the maintenance of basal blood pressure during health. This is consistent with oxidants playing important homeostatic regulatory roles and not simply causing oxidative damage. Keywords: redox-signalling, oxidant-sensing, kinase, blood pressure doi:10.1016/j.freeradbiomed.2012.08.116

doi:10.1016/j.freeradbiomed.2012.08.115

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