Proteotoxic Cardiac Disease

90 Contrasting Regulation of NOX4 versus NOX2 in Patients with Aortic Stenosis María de Ujué Moreno1, Begoña López1, Ana Fortuño1, Arantxa González1, Gorka San José1, Ajay M Shah2, Javier Díez1, and Guillermo Zalba1 1 Centre for Applied Medical Research, University of Navarra, 2 Spain, Kings’ College London, United Kingdom Introduction: Oxidative stress is critically implicated in cardiac alterations induced by pressure overload. the NADPH oxidases are a key family of reactive oxygen species (ROS)-producing enzymes. Depending on the NOX subunit, the NADPH oxidases may produce different ROS, be specifically expressed and differentially contribute to pathophysiology. Animal models of pressure overload show a detrimental role of NOX2, whereas NOX4 may be protective, regulating myocardial angiogenesis. Human cardiac studies are scarce, indicating that NOX2 may be detrimental whereas information on NOX4 is lacking. Hypothesis: We hypothesize that cardiac NOX2 and NOX4 may be differentially implicated in human aortic stenosis. Methods: Left ventricular biopsies from 34 patients with aortic stenosis were obtained during surgery for valve replacement. Cardiac function was assessed by echocardiography. Cardiac samples from 9 subjects deceased of non-cardiovascular diseases were also analysed. NOX2 and NOX4 were localised by immunohistochemistry and quantified by western blot. Myocardial structure, including capillary density, was also assessed. Results: NOX4 was detected in cardiomyocytes. NOX4 levels were 5-fold reduced in patients compared to controls. in addition, NOX4 levels directly correlated with parameters of cardiac function (ejection fraction, shortening fraction, deceleration time). Capillary density was reduced in patients compared to controls. Interestingly, NOX4 levels directly correlated with capillary density (r=0.389, P=0.023), which in turn associated with cardiac function. NOX2 was detected in fibroblasts, white cells and cardiomyocytes. NOX2 levels were 10-fold increased in patients compared to controls. NOX2 did not correlate with parameters of cardiac function or myocardial structure. Conclusions: Our results indicate that the diverse NOX isoforms may be differentially involved in cardiac chronic pressure overload in humans. Whereas a deficiency of NOX4 may contribute to impaired cardiac microvascularization and function, the consequences of an excess of NOX2 remain to be determined. Further studies are required to elucidate the molecular mechanisms linking changes in NOX proteins with cardiac alterations in aortic stenosis.

doi : 10.1016/j.freeradbiomed.2011.10.115

91 Gluatredoxin 1 in Vivo Impairs Angiogenesis After Mouse Hind-Limb Ischemia Colin Edward Murdoch1, Leah Mycoff1, Priyanka Bearelly1, Richard a Cohen1, and Reiko Matsui1 1 Boston University Glutaredoxin-1 (Glrx) is a small cytosolic enzyme which catalyzes the reduction of glutathione-protein mixed disulfides. in cultured endothelial cells, we have previously demonstrated that overexpression of Glrx was shown to inhibit p21ras- and VEGFinduced migration. Moreover, mice fed high fat high sucrose diet showed an increase in Glrx gene expression in VEGF-matrigel plugs implanted subcutaneously, suggesting a role for Glrx in impaired angiogenesis. We therefore evaluated the in vivo effects of Glrx overexpression on angiogenesis using a model of hind limb ischemia in Glrx transgenic (TG) mice (from Dr.Ho YS). Recovery of blood flow was serially monitored after femoral artery

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ligation by laser Doppler (Moor Instruments). Glrx TG mice displayed a blunted recovery to hindlimb ischemia compared with Wt littermates over 2 weeks (0.2 vs. 0.5 in ischemic/non ischemic blood flow ratio; P<0.05; n>5). To further explore the role of Glrx in VEGF-induced gene expression we infected mouse cardiac endothelial cells with adenovirus containing Glrx (adGlrx) or LacZ (adLacZ) vector and stimulated with VEGF (50 ng/ml, 6h) . Gene expression was assessed by quantitative PCR with TaqMan assay using StepOne (Applied Biosystem). VEGF caused an increase in mRNA levels of the NADPH oxidase isoforms Nox2 and Nox4 (83% and 70% increase respectively; P<0.05; n=6) in adLacZ endothelial cells, which was ablated in the adGRX group (14% decrease and 7% increase respectively; adGlrx, +VEGF cf. –VEGF; P=ns). Other angiogenesis-related genes (angiopoietin 1, eNOS) and oxidant-regulating gene (p47phox) did not show significant differences between adLacZ and adGlrx. in addition, VEGF(50 ng/ml, 30-60 min) induced phosphorylation of ERK in endothelial cells was not impaired by adGlrx. Together this data suggests that Grx1 may negatively regulate angiogenesis posthind limb ischemia, likely through regulating endothelial genes, and that Glrx upregulation may be critical to the impaired angiogenesis observed in pathological conditions including metabolic disorders.

doi : 10.1016/j.freeradbiomed.2011.10.116

92 Reductive Stress: a Paradigm Change in Protein Aggregation/Proteotoxic Cardiac Disease Sellamuthu S. Gounder1, Corey J Miller1, Donna D Zhang2, John R Hoidal3, and Rajasekaran Namakkal Soorappan1 1 Divisions of Cardiology & Pulmonary, Department of Medicine, 2 University of Utah, Department of Pharmacology, University of 3 Arizona, Division of Pulmonary, Department of Medicine, University of Utah Background: Reductive stress (RS) is an underrepresented phenomenon in the fields of redox biochemistry and cardiovascular pathology. We recently discovered that RS is coupled with mutant protein aggregation cardiac hypertrophy/cardiomyopathy (MPAC), but the causal mechanisms are unknown. Here we tested a hypothesis that constitutive activation of Nrf2 (Nuclear erythroid-2 like factor 2) will increase intracellular reducing power, which leads to endoplasmic reticulum (ER) stress, and thereby promotes unfolded/misfolded protein response and protein aggregation. Methods: Age matched (3 and 6 months) MPAC-TG and nontransgenic (NTG) mice (n=6/group/experiment) were used to asses the role of Nrf2 on RS. for in vitro studies, wild-type (WT) -/and Keap1 mouse embryonic fibroblasts (MEFs) were transfected with mutant-R120G-CryAB plasmids and used to determine the redox state (GSH/GSSG content), ROS levels (by EPR), rate of protein aggregation, ER-stress, ubiquitination (Western blotting) and mRNA expression for major antioxidants. Results: We found that sustained activation of Nrf2 contributes to RS in the mutant protein overexpressing hearts. Sustained nuclear translocation of Nrf2 occurs due to ROS generation and Keap1 sequestration, which resulted in RS in MPAC-TG mice. in -/vitro experiments using Keap1 cells reveled >3.0 fold increase in Nrf2 protein and its binding activity with antioxidant response elements (ARE) when compared to WT MEFs. Reduced glutathione (GSH) content and GSH/GSSG ratio were significantly increased to maintain a “highly reducing environment (RS)” in the -/Keap1 when compared to WT MEFs. in association with ERstress (elevated Grp78 & Grp94), increased ubiquitination and aggregation of proteins were observed in the Nrf2 overexpressing -/(Keap1 ) MEFs transfected with plasmids for mutant protein, suggesting enhanced reducing capacity promotes proteotoxicity. -/Further, we observed that RS was sustained in Keap1 MEFs through transcriptional upregulation of major antioxidant genes

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that contribute to redox metabolism, including GSH biosynthesis. Conclusion: Our results demonstrate that sustained RS promotes ER stress, impairs ubiquitin proteasome function, and leads to aggregation of misfolded and/or unfolded proteins. Maintaining a redox homeostasis by manipulating Nrf2 signaling is of potent therapeutic significance.

doi : 10.1016/j.freeradbiomed.2011.10.117

93 Plasma Levels of Myeloperoxidase (MPO) and Thiocyanate (SCN-) Predict Mortality in Patients Following Acute Myocardial Infarction Polina Nedoboy1, Philip E Morgan1, Tessa J Mocatta2, Mark a Richards2, Christine C Winterbourn2, and Michael J Davies1 1 2 The Heart Research Institute, Sydney, Australia, University of Otago, Christchurch, New Zealand Myeloperoxidase (MPO), a heme enzyme secreted by activated – phagocytes at sites of inflammation, catalyses the oxidation of Cl – – , Br and SCN to the powerful oxidants HOCl, HOBr and HOSCN respectively. Previous studies have shown that elevated MPO levels are present in all grades of human atherosclerotic lesions, and are both diagnostic of cardiovascular disease, and prognostic of long-term post-myocardial infarction (MI) mortality. the role of HOSCN produced by MPO in biological damage is however – unclear. SCN is a preferred substrate of MPO, and higher levels of this anion are present in the plasma of smokers, suggesting that HOSCN formation may contribute to the enhanced risk of atherosclerosis (and other diseases) in smokers. In this study we investigated potential associations between – plasma levels of SCN , MPO, and other biochemical parameters – – (Br , NO2 , protein carbonyls and thiols) and 12-year mortality retrospectively in 178 patients after an acute myocardial infarction (MI). the smokers had higher plasma SCN levels than nonsmokers (106±8.0 μM vs. 48±3.0 μM), with these correlating positively with increased MPO-stimulated thiol oxidation (p < 0.012 , Spearman r=0.18). High MPO levels correlated with higher rates of follow-up, all-cause, mortality (p < 0.001), but no – – significant correlations were detected with plasma Br , NO2 , protein carbonyls or absolute thiol levels. Interestingly, subjects – with below median MPO levels and above median SCN concentrations had significantly lower mortality rates than either group with high MPO levels (p < 0.001), and the cohort with below – median MPO levels and below median SCN (p < 0.03). One – interpretation of these data is that high plasma levels of SCN , as seen in smokers and those on particular diets, provide protection by decreasing damage induced by the highly damaging effects of MPO-derived HOCl and HOBr.

to laminar shear stress. CK-1 mediates shear stress-stimulated ERK/Akt/eNOS activations. Overexpression of CK-1 was shown to hyperactivate ERK/eNOS when bovine aortic endothelial cells (BAEC) were exposed to shear stress, whereas CK-1 knockdown was revealed to inhibit the shear stress-activated ERK/eNOS. We also found that N-terminal head domain of CK-1 plays an important role in the shear stress-induced activations of ERK/eNOS. Additionally we dissected the role of CK-1 for endothelial NO production in static condition. Interestingly, CK-1 inhibits calcium/ionophore-stimulated NO production in the static condition, indicating that CK-1 differentially regulates NO production according to static or shear exposure. Together, our data address that CK-1 is a key differential regulator for NO production between static and shear conditions. Keywords : Cytokeratin-1, KRS, Shear stress, eNOS, endothelial cells.

doi : 10.1016/j.freeradbiomed.2011.10.119

95 Renal Mitochondrial Dysfunction: Unraveling the Mystery of Sepsis Induced Renal Injury

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Naeem K Patil1, Nirmala Parajuli1, Philip R Mayeux1, and Lee Ann MacMillan-Crow1 1 University of Arkansas for Medical Sciences Sepsis which is, caused by microbial infection is characterized by a severe systemic inflammatory response. This often leads to multiple organ failure and ultimately death. the majority of sepsis patients develop acute kidney injury (AKI) and sepsis complicated by AKI is associated with 70% mortality as opposed to 28.6% mortality with sepsis alone or 45% mortality with AKI alone. Currently, there is no effective therapy to treat or prevent sepsis and clinicians rely only on supportive care. It is imperative to know the early pathophysiological events in sepsis induced AKI to develop new therapeutic modalities to treat and/or prevent AKI. Recent experimental evidence suggests the role of mitochondrial dysfunction in sepsis induced organ damage. Therefore we are interested in studying the alterations in renal mitochondrial physiology during sepsis. Our preliminary data reveal that the key mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), is inactivated in the kidney without a change in the protein levels, as early as 4 hours following sepsis using a cecal ligation puncture (CLP) mouse model. Interestingly, treatment of renal lysates with dithiothreitol restored MnSOD activity, which suggests a role for cysteine residues playing a role in the observed inactivation. in addition, septic mice (4 hr) had increased levels of nitrotyrosine staining in renal tissues. Mitochondrial respiration was measured in renal tissue using high resolution respirometry. the freshly isolated renal tissue biopsy specimen from septic mice had impaired mitochondrial complex respiration as compared to sham controls. Therefore, our data demonstrate the role of early renal mitochondrial dysfunction as one of the contributing factors involved in sepsis induced AKI. We hypothesize that renal mitochondrial dysfunction acts as a trigger for cellular damage in sepsis, leading to AKI and organ failure.

Cytokeratin-1 Regulates Shear Stress-Induced Nitric Oxide Production in Endothelial Cells

do i : 10.1016/j.freeradbiomed.2011.10.120

doi : 10.1016/j.freeradbiomed.2011.10.118

Sunyoung Ahn1, and Heonyong Park1 1 Dankook University Atherosclerotic lesions occur dominantly in vessel areas exposed to low or disturbed shear stress. Accordingly, laminar shear stress is well-known as an anti-atherogenic factor. Recently, we have identified a novel shear sensing molecule by using lysyl tRNA synthetase (KRS), an established pro-inflammatory protein. in endothelial cells, cytokeratin-1 (CK-1) appeared to interact with KRS and activate a variety of cell signal transductions in response

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