http://hdl.handle.net/10027/7416 Thu, 23 May 2013 14:27:06 GMT 2013-05-23T14:27:06Z http://hdl.handle.net/10027/8786 Reactive oxygen species-targeted therapeutic interventions for atrial fibrillation Sovari, Ali A.; DudleyJr., Samuel C. Atrial fibrillation (AF) is the most common arrhythmia that requires medical attention, and its incidence is increasing. Current ion channel blockade therapies and catheter ablation have significant limitations in treatment of AF, mainly because they do not address the underlying pathophysiology of the disease. Oxidative stress has been implicated as a major underlying pathology that promotes AF; however, conventional antioxidants have not shown impressive therapeutic effects. A more careful design of antioxidant therapies and better selection of patients likely are required to treat effectively AF with antioxidant agents. Current evidence suggest inhibition of prominent cardiac sources of reactive oxygen species (ROS) such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and targeting subcellular compartments with the highest levels of ROS may prove to be effective therapies for AF. Increased serum markers of oxidative stress may be an important guide in selecting the AF patients who will most likely respond to antioxidant therapy. This Document is Protected by copyright and was first published by Frontiers. All rights reserved. It is reproduced with permission Sun, 01 Jan 2012 06:00:00 GMT http://hdl.handle.net/10027/8786 2012-01-01T06:00:00Z http://hdl.handle.net/10027/8291 A Novel, In-Solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Buttrick The molecular conformation of the cardiac myosin motor is modulated by inter-molecular interactions among the heavy chain, the light chains, myosin binding protein-C (MyBP-C) and titin, and is governed by post-translational modifications (PTMs). In-gel digestion followed by liquid chromatography mass spectrometry (LC/MS/MS) has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based workflow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Sub-cellular fractionation followed by OFFGEL electrophoresis (OGE) resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain (MHC), and MyBPC of the thick filament. Further purification of RLC using reverse phase (RP) -HPLC separation and UV detection enriched for RLC PTMs at the intact protein level, and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OGE fractions were un-phosphorylated (78.8%), singly- (18.1%) and doubly-phosphorylated (3.1%) RLC. The novel aspects of this study are: 1) milligram amounts of endogenous cardiac sarcomeric sub-proteome were focused with resolution comparable to 2DE, 2) separation and quantification of post-translationally modified variants was achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in-solution, 4) endogenous charge variants of RLC were separated; a novel doublyphosphorylated form was identified in mouse, and singly-phosphorylated, singly-deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method. This research was originally published in Molecular and Cellular Proteomics. Sarah B. Scruggs, Rick Reisdorph, Mike L. Armstrong, Chad M. Warren1, Nichole Reisdorph, R. John Solaro, Peter M. Buttrick. A Novel, In-Solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain. Molecular and Cellular Proteomics. 2010. 9:1804-1818. © the American Society for Biochemistry and Molecular Biology DOI: 10.1074/mcp.M110.000075 Sat, 01 May 2010 05:00:00 GMT http://hdl.handle.net/10027/8291 2010-05-01T05:00:00Z http://hdl.handle.net/10027/8266 Extracellular SOD-Derived H2O2 Promotes VEGF Signaling in Caveolae/Lipid Rafts and Post-Ischemic Angiogenesis in Mice Oshikawa, Jin; Urao, Norifumi; Kim, Ha Won; Kaplan, Nihal; Razvi, Masooma; McKinney, Ronald; Poole, Leslie B.; Fukai, Tohru; Ushio-Fukai, Masuko Reactive oxygen species (ROS), in particular, H2O2, is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H2O2, and anchors to EC surface through heparinbinding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSODderived H2O2 regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSODDHBD), increases H2O2 levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H2O2 in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-DHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H2O2. Either exogenous H2O2 (,500 mM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H2O2 to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSODinduced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H2O2 generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases. © 2010 Oshikawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The original version is available through the Public Library of Science at DOI: 10.1371/journal.pone.0010189. Wed, 21 Apr 2010 05:00:00 GMT http://hdl.handle.net/10027/8266 2010-04-21T05:00:00Z http://hdl.handle.net/10027/8242 Superoxide Dismutases: Role in Redox Signaling, Vascular Function and Diseases Fukai, Tohru; Ushio-Fukai, Masuko Excessive reactive oxygen species Revised abstract, especially superoxide anion (O2•−), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against O2•−, which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of O2•− H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies. Antioxid. Redox Signal. 15, 000–000. This is a copy of an article published in the Antioxidants and Redox Signaling © 2011 copyright Mary Ann Liebert, Inc.; Antioxidants and Redox Signaling is available online at: http://www.liebertonline.com. DOI: 10.1089/ars.2011.399 Thu, 07 Apr 2011 05:00:00 GMT http://hdl.handle.net/10027/8242 2011-04-07T05:00:00Z