- Email: [email protected]
Downregulation of SIRT3 Exerts Mitochondrial Dysfunction in Cultured Cardiomyocytes
the vulnerability for ventricular arrhythmias in a murine model of ischemic cardiomyopathy. Whereas no differences were observed in infarct size, characteristics of electrical conduction pointed to aggravated adverse electrical or structural remodeling in WT as compared to Mpo-/- mice. The results underline the important role of inflammatory processes in cardiac remodeling upon myocardial infarction and fibroblast activation and identify MPO as a potential therapeutic target to prevent ventricular arrhythmias in ischemic cardiomyopathy. doi: 10.1016/j.freeradbiomed.2014.10.415
101 Superoxide and Superoxide Dismutase Levels in HighRisk Pregnant Women: A Pilot Feasibility Study Tiffany A Moore1, Iman Ahmad1, Jocelyn Jones1, Adam Case1, and Matthew Zimmerman1 1 University of Nebraska Medical Center, USA Previous studies of the perinatal population have suggested complications, such as pre-eclampsia, are correlated with biomarkers of oxidative stress. While these biomarkers are an indicator of an aberrant redox environment, levels of specific radical species in this high-risk patient population remain unknown. Understanding that superoxide (O2Ɣ-) is a precursor free radical to many other damaging reactive oxygen species, the goals of this pilot feasibility study were to: 1) quantify O2Ɣ- levels and total superoxide dismutase (SOD) activity in the blood of high-risk pregnant women; 2) compare these levels with perinatal outcomes. Whole blood was obtained by venipuncture from 4 pregnant women between 15-19 weeks gestation recruited from a maternal-fetal-medicine clinic at an academic medical center. Thirty minutes after FROOHFWLRQ ȝO of whole blood was incubated with a O2Ɣ--sensitive electron paramagnetic resonance (EPR) spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5tetramethylpyrrolidine (CMH), for 30 minutes at 37.5°C then frozen in liquid nitrogen. Superoxide levels were determined using a Bruker eScan EPR spectrometer and expressed as EPR DUELWUDU\ XQLWV $8 $ VHSDUDWH ȝO of whole blood was centrifuged for 2 minutes at 1.5xg, and protein was obtained from the separated red blood cells (RBC). Total SOD activity was measured from the RBC protein using the Dojindo SOD activity assay. of the four recruited patients, one developed postpartum pre-eclampsia. Interestingly, the CMH oxidation levels (3.9x106AU) and SOD activity levels (129,000 U/ml) for the one patient with postpartum pre-eclampsia were considerably HOHYDWHGFRPSDUHGWRWKHUHPDLQLQJWKUHHSDWLHQWV¶DYHUDJH&0+ oxidation levels (2.5x106 ± 0.30x106 AU) and SOD activity levels (31,000 ± 10,000 U/ml) that did not develop any pre-eclampsia. This is the first-known study to examine circulating redox status in whole blood of high-risk pregnant women using the gold-standard for detecting O2Ɣ- levels, that is, EPR spectroscopy. A larger study designed to examine the redox status in high-risk pregnant women over time, in their newborn infants, and with perinatal outcomes is currently being conducted. doi: 10.1016/j.freeradbiomed.2014.10.416
102 Effect of Chronic Ethanol Consumption on the Increased Oxidative Stress in Rat Corpus Cavernosum Tissue Jaqueline Jóice Muniz1, Leticia Nogueira Leite1, Riccardo Lacchini1, José Eduardo Tanus-Santos1, and Carlos Renato Tirapelli1 1 University of Sao Paulo, Brazil The aim of this study was to assess the effects of chronic ethanol consumption on the endothelinergic system, mitogen-activated protein kinase (MAPK) pathway, and on the oxidative stress in cavernosal tissue as factors involved in erectile dysfunction (ED). All protocols were approved by the local Ethics Committee (12.1.317.53.9). Male Wistar rats were divided in two groups: ethanol group±treated with ethanol (20% vol/vol) for 6 weeks; control group±water for 6 weeks. In cumulative concentrationresponse curves for Endothelin 1 (ET-1), the ET-1-induced contraction was higher in ethanol-treated rats (36.1 ± 2.7% KCl 120mM; n=5) compared to control group (20.7 ± 0.9% KCl 120mM; n=5). In ethanol group, the contraction induced by ET-1 was significantly reduced in the presence of apocynin (100μmol/L), an inhibitor of NAD(P)H oxidase, and SP600125 (100 μmol/L), an inhibitor of SAPK/JNK. Plasma antioxidant activity was higher in ethanol group (4.81± 0.19 mmol/L; n=11) than control group (2.52±0.17 mmol/L; n=10). Catalase levels were higher in ethanol group (0.062±0.005 U/mL; n=10 and 0.090±0.015 U/mg protein; n=9) when compared to control group (0.043±0.004 U/mL; n=10 and 0.037±0.007 U/mg protein; n=9) in plasma and cavernosal tissue, respectively. Superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) levels in the cavernosal tissue were lower in ethanol group (3.13±0.63 pmol/μg protein; n=7 and 3.77±0.32 pmol/μg protein; n=8) when compared to control group (5.12±0.53 pmol/μg protein; n=7 and 6.15±0.76 pmol/μg protein; n=8), respectively. No difference was found in level of glutathione and mRNA of the p38MAPK, SAPK/JNK and ERK1/2 between groups. These results show that the chronic ethanol consumption affects intracellular pathways activated by ET-1 and contributes to increased oxidative stress in cavernosal tissue, which can lead to ED. Financial Support: FAPESP doi: 10.1016/j.freeradbiomed.2014.10.417
103 Downregulation of SIRT3 Exerts Mitochondrial Dysfunction in Cultured Cardiomyocytes Rebecca Maria Parodi-Rullán1, Sergiy Nadtochiy2, Paul Brookes2, and Sabzali Javadov1 1 University of Puerto Rico School of Medicine, Puerto Rico, 2 University of Rochester Medical Center, USA Background: Sirtuins are NAD+dependent deacetylases that regulate protein activity through acetylation/deacetylation. Mitochondria contain three of seven sirtuins, and SIRT3 is a major mitochondrial isoform. Lack of specific pharmacological inhibitors for SIRT3 complicates elucidating the role of the sirtuin in mitochondrial metabolism and function. In this study, we: 1) developed a method for downregulation of SIRT3 expression using the siRNA technique in H9c2 cardiomyocytes, and 2) assessed the effect of SIRT3 genetic ablation on mitochondrial function. Methods: To optimize the conditions for silencing, 1x105, 2x105or 3x105 cells per a 10-cm dish and 2.4x103 or 10x103 cells per well for the 24-well plate were transfected with 100 nm or 200 nm siRNA for 24, 48 or 72h. Protein levels of SIRT3 were analyzed by SDS-PAGE Western blotting using SIRT3 antibodies. Once an optimal condition with maximum SIRT3 silencing was achieved, mitochondrial membrane potential was assessed in 24-
SFRBM 2014
S51
well plates using the membrane-permeant and mitochondrial membrane potential sensitive fluorescent dye, JC-1. Results: Maximum SIRT3 silencing (41% reduction of protein expression, P<0.02 vs. negative control) in H9c2 cells was observed at 72h when 1x105 cells per 10-cm dish and 10x103 cells per well for the 24-well were plated. Downregulation of SIRT3 induced depolarization of the mitochondrial membrane that demonstrated a 30% lower (P<0.03) membrane potential than control cells. Conclusion: Genetic downregulation can substitute pharmacological inhibition of SIRT3 to elucidate the relationship between mitochondrial dysfunction and protein acetylation in response to oxidative stress in cultured cardiomyocytes. The work was supported by the NHLBI NIH grant (SC1HL118669) to Sabzali Javadov, and the SFRBM 2013 Research MiniFellowship, and the MBRS-RISE Program grant (R25-GM061838) to Rebecca Parodi-Rullán. doi: 10.1016/j.freeradbiomed.2014.10.418
104 Superoxide Generation and Oxidative Tissue Damage in Bicuspid Aortic Valve-Associated Aortopathy Julie A Phillippi1, Benjamin R Green1, Mary P Kotlarczyk1, Riley M Hermmann1, Marie Billaud1, Jennifer C Hill1, Murugesan Velayutham1, Arturo J Cardounel1, Nandiezhda Cantu-Medéllin1, Eric E Kelley1, and Thomas G Gleason1 1 University of Pittsburgh, USA Dissection of the aorta is a medical emergency with >90% mortality in the absence of surgical intervention. Presence of the congenital defect of bicuspid aortic valve (BAV) portends an increased risk of dissection and is associated with aneurysm localized to the proximal ascending aorta. Risk assessment for aortic catastrophe in BAV patients is challenging due to a lack of knowledge in the governing mechanisms of the associated aortopathy. Prior work from our group identified increased susceptibility to peroxide-induced cell death in aortic smooth muscle cells (SMCs) from BAV patients. In the present study, we hypothesized that the susceptibility to peroxide in SMC from BAV patients causes more superoxide-mediated oxidative damage to the aortic media compared to non-BAV patients. Aortic tissue extracts and primary SMCs were isolated from patients undergoing elective ascending aortic/aortic valve replacement and during heart transplants with IRB approval and informed patient consent. HPLC-based detection of the superoxide-specific oxidation product 2-OH-E+ in hydroethidine-exposed fresh aortic specimens revealed elevated superoxide generation in BAV specimens when compared with TAV (n=92, p<0.03). Using electron paramagnetic resonance spectroscopy with the cellpermeable 1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetra methylpyrrolidine (CMH) spin probe, SOD-inhibited CMH oxidation was increased in the presence of peroxide when compared with non-treated BAV SMCs (n=3, p<0.03). Protein carbonyl content was increased in medial extracts from nonaneurysmal BAV patients when compared with normal specimens (n=45, p<0.002). The results show that superoxide is a prominent peroxide-induced reactive oxygen species generated by SMCs and its accumulation is associated with increased medial layer protein oxidation in BAV specimens prior to aneurysm formation. Ongoing studies are focused on defining mechanisms of superoxide generation and the impact on wall biomechanics. These findings improve our understanding of free radical-related mechanisms involved in BAV-associated aortopathy. Importantly, this could help to develop novel interventions for BAV patients who are at risk for aortic catastrophe.
S52
doi: 10.1016/j.freeradbiomed.2014.10.419
105 The Extracellular Superoxide Dismutase Alters Myocardial Rescue Pathways in Ischaemic Left Ventricular Tissue Antonio Pinto1, Markus Kornfeld1, Hug Aubin1, Payam Akhyari1, and Artur Lichtenberg1 1 Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Düsseldorf, Germany The extracellular superoxide dismutase (SOD3) is a secreted and protective enzyme in the cardiovascular system. Its main function is the detoxification of superoxide. In myocardial ischaemia superoxide production is elevated and contributes to ischaemia and reperfusion injury. Therefore in a rat mutagenesis model we investigate the impact of SOD3 inactivity on signal transduction in the setting of myocardial ischaemia. SOD3 inactivity has been achieved by N-Ethyl-N-nitrosourea mutagenesis of DahlSS rats (PhysGen Program, Medical College of Wisconsin), shifting glutamate 124 to asparte in the catalytic triad (SOD3-E124D). Myocardial ischaemia was achieved by ligating the LAD in SOD3-E124D and DahlSS rats (control group) after 10min or 24h of occlusion (n = 5, every group). SOD3 expression in heart and plasma, and influence on associated signal transduction pathways were analysed by immunoblotting. SOD3 expression was not altered in plasma after 10min of occlusion. A decrease of SOD3 in the ischaemic zones of SOD3E124D rats was detected. 3-nitrotyrosine in ischaemic regions was elevated after 10min, this effect was more pronounced in SOD3-E124D rats. Concomitantly phosphorylation of p38 was drastically induced. Subsequent downregulation of eNOS was more accentuated in SOD3-E124D. Sustained myocardial ischaemia for 24h elevated STAT3 pathway activation, while Akt phosphorylation was depressed in SOD3-E124D rats. Plasma and tissue levels of SOD3 were differently induced in SOD3E124D and DahlSS after 24h of ischemia. Upon echocardiography LV function was by trend lower in SOD3E124D as compared to Dahl SS. SOD3 expression is induced by sustained ischaemia. Superoxide is a major contributor to rescue pathway activation and influences nitric oxide levels. Inactivity of SOD3 affects molecular and physiological parameters in acute and sustained ischaemia. doi: 10.1016/j.freeradbiomed.2014.10.420
106 Nitrite Activates Protein Kinase A and Increases AKinase Anchoring Protein Expression to Regulate Mitochondrial Function in Non-Hypoxic Conditions Rafael Portella1, Christelle Pride1, José Eduardo Tanus-Santos2, and Sruti Shiva1 1 University of Pittsburgh, USA, 2University of Sao Paulo, Brazil Nitrite has emerged as an important signaling molecule in the past decade. The majority of physiological effects mediated by nitrite are thought to be dependent on the reduction of nitrite to nitric oxide in conditions of low pH and oxygen tension. However, we have shown that nitrite confers cardioprotection when administered prior to an ischemic episode. This cardioprotection is dependent on the nitrite-mediated normoxic activation of protein kinase A (PKA), which modulates mitochondrial morphology and function. However, the mechanism by which nitrite activates PKA and its ability to target PKA to the mitochondrion is unknown. In
SFRBM 2014
101 Superoxide and Superoxide Dismutase Levels in HighRisk Pregnant Women: A Pilot Feasibility Study Tiffany A Moore1, Iman Ahmad1, Jocelyn Jones1, Adam Case1, and Matthew Zimmerman1 1 University of Nebraska Medical Center, USA Previous studies of the perinatal population have suggested complications, such as pre-eclampsia, are correlated with biomarkers of oxidative stress. While these biomarkers are an indicator of an aberrant redox environment, levels of specific radical species in this high-risk patient population remain unknown. Understanding that superoxide (O2Ɣ-) is a precursor free radical to many other damaging reactive oxygen species, the goals of this pilot feasibility study were to: 1) quantify O2Ɣ- levels and total superoxide dismutase (SOD) activity in the blood of high-risk pregnant women; 2) compare these levels with perinatal outcomes. Whole blood was obtained by venipuncture from 4 pregnant women between 15-19 weeks gestation recruited from a maternal-fetal-medicine clinic at an academic medical center. Thirty minutes after FROOHFWLRQ ȝO of whole blood was incubated with a O2Ɣ--sensitive electron paramagnetic resonance (EPR) spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5tetramethylpyrrolidine (CMH), for 30 minutes at 37.5°C then frozen in liquid nitrogen. Superoxide levels were determined using a Bruker eScan EPR spectrometer and expressed as EPR DUELWUDU\ XQLWV $8 $ VHSDUDWH ȝO of whole blood was centrifuged for 2 minutes at 1.5xg, and protein was obtained from the separated red blood cells (RBC). Total SOD activity was measured from the RBC protein using the Dojindo SOD activity assay. of the four recruited patients, one developed postpartum pre-eclampsia. Interestingly, the CMH oxidation levels (3.9x106AU) and SOD activity levels (129,000 U/ml) for the one patient with postpartum pre-eclampsia were considerably HOHYDWHGFRPSDUHGWRWKHUHPDLQLQJWKUHHSDWLHQWV¶DYHUDJH&0+ oxidation levels (2.5x106 ± 0.30x106 AU) and SOD activity levels (31,000 ± 10,000 U/ml) that did not develop any pre-eclampsia. This is the first-known study to examine circulating redox status in whole blood of high-risk pregnant women using the gold-standard for detecting O2Ɣ- levels, that is, EPR spectroscopy. A larger study designed to examine the redox status in high-risk pregnant women over time, in their newborn infants, and with perinatal outcomes is currently being conducted. doi: 10.1016/j.freeradbiomed.2014.10.416
102 Effect of Chronic Ethanol Consumption on the Increased Oxidative Stress in Rat Corpus Cavernosum Tissue Jaqueline Jóice Muniz1, Leticia Nogueira Leite1, Riccardo Lacchini1, José Eduardo Tanus-Santos1, and Carlos Renato Tirapelli1 1 University of Sao Paulo, Brazil The aim of this study was to assess the effects of chronic ethanol consumption on the endothelinergic system, mitogen-activated protein kinase (MAPK) pathway, and on the oxidative stress in cavernosal tissue as factors involved in erectile dysfunction (ED). All protocols were approved by the local Ethics Committee (12.1.317.53.9). Male Wistar rats were divided in two groups: ethanol group±treated with ethanol (20% vol/vol) for 6 weeks; control group±water for 6 weeks. In cumulative concentrationresponse curves for Endothelin 1 (ET-1), the ET-1-induced contraction was higher in ethanol-treated rats (36.1 ± 2.7% KCl 120mM; n=5) compared to control group (20.7 ± 0.9% KCl 120mM; n=5). In ethanol group, the contraction induced by ET-1 was significantly reduced in the presence of apocynin (100μmol/L), an inhibitor of NAD(P)H oxidase, and SP600125 (100 μmol/L), an inhibitor of SAPK/JNK. Plasma antioxidant activity was higher in ethanol group (4.81± 0.19 mmol/L; n=11) than control group (2.52±0.17 mmol/L; n=10). Catalase levels were higher in ethanol group (0.062±0.005 U/mL; n=10 and 0.090±0.015 U/mg protein; n=9) when compared to control group (0.043±0.004 U/mL; n=10 and 0.037±0.007 U/mg protein; n=9) in plasma and cavernosal tissue, respectively. Superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) levels in the cavernosal tissue were lower in ethanol group (3.13±0.63 pmol/μg protein; n=7 and 3.77±0.32 pmol/μg protein; n=8) when compared to control group (5.12±0.53 pmol/μg protein; n=7 and 6.15±0.76 pmol/μg protein; n=8), respectively. No difference was found in level of glutathione and mRNA of the p38MAPK, SAPK/JNK and ERK1/2 between groups. These results show that the chronic ethanol consumption affects intracellular pathways activated by ET-1 and contributes to increased oxidative stress in cavernosal tissue, which can lead to ED. Financial Support: FAPESP doi: 10.1016/j.freeradbiomed.2014.10.417
103 Downregulation of SIRT3 Exerts Mitochondrial Dysfunction in Cultured Cardiomyocytes Rebecca Maria Parodi-Rullán1, Sergiy Nadtochiy2, Paul Brookes2, and Sabzali Javadov1 1 University of Puerto Rico School of Medicine, Puerto Rico, 2 University of Rochester Medical Center, USA Background: Sirtuins are NAD+dependent deacetylases that regulate protein activity through acetylation/deacetylation. Mitochondria contain three of seven sirtuins, and SIRT3 is a major mitochondrial isoform. Lack of specific pharmacological inhibitors for SIRT3 complicates elucidating the role of the sirtuin in mitochondrial metabolism and function. In this study, we: 1) developed a method for downregulation of SIRT3 expression using the siRNA technique in H9c2 cardiomyocytes, and 2) assessed the effect of SIRT3 genetic ablation on mitochondrial function. Methods: To optimize the conditions for silencing, 1x105, 2x105or 3x105 cells per a 10-cm dish and 2.4x103 or 10x103 cells per well for the 24-well plate were transfected with 100 nm or 200 nm siRNA for 24, 48 or 72h. Protein levels of SIRT3 were analyzed by SDS-PAGE Western blotting using SIRT3 antibodies. Once an optimal condition with maximum SIRT3 silencing was achieved, mitochondrial membrane potential was assessed in 24-
SFRBM 2014
S51
well plates using the membrane-permeant and mitochondrial membrane potential sensitive fluorescent dye, JC-1. Results: Maximum SIRT3 silencing (41% reduction of protein expression, P<0.02 vs. negative control) in H9c2 cells was observed at 72h when 1x105 cells per 10-cm dish and 10x103 cells per well for the 24-well were plated. Downregulation of SIRT3 induced depolarization of the mitochondrial membrane that demonstrated a 30% lower (P<0.03) membrane potential than control cells. Conclusion: Genetic downregulation can substitute pharmacological inhibition of SIRT3 to elucidate the relationship between mitochondrial dysfunction and protein acetylation in response to oxidative stress in cultured cardiomyocytes. The work was supported by the NHLBI NIH grant (SC1HL118669) to Sabzali Javadov, and the SFRBM 2013 Research MiniFellowship, and the MBRS-RISE Program grant (R25-GM061838) to Rebecca Parodi-Rullán. doi: 10.1016/j.freeradbiomed.2014.10.418
104 Superoxide Generation and Oxidative Tissue Damage in Bicuspid Aortic Valve-Associated Aortopathy Julie A Phillippi1, Benjamin R Green1, Mary P Kotlarczyk1, Riley M Hermmann1, Marie Billaud1, Jennifer C Hill1, Murugesan Velayutham1, Arturo J Cardounel1, Nandiezhda Cantu-Medéllin1, Eric E Kelley1, and Thomas G Gleason1 1 University of Pittsburgh, USA Dissection of the aorta is a medical emergency with >90% mortality in the absence of surgical intervention. Presence of the congenital defect of bicuspid aortic valve (BAV) portends an increased risk of dissection and is associated with aneurysm localized to the proximal ascending aorta. Risk assessment for aortic catastrophe in BAV patients is challenging due to a lack of knowledge in the governing mechanisms of the associated aortopathy. Prior work from our group identified increased susceptibility to peroxide-induced cell death in aortic smooth muscle cells (SMCs) from BAV patients. In the present study, we hypothesized that the susceptibility to peroxide in SMC from BAV patients causes more superoxide-mediated oxidative damage to the aortic media compared to non-BAV patients. Aortic tissue extracts and primary SMCs were isolated from patients undergoing elective ascending aortic/aortic valve replacement and during heart transplants with IRB approval and informed patient consent. HPLC-based detection of the superoxide-specific oxidation product 2-OH-E+ in hydroethidine-exposed fresh aortic specimens revealed elevated superoxide generation in BAV specimens when compared with TAV (n=92, p<0.03). Using electron paramagnetic resonance spectroscopy with the cellpermeable 1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetra methylpyrrolidine (CMH) spin probe, SOD-inhibited CMH oxidation was increased in the presence of peroxide when compared with non-treated BAV SMCs (n=3, p<0.03). Protein carbonyl content was increased in medial extracts from nonaneurysmal BAV patients when compared with normal specimens (n=45, p<0.002). The results show that superoxide is a prominent peroxide-induced reactive oxygen species generated by SMCs and its accumulation is associated with increased medial layer protein oxidation in BAV specimens prior to aneurysm formation. Ongoing studies are focused on defining mechanisms of superoxide generation and the impact on wall biomechanics. These findings improve our understanding of free radical-related mechanisms involved in BAV-associated aortopathy. Importantly, this could help to develop novel interventions for BAV patients who are at risk for aortic catastrophe.
S52
doi: 10.1016/j.freeradbiomed.2014.10.419
105 The Extracellular Superoxide Dismutase Alters Myocardial Rescue Pathways in Ischaemic Left Ventricular Tissue Antonio Pinto1, Markus Kornfeld1, Hug Aubin1, Payam Akhyari1, and Artur Lichtenberg1 1 Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Düsseldorf, Germany The extracellular superoxide dismutase (SOD3) is a secreted and protective enzyme in the cardiovascular system. Its main function is the detoxification of superoxide. In myocardial ischaemia superoxide production is elevated and contributes to ischaemia and reperfusion injury. Therefore in a rat mutagenesis model we investigate the impact of SOD3 inactivity on signal transduction in the setting of myocardial ischaemia. SOD3 inactivity has been achieved by N-Ethyl-N-nitrosourea mutagenesis of DahlSS rats (PhysGen Program, Medical College of Wisconsin), shifting glutamate 124 to asparte in the catalytic triad (SOD3-E124D). Myocardial ischaemia was achieved by ligating the LAD in SOD3-E124D and DahlSS rats (control group) after 10min or 24h of occlusion (n = 5, every group). SOD3 expression in heart and plasma, and influence on associated signal transduction pathways were analysed by immunoblotting. SOD3 expression was not altered in plasma after 10min of occlusion. A decrease of SOD3 in the ischaemic zones of SOD3E124D rats was detected. 3-nitrotyrosine in ischaemic regions was elevated after 10min, this effect was more pronounced in SOD3-E124D rats. Concomitantly phosphorylation of p38 was drastically induced. Subsequent downregulation of eNOS was more accentuated in SOD3-E124D. Sustained myocardial ischaemia for 24h elevated STAT3 pathway activation, while Akt phosphorylation was depressed in SOD3-E124D rats. Plasma and tissue levels of SOD3 were differently induced in SOD3E124D and DahlSS after 24h of ischemia. Upon echocardiography LV function was by trend lower in SOD3E124D as compared to Dahl SS. SOD3 expression is induced by sustained ischaemia. Superoxide is a major contributor to rescue pathway activation and influences nitric oxide levels. Inactivity of SOD3 affects molecular and physiological parameters in acute and sustained ischaemia. doi: 10.1016/j.freeradbiomed.2014.10.420
106 Nitrite Activates Protein Kinase A and Increases AKinase Anchoring Protein Expression to Regulate Mitochondrial Function in Non-Hypoxic Conditions Rafael Portella1, Christelle Pride1, José Eduardo Tanus-Santos2, and Sruti Shiva1 1 University of Pittsburgh, USA, 2University of Sao Paulo, Brazil Nitrite has emerged as an important signaling molecule in the past decade. The majority of physiological effects mediated by nitrite are thought to be dependent on the reduction of nitrite to nitric oxide in conditions of low pH and oxygen tension. However, we have shown that nitrite confers cardioprotection when administered prior to an ischemic episode. This cardioprotection is dependent on the nitrite-mediated normoxic activation of protein kinase A (PKA), which modulates mitochondrial morphology and function. However, the mechanism by which nitrite activates PKA and its ability to target PKA to the mitochondrion is unknown. In
SFRBM 2014