- Email: [email protected]
0333 : Tissue specificity of the membrane vs nuclear actions of estrogen receptor alpha: insights from targeted mutations in mouse models
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Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
risk factor for endothelial dysfunction. We addressed the relationship between ADMA plasma levels and AF occurrence in AMI. Methods 273 patients hospitalized for AMI were included. Continuous electrocardiographic monitoring (CEM) ≥48 hours was recorded and ADMA was measured by High Performance Liquid Chromatography on admission blood sample. Results The incidence of silent and symptomatic AF was 39(14%) and 29 (11%), respectively. AF patients were markedly older than patients without AF (≈ 20 y). There was a trend towards higher ADMA levels in patients with symptomatic AF than in patients with silent AF or no AF (0.53 vs 0.49 and 0.49 μmol/L, respectively). After matching on age, we found that patients with symptomatic AF had a higher heart rate on admission and a higher rate of patients with LV dysfunction (28% vs. 3%, p=0.025). Patients who developed symptomatic AF had a higher ADMA level (0.53 vs. 0.43 μmol/L; p=0.001). Multivariate logistic regression analysis to estimate symptomatic AF occurrence showed that ADMA was independently associated with symptomatic AF (OR: 2.46 [1.21-5.00], p=0.013) beyond history of AF, LVEF<40% and elevated HR. Conclusion We show that high ADMA level is associated with the occurrence of AF. Although no causative role can be concluded from our observational study, our work further supports the hypothesis that endothelial dysfunction is involved in the pathogenesis of AF in AMI. The author hereby declares no conflict of interest
0333 Tissue specificity of the membrane vs nuclear actions of estrogen receptor alpha: insights from targeted mutations in mouse models Jean-François Arnal *, Coralie Fontaine, Marie-Cecile Valera, Marine Adlanmerini, Pierre Gourdy, Françoise Lenfant CHU Toulouse, Rangueil, INSERM U1048 – I2MR, Toulouse, France * Corresponding author: [email protected] (Jean-Francois Arnal) Thanks to unique mouse models, we demonstrated that the estrogen receptor alpha (ERa), but not ERb, is absolutely necessary for most of the arterial and metabolic actions of 17b-estradiol (E2). Estrogens also elicit deleterious effects on the uterus and breast (partly via their proliferative effect which increases the risk of cancer) as well as increased risk of venous thromboembolism, which are the two main limitations of classic estrogen therapies. ER contains two independent transactivation functions AF-1 and AF-2, and our team dissected for the first time in vivo the roles of functions ERaAF-1 and AF-2, allowing to define the key role of these nuclear actions in several tissues. More recently, we reported the key role of the ERa fraction localized to the plasma membrane which is absolutely necessary for the protective endothelial actions of E2 as well as, more unexpectedly, for female fertility. One mystery of estrogens biology is that selective ER modulators (SERMs, such as tamoxifen, raloxifen) have a highly tissue-specific action, behaving as agonists in some tissues (such as bone) and as antagonist in others (breast). We will report how these mouse models allowed us to characterize: 1) the tissue-specific roles of ERa : membrane ERa mediates the effect of E2 in the endothelium (acceleration of reendothelialization, activation of endothelial NO synthase), whereas nuclear ERa mediates the effect of E2 in the uterus. 2) a new natural SERM (Estetrol) currently tested in a phase III clinical trial as a safer oral contraception or hormonal treatment of menopause, as this molecule does not alter the hepatic-derived coagulation factors, and thereby could not increase the venous thrombosis risk. 3) an Estrogen Dendrimer Conjugate, a selective activator of membrane ERa, which could be a new endothelial protective SERM. The author declares a conflict of interest: JF Arnal'team received a financial support from Uteron/Mithra to study the mechanisms of action of Estetrol.
0546 Mitochondrial induced oxidative stress triggers Ca2+-dependent proteolysis and underlie VIDD Haikel Dridi * Université de Montpellier, INSERM U1046, Montpellier, France * Corresponding author: [email protected] (Haikel Dridi) Rationale In rodents, prolonged controlled mechanical ventilation (CMV) and associated diaphragm inactivity elicits complex pathologic changes
referred to as ventilator-induced diaphragm dysfunction (VIDD). Previous workers have demonstrated that VIDD is characterized by increased mitochondrial generation of reactive oxygen species (ROS) and redox-induced dysfunction in calcium release. Powers et al., (2011) have demonstrated that SS-31, a mitochondrial targeted antioxidant, markedly attenuates mitochondrial ROS generation. Since, the calcium-release pathology associated with VIDD is thought to be secondary to myofiber redox changes, we hypothesized that SS-31 should markedly attenuate these abnormalities in myofiber calcium regulation. Objective To test the hypothesis that SS-31, a mitochondrial-targeted antioxidant, attenuates the effect of prolonged CMV on diaphragm myofiber calcium homeostasis. Measurements and main results Specific force of diaphragm fibers decreased significantly 28 ±5%, (n=10) following 12hrs of mechanical ventilation compared to fibers from unventilated control mice. These changes were associated with a significant decrease in cross-sectional area (574 ±19 versus 673± 21 μm m2), an increase in calpain activity, the oxidation of the RyR1 macromolecular complexed, RyR1 phosphorylation at Ser-2844 and the depletion of the stabilizing subunit calstabin1 resulting in an increase in sarcoplasmic reticulum (SR) Ca2+ leak. Diaphragm from mice treated with SS-31 were protected against all of these changes. On the basis of these findings we propose that SR Ca2+ leak via RyR1 due to mitochondrial induced oxidative stress, triggers Ca2+-dependent proteolysis and underlie VIDD. These results emphasize the need to develop therapeutic interventions that mitigate mitochondrial dysfunctional thereby reduce mechanical ventilation-induced diaphragmatic weakness. The author hereby declares no conflict of interest
0460 Nitric oxide and hemoglobin form a paramagnetic compound quantifiable by Electron Paramagnetic Resonance (EPR) spectroscopy in venous erythrocytes that reflects vascular NO bioavailability in vivo Flavia Dei Zotti *, Irina Lobysheva, Jean-Luc Balligand Université Catholique de Louvain, Bruxelles, Belgique * Corresponding author: [email protected] (Flavia Dei Zotti) Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo.We developed a modified subtraction method using EPR to quantify it in RBCs from mouse,rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS(+/+) and eNOS(-/-)mice in vitro and its sensitivity to NOS or arginase inhibitors.Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS(+/+) but insensitive to these modulators in eNOS(-/-)RBCs. We found that upon exposure to exogenous NO-donor,the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air),and preserved at 21% of O2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely,CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence.This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H2O2.We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers;N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H2O2.HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo. The author hereby declares no conflict of interest
© Elsevier Masson SAS. All rights reserved.
Archives of Cardiovascular Diseases Supplements (2016) 8, 216-218
risk factor for endothelial dysfunction. We addressed the relationship between ADMA plasma levels and AF occurrence in AMI. Methods 273 patients hospitalized for AMI were included. Continuous electrocardiographic monitoring (CEM) ≥48 hours was recorded and ADMA was measured by High Performance Liquid Chromatography on admission blood sample. Results The incidence of silent and symptomatic AF was 39(14%) and 29 (11%), respectively. AF patients were markedly older than patients without AF (≈ 20 y). There was a trend towards higher ADMA levels in patients with symptomatic AF than in patients with silent AF or no AF (0.53 vs 0.49 and 0.49 μmol/L, respectively). After matching on age, we found that patients with symptomatic AF had a higher heart rate on admission and a higher rate of patients with LV dysfunction (28% vs. 3%, p=0.025). Patients who developed symptomatic AF had a higher ADMA level (0.53 vs. 0.43 μmol/L; p=0.001). Multivariate logistic regression analysis to estimate symptomatic AF occurrence showed that ADMA was independently associated with symptomatic AF (OR: 2.46 [1.21-5.00], p=0.013) beyond history of AF, LVEF<40% and elevated HR. Conclusion We show that high ADMA level is associated with the occurrence of AF. Although no causative role can be concluded from our observational study, our work further supports the hypothesis that endothelial dysfunction is involved in the pathogenesis of AF in AMI. The author hereby declares no conflict of interest
0333 Tissue specificity of the membrane vs nuclear actions of estrogen receptor alpha: insights from targeted mutations in mouse models Jean-François Arnal *, Coralie Fontaine, Marie-Cecile Valera, Marine Adlanmerini, Pierre Gourdy, Françoise Lenfant CHU Toulouse, Rangueil, INSERM U1048 – I2MR, Toulouse, France * Corresponding author: [email protected] (Jean-Francois Arnal) Thanks to unique mouse models, we demonstrated that the estrogen receptor alpha (ERa), but not ERb, is absolutely necessary for most of the arterial and metabolic actions of 17b-estradiol (E2). Estrogens also elicit deleterious effects on the uterus and breast (partly via their proliferative effect which increases the risk of cancer) as well as increased risk of venous thromboembolism, which are the two main limitations of classic estrogen therapies. ER contains two independent transactivation functions AF-1 and AF-2, and our team dissected for the first time in vivo the roles of functions ERaAF-1 and AF-2, allowing to define the key role of these nuclear actions in several tissues. More recently, we reported the key role of the ERa fraction localized to the plasma membrane which is absolutely necessary for the protective endothelial actions of E2 as well as, more unexpectedly, for female fertility. One mystery of estrogens biology is that selective ER modulators (SERMs, such as tamoxifen, raloxifen) have a highly tissue-specific action, behaving as agonists in some tissues (such as bone) and as antagonist in others (breast). We will report how these mouse models allowed us to characterize: 1) the tissue-specific roles of ERa : membrane ERa mediates the effect of E2 in the endothelium (acceleration of reendothelialization, activation of endothelial NO synthase), whereas nuclear ERa mediates the effect of E2 in the uterus. 2) a new natural SERM (Estetrol) currently tested in a phase III clinical trial as a safer oral contraception or hormonal treatment of menopause, as this molecule does not alter the hepatic-derived coagulation factors, and thereby could not increase the venous thrombosis risk. 3) an Estrogen Dendrimer Conjugate, a selective activator of membrane ERa, which could be a new endothelial protective SERM. The author declares a conflict of interest: JF Arnal'team received a financial support from Uteron/Mithra to study the mechanisms of action of Estetrol.
0546 Mitochondrial induced oxidative stress triggers Ca2+-dependent proteolysis and underlie VIDD Haikel Dridi * Université de Montpellier, INSERM U1046, Montpellier, France * Corresponding author: [email protected] (Haikel Dridi) Rationale In rodents, prolonged controlled mechanical ventilation (CMV) and associated diaphragm inactivity elicits complex pathologic changes
referred to as ventilator-induced diaphragm dysfunction (VIDD). Previous workers have demonstrated that VIDD is characterized by increased mitochondrial generation of reactive oxygen species (ROS) and redox-induced dysfunction in calcium release. Powers et al., (2011) have demonstrated that SS-31, a mitochondrial targeted antioxidant, markedly attenuates mitochondrial ROS generation. Since, the calcium-release pathology associated with VIDD is thought to be secondary to myofiber redox changes, we hypothesized that SS-31 should markedly attenuate these abnormalities in myofiber calcium regulation. Objective To test the hypothesis that SS-31, a mitochondrial-targeted antioxidant, attenuates the effect of prolonged CMV on diaphragm myofiber calcium homeostasis. Measurements and main results Specific force of diaphragm fibers decreased significantly 28 ±5%, (n=10) following 12hrs of mechanical ventilation compared to fibers from unventilated control mice. These changes were associated with a significant decrease in cross-sectional area (574 ±19 versus 673± 21 μm m2), an increase in calpain activity, the oxidation of the RyR1 macromolecular complexed, RyR1 phosphorylation at Ser-2844 and the depletion of the stabilizing subunit calstabin1 resulting in an increase in sarcoplasmic reticulum (SR) Ca2+ leak. Diaphragm from mice treated with SS-31 were protected against all of these changes. On the basis of these findings we propose that SR Ca2+ leak via RyR1 due to mitochondrial induced oxidative stress, triggers Ca2+-dependent proteolysis and underlie VIDD. These results emphasize the need to develop therapeutic interventions that mitigate mitochondrial dysfunctional thereby reduce mechanical ventilation-induced diaphragmatic weakness. The author hereby declares no conflict of interest
0460 Nitric oxide and hemoglobin form a paramagnetic compound quantifiable by Electron Paramagnetic Resonance (EPR) spectroscopy in venous erythrocytes that reflects vascular NO bioavailability in vivo Flavia Dei Zotti *, Irina Lobysheva, Jean-Luc Balligand Université Catholique de Louvain, Bruxelles, Belgique * Corresponding author: [email protected] (Flavia Dei Zotti) Reduced bioavailability of nitric oxide is a hallmark of endothelial dysfunction in metabolic and cardiovascular diseases, but its quantification in circulating blood remains a challenge.NO can form iron-nitrosyl complexes with hemoglobin(5-coordinate-α-HbNO) in erythrocytes(RBCs).We hypothesized that this complex reflects bioavailability of vascular NO and endothelial function in vivo.We developed a modified subtraction method using EPR to quantify it in RBCs from mouse,rat and human venous blood. NO could be supplied from vascular or erythrocytic NOS.We detected eNOS proteins in rodent and human RBC.To test eNOS functionality, we measured nitrite/ nitrate production and HbNO formation in human RBCs and from eNOS(+/+) and eNOS(-/-)mice in vitro and its sensitivity to NOS or arginase inhibitors.Nitrite and HbNO signals increased after arginase inhibition and were abrogated upon NOS inhibition in human and eNOS(+/+) but insensitive to these modulators in eNOS(-/-)RBCs. We found that upon exposure to exogenous NO-donor,the formation of HbNO was higher in hypoxic conditions(1%O2:0.018+/–0.002 compared to room air 0.0036+/–0.0004 μmol HbNO/μmolNO-donor).Accordingly, the stability of pre-formed HbNO was higher under hypoxia(17% degradation after 30 min vs 49% at room air),and preserved at 21% of O2 by incubation with catalase(CAT:2μM HbNO vs 0.5μM HbNO in untreated controls).Conversely,CAT inhibition increased ROS formation in RBCs, measured by FACS analysis of DCFDA fluorescence.This suggested that HbNO formation is sensitive to oxidative degradation, possibly by H2O2.We compared circulating HbNO levels in venous RBCs from normal volunteers or patients with cardiovascular diseases and found decreased HbNO in patients(0.141+/–11μM vs 0.22+/–12μM in volunteers;N=38 and48).We conclude that HbNO reflects exposure of RBCs to NO in vivo and is sensitive to oxidative degradation by H2O2.HbNO could be developed as a biomarker of NO bioavailability and/or oxidative stress ex vivo. The author hereby declares no conflict of interest
© Elsevier Masson SAS. All rights reserved.