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Major role of HIF-1 in endoplasmic reticulum stress and calcium alterations, leading to increased susceptibility to myocardial ischemiareperfusion, induced by chronic intermittent hypoxia
UntitledBook1.book Page 185 Monday, March 20, 2017 2:28 PM
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Archives of Cardiovascular Diseases Supplements (2017) 9, 185-186
oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMC protected against AKI. Furthermore, genetic deletion of MR in SMC blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the pig model of AKI. Altogether, we show that MR antagonism or deletion of the MR gene in SMC limit the renal injury induced by IR through effects on Rac1-mediated MR signalling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.
Topic 17 – Ischemia/reperfusion, stroke April 07th, Friday 2017
025
The author hereby declares no conflict of interest
Major role of HIF-1 in endoplasmic reticulum stress and calcium alterations, leading to increased susceptibility to myocardial ischemiareperfusion, induced by chronic intermittent hypoxia S. Moulin* (1), A. Thomas (2), J. Morand (1), C. Arnaud (1), D. GodinRibuot (1), E. Belaidi-Corsat (1) (1) Laboratoire HP2 Inserm U1042, Grenoble, France – (2) Cedars-Sinai Heart Institute, Los Angeles, Etats-Unis *Corresponding author: [email protected] Chronic intermittent hypoxia (IH) is described as a major detrimental factor leading to cardiovascular morbi-mortality in obstructive sleep apnea (OSA) patients. Previously, we demonstrated that IH increases myocardial susceptibility to ischemia-reperfusion (I/R) through endoplasmic reticulum (ER) stress-induced hypoxia inducible factor-1 (HIF-1). In view of HIF-1 involvement in response to IH, the aim of this study was to observe whether it could promote 1) susceptibility to I/R, 2) ER stress and 3) calcium (Ca2+) homeostasis alterations via mitochondria-associated ER membranes (MAM). Wild type and HIF-1α+/- mice were 14-days exposed to IH (21-5% FiO2, 60s-cycles, 8h/day), or normoxia (N). At the end of exposure, infarct size was measured following I/R (45/90min). ER stress and MAM integrity were assessed by Western blot, immunoprecipitation, qRT-PCR, interorganelle Ca2+ exchange and Duolink Proximity ligation assay on isolate adult cardiomyocytes. IH increased myocardial infarct (IH: 59.5±6 vs. N: 34.9±4.1, p<0.01). IH induced ER stress (increase in glucose-regulated protein kinase (GRP) 78 and C/EBP homologous protein pro-apoptotic pathway), associated with an alteration in Ca2+ exchange (Ca2+ overload in cytosol and ER). We also observed MAM alteration characterized by a decrease in GRP75, whereas others “MAM actors” increased. All of IH-induced modifications are abolished by HIF-1 partial deletion (I/R, IH/HIF-1α+/-: 37.1±5.1 vs. IH/WT, p<0.05). Thus, HIF-1 plays a pivotal role in the IH-induced ER stress and Ca2+ alterations, accentuating the IH-induced susceptibility to myocardial I/R. This study suggests that HIF-1 should be considered as a potential therapeutic target to prevent myocardial ischemia damages in OSA patients. The author hereby declares no conflict of interest
080 Benefit of Mineralocorticoid Receptor antagonism in ischemia-reperfusion induced acute kidney injury: role of vascular smooth muscle Rac1. G. Andre-Gregoire* (1-2), J. Barrera-Chimal (1), A. Nguyen Dinh Cat (1), S. Lechner (1), J. Cau (3), S. Prince (1), G. Loirand (4), V. Sauzeau (5), T. Hauet (3), F. Jaisser (1) (1) Centre de Recherche des Cordeliers, Inserm UMR1138, Paris – (2) INSERM U892 CNRS 6299, Centre de Recherche en Cancérologie Nantes-Angers, Nantes – (3) IRTOMIT Inserm UMR1082, Université de Poitiers, CHU de Poitiers, Poitiers – (4) L’institut du thorax, Inserm UMR1087 – (5) Inserm 1087, Nantes, France *Corresponding author: [email protected] Acute kidney injury (AKI) is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig model of human AKI. Our results show that, in mice, MR deficiency in smooth muscle cells (SMC) protects against kidney IR injury. MR blockade or genetic deletion of MR in SMC is associated with weaker
372 A new and economic device for cellular intermittent hypoxia M. Minoves (1-2), J. Morand (1-2), J. Polak (3), F. Perriot (1-2), E. Lemarié (1-2), B. Gonthier (1-2), JB. Menut (4), JL. Pépin (1-2), D. Godin-Ribuot (1-2), A. Briançon-Marjollet* (1-2) (1) Université Grenoble Alpes, Grenoble Cedex 9 – (2) INSERM U1042 Laboratoire HP2, Grenoble, France – (3) Center for Research on Diabetes, Metabolism and Nutrition, Third Faculty of medicine, Charles University, Prague, République tchéque – (4) SMTEC, Nyon, Suisse – (5) Laboratoire HP2 – (6) Laboratoire HP2 Inserm U1042, Grenoble, France *Corresponding author: [email protected] Performing rapid hypoxia-reoxygenation cycles in cell cultures is a technical challenge that needs to be overcome in order to develop pertinent cell models in the field of hypoxia/ischemia research. In particular, the cell models mimicking the pattern of intermittent hypoxia characteristic of obstructive sleep apnea (OSA) that have been developed in some laboratories have several limitations. Our goal was to develop a new intermittent hypoxia device for cell culture, allowing exposure of several culture dishes to rapid intermittent hypoxia cycles with minimal gas consumption. We used a commercially-available gas blender combined with gas-permeable plates and custom-made plate holders. With gas flows as low as 200mL/ min, the device enables oxygen cycles between around 25 and 125mmHg in the culture medium, thus allowing high amplitudes of oxygen variation. During the normoxic phase (16% O2), a plateau above 110mmHg was reached in less than one minute, while the descending phase was slower. Extending the hypoxic phase to 5 minutes at 2% O2 decreased oxygen level down to 25 mmHg in the medium. The cycles were only modestly altered by the volume of culture medium or the presence of cells. In contrast, the nadir during hypoxic phases changed when cycles were measured at different heights above the bottom of the plates. Finally, we validate this new model by showing that a 6h exposure to intermittent hypoxia induced an increase of HIF-1α expression in primary endothelial cells (HAEC). Thus, this new device can perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several plates or dishes simultaneously. This device will allow the investigation of the cellular effects of intermittent hypoxia cycles relevant to OSA pathophysiology. Moreover, it can also be useful for other applications such as finelycontrolled ischemia-reperfusion studies in vitro. The author hereby declares no conflict of interest
413 A novel cardioprotection strategy: targeting the ER via the TRANSLOCON, a calcium leak channel. R. Al-Mawla* (1-2), L. Paita (1-2), M. Ovize(3-1), S. Ducreux (1-2), F. Van-Coppenolle (1-2) (1) INSERM 1060, CarMen, IHU Opera, Bron – (2) Université Claude Bernard Lyon 1, Lyon – (3) Novartis, Sainte foy les Lyon, France *Corresponding author: [email protected] Background Reperfusion after a prolonged ischemia can cause irreversible cellular damages including through the alteration of calcium homeostasis. Under resting conditions, calcium pumps such as SERCA and calcium release channels such as IP3R, RyR, and leak channels contribute to calcium homeo-
© Elsevier Masson SAS. All rights reserved.
185
Archives of Cardiovascular Diseases Supplements (2017) 9, 185-186
oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMC protected against AKI. Furthermore, genetic deletion of MR in SMC blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the pig model of AKI. Altogether, we show that MR antagonism or deletion of the MR gene in SMC limit the renal injury induced by IR through effects on Rac1-mediated MR signalling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.
Topic 17 – Ischemia/reperfusion, stroke April 07th, Friday 2017
025
The author hereby declares no conflict of interest
Major role of HIF-1 in endoplasmic reticulum stress and calcium alterations, leading to increased susceptibility to myocardial ischemiareperfusion, induced by chronic intermittent hypoxia S. Moulin* (1), A. Thomas (2), J. Morand (1), C. Arnaud (1), D. GodinRibuot (1), E. Belaidi-Corsat (1) (1) Laboratoire HP2 Inserm U1042, Grenoble, France – (2) Cedars-Sinai Heart Institute, Los Angeles, Etats-Unis *Corresponding author: [email protected] Chronic intermittent hypoxia (IH) is described as a major detrimental factor leading to cardiovascular morbi-mortality in obstructive sleep apnea (OSA) patients. Previously, we demonstrated that IH increases myocardial susceptibility to ischemia-reperfusion (I/R) through endoplasmic reticulum (ER) stress-induced hypoxia inducible factor-1 (HIF-1). In view of HIF-1 involvement in response to IH, the aim of this study was to observe whether it could promote 1) susceptibility to I/R, 2) ER stress and 3) calcium (Ca2+) homeostasis alterations via mitochondria-associated ER membranes (MAM). Wild type and HIF-1α+/- mice were 14-days exposed to IH (21-5% FiO2, 60s-cycles, 8h/day), or normoxia (N). At the end of exposure, infarct size was measured following I/R (45/90min). ER stress and MAM integrity were assessed by Western blot, immunoprecipitation, qRT-PCR, interorganelle Ca2+ exchange and Duolink Proximity ligation assay on isolate adult cardiomyocytes. IH increased myocardial infarct (IH: 59.5±6 vs. N: 34.9±4.1, p<0.01). IH induced ER stress (increase in glucose-regulated protein kinase (GRP) 78 and C/EBP homologous protein pro-apoptotic pathway), associated with an alteration in Ca2+ exchange (Ca2+ overload in cytosol and ER). We also observed MAM alteration characterized by a decrease in GRP75, whereas others “MAM actors” increased. All of IH-induced modifications are abolished by HIF-1 partial deletion (I/R, IH/HIF-1α+/-: 37.1±5.1 vs. IH/WT, p<0.05). Thus, HIF-1 plays a pivotal role in the IH-induced ER stress and Ca2+ alterations, accentuating the IH-induced susceptibility to myocardial I/R. This study suggests that HIF-1 should be considered as a potential therapeutic target to prevent myocardial ischemia damages in OSA patients. The author hereby declares no conflict of interest
080 Benefit of Mineralocorticoid Receptor antagonism in ischemia-reperfusion induced acute kidney injury: role of vascular smooth muscle Rac1. G. Andre-Gregoire* (1-2), J. Barrera-Chimal (1), A. Nguyen Dinh Cat (1), S. Lechner (1), J. Cau (3), S. Prince (1), G. Loirand (4), V. Sauzeau (5), T. Hauet (3), F. Jaisser (1) (1) Centre de Recherche des Cordeliers, Inserm UMR1138, Paris – (2) INSERM U892 CNRS 6299, Centre de Recherche en Cancérologie Nantes-Angers, Nantes – (3) IRTOMIT Inserm UMR1082, Université de Poitiers, CHU de Poitiers, Poitiers – (4) L’institut du thorax, Inserm UMR1087 – (5) Inserm 1087, Nantes, France *Corresponding author: [email protected] Acute kidney injury (AKI) is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig model of human AKI. Our results show that, in mice, MR deficiency in smooth muscle cells (SMC) protects against kidney IR injury. MR blockade or genetic deletion of MR in SMC is associated with weaker
372 A new and economic device for cellular intermittent hypoxia M. Minoves (1-2), J. Morand (1-2), J. Polak (3), F. Perriot (1-2), E. Lemarié (1-2), B. Gonthier (1-2), JB. Menut (4), JL. Pépin (1-2), D. Godin-Ribuot (1-2), A. Briançon-Marjollet* (1-2) (1) Université Grenoble Alpes, Grenoble Cedex 9 – (2) INSERM U1042 Laboratoire HP2, Grenoble, France – (3) Center for Research on Diabetes, Metabolism and Nutrition, Third Faculty of medicine, Charles University, Prague, République tchéque – (4) SMTEC, Nyon, Suisse – (5) Laboratoire HP2 – (6) Laboratoire HP2 Inserm U1042, Grenoble, France *Corresponding author: [email protected] Performing rapid hypoxia-reoxygenation cycles in cell cultures is a technical challenge that needs to be overcome in order to develop pertinent cell models in the field of hypoxia/ischemia research. In particular, the cell models mimicking the pattern of intermittent hypoxia characteristic of obstructive sleep apnea (OSA) that have been developed in some laboratories have several limitations. Our goal was to develop a new intermittent hypoxia device for cell culture, allowing exposure of several culture dishes to rapid intermittent hypoxia cycles with minimal gas consumption. We used a commercially-available gas blender combined with gas-permeable plates and custom-made plate holders. With gas flows as low as 200mL/ min, the device enables oxygen cycles between around 25 and 125mmHg in the culture medium, thus allowing high amplitudes of oxygen variation. During the normoxic phase (16% O2), a plateau above 110mmHg was reached in less than one minute, while the descending phase was slower. Extending the hypoxic phase to 5 minutes at 2% O2 decreased oxygen level down to 25 mmHg in the medium. The cycles were only modestly altered by the volume of culture medium or the presence of cells. In contrast, the nadir during hypoxic phases changed when cycles were measured at different heights above the bottom of the plates. Finally, we validate this new model by showing that a 6h exposure to intermittent hypoxia induced an increase of HIF-1α expression in primary endothelial cells (HAEC). Thus, this new device can perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several plates or dishes simultaneously. This device will allow the investigation of the cellular effects of intermittent hypoxia cycles relevant to OSA pathophysiology. Moreover, it can also be useful for other applications such as finelycontrolled ischemia-reperfusion studies in vitro. The author hereby declares no conflict of interest
413 A novel cardioprotection strategy: targeting the ER via the TRANSLOCON, a calcium leak channel. R. Al-Mawla* (1-2), L. Paita (1-2), M. Ovize(3-1), S. Ducreux (1-2), F. Van-Coppenolle (1-2) (1) INSERM 1060, CarMen, IHU Opera, Bron – (2) Université Claude Bernard Lyon 1, Lyon – (3) Novartis, Sainte foy les Lyon, France *Corresponding author: [email protected] Background Reperfusion after a prolonged ischemia can cause irreversible cellular damages including through the alteration of calcium homeostasis. Under resting conditions, calcium pumps such as SERCA and calcium release channels such as IP3R, RyR, and leak channels contribute to calcium homeo-
© Elsevier Masson SAS. All rights reserved.