Hypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO, and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4'-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4'-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4'-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity.

Several studies have reported that CORM-3, a water-soluble carbon monoxide releasing molecule, elicits cardioprotection against myocardial infarction but the mechanism remains to be investigated. Numerous reports indicate that inhibition of pH regulators, the Na+/H+ exchanger (NHE) and Na+/HCO3- symporter (NBC), protect cardiomyocytes from hypoxia/reoxygenation injury by delaying the intracellular pH (pHi) recovery at reperfusion. Our goal was to explore whether CORM-3-mediated cytoprotection involves the modulation of pH regulation. When added at reoxygenation, CORM-3 (50 mu M) reduced the mortality of cardiomyocytes exposed to 3 h of hypoxia and 2 h of reoxygenation in HCO3--buffered solution. This effect was lost when using inactive iCORM-3, which is depleted of CO and used as control, thus implicating CO as the mediator of this cardioprotection. Interestingly, the cardioprotective effect of CORM-3 was abolished by switching to a bicarbonate-free medium. This effect of CORM-3 was also inhibited by 5-hydroxydecanoate, a mitochondrial ATP-dependent K+(mK(ATP)) channel inhibitor (500 mu M) or PD098059, a MEK1/2 inhibitor (10 mu M). In additional experiments and in the absence of hypoxia-reoxygenation, intracellular pH was monitored in cardiomyocytes exposed to cariporide to block NHE activity. CORM-3 inhibited alkalinisation and this effect was blocked by PD098059 and 5-HD. In conclusion, CORM-3 protects the cardiomyocyte against hypoxia-reoxygenation injury by inhibiting a bicarbonate transporter at reoxygenation, probably the Na+/HCO3- symporter. This cardioprotective effect of CORM-3 requires the activation of mK ATP channels and the activation of MEK1/2.

Aims Recent studies reported that cAMP-binding protein Epac1-deficient mice were protected against various forms of cardiac stress, suggesting that pharmacological inhibition of Epac1 could be beneficial for the treatment of cardiac diseases. To test this assumption, we characterized an Epac1-selective inhibitory compound and investigated its potential cardioprotective properties. Methods and results We used the Epac1-BRET (bioluminescence resonance energy transfer) for searching for non-cyclic nucleotide Epac1 modulators. A thieno[2,3-b]pyridine derivative, designated as AM-001 was identified as a non-competitive inhibitor of Epac1. AM-001 has no antagonist effect on Epac2 or protein kinase A activity. This small molecule prevents the activation of the Epac1 downstream effector Rap1 in cultured cells, in response to the Epac1 preferential agonist, 8-CPT-AM. In addition, we found that AM-001 inhibited Epac1-dependent deleterious effects such as cardiomyocyte hypertrophy and death. Importantly, AM-001-mediated inhibition of Epac1 reduces infarct size after mouse myocardial ischaemia/reperfusion injury. Finally, AM-001 attenuates cardiac hypertrophy, inflammation and fibrosis, and improves cardiac function during chronic beta-adrenergic receptor activation with isoprenaline (ISO) in mice. At the molecular level, ISO increased Epac1-G protein-coupled receptor kinase 5 (GRK5) interaction and induced GRK5 nuclear import and histone deacetylase type 5 (HDAC5) nuclear export to promote the activity of the prohypertrophic transcription factor, myocyte enhancer factor 2 (MEF2). Inversely, AM-001 prevented the non-canonical action of GRK5 on HDAC5 cytoplasmic shuttle to down-regulate MEF2 transcriptional activity. Conclusion Our study represents a 'proof-of-concept' for the therapeutic effectiveness of inhibiting Epac1 activity in cardiac disease using small-molecule pharmacotherapy.

H11 kinase/Hsp22 (Hsp22) is a small heat shock protein, which, when overexpressed cardiac specifically in transgenic (TG) mice, induces stable left ventricular (LV) hypertrophy. Hsp22 also increases oxidative phosphorylation and mitochondrial reactive oxygen species (ROS) production, mechanisms mediating LV hypertrophy, senescence and reduced lifespan. Therefore, we investigated whether ROS production mediates LV hypertrophy, senescence and reduced life span in Hsp22 TG mice. Survival curves revealed that TG mice had a 48% reduction in their mean life span compared to wild type (WT) mice. This was associated with a significant increase in senescence markers, such as p16, p19 mRNA levels as well as the percentage of β-galactosidase positive cells and telomerase activity. Oxidized (GSSG)/reduced (GSH) glutathione ratio, an indicator of oxidative stress, and ROS production from 3 major cellular sources was measured in cardiac tissue. Hearts from TG mice exhibited a decrease in GSH/GSSG ratio together with increased ROS production from all sources. To study the role of ROS, mice were treated with the antioxidant Tempol from weaning to their sacrifice. Chronic Tempol treatment abolished oxidative stress and overproduction of ROS, and reduced myocardial hypertrophy and Akt phosphorylation in TG mice. Tempol also significantly extended life span and prevented aging markers in TG mice. Taken together these results show that overexpression of Hsp22 increases oxidative stress responsible for the induction of hypertrophy and senescence and ultimately reduction in life span.

Introduction: P16Ink4a is a key factor in the control of the cell cycle and governs the stem cells self-renewal and differentiation in several tissues. To investigate the role of p16 in type 2 alveolar epithelial stem cell proliferation and in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Methods: Left side pneumonectomy of C57Bl/6 wild type (WT) or p16-/- mice was performed after thoracotomy, while sham mice went through thoracotomy only. Ten days after surgery morphometric analysis (mean linear intercept, alveolar surface), septal thickness, collagen deposition (Red Sirius), alveolar type 2 cells and myofibroblasts content were quantified (by immunohistochemistry for proSPC and αSMA respectively). Results: Pneumonectomised WT mice versus sham WT mice showed an increase volume of the remaining right lung, and an increase of alveolar surface but no modification of the mean linear intercept. Pneumonectomised p16-/- mice versus sham p16-/- have no modification of lung volume or alveolar surface, but increased septal thickness. In pneumonectomised group, p16-/- mice exhibited an increased number of lung cells especially alveolar type 2 cells as compared to WT mice. Collagen deposition (red Sirius) and myofibroblast content (αSMA) were not different between the groups. Conclusion: P16 deletion promotes epithelial type 2 cells differentiation during lung regeneration after pneumonectomy. P16 inhibition may be a tool to initiate and sustain alveologenesis in adults.

A major cause of cell death during myocardial ischemia-reperfusion is mitochondrial dysfunction. We previously showed that the reperfusion of an ischemic myocardium was associated with an accumulation of cholesterol into mitochondria and a concomitant strong generation of auto-oxidized oxysterols. The inhibition of mitochondrial accumulation of cholesterol abolished the formation of oxysterols and prevented mitochondrial injury at reperfusion. The aim of this study was to investigate the impact of hypercholesterolemia on sterol and oxysterol accumulation in rat cardiac cytosols and mitochondria and to analyse the effect of the translocator protein ligand 4'-chlorodiazepam on this accumulation and mitochondrial function. Hypercholesterolemic ZDF fa/fa rats or normocholesterolemic lean rats were submitted to 30min of coronary artery occlusion followed by 15min reperfusion where cardiac cytosols and mitochondria were isolated. Hypercholesterolemia increased the cellular cardiac concentrations of cholesterol, cholesterol precursors and oxysterols both in cytosol and mitochondria in non-ischemic conditions. It also amplified the accumulation of all these compounds in cardiac cells and the alteration of mitochondrial function with ischemia-reperfusion. Administration of 4'-chlorodiazepam to ZDF fa/fa rats had no effect on the enhancement of sterols and oxysterols observed in the cytosols but inhibited cholesterol transfer to the mitochondria. It also alleviated the mitochondrial accumulation of all the investigated sterols and oxysterols. This was associated with a restoration of oxidative phosphorylation and a prevention of mitochondrial transition pore opening. The inhibition of cholesterol accumulation with TSPO ligands represents an interesting strategy to protect the mitochondria during ischemia-reperfusion in hypercholesterolemic conditions.

Rationale: Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood. Objective: To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury. Methods and Results: We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation ( Epac1 −/− ) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation–induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation–induced cell death. Mechanistically, Epac1 favors Ca 2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca 2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca 2+ /calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte. Conclusions: Our results reveal the existence, within mitochondria, of different cAMP–Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage.

Alternative Ways to Die Session held on 8 July 2016 doi:10.1093/cvr/cvw121 5 Epac1 deletion prevents cardiomyocyte apoptosis during ischemia/reperfusion M. Laudette1; L. Fazal1; S. Pons2; F. Tortosa1; P. Sicard1; J. Mialet-Perez1; B. Ghaleh2; F. Lezoualc’h1 Inserm-UPS U1048, Institute of Cardiovascular and Metabolic Diseases (I2MC), Toulouse, France; University Hospital Henri Mondor, Inserm U955, Team 3, Creteil, France Introduction: Early coronary reperfusion has been established as the best therapeutic strategy to limit infarct size and improve prognosis. Therefore, elucidating the mechanisms underlying cardiomyocyte death may yield novel therapeutic targets to limit ischemia reperfusion (I/R) injury. I/R is accompanied and influenced by perturbations of the b-adrenergic receptor pathway which acts through cAMP dependent signaling cascade to modulate cardiac function and remodeling. Purpose: Although the involvement of the cAMP-binding protein Epac1 in cardiac hypertrophy and arrhythmia has been recently described, its role in I/R induced-cardiomyocyte death has not yet been investigated. Methods: Isolated adult cardiomyocytes from Epac1 knock-out (Epac1-/-) mice or wild-type (WT) littermates were exposed to hypoxia (HX) for 4h and 2h of reoxygenation period (HX+R). Cell death was determined by Trypan blue staining and LDH release. The mitochondrial permeability transition pore (mPTP) opening was monitored by the calcein loading CoCl(2)-quenching technique. The area at risk was examined by Evans blue and infarct size was evaluated by TTC staining. Results: Our data showed that HX+R-induced cardiomyocyte death were significantly prevented in adult cardiomyocytes isolated from Epac1-/mice. In addition, we found that the increased expression of apoptotic markers (Bax, cleaved Caspase-9,Caspase-3) during HX+R conditions were also inhibited in Epac1-/cardiomyocytes compared to WT cells. Interestingly, HX+R induced a decrease in calcein fluorescence corresponding to mPTP opening (56%+1.1 vs control cells set at 100%) in WT cardiomyocytes while genetic deletion of Epac1 prevented mPTP opening in HX+R conditions. Concomitantly, we found that the infarct size was significantly reduced in the Epac1-/mice compared to the WT animals (53+4 % vs 33+4%, p,0.01) despite the same area at risk. Conclusion: Epac1 deletion confers resistance to I/R injury via the inhibition of a mitochondrial death signaling. Our study shed light the therapeutic potential of the inhibition of Epac1 and, the development of Epac1 inhibitors as new drugs to treat I/R injury. 6 Subcellular redistribution of mitogen and stress activated kinase 1 (MSK1) contributes to protection against oxidative stressinduced apoptosis in cardiac myocytes A. Apostolopoulos; K. Mellidis; E. Barlaka; A. Moraiti; A. Lazou Aristotle University of Thessaloniki, School of Biology, Thessaloniki, Greece MSK1 is a predominantly nuclear kinase known to associate with a variety of nuclear targets, including transcription factors and chromatin-associated proteins, in several tissues. However, scarce evidence indicates that this kinase may also have cytoplasmic targets. In the heart, we have previously shown that MSK1 phosphorylates CREB and contributes to the hypertrophic response. In this study, we aimed to determine the subcellular distribution of MSK1 and its role in response to oxidative stress in cardiac myocytes. Exposure of cardiac myocytes to H2O2 or simulated ischemia (SI) resulted in increased phosphorylation of MSK1, which is mediated by p38 MAPK and/or ERK1/2. Subcellular localization of endogenous MSK1 was determined under confocal laser scanning microscope using immunofluorescence and antibodies to both totaland phosphoprotein. Upon exposure to oxidative stress, a significant percentage of nuclear MSK1 was translocated to the cytoplasm. In addition, pretreatement of cardiac myocytes with the MSK1 inhibitor SB747651A resulted in increased levels of apoptosis either at basal conditions or after oxidative stress. Furthermore, inhibition of MSK1 downregulated the antiapoptotic Bcl-2 protein. The results demonstrate that, altered distribution of MSK1 may lead to modulation of cytoplasmic targets, including apoptosis associated proteins, contributing to cardioprotection during oxidative stress. 7 Excessive ROS production in mitochondria switches off protective mitochondrial kinase signaling M. Tanno1; W. Ohwada1; T. Yano1; T. Miki1; A. Kuno2; S. Ishikawa1; Y. Tatekoshi1; K. Nishizawa1; M. Mizuno1; T. Miura1 Sapporo Medical University, Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo, Japan; Sapporo Medical University, Department of Pharmacology, Sapporo, Japan Background: Accumulating evidence indicates that both anti-survival and pro-survival signaling mechanisms are localized within mitochondria, among which glycogen synthase kinase 3-b (GSK-3b) plays crucial roles in regulation of the mitochondrial permeability transition pore (mPTP). We recently demonstrated that GSK-3b translocates to the mitochondria in response to oxidative stress, promoting production of excess reactive oxygen species (ROS) and opening of the mPTP. Here, we examined how ROS modifiy mitochondrial pro-survival kinases. Methods and Results: Isolated mitochondria from HEK293 cells were incubated in a vehicle or trypsin at 1 1,000 mmol/L. Immunoblot analysis revealed that TOM20, an outer membrane (OM) protein, cytochrome oxidase IV, an inner membrane (IM) protein, and cyclophilin D, a matrix protein, were digested by 1, 100 and 1,000 mmol/L of trypsin, respectively, confirming stepwise digestion of mitochondrial membranes. At baseline, 56%, 12% and 21% of mitochondrial GSK-3bwere localized in OM, IM and matrix, respectively, the other 11% presumably being in the intermembrane space. A similar pattern of intra-mitochondrial localization was observed for ERK. In contrast, only 10% of mitochondrial Akt was observed in OM, and 32% and 26% of Akt were in IM and matrix, respectively. Treatment with IGF-1 (10 nmol/L, 45 min) increased total mitochondrial GSK-3b by 1.4 fold, with its increase in IM being prominent (2.6 fold), compared to those in vehicle-treated cells. The level of protective Ser9-phospho GSK-3b in mitochondria was increased by 1.5 fold, and a marked increase in IM by 13.5 fold was observed. IGF-1 increased phospho-Akt in OM and IM by 3.4 and 3.6 fold, respectively, and phospho-ERK in OM and IM by 1.8 and 2.9 fold, respectively, though such an increase was not observed for phosphorylation of both kinases in the matrix. Addition of antimycin A (40 mmol/L, 30min), a mitochondrial ROS inducer, to IGF-1 eliminated phosphorylation of Akt, ERK and GSK-3b in all compartments. AA did not change intra-mitochondrial localization of Ak and ERK but slightly increased total GSK-3b level. Conclusion: The present results suggest that Akt and ERK in OM and/or IM contribute to protective Ser9-phosphorylation of GSK-3b in mitochondria and that excessive mitochondrial ROS production switches off the protective signaling by dephosphorylation of mitochondrial Akt and ERK possibly via activation of phosphatases. Cardiovascular Research Supplements (2016) 111, S1 Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: journals.permissions@oup.com. by gest on D ecem er 0, 2016 D ow nladed fom

Alternative Ways to Die Session held on 8 July 2016 doi:10.1093/cvr/cvw121 5 Epac1 deletion prevents cardiomyocyte apoptosis during ischemia/reperfusion M. Laudette1; L. Fazal1; S. Pons2; F. Tortosa1; P. Sicard1; J. Mialet-Perez1; B. Ghaleh2; F. Lezoualc’h1 Inserm-UPS U1048, Institute of Cardiovascular and Metabolic Diseases (I2MC), Toulouse, France; University Hospital Henri Mondor, Inserm U955, Team 3, Creteil, France Introduction: Early coronary reperfusion has been established as the best therapeutic strategy to limit infarct size and improve prognosis. Therefore, elucidating the mechanisms underlying cardiomyocyte death may yield novel therapeutic targets to limit ischemia reperfusion (I/R) injury. I/R is accompanied and influenced by perturbations of the b-adrenergic receptor pathway which acts through cAMP dependent signaling cascade to modulate cardiac function and remodeling. Purpose: Although the involvement of the cAMP-binding protein Epac1 in cardiac hypertrophy and arrhythmia has been recently described, its role in I/R induced-cardiomyocyte death has not yet been investigated. Methods: Isolated adult cardiomyocytes from Epac1 knock-out (Epac1-/-) mice or wild-type (WT) littermates were exposed to hypoxia (HX) for 4h and 2h of reoxygenation period (HX+R). Cell death was determined by Trypan blue staining and LDH release. The mitochondrial permeability transition pore (mPTP) opening was monitored by the calcein loading CoCl(2)-quenching technique. The area at risk was examined by Evans blue and infarct size was evaluated by TTC staining. Results: Our data showed that HX+R-induced cardiomyocyte death were significantly prevented in adult cardiomyocytes isolated from Epac1-/mice. In addition, we found that the increased expression of apoptotic markers (Bax, cleaved Caspase-9,Caspase-3) during HX+R conditions were also inhibited in Epac1-/cardiomyocytes compared to WT cells. Interestingly, HX+R induced a decrease in calcein fluorescence corresponding to mPTP opening (56%+1.1 vs control cells set at 100%) in WT cardiomyocytes while genetic deletion of Epac1 prevented mPTP opening in HX+R conditions. Concomitantly, we found that the infarct size was significantly reduced in the Epac1-/mice compared to the WT animals (53+4 % vs 33+4%, p,0.01) despite the same area at risk. Conclusion: Epac1 deletion confers resistance to I/R injury via the inhibition of a mitochondrial death signaling. Our study shed light the therapeutic potential of the inhibition of Epac1 and, the development of Epac1 inhibitors as new drugs to treat I/R injury. 6 Subcellular redistribution of mitogen and stress activated kinase 1 (MSK1) contributes to protection against oxidative stressinduced apoptosis in cardiac myocytes A. Apostolopoulos; K. Mellidis; E. Barlaka; A. Moraiti; A. Lazou Aristotle University of Thessaloniki, School of Biology, Thessaloniki, Greece MSK1 is a predominantly nuclear kinase known to associate with a variety of nuclear targets, including transcription factors and chromatin-associated proteins, in several tissues. However, scarce evidence indicates that this kinase may also have cytoplasmic targets. In the heart, we have previously shown that MSK1 phosphorylates CREB and contributes to the hypertrophic response. In this study, we aimed to determine the subcellular distribution of MSK1 and its role in response to oxidative stress in cardiac myocytes. Exposure of cardiac myocytes to H2O2 or simulated ischemia (SI) resulted in increased phosphorylation of MSK1, which is mediated by p38 MAPK and/or ERK1/2. Subcellular localization of endogenous MSK1 was determined under confocal laser scanning microscope using immunofluorescence and antibodies to both totaland phosphoprotein. Upon exposure to oxidative stress, a significant percentage of nuclear MSK1 was translocated to the cytoplasm. In addition, pretreatement of cardiac myocytes with the MSK1 inhibitor SB747651A resulted in increased levels of apoptosis either at basal conditions or after oxidative stress. Furthermore, inhibition of MSK1 downregulated the antiapoptotic Bcl-2 protein. The results demonstrate that, altered distribution of MSK1 may lead to modulation of cytoplasmic targets, including apoptosis associated proteins, contributing to cardioprotection during oxidative stress. 7 Excessive ROS production in mitochondria switches off protective mitochondrial kinase signaling M. Tanno1; W. Ohwada1; T. Yano1; T. Miki1; A. Kuno2; S. Ishikawa1; Y. Tatekoshi1; K. Nishizawa1; M. Mizuno1; T. Miura1 Sapporo Medical University, Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo, Japan; Sapporo Medical University, Department of Pharmacology, Sapporo, Japan Background: Accumulating evidence indicates that both anti-survival and pro-survival signaling mechanisms are localized within mitochondria, among which glycogen synthase kinase 3-b (GSK-3b) plays crucial roles in regulation of the mitochondrial permeability transition pore (mPTP). We recently demonstrated that GSK-3b translocates to the mitochondria in response to oxidative stress, promoting production of excess reactive oxygen species (ROS) and opening of the mPTP. Here, we examined how ROS modifiy mitochondrial pro-survival kinases. Methods and Results: Isolated mitochondria from HEK293 cells were incubated in a vehicle or trypsin at 1 1,000 mmol/L. Immunoblot analysis revealed that TOM20, an outer membrane (OM) protein, cytochrome oxidase IV, an inner membrane (IM) protein, and cyclophilin D, a matrix protein, were digested by 1, 100 and 1,000 mmol/L of trypsin, respectively, confirming stepwise digestion of mitochondrial membranes. At baseline, 56%, 12% and 21% of mitochondrial GSK-3bwere localized in OM, IM and matrix, respectively, the other 11% presumably being in the intermembrane space. A similar pattern of intra-mitochondrial localization was observed for ERK. In contrast, only 10% of mitochondrial Akt was observed in OM, and 32% and 26% of Akt were in IM and matrix, respectively. Treatment with IGF-1 (10 nmol/L, 45 min) increased total mitochondrial GSK-3b by 1.4 fold, with its increase in IM being prominent (2.6 fold), compared to those in vehicle-treated cells. The level of protective Ser9-phospho GSK-3b in mitochondria was increased by 1.5 fold, and a marked increase in IM by 13.5 fold was observed. IGF-1 increased phospho-Akt in OM and IM by 3.4 and 3.6 fold, respectively, and phospho-ERK in OM and IM by 1.8 and 2.9 fold, respectively, though such an increase was not observed for phosphorylation of both kinases in the matrix. Addition of antimycin A (40 mmol/L, 30min), a mitochondrial ROS inducer, to IGF-1 eliminated phosphorylation of Akt, ERK and GSK-3b in all compartments. AA did not change intra-mitochondrial localization of Ak and ERK but slightly increased total GSK-3b level. Conclusion: The present results suggest that Akt and ERK in OM and/or IM contribute to protective Ser9-phosphorylation of GSK-3b in mitochondria and that excessive mitochondrial ROS production switches off the protective signaling by dephosphorylation of mitochondrial Akt and ERK possibly via activation of phosphatases. Cardiovascular Research Supplements (2016) 111, S1 Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: journals.permissions@oup.com. by gest on N ovem er 2, 2016 D ow nladed fom