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Hydrogen sulfide prevents doxorubicin-induced apoptosis of H9c2 cardiomyocytes through activation of AMPK
Abstracts/Nitric Oxide 47 (2015) S14–S60
PP106 Redox activities of hydrogen sulfide and polysulfides; implications in oxygen sensing Eric R. DeLeon a,b, Evelyn S. Huang b, Yan Gao a, Kenneth R. Olson a,b a Indiana University School of Medicine – South Bend, South Bend, IN, USA b University of Notre Dame, Notre Dame, IN, USA Introduction: Vascular oxygen sensing has been attributed to hypoxic generation of reactive oxygen species (ROS) which oxidize the cytosol and reduce the mitochondrial matrix (Waypa et al., 2010 [1]). However, H2S and polysulfides (H2Sn) have also been shown to affect redox status (Greiner et al., 2013 [2]). We hypothesize that reactive sulfur species (RSS) have the same effect on redox status as ROS and propose that this supports our hypothesis that the metabolism of H2S is the vascular oxygen sensor (Olson, 2015 [3]). Methods: In this study, we examined the effects of H2S (Na2S) and mixed H2Sn (K2Sn) on redox status of the redox sensitive green fluorescent protein (roGFP) purified from Escherichia coli (roGFP-E) using ~40% and 100% oxidized protein incubated in either normoxia (21% O2) or anoxia (0% O2). We also examined the effects of H2S and H2Sn on roGFP inserted into either the cytoplasm (roGFP-C) or mitochondrial matrix (roGFP-M) of HEK-293 cells. Results: 1–300 μM H2S had no effect on roGFP-E whereas 40% oxidized roGFP-E was further oxidized by 1 mM H2S and completely oxidized by H2S >1 mM in both 21 and 0% O2. H2Sn oxidized roGFP-E at considerably lower concentrations (>3 μM) and independently of O2 availability. We saw no evidence for roGFP-E reduction by either H2S or H2Sn. Conversely, 10 μM H2S and above oxidized roGFP-C and reduced roGFP-M in-vitro; H2Sn reduced roGFP-C and roGFP-M at concentrations <1 mM and oxidized them at concentrations >1 mM. Conclusions: 1. H2S is not an efficient reductant, particularly in cells. 2. The time course of H2S activation is inconsistent with a preexisting polysulfide contaminant, but because H2S cannot be an oxidant, we conclude that H2S must somehow react with roGFP-E to produce an oxidant, probably a polysulfide. 3. The oxidizing ability of H2S is O2-independent. 4. The increased efficacy of H2S compared to H2Sn in HEK-293 cells may be due to rapid permeation of H2S. 5. The effects of hypoxia on intracellular redox status is mimicked by exogenous H 2 S, providing additional support to our hypothesis of H2S metabolism as the O2 sensor. Support: NSF Grant Nos. IOS1051627, IOS1446310. References [1] G.B. Waypa, J.D. Marks, R. Guzy, P.T. Mungai, J. Schriewer, D. Dokic, et al., Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells, Circ. Res. 106 (3) (2010) 526–535, doi:10.1161/CIRCRESAHA.109.206334. [2] R. Greiner, Z. Pálinkás, K. Bäsell, D. Becher, H. Antelmann, P. Nagy, et al., Hydrogen sulfide as an oxygen sensor, Antioxid. Redox Signal. 19 (15) (2013) 1749–1765, doi:10.1089/ars.2012.5041 [Epub 2013 Jun 20]. [3] K.R. Olson, Antioxid. Redox Signal. 22 (5) (2015) 377–397, doi:10.1089/ ars.2014.5930 [Epub 2014 Jul 30]. http://dx.doi.org/10.1016/j.niox.2015.02.136
PP107 Hydrogen sulfide prevents doxorubicin-induced apoptosis of H9c2 cardiomyocytes through activation of AMPK Yuan Zhang a, Shun-Lin Qu a, Zhi-Han Tang a, Zhong Ren a, Mi-Hua Liu a, Xin-Yuan Luo a, Lu-Shan Liu a, Yu Gui b, Xi-Long Zheng b, Zhi-Sheng Jiang a
S57
a Institute of Cardiovascular Disease and Key Lab for Arteriosclerology of Hunan Province, University of South China, Hengyang City, Hunan Province, China b The Smooth Muscle Research Group, Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Canada
Background and objective: Doxorubicin induces apoptosis of cardiomyocytes, which plays an important role in its myocardial toxicity. Hydrogen sulfide is the third endogenous gasotransmitter after nitric oxide and carbon monoxide, and has a protective effect on cardiomyocytes. In addition, adenosine monophosphate activated protein kinase (AMPK) is not only a cellular energy, but also a pressure sensor and regulator. AMPK regulates reactive oxygen species/redox balance, cell apoptosis and proliferation to maintain cellular homeostasis. In this study, we aimed to explore the role of AMPK activation in the antagonist influence of hydrogen sulfide against apoptosis of H9c2 cardiomyocytes induced by doxorubicin. Methods and results: H9c2 cardiomyocytes with or without pretreatment of 100 μmol/L NaHS were exposed to 5 μmol/L doxorubicin for 24 h, followed by western blot or dichlorofluorescein diacetate (DCFH-DA) analyses. Our results showed that in NaHS+doxorubicin group the ratio of p-AMPK/AMPK was 1.4-fold higher (p < 0.05), and the expression of p53 protein and the level of ROS were decreased by 51.4% (p < 0.05) and 40% (p < 0.01), respectively, compared with those in doxorubicin treatment. While in compound C (AMPK inhibitor) + NaHS+doxorubicin group, the ratio of p-AMPK/AMPK was reduced by 25.8% (p < 0.05), and the expression of p53 protein, ROS level and cell apoptosis rate measured by Hoechst 33258 nuclear staining were elevated by 1.9-fold (p < 0.05), 1.4-fold (p < 0.05) and 1.6-fold (p < 0.05), respectively, compared with those in the NaHS+doxorubicin group. Conclusion: Hydrogen sulfide prevents doxorubicin-induced apoptosis of H9c2 cardiomyocytes likely through activation of AMPK. Keywords: Hydrogen sulfide; H9c2 cardiomyocytes; apoptosis; AMPK http://dx.doi.org/10.1016/j.niox.2015.02.137
PP108 Dysregulation of placental cystathionine-β-synthase promotes fetal growth restriction Keqing Wang, Meng Cai, Wijdan Abu-Alkheir, Shakil Ahmad, Asif Ahmed Vascular Biology Laboratory, Aston Medical School, Aston University, Birmingham, UK Fetal growth restriction (FGR) is characterized by the birth weight and body mass below the tenth percentile for gestational age. FGR is a major cause of perinatal morbidity and mortality and babies born with FGR are prone to develop cardiovascular diseases later in life. The underlying pathology of FGR is inadequate placental transfer of nutrients from mother to fetus, which can be caused by placental insufficiency. Hydrogen sulfide (H2S), a gaseous messenger is produced endogenously by cystathionine-lyase (Cth), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), which are present in human placenta. Recently, we demonstrated that the dysregulation of H2S/Cth pathway is associated with preeclampsia and blockade of CSE activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in H2S pathways promote FGR and H2S donor restores fetal growth in mice where CBS or CSE activity has
PP106 Redox activities of hydrogen sulfide and polysulfides; implications in oxygen sensing Eric R. DeLeon a,b, Evelyn S. Huang b, Yan Gao a, Kenneth R. Olson a,b a Indiana University School of Medicine – South Bend, South Bend, IN, USA b University of Notre Dame, Notre Dame, IN, USA Introduction: Vascular oxygen sensing has been attributed to hypoxic generation of reactive oxygen species (ROS) which oxidize the cytosol and reduce the mitochondrial matrix (Waypa et al., 2010 [1]). However, H2S and polysulfides (H2Sn) have also been shown to affect redox status (Greiner et al., 2013 [2]). We hypothesize that reactive sulfur species (RSS) have the same effect on redox status as ROS and propose that this supports our hypothesis that the metabolism of H2S is the vascular oxygen sensor (Olson, 2015 [3]). Methods: In this study, we examined the effects of H2S (Na2S) and mixed H2Sn (K2Sn) on redox status of the redox sensitive green fluorescent protein (roGFP) purified from Escherichia coli (roGFP-E) using ~40% and 100% oxidized protein incubated in either normoxia (21% O2) or anoxia (0% O2). We also examined the effects of H2S and H2Sn on roGFP inserted into either the cytoplasm (roGFP-C) or mitochondrial matrix (roGFP-M) of HEK-293 cells. Results: 1–300 μM H2S had no effect on roGFP-E whereas 40% oxidized roGFP-E was further oxidized by 1 mM H2S and completely oxidized by H2S >1 mM in both 21 and 0% O2. H2Sn oxidized roGFP-E at considerably lower concentrations (>3 μM) and independently of O2 availability. We saw no evidence for roGFP-E reduction by either H2S or H2Sn. Conversely, 10 μM H2S and above oxidized roGFP-C and reduced roGFP-M in-vitro; H2Sn reduced roGFP-C and roGFP-M at concentrations <1 mM and oxidized them at concentrations >1 mM. Conclusions: 1. H2S is not an efficient reductant, particularly in cells. 2. The time course of H2S activation is inconsistent with a preexisting polysulfide contaminant, but because H2S cannot be an oxidant, we conclude that H2S must somehow react with roGFP-E to produce an oxidant, probably a polysulfide. 3. The oxidizing ability of H2S is O2-independent. 4. The increased efficacy of H2S compared to H2Sn in HEK-293 cells may be due to rapid permeation of H2S. 5. The effects of hypoxia on intracellular redox status is mimicked by exogenous H 2 S, providing additional support to our hypothesis of H2S metabolism as the O2 sensor. Support: NSF Grant Nos. IOS1051627, IOS1446310. References [1] G.B. Waypa, J.D. Marks, R. Guzy, P.T. Mungai, J. Schriewer, D. Dokic, et al., Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells, Circ. Res. 106 (3) (2010) 526–535, doi:10.1161/CIRCRESAHA.109.206334. [2] R. Greiner, Z. Pálinkás, K. Bäsell, D. Becher, H. Antelmann, P. Nagy, et al., Hydrogen sulfide as an oxygen sensor, Antioxid. Redox Signal. 19 (15) (2013) 1749–1765, doi:10.1089/ars.2012.5041 [Epub 2013 Jun 20]. [3] K.R. Olson, Antioxid. Redox Signal. 22 (5) (2015) 377–397, doi:10.1089/ ars.2014.5930 [Epub 2014 Jul 30]. http://dx.doi.org/10.1016/j.niox.2015.02.136
PP107 Hydrogen sulfide prevents doxorubicin-induced apoptosis of H9c2 cardiomyocytes through activation of AMPK Yuan Zhang a, Shun-Lin Qu a, Zhi-Han Tang a, Zhong Ren a, Mi-Hua Liu a, Xin-Yuan Luo a, Lu-Shan Liu a, Yu Gui b, Xi-Long Zheng b, Zhi-Sheng Jiang a
S57
a Institute of Cardiovascular Disease and Key Lab for Arteriosclerology of Hunan Province, University of South China, Hengyang City, Hunan Province, China b The Smooth Muscle Research Group, Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Canada
Background and objective: Doxorubicin induces apoptosis of cardiomyocytes, which plays an important role in its myocardial toxicity. Hydrogen sulfide is the third endogenous gasotransmitter after nitric oxide and carbon monoxide, and has a protective effect on cardiomyocytes. In addition, adenosine monophosphate activated protein kinase (AMPK) is not only a cellular energy, but also a pressure sensor and regulator. AMPK regulates reactive oxygen species/redox balance, cell apoptosis and proliferation to maintain cellular homeostasis. In this study, we aimed to explore the role of AMPK activation in the antagonist influence of hydrogen sulfide against apoptosis of H9c2 cardiomyocytes induced by doxorubicin. Methods and results: H9c2 cardiomyocytes with or without pretreatment of 100 μmol/L NaHS were exposed to 5 μmol/L doxorubicin for 24 h, followed by western blot or dichlorofluorescein diacetate (DCFH-DA) analyses. Our results showed that in NaHS+doxorubicin group the ratio of p-AMPK/AMPK was 1.4-fold higher (p < 0.05), and the expression of p53 protein and the level of ROS were decreased by 51.4% (p < 0.05) and 40% (p < 0.01), respectively, compared with those in doxorubicin treatment. While in compound C (AMPK inhibitor) + NaHS+doxorubicin group, the ratio of p-AMPK/AMPK was reduced by 25.8% (p < 0.05), and the expression of p53 protein, ROS level and cell apoptosis rate measured by Hoechst 33258 nuclear staining were elevated by 1.9-fold (p < 0.05), 1.4-fold (p < 0.05) and 1.6-fold (p < 0.05), respectively, compared with those in the NaHS+doxorubicin group. Conclusion: Hydrogen sulfide prevents doxorubicin-induced apoptosis of H9c2 cardiomyocytes likely through activation of AMPK. Keywords: Hydrogen sulfide; H9c2 cardiomyocytes; apoptosis; AMPK http://dx.doi.org/10.1016/j.niox.2015.02.137
PP108 Dysregulation of placental cystathionine-β-synthase promotes fetal growth restriction Keqing Wang, Meng Cai, Wijdan Abu-Alkheir, Shakil Ahmad, Asif Ahmed Vascular Biology Laboratory, Aston Medical School, Aston University, Birmingham, UK Fetal growth restriction (FGR) is characterized by the birth weight and body mass below the tenth percentile for gestational age. FGR is a major cause of perinatal morbidity and mortality and babies born with FGR are prone to develop cardiovascular diseases later in life. The underlying pathology of FGR is inadequate placental transfer of nutrients from mother to fetus, which can be caused by placental insufficiency. Hydrogen sulfide (H2S), a gaseous messenger is produced endogenously by cystathionine-lyase (Cth), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), which are present in human placenta. Recently, we demonstrated that the dysregulation of H2S/Cth pathway is associated with preeclampsia and blockade of CSE activity induces preeclampsia-like condition in pregnant mice. We hypothesized that defect in H2S pathways promote FGR and H2S donor restores fetal growth in mice where CBS or CSE activity has