Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte ApoptosisviaAkt-Dependent Enhancement of iNOS Expression

The present study explored the potential causal link between ischemia-driven cyclooxygenase-2 (COX-2) expression and enhanced apoptosis during myocardial ischemia/reperfusion (I/R) by using H9C2 cardiomyocytes and primary rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that H/R resulted in higher COX-2 expression than that of controls, which was prevented by pretreatment with Helenalin (NFκB specific inhibitor). Furthermore, pretreatment with NS398 (COX-2 specific inhibitor) significantly attenuated H/R-induced cell injury [lower lactate dehydrogenase (LDH) leakage and enhanced cell viability] and apoptosis (higher Bcl2 expression and lower level of cleaved caspases-3 and TUNEL-positive cells) in cardiomyocytes. The amelioration of posthypoxic apoptotic cell death was paralleled by significant attenuation of H/R-induced increases in proinflammatory cytokines [interleukin 6 (IL6) and tumor necrosis factor (TNFα)] and reactive oxygen species (ROS) production and by higher protein expression of phosphorylated Akt and inducible nitric oxide synthase (iNOS) and enhanced nitric oxide production. Moreover, the application of LY294002 (Akt-specific inhibitor) or 1400W (iNOS-selective inhibitor) cancelled the cellular protective effects of NS398. Findings from the current study suggest that activation of NFκB during cardiomyocyte H/R induces the expression of COX-2 and that higher COX-2 expression during H/R exacerbates cardiomyocyte H/R injuryviamechanisms that involve cross talks among inflammation, ROS, and Akt/iNOS/NO signaling.

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miR-181c-5p Exacerbates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Targeting PTPN4

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/1957920 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 8

Author(s):

Liang Ge ◽

Yin Cai ◽

Fan Ying ◽

Hao Liu ◽

Dengwen Zhang ◽

...

Keyword(s):

Cell Death ◽

Cell Apoptosis ◽

Cell Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Control Cell ◽

Luciferase Reporter ◽

Apoptosis Pathway ◽

H9c2 Cardiomyocytes ◽

Hypoxia Reoxygenation

Background. Activation of cell apoptosis is a major form of cell death during myocardial ischemia/reperfusion injury (I/RI). Therefore, examining ways to control cell apoptosis has important clinical significance for improving postischemic recovery. Clinical evidence demonstrated that miR-181c-5p was significantly upregulated in the early phase of myocardial infarction. However, whether or not miR-181c-5p mediates cardiac I/RI through cell apoptosis pathway is unknown. Thus, the present study is aimed at investigating the role and the possible mechanism of miR-181c-5p in apoptosis during I/R injury by using H9C2 cardiomyocytes. Methods and Results. The rat origin H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 6 hours reoxygenation) to induce cell injury. The results showed that H/R significantly increased the expression of miR-181c-5p but not miR-181c-3p in H9C2 cells. In line with this, in an in vivo rat cardiac I/RI model, miR-181c-5p expression was also significantly increased. The overexpression of miR-181c-5p by its agomir transfection significantly aggravated H/R-induced cell injury (increased lactate dehydrogenase level and reduced cell viability) and exacerbated H/R-induced cell apoptosis (greater cleaved caspases 3 expression, Bax/Bcl-2 and more TUNEL-positive cells). In contrast, inhibition of miR-181c-5p in vitro had the opposite effect. By using computational prediction algorithms, protein tyrosine phosphatase nonreceptor type 4 (PTPN4) was predicted as a potential target gene of miR-181c-5p and was verified by the luciferase reporter assay. The overexpression of miR-181c-5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes. Moreover, knockdown of PTPN4 significantly aggravated H/R-induced enhancement of LDH level, cleaved caspase 3 expression, and apoptotic cell death, which mimicked the proapoptotic effects of miR-181c-5p in H9C2 cardiomyocytes. Conclusions. These findings suggested that miR-181c-5p exacerbates H/R-induced cardiomyocyte injury and apoptosis via targeting PTPN4 and that miR-181c-5p/PTPN4 signaling may yield novel strategies to combat myocardial I/R injury.

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Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01003.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. H1487-H1495 ◽

Cited By ~ 56

Author(s):

Emmanuel Eroume A. Egom ◽

Tamer M. A. Mohamed ◽

Mamas A. Mamas ◽

Ying Shi ◽

Wei Liu ◽

...

Keyword(s):

Nitric Oxide ◽

Cardiac Myocytes ◽

Coronary Sinus ◽

Cell Injury ◽

Ischemia Reperfusion ◽

Cardiac Ischemia ◽

Protective Effects ◽

Lactate Dehydrogenase Activity ◽

Nitric Oxide Release ◽

Sphingosine 1 Phosphate

We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus ( n = 7) or femoral vein ( n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min ( n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h ( n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.

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Abstract 319: Mirna-125b Reduces Ischemia / Reperfusion (i/r)-induced Apoptosis Through Targeting Multiple Targets in Intrinsic and Extrinsic Apoptosis Pathways

Circulation Research ◽

10.1161/res.117.suppl_1.319 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Peipei Wang ◽

Qiying Chen ◽

Yue Zhou ◽

Arthur M Richards

Keyword(s):

Cell Count ◽

Cell Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Luciferase Reporter ◽

Protective Effects ◽

Extrinsic Pathway ◽

Multiple Genes ◽

Induced Apoptosis

Apoptosis is mediated through extrinsic and intrinsic pathways, both play a role in ischemia/reperfusion (I/R) injury. Predicted targets for miRNA-125b include extrinsic pathway mediators Traf6 and Tnfrsf1b, and intrinsic mitochondria regulators Bcl-2 family pro-apoptotic effectors Bak1 and BH3-only facilitators Bim, Bmf, Puma. We hypothesized that miRNA-125b directly targets multiple genes to reduce I/R-induced apoptosis. Myoblast H9c2 cells underwent 16 hours 0.2% O 2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R) and were transfected with miRNA-125b mimic vs. scrambled mimic control (25 nmol, miR-125b-M vs. MC) and miR-125b inhibitor vs. inhibitor control (miR-125b-I vs. IC). Cell count/viability, WST assay, cell injury-induced LDH release and apoptotic marker Casp3/7 were measured. Cells were trypsinized for assessment of apoptosis (7-AAD and annexin V double staining) and lysed for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Bak1 plasmids were cloned and transfected into H9c2 for recovery studies. The effects were verified in neonatal rat ventricular myocytes (NRVM). miRNA-125b-M significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-125b-M vs. MC p<0.05). qPCR confirmed that (1) miR-125b expression was reduced in H-R; (2) RISC-loaded (immunoprecipitation pull-down Ago-2) miR-125b increased by ~35 fold and reduced to ~3% following mimic and inhibitor transfection respectively; (3) multiple apoptosis-related genes were reduced by miR-125b-M, Bak1, Bmf, Bim, Puma, Traf6 and Tnfrsf1b. All changes were confirmed by WB. Luciferase reporter assays indicated miR-125b bound to the 3’-UTR of all genes tested except Traf6. Total apoptotic cell numbers and Casp3/7 release were significantly reduced by miR-125b-M. The protective effect of miRNA-125b was partially abolished by Bak1 overexpression (pCMV-Myc-Bak1 and miR-125b co-transfection). Protective effects of miRNA-125b were further verified in NRVM. MiRNA-125b inhibitor reversed protective effects and target changes at mRNA and protein level. miR-125b is powerfully cardioprotective in I/R injury due to directly targeting multiple genes in the extrinsic and intrinsic apoptotic pathways.

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Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/3245483 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Qihui Jin ◽

Yanhong Jiang ◽

Lizhong Fu ◽

Yanqiu Zheng ◽

Yuxia Ding ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Injury ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion ◽

Control Group ◽

Protective Effects ◽

H9c2 Cells ◽

Cytochrome C Release ◽

Mitochondrial Ros ◽

Hypoxia Reoxygenation

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

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Glaucocalyxin A Protects H9c2 Cells Against Hypoxia/Reoxygenation-Induced Injury Through the Activation of Akt/Nrf2/HO-1 Pathway

Cell Transplantation ◽

10.1177/0963689720967672 ◽

2020 ◽

Vol 29 ◽

pp. 096368972096767

Author(s):

Zhuo Peng ◽

Rui Zhang ◽

Longfei Pan ◽

Honghong Pei ◽

Zequn Niu ◽

...

Keyword(s):

Signaling Pathway ◽

Ischemia Reperfusion ◽

Biologically Active ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Protective Effects ◽

H9c2 Cells ◽

Rat Cardiomyocytes ◽

Myocardial Ischemia Reperfusion ◽

Hypoxia Reoxygenation

Myocardial infarction (MI) is one of the most serious cardiovascular diseases associated with myocardial ischemia/reperfusion (I/R) injury. Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid that has been found to ameliorate myocardial I/R injury in mice. However, the mechanism has not been fully investigated. In the present study, we aimed to investigate the effect of GLA on rat cardiomyocytes H9c2 cells exposed to hypoxia/reoxygenation (H/R). The results showed that GLA treatment improved cell viability of H/R-stimulated H9c2 cells. Administration with GLA suppressed the H/R-stimulated reactive oxygen species (ROS) production in H9c2 cells. GLA also elevated the activities of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase in H/R-stimulated H9c2 cells. Moreover, GLA prevented H/R-stimulated cell apoptosis in H9c2 cells, as evidenced by increased bcl-2 expression, decreased bax expression, as well as reduced caspase-3 activity. Furthermore, GLA enhanced the activation of protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in H9c2 cells exposed to H/R. Additionally, treatment with LY294002 reserved the protective effects of GLA on H/R-stimulated oxidative injury in H9c2 cells. In conclusion, these findings suggested that GLA protected H9c2 cells from H/R-stimulated oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. Thus, GLA might be a promising therapeutic agent for the prevention and treatment of myocardial I/R.

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CircHIPK3 regulates the autophagy and apoptosis of hypoxia/reoxygenation-stimulated cardiomyocytes via the miR-20b-5p/ATG7 axis

Cell Death Discovery ◽

10.1038/s41420-021-00448-6 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Zhimei Qiu ◽

Yan Wang ◽

Weiwei Liu ◽

Chaofu Li ◽

Ranzun Zhao ◽

...

Keyword(s):

Cell Injury ◽

Ischemia Reperfusion ◽

Circular Rna ◽

Control Group ◽

Loss Of Function ◽

Cardiomyocyte Autophagy ◽

Myocardial Ischemia Reperfusion ◽

Injury Research ◽

Hypoxia Reoxygenation

AbstractAutophagy and apoptosis are involved in myocardial ischemia/reperfusion (I/R) injury. Research indicates that circular RNA HIPK3 (circHIPK3) is crucial to cell autophagy and apoptosis in various cancer types. However, the role of circHIPK3 in the regulation of cardiomyocyte autophagy and apoptosis during I/R remains unknown. Our study aimed to examine the regulatory effect of circHIPK3 during myocardial I/R and investigate its mechanism in cardiomyocyte autophagy and apoptosis. Methods and results. The expression of circHIPK3 was upregulated during myocardial I/R injury and hypoxia/reoxygenation (H/R) injury of cardiomyocytes. To study the potential role of circHIPK3 in myocardial H/R injury, we performed gain-of-function and loss-of-function analyses of circHIPK3 in cardiomyocytes. Overexpression of circHIPK3 significantly promoted H/R-induced cardiomyocyte autophagy and cell injury (increased intracellular reactive oxygen species (ROS) and apoptosis) compared to those in the control group, while silencing of circHIPK3 showed the opposite effect. Further research found that circHIPK3 acted as an endogenous miR-20b-5p sponge to sequester and inhibit miR-20b-5p activity, resulting in increased ATG7 expression. In addition, miR-20b-5p inhibitors reversed the decrease in ATG7 induced by silencing circHIPK3. Conclusions. CircHIPK3 can accelerate cardiomyocyte autophagy and apoptosis during myocardial I/R injury through the miR-20b-5p/ATG7 axis. These data suggest that circHIPK3 may serve as a potential therapeutic target for I/R.

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Hypoxia compared with normoxia alters the effects of nitric oxide in ischemia-reperfusion lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.3.l504 ◽

1997 ◽

Vol 273 (3) ◽

pp. L504-L512 ◽

Cited By ~ 2

Author(s):

Y. C. Huang ◽

P. W. Fisher ◽

E. Nozik-Grayck ◽

C. A. Piantadosi

Keyword(s):

Nitric Oxide ◽

Lung Injury ◽

Average Rate ◽

Ischemia Reperfusion ◽

Oxidant Stress ◽

No Production ◽

Protective Effects ◽

Lung Weight ◽

Edema Formation ◽

No Donors

Because both the biosynthesis of nitric oxide (NO.) and its metabolic fate are related to molecular O2, we hypothesized that hypoxia would alter the effects of NO. during ischemia-reperfusion (IR) in the lung. In this study, buffer-perfused lungs from rabbits underwent either normoxic IR (AI), in which lungs were ventilated with 21% O2 during ischemia and reperfusion, or hypoxic IR (NI), in which lungs were ventilated with 95% N2 during ischemia followed by reoxygenation with 21% O2. Lung weight gain (WG) and pulmonary artery pressure (Ppa) were monitored continuously, and microvascular pressure (Pmv) was measured after reperfusion to calculate pulmonary vascular resistance. We found that both AI and NI produced acute lung injury, as shown by increased WG and Ppa during reperfusion. In AI, where perfusate PO2 was > 100 mmHg, the administration of the NO. synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) before ischemia worsened WG and Ppa. Pmv also increased, suggesting a hydrostatic mechanism involved in edema formation. The effects of L-NAME could be attenuated by giving L-arginine and exogenous NO. donors before ischemia or before reperfusion. Partial protection was also provided by superoxide dismutase. In contrast, lung injury in NI at perfusate PO2 of 25-30 mmHg was attenuated by L-NAME; this effect could be reversed by L-arginine. Exogenous NO. donors given either before ischemia or before reperfusion, however, did not increase lung injury. NO. production was measured by quantifying the total nitrogen oxides (NOx) accumulating in the perfusate. The average rate of NOx accumulation was greater in AI than in NI. We conclude that hypoxia prevented the protective effects of NO on AI lung injury. The effects of hypoxia may be related to lower NO. production relative to oxidant stress during IR and/or altered metabolic fates of NO.-mediated production of peroxynitrite by hypoxic ischemia.

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Nitric oxide preconditioning regulates endothelial monolayer integrity via the heat shock protein 90-soluble guanylate cyclase pathway

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00498.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. H893-H903 ◽

Cited By ~ 14

Author(s):

Galina N. Antonova ◽

Connie M. Snead ◽

Alexander S. Antonov ◽

Christiana Dimitropoulou ◽

Richard C. Venema ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Heat Shock ◽

Heat Shock Protein ◽

Cell Injury ◽

Soluble Guanylate Cyclase ◽

Heat Shock Protein 90 ◽

Protective Effects ◽

No Donor ◽

Spermine Nonoate

Large (pathological) amounts of nitric oxide (NO) induce cell injury, whereas low (physiological) NO concentrations often ameliorate cell injury. We tested the hypotheses that pretreatment of endothelial cells with low concentrations of NO (preconditioning) would prevent injury induced by high NO concentrations. Apoptosis, induced in bovine aortic endothelial cells (BAECs) by exposing them to either 4 mM sodium nitroprusside (SNP) or 0.5 mM N-(2-aminoethyl)- N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) for 8 h, was abolished by 24-h pretreatment with either 100 μM SNP, 10 μM spermine NONOate, or 100 μM 8-bromo-cGMP (8-Br-cGMP). Repair of BAECs following wounding, measured as the recovery rate of transendothelial electrical resistance, was delayed by 8-h exposure to 4 mM SNP, and this delay was significantly attenuated by 24-h pretreatment with 100 μM SNP. NO preconditioning produced increased association and expression of soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90). The protective effect of NO preconditioning, but not the injurious effect of 4 mM SNP, was abolished by either a sGC activity inhibitor 1H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (ODQ) or a HSP90 binding inhibitor (radicicol) and was mimicked by 8-Br-cGMP. We conclude that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the HSP90/sGC pathway. HSP90/sGC may thus play a role in the protective effects of NO-generating drugs from injurious stimuli.

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Myostatin Deficiency Protects C2C12 Cells from Oxidative Stress by Inhibiting Intrinsic Activation of Apoptosis

Cells ◽

10.3390/cells10071680 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1680

Author(s):

Marius Drysch ◽

Sonja Verena Schmidt ◽

Mustafa Becerikli ◽

Felix Reinkemeier ◽

Stephanie Dittfeld ◽

...

Keyword(s):

Intracellular Signaling ◽

Ischemia Reperfusion ◽

C2c12 Cell ◽

C2c12 Cells ◽

Ros Generation ◽

Protective Effects ◽

Activity Increase ◽

Mitogen Activated Protein ◽

Myostatin Inhibition ◽

Hypoxia Reoxygenation

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a Myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn−/− cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn−/− cells and could serve as basis for further research.

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Abstract 13879: Ischemic Postconditioning Confers Cardioprotection through Adiponectin-dependent Mitochondrial STAT3 Activation in Mice

Circulation ◽

10.1161/circ.130.suppl_2.13879 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Haobo Li ◽

Michael G Irwin ◽

Zhengyuan Xia

Keyword(s):

Cell Injury ◽

Troponin I ◽

Ischemia Reperfusion Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Cardiac Injury ◽

Systolic Pressure ◽

Gene Knockdown ◽

Area At Risk ◽

In Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) plays a key role in postconditioning (IPo) mediated protection against myocardial ischemia reperfusion injury, but the mechanism by which IPo activates STAT3 is unknown. Adiponectin (APN), a protein with anti-ischemic properties, activates STAT3. We hypothesized that IPo activates mitochondrial STAT3 (MitoSTAT3) via APN signaling. Methods and Results: Wild type (WT) and APN knockout (KO) mice were either sham operated or subjected to 30 min of coronary artery occlusion followed by 2 hours of reperfusion with or without IPo (3 cycles of 10 seconds reperfusion and 10 seconds reocclusion; n=8/group). At the end of reperfusion, KO mice exhibited more severe myocardial injury evidenced as increased infarct size (% of area at risk) 49.2±2.0 vs WT 39.4±3.5, P <0.01; plasma troponin I (ng/ml): KO 72.8±7.6 vs WT 45.7±4.0, P <0.01; worse cardiac function (lower dP/dt max and end-systolic pressure-volume relation, P <0.05); more severely impaired mitochondrial function (reductions in complex IV and complex V protein expression) and more severe reduction of MitoSTAT3 phosphorylation (activation) at site Ser727, P <0.01. IPo significantly attenuated post-ischemic cardiac injury and dysfunction with a concomitant increase in phosphorylated MitoSTAT3 and attenuation of mitochondrial dysfunction in WT (all P <0.05) but not in KO mice. In cultured cardiac H9C2 cells, hypoxic postconditioning (HPo, 3 cycles of 5 min hypoxia and 5 min reoxygenation) significantly attenuated hypoxia/reoxygenation (HR, 3 hours hypoxia/3 hours reoxygenation) induced cell injury (increased apoptotic cell death as % of HR): HR 100.2±0.4 vs HPo 78.2±4.8, P <0.05) and reduced mitochondrial transmembrane potential (% total cells, HR 37.2±4.9 vs HPo 23.5±3.7, P <0.01). APN, adiponectin receptor 1 (AdipoR1), or STAT3 gene knockdown but not AdipoR2 gene knockdown, respectively, abolished HPo cellular protection (all P <0.05 vs. HPo). APN supplementation (10μg/ml) restored HPo protection in cells with APN knockdown but not in cells with AdipoR1or STAT3 gene knockdown. Conclusion: Adiponectin and AdipoR1 signaling are required for IPo to activate myocardial mitochondrial STAT3 to confer cardioprotection.

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