Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O2·−). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O2·− generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91phox, O2·−, and P21-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

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Cardioprotective Natural Compound Pinocembrin Attenuates Acute Ischemic Myocardial Injury via Enhancing Glycolysis

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/4850328 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Yanjun Zheng ◽

Guoqing Wan ◽

Bo Yang ◽

Xuefeng Gu ◽

Jingrong Lin

Keyword(s):

Contractile Function ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Glycolytic Enzyme ◽

Isolated Rat Heart ◽

Cardiac Contractile Function ◽

Direct Stimulation ◽

Underlying Mechanisms

Purpose. Emerging evidence has shown that pinocembrin protects the myocardium from ischemic injury in animals. However, it is unknown whether it has cardioprotection when given at the onset of reperfusion. Also, mechanisms mediating the cardioprotective actions of pinocembrin were largely unknown. Thus, this study is aimed at investigating the effects of pinocembrin postconditioning on ischemia-reperfusion (I/R) injury and the underlying mechanisms. Methods. The in vivo mouse model of myocardial I/R injury, ex vivo isolated rat heart with global I/R, and in vitro hypoxia/reoxygenation (H/R) injury model for primary cardiomyocytes were used. Results. We found that pinocembrin postconditioning significantly reduced the infarct size and improved cardiac contractile function after acute myocardial I/R. Mechanically, in primary cardiomyocytes, we found that pinocembrin may confer protection in part via direct stimulation of cardiac glycolysis via promoting the expression of the glycolytic enzyme, PFKFB3. Besides, PFKFB3 inhibition abolished pinocembrin-induced glycolysis and protection in cardiomyocytes. More importantly, PFKFB3 knockdown via cardiotropic adeno-associated virus (AAV) abrogated cardioprotective effects of pinocembrin. Moreover, we demonstrated that HIF1α is a key transcription factor driving pinocembrin-induced PFKFB3 expression in cardiomyocytes. Conclusions. In conclusion, these results established that the acute cardioprotective benefits of pinocembrin are mediated in part via enhancing PFKFB3-mediated glycolysis via HIF1α, which may provide a new therapeutic target to impede the progression of myocardial I/R injury.

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Sphk1-induced Autophagy in Microglia Promotes Neuronal Injury Following Cerebral Ischemia-Reperfusion

10.21203/rs.3.rs-91557/v1 ◽

2020 ◽

Author(s):

Manhua Lv ◽

Yongjia Jiang ◽

Dayong Zhang ◽

Dan Yao ◽

Yuefeng Cheng ◽

...

Keyword(s):

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Neuronal Injury ◽

Glucose Deprivation ◽

Enzyme Linked Immunosorbent Assay ◽

Cerebral Ischemia Reperfusion ◽

The Impact

Abstract Background: Microglial hyperactivation driven by SphK1/S1P signaling and consequent inflammatory mediator production is a key driver of cerebral ischemia-reperfusion injury (CIRI). While SphK1 reportedly controls autophagy and microglial activation, it remains uncertain as to whether it is similarly able to regulate damage mediated by CIRI-activated microglia. Methods: In the present study, we utilized both an in vitro oxygen-glucose deprivation reperfusion (OGDR) model and an in vivo rat model of focal CIRI to test whether Sphk1 and autophagy is expressed in microglia. Western blot analysis was used to estimate the autophagy protein level (LC3 and SQSTM ) at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-1β, IL-6 and TNF-α in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit and flow cytometry to detect apoptotic neurons.Results: We determined that in the context of CIRI, microglia upregulated SphK1 and induced autophagy, while inhibiting these changes by lentivirus targeting SphK1 significantly decreased expression of autophagy . Moreover, we determined that autophagic body formation was enhanced in cerebral tissues following I/R. We also explored the impact of SphK1-induced autophagy on microglial inflammatory cytokine production and associated neuronal apoptosis using an in vitro OGDR model system. At a mechanistic level, we found that SphK1 promotes autophagy via the tumor necrosis factor receptor-associated factor 2 (TRAF2) pathway. Conclusion: These results reveal a novel mechanism whereby SphK1-induced autophagy in microglia can contribute to the pathogenesis of CIRI, potentially highlighting novel avenues for future therapeutic intervention in IS patients.

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Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models

AJP Renal Physiology ◽

10.1152/ajprenal.90422.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. F177-F190 ◽

Cited By ~ 15

Author(s):

Frederic Favreau ◽

Ludivine Rossard ◽

Keqiang Zhang ◽

Thibault Desurmont ◽

Emilie Manguy ◽

...

Keyword(s):

Proximal Tubule ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Regulatory Protein ◽

Translocator Protein ◽

Mitochondrial Functions ◽

The Impact ◽

Tspo Expression

Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18-kDa drug- and cholesterol-binding protein localized to the outer mitochondrial membrane and implicated in a variety of cell and mitochondrial functions. To determine the role of TSPO in ischemia-reperfusion injury (IRI), we used both in vivo and in vitro porcine models: an in vivo renal ischemia model where different conservation modalities were tested and an in vitro model where TSPO-transfected porcine proximal tubule LLC-PK1cells were exposed to hypoxia and oxidative stress. The expression of TSPO and its partners in steroidogenic cells, steroidogenic acute regulatory protein (StAR) and cytochrome P-450 side chain cleavage CYP11A1, as well as the impact of TSPO overexpression and exposure to TSPO ligands in vitro in hypoxia-ischemia conditions were investigated. Hypoxia induced caspase activation, reduction of ATP content, and LLC-PK1cell death. Transfection and overexpression of TSPO rescued the cells from the detrimental effects of hypoxia and reoxygenation. Moreover, TSPO overexpression was accompanied by a reduction of H2O2-induced necrosis. TSPO drug ligands did not affect TSPO-mediated functions. In vivo, TSPO expression was modulated by IRI and during regeneration particularly in proximal tubule cells, which do not express this protein at the basal level. Under the same conditions, StAR and CYP11A1 protein and gene expression was reduced without apparent relation to TSPO changes. Pregnenolone was identified and measured in the pig kidney. Pregnenolone synthesis was not affected by the experimental conditions used. Taken together, these results indicate that changes in TSPO expression in kidney regenerating tissue could be important for renal protection and maintenance of kidney function.

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N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

Cell Death and Disease ◽

10.1038/s41419-021-03622-x ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Ying Dong Du ◽

Wen Yuan Guo ◽

Cong Hui Han ◽

Ying Wang ◽

Xiao Song Chen ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Mitochondrial Fragmentation ◽

Hepatic Ischemia ◽

Adeno Associated Virus ◽

Downstream Target ◽

Hepatic Ischemia Reperfusion

AbstractDespite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.

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Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

Molecular Medicine ◽

10.1186/s10020-021-00332-0 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Jian-Ping Zhang ◽

Wei-Jing Zhang ◽

Miao Yang ◽

Hua Fang

Keyword(s):

Cell Apoptosis ◽

Ischemia Reperfusion Injury ◽

Glycogen Synthase ◽

Ischemia Reperfusion ◽

Inflammatory Factors ◽

Therapeutic Effects ◽

Protective Effects ◽

Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

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LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽

10.1007/s13577-021-00527-x ◽

2021 ◽

Author(s):

Jiaying Zhu ◽

Zhu Zhu ◽

Yipin Ren ◽

Yukang Dong ◽

Yaqi Li ◽

...

Keyword(s):

Cerebral Ischemia ◽

Nuclear Translocation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Mice Model ◽

Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

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Molecular Aspects of Volatile Anesthetic-Induced Organ Protection and Its Potential in Kidney Transplantation

International Journal of Molecular Sciences ◽

10.3390/ijms22052727 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2727

Author(s):

Gertrude J. Nieuwenhuijs-Moeke ◽

Dirk J. Bosch ◽

Henri G.D. Leuvenink

Keyword(s):

Kidney Transplantation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Animal Experiments ◽

Volatile Anesthetic ◽

Organ Protection ◽

Graft Outcome ◽

Molecular Aspects

Ischemia reperfusion injury (IRI) is inevitable in kidney transplantation and negatively impacts graft and patient outcome. Reperfusion takes place in the recipient and most of the injury following ischemia and reperfusion occurs during this reperfusion phase; therefore, the intra-operative period seems an attractive window of opportunity to modulate IRI and improve short- and potentially long-term graft outcome. Commonly used volatile anesthetics such as sevoflurane and isoflurane have been shown to interfere with many of the pathophysiological processes involved in the injurious cascade of IRI. Therefore, volatile anesthetic (VA) agents might be the preferred anesthetics used during the transplantation procedure. This review highlights the molecular and cellular protective points of engagement of VA shown in in vitro studies and in vivo animal experiments, and the potential translation of these results to the clinical setting of kidney transplantation.

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In vivo and In vitro PPAR-y Activation Decreases M1-Macrophage Polarization and Improves Liver Ischemia Reperfusion Injury

Transplantation Journal ◽

10.1097/01.tp.0000543674.85457.9b ◽

2018 ◽

Vol 102 ◽

pp. S708

Author(s):

Ivan Linares ◽

Agata Bartczak ◽

Kaveh Farrokhi ◽

Dagmar Kollmann ◽

Moritz Kaths ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Macrophage Polarization ◽

Liver Ischemia ◽

M1 Macrophage

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Poly-IC Preconditioning Protects against Cerebral and Renal Ischemia-Reperfusion Injury

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2011.160 ◽

2011 ◽

Vol 32 (2) ◽

pp. 242-247 ◽

Cited By ~ 29

Author(s):

Amy E B Packard ◽

Jason C Hedges ◽

Frances R Bahjat ◽

Susan L Stevens ◽

Michael J Conlin ◽

...

Keyword(s):

Reperfusion Injury ◽

Inflammatory Responses ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Renal Ischemia ◽

Cortical Cells ◽

Polycytidylic Acid ◽

Populations At Risk

Preconditioning induces ischemic tolerance, which confers robust protection against ischemic damage. We show marked protection with polyinosinic polycytidylic acid (poly-IC) preconditioning in three models of murine ischemia-reperfusion injury. Poly-IC preconditioning induced protection against ischemia modeled in vitro in brain cortical cells and in vivo in models of brain ischemia and renal ischemia. Further, unlike other Toll-like receptor (TLR) ligands, which generally induce significant inflammatory responses, poly-IC elicits only modest systemic inflammation. Results show that poly-IC is a new powerful prophylactic treatment that offers promise as a clinical therapeutic strategy to minimize damage in patient populations at risk of ischemic injury.

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Abstract 14391: Transient Cell Cycle Induction in Cardiomyocytes is a Promising Approach to Treat Ischemia Induced Heart Failure

Circulation ◽

10.1161/circ.142.suppl_3.14391 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Riham Abouleisa ◽

Qinghui Ou ◽

Xian-liang Tang ◽

Mitesh Solanki ◽

Yiru Guo ◽

...

Keyword(s):

Cell Cycle ◽

Cardiac Function ◽

Single Cell ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Cardiomyocyte Proliferation ◽

Specific Expression ◽

Control Virus

Rationale: The regenerative capacity of the heart to repair itself after myocardial infarction (MI)is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 andCdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in vitro and in vivo andimproves cardiac function after MI. However, its clinical application is limited due to the concernsfor tumorigenic potential in other organs. Objectives: To first, identify on a single cell transcriptomic basis the necessary reprogrammingsteps that cardiomyocytes need to undertake to progress through the proliferation processfollowing 4F overexpression, and then, to determine the pre-clinical efficacy of transient andcardiomyocyte specific expression of 4F in improving cardiac function after MI in small and largeanimals. Methods and Results: Temporal bulk and single cell RNAseq of mature hiPS-CMs treated with4F or LacZ control for 24, 48, or 72 h revealed full cell cycle reprogramming in 15% of thecardiomyocyte population which was associated with sarcomere disassembly and metabolicreprogramming. Transient overexpression of 4F specifically in cardiomyocytes was achievedusing non-integrating lentivirus (NIL) driven by TNNT2 (TNNT2-4F-NIL). One week after inductionof ischemia-reperfusion injury in rats or pigs, TNNT2-4F-NIL or control virus was injectedintramyocardially. Compared with controls, rats or pigs treated with TNNT2-4F-NIL showed a 20-30% significant improvement in ejection fraction and scar size four weeks after treatment, asassessed by echocardiography and histological analysis. Quantification of cardiomyocyteproliferation in pigs using a novel cytokinesis reporter showed that ~10% of the cardiomyocyteswithin the injection site were labelled as daughter cells following injection with TNNT2-4F-NILcompared with ~0.5% background labelling in control groups. Conclusions: We provide the first understanding of the process of forced cardiomyocyteproliferation and advanced the clinical applicability of this approach through minimization ofoncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specificviral construct.

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