scholarly journals Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1309
Author(s):  
Sandra Funcke ◽  
Tessa R. Werner ◽  
Marc Hein ◽  
Bärbel M. Ulmer ◽  
Arne Hansen ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Ischemia Reperfusion ◽  
Heart Tissue ◽  
Contractile Force ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Engineered Heart Tissue ◽  
Hypoxia Reoxygenation

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

scholarly journals Overexpression of TIMP3 Protects Against Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Apoptosis Through ROS/Mapks Pathway

2017 ◽  
Vol 44 (3) ◽  
pp. 1011-1023 ◽  
Author(s):  
Hui Liu ◽  
Xibo Jing ◽  
Aiqiao Dong ◽  
Baobao Bai ◽  
Haiyan Wang
Keyword(s):  
Myocardial Infarct ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Tunel Staining ◽  
Ros Production ◽  
Rat Cardiomyocytes ◽  
Doxorubicin Treatment ◽  
Myocardial Apoptosis

Background/Aims: Myocardial ischemia/reperfusion (I/R) injury remains a great challenge in clinical therapy. Tissue inhibitor of metalloproteinases 3 (TIMP3) plays a crucial role in heart physiological and pathophysiological processes. However, the effects of TIMP3 on I/R injury remain unknown. Methods: C57BL/6 mice were infected with TIMP3 adenovirus by local delivery in myocardium followed by I/R operation or doxorubicin treatment. Neonatal rat cardiomyocytes were pretreated with TIMP3 adenovirus prior to anoxia/reoxygenation (A/R) treatment in vitro. Histology, echocardiography, in vivo phenotypical analysis, flow cytometry and western blotting were used to investigate the altered cardiac function and underlying mechanisms. Results: The results showed that upregulation of TIMP3 in myocardium markedly inhibited myocardial infarct areas and the cardiac dysfunction induced by I/R or by doxorubicin treatment. TUNEL staining revealed that TIMP3 overexpression attenuated I/R-induced myocardial apoptosis, accompanied by decreased Bax/Bcl-2 ratio, Cleaved Caspase-3 and Cleaved Caspase-9 expression. In vitro, A/R-induced cardiomyocyte apoptosis was abrogated by pharmacological inhibition of reactive oxygen species (ROS) production or MAPKs signaling. Attenuation of ROS production reversed A/R-induced MAPKs activation, whereas MAPKs inhibitors showed on effect on ROS production. Furthermore, in vivo or in vitro overexpression of TIMP3 significantly inhibited I/R- or A/R-induced ROS production and MAPKs activation. Conclusion: Our findings demonstrate that TIMP3 upregulation protects against cardiac I/R injury through inhibiting myocardial apoptosis. The mechanism may be related to inhibition of ROS-initiated MAPKs pathway. This study suggests that TIMP3 may be a potential therapeutic target for the treatment of I/R injury.

Abstract 1471: Development and Characterization of Force Generating Human Engineered Heart Tissue from Embryonic Stem Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Christina Rogge ◽  
Michael Didié ◽  
Erich Wettwer ◽  
Ursula Ravens ◽  
Ralph Graichen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Heart Tissue ◽  
Cardiac Repair ◽  
Embryoid Bodies ◽  
Laser Scanning Microscopy ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Engineered Heart Tissue

Engineered Heart Tissue (EHT) from neonatal rat cardiomyocytes has been used successfully as in vitro model and in cardiac repair. Here, we hypothesized that human embryonic stem cells (hESC) can be used to generate EHT with properties of native myocardium. Methods: hESC (hES3-ENVY) were differentiated in embryoid bodies, enzymatically dispersed, and subjected to EHT-generation in circular casting molds (1.5x10 6 cells, 0.4 mg collagen, 10% Matrigel/EHT; inner/outer diameter - 2/4 mm). Contractile function was assessed 10 days after casting under isometric conditions (37°C, 1.5 Hz, Tyrode’s solution). Action potentials (AP) were recorded in spontaneously contracting EHTs with intracellular electrodes (37°C, Tyrode’s solution). Calcium gradients were assessed by confocal laser scanning microscopy (CLSM) after rhod-2 loading. EHT-morphology was examined by CLSM and electron microscopy (EM). Results: hESC-EHTs contracted synchronously and spontaneously at 1.1±0.1 Hz (n=3). Increasing concentrations of extracellular calcium (0.2–2.4 mM) enhanced force of contraction from 53±8 to 199±22 μN (n=8, p<0.05; EC 50 : 0.8±0.04 mM). Isoprenaline (1 μM) at 0.4 mM calcium increased twitch tension from 61±7 to 108±15 μN (n=8, p<0.05) and shortened relaxation time from 111±6 to 87±4 ms (n=3, p<0.05). Cardiomyocytes within EHTs formed a functional syncytium composed of predominantly oriented muscle strands with a high degree of sarcomere differentiation (CLSM, EM). Cell-cell contacts through adherens junctions were identified by EM. Synchronous calcium gradient spread in spontaneously contracting EHTs indicated electrical coupling of individual cells within the multicellular constructs. AP recordings identified pacemaker cells (spontaneous diastolic depolarization) and cells with a flat phase 4 of the AP (working myocardium-like cells). Pharmacological studies demonstrated the presence and functional relevance of I Na (10–30 μM flecainide), I Ca (1 μM nisoldipine), and I Kr (1–5 μM E4031). Conclusion: Human force-generating EHT with functional and morphological properties of native myocardium can be generated. Ultimately, hESC-EHTs may constitute a model system for substance screening and could further be utilized in cardiac repair.

scholarly journals Initiation and propagation of ectopic waves: insights from an in vitro model of ischemia-reperfusion injury

2002 ◽  
Vol 283 (2) ◽  
pp. H741-H749 ◽  
Author(s):  
Ara Arutunyan ◽  
Luther M. Swift ◽  
Narine Sarvazyan
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Border Zone ◽  
Ectopic Activity ◽  
Functional Layer ◽  
Rat Cardiomyocytes ◽  
Coupled Cells ◽  
Vitro Model

The objective of the present study was to directly visualize ectopic activity associated with ischemia-reperfusion and its progression to arrhythmia. To accomplish this goal, we employed a two-dimensional network of neonatal rat cardiomyocytes and a recently developed model of localized ischemia-reperfusion. Washout of the ischemia-like solution resulted in tachyarrhythmic episodes lasting 15–200 s. These episodes were preceded by the appearance of multiple ectopic sources and propagation of ectopic activity along the border of the former ischemic zone. The ectopic sources exhibited a slow rise in diastolic calcium, which disappeared upon return to the original pacing pattern. Border zone propagation of ectopic activity was followed by its escape into the surrounding control network, generating arrhythmias. Together, these observations suggest that upon reperfusion, a distinct layer, which consists of ectopically active, poorly coupled cells, is formed transiently over an injured area. Despite being neighbored by a conductive and excitable tissue, this transient functional layer is capable of sustaining autonomous waves and serving as a special conductive medium through which ectopic activity can propagate before spreading into the surrounding healthy tissue.

scholarly journals Beta-1-Adrenergic Receptors Mediate Nrf2-HO-1-HMGB1 Axis Regulation to Attenuate Hypoxia/Reoxygenation-Induced Cardiomyocytes Injury in Vitro

2015 ◽  
Vol 35 (2) ◽  
pp. 767-777 ◽  
Author(s):  
Jichun Wang ◽  
Xiaorong Hu ◽  
Jing Xie ◽  
Weipan Xu ◽  
Hong Jiang
Keyword(s):  
Adrenergic Receptors ◽  
Pi3k Inhibitor ◽  
Neonatal Rat ◽  
Control Group ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Cardiomyocytes ◽  
Nf Kappab ◽  
Cultured Cardiomyocytes ◽  
Hypoxia Reoxygenation

Backgroud/Aims: The aim of the study was to evaluate the effects of beta1-adrenergic receptors (β1-ARs) -mediated nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1)-high mobility group box 1 protein (HMGB1) axis regulation in hypoxia/reoxygenation (H/R)-induced neonatal rat cardiomyocytes. Methods: The neonatal cultured cardiomyocytes were concentration-dependently pretreated by dobutamine (DOB), a selective β1-adrenergic receptor agonist, in the absence and/or presence of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), SB203580 (a p38mitogen-activated-protein kinase (p38MAPK) inhibitor), Nrf2siRNA and HO-1siRNA, respectively, and then treated by H/R. The effects and mechanisms by which H/R-induced cardiomyocytes injury were evaluated. Results: Significant increase of HO-1 was found in neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Significant change for Nrf2 translocation was also revealed in neonatal cultured cardiomyocytes treated with DOB. Insignificant decreases of NF-kappaB p65 activation and HMGB1 release were observed in H/R-induced neonatal cultured cardiomyocytes treated with DOB, when compared to the control group. Importantly, DOB treatment significantly increased the cell viability and decreased the levels of LDH and MDA in H/R-induced cardiomyocytes injury. However, DOB failed to increase HO-1, inhibit NF-kappaB p65 activation, prevent HMGB1 release and attenuate H/R-induced cardiomyocytes injury when the cultured cardiomyocytes were pretreated by Nrf2siRNA, HO-1siRNA, PI3K inhibitor (LY294002) and p38MAPK inhibitor (SB203580), respectively. Conclusions: β1-ARs-mediated Nrf2-HO-1-HMGB1 axis regulation plays a critical protective role in H/R-induced neonatal rat cardiomyocytes injury in vitro via PI3K/p38MAPK signaling pathway.

MicroRNA-34a protects myocardial cells against ischemia–reperfusion injury through inhibiting autophagy via regulating TNFα expression

2018 ◽  
Vol 96 (3) ◽  
pp. 349-354 ◽  
Author(s):  
Haifeng Shao ◽  
Lili Yang ◽  
Li Wang ◽  
Bozan Tang ◽  
Jian Wang ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Heart Tissue ◽  
Neonatal Rat ◽  
Cell Counting ◽  
In Vivo Model ◽  
Myocardial Cells ◽  
Rat Cardiomyocytes ◽  
Cardiac Tissues

Background: ischemia–reperfusion (I/R) is a consequence of restored blood supply after myocardial infarction. Myocardial I/R injury can be alleviated by reducing autophagy in heart tissue. MicroRNA-34a (miR-34a) has been shown to regulate autophagy in a renal model of I/R, but it is not known whether it can protect cardiac tissues from I/R injury. This study investigated how miR-34a protects myocardial cells from I/R injury by inhibiting autophagy via regulation of tumor necrosis factor α (TNFα). Methods: we constructed an I/R model in vivo using Langendorff perfusion, and we constructed an in vivo model by treating neonatal rat cardiomyocytes (NRCMs) with hypoxia–reoxygenation (H/R method). Transfected adenoviral-overexpressed miR-34a mimics and controlled NRCMs after H/R. We analyzed cell viability using the MTT assay and a cell counting kit-8 (CCK-8) assay. Changes in the rate of apoptosis were detected by flow cytometry. We investigated the effect mechanisms of miR-34a with Western blot and luciferase assays. Results: miR-34a expression decreased after in vivo reperfusion of the myocardial cells and heart tissues of neonatal rats. MiR-34a reduced apoptosis of the NRCMs and autophagy levels, simultaneously, after H/R injury. Further, miR-34a decreased the expression of Lc3-II and p62, indicating that miR-34a reduces myocardial I/R injury by decreasing TNFα expression. Conclusion: miR-34a can inhibit autophagy levels after I/R by targeting TNFα, thereby reducing myocardial injury.

Argon Exposure Induces Postconditioning in Myocardial Ischemia–Reperfusion

2017 ◽  
Vol 22 (6) ◽  
pp. 564-573 ◽  
Author(s):  
Sandrine Lemoine ◽  
Katrien Blanchart ◽  
Mathieu Souplis ◽  
Adrien Lemaitre ◽  
Damien Legallois ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Ischemia Reperfusion ◽  
Contractile Force ◽  
Left Ventricular ◽  
Border Zone ◽  
Left Ventricular Ejection ◽  
Human Atrial Appendages ◽  
Hypoxia Reoxygenation

Background and Purpose: Cardioprotection against ischemia–reperfusion (I/R) damages remains a major concern during prehospital management of acute myocardial infarction. Noble gases have shown beneficial effects in preconditioning studies. Because emergency proceedings in the context of myocardial infarction require postconditioning strategies, we evaluated the effects of argon in such protocols on mammalian cardiac tissue. Experimental Approaches: In rat, cardiac I/R was induced in vivo by transient coronary artery ligature and cardiac functions were evaluated by magnetic resonance imaging. Hypoxia–reoxygenation (H/R)-induced arrhythmias were evaluated in vitro using intracellular microelectrodes on both rat-isolated ventricle and a model of border zone in guinea pig ventricle. Hypoxia–reoxygenation loss of contractile force was assessed in human atrial appendages. In those models, postconditioning was induced by 5 minutes application of argon at the time of reperfusion. Key Results: In the in vivo model, I/R produced left ventricular ejection fraction decrease (24%) and wall motion score increase (36%) which was prevented when argon was applied in postconditioning. In vitro, argon postconditioning abolished H/R-induced arrhythmias such as early after depolarizations, conduction blocks, and reentries. Recovery of contractile force in human atrial appendages after H/R was enhanced in the argon group, increasing from 51% ± 2% in the nonconditioned group to 83% ± 7% in the argon-treated group ( P < .001). This effect of argon was abolished in the presence of wortmannin and PD98059 which inhibit prosurvival phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and MEK/extracellular receptor kinase 1/2 (ERK 1/2), respectively, or in the presence of the mitochondrial permeability transition pore opener atractyloside, suggesting the involvement of the reperfusion injury salvage kinase pathway. Conclusion and Implications: Argon has strong cardioprotective properties when applied in conditions of postconditioning and thus appears as a potential therapeutic tool in I/R situations.

scholarly journals Dexmedetomidine Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury by Suppressing TLR4-MyD88-NF-κB Signaling

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Jin-meng Gao ◽  
Xiao-wen Meng ◽  
Juan Zhang ◽  
Wei-rong Chen ◽  
Fan Xia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Ischemia Reperfusion Injury ◽  
Cell Damage ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Rat Cardiomyocytes ◽  
Knock Down ◽  
Ldh Activity ◽  
Hypoxia Reoxygenation

Objective. We previously reported that dexmedetomidine (DEX) offers cardioprotection against ischemia/reperfusion injury in rats. Here, we evaluated the role of toll-like receptors 4- (TLR4-) myeloid differentiation primary response 88- (MyD88-) nuclear factor-kappa B (NF-κB) signaling in DEX-mediated protection of cardiomyocytes usingin vitromodels of hypoxia/reoxygenation (H/R).Methods. The experiments were carried out in H9C2 cells and in primary neonatal rat cardiomyocytes. Cells pretreated with vehicle or DEX were exposed to hypoxia for 1 h followed by reoxygenation for 12 h. We analyzed cell viability and lactate dehydrogenase (LDH) activity and measured tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and IL-1βmRNA levels, TLR4, MyD88, and nuclear NF-κB p65 protein expression and NF-κB p65 nuclear localization. TLR4 knock-down by TLR4 siRNA transfection and overexpression by TLR4 DNA transfection were used to further confirm our findings.Results. DEX protected against H/R-induced cell damage and inflammation, as evidenced by increased cell survival rates, decreased LDH activity, and decreased TNF-α, IL-6, and IL-1βmRNA levels, as well as TLR4 and NF-κB protein expression. TLR4 knock-down partially prevented cell damage following H/R injury, while overexpression of TLR4 abolished the DEX-mediated protective effects.Conclusions. DEX pretreatment protects rat cardiomyocytes against H/R injury. This effect is partly mediated by TLR4 suppression via TLR4-MyD88-NF-κB signaling.

scholarly journals CTRP5-Overexpression Attenuated Ischemia-Reperfusion Associated Heart Injuries and Improved Infarction Induced Heart Failure

2020 ◽  
Vol 11 ◽  
Author(s):  
Meng Peng ◽  
Yuan Liu ◽  
Xiang-qin Zhang ◽  
Ya-wei Xu ◽  
Yin-tao Zhao ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Related Protein ◽  
Rat Cardiomyocytes ◽  
Knock Out ◽  
Cardiac Functions

Aims: C1q/tumor necrosis factor (TNF)-related protein 5 (CTRP5) belongs to the C1q/TNF-α related protein family and regulates glucose, lipid metabolism, and inflammation production. However, the roles of CTRP5 in ischemia/reperfusion (I/R) associated with cardiac injuries and heart failure (HF) needs to be elaborated. This study aimed to investigate the roles of CTRP5 in I/R associated cardiac injuries and heart failure.Materials and Methods: Adeno-associated virus serum type 9 （AAV9）vectors were established for CTRP5 overexpression in a mouse heart (AAV9-CTRP5 mouse). AAV9-CTRP5, AMPKα2 global knock out （AMPKα2−/−）and AAV9-CTRP5+ AMPKα2−/− mice were used to establish cardiac I/R or infarction associated HF models to investigate the roles and mechanisms of CTRP5 in vivo. Isolated neonatal rat cardiomyocytes (NRCMS) transfected with or without CTRP5 adenovirus were used to establish a hypoxia/reoxygenation (H/O) model to study the roles and mechanisms of CTRP5 in vitro.Key Findings: CTRP5 was up-regulated after MI but was quickly down-regulated. CTRP5 overexpression significantly decreased I/R induced IA/AAR and cardiomyocyte apoptosis, and attenuated infarction area, and improved cardiac functions. Mechanistically, CTRP5 overexpression markedly increased AMPKα2 and ACC phosphorylation and PGC1-α expression but inhibited mTORC1 phosphorylation. In in vitro experiments, CTRP5 overexpression could also enhance AMPKα2 and ACC phosphorylation and protect against H/O induced cardiomyocytes apoptosis. Finally, we showed that CTPR5 overexpression could not protect against I/R associated cardiac injuries and HF in AMPKα2−/− mice.Significance: CTRP5 overexpression protected against I/R induced mouse cardiac injuries and attenuated myocardial infarction induced cardiac dysfunction by activating the AMPKαsignaling pathway.

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