Simulated Ischemia Research Articles

B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction-Related Adverse Cardiac Remodeling in Mice.

BackgroundHuman relaxin‐2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia‐reperfusion injury. B7‐33, a synthetically designed peptide analogous to B‐chain of relaxin‐2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin‐2) by preferentially phosphorylating the mitogen‐activated protein kinase extracellular signal‐regulated kinase 1/2. We sought to investigate the effects of B7‐33 treatment post ischemia‐reperfusion injury in mice.Methods and ResultsAdult male CD1 mice were subjected to ischemia‐reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7‐33 significantly reduced infarct size (21.99% versus 45.32%; P=0.02) and preserved fractional shortening (29% versus 23%; P=0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7‐33 and vehicle groups, respectively). In vitro, primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia‐reperfusion injury (simulated ischemia reoxygenation). B7‐33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7‐33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP78 in an extracellular signal‐regulated kinase 1/2–dependent manner.ConclusionsB7‐33 confers acute cardioprotection and limits myocardial infarction–related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.

Amino acids‐induced cardiac protection by promoting mitochondrial function via mTOR and Opa‐1 signaling

ObjectsAmino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs.MethodsCardiac myocytes were isolated by cardiac retrograde aortic perfusion and collagenase and were subjected to simulated ischemia and cell death was quantified. Immunoblot and real time PCR were performed as biochemical assay and mitochondrial function was assessed using Seahorse XF cell Mito test.ResultsIn myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA‐induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). To elucidate the mechanism, we assessed changes in the mitochondrial fusion regulators Opa‐1, Mfn‐1, and Mfn‐2. BCAA treatment increased expression of Opa‐1, but not Mfn‐1 or Mfn‐2. Enhanced mitochondrial fusion also improved mitochondrial function including oxygen consumption rate.ConclusionsBCAA acutely regulates mitochondrial metabolism in cardiomyocytes and increased mitochondrial fusion, Opa‐1, and the Akt‐mTOR pathway, offering a novel target for prevention and treatment of coronary disease.Support or Funding InformationThis study was supported by Japan Society of Promotion Science #19K09353 to YMT.

Myocardial Ischemia‐Reperfusion Injury: Does Disruption of Mitochondrial Cristae Integrity Play a Requisite Mechanistic Role?

Lethal myocardial ischemia‐reperfusion (IR) injury (i.e., cardiomyocyte death resulting in ‘heart attack’ or myocardial infarction, MI) is the leading global cause of death and disability. Despite improvements in the clinical management of MI, the molecular mechanisms responsible for lethal IR injury are incompletely understood. Data from our group and others have revealed that cardiomyocytes subjected to IR display a loss in integrity of optic atrophy protein (OPA1), the master‐regulator of fusion at the inner mitochondrial membrane and putative gate‐keeper of cristae junction integrity. Our goal in the current study was to establish whether disruption of OPA1 integrity plays a causal role in determining cardiomyocyte fate. To test this concept, HL‐1 cardiomyocytes were subjected to 2.5 hours of simulated ischemia + reperfusion. In Protocol 1, the cleavage and subsequent release of OPA1 from mitochondria into the cytosol was assessed by immunoblotting at 30 min post‐R. As expected, IR was associated with degradation of OPA1 (in particular, the long (L) forms of the protein critical for inner mitochondrial membrane architecture), and a >15‐fold increase in cytosolic expression of the lower molecular weight short (S) forms (data not shown). In Protocol 2, to demonstrate cause‐and‐effect, HL‐1 cells were transfected with either siRNA targeting OPA1 or scrambled peptide before undergoing 2.5 hours I + 24 hours R or a time‐matched normoxic period. Additional non‐transfected cells served as controls. Using 50 nM siRNA, near‐total knockdown (KD) of OPA1 (most notably, the critical L‐forms) was achieved (Figure 1). In the normoxic cohorts, cardiomyocyte viability (quantified using the MTT assay) was modestly but significantly reduced in siRNA‐transfected cells (Figure 2 – left: *p<0.05 versus normoxic non‐transfected cells). However, contrary to the premise of our study, OPA1 knockdown had no effect on viability in cells subjected to IR: i.e., did not exacerbate IR‐induced cardiomyocyte death (Figure 2 – right). These data suggest that maintenance of OPA1 integrity does not have a requisite, causal role in mediating lethal myocardial ischemia‐reperfusion injury.Support or Funding InformationDetroit Cardiovascular Training Grant T32 (NIH 2T32HL120822) through the National Heart, Lung, and Blood Institute ‐ Wayne State UniversityOPA1 Knockdown with siRNAFigure 1HL‐1 Cell viability with OPA1 KnockdownFigure 2

Small extracellular vesicles secreted from human amniotic fluid mesenchymal stromal cells possess cardioprotective and promigratory potential

Mesenchymal stromal cells (MSCs) exhibit antiapoptotic and proangiogenic functions in models of myocardial infarction which may be mediated by secreted small extracellular vesicles (sEVs). However, MSCs have frequently been harvested from aged or diseased patients, while the isolated sEVs often contain high levels of impurities. Here, we studied the cardioprotective and proangiogenic activities of size-exclusion chromatography-purified sEVs secreted from human foetal amniotic fluid stem cells (SS-hAFSCs), possessing superior functional potential to that of adult MSCs. We demonstrated for the first time that highly pure (up to 1.7 × 1010 particles/µg protein) and thoroughly characterised SS-hAFSC sEVs protect rat hearts from ischaemia–reperfusion injury in vivo when administered intravenously prior to reperfusion (38 ± 9% infarct size reduction, p < 0.05). SS-hAFSC sEVs did not protect isolated primary cardiomyocytes in models of simulated ischaemia–reperfusion injury in vitro, indicative of indirect cardioprotective effects. SS-hAFSC sEVs were not proangiogenic in vitro, although they markedly stimulated endothelial cell migration. Additionally, sEVs were entirely responsible for the promigratory effects of the medium conditioned by SS-hAFSC. Mechanistically, sEV-induced chemotaxis involved phosphatidylinositol 3-kinase (PI3K) signalling, as its pharmacological inhibition in treated endothelial cells reduced migration by 54 ± 7% (p < 0.001). Together, these data indicate that SS-hAFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting.

Study of novel deformable image registration in myocardial perfusion single-photon emission computed tomography.

In the present study, deformable image registration (DIR) technology was applied to gated myocardial perfusion single-photon emission computed tomography (G-MPS) reconstructed images in distorting all image phases. We aimed to define a new method of end-diastole compatible image registration and verify the clinical usability for any cardiac volume. Projection images were created using the Monte Carlo simulation. All image phases were shifted to fit the end-diastole phase by applying DIR to images that were reconstructed from projection images. Defect ratios were subsequently evaluated using the simulated images of the anterior wall simulated ischemia. Furthermore, receiver operating characteristic (ROC) analysis was performed for the clinical evaluation of DIR and nongated images. To this end, normal volume and small hearts of 33 patients without coronary artery disease and 55 with single vessel disease (coronary stenosis > 70%) were evaluated. Defect ratio analysis for voxel values of 25-100 were 75.7-21.3 for nongated and 74.7-15.6 for DIR images. For normal cardiac volume, the area under the ROC curve was 0.901 ± 0.088 for nongated and 0.925 ± 0.073 for DIR images (P = 0.078). Finally, for small cardiac volume, the area under the ROC curve was 0.651 ± 0.124 for nongated and 0.815 ± 0.119 for DIR (P < 0.01). In the present study, we developed a new registration technique by applying DIR to G-MPS images. When optimal DIR conditions were applied, the resolution of G-MPS images was improved. Furthermore, the diagnostic ability was improved in cases of small cardiac volume.

Branched-chain amino acids-induced cardiac protection against ischemia/reperfusion injury

AimsAmino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs. Main methodsAn in vivo model of I/R injury was tested in control, mTOR+/+, and mTOR+/−. Mice were randomly assigned to receive BCAAs, rapamycin, or BCAAs + rapamycin. Furthermore, isolated cardiomyocytes were subjected to simulated ischemia and cell death was quantified. Biochemical and mitochondrial swelling assays were also performed. Key findingsMice treated with BCAAs had a significant reduction in infarct size as a percentage of the area at risk compared to controls (34.1 ± 3.9% vs. 44.7 ± 2.6%, P = 0.001), whereas mice treated with the mTOR inhibitor rapamycin were not protected by BCAA administration (42.2 ± 6.5%, vs. control, P = 0.015). This protection was not detected in our hetero knockout mice of mTOR. Western blot analysis revealed no change in AKT signaling whereas activation of mTOR was identified. Furthermore, BCAAs prevented swelling which was reversed by the addition of rapamycin. In myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA-induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). SignificanceBCAA treatment exhibits a protective effect in myocardial I/R injury and that mTOR plays an important role in this preconditioning effect.

Endothelial protective effect of rapamycin against simulated ischemia injury through up-regulation of autophagy and inhibition of endoplasmic reticulum stress

IntroductionRapamycin has been shown to have cytoprotective properties in some experimental models of ischemia. However, the precise molecular mechanisms underlying the positive effect of rapamycin on endothelial cells in ischemic injury remain unknown. It is very important because endothelial cells are firstly exposed to ischemia and play an important role in ischemic organ damage. Autophagy and endoplasmic reticulum stress are suggested to be implicated in hypoxic/ischemic injury of endothelial cells. This study aims to explore whether the endothelial protective effect of rapamycin is associated with exacerbation of autophagy and attenuation of endoplasmic reticulum stress.Material and methodsThe protective effects of rapamycin against oxygen and glucose deprivation (OGD)-induced cell injury were explored in human vascular endothelial cells (HUVECs). Cell viability was measured by MTT assay. The protein levels of Beclin 1, p62, p-mTOR, p-S6K, p-4EBP, GRP78, p-PERK and p-IRE1 were analyzed using immunoblotting.ResultsRapamycin in the simulated ischemia model increased the cell viability, indicating its cytoprotective effect (p &lt; 0.05). Experiments with 3-methyladenine as an inhibitor of autophagy and thapsigargin as an inducer of endoplasmic reticulum stress support that rapamycin exerts endothelial protective effects against OGD-induced damage via autophagy – endoplasmic reticulum stress pathway.ConclusionsThis study demonstrated that rapamycin protects ischemic HUVECs via down-regulation of the mTOR pathway, enhancement of autophagy and inhibition of endoplasmic reticulum stress.

MiR-208a participates with sevoflurane post-conditioning in protecting neonatal rat cardiomyocytes with simulated ischemia-reperfusion injury via PI3K/AKT signaling pathway.

We aimed to evaluate the role of miR-208a in sevoflurane post-conditioning protecting neonatal rat cardiomyocytes with simulated ischemia reperfusion injury (SI/RI) via PI3K/AKT signaling pathway. The cardiomyocytes of healthy neonatal rats were extracted as the Normal group. Other cardiomyocytes were used to establish the SI/RI model. Sevoflurane post-conditioning, miR-208a inhibitor, or PI3K/AKT pathway activator were used in the treatment of cardiomyocytes. The cell viability, cell cycle, apoptosis, levels of superoxide dismutase (SOD), malondialdehyde (MDA), miR-208a and mRNA, expression of PI3K, AKT, and autophagy-related factors in each group were measured and compared. Monodansylcadaverine (MDC) was used to measure the fluorescence intensity of autophagosomes. In neonatal rat cardiomyocytes with SI/RI, the expression of miR-208a and MDA, apoptosis and the expression of autophagy-related factors increased, with PI3K/AKT pathway inhibited, SOD level decreased, cell viability reduced, and the fluorescence intensity of autophagosomes enhanced (all p<0.05). Sevoflurane post-conditioning can promote the increase of SOD and the decrease of MDA in cardiomyocytes with SI/RI, with PI3K/AKT pathway activated, viability of cardiomyocytes enhanced, apoptosis reduced, the expression of autophagy-related factors and the fluorescence intensity of autophagosomes inhibited (all p<0.05). Sevoflurane post-conditioning combined with miR-208a inhibitor or PI3K/AKT pathway activator further promoted the above-mentioned effects (all p<0.05). Our results indicate that the inhibited expression of miR-208a suppresses the expression of autophagy-related factors, enhances cell viability of cardiomyocytes, and reduces apoptosis, thereby protecting neonatal rat cardiomyocytes with SI/RI.

Effects of tetracycline on myocardial infarct size in obese rats with chemically-induced colitis

Recent evidence suggests that antibiotic-induced changes in the composition of intestinal microflora, as well as the systemic immunoendocrine effects that result from them, can modulate myocardial tolerance to ischemia-reperfusion injury. The aim of this study was to investigate the effects of tetracycline (TTC) on myocardial infarct size in the isolated hearts obtained from obese rats with chemically-induced colitis (CIC). The association between TTC-induced changes in infarct size and intestinal microbiome composition as well as plasma levels of cytokines and short-chain fatty acids (SCFAs) was also studied. Obesity was induced in Wistar rats by feeding them a high-fat, high-carbohydrate diet for five weeks. A single rectal administration of 3% acetic acid (2 mL) to the rats resulted in CIC. Healthy rats as well as obese rats with CIC received TTC (15 mg daily for 3 days) via gavage. The rats were euthanized, after which isolated heart perfusion with simulated global ischemia and reperfusion was performed. Infarct size was determined histochemically. Lipopolysaccharide (LPS) and cytokine levels in plasma were measured by enzyme-linked immunosorbent assay, whereas SCFA levels in plasma were measured by gas chromatography/mass spectrometry. The intestinal microbiome was analyzed using reverse transcription polymerase chain reaction. The treatment with TTC resulted in significant infarct size limitation (50 ± 7 vs. 62 ± 4% for the control mice, p < 0.05) in the hearts from intact animals. However, infarct size was not different between the control rats and the obese rats with CIC. Furthermore, infarct size was significantly larger in TTC-treated obese rats with CIC than it was in the control animals (77 ± 5%, p < 0.05). The concentrations of proinflammatory cytokines and LPS in serum were elevated in the obese rats with CIC. Compared to the control rats, the rats with both obesity and CIC had lower counts of Lactobacillus and Bifidobacterium spp. but higher counts of Escherichia coli. The effects of TTC on infarct size were not associated with specific changes in SCFA levels. TTC reduced infarct size in the healthy rats. However, this effect was reversed in the obese animals with CIC. Additionally, it was associated with specific changes in gut microbiota and significantly elevated levels of cytokines and LPS.

Effects of Atorvastatin on Transient Sodium Currents in Rat Normal, Simulated Ischemia, and Reperfusion Ventricular Myocytes

Objectives: (1) To detect the whether the effects of simulated ischemia on I_Na of rat left ventricular myocytes in a time-dependent manner and the effects of atorvastatin on ischemia I_Na; (2) To investigate the effects of atorvastatin on I_Na of rat-simulated ischemia/reperfusion (I/R) ventricular cells. Materials and Methods: Ventricular cells were enzymatically isolated by Langendorff perfusion system. Whole-cell patch clamp was applied to detect I_Na level. Some elements of extracellular fluid were hanged to simulate the status of normal, I and R condition. Then the effects of atorvastatin on I_Na were observed. Results: (1) During simulated reperfusion, I_Na decreased and atorvastatin further suppressed the reduction degree. (2) At test potential –40 mV, no difference was detected among peak I_Na amplitude of ischemia for 20 min, reperfusion phase 3/5/7/9 min in continuous ischemia (I) group (p = 0.275). In I/R group, peak I_Na amplitude continuously decreased at 3 min (p = 0.005) and 9 min (p = 0.041). In atorvastatin intervention + I/R (Statin + I/R) group, peak I_Na amplitude at reperfusion 3 min decreased compared with ischemia phase (p = 0.000), while no significant difference was detected between 3 and 9 min (p = 0.858). The differences were significant at the same time point between groups. At reperfusion 3/5/7/9 min, peak I_Na of the I/R group was lower than the ischemia group (all p = 0.000), same as the Statin + I/R group (p = 0.000, p = 0.003, p = 0.006, and p = 0.001). Peak I_Na of the Statin + I/R group was higher than the I/R group at the same time point (p = 0.011, p = 0.033, p = 0.003, p = 0.003). There was no change in the I group during reperfusion phase (p > 0.05). In I/R group, V_1/2 (mV) shifted from –58.87 ± 3.36 to –54.33 ± 2.40, k (mV) shifted from 1.25 ± 0.59 to 1.91 ± 0.84 (p < 0.05). In the Statin + I/R group, V_1/2 (mV) increased from –57.80 ± 2.97 to –52.76 ± 3.14 (p < 0.01), no change was observed in k (p > 0.05). Conclusions: (1) In the status of reperfusion, I_Na decreased more than that in the status of ischemia. (2) Atorvastatin protected the cells from reduction of I_Na during long-time simulated (>15 min) I/R. (3) Overall, atorvastatin affected I_Na of the normal, simulated ischemic/reperfusion cell in rat left ventricle by blocking sodium channel ­directly.

Effects of atorvastatin on time-dependent change of fast sodium current in simulated acute ischaemic ventricular myocytes.

Our previous experiments showed that the transient sodium current (INa) was abnormally increased in early ischaemia and atorvastatin could inhibit INa. The aim of this study was to observe the time-dependent effects of simulated ischaemia on INa and characterise the direct effects of atorvastatin on ischaemic INa. Left ventricular myocytes were isolated from Wistar rats and randomly divided into two groups: a control group (normal to simulated ischaemia) and a statin group (normal to simulated ischaemia with 5 µmol/l atorvastatin). The INa was recorded under normal conditions (as baseline) by whole-cell patch clamp and recorded from three to 21 minutes in the next phase of simulated ischaemic conditions. In the control group, normalised INa (at -40 mV) was increased to the peak (1.15 ± 0.08 mA) at three minutes of ischaemia compared with baseline (0.95 ± 0.04 mA, p < 0.01), it subsequently returned to baseline levels at nine and 11 minutes of ischaemia (0.98 ± 0.12 and 0.92 ± 0.12 mA, respectively), and persistently decreased with prolonged ischaemic time. In the statin group, there were no differences between baseline and the early stages of ischaemia (0.97 ± 0.04 mA at baseline vs 0.92 ± 0.12 mA in ischaemia for three minutes, p > 0.05). Our results suggest that, in the early stages of ischaemia, changes in INa in ventricular myocytes are time-dependent, showing an initial increase followed by a decrease, while atorvastatin inhibited the transient increase in INa and made the change more gradual.

Abstract 163: NFkB Promotes Oxidative Stress-induced Necrosis and Ischemia Reperfusion Injury Through NRF2-ARE Pathway

Objective: Despite the well-studied pro-survival function of nuclear factor-κB (NFκB), recent studies suggest that NFκB may also play a pathogenic role in myocardial ischemia injury and adverse remodeling. This study aims to define a new pro-cell death role of NFκB in response to oxidative stress and the functional implications in ischemia reperfusion (I/R) injury. Methods and Results: We identified an unexpected pro-cell death role of NFκB in oxidative stress-induced necrosis, and provide new mechanistic evidence that NFκB, in cooperation with HDAC3, negatively regulates NRF2-ARE anti-oxidative signaling through transcriptional silencing. Genetic deletion of NFκB-p65 inhibits, whereas activation of NFκB promotes, oxidative stress-induced cell death and HMGB1 release, a biomarker of necrosis. Moreover, simulated ischemia reperfusion (sI/R) and doxorubicin (Dox) treatment both induce NFκB-luciferase activity in cardiomyocytes, and inhibition of NFκB diminishes sI/R- and Dox- induced necrosis. Importantly, NFκB negatively regulates NRF2-ARE activity and the expression of anti-oxidant proteins. Mechanistically, co-immunoprecipitation reveals that p65 is required for the association between NRF2 and HDAC3 and transcriptional silencing of NRF2-ARE activity. Further, the ability of HDAC3 to repress NRF2-ARE activity is lost in p65-/- cells. The HADC inhibitor TSA and NFκB inhibitor BMS-345541 both increase NRF2-ARE activity and promote cell survival following sI/R. In vivo , NFκB transcriptional activity in the heart is significantly elevated after I/R injury, which is abolished by cardiac-specific deletion of p65. Moreover, ablation of p65 using p65 fl/fl-Nkx-Cre mice reduces myocardial infarct size after acute I/R, and prevents chronic remodeling and contractile dysfunction after myocardial infarction. Conclusions: Our results identified NFκB as a key regulator of oxidative stress-induced necrosis by suppressing the NRF2-ARE anti-oxidant pathway through an HDAC3-dependent mechanism. Ablation of NFκB-p65 attenuates oxidative stress-induced necrosis and I/R injury, suggesting a new pathogenic role of the NFκB pathway, and thus a therapeutic target, in myocardial ischemic injury and remodeling.

The Endocannabinoid Anandamide regulates oxytocin receptor in human placenta

Previous studies have shown that modulation of the receptor-mediated endocannabinoid system during ischemia injury can induce potent neuroprotective effects. However, little is known about whether cannabinoid-2 (CB2) receptor agonist would produce a protective effect on blood–spinal cord barrier (BSCB) during ischemia. Using an in vivo transient spinal cord ischemia model in rats, JWH-015 (1 mg/kg, i.p.), a CB2 receptor selective agonist, or vehicles were injected 20 min before ischemia. The effects of JWH-015 on BSCB permeability, the major structural protein for the formation of caveolae, caveolin-1 (cav-1), tight junction (TJ) protein Occludin and zona occludens protein-1 (ZO-1) were examined at day 1, day 3 and day 7 of reperfusion after transient spinal cord ischemia in rats. Here we demonstrated that JWH-015 significantly down-regulated the expression of cav-1, up-regulated the expression of TJ proteins, and then decreased the permeability of BSCB compared with control group. In addition, using an in vitro BBB model, oxygen glucose deprivation (OGD) was applied to simulate spinal cord ischemia in vitro in Human brain microvascular endothelial cells (HBMECs). JWH-015 greatly increased the transepithelial electrical resistance (TEER) and changed the distribution of ZO-1 and Occludin. Moreover, JWH-015 induced the expression of p-PKB and p-FoxO1 protein and decreased the expression of cav-1, which were greatly reversed by ROS inhibitor or PI3K inhibitor. Taken together, all of these results suggested that JWH-015 might regulate the BSCB permeability and this effect could be related to paracellular and transcellular pathway. And pharmacological CB2R ligands offer a new strategy for BSCB protection during ischemic injury.

A novel form of glycolytic metabolism-dependent cardioprotection revealed by PKCα and β inhibition.

Acute hyperglycaemia at the time of a heart attack worsens the outcome for the patient. Acute hyperglycaemia is not limited to diabetic patients and can be due to a stress response in non-diabetics. This study suggests that the damaging cardiac effects of hyperglycaemia can be reversed by selective PKC inhibition. If PKCα/β isoforms are inhibited, then high glucose itself becomes protective against ischaemic damage. Selective PKC inhibition may therefore be a useful therapeutic tool to limit the damage that can occur during a heart attack by stress-induced hyperglycaemia. Hyperglycaemia has a powerful association with adverse prognosis for patients with acute coronary syndromes (ACS). Previous work shows that high glucose prevents ischaemic preconditioning and causes electrical and mechanical disruption via protein kinase C α/β (PKCα/β) activation. The present study aimed to: (i) determine whether the adverse clinical association of hyperglycaemia in ACS can be replicated in preclinical cellular models of ACS and (ii) determine the importance of PKCα/β activation to the deleterious effect of glucose. Freshly isolated rat, guinea pig or rabbit cardiomyocytes were exposed to simulated ischaemia after incubation in the presence of normal (5mm) or high (20mm) glucose in the absence or presence of small molecule or tat-peptide-linked PKCαβ inhibitors. In each of the four conditions, the following hallmarks of cardioprotection were recorded using electrophysiology or fluorescence imaging: cardiomyocyte contraction and survival, action potential stability and time to failure, intracellular calcium and ATP, mitochondrial depolarization, ischaemia-sensitive leak current, and time to Kir 6.2 opening. High glucose alone resulted in decreased cardiomyocyte contraction and survival; however, it also imparted cardioprotection in the presence of PKCα/β inhibitors. This cardioprotective phenotype displayed improvements in all of the measured parameters and decreased myocardium damage during whole heart coronary ligation experiments. High glucose is deleterious to cellular and whole-heart models of simulated ischaemia, in keeping with the clinical association of hyperglycaemia with an adverse outcome in ACS. PKCαβ inhibition revealed high glucose to show a cardioprotective phenotype in this setting. The results of the present study suggest the potential for the therapeutic application of PKCαβ inhibition in ACS associated with hyperglycaemia.

L-Alpha-glycerylphosphorylcholine can be cytoprotective or cytotoxic in neonatal rat cardiac myocytes: a double-edged sword phenomenon

l-Alpha-glycerylphosphorylcholine (GPC) is a widely used food supplement. GPC has been shown to exert beneficial effects in several organs; however, the cardiac effects of GPC have yet to be investigated. The aim of the present study was therefore to map out the effects of GPC on cardiac myocytes, with or without ischemia–reperfusion insult. Neonatal rat cardiac myocytes were treated with GPC at 1, 10, 80, and 100 µM concentrations for 15 min, 3 h, or 24 h, respectively. Cell viability by calcein assay and the degree of oxidative stress by DHE (superoxide level) and H2DCF (total ROS accumulation) staining were measured. In separate experiments, cardiomyocytes were pre-treated with the optimal concentration of GPC for 3 h and then cells were exposed to 4 h of simulated ischemia followed by 2 h of reperfusion (SI/R). Cell viability was measured at the end of the SI/R protocol. In normoxic conditions, the 15-min and the 3-h GPC treatment did not affect cell viability, total ROS, and superoxide levels. Under SI/R conditions, the 3-h GPC treatment protected the cardiac myocytes from SI/R-induced cell death and did not alter the level of oxidative stress. The 24-h GPC treatment in normoxic conditions resulted in significant cell death and increased oxidative stress at each concentration. Here we provide the first evidence for the cytoprotective effect of short-term GPC treatment. However, long-term administration of GPC may exert cytotoxicity in a wide concentration range in cardiac myocytes. These results may draw attention to a comprehensive cardiac safety protocol for the testing of GPC.

Mitochondrial Reactive Oxygen Species Generated at the Complex-II Matrix or Intermembrane Space Microdomain Have Distinct Effects on Redox Signaling and Stress Sensitivity in Caenorhabditis elegans.

Aims: How mitochondrial reactive oxygen species (ROS) impact physiological function may depend on the quantity of ROS generated or removed, and the subcellular microdomain in which this occurs. However, pharmacological tools currently available to alter ROS production in vivo lack precise spatial and temporal control. Results: We used CRISPR/Cas9 to fuse the light-sensitive ROS-generating protein, SuperNova to the C-terminus of mitochondrial complex II succinate dehydrogenase subunits B (SDHB-1::SuperNova) and C (SDHC-1::SuperNova) in Caenorhabditis elegans to localize SuperNova to the matrix-side of the inner mitochondrial membrane, and to the intermembrane space (IMS), respectively. The presence of the SuperNova protein did not impact complex II activity, mitochondrial respiration, or C. elegans development rate under dark conditions. ROS production by SuperNova protein in vitro in the form of superoxide (O2˙-) was both specific and proportional to total light irradiance in the 540-590 nm spectra, and was unaffected by varying the buffer pH to resemble the mitochondrial matrix or IMS environments. We then determined using SuperNova whether stoichiometric ROS generation in the mitochondrial matrix or IMS had distinct effects on redox signaling in vivo. Phosphorylation of PMK-1 (a p38 MAPK homolog) and transcriptional activity of SKN-1 (an Nrf2 homolog) were each dependent on both the site and duration of ROS production, with matrix-generated ROS having more prominent effects. Furthermore, matrix- but not IMS-generated ROS attenuated susceptibility to simulated ischemia reperfusion injury in C. elegans. Innovation and Conclusion: Overall, these data demonstrate that the physiological output of ROS depends on the microdomain in which it is produced. Antioxid. Redox Signal. 31, 594-607.

Tao-Hong-Si-Wu decoction reduces ischemia reperfusion rat myoblast cells calcium overloading and inflammation through the Wnt/IP3R/CAMKII pathway.

Limb ischemia reperfusion (LIRI) injury is associated with serious local and systemic effects. Reperfusion may augment tissue injury in excess of that produced by ischemia alone. Calcium overloading and inflammation are considered to be two of the pathological mechanisms of limb ischemia/reperfusion (I/R) injury. Tao-Hong-Si-Wu decoction (THSWD) is a traditional Chinese herbal medicine with a powerful anti-inflammatory properties. We studied the probable restorative effect of THSWD on limb I/R-induced calcium overloading and inflammation in myoblast obtained from gastrocnemius muscle tissues of Sprague-Dawley rats (Frizzled Z5,a wnt5a blocker; KN-93, a calmodulin-dependent protein kinase II (CamkII) blocker; XeC, a IP3R blocker as positive controls). The simulated ischemia and reperfusion(I/R) solutions were used to imitate LIRI environment. The results showed that after I/R treatment, the secretion of proinflammatory factors (TNF-α and IL-1β) and Wnt5a/Ca2+ signal molecules (wnt5a, camkII, and IP3R) upregulated significantly, the Ca2+ concentration enhanced too in myoblast cells. THSWD pretreatment decreased the secretion of TNF-α and IL-1β, Ca2+ concentration; and abated the Wnt5a/Ca2+ signal molecules of wnt5a, camkII and IP3R expression activated by I/R injury; but could not abated the Wnt11 and protein kinase C (PKC) expression significantly, the results was similar with Frizzled Z5 treatment cells. Our research illustrated that THSWD may have a mitigating effect on LIRI targeting Wnt/IP3R/CAMKII but not Wnt/IP3R/PKC signaling pathway for the first time. This study may encourage the use of THSWD in the critical clinical settings with LIRI.

Modulation of Electron Transport by Metformin in Cardiac Protection: Role of Complex I

RationaleModulation of mitochondrial complex I during reperfusion reduces cardiac injury following ischemia. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with reactive oxygen species (ROS) production. Metformin treatment may preserve the D form.ObjectiveWe hypothesize that maintenance of deactivation of complex I during early reperfusion will protect cardiomyocytes. The goal is to evaluate the potential of acute, high dose Metformin treatment to modulate complex I at the onset of reperfusion/reoxygenation and decrease subsequent cellular injury.MethodsH9c2 cells underwent 6 hr. simulated ischemia and 24 hr. reoxygenation with or without metformin treatment (1 mM) at the onset of reoxygenation. Cell death, ROS and thiol modification were studied. Additional experiments were performed with or without siRNA knockdown of AMPKa2 catalytic subunit.ResultsMetformin selectively inhibited complex I oxidative phosphorylation measured at reoxygenation (Metformin: 0.34±0.1 vs control: 0.59±0.05 nmolO2/min/106cells; ±SE; n=5–10; p&lt;0.05) with decreased total cell death following simulated ischemia and reoxygenation (Metformin: 8.3±0.2 vs. control: 14.4±0.6%; n=3–6; p&lt;0.05). Complex II driven oxidative phosphorylation was not reduced by metformin, supporting selectively to complex I. Apoptosis assessed by annexin‐V labeling and flow cytometry was also decreased by metformin treatment. The “burst” of ROS at reoxygenation was decreased and free thiol group content was preserved by metformin treatment at reoxygenation. Metformin decreased cell death in AMPK knockdown cells.ConclusionMetformin blockade of complex I at the onset of reoxygenation reduces cell injury. Metformin specifically inhibited complex I at reoxygenation with less oxidative injury, in line with protection against D→A reactivation of complex I. Metformin protection was AMPK independent, supporting a complex I mechanism of protection. Acute, high dose Metformin is a potentially safe translational complex I inhibitor to treat ischemia‐reperfusion injury.Support or Funding InformationThis work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs; National Institutes of Health NIH R21AG054975‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of the RNA binding protein HuR protects against cardiac ischemia/reperfusion injury by reducing inflammatory gene expression

Despite medical advances, cardiac ischemia/reperfusion (I/R) injury remains a leading cause of morbidity and a huge economic burden in the United States. RNA binding proteins are becoming recognized as potential mediators of cardiac physiology and pathology, but the role of HuR, an RNA binding protein highly expressed in myocytes, in acute cardiac I/R injury is unknown. HuR has been shown in other tissues to be a critical post‐transcriptional mediator of pro‐inflammatory chemokine and cytokine gene expression, and our lab has shown HuR to be activated (nuclear‐to‐cytoplasmic translocation) in cardiomyocytes 2 hours post‐ischemia/reperfusion injury.To address the functional role of HuR in I/R, cardiomyocyte‐specific HuR deletion mice (iCM‐HuR−/−) were subjected to 30 minutes of LAD (left anterior descending) coronary artery ligation followed by 24 hours of reperfusion. In parallel, a separate group of wild‐type mice were subjected to 30 minutes ischemia with a pharmacological inhibitor of HuR given just prior to reperfusion. Analysis of infarct size showed a smaller infarct with both HuR genetic deletion (41% infarct/risk in iCM‐HuR−/− vs. 51% infarct/risk in control, N=3, P&lt;0.05) and pharmacological inhibition (~11% decrease in infarct size compared to vehicle, N=4, P=0.069). We were also able to recapitulate this protection in vitro using neonatal rat ventricular myocytes (NRVMs) and H9C2s (myoblast derived rat left ventricular cardiomyocytes) subjected to 6 hrs of simulated ischemia (glucose deprivation and hypoxia at ≤1% O2) followed by 2 or 24 hrs of reperfusion (reoxygenation and reintroduction of glucose/serum) by showing that siRNA‐mediated knockdown or pharmacological inhibition of HuR significantly reduced cell death and caspase‐3 activation. Analysis of inflammatory gene expression demonstrates a significant blunting of IL‐6 expression at just two hours post‐reperfusion both in vivo and in vitro, suggesting that HuR activity is necessary for the early induction of inflammatory gene expression networks.In conclusion, our results suggest that inhibition of HuR is protective against cardiac I/R injury through a reduction in inflammatory gene expression. Work is still ongoing to identify the specific means by which HuR is activated and mediating expression of inflammatory cytokines and chemokines.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sulodexide up-regulates glutathione S-transferase P1 by enhancing Nrf2 expression and translocation in human umbilical vein endothelial cells injured by oxygen glucose deprivation.

IntroductionSulodexide (SDX) is used for the treatment of many vascular disorders due to its anticoagulant, anti-inflammatory and anti-atherosclerotic properties. However, the detailed molecular mechanism of its endothelioprotective action is still not completely understood. There is increasing evidence suggesting that antioxidant enzymes play an important role in anti-ischemic properties of SDX. We postulate that up-regulation of glutathione-S-transferase P1 (GSTP1) mediated by the transcription factor Nrf2 could be associated with the antioxidant effect of SDX on vascular endothelial cells.Material and methodsIn the present study, we investigated whether SDX affects GSTP1 and Nrf2 in oxygen glucose deprivation (OGD) treated human umbilical vein endothelial cells (HUVECs). The cells treated with/without SDX (0.5 LRU/ml) were subjected to OGD for 1–6 h. To study the influence of SDX on the Nrf2 nucleus accumulation, the cells were incubated with 0.5 LRU/ml SDX in OGD for 1 h.ResultsWe found that after short-term OGD (1–3 h), the drug increased the expression of both GSTP1 and Nrf2 mRNA/protein in HUVECs (p < 0.05), as determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). SDX treatment also enhanced the nuclear accumulation of Nrf2 in HUVECs after 1 h of OGD (p < 0.05).ConclusionsSDX induces a rapid onset of the antioxidant response by up-regulating the expression of GSTP1 and Nrf2 in endothelial cells subjected to in vitro simulated ischemia.