Min Of Coronary Artery Occlusion Research Articles

A Comparative Study: Cardioprotective Effects of High-Intensity Interval Training Versus Ischaemic Preconditioning in Rat Myocardial Ischaemia-Reperfusion.

(1) Background: Years of research have identified ischemic preconditioning (IPC) as a crucial endogenous protective mechanism against myocardial ischemia-reperfusion injury, enhancing the myocardial cell's tolerance to subsequent ischemic damage. High-intensity interval training (HIIT) is promoted by athletes because it reduces exercise duration and improves metabolic response and cardiopulmonary function. Our objective was to evaluate and compare whether HIIT and IPC could reduce myocardial ischemia and reperfusion injury in rats. (2) Methods: Male Sprague-Dawley rats were divided into four groups: sham surgery, coronary artery occlusion (CAO), high-intensity interval training (HIIT), and ischemic preconditioning (IPC). The CAO, HIIT, and IPC groups experienced 40 min of coronary artery occlusion followed by 3 h of reperfusion to induce myocardial ischemia-reperfusion injury. Subsequently, the rats were sacrificed, and blood samples along with cardiac tissues were examined. The HIIT group received 4 weeks of training before surgery, and the IPC group underwent preconditioning before the ischemia-reperfusion procedure. (3) Results: The HIIT and IPC interventions significantly reduced the extent of the myocardial infarction size and the levels of serum troponin I and lactate dehydrogenase. Through these two interventions, serum pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were significantly decreased, while the anti-inflammatory cytokine IL-10 was increased. Furthermore, the expression of pro-apoptotic proteins PTEN, caspase-3, TNF-α, and Bax in the myocardium was reduced, and the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) was increased, ultimately reducing cellular apoptosis in the myocardium. In conclusion, both HIIT and IPC demonstrated effective strategies with potential for mitigating myocardial ischemia-reperfusion injury for the heart.

Blockage of PHLPP1 protects against myocardial ischemia/reperfusion injury in diabetic mice via activation of STAT3 signaling.

Diabetes can exacerbate myocardial ischemia/reperfusion (IR) injury. However, the sensitivity to IR injury and the underlying mechanisms in diabetic hearts remain unclear. Inhibition of PH domain leucine-rich repeating protein phosphatase (PHLPP1) could reduce myocardial IR injury, our previous study demonstrated that the expression of PHLPP1 was upregulated in diabetic myocardial IR model. Thus, this study aimed to investigate the mechanism of PHLPP1 in diabetic myocardial IR injury. Nondiabetic and diabetic C57BL/6 mice underwent 45min of coronary artery occlusion followed by 2h of reperfusion. Male C57BL/6 mice were injected with streptozotocin for five consecutive days to establish a diabetes model. H9c2 cells were exposed to normal or high glucose and subjected to 4h of hypoxia followed by 4h of reoxygenation. Diabetes or hyperglycemia increased postischemic infarct size, cellular injury, release of creatine kinase-MB, apoptosis, and oxidative stress, while exacerbating mitochondrial dysfunction. This was accompanied by enhanced expression of PHLPP1 and decreased levels of p-STAT3 and p-Akt. These effects were counteracted by PHLPP1 knockdown. Moreover, PHLPP1 knockdown resulted in an increase in mitochondrial translocation of p-STAT3 Ser727 and nuclear translocation of p-STAT3 Tyr705 and p-STAT3 Ser727. However, the effect of PHLPP1 knockdown in reducing posthypoxic cellular damage was nullified by either Stattic or LY294002. Additionally, a co-immunoprecipitation assay indicated a direct interaction between PHLPP1 and p-STAT3 Ser727, but not p-STAT3 Tyr705. The abnormal expression of PHLPP1 plays a significant role in exacerbating myocardial IR injury in diabetic mice. Knockdown of PHLPP1 to activate the STAT3 signaling pathway may represent a novel strategy for alleviating myocardial IR injury in diabetes.

Abstract 13767: A Novel Pharmacological Inhibitor of Type 5 Adenylyl Cyclase Inhibitor Enhances Exercise Capacity and Protects Against Glucose Intolerance and Myocardial Ischemia

Disruption of adenylyl cyclase 5 (AC5 KO) is a healthful longevity model; not only do the AC5 KO mice live a third longer than wild type (WT) mice, but are also protected against heart failure, obesity and diabetes and exercise intolerance. To translate to the clinics, we developed a new drug, C90, which inhibits AC5, and examined its effects on cardioprotection in pigs, exercise capacity and glucose tolerance in mice. C90 (30 mg/kg/day) or vehicle were chronically administered to age-matched C57/BL6 male mice via osmotic pump. After baseline exercise, the WT mice, all performing similarly, were divided into two groups; one that received C90 for 14 days and the other receiving vehicle for 14 days. After 14 days of C90 treatment, WT mice exhibited greater exercise capacity reflected by longer running distance (384 ± 27 m vs. 253 ± 16 m, p<0.05) and greater work to exhaustion (18.1 ± 1.5 J vs. 12.4 ± 0.7 J, p<0.05) than mice receiving vehicle. Sensitivity to glucose was measured using a glucose tolerance test in mice. Following 14-days treatment with C90 (30 mg/kg/day), the mice exhibited an overall improvement in glucose tolerance, measured as area under the curve (A.U.) for glucose levels following insulin injection, when compared to vehicle (6414 ± 890 A.U. vs. 9658 ± 1039 A.U., p<0.04). Furthermore, C90-treated mice had a lower fasting glucose level on a regular diet, when compared to vehicle (113 ± 6.5 mg/dl vs. 129 ± 4.2 mg/dl, p<0.05). The cardioprotective effects of C90 were previously demonstrated in mice. In the current study, we investigated the cardioprotective effects of C90 in a porcine model of ischemia/reperfusion (I/R). Ischemia was induced for 60 min coronary artery occlusion, followed by 3-hours reperfusion. C90 (1.2 mg/kg), infused 5 min following the start of the reperfusion led to a decreased infarct size/area at risk (INF/AAR) (21.6 ± 4.3% vs. 36.8 ± 2.9%, p<0.05) when compared to vehicle, with no difference between AAR/left ventricle between the C90 and vehicle groups. In view of its lower level of toxicity than other AC5 inhibitors, C90 is an excellent candidate for clinical development for pharmacological treatment of myocardial ischemia, glucose intolerance and exercise intolerance.

ECG Markers of Acute Melatonin Treatment in a Porcine Model of Acute Myocardial Ischemia.

In myocardial ischemia, melatonin confers antiarrhythmic action, but its electrocardiographic expression is unclear. We aimed to evaluate the effects of melatonin treatment on electrocardiogram (ECG) parameters reflecting major arrhythmogenic factors and to test the association of these parameters with ventricular fibrillation (VF) incidence. Myocardial ischemia was induced by 40 min coronary artery occlusion in 25 anesthetized pigs. After induction of ischemia, 12 and 13 animals were given melatonin or placebo, respectively. Twelve-lead ECGs were recorded and durations of QRS, QT, Tpeak-Tend intervals and extrasystolic burden were measured at baseline and during occlusion. During ischemia, VF episodes clustered into early and delayed phases (<10 and >20 min, respectively), and QRS duration was associated with VF incidence. QT interval and extrasystolic burden did not differ between the groups. The Tpeak-Tend interval was progressively prolonged, and the prolongation was less pronounced in the treated animals. QRS duration increased, demonstrating two maxima (5–10 and 25 min, respectively). In the melatonin group, the earlier maximum was blunted, and VF development in this period was prevented. Thus, acute melatonin treatment prevented excessive prolongation of the QRS and Tpeak-Tend intervals in the porcine myocardial infarction model, and QRS duration can be used for the assessment of antiarrhythmic action of melatonin.

Human Muse cells reduce myocardial infarct size and improve cardiac function without causing arrythmias in a swine model of acute myocardial infarction.

We recently reported that multilineage-differentiating stress enduring (Muse) cells intravenously administered after acute myocardial infarction (AMI), selectively engrafted to the infarct area, spontaneously differentiated into cardiomyocytes and vessels, reduced the infarct size, improved the left ventricular (LV) function and remodeling in rabbits. We aimed to clarify the efficiency of Muse cells in a larger animal AMI model of mini-pigs using a semi-clinical grade human Muse cell product. Mini-pigs underwent 30 min of coronary artery occlusion followed by 2 weeks of reperfusion. Semi-clinical grade human Muse cell product (1x107, Muse group, n = 5) or saline (Vehicle group, n = 7) were intravenously administered at 24 h after reperfusion. The infarct size, LV function and remodeling were evaluated by echocardiography. Arrhythmias were evaluated by an implantable loop recorder. The infarct size was significantly smaller in the Muse group (10.5±3.3%) than in the Vehicle group (21.0±2.0%). Both the LV ejection fraction and fractional shortening were significantly greater in the Muse group than in the Vehicle group. The LV end-systolic and end-diastolic dimensions were significantly smaller in the Muse group than in the Vehicle group. Human Muse cells homed into the infarct border area and expressed cardiac troponin I and vascular endothelial CD31. No arrhythmias and no blood test abnormality were observed. Muse cell product might be promising for AMI therapy based on the efficiency and safety in a mini-pig AMI.

Cardiac contractile dysfunction, during and following ischaemia, is attenuated by low-dose dietary fish oil in rats.

Supplementing animal diets with high-dose fish oil, rich in long chain omega-3 (ω-3) docosahexaenoic acid (DHA), enhances cardiac contractile efficiency and attenuates dysfunction, attributable to ischaemia. However, it remains unclear whether smaller doses, equivalent to what is achievable via regular fish consumption in the human diet, offer similar protection. Male Sprague-Dawley (12-15w) rats were fed isoenergetic diets (ad libitum) containing 10% fat by weight (22% energy) for 4-5w. Control diet (CON) contained 5.5% beef tallow; 2.5% ω-6 sunflower seed oil; 2% olive oil. Fish oil diets included high-DHA tuna oil exchanged for olive oil to provide 0.32% (FO1; human equivalent EPA + DHA 570mg/d) or 1.25% (FO2; equivalent EPA + DHA 2.3g/d) wt/wt dose of fish oil. Anaesthetised rats (pentobarbital: 60mg/kg i.p.) were subjected to 45min coronary artery occlusion then reperfusion in vivo as a whole animal model of regional myocardial ischaemia, with left ventricular haemodynamic function measured by conductance catheter. Ischaemia-induced reductions in rate pressure product recovered faster in the FO2 group and post-ischaemic left ventricular pressure-volume loop integrity (shifted downwards and right in CON) was partially protected in both fish oil groups. Ischaemia-induced contractile dysfunction in rats is limited from fish oil doses equivalent to regular consumption of fish in the human diet. These observations highlight plausible and clinically relevant physiological changes that rationalise nutritional conditioning of the heart with DHA for on-going cardioprotection.

Sevoflurane as opposed to propofol anesthesia preserves mitochondrial function and alleviates myocardial ischemia/reperfusion injury

BackgroundPharmacological interventions reducing myocardial ischemia and reperfusion (I/R) injury include the administration of anesthetics. Both sevoflurane as well as propofol have been shown to elicit cardiac protection via distinct molecular mechanisms. We investigated the hypothesis that sevoflurane in contrary to propofol anesthesia elicits cardiac protection against I/R-injury via mitochondrial mechanisms of disease. MethodsMale New Zealand white rabbits (n = 42) were subjected 30 min of coronary artery occlusion followed by 3 h of reperfusion. After induction with pentobarbital, the animals either received sevoflurane or propofol to maintain general anesthesia. Infarct size was determined gravimetrically after triphenyltetrazolium chlorid-staining. Cardiac mitochondria were isolated and mitochondrial oxygen consumption was measured using a Clark electrode. Mitochondrial respiratory chain complex activities (I-IV) were analyzed utilizing specific assays. Data are mean ± SD. ResultsSevoflurane anesthesia significantly decreased the resulting myocardial infarct size compared to propofol anesthesia (p = 0.0275 vs. propofol). Mitochondria from animals receiving propofol anesthesia showed a significantly reduced mitochondrial respiratory control ratio (p = 0.01909 vs. sham) and impaired activities of respiratory complex I (p = 0.0147 vs. sham; p < 0.01 vs. sevoflurane) as well as respiratory complex IV (p = 0.0181 vs. sham). Mitochondrial dysfunction was absent in sevoflurane anesthesized animals. Furthermore, a significantly higher portion of complex I was found to be in its deactive form during I/R-injury in animals receiving sevoflurane anesthesia (p = 0.0123 vs. propofol). ConclusionsSevoflurane as opposed to propofol anesthesia preserved mitochondrial respiration and elicited cardiac protection against I/R-injury.

Cardioprotective effect of sonic hedgehog ligand in pig models of ischemia reperfusion.

Sonic hedgehog (SHH) signaling pathway is involved in embryonic tissue patterning and development. Our previous work identified, in small rodent model of ischemia reperfusion, SHH as a specific efficient tool to reduce infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. The goal of the present study was to provide a proof of concept of the cardioprotective effect of SHH ligand in a porcine model of acute ischemia.Methods: The antiarrhythmic effect of SHH, either by a recombinant peptide (N-SHH) or shed membrane microparticles harboring SHH ligand (MPsSHH+), was evaluated in a first set of pigs following a short (25 min) coronary artery occlusion (CAO) followed by 24 hours-reperfusion (CAR) (Protocol A). The infarct-limiting effect was evaluated on a second set of pigs with 40 min of coronary artery occlusion followed by 24 hours reperfusion (Protocol B). Electrocardiogram (ECG) was recorded and arrhythmia's scores were evaluated. Area at risk and myocardial infarct size were quantified.Results: In protocol A, administration of N-SHH 15 min. after the onset of coronary occlusion significantly reduced the occurrence of ventricular fibrillation compared to control group. Evaluation of arrhythmic score showed that N-SHH treatment significantly reduced the overall occurrence of arrhythmias. In protocol B, massive infarction was observed in control animals. Either N-SHH or MPsSHH+ treatment reduced significantly the infarct size with a concomitant increase of salvaged area. The reduction in infarct size was both accompanied by a significant decrease in systemic biomarkers of myocardial injury, i.e., cardiac troponin I and fatty acid-binding protein and an increase of eNOS activation.Conclusions: We show for the first time in a large mammalian model that the activation of the SHH pathway by N-SHH or MPsSHH+ offers a potent protection of the heart to ischemia-reperfusion by preventing the reperfusion arrhythmias, reducing the infarct area and the circulating levels of biomarkers for myocardial injury. These data open up potentially theranostic prospects for patients suffering from myocardial infarction to prevent the occurrence of arrhythmias and reduce myocardial tissue damage.

A Novel Drug to Reduce Myocardial Infarct Size, Even When Administered After Coronary Artery Reperfusion

Despite the thousands of studies on drug therapy designed to reduce myocardial infarct size (IS), almost none have been successfully translated to the clinics. A major reason for failure is that many of these drugs are only effective when administered before coronary artery reperfusion (CAR), which limits their utility in the clinical setting, where CAR must be instituted immediately in patients after the myocardial infarction (MI) diagnosis has been made, not leaving time for drug therapy. It is also important that the drug does not increase myocardial oxygen consumption. In our prior work on the adenylyl cyclase type 5 (AC5) knock out (KO) mouse model, we found that it enhanced longevity, protected against diabetes and obesity, improved exercise performance and reduced IS after coronary artery occlusion (CAO) and CAR. Based on these studies, and since it is difficult to delete genes in humans, we developed a novel pharmacological candidate to inhibit AC5, named C90. C90 reduced AC activity and cAMP in response to forskolin more than vehicle, (12.9±1.9 vs. 14.2 ± 3.1 pmol/mg/min). However, in AC5 KO C90 no longer reduced cAMP in response to forskolin, indicating its selectivity for AC5 inhibition. The goal of this investigation was to examine the new AC5 inhibitor, C90, and compare its efficacy in reducing IS when the drug was delivered after CAR with that of adenosine, the gold standard for ischemic cardioprotection. Wild type (WT) mice were subjected to 30 min CAO followed by 24 hours CAR. C90 or vehicle was then administered at 5 min after CAR by intravenous (IV) administration at doses of 0.01, 0.03, 0.06, 0.6, and 1.2 mg/kg. The area at risk was similar in all groups, determined by triphenyltetrazolium chloride (TTC) staining. IS was reduced in a dose‐response manner by C90. IS was reduced by 32% at a dose of 0.06 mg/kg of C90, whereas the same dose of adenosine reduced IS similarly when administered before CAR but failed to reduce IS when administered after CAR. C90 further reduced IS by 64% at a dose of 0.6 mg/kg and by 71% at a dose of 1.2 mg/kg. When C90 was delivered at 10 min after CAR, it still IS by 50% and 53% at doses of 0.6 mg/kg and 1.2 mg/kg, respectively. In addition, LV ejection fraction (LV EF) was also examined at 4 weeks after myocardial infarction in mice, and was preserved in the C90 group (LVEF = 65±1.6%) vs. the vehicle group (LVEF = 52±2.9%). C90 also reduced IS by 49% in the Watanabe rabbit, model of atherosclerosis. Thus, C90, an inhibitor of AC5, is a novel target for clinical development, since it reduces myocardial IS when delivered after reperfusion, a key feature for patients coming to the emergency room, where reperfusion must be carried out before additional therapy. An additional advantageous feature of C90 and inhibition of AC5, is that it reduces beta‐adrenergic receptor signaling and myocardial oxygen consumption, which are important for patients with heart disease.Support or Funding InformationNIH R21AG053514; NIH R44 HL142390; NIH R44HL112512This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardioprotection of benzolamide in a regional ischemia model: Role of eNOS/NO

BackgroundRecent studies from our laboratory show the cardioprotective action of benzolamide (BZ, carbonic anhydrase inhibitor) against ischemia-reperfusion injury. However, the mechanisms involved have not been fully elucidated. ObjectiveTo examine the participation of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) in the effects of BZ in a model of regional ischemia. MethodsIsolated rat hearts perfused by Langendorff technique were submitted to 40 min of coronary artery occlusion followed by 60 min of reperfusion (IC). Other hearts received BZ during the first 10 min of reperfusion in absence or presence of L-NAME, NOS inhibitor. The infarct size (IS) and the post-ischemic recovery of myocardial function were measured. Oxidative/nitrosative damage were assessed by reduced glutathione (GSH) content, thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine levels. The expression of phosphorylated forms of Akt, p38MAPK and eNOS, and the concentration of inducible nitric oxide synthase (iNOS) were also determined. ResultsBZ significantly decreased IS (6.2 ± 0.5% vs. 34 ± 4%), improved post-ischemic contractility, preserved GSH levels and diminished TBARS and 3-nitrotyrosine. In IC hearts, P-Akt, P-p38MAPK and P-eNOS decreased and iNOS increased. After BZ addition the levels of P-kinases and P-eNOS increased and iNOS decreased. Except for P-Akt, P-p38MAPK and iNOS, the effects of BZ were abolished by L-NAME. ConclusionsOur data demonstrate that the treatment with BZ at the onset of reperfusion was effective to reduce cell death, contractile dysfunction and oxidative/nitrosative damage produced by coronary artery occlusion. These BZ-mediated beneficial actions appear mediated by eNOS/NO-dependent pathways.

Classic Papers Revisited: An Early Study of Cardioprotection by Volatile Anesthetics

Abstract Recovery of Contractile Function of Stunned Myocardium in Chronically Instrumented Dogs Is Enhanced by Halothane or Isoflurane. By Warltier DC, al-Wathiqui MH, Kampine JP, and Schmeling WT. Anesthesiology 1988; 69:552–65. Reprinted with permission. Following brief periods (5–15 min) of total coronary artery occlusion and subsequent reperfusion, despite an absence of tissue necrosis, a decrement in contractile function of the postischemic myocardium may nevertheless be present for prolonged periods. This has been termed “stunned” myocardium to differentiate the condition from ischemia or infarction. Because the influence of volatile anesthetics on the recovery of postischemic, reperfused myocardium has yet to be studied, the purpose of this investigation was to compare the effects of halothane and isoflurane on systemic and regional hemodynamics following a brief coronary artery occlusion and reperfusion. Nine groups comprising 79 experiments were completed in 42 chronically instrumented dogs. In awake, unsedated dogs a 15-min coronary artery occlusion resulted in paradoxical systolic lengthening in the ischemic zone. Following reperfusion active systolic shortening slowly returned toward control levels but remained approximately 50% depressed from control at 5 h. In contrast, dogs anesthetized with halothane or isoflurane (2% inspired concentration) demonstrated complete recovery of function 3–5 h following reperfusion. Because the anesthetics directly depressed contractile function, additional experiments were conducted in which a 15-minute coronary artery occlusion was produced during volatile anesthesia; however, each animal was allowed to emerge from the anesthetized state at the onset of reperfusion. Similar results were obtained in these experiments, demonstrating total recovery of contractile function within 3–5 h following reperfusion. Thus, despite comparable degrees of contractile dysfunction during coronary artery occlusion in awake and anesthetized dogs, the present results demonstrate that halothane and isoflurane produce marked improvement in the recovery of segment function following a transient ischemic episode. Therefore, volatile anesthetics may attenuate postischemic left ventricular dysfunction occurring intraoperatively and enhance recovery of regional wall motion abnormalities during reperfusion.

Vascular endothelial growth factor regulation of endothelial nitric oxide synthase phosphorylation is involved in isoflurane cardiac preconditioning.

Previous studies indicate that nitric oxide derived from endothelial nitric oxide synthase (eNOS) serves as both trigger and mediator in anaesthetic cardiac preconditioning. The mechanisms underlying regulation of eNOS by volatile anaesthetics have not been fully understood. Therefore, this study examined the role of vascular endothelial growth factor (VEGF) in isoflurane cardiac preconditioning. Wistar rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Isoflurane given prior to ischaemia/reperfusion significantly decreased myocardial infarct size from 60 ± 1% in control to 40 ± 3% (n = 8 rats/group, P < 0.05). The beneficial effects of isoflurane were blocked by neutralizing antibody against VEGF (nVEGF). Coronary arterial endothelial cells (ECs) alone or together with cardiomyocytes (CMs) were subjected to hypoxia/reoxygenation injury. The expression of VEGF and eNOS was analysed by western blot, and nitric oxide was measured by ozone-based chemiluminescence. In co-cultured CMs and ECs, isoflurane administered before hypoxia/reoxygenation attenuated lactate dehydrogenase activity and increased the ratio of phosphorylated eNOS/eNOS and nitric oxide production. The protective effect of isoflurane on CMs was compromised by nVEGF and after VEGF in ECs was inhibited with hypoxia inducible factor-1α short hairpin RNA (shRNA). The negative effect of hypoxia inducible factor-1α shRNA was restored by recombinant VEGF. Isoflurane cardiac preconditioning is associated with VEGF regulation of phosphorylation of eNOS and nitric oxide production.

GYY4137 protects against myocardial ischemia/reperfusion injury via activation of the PHLPP-1/Akt/Nrf2 signaling pathway in diabetic mice

BackgroundThis study explores the protective effects of a hydrogen sulfide donor, morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate (GYY4137), in the hearts of diabetic mice that had been subjected to myocardial ischemia/reperfusion injury. Diabetes impairs the Akt pathway, in which the Akt protein is dephosphorylated and inactivated by PH domain leucine-rich repeat protein phosphatase-1 (PHLPP-1). However, the function of PHLPP-1 and molecular mechanism that underlies the cardiac protection exerted by GYY4137 remains unknown. MethodsDiabetic or nondiabetic mice were subjected to 45 min of coronary artery occlusion followed by 2 h of reperfusion. H9c2 cells were cultured with normal or high glucose and then subjected to 3 h of hypoxia followed by 6 h of reoxygenation. Pretreatment with GYY4137 was performed in a randomized manner before ischemia/reperfusion or hypoxia/reoxygenation. The infarct size, cardiomyocyte apoptosis, and oxidative stress were measured. Western blotting was conducted to elucidate the protective mechanism. ResultsDiabetic mice or H9c2 cells exposed to high glucose displayed a larger infarct size, more severe cardiomyocyte apoptosis, lower cell viability, and increased oxidative stress, which were associated with increased levels of PHLPP-1 and reduced levels of p-Akt and nuclear factor-erythroid-2-related factor 2 (Nrf2) protein expression. These changes were prevented/reversed by GYYG4137 pretreatment. At the cellular level, PHLPP-1 siRNA attenuated cellular injury, and this was associated with increased p-Akt and nuclear Nrf2 protein, whereas the decrement of Akt phosphorylation induced by LY294002 augmented cellular injury and decreased nuclear Nrf2. ConclusionsGYY4137 activates the PHLPP-1/Akt/Nrf2 pathway to protect against diabetic myocardial ischemia/reperfusion injury.

A TSPO ligand prevents mitochondrial sterol accumulation and dysfunction during myocardial ischemia-reperfusion in hypercholesterolemic rats

A major cause of cell death during myocardial ischemia-reperfusion is mitochondrial dysfunction. We previously showed that the reperfusion of an ischemic myocardium was associated with an accumulation of cholesterol into mitochondria and a concomitant strong generation of auto-oxidized oxysterols. The inhibition of mitochondrial accumulation of cholesterol abolished the formation of oxysterols and prevented mitochondrial injury at reperfusion. The aim of this study was to investigate the impact of hypercholesterolemia on sterol and oxysterol accumulation in rat cardiac cytosols and mitochondria and to analyse the effect of the translocator protein ligand 4′-chlorodiazepam on this accumulation and mitochondrial function. Hypercholesterolemic ZDF fa/fa rats or normocholesterolemic lean rats were submitted to 30min of coronary artery occlusion followed by 15min reperfusion where cardiac cytosols and mitochondria were isolated. Hypercholesterolemia increased the cellular cardiac concentrations of cholesterol, cholesterol precursors and oxysterols both in cytosol and mitochondria in non-ischemic conditions. It also amplified the accumulation of all these compounds in cardiac cells and the alteration of mitochondrial function with ischemia-reperfusion. Administration of 4′-chlorodiazepam to ZDF fa/fa rats had no effect on the enhancement of sterols and oxysterols observed in the cytosols but inhibited cholesterol transfer to the mitochondria. It also alleviated the mitochondrial accumulation of all the investigated sterols and oxysterols. This was associated with a restoration of oxidative phosphorylation and a prevention of mitochondrial transition pore opening.The inhibition of cholesterol accumulation with TSPO ligands represents an interesting strategy to protect the mitochondria during ischemia-reperfusion in hypercholesterolemic conditions.

DJ-1 overexpression restores ischaemic post-conditioning-mediated cardioprotection in diabetic rats: role of autophagy.

IPO (ischaemic post-conditioning) is a promising method of alleviating myocardial IR (ischaemia-reperfusion) injury; however, IPO-mediated cardioprotection is lost in diabetic hearts via mechanisms that remain largely unclear. We hypothesized that decreased cardiac expression of DJ-1, a positive modulator of autophagy, compromises the effectiveness of IPO-induced cardioprotection in diabetic rats. Diabetic rats subjected to myocardial IR (30 min of coronary artery occlusion followed by 120 min of reperfusion) exhibited more severe myocardial injury, less cardiac autophagy, lower DJ-1 expression and AMPK (adenosine monophosphate-activated protein kinase)/mTOR (mammalian target of rapamycin) pathway activity than non-diabetic rats. IPO significantly attenuated myocardial injury and up-regulated cardiac DJ-1 expression, AMPK/mTOR activity and autophagy in non-diabetic rats but not in diabetic rats. AAV9 (adeno-associated virus 9)-mediated cardiac DJ-1 overexpression as well as pretreatment with the autophagy inducer rapamycin restored IPO-induced cardioprotection in diabetic rats, an effect accompanied by AMPK/mTOR activation and autophagy up-regulation. Combining HPO (hypoxic post-conditioning) with DJ-1 overexpression markedly attenuated HR (hypoxia-reoxygenation) injury in H9c2 cells with high glucose (HG, 30 mM) exposure, accompanied by AMPK/mTOR signalling activation and autophagy up-regulation. The DJ-1 overexpression-mediated preservation of HPO-induced cardioprotection was completely inhibited by the AMPK inhibitor compound C (CC) and the autophagy inhibitor 3-MA (3-methyladenine). Thus, decreased cardiac DJ-1 expression, which results in impaired AMPK/mTOR signalling and decreased autophagy, could be a major mechanism underlying the loss of IPO-induced cardioprotection in diabetes.

Selective replacement of mitochondrial DNA increases the cardioprotective effect of chronic continuous hypoxia in spontaneously hypertensive rats.

Mitochondria play an essential role in improved cardiac ischaemic tolerance conferred by adaptation to chronic hypoxia. In the present study, we analysed the effects of continuous normobaric hypoxia (CNH) on mitochondrial functions, including the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening, and infarct size (IS) in hearts of spontaneously hypertensive rats (SHR) and the conplastic SHR-mtBN strain, characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain. Rats were adapted to CNH (10% O2, 3weeks) or kept at room air as normoxic controls. In the left ventricular mitochondria, respiration and cytochrome c oxidase (COX) activity were measured using an Oxygraph-2k and the sensitivity of MPTP opening was assessed spectrophotometrically as Ca2+-induced swelling. Myocardial infarction was analysed in anaesthetized open-chest rats subjected to 20min of coronary artery occlusion and 3h of reperfusion. The IS reached 68±3.0% and 65±5% of the area at risk in normoxic SHR and SHR-mtBN strains, respectively. CNH significantly decreased myocardial infarction to 46±3% in SHR. In hypoxic SHR-mtBN strain, IS reached 33±2% and was significantly smaller compared with hypoxic SHR. Mitochondria isolated from hypoxic hearts of both strains had increased detergent-stimulated COX activity and were less sensitive to MPTP opening. The maximum swelling rate was significantly lower in hypoxic SHR-mtBN strain compared with hypoxic SHR, and positively correlated with myocardial infarction in all experimental groups. In conclusion, the mitochondrial genome of SHR modulates the IS-limiting effect of adaptation to CNH by affecting mitochondrial energetics and MPTP sensitivity to opening.

Fundamental hemodynamic mechanisms mediating the response to myocardial ischemia in conscious paraplegic mice: cardiac output versus peripheral resistance.

Autonomic dysfunction, a relative sedentary lifestyle, a reduced muscle mass and increased adiposity leads to metabolic abnormalities that accelerate the development of coronary artery disease (CAD) in individuals living with spinal cord injury (SCI). An untoward cardiac incident is related to the degree of CAD, suggesting that the occurrence of a significant cardiac event is significantly higher for individuals with SCI. Thus, understanding the fundamental hemodynamic mechanisms mediating the response to myocardial ischemia has the potential to positively impact individuals and families living with SCI. Accordingly, we systematically investigated if thoracic level 5 spinal cord transection (T5X; paraplegia) alters the arterial blood pressure response to coronary artery occlusion and if the different arterial blood pressure responses to coronary artery occlusion between intact and paraplegic mice are mediated by changes in cardiac output and or systemic peripheral resistance and whether differences in cardiac output are caused by changes in heart rate and or stroke volume. To achieve this goal, the tolerance to 3 min of coronary artery occlusion was determined in conscious intact and paraplegic mice. Paraplegic mice had an impaired ability to maintain arterial blood pressure during coronary artery occlusion as arterial pressure fell to near lethal levels by 1.38 ± 0.64 min. The lower arterial pressure was mediated by a lower cardiac output as systemic peripheral resistance was elevated in paraplegic mice. The lower cardiac output was mediated by a reduced heart rate and stroke volume. These results indicate that in paraplegic mice, the arterial pressure response to coronary artery occlusion is hemodynamically mediated primarily by cardiac output which is determined by heart rate and stroke volume.

Delayed therapeutic hypothermia protects against the myocardial no-reflow phenomenon independently of myocardial infarct size in a rat ischemia/reperfusion model

BackgroundAdjunctive therapies, given in addition to reperfusion to reduce myocardial infarct size, have been disappointing based on clinical trials. New therapeutic targets independent of infarct size modification are needed. The no-reflow phenomenon occurs commonly after the infarct-related coronary artery is opened and predicts poor clinical outcome. We investigated the effects of a single application of delayed (post-reperfusion) therapeutic hypothermia (TH) in a rat model of coronary artery occlusion/reperfusion. MethodsRats were subjected to 60min of coronary artery occlusion followed by 3h of reperfusion. Rats were divided into normothermic (n=5) and TH (n=5) groups. In the TH, hypothermia was initiated at 1min after coronary artery reperfusion by pumping room-temperature (22°C) saline into and out of the thoracic cavity for 1h. This decreased intrathoracic temperature to around 26°C within 12min. At 3h after reperfusion, hearts were excised for infarct size and no-reflow zone measurement. ResultsIschemic risk area and infarct size were similar between the 2 groups. No-reflow area (expressed as % of risk area) was significantly reduced in TH group (18.0±4.4%) compared with normothermic group (39.5±2.9%,P=0.005). When expressed as % of necrotic area, no-reflow area was reduced by more than half in TH group (25.5±6.4%) versus innormothermic group (54.4±5.3%,P=0.01). ConclusionsIn this preliminary study, hypothermia initiated after reperfusion following 60min of coronary artery occlusion had no effect on infarct size yet substantially reduced the extent of no-reflow.

Remote Limb Ischaemic Postconditioning Protects Against Myocardial Ischaemia/Reperfusion Injury in Mice: Activation of JAK/STAT3-Mediated Nrf2-Antioxidant Signalling

Background: This study aimed to evaluate the protective effect and mechanisms of remote limb ischaemic postconditioning (RIPostC) against myocardial ischaemia/reperfusion (IR) injury. Methods: Male mice underwent 45 min of coronary artery occlusion followed by 2 h of reperfusion. RIPostC was achieved by three cycles of 5 min of ischaemia and 5 min of reperfusion in the left hind limb at the start of the reperfusion period. After 2 h of cardiac reperfusion, myocardial infarct size, cardiac enzyme release, apoptosis and oxidative stress were assessed. Protein expression and phosphorylation were measured by Western blotting. Results: RIPostC significantly decreased cardiac IR injury, as reflected by reduced infarct size and cellular apoptosis (22.9 ± 3.3% vs 40.9 ± 6.2% and 13.4% ± 3.1% vs 26.2% ± 3.1%, respectively, both P < 0.01) as well as plasma creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) release (21.97 ± 4.08 vs 35.86 ± 2.91 ng/ml and 6.17 ± 0.58 vs 8.37 ± 0.89 U/ml, respectively, both P < 0.01) compared with the IR group. RIPostC significantly increased the phosphorylation of myocardial STAT3, Akt and eNOS (P < 0.01). In addition, RIPostC elevated the nuclear translocation of Nrf2 and the expression of HO-1 and reduced myocardial oxidative stress (P < 0.05). Interestingly, pretreatment with the JAK/STAT3 inhibitor AG490 blocked the cardioprotective effect of RIPostC accompanied by decreased phosphorylation of myocardial STAT3, Akt and eNOS (P < 0.05), decreased nuclear translocation of Nrf2 and expression of HO-1, as well as increased oxidative stress (P < 0.05). Conclusion: RIPostC attenuates apoptosis and protects against myocardial IR injury, possibly through the activation of JAK/STAT3-mediated Nrf2-antioxidant signalling.

Abstract 12589: Delayed Therapeutic Hypothermia Protects Against the Myocardial No-reflow Phenomenon Independent of Infarct Size in a Rat Ischemia/reperfusion Model

Background: Adjunctive therapy in addition to reperfusion to reduce myocardial infarct size has been disappointing based on clinical trials. New therapeutic targets independent of infarct size modification are needed. No-reflow predicts poor clinical outcome. In this study, we investigate the effects of a single application of delayed (post-reperfusion) therapeutic hypothermia (TH) on no-reflow in rats. Methods: Rats were subjected to 60 min of coronary artery occlusion followed by 3 hours of reperfusion. Rats were divided into normothermic (n=5) and TH (n=5) groups. In the TH, hypothermia was initiated at 1 minute after coronary artery reperfusion by pumping room-temperature (22 °C) saline into and out of the thoracic cavity for 1 hour. This decreased intra-thoracic temperature to around 27 °C within a few minutes. At 3 hours after reperfusion, hearts were excised for infarct size (tetrazolium staining) and no-reflow zone (Thioflavin S technique) measurement. Results: Ischemic risk area (expressed as % of LV area, assessed by blue dye technique) was 52.4 ± 2.9% in normothermic group and 44.8±3.7% in TH group (p=0.15). Infarct size (expressed as % of risk area) was similar between normothermic (73.8 ±3.8% ) and TH (72.0± 4.0%, p = 0.76) groups. No-reflow area (expressed as % of risk area) was significantly reduced in TH group (18.0 ± 4.4%) compared with normothermic group (39.5 ± 2.9%, P = 0.005). When expressed as % of necrotic area, no-reflow area was reduced by more than half in TH group (25.5 ± 6.4%) versus in normothermic group (54.4 ± 5.3%, P = 0.01). Conclusions: A single delayed TH markedly diminishes reperfusion induced microvascular damage and no-reflow even when initiated after reperfusion, and independent of infarct size.