Min Of Global Ischemia Research Articles

Discussion of the best time to use dobutamine after myocardium ischemia/reperfusion

Objective Using isolated rat heart ischemia/reperfusion injury(I/RI) model to compare the effects of dobutamine giving at different time points after reperfusion on cardiac function and myocardial injury and to explore the best time to use dobutamine after myocardium ischemia/reperfusion(I/R). Methods Thirty-six male SD rats, establishing isolated heart I/RI model in Langendorff perfusion apparatus with 15 min equilibrium, 30 min global ischemia and followed by 60 min reperfusion, were randomly divided into four groups(n=9). Different treatment was taken in reperfusion period. Group I/R: reperfused with Kreb's-Henseleit(K-H) solution, dobutamine group one(group D1): received dobutamine for 30 min after 5 min of reperfusion, other time perfused with K-H solution. Dobutamine group two (group D2): given dobutamine for 30 min after 15 min of reperfusion, other time perfused with K-H solution. Dobutamine group three(group D3): administrated dobutamine for 30 min after 25 min of reperfusion, other time perfused with K-H solution. Dobutamine infusion dose was 10 μg·kg-1·min-1. Heart function indexes of each group at the end of balance reperfusion(T0) and 10 min (T1), 20 min(T2), 30 min(T3), 60 min(T4) of reperfusion: HR, left ventricular end diastolic pressure(LVEDP), left ventricular developed pressure(LVDP), the maximum rate of left ventricular pressure change(±dp/dtmax) and coronary flow(CF) were recorded. The coronary flow at T0-T4 time points was used to measure the activity of lactate dehydrogenase (LDH) and creatine kinase(CK) according to the test methods attached to LDH and CK test kit. Myocardial infarct size(MIS) were measured with 2,3,5-triphenyl tetrazolium chloride (TTC) staining method. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2a) and ryanodine receptors(RyR2) expression were determined by Western bloting. Results After giving dobutamine, HR, LVEDP, LDH and CK activity and MIS of group D1 were higher than those in group I/R(P 0.05). SERCA2a expression between the above groups was no significant difference. While RyR2 expression was higher in groups with dobutamin compared to group I/R. There was no significant difference in above data between group D2 and D3 after using dobutamine. Conclusions Giving dobutamine after 15 min of myocardial I/R in rats is superior to the other time points. Using dobutamine at this time point can timely and effectively improve HR, increase CF, improve myocardial contractile function, and does not aggravate myocardial injury. Key words: Myocardial; Ischemia/reperfusion injury; Dobutamine

Effect of sevoflurane postconditioning on mitochondrial connexin 43 during myocardial ischemia-reperfusion in rats and the role of mito-KATP channels: an in vitro experiment

Objective To investigate the effect of sevoflurane postconditioning on mitochondrial connexin 43 (Cx43) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts and the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in it. Methods Forty-five adult male Sprague-Dawley rats, weighing 200-250 g, were used in the study.Their hearts were excised and retrogradely perfused with K-H solution in a Langendorff apparatus.The 45 isolated hearts were assigned into 5 groups (n=9 each) using a random number table: control group (group C), I/R group, sevoflurane postconditioning group (group Sev), and sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD, a specific mito-KATP channel blocker) group (group Sev+ 5-HD) and I/R plus 5-HD group (group I/R+ 5-HD). The hearts were subjected to 20 min of global ischemia followed by 90 min of reperfusion to establish the model of myocardial I/R injury.From the beginning of reperfusion, the hearts were perfused with K-H solution saturated with 3% sevoflurane for 15 min in group Sev, with K-H solution saturated with 3% sevoflurane and containing 100 μmol/L 5-HD in group Sev+ 5-HD, and with K-H solution containing 100 μmol/L 5-HD in group 5-HD.The heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) and the maximum rate of increase and decrease of ventricular pressure (±dp/dtmax) were recorded at the end of equilibration (T1) and 30 and 60 min of reperfusion (T2, 3). At 90 min of reperfusion, the myocardial infarct size was measured by TTC staining, and the expression of total Cx43 (tCx43) and phosphorylated Cx43 (p-Cx43) in mitochondria was determined by Western blot analysis.The percentage of myocardial infarct size and p-Cx43/tCx43 ratio were calculated. Results Compared with group C, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, and the percentage of myocardial infarct size was increased in the other 4 groups, the expression of mitochondrial tCx43 and p-Cx43 was significantly down-regulated in I/R, Sev+ 5-HD and 5-HD groups (P 0.05). Compared with group I/R, the HR, LVDP and ±dp/dtmax were significantly increased, and the LVEDP was decreased at T2, 3, the percentage of myocardial infarct size was decreased, and the expression of mitochondrial tCx43 and p-Cx43 was up-regulated in group Sev (P 0.05). Compared with group Sev, the HR, LVDP and ±dp/dtmax were significantly decreased, and the LVEDP was increased at T2, 3, the percentage of myocardial infarct size was increased, and the expression of mitochondrial tCx43 and p-Cx43 was down-regulated in group Sev+ 5-HD (P 0.05). Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R injury may be related to induction of mito-KATP channel opening and up-regulation of the expression of mitochondrial Cx43 in cardiomyocytes of rats. Key words: Anesthetics, inhalation; Ischemic postconditioning; Myocardial reperfusion injury; Mitochondria; Connexin 43; KATP channels

Cardioprotective Effects of Mitochondria-Targeted Peptide SBT-20 in two Different Models of Rat Ischemia/Reperfusion.

Dysfunctional mitochondria are considered to be the major source of intracellular reactive oxygen species and play a central role in the pathophysiology of myocardial ischemia/reperfusion. This study sought to determine effects of mitochondria-targeted cytoprotective peptide SBT-20 on myocardial infarct size in two different models of ischemia/reperfusion. For in vivo studies, anesthetized Sprague Dawley rats were subjected to 30min of coronary artery occlusion followed by 3h of reperfusion. Rats received saline (control), low dose SBT-20 (0.3mg/kg/h) or high dose SBT-20 (3mg/kg/h) treatment (n=15 rats in each group). Saline or SBT-20 were delivered into the jugular vein starting 5min after coronary artery occlusion and were continued for one hour post coronary artery reperfusion. Body temperature, heart rate and blood pressure were monitored during the procedure. At the end of 3h reperfusion, the ischemic risk area, no-reflow area, and infarct size were measured. In separate in vitro studies, isolated rat hearts were exposed to 20min global ischemia, followed by SBT-20 administration (1μM) or no SBT-20 (control) throughout the 2h reperfusion. In vitro studies were conducted in cells and heart mitochondria to ascertain the mitochondrial effects of SBT-20 on mitochondrial respiration and reactive oxygen species production. In the in vivo study, the ischemic risk areas (as a percentage of the left ventricle) were similar among the saline (49.5±2.3%), low dose SBT-20 (48.6±2.1%), and high dose SBT-20 groups (48.7±3.0%). Treatment with SBT-20 significantly reduced infarct size ( as a percentage of risk area) in low dose (62.1±4.4%) and high dose (64.0±4.9%) compared with saline treatment (77.6±2.6%, p=0.001 for both doses). There was no difference in infarct size between low and high dose SBT-20 treatment. The no-reflow areas (as a percentage of the risk area) were comparable among the saline (23.9±1.7%), low dose SBT-20 (23.7±2.8%), and high dose groups (25.0±2.1%). Body temperature, heart rate and blood pressure were comparable among the 3 groups at baseline, during ischemia, and at the end of 3h of reperfusion. In the in vitro study, infarct size was reduced from 43.3±2.6% in control group (n=11) to 17.2±2.8% in the SBT-20 treatment group (n=5, p<0.05). There were no benefits of SBT-20 on recovery of left ventricular developed pressure, coronary flow, or maximal rates of contraction/relaxation. In cell studies, treatment with SBT-20 significantly improved maximal mitochondrial respiration in response to an H2O2 challenge. In isolated mitochondria, reactive oxygen species production was significantly blunted following treatment with SBT-20. In summary, SBT-20 significantly reduced infarct size in two different models of myocardial injury, but did not affect hemodynamics or no-reflow area in rat heart. The reduction in injury is postulated to involve stabilization of mitochondrial function and reduced mitochondrial production of ROS.

P15 G-protein coupled receptor-55 plays a cardioprotective role in the response to myocardial ischemia/reperfusion injury in the setting of obesity

Activation of G-protein coupled receptor 55 (GPR55) by endogenous ligand lysophosphatidylinositol (LPI) stimulates numerous pathways, including intracellular calcium release and regulation of gene expression via ERK1/2 MAP kinase activation. The GPR55/LPI system is upregulated in the state of human obesity, as shown by increased adipose tissue mRNA and protein levels of GPR55 alongside elevated plasma concentrations of LPI. Our group has recently shown that exogenous administration of LPI prior to ischaemia/reperfusion (I/R) results in an increased infarct size through GPR55 activation of RhoA-ROCK, therefore we hypothesise that, obese individuals may suffer a worse outcome following myocardial infarction due to GPR55/LPI upregulation. In the present study, male GPR55-/- and C57Bl/6J mice were fed either a standard chow (SD) or high fat diet (HFD) for 12 weeks, after which mice were anaesthetised and the heart removed and retrogradely perfused using a Langendorff setup. Hearts were stabilised for 15 min followed by 30 min global ischaemia and 30 min reperfusion; hearts were then stained using triphenyltetrazolium chloride to determine infarct size. Hearts from wild type mice fed a HFD exhibited significantly smaller myocardial infarcts induced by I/R compared to SD fed controls. However, this effect was abrogated in the GPR55-/- mice, suggesting that rather than suffering a worse outcome as hypothesised, endogenous (unlike exogenous) LPI may play a protective role through GPR55. To gain insight into possible mechanisms underlying this protection, studies are currently underway to measure tissue levels of LPI and markers of the RISK pathway activation in infarcted myocardial tissue.

Effect of oleuropein on myocardial dysfunction and oxidative stress induced by ischemic-reperfusion injury in isolated rat heart

BackgroundStudies have reported antioxidant effect of oleuropein in isolated rat heart. ObjectiveThis study was conducted to investigate whether perfusion of isolated rat heart with oleuropein, before induction of ischemia or at the onset of reperfusion, had any effect on the hemodynamic parameters, infarct size and biochemical factors following ischemic – reperfusion injury. Materials and methodsForty-eight male Wistar rats were divided into 6 groups: the control groups (Con-P and Con-T groups), O10-P and O50-P groups perfused with 10 and 50 μg/g heart oleuropein 5 min before the induction of ischemia and O10-T and O50-T groups perfused with 10 and 50 μg/g heart oleuropein at the beginning of the reperfusion, respectively. All hearts were subjected to 30 min global ischemia and 90 min reperfusion. Hemodynamic parameters were monitored throughout the experiment. The creatine kinase (CK) and malondialdehyde (MDA) level of coronary outflow were assayed and the infarct size measured at the end of reperfusion. ResultsWe found hemodynamic parameters namely heart rate, left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), ±dp/dt and coronary outflow significantly improved in all groups that received oleuropein compared to the control groups. Also, the infarct size was smaller and the coronary outflow levels of CK and MDA were lower in the oleuropein groups compared to the control groups. ConclusionsThe findings suggest that perfusion of isolated rat heart with oleuropein would lead to improved myocardial dysfunction following ischemic-reperfusion injury. Our findings confirm the antioxidant potential of oleuropein.

Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

BackgroundEstrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.MethodsWe randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.ResultsCompared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.ConclusionsActivation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0104-8) contains supplementary material, which is available to authorized users.

Cardiomyocyte mitochondria as targets of humoral factors released by remote ischemic preconditioning.

IntroductionRemote ischemic preconditioning (RIPC) reduces myocardial infarct size, and protection can be transferred with plasma to other individuals, even across species. Mitochondria are the end-effectors of cardioprotection by local ischemic conditioning maneuvers. We have now analyzed mitochondrial function in response to RIPC.Material and methodsPlasma from pigs undergoing placebo or RIPC (infarct size reduction by 67% in RIPC pigs compared to placebo) was transferred to isolated perfused rat hearts subjected to 30 min global ischemia followed by 120 min reperfusion for infarct size measurement. Additional experiments were terminated at 10 min reperfusion to isolate mitochondria for functional measurements. Effects of RIPC pig plasma were compared to local ischemic preconditioning (IPC) or to infusion of tumor necrosis factor α (TNF-α).ResultsIschemia/reperfusion (I/R) induced an infarct of 41 ±2% of total ventricular mass. Placebo pig plasma did not affect infarct size (38 ±1, p = 0.13). The RIPC pig plasma reduced infarct size (27 ±2, p < 0.001), as did IPC (20 ±1, p < 0.001) and TNF-α (28 ±2, p < 0.001). Associated with cardioprotection, reductions of mitochondrial adenosine diphosphate (ADP)-stimulated respiration, adenosine triphosphate (ATP) production and calcium retention capacity (CRC) by I/R and placebo pig plasma were prevented by RIPC pig plasma, as they were by IPC and TNF-α. Mitochondrial reactive oxygen species production (nmol H2O2/100 µg protein) induced by I/R (272 ±34) was comparable in response to placebo pig plasma (234 ±28, p = 0.37) and was reduced by RIPC pig plasma (83 ±15, p < 0.001) as well as by IPC (78 ±21, p < 0.001) and TNF-α (125 ±42, p = 0.002).ConclusionsIn rat myocardium, mitochondria are an intracellular target of protection induced by humoral factors retrieved from pigs undergoing RIPC.

Activation of CRHR1 receptors regulates social and depressive-like behaviors and expression of BDNF and TrkB in mesocorticolimbic regions following global cerebral ischemia

Increased HPA axis activation and CRH release characterize the brain's response to global cerebral ischemia. Recently, CRH via activation of CRH type 1 receptors (CRHR1) has been shown to regulate Brain Derived Neurotrophic Factor (BDNF) secretion and emotional behavior. The current study investigates the impact of CRHR1 blockade on BDNF/TrkB signaling expression in the mesolimbic circuitry, and social and depressive-like behavior following global ischemia. Adult male Wistar rats were injected with Antalarmin (2μg/μl) or a vehicle 30min prior to 10min global cerebral ischemia (4VO model) or sham occlusion. The Three Chamber Social Approach Test (SIT) assessed sociability and preference for social novelty, and the novelty suppressed feeding test (NSFT), forced swim test (FST), and sucrose preference test characterized anxiety and depression. Corticosterone levels and organ (thymus, seminal and adrenal glands) weights were determined as additional physiological indices of stress. Immunohistochemistry, Western blot and Rt-PCR were used to assess BDNF and TrkB receptor levels in subregions of the medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and ventral tegmental area (VTA) 30days post-ischemia. Our findings indicate reduced BDNF and TrkB protein and mRNA expression in the mPFC post-ischemia, while heightened levels were found in the NAc. Ischemia increased immobility in the FST and reduced sucrose preference and led to reduced latency to feed in the NSFT and heightened sociability and social novelty preference in the SIT. Antalarmin treatment normalized post-ischemic biochemical/behavioral changes. Our findings support lasting effects of CRHR1 activation on brain plasticity markers, likely playing a role in emotional impairments following cardio- or cerebro-vascular accidents.

NFATc3 is necessary for macrophage iNOS expression during myocardial ischemia and reperfusion

In the present study, we sought to examine whether inhibition NFATc3, improves cardiac function and reduces heart damage during IR. Hearts of male C57BL/J6 and NFATc3 knockout (KO) mice perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion. Our data show that NFATc3 regulates expression of inducible nitric oxide synthase (iNOS) in macrophages stimulated after I/R. In the NFATc3 knockout (KO) mice, left ventricular developed pressure, rate pressure product, contraction and relaxation rates and coronary flow significantly improved following reperfusion compared with C57BL/J6 mice.

Ex Vivo Treatment with a Polyphenol-Enriched Cocoa Extract Ameliorates Myocardial Infarct and Postischemic Mitochondrial Injury in Normotensive and Hypertensive Rats.

Our objective was to determine the effects of a polyphenol-enriched cocoa extract (PCE) on myocardial postischemic alterations in normotensive (Wistar rats, W) and spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 110 min of perfusion or 20 min stabilization, 30 min global ischemia, and 60 min reperfusion (R). Other hearts were treated with PCE at the onset of R. Infarct size, the reduced glutathione (GSH), and the expression of phospho-Akt, P-GSK-3β, and P-eNOS were assessed. In isolated mitochondria, the Ca(2+)-mediated response of mitochondrial permeability transition pore (mPTP), membrane potential (Δψm), and superoxide production were determined. PCE decreased infarct size, partly preserved GSH, increased the P-Akt, P-GSK-3β, and P-eNOS contents, improved mPTP response to Ca(2+), decreased the superoxide production, and restored Δψm. These data show that PCE decreases the cardiac postischemic damage in W rats and SHR and suggest that Akt/GSK-3β/eNOS dependent pathways are involved.

0132 : Oxidative stress and cardio-metabolic alterations induced by postnatal programming can be reversed in adulthood by a short-term moderate caloric restriction

Postnatal overfeeding (PNOF) in rodents induces early programming of cardio-metabolic risk. Our aim was to determine if a moderate diet restriction could restore cardio-metabolic alterations induced by PNOF. Immediately after birth, litters of C57BL/6 mice were either maintained at 9 (normal litter, NL), or reduced to 3 (small litter, SL) to induce PNOF. At weaning, all mice received a standard diet ad libitum (AL). At 6 month of age, half of the NL and SL mice were assigned to a moderate 20% calorie restriction (CR: NLCR, SLCR) for one month, while the other mice continued to eat AL (AL: NLAL, SLAL). Glucose and insulin tolerance tests, cardiac function (echocardiography), body composition (Echo-MRI), cardiac sensitivity to ischemia-reperfusion injury, mitochondrial function, reactive oxygen species (ROS) generation (EPR spectroscopy) and insulin signaling were assessed before and/or after one month of CR. Adult SL mice presented overweight, fat accumulation, hyperleptinemia, glucose intolerance, insulin resistance and decreased left ventricular ejection fraction (LVEF). After one month of moderate CR, body weight of SLCR was normalized to this of NLAL however their fat mass and leptinemia were not decreased. Glucose metabolism was improved and LVEF was increased In SLCR. After 30 min of global ischemia, hearts isolated from SLCR mice showed better recovery and smaller infarct size than this of others groups. CR increased the cardiac mitochondrial respiratory rate in SLCR mice whereas cardiac ROS production was significantly decreased in SLCR mice. Insulin signaling in heart was affected neither by PNOF nor by CR. Intriguingly, no difference was observed in NLCR mice for most of the parameters investigated. Our results confirmed the programming of early overfeeding on metabolic and cardiac function. A short-term moderate CR in not only normalized body weight in SL mice but also ameliorate the metabolic programming and reverse the cardiac dysfunction induced by PNOF. The author hereby declares no conflict of interest

Inhibition of SGLT1 abrogates preconditioning-induced cardioprotection against ischemia-reperfusion injury

BackgroundRecently, we reported Na+/glucose co-transporter (SGLT1) expression in mouse and human heart. We speculated that SGLT1 might play an important role in ischemic preconditioning-induced cardioprotection. Therefore, the present study was designed to find the role of SGLT1 in ischemic preconditioning-induced cardioprotection. MethodsHearts isolated from SD male rats were subjected to either ischemia-reperfusion injury (I/R) (15 min global ischemia followed by 20 min reperfusion) or ischemic preconditioning (IPC) (3 cycles of 2 min global ischemia separated by 3 min reperfusion) followed by I/R in presence and absence of phlorizin, an SGLT1 inhibitor. ResultsIPC increased membrane SGLT1 expression in rat heart as observed by immunoblotting and immunohistochemistry. Hearts from I/R group showed significant increase in oxidative stress levels and marked myocardial injury as compared to control. We also observed significant increase in apoptotic parameters in I/R heart, as measured by caspase-3 activity, TUNEL positive nuclei and gene expression analysis. Significant improvement in oxidative stress, apoptosis parameters and cardiac injury was observed in I/R hearts when subjected to IPC. However, all beneficial effects of preconditioning were lost when hearts were pre-treated with phlorizin. ConclusionPresent study indicated that inhibition of SGLT1 by phlorizin abrogated the beneficial effect of ischemic-preconditioning and for the first time, provides evidence that SGLT1 plays a crucial role in ischemic preconditioning-induced cardioprotection.

Cyclosporine-A mimicked the ischemic pre- and postconditioning-mediated cardioprotection in hypertensive rats: Role of PKCε

Our aim was to assess the action of cyclosporine-A (CsA) against reperfusion injury in spontaneously hypertensive rats (SHR) compared to the effects of ischemic pre- (IP) and postconditioning (IPC), examining the role played by PKCε. Isolated hearts were submitted to the following protocols: IC: 45min global ischemia (GI) and 1h reperfusion (R); IP: a cycle of 5min GI and 10min of R prior to 45min-GI; and IPC: three cycles of 30s-GI/30s-R at the start of R. Other hearts of the IC, IP and IPC groups received CsA (mitochondrial permeability transition pore inhibitor) or chelerythrine (Che, non-selective PKC inhibitor). Infarct size (IS) was assessed. TBARS and reduced glutathione (GSH) content — as parameters of oxidative damage, the expression of P-Akt, P-GSK-3β, P-PKCε and cytochrome c (Cyc) release — as an index of mitochondrial permeability and the response of isolated mitochondria to Ca2+ were also measured. IS similarly decreased in preconditioned, postconditioned and CsA treated heart showing the highest values in the combinations IP+CsA and IPC+CsA. TBARS decreased and GSH was partially preserved after all interventions. The content of P-Akt, P-GSK-3β and P-PKCε increased in cytosol and decreased in mitochondria after IP and IPC. In CsA treated hearts these enzymes increased in both fractions reaching the highest values. Cyc release was attenuated and the response of mitochondria to Ca2+ was improved by the interventions. The beneficial effects of IP and IPC were annulled when PKC was inhibited with Che. A PKCε/VDAC association was also detected. These data show that, in SHR, the CsA treatment mimicked and reinforced the cardioprotective action afforded by IP and IPC in which PKCε-mediated attenuation of mitochondrial permeability appears as the main mechanism involved.

Protective Role of Ramipril and Candesartan against Myocardial Ischemic Reperfusion Injury: A Biochemical and Transmission Electron Microscopical Study.

The present study was designed to investigate the role of combined administration of Ramipril and Candesartan against in vitro myocardial ischemic reperfusion injury in rat. Male Wistar albino rats were divided into five groups (n = 6) and treated with saline (10 mL/kg), Ramipril (2 mg/kg), Candesartan (1 mg/kg), and the combination of both drugs, respectively 24 h before induction of global ischemia (5 min of stabilization, 9 min of global ischemia, and 12 min of reflow). Combination of Ramipril and Candesartan when compared to the monotherapy significantly increased the levels of superoxide dismutase, reduced glutathione, catalase, and nitric oxide and decreased the levels of thiobarbituric acid reactive substances. In addition, the superior protective role of combination of Ramipril and Candesartan on ischemia induced myocardial damage was further confirmed by well preserved myocardial tissue architecture in light microscopy and transmission electron microscopy analysis studies. The combination was proved to be effective in salvaging the myocardial tissue against ischemic reperfusion injury when compared to the monotherapy of individual drugs and further investigations on protective mechanism of drugs by increasing the nitric oxide level at molecular levels are needed.

Abstract 18010: Temporal Kinetics of Remote Ischemic Preconditioning: Transfer of Protection With Plasma Dialysate From Healthy Volunteers to an Isolated Mouse Heart

Objective: We aimed to analyze the temporal kinetics of cardioprotection by humoral factor(s) in healthy volunteers undergoing remote ischemic preconditioning (RIPC), a maneuver known to protect against myocardial injury, using infarct size in an isolated mouse heart as read-out. Methods: Ten healthy volunteers underwent RIPC by 3 cycles of 5 min upper-limb blood pressure cuff inflation and 5 min deflation. Venous blood samples were obtained at baseline before RIPC and after 5 and 30 min, 1, 6 and 24 h and daily up to 7 days. Plasma was dialyzed against the 20-fold volume of Krebs-Henseleit buffer for 24 h (cut-off: 12-14 kDa). Isolated mouse hearts were mounted on a Langendorff apparatus and perfused with Krebs-Henseleit buffer at a pressure of 100 mmHg. Before undergoing 20 min global ischemia and 120 min reperfusion, hearts were perfused with dialysate for 15 min. Infarct size was assessed by TTC-staining. The cardioprotective potential of the plasma dialysate obtained 5 min after RIPC was further evaluated by decreasing its dilution (1:10) in those samples, which did not induce cardioprotection in a dilution of 1:20. Results: The cardioprotective effect of RIPC was present in all cases 30 min after RIPC, lasted for at least 6 days and vanished thereafter (see Figure). Plasma taken 5 min after RIPC induced cardioprotection in only 50 % of cases, when using a dialysate with a 1:20 dilution. Decreasing the dilution to 1:10 induced cardioprotection in all cases (Figure insert). Conclusion: A single RIPC-maneuver induces the release of (a) dialyzable, humoral factor(s), which reduce(s) infarct size no later than 5 min after RIPC and remain(s) operative for up to 6 days. These results imply that cardioprotection is at least in part effected by (a) factor(s) which is/are quickly released/activated, long-lasting and/or continuously produced for quite a while after RIPC.

Partial inhibition of the ubiquitin–proteasome system ameliorates cardiac dysfunction following ischemia–reperfusion in the presence of high glucose

BackgroundAcute hyperglycemia co-presenting with myocardial infarction (in diabetic and non-diabetic individuals) is often associated with a poor prognosis. Although acute hyperglycemia induces oxidative stress that can lead to dysregulation of the ubiquitin–proteasome system (UPS), it is unclear whether increased/decreased UPS is detrimental with ischemia–reperfusion under such conditions. As our earlier data implicated the UPS in cardiac damage, we hypothesized that its inhibition results in cardioprotection with ischemia–reperfusion performed under conditions that simulate acute hyperglycemia.MethodsEx vivo rat heart perfusions were performed with Krebs–Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min stabilization, followed by 20 min global ischemia and 60 min reperfusion ± 5 µM lactacystin and 10 µM MG-132, respectively. The UPS inhibitors were added during the first 20 min of the reperfusion phase and various cardiac functional parameters evaluated. In parallel experiments, infarct sizes were assessed following 20 min regional ischemia and 120 min reperfusion ± each of the respective UPS inhibitors (added during reperfusion). Heart tissues were collected and analyzed for markers of oxidative stress, UPS activation, inflammation and autophagy.ResultsThe proteasome inhibitor doses and treatment duration here employed resulted in partial UPS inhibition during the reperfusion phase. Both lactacystin and MG-132 administration resulted in cardioprotection in our experimental system, with MG-132 showing a greater effect. The proteasome inhibitors also enhanced cardiac superoxide dismutase protein levels (SOD1, SOD2), attenuated pro-inflammatory effects and caused an upregulation of autophagic markers.ConclusionsThis study established that partial proteasome inhibition elicits cardioprotection in hearts exposed to ischemia–reperfusion with acute simulated hyperglycemia. These data reveal that protease inhibition triggered three major protective effects, i.e. (a) enhancing myocardial anti-oxidant defenses, (b) attenuating inflammation, and (c) increasing the autophagic response. Thus the UPS emerges as a unique therapeutic target for the treatment of ischemic heart disease under such conditions.

Abstract 398: Histone Demethylase Kdm6a Contributes To X Chromosome Dependent Ischemia/reperfusion Injury Via Epigenetic Regulation

Introduction: Sex differences in susceptibility to ischemia/reperfusion (I/R) injury have been mostly attributed to sex hormones. Recently we examined the role of sex chromosomes in sex differences in myocardial I/R injury. We discovered that gonadectomized mice with two X chromosomes (XX or XXY) have ~50% larger infarct size after I/R injury, compared to mice with one X chromosome (XY or XO). Only few X genes escape X inactivation and are expressed higher in XX than XY individuals. Here we examined the role of “X escapee” histone demethylase Kdm6a which is important in cardiac development. Methods: Female mice with a heterozygous global knockout of Kdm6a (Kdm6a+/-) and with 2 copies of Kdm6a (Kdm6a+/+, regular WT) were used. Isolated mouse hearts were subjected to 30 min global normothermic ischemia followed by 60 min reperfusion. RNA-Seq analysis was performed by comparing gene expression in hearts of Kdm6a+/+ vs. Kdm6a+/- females at baseline before ischemia. We calculated an unbiased composite score of relevance in which the level of significance of the Kdm6a effect on expression (p value) was integrated with the size of the KDM6A effect on expression (fold change), and with the amount of H3K27me3 mark found on the genes in the heart based on online ChIP-Seq data. Two way ANOVA was used for statistical analysis. P&lt;0.05 was considered statistically significant. Values are expressed as mean± SE. Results: Kdm6a+/+ female mice had significantly lower heart functional recovery compared to their littermate Kdm6a+/- (LVDP: 46.7±9.8% vs. 79.8±3.5%; RPP: 44.1±10.5% vs. 76.2±8.5%, n=6-8 mice/group, p&lt;0.01). The integration of our RNA-Seq data using the hearts of female mice with 2 vs. 1 copy of Kdm6a (n=4 samples per group) with online datasets measuring the H3K27me3 mark, sex differences in expression in humans and mice, and involvement in ischemic heart failure, revealed carbonic anhydrase-3 (Car3) as the most interesting candidate (upregulated ~7 fold in the hearts of Kdm6a+/+ vs. Kdm6a+/- female mice) at baseline. Car3 encodes one isoform of carbonic anhydrase, involved in pH regulation, which is a critical part of I/R injury. Conclusion: Histone demethylase KDM6A contributes to X chromosome dependent I/R injury via epigenetic regulation

Tolerance to ischaemic injury in remodelled mouse hearts: less ischaemic glycogenolysis and preserved metabolic efficiency.

Post-infarction remodelled failing hearts have reduced metabolic efficiency. Paradoxically, they have increased tolerance to further ischaemic injury. This study was designed to investigate the metabolic mechanisms that may contribute to this phenomenon and to examine the relationship between ischaemic tolerance and metabolic efficiency during post-ischaemic reperfusion. Male C57BL/6 mice were subjected to coronary artery ligation (CAL) or SHAM surgery. After 4 weeks, in vivo mechanical function was assessed by echocardiography, and then isolated working hearts were perfused in this sequence: 45 min aerobic, 15 min global no-flow ischaemia, and 30 min aerobic reperfusion. Left ventricular (LV) function, metabolic rates, and metabolic efficiency were measured. Relative to SHAM, both in vivo and in vitro CAL hearts had depressed cardiac function under aerobic conditions (45 and 36%, respectively), but they had a greater recovery of LV function during post-ischaemic reperfusion (67 vs. 49%, P < 0.05). While metabolic efficiency (LV work per ATP produced) was 50% lower during reperfusion of SHAM hearts, metabolic efficiency in CAL hearts did not decrease. During ischaemia, glycogenolysis was 28% lower in CAL hearts, indicative of lower ischaemic proton production. There were no differences in mitochondrial abundance, calcium handling proteins, or key metabolic enzymes. Compared with SHAM, remodelled CAL hearts are more tolerant to ischaemic injury and undergo no further deterioration of metabolic efficiency during reperfusion. Less glycogen utilization in CAL hearts during ischaemia may contribute to increased ischaemic tolerance by limiting ischaemic proton production that may improve ion homeostasis during early reperfusion.

Acute inhibition of monoamine oxidase and ischemic preconditioning in isolated rat hearts: interference with postischemic functional recovery but no effect on infarct size reduction.

Monoamine oxidases (MAOs) have recently emerged as important mitochondrial sources of oxidative stress in the cardiovascular system. Generation of reactive oxygen species during the brief episodes of ischemic preconditioning (IPC) is responsible for the cardioprotection at reperfusion. The aim of this study was to assess the effects of two MAO inhibitors (clorgyline and pargyline) on the IPC-related protection in isolated rat hearts. Animals subjected to 30 min global ischemia and 120 min reperfusion were assigned to the following groups: (i) Control, no additional intervention; (ii) IPC, 3 cycles of 5 min ischemia and 5 min reperfusion before the index ischemia; (iii) IPC-clorgyline, IPC protocol bracketed for 5 min with clorgyline (50 μmol/L); (iv) IPC-pargyline, IPC protocol bracketed for 5 min with pargyline (0.5 mmol/L). The postischemic functional recovery was assessed by the left ventricular developed pressure (LVDP) and the indices of contractility (+dLVP/dt max) and relaxation (-dLVP/dt max). Infarct size (IS) was quantified by TTC staining. In both genders, IPC significantly improved functional recovery that was further enhanced in the presence of either clorgyline or pargyline. IS reduction was comparable among all the preconditioned groups, regardless of the presence of MAO inhibitors. In isolated rat hearts, acute inhibition of MAOs potentiates the IPC-induced postischemic functional recovery without interfering with the anti-necrotic protection.

TGF-β improves myocardial function and prevents apoptosis induced by anoxia-reoxygenation, through the reduction of endoplasmic reticulum stress.

Transforming growth factor-β (TGF-β) is known for its role in ventricular remodeling, inflammatory response, cell survival, and apoptosis. However, its role in improving myocardial function in rat hearts subjected to ischemia-reperfusion (I/R) and protecting against apoptosis induced in cardiomyocytes by anoxia-reoxygenation (A/R) has not been elucidated. This study investigated the protective effects and molecular mechanisms of TGF-β on myocardial function and cardiomyocyte apoptosis. We used TUNEL staining, we tested cell viability, and we measured mitochondrial membrane potential and levels of mitochondrial ROS after 6 h of simulated anoxia together with various durations of simulated reoxygenation in H9c2 cells. We further observed the contractile function in rat hearts after they were subjected to 30 min global ischemia and 180 min reperfusion. Pretreatment with TGF-β markedly inhibited apoptosis in H9c2 cells, as evidenced by increased cell viability and decreased numbers of TUNEL-positive cells, maintained mitochondrial membrane potential, and diminished mitochondrial production of reactive oxygen species (ROS). These changes were associated with the inhibition of endoplasmic reticulum (ER) stress-dependent markers of apoptosis (GRP78, CHOP, caspase-12, and JNK), and the modulation of the expression of Bcl2/Bax. Furthermore, TGF-β improved I/R-induced myocardial contractile dysfunction. All of these protective effects were concentration-dependent. Our results show that TGF-β prevents A/R-induced apoptosis of cardiomyocytes and improves myocardial function in rat hearts injured by I/R.