No-flow Ischemia Research Articles

Intraluminal oxygen can keep small bowel mucosa intact in a segmental ischemia model

Intestinal preservation for transplantation is accompanied by hypoperfusion with long periods of ischemia with total blood cessation and absolute withdrawal of oxygen leading to structural damage. The application of intraluminal oxygen has been successfully tested in small-animal series during storage and transport of the organ but have been so far clinically unrelatable. In this study, we tested whether a simple and clinically approachable method of intraluminal oxygen application could prevent ischemic damage in a large animal model, during warm ischemia time. We utilised a local no-flow ischemia model of the small intestine in pigs. A low-flow and high-pressure intraluminal oxygen deliverance system was applied in 6 pigs and 6 pigs served as a control group. Mucosal histopathology, hypoxia and barrier markers were evaluated after two hours of no-flow conditions, in both treatment and sham groups, and in healthy tissue. Macro- and microscopically, the luminal oxygen delivered treatment group showed preserved small bowel’s appearance, viability, and mucosal integrity. A gradual deterioration of histopathology and barrier markers and increase in hypoxia-inducible factor 1-α expression towards the sites most distant from the oxygen application was observed. Intraluminal low-flow, high oxygen delivery can preserve the intestinal mucosa during total ischemia of the small intestine. This finding can be incorporated in methods to overcome small bowel ischemia and improve intestinal preservation for transplantation.

Cardioprotective Effect against Ischemia-Reperfusion Injury of PAK-200, a Dihydropyridine Analog with an Inhibitory Effect on Cl- but Not Ca2+ Current.

We examined the effects of a dihydropyridine analog, PAK-200, on guinea pig myocardium during experimental ischemia and reperfusion. In isolated ventricular cardiomyocytes, PAK-200 (1 μM) had no effect on the basal peak inward and steady-state currents but inhibited the isoprenaline-induced time-independent Cl- current. In the right atria, PAK-200 had no effect on the beating rate and the chronotropic response to isoprenaline. In an ischemia-reperfusion model with coronary-perfused right ventricular tissue, a decrease in contractile force and a rise in tension were observed during a period of 30-min no-flow ischemia. Upon reperfusion, contractile force returned to less than 50% of preischemic values. PAK-200 had no effect on the decline in contractile force during the no-flow ischemia but reduced the rise in resting tension. PAK-200 significantly improved the recovery of contractile force after reperfusion to about 70% of the preischemic value. PAK-200 was also shown to attenuate the decrease in tissue ATP during ischemia. Treatment of ventricular myocytes with an ischemia-mimetic solution resulted in depolarization of the mitochondrial membrane potential and an increase in cytoplasmic and mitochondrial Ca2+ concentrations. PAK-200 significantly delayed these changes. Thus, PAK-200 inhibits the cAMP-activated chloride current in cardiac muscle and may have protective effects against ischemia-reperfusion injury through novel mechanisms.

Myocardial Recovery, Metabolism, and Structure after Cardiac Arrest with Cardioplexol.

Clinical studies indicate encouraging cardioprotective potential for Cardioplexol. Its cardioprotective capacities during 45 minutes of ischemia compared with pure no-flow ischemia or during 90 minutes of ischemia compared with Calafiore cardioplegia were investigated experimentally. Forty-four rat hearts were isolated and inserted into a blood-perfused pressure-controlled Langendorff apparatus. In a first step, cardiac arrest was induced by Cardioplexol or pure no-flow ischemia lasting 45 minutes. In a second step, cardiac arrest was induced by Cardioplexol or Calafiore cardioplegia lasting 90 minutes. For both experimental steps, cardiac function, metabolic parameters, and troponin I levels were evaluated during 90 minutes of reperfusion. At the end of reperfusion, hearts were fixed, and ultrastructural integrity was examined by electron microscopy. Step 1: after 90 minutes of reperfusion, hearts exposed to Cardioplexol had significantly higher left ventricular developed pressure (CP-45': 74%BL vs. no-flow-45': 45%BL; p = 0.046) and significantly better maximal left ventricular relaxation (CP-45': 84%BL vs. no-flow-45': 51%BL; p = 0.012). Oxygen consumption, lactate production, and troponin levels were similar in both groups. Step 2: left ventricular developed pressure was lower (22 vs. 48% of BL; p = 0.001) and coronary flow was lower (24 vs. 53% of BL; p = 0.002) when Cardioplexol was used compared with Calafiore cardioplegia. Troponin I levels were significantly higher under Cardioplexol (358.9 vs. 106.1 ng/mL; p = 0.016). Cardioplexol significantly improves functional recovery after 45 minutes of ischemia compared with pure ischemia. However, Cardioplexol protects the myocardium from ischemia/reperfusion-related damage after 90 minutes of ischemia worse than Calafiore cardioplegia.

Soluble Urokinase Plasminogen Activator Receptor (suPAR) Promotes Cardioprotective Intracellular Signalling in Myocardial Ischaemia

Soluble urokinase Plasminogen Activator Receptor (suPAR) is an emerging biomarker of inflammation capable of predicting cardiovascular outcomes. However, the biological activity of suPAR on the heart is poorly understood. Potential cardiac intracellular signalling pathways triggered by suPAR were explored. Isolated Langendorff rat hearts undergoing global ischaemia received treatment with either suPAR infusion or perfusion buffer (controls). Proteins were extracted from hearts for each treatment group, and Troponin T in rat outflow perfusates following ischaemia were measured. The phosphorylation status of each kinase was characterised using a phospho-specific antibody and a ratio of phosphorylation to the total protein was calculated. Analysis between treatment groups was performed with unpaired t-test with Welch’s correction. Hearts receiving suPAR treatment after ischaemia showed a 1.4-fold increase in Akt phosphorylation, and a 1.6-fold increase in Akt phosphorylation if suPAR was given before ischaemia. The ratio of p-Akt/Akt was 1.83±0.14 (n=7) in the post-ischaemic suPAR treatment group vs 1.3±0.04 (n=6) in controls (p=0.007). Pre-ischaemic suPAR treatment gave a p-Akt/Akt ratio of 1.08±0.03 (n=8) vs 0.67±0.11 (n=5) in controls (p=0.01). Upstream signalling ERK1 and ERK2 phosphorylation also increased at 2.6-fold (p=0.009) and 1.9-fold (p=0.01), respectively, in suPAR treated heart vs controls. Finally, suPAR treated hearts obtained lower Troponin T values compared with controls after recovery from ischaemia (p<0.001). suPAR infusion promoted the phosphorylation of ERK and Akt kinases in isolated rat heart tissue and reduced related cardiac troponin release after a period of global no-flow ischaemia. These actions suggest that suPAR is a novel activator of the reperfusion injury salvage kinase (RISK) pathway.

Role of the protein kinase A signaling pathway and identification of mediators in the cardioprotective effects of enteral lactoferrin for ischemia-reperfusion injury in an isolated rat heart model

ObjectiveLactoferrin is an iron-binding glycoprotein. Enteral lactoferrin attenuates myocardial ischemia-reperfusion (IR) injury, but the underlying mechanism remains unknown. The aim of this study was to investigate protein kinase A (PKA) signaling pathway activation and levels of serum glucagonlike peptide-1 (GLP-1), secreted by intestinal endocrine L cells, and adiponectin, secreted by adipose tissue, after enteral lactoferrin administration. MethodsHearts (N = 32) were excised from Wistar rats and perfused using a Langendorff system. To assess the role of the PKA pathway in the cardioprotective effects of lactoferrin, an inhibitor of PKA (H89) was applied before no-flow ischemia. Rats were randomly divided into four groups: control, lactoferrin (LF), control+H89, and LF+H89. The control and control+H89 groups were administered normal saline by gavage, and the LF and L +H89 groups were administered bovine lactoferrin (1000 mg/kg) by gavage 15 min before intraperitoneal pentobarbital injection. Muscle sampling was performed at the end of reperfusion. When rats were sacrificed, blood was sampled to measure hormone levels. The primary outcome was maximum left ventricular pressure derivative (LV dP/dt max) 15 min after reperfusion. ResultsLV dP/dt max at 10 and 15 min after reperfusion was significantly higher in the LF group than in the control group (P < 0.05), and the effect was diminished by H89. The PKA pathway was significantly activated in the LF group. Enteral lactoferrin increased serum GLP-1 but not serum adiponectin levels. ConclusionsEnteral lactoferrin induces cardioprotective effects against myocardial IR injury via the PKA signaling pathway and increases serum GLP-1 levels.

Intra-ischemic hypothermia cardioprotection involves modulation of PTEN/Akt/ERK signaling and fatty acid oxidation.

Therapeutic hypothermia (TH) provides cardioprotection from ischemia/reperfusion (I/R) injury. However, it remains unknown how TH regulates metabolic recovery. We tested the hypothesis that TH modulates PTEN, Akt, and ERK1/2, and improves metabolic recovery through mitigation of fatty acid oxidation and taurine release. Left ventricular function was monitored continuously in isolated rat hearts subjected to 20 min of global, no-flow ischemia. Moderate cooling (30°C) was applied at the start of ischemia and hearts were rewarmed after 10min of reperfusion. The effect of TH on protein phosphorylation and expression at 0 and 30 min of reperfusion was investigated by western blot analysis. Post-ischemic cardiac metabolism was investigated by 13 C-NMR. TH enhanced recovery of cardiac function, reduced taurine release, and enhanced PTEN phosphorylation and expression. Phosphorylation of Akt and ERK1/2 was increased at the end of ischemia but decreased at the end of reperfusion. On NMR analysis, TH-treated hearts displayed decreased fatty acid oxidation. Direct cardioprotection by moderate intra-ischemic TH is associated with decreased fatty acid oxidation, reduced taurine release, enhanced PTEN phosphorylation and expression, and enhanced activation of both Akt and ERK1/2 prior to reperfusion.

Abstract 103: Regulation Of Cardiac PTEN/Akt Signaling By Moderate Intra-Ischemic Therapeutic Hypothermia

Introduction: Previous studies demonstrate a critical role for Akt in mediating cardioprotection by therapeutic hypothermia. However, the mechanism of hypothermia-induced Akt activation remains poorly understood. Akt activation is determined in part by PTEN activity and degradation. Hypothesis: We hypothesized that moderate intra-ischemic cooling activates Akt in the ischemic-reperfused heart due to enhanced PTEN degradation. Methods: Rat hearts were isolated and perfused media containing 5 mM glucose. LV function was monitored continuously by an intraventricular balloon. Rate-pressure product (RPP=heart rate x LV developed pressure) was monitored as an index of cardiac function. Hearts in the normothermic control group (I/R) were subjected to 20 min. of global, no-flow ischemia followed by reperfusion at 37 °C. In the hypothermia intervention group (I/R+TH), hearts were cooled to 30 °C at the start of ischemia and then rewarmed 10 min. after reperfusion. Coronary perfusate was collected from the pulmonary artery at preischemic baseline and 10 min. after reperfusion. Ventricular tissue was harvested at 0 or 30 min. after reperfusion for western blotting. Results: RPP was significantly higher in the I/R+TH group (n=7) compared to the I/R group (n=8) at 30 min. post-reperfusion (24,725 ± 1,112 mmHg*beats/min. vs. 18,237 ± 2,223 mmHg*beats/min., p&lt;0.05). Coronary perfusate taurine concentration was significantly higher in the I/R group compared to the I/R+TH group at 10 min. post-reperfusion (19 ± 4 μM vs. 5 ± 1 μM, p&lt;0.01). Akt serine473 phosphorylation was significantly higher in the I/R+TH group compared to the I/R group at 0 min. post-reperfusion (p&lt;0.01), but significantly lower at 30 min. post-reperfusion (p&lt;0.01). Additionally, TH enhanced total PTEN expression at 0 min. (p&lt;0.05) and 30 min. (p&lt;0.01) post-reperfusion without affecting the ratio of phospho-PTEN/PTEN. Conclusion: Intra-ischemic cooling was associated with temporal changes in Akt phosphorylation despite enhanced PTEN expression. Additionally, modulation of cardiac taurine release by hypothermia should be carefully considered in future studies assessing taurine as a biomarker for cardiac arrest and ischemic heart pathologies.

Abstract 114: Postconditioning With Argon Has No Direct Lung-protective Effect Against Ischemia Reperfusion In A Rat Isolated Lung Model

Cardiac arrest (CA) remains a significant medical problem with stagnating low survival rates. Ventilation with the noble gas Argon (Ar) after return of spontaneous circulation improved neurologically favorable survival in a porcine CA model. We hypothesized that postconditioning with Ar attenuates global ischemia reperfusion (IR) injury in a rat isolated lung model. Following Institutional Animal Care and Use Committee approval and conforming with the "Position of the American Heart Association on Research Animal Use", lungs of adult male Sprague Dawley rats (250-350 g) were isolated and positive-pressure ventilated (10 mL/kg body weight [BW]) at a respiration rate of 30 breaths/minute. Lungs were perfused with physiological crystalloid buffer at a constant flow of 40 mL/kg BW. The control (C) group (n=6-7) was ventilated with 65% N 2 , 5% CO 2 , 30% O 2 , the Ar group (n=6) with 65% Ar, 5% CO 2 , 30% O 2 before global no-flow ischemia for 8 min followed by reperfusion. Mean arterial (MAP) and airway pressures (AWP) were recorded continuously. Lung viability as survival time until development of fulminant pulmonary edema was noted. Data are mean +/- SEM. Statistics: Student's t-Test with *p&lt; 0.05. IR significantly increased MAP and AWP throughout reperfusion compared to baseline; contrary to our hypothesis, though, ventilation with Ar did not signficantly increase survival time (panel A) or lower MAP (B) or AWP (C) of the isolated lung during reperfusion in this model. In-vivo models may be better-suited to assess Ar’s organ-protective effects following CA.

Cardioprotective effects of enteral vs. parenteral lactoferrin administration on myocardial ischemia-reperfusion injury in a rat model of stunned myocardium

Background:Lactoferrin, an iron-binding glycoprotein, is known to have protective effects against intestinal and cerebral ischemia-reperfusion (IR) injuries; however, its cardioprotective effects against the stunned myocardium are unknown. This study aimed to test the hypothesis that lactoferrin has cardioprotective effects against stunned myocardium.Methods:Using isolated rat hearts (Langendorff system), we determined the effects of lactoferrin administered enterally and by direct cardiac perfusion. Rat hearts were perfused using the Langendorff system, and two experiments were performed. In experiment 1, the hearts were divided into the enteral lactoferrin (E-LF) 7.5 m, 15 m, 30 m, and 60 m groups, where lactoferrin (1000 mg/kg) was administered enterally 7.5, 15, 30, and 60 min, respectively, before perfusion; and a control group, where saline was administered 30 min before perfusion. In experiment 2, hearts were allocated to the perfusate lactoferrin (P-LF) 15 and 100 groups, where 15 mg/L and 100 mg/L lactoferrin were respectively added to the perfusate, and a control group. Each group was perfused for 20 min prior to 15 min of no-flow ischemia with pacing, followed by 20 min of reperfusion. The primary outcome was the maximum left ventricular derivative of pressure development (LV dP/dt max) 15 min after reperfusion. Myocardial phospho-protein kinase B (p-Akt) was assayed using western blotting.Results:The LV dP/dt max 15 min after reperfusion in the E-LF 15 and 30 m groups was significantly higher than that in the control group. However, the effects disappeared in the E-LF 60 m group. In the second experiment, there were no significant differences in LV dP/dt max. Myocardial p-Akt was not significantly activated in any lactoferrin group.Conclusion:Cardioprotection was observed 15–30 min after enteral lactoferrin but not by direct cardiac perfusion with lactoferrin. Myocardial p-Akt was not associated with the cardioprotective effect. The cardioprotective effect may be induced by enteral lactoferrin-induced substances.

L-2-Hydroxyglutarate Protects Against Cardiac Injury via Metabolic Remodeling.

L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase]-/-) or heterozygous (L2HGDH+/-) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH+/+) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [13C]- and [31P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH-/- and L2HGDH+/- hearts compared with L2HGDH+/+ hearts under ischemic stress conditions. L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.

Low Flow versus No Flow: Ischaemia Reperfusion Injury Following Different Experimental Models in the Equine Small Intestine

Simple SummaryOne of the main causes of colic in horses is the occlusion of the intestinal blood vessels after displacement or entrapment of the small intestine. In search of new therapies to treat this lethal disease, experimental models have been used to simulate the clinical situation. Both low flow (LF) models with partial blood flow occlusion as well as no flow (NF) models with complete occlusion have been implemented in different studies. This has led to conflicting results and comparative studies are lacking. The objective of this study was to characterize the development of intestinal injury over time in two different experimental models implementing either partial or complete vessel occlusion. Under general anaesthesia, local intestinal blood flow was reduced by 80% in seven horses (LF), and by 100% in another seven horses (NF). The LF group exhibited more bleeding in the intestinal wall and a relatively high variability in intestinal oxygen levels and tissue damage. The NF group showed lower oxygen levels and decreased barrier function of the intestinal wall. These results aid in the selection of the suitable experimental model for future studies. The high variability following LF suggests that an NF model may produce more consistent intestinal damage.In experimental studies investigating strangulating intestinal lesions in horses, different ischaemia models have been used with diverging results. Therefore, the aim was to comparatively describe ischaemia reperfusion injury (IRI) in a low flow (LF) and no flow (NF) model. Under general anaesthesia, 120 min of jejunal ischaemia followed by 120 min of reperfusion was induced in 14 warmbloods. During ischaemia, blood flow was reduced by 80% (LF, n = 7) or by 100% (NF, n = 7). Intestinal blood flow and oxygen saturation were measured by Laser Doppler fluxmetry and spectrophotometry. Clinical, histological, immunohistochemical and Ussing chamber analyses were performed on intestinal samples collected hourly. Tissue oxygen saturation was significantly lower in NF ischaemia. The LF group exhibited high variability in oxygen saturation and mucosal damage. Histologically, more haemorrhage was found in the LF group at all time points. Cleaved-caspase-3 and calprotectin-stained cells increased during reperfusion in both groups. After NF ischaemia, the tissue conductance was significantly higher during reperfusion. These results aid in the selection of suitable experimental models for future studies. Although the LF model has been suggested to be more representative for clinical strangulating small intestinal disease, the NF model produced more consistent IRI.

Matrix Metalloproteinase-2 Inhibition in Acute Ischemia-Reperfusion Heart Injury-Cardioprotective Properties of Carvedilol.

Matrix metalloproteinase 2 (MMP-2) is activated in hearts upon ischemia-reperfusion (IR) injury and cleaves sarcomeric proteins. It was shown that carvedilol and nebivolol reduced the activity of different MMPs. Hence, we hypothesized that they could reduce MMPs activation in myocytes, and therefore, protect against cardiac contractile dysfunction related with IR injury. Isolated rat hearts were subjected to either control aerobic perfusion or IR injury: 25 min of aerobic perfusion, followed by 20 min global, no-flow ischemia, and reperfusion for 30 min. The effects of carvedilol, nebivolol, or metoprolol were evaluated in hearts subjected to IR injury. Cardiac mechanical function and MMP-2 activity in the heart homogenates and coronary effluent were assessed along with troponin I content in the former. Only carvedilol improved the recovery of mechanical function at the end of reperfusion compared to IR injury hearts. IR injury induced the activation and release of MMP-2 into the coronary effluent during reperfusion. MMP-2 activity in the coronary effluent increased in the IR injury group and this was prevented by carvedilol. Troponin I levels decreased by 73% in IR hearts and this was abolished by carvedilol. Conclusions: These data suggest that the cardioprotective effect of carvedilol in myocardial IR injury may be mediated by inhibiting MMP-2 activation.

Abstract 330: No Direct Mitochondrial Protection Against Ischemia Reperfusion Injury in Rat Isolated Hearts by P188 Postconditioning

Introduction: Myocardial infarction and cardiac arrest lead to ischemia-reperfusion (IR) injury in the heart. Timely reperfusion through percutaneous coronary intervention and cardiopulmonary resuscitation, respectively, reduces ischemia but also exacerbates myocardial injury. Maintaining mitochondrial function is crucial in maintaining cardiomyocyte function in IR injury. Poloxamer 188 (P188) is a triblock copolymer that has shown protective effects in in-vitro, ex-vivo and in-vivo myocardial IR models. P188 is thought to improve cellular and mitochondrial function during IR by stabilizing membranes. Hypothesis: P188 postconditioning has direct protective effects on mitochondrial function as assessed by ATP synthesis, oxygen consumption and calcium retention capacity (CRC). Methods: After approval by the local authorities, hearts of 42 adult male Sprague-Dawley rats were isolated and perfused ex-vivo with oxygenated Krebs Buffer (KB) for 20 min before 30 min of no-flow ischemia. Hearts were reperfused for 10 min with KB. Cardiac mitochondria were isolated with 1 mM P188 vs 1 mM polyethylene glycol (PEG) vs vehicle by differential centrifugation. Mitochondrial function was assessed for complex I and II substrates of the respiratory chain. Statistics: Kruskal-Wallis with Dunn’s posthoc testing; alpha=0.05. Results: Mitochondrial function decreased significantly after ischemia and showed improvement with reperfusion. P188 did not result in significant improvements in mitochondrial ATP synthesis, oxygen consumption and CRC function after IR, and neither did PEG. Conclusions: P188 does not have a direct protective effect on mitochondria in this model. This might be owed to the fact that no additional damage could be observed after reperfusion which is the type of injury targeted by P188 post-conditioning.

Ameliorating Effect of Klotho Protein on Rat Heart during I/R Injury.

An essential procedure for the treatment of myocardial infarction is restoration of blood flow in the obstructed infarct artery, which may cause ischaemia/reperfusion (I/R) injury. Heart I/R injury manifests in oxidative stress, metabolic and morphological disorders, or cardiac contractile dysfunction. Klotho protein was found to be produced in the heart tissue and participate in antioxidation or ion homeostasis. The aim of this study was to examine an influence of Klotho protein on the heart subjected to I/R injury. Wistar rats served as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischaemia, and isolated rat cardiomyocytes underwent chemical I/R in vitro, with or without recombinant Klotho protein administration. Haemodynamic parameters of heart function, cell contractility, markers of I/R injury and oxidative stress, and the level of contractile proteins such as myosin light chain 1 (MLC1) and troponin I (TnI) were measured. The treatment of hearts subjected to I/R injury with Klotho protein resulted in a recovery of heart mechanical function and ameliorated myocyte contractility. This improvement was associated with decreased tissue injury, enhanced antioxidant capacity, and reduced release of MLC1 and TnI. The present research showed the contribution of Klotho to cardioprevention during I/R. Thus, Klotho protein may support the protection from I/R injury and prevention of contractile dysfunction in the rat heart.

Attenuating loss of cardiac conduction during no-flow ischemia through changes in perfusate sodium and calcium.

Myocardial ischemia leads to conduction slowing, cell-to-cell uncoupling, and arrhythmias. We previously demonstrated that varying perfusate sodium (Na+) and calcium (Ca2+) attenuates conduction slowing and arrhythmias during simulated ischemia with continuous perfusion. Cardioprotection was selectively associated with widening of the perinexus, a gap junction adjacent nanodomain important to ephaptic coupling. It is unknown whether perfusate composition affects the perinexus or ischemic conduction during nonsimulated ischemia, when coronary flow is reduced or halted. We hypothesized that altering preischemic perfusate composition could facilitate perinexal expansion and attenuate conduction slowing during global ischemia. To test this hypothesis, ex vivo guinea pig hearts (n = 49) were Langendorff perfused with 145 or 153 mM Na+ and 1.25 or 2.0 mM Ca2+ and optically mapped during 30 min of no-flow ischemia. Altering Na+ and Ca2+ did not substantially affect baseline conduction. Increasing Na+ and decreasing Ca2+ both lowered pacing thresholds, whereas increasing Ca2+ narrowed perinexal width (Wp). A least squares mean estimate revealed that reduced perfusate Na+ and Ca2+ resulted in the most severe conduction slowing during ischemia. Increasing Na+ alone modestly attenuated conduction slowing, yet significantly delayed the median time to conduction block (10 to 16 min). Increasing both Na+ and Ca2+ selectively widened Wp during ischemia (22.7 vs. 15.7 nm) and attenuated conduction slowing to the greatest extent. Neither repolarization nor levels of total or phosphorylated connexin43 correlated with conduction slowing or block. Thus, perfusate-dependent widening of the perinexus preserved ischemic conduction and may be an adaptive response to ischemic stress.NEW & NOTEWORTHY Conduction slowing during acute ischemia creates an arrhythmogenic substrate. We have shown that extracellular ionic concentrations can alter conduction by modulating ephaptic coupling. Here, we demonstrate increased extracellular sodium and calcium significantly attenuate conduction slowing during no-flow ischemia. This effect was associated with selective widening of the perinexus, an intercalated disc nanodomain and putative cardiac ephapse. These findings suggest that acute changes in ephaptic coupling may serve as an adaptive response to ischemic stress.

Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury.

Objectives Coronary reperfusion procedure leads to ischemia/reperfusion injury of the heart (IRI). IRI arises from increased degradation of myosin light chains and increased activity of matrix metalloproteinase 2 (MMP-2). Increased production of toxic peroxynitrite (ONOO−) during oxidative stress is a source of increased nitration/nitrosylation of contractile proteins, which enhance their degradation through MMP-2. Hence, an imbalance in nitric oxide (NO) metabolism along with oxidative stress is an important factor contributing to pathophysiology of cardiovascular disorders, including myocardial infarction. The aim of the current study was to provide an important insight into understanding the interaction of iNOS, eNOS, and ADMA during oxidative stress and to propose the beneficial therapy to modulate this interaction. Material and Methods. Pathogen-free Wistar rats were used in this study as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischemia with or without administration of DOXY (1 µM), ML-7 (0.5 µM), and L-NAME (2 µM) mixture. Haemodynamic parameters of heart function, markers of I/R injury, tissue expression of iNOS, eNOS, and phospho-eNOS, asymmetric dimethylarginine, and NO production as well as MMP-2 activity were measured. Results Mechanical heart function and coronary flow (CF) were decreased in the hearts subjected to I/R. Treatment of the hearts with the tested mixture resulted in a recovery of mechanical function due to decreased activity of MMP‐2. An infusion of Doxy, ML-7, and L-NAME mixture into I/R hearts decreased the expression of iNOS, eNOS, and phospho-eNOS and in consequence reduced ADMA expression. Decreased ADMA production led to enhanced NO synthesis and improvement of cardiac function at 85% of aerobic control. Conclusions Synergistic effect of the multidrug therapy with the subthreshold doses allows addressing a few pathways of I/R injury simultaneously to achieve protection of cardiac function during I/R.

Repolarization and arrhythmia modulation by K-ATP channel activation in the porcine right ventricle

The RVOT is involved in arrhythmias in several diseases such as the Brugada syndrome (BrS). In a subpopulation of patients with BrS and early repolarization (ER) syndrome, a K-ATP gain-of-function (GOF) mutation has previously been described and was suggested to contribute to arrhythmogenicity in this context. To determine the role of regional K-ATP channels expression on RV repolarization heterogeneity and in arrhythmia occurrence and modulation. Pig right ventricles (RV) were perfused by both right and left coronary arteries, paced at 1 or 1.5 Hz and the electrical activity optically-mapped (di-4-ANEPPS, 20 μM). The preparation was perfused with pinacidil (PINA, 20 μM), and diazoxide (DIAZ, 100 μM), and subjected to 20 min no-flow (NF) ischemia episodes followed by 20 min reperfusion (RF). Arrhythmia susceptibility was assessed using a 20 Hz burst protocol while spontaneous arrhythmias were also recorded. Protein expression was assessed by western blot. PINA-induced action potential duration (APD) shortening was larger in RVOT than in the free wall (FW). DIAZ induced a slight but significant reduction in APD across the RV. SUR2A expression was greater in the RVOT epicardium vs. FW but Kir6.1/2 and SUR1 expression was similar in both regions. PINA induced spontaneous ventricular fibrillation (VF) in 80% of the preparations with higher dominant frequency (DF) and regularity index in the RVOT than the FW. DF of burst-induced VF were higher in PINA but not in DIAZ compared to CTRL VF. Similarly to PINA, NF-induced APD shortening was greater in the RVOT than in the FW. No spontaneous arrhythmia occurred during NF but slow ventricular tachycardias arose in 36% of the preparations during RF. K-ATP activation leads to an increase in RV repolarization dispersion which is likely to contribute to arrhythmogenicity in BrS or ER patients with a K-ATP channel GOF mutation and may underlie BrS phenocopy induced by RV ischemia.

Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation

We have previously demonstrated that inorganic polyphosphate (polyP) is a potent activator of the mitochondrial permeability transition pore (mPTP) in cardiac myocytes. PolyP depletion protected against Ca2+-induced mPTP opening, however it did not prevent and even exacerbated cell death during ischemia-reperfusion (I/R). The central goal of this study was to investigate potential molecular mechanisms underlying these dichotomous effects of polyP on mitochondrial function. We utilized a Langendorff-perfused heart model of I/R to monitor changes in polyP size and chain length at baseline, 20 min no-flow ischemia, and 15 min reperfusion. Freshly isolated cardiac myocytes and mitochondria from C57BL/6J (WT) and cyclophilin D knock-out (CypD KO) mice were used to measure polyP uptake, mPTP activity, mitochondrial membrane potential, respiration and ATP generation. We found that I/R induced a significant decrease in polyP chain length. We, therefore, tested, the ability of synthetic polyPs with different chain length to accumulate in mitochondria and induce mPTP. Both short and long chain polyPs accumulated in mitochondria in oligomycin-sensitive manner implicating potential involvement of mitochondrial ATP synthase in polyP transport. Notably, only short-chain polyP activated mPTP in WT myocytes, and this effect was prevented by mPTP inhibitor cyclosprorin A and absent in CypD KO myocytes. To the contrary, long-chain polyP suppressed mPTP activation, and enhanced ADP-linked respiration and ATP production. Our data indicate that 1) effect of polyP on cardiac function strongly depends on polymer chain length; and 2) short-chain polyPs (as increased in ischemia-reperfusion) induce mPTP and mitochondrial uncoupling, while long-chain polyPs contribute to energy generation and cell metabolism.

Abstract 251: Comparison of Ischemia-Reperfusion Injury in Langendorff-Prepared Hearts From Zucker Diabetic Fatty and Lean Rats

Introduction: The Zucker Diabetic Fatty (ZDF) rat is a relevant model for assessing ischemia-reperfusion (IR) injury, due to it having comorbidities associated with a higher risk for cardiac arrest and poorer outcomes following cardiopulmonary resuscitation in humans. Hypothesis: Based on previous studies in the literature, we hypothesize that ZDF rats would have similar outcomes to lean rats subjected to IR when accounting for perfusion buffer osmolarity. Methods: ZDF (n=4) and lean control (n=6) rats were anesthetized with pentobarbital, intubated, and mechanically ventilated and monitored for 3-hours, with blood glucose (BG) readings taken before and after ventilation. They were then decapitated and a thoracotomy performed to isolate the Langendorff-prepared hearts. Krebs buffer with the osmolarity adjusted to each rat’s BG level using glucose was used to perfuse the hearts. Following a 20-minute stabilization period, the hearts were subjected to a 30-minute period of global no-flow ischemia followed by 120-minutes of reperfusion. Spontaneous heart rate, isovolumetric left ventricular pressure (LVP), and mitochondrial redox state were measured continuously. Data are mean ± SD. Statistics: two-tailed t-test, p&lt;0.05 Results: There was no significant difference in average body weight between the two groups; however, ZDF rats showed significantly higher BG levels (504±52 vs 174±14 mg/dl) than their lean littermates. ZDF rats had a greater percent change from baseline systolic LVP upon reperfusion than lean rats (133±33.3 vs 84.9±16.5 %), with the lean rats never returning to baseline. Diastolic contracture occurred more in lean rats during ischemia, but trended towards a lower contracture during reperfusion. No significant difference was seen in developed LVP, rate pressure products, or mitochondrial redox states, although lean rats trended towards better diastolic contractility and relaxation. Conclusion: Lean rats showed marginally better outcomes in heart function than their ZDF littermates. Future studies will focus on potential mechanisms of Type 2 diabetes that may exacerbate IR injuries.

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.