Low-flow Ischemia Research Articles

Upregulation of Hsp27 via further inhibition of histone H2A ubiquitination confers protection against myocardial ischemia/reperfusion injury by promoting glycolysis and enhancing mitochondrial function

Research suggests that ischemic glycolysis improves myocardial tolerance to anoxia and low-flow ischemia. The rate of glycolysis during ischemia reflects the severity of the injury caused by ischemia and subsequent functional recovery following reperfusion. Histone H2AK119 ubiquitination (H2Aub) is a common modification that is primarily associated with gene silencing. Recent studies have demonstrated that H2Aub contributes to the development of cardiovascular diseases. However, the underlying mechanism remains unclear. This study identified Hsp27 (heat shock protein 27) as a H2Aub binding protein and explored its involvement in mediating glycolysis and mitochondrial function. Functional studies revealed that inhibition of PRC1 (polycomb repressive complex 1) decreased H2Aub occupancy and promoted Hsp27 expression through inhibiting ubiquitination. Additionally, it increased glycolysis by activating the NF-κB/PFKFB3 signaling pathway during myocardial ischemia. Furthermore, Hsp27 reduced mitochondrial ROS production by chaperoning COQ9, and suppressed ferroptosis during reperfusion. A delivery system was developed based on PCL-PEG-MAL (PPM)-PCM-SH (CWLSEAGPVVTVRALRGTGSW) to deliver PRT4165 (PRT), a potent inhibitor of PRC1, to damaged myocardium, resulting in decreased H2Aub. These findings revealed a novel epigenetic mechanism connecting glycolysis and ferroptosis in protecting the myocardium against ischemia/reperfusion injury.

Pharmacological Cardioprotection against Ischemia Reperfusion Injury-The Search for a Clinical Effective Therapy.

Pharmacological conditioning aims to protect the heart from myocardial ischemia-reperfusion injury (IRI). Despite extensive research in this area, today, a significant gap remains between experimental findings and clinical practice. This review provides an update on recent developments in pharmacological conditioning in the experimental setting and summarizes the clinical evidence of these cardioprotective strategies in the perioperative setting. We start describing the crucial cellular processes during ischemia and reperfusion that drive acute IRI through changes in critical compounds (∆GATP, Na+, Ca2+, pH, glycogen, succinate, glucose-6-phosphate, mitoHKII, acylcarnitines, BH4, and NAD+). These compounds all precipitate common end-effector mechanisms of IRI, such as reactive oxygen species (ROS) generation, Ca2+ overload, and mitochondrial permeability transition pore opening (mPTP). We further discuss novel promising interventions targeting these processes, with emphasis on cardiomyocytes and the endothelium. The limited translatability from basic research to clinical practice is likely due to the lack of comorbidities, comedications, and peri-operative treatments in preclinical animal models, employing only monotherapy/monointervention, and the use of no-flow (always in preclinical models) versus low-flow ischemia (often in humans). Future research should focus on improved matching between preclinical models and clinical reality, and on aligning multitarget therapy with optimized dosing and timing towards the human condition.

The Acute Effect of Fructose on Cardiac Hemodynamic Responses and Infarcted Area in Isolated Rat Heart During Ischemia-Reperfusion

Introduction: This study aimed to investigate the effects of fructose on cardiac hemodynamics and infarct size and the role of the antioxidant mechanism in these effects in isolated rat hearts undergoing ischemiareperfusion. Patients and Methods: Isolated hearts obtained from female Wistar rats were perfused with Krebs-Henseleit solution containing 12 mM glucose or solution containing 12 mM fructose or 48 mM fructose and underwent lowflow ischemia followed by reperfusion on the Langendorff apparatus. Left ventricular developed pressure (LVDP), timedependent pressure changes (dp/dt max, dp/dt min) and heart rates were recorded at the 1st, 15th and 120th minutes of reperfusion following ischemia, and the percentage of the infarct area and the size of the risk area were determined. At the end of the reperfusion, total oxidant capacity (TOS), malondialdehyde (MDA) and glutathione (GSH) levels were examined in perfusion fluid samples. Results: Basal dp/dt max values were lower in the high fructose group compared to the glucose group (p= 0.032). When the hearts were perfused with 12 mM fructose, a significant increase was observed in the percentage of the ischemic area and risk area compared to equivalent glucose and high fructose (p< 0.001 and p< 0.001, respectively). MDA, GSH and TOS values were comparable in all groups. Conclusion: The present study shows that fructose perfusion plays a role in reduced ventricular contractile function relative to glucose in isolated rat hearts. This reduction triggered by fructose appears to be independent of antioxidant mechanisms. Furthermore, fructose perfusion at glucose-equivalent concentration causes a greater increase in infarct area in ischemic hearts, whereas an increase in fructose concentration appears to prevent this effect.

Adaptive mechanisms in no flow vs. low flow ischemia in equine jejunum epithelium: Different paths to the same destination.

Intestinal ischemia reperfusion injury (IRI) is a frequent complication of equine colic. Several mechanisms may be involved in adaptation of the intestinal epithelium to IRI and might infer therapeutic potential, including hypoxia-inducible factor (HIF) 1α, AMP-activated protein kinase (AMPK), nuclear factor-erythroid 2-related factor 2 (NRF2), and induction of autophagy. However, the mechanisms supporting adaptation and thus cellular survival are not completely understood yet. We investigated the activation of specific adaptation mechanisms in both no and low flow ischemia and reperfusion simulated in equine jejunum epithelium in vivo. We found an activation of HIF1α in no and low flow ischemia as indicated by increased levels of HIF1α target genes and phosphorylation of AMPKα tended to increase during ischemia. Furthermore, the protein expression of the autophagy marker LC3B in combination with decreased expression of nuclear-encoded mitochondrial genes indicates an increased rate of mitophagy in equine intestinal IRI, possibly preventing damage by mitochondria-derived reactive oxygen species (ROS). Interestingly, ROS levels were increased only shortly after the onset of low flow ischemia, which may be explained by an increased antioxidative defense, although NFR2 was not activated in this setup. In conclusion, we could demonstrate that a variety of adaptation mechanisms manipulating different aspects of cellular homeostasis are activated in IRI irrespective of the ischemia model, and that mitophagy might be an important factor for epithelial survival following small intestinal ischemia in horses that should be investigated further.

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.

MiR-126-HMGB1-HIF-1 Axis Regulates Endothelial Cell Inflammation during Exposure to Hypoxia-Acidosis.

Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3′UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.

Co-Polarized [1-13C]Pyruvate and [1,3-13C2]Acetoacetate Provide a Simultaneous View of Cytosolic and Mitochondrial Redox in a Single Experiment.

Cellular redox is intricately linked to energy production and normal cell function. Although the redox states of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissues is difficult. Here, we employed 13C magnetic resonance spectroscopy (MRS) and co-polarized [1-13C]pyruvate and [1,3-13C2]acetoacetate ([1,3-13C2]AcAc) to monitor production of hyperpolarized (HP) lactate and β-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [Pi]/[ATP] measured by 31P NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-13C]pyruvate to [1-13C]lactate and [1,3-13C2]AcAc to [1,3-13C2]β-hydroxybutyrate ([1,3-13C2]β-HB) was readily detected. A significant increase in HP [1,3-13C2]β-HB but not [1-13C]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-13C]lactate or [1,3-13C2]β-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissues using co-polarized [1-13C]pyruvate and [1,3-13C2]AcAc and 13C MRS and that changes in mitochondrial redox may precede changes in cytosolic redox.

β-hydroxybutyrate accumulates in the rat heart during low-flow ischaemia with implications for functional recovery.

Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.

B-AB01-04 INTERACTION BETWEEN ISCHEMIA AND SYMPATHETIC ACTIVITY ON CARDIAC ELECTROPHYSIOLOGY AND ARRHYTHMIA IN THE INNERVATED LANGENDORFF-PERFUSED MOUSE HEART

Sympathetic activation of the heart plays a key role in the modulation of cardiac electrophysiology and arrhythmogenesis. It is generally believed that sympathetic activation is proarrhythmic in the setting of cardiac ischemia. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac electrophysiological activities during ischemia. To investigate the effects of sympathetic activation in the innervated Langendorff-perfused mouse heart subjected to acute ischemia. The heart and posterior thoracic cavity from mice (C57Bl6, N=4) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2+ to study the effects of sympathetic nerve stimulation (SNS). SNS, achieved through spinal cord stimulation at T1-T3, was performed before, during and immediately after 5 min of low-flow ischemia (10% of pre-ischemic flow rate). Early afterdepolarizations (EADs) and ventricular tachycardia (VT) occurred in all 4 animals within 5 min of ischemia and immediately upon reperfusion. Both SNS and ventricular pacing, during either ischemia or reperfusion, completely abolished EADs and VT induced by global low-flow ischemia and reperfusion (Fig). In the isolated innervated mouse heart, we showed that sympathetic stimulation protects against ischemia-induced cardiac arrhythmias potentially by over-driving ventricular ectopic activity.

Necrotizing Esophagitis: A Big Squeeze?

Necrotizing esophagitis is rare and poorly understood. The etiologies reported in what little has been published (i.e., gastroesophageal reflux exacerbated by gastric outlet obstruction and low-flow ischemia) seem somewhat simplistic and lack any direct evidence. The following paper illustrates a recent clinical case while laying out arguments supporting esophageal spasm as a possible contributing factor.

P0519HEXARELIN PROTECTS ISCHEMIA REPERFUSION INDUCED ACUTE KIDNEY INJURY BY TARGETING 14-3-3Σ

Abstract Background and Aims Acute kidney injury (AKI), manifested as a sudden decline in renal function on the order of days or a week, is one of the most common complications and is strongly associated with renal prognosis, medical expenses and length of stay of hospitalized patients. However, incidence of AKI can be reduced by early intervention. Hexarelin is a synthetic analogue of ghrelin with a potent GH releasing activity, which is also found to exhibited a significant protectant activity against postischemic ventricular dysfunction in isolated perfused hearts subjected to low flow ischemia and reperfusion. This study aims to explore the effects of hexarelin on ischemia reperfusion induced acute kidney injury (I/R-AKI) and the underlying mechanism of it. Method Thirty healthy male SPF SD aged at 8 weeks weighing 250-300g rats were divided into 5 groups including normal group, sham operation group, acute kidney injury (AKI) group, acute kidney injury group after hexarelin (HEX + AKI), and acute kidney injury group after saline (Saline + AKI). Bilateral kidney artery clamping was applied for 50 min to induce AKI. The drug-administered group and the saline-controlled group rats were treated with intraperitoneal injections 7 days before modelling and the Hex + AKI group and the saline + AKI group were given Hexarelin (dose 100 μg/kg. d) and equal volume of saline, respectively. To explore the underlying mechanism of HEX on I/R-AKI, gene expression profiling of AKI samples were searched from Pubmed, GEO dataset. Differential expression genes were used to predict canonical signal transduction pathways and signalling networks. Molecular docking was used to predict the combination of hub proteins and HEX. Results 1) Renal function in HEX-pretreated rats was significantly improved compared with control group: HE results showed that renal tubules were less dilated and rebuilt after pretreatment, and the infiltration area was significantly reduced; serum creatinine (P = 0.0005 <0.05), urea nitrogen (P <0.0001) concentration were significantly reduced; RT-qPCR showed that relative expression of Kim-1 (P<0.0001), caspase3 (P = 0.007), Bax (P <0.0001), Bad (P = 0.0168 <0.05) in HEX+AKI group were significantly decreased while the relative expression of Bcl-2 (P <0.05) was significantly increased compared with saline treated rats; 2) Microarray data analysis showed that 63 differential expression genes including SFN, Cyr61, Kim-1 were hub genes in the development of I/R-AKI; 3) Three-dimensional structure of the hub proteins HEX were obtained from the SWISS-MODEL database and the PubChem compound database, and Autodock software was used to calculate the molecular docking between proteins and HEX. Results showed that HEX and the protein that encoded by the gene SFN (SFN, 14-3-3σ) binding stably with a binding energy of -8.51; Conclusion Hexarelin protects ischemia reperfusion induced acute kidney injury by targeting 14-3-3σ.

The Amplitude-Normalized Area of a Bipolar Electrogram as a Measure of Local Conduction Delay in the Heart.

BackgroundRe-entrant ventricular tachycardia may be non-inducible or haemodynamically compromising, requiring assessment of the electrophysiological properties of the myocardium during sinus rhythm (i.e., substrate mapping). Areas of heart tissue with slow conduction can act as a critical isthmus for re-entrant electrical excitation and are a potential target for ablation therapy.AimTo develop and validate a novel metric of local conduction delay in the heart, the amplitude-normalized electrogram area (norm_EA).MethodsA computational model of a propagating mouse action potential was used to establish the impact of altering sodium channel conductance, intracellular conductivity, fibrosis density, and electrode size/orientation on bipolar electrogram morphology. Findings were then validated in experimental studies in mouse and guinea pig hearts instrumented for the recording of bipolar electrograms from a multipolar linear mapping catheter. norm_EA was calculated by integrating the absolute area of a bipolar electrogram divided by the electrogram amplitude. Electrogram metrics were correlated with the local conduction delay during sodium channel block, gap junction inhibition, and acute ischemia.ResultsIn computational simulations, reducing sodium channel conductance and intracellular conductivity resulted in a decrease in signal amplitude and increase in norm_EA (reflecting a broadening of electrogram morphology). For larger electrodes (3 mm diameter/7.1 mm2 area), the change in norm_EA was essentially linear with the change in local conduction delay. Experimental studies supported this finding, showing that the magnitude of change in norm_EA induced by flecainide (1–4 μM), carbenoxolone (10–50 μM), and low-flow ischemia (25% of initial flow rate) was linearly correlated with the local conduction delay in each condition (r2 = 0.92). Qualitatively similar effects were observed in guinea pig hearts perfused with flecainide. Increasing fibrosis density in the computational model also resulted in a decrease in signal amplitude and increase in norm_EA. However, this remains to be validated using experimental/clinical data of chronic infarct.Conclusionnorm_EA is a quantitative measure of local conduction delay between the electrode pair that generates a bipolar electrogram, which may have utility in electrophysiological substrate mapping of non-inducible or haemodynamically compromising tachyarrhythmia.

Neuroprotective effect of low-intensity transcranial ultrasound stimulation in endothelin-1-induced middle cerebral artery occlusion in rats

BackgroundIschemic stroke is one of the leading causes of death and disability worldwide. Low-intensity transcranial ultrasound stimulation (LITUS) is a promising neuroprotective treatment for ischemic stroke. Diffusion-weighted imaging (DWI) can be highly sensitive in the detection of ischemic brain injury. Relative apparent diffusion coefficient (rADC) values can be used to evaluate the effect of LITUS on ischemic stroke. PurposeThe aim of this study was to determine the neuroprotective effect of LITUS at different time points using endothelin-1-induced middle cerebral artery occlusion in rats as a model of ischemic stroke. MethodsEndothelin-1 (ET-1) was injected into the cerebral parenchyma near the middle cerebral artery, which induced focal, reversible, low-flow ischemia in rats. After occlusion of the middle cerebral artery for 30 min, 120 min, and 240 min, LITUS stimulation was used respectively. DWI was performed at 1, 3, 6, 12, 18, 24, 48, and 72 h after ischemia using a 3 T scanner. The rADC values were calculated, and functional outcomes assessed using neurobehavioral scores after ischemia. Nissl staining and estimation of Na+–K+-ATPase activity were used to assess the neuropathology after completing the last Magnetic Resonance Imaging (MRI) examination. ResultsEndothelin-1-induced occlusion of the middle cerebral artery resulted in significant dysfunction and neuronal damage in rats. Rats that received LITUS exhibited reduced damage of the affected brain tissue after cerebral ischemia. The greatest protective effect was found when LITUS stimulation occurred 30 min after cerebral ischemia. ConclusionsImaging, behavioral, and histological results suggested that LITUS stimulation after an ischemic stroke produced significant neuroprotective effects.

A Rare Case of Recurrent Idiopathic Low-flow Priapism that Developed into a High-flow Priapism

Priapism, according to the American Urological Association is defined as a persistent penile erection that continues hours beyond sexual stimulation; typically, greater than 4 hours. Although priapism is a rare condition and has an unpredictable course in most presentation, it affects 5.36 per 100,000 male subjects per year [1]. Priapism is a urological emergency and delay in treatment or refractory cases can result in cavernous smooth muscle necrosis, fibrosis and penile shortening [2]. There are 2 categories of priapism-namely low-flow (ischemia, veno-occlusive) and high-flow (non-ischemic, arterial) [3,4]. There is a subset of ischemic priapism known as stuttering priapism which presents with recurrent incidences of ischemic priapism varying in length and is usually self-limiting [4]. Low-flow priapism occurs when an occlusive process inhibits the relaxation of the corpus cavernosum, thus the outflow of blood is impaired. The conditions associated with low-flow/ischemic priapism are as follows: sickle cell disease, vasoactive drugs, neoplastic diseases of the penis, urethra, prostate, bladder, kidney, gastrointestinal tract, leukemia, polycythemia, traumatic injury, hyperlipidemic parenteral nutrition, hemodialysis, heparin treatment, Fabry disease and neurologic conditions [3]. On the other hand, high-flow priapism occurs when there is increased arterial blood flow or pooling of blood. Conditions associated with high-flow priapism include traumatic arterio-cavernous fistula, vasoactive drugs, penile revascularization surgery, and neurologic conditions [3]. The mechanism of penile erection is a multifocal phenomenon that involves the nervous system, molecules (nitric oxide, cGMP, calcium), enzymes, and blood vessels. We present an interesting case of a patient with a history of recurrent priapism who converted from a low-flow priapism to a high-flow priapism, thought to be secondary to an arterio-cavernous fistula. Upon further review of PubMed and NIH database, there has been only few of such cases reported. We discuss the diagnostic process and management of high-flow priapism in this report.

Examination of the Effects of Conduction Slowing on the Upstroke of Optically Recorded Action Potentials.

IntroductionThe upstroke of optical action potentials (APs) recorded from intact hearts are generally recognized to be slower than those recorded with microelectrodes. This is thought to reflect spatial signal averaging within the volume of tissue that makes up the optical signal. However, to date, there has been no direct experimental study on the relationship between conduction velocity (CV) and optical AP upstroke morphology in the intact heart. Notably, it is known that sodium channel block and gap junction inhibition, which both slow CV, exert differential effects on the upstroke velocity of microelectrode-recorded APs. Whether such differences are evident in optical APs is not known. The present study sought to determine the relationship between tissue CV and optical AP upstroke velocity in intact mouse hearts.Materials and MethodsIsolated, perfused mouse hearts were stained with the potentiometric dye Rh-237. Fluorescent signals were recorded from across the anterior surface of the left and right ventricles during constant pacing. Maximum rate of change in fluorescence (dF/dtmax) and tissue CV were assessed in control conditions, during an acute period of low-flow ischemia, and following perfusion of flecainide (1–3 μmol/L), a sodium channel blocker, or carbenoxolone (10–50 μmol/L), a gap junction inhibitor.ResultsDuring epicardial pacing, an anisotropic pattern was observed in both activation and dF/dtmax maps, with more rapid optical AP upstroke velocities orientated along the fastest conduction paths (and vice versa). Low-flow ischemia resulted in a time-dependent slowing of ventricular CV, which was accompanied by a concomitant reduction in optical AP upstroke velocity. All values returned to baseline on tissue reperfusion. Both flecainide and carbenoxolone were associated with a concentration-dependent reduction in CV and decrease in optical AP upstroke velocity, despite distinct mechanisms of action. Similar responses to carbenoxolone were observed for low- (156 μm pixel with) and high- (20 μm pixel width) magnification recordings. Comparison of data from all interventions revealed a linear relationship between CV and upstroke dF/dt.ConclusionIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition.

Abstract 565: Response of the Ischemic Myocardium to Adrenergic Stimulation as Detected by Hyperpolarized [1- 13 C]pyruvate

Metabolic imaging by nuclear methods is widely used to access ischemic myocardium but provide limited information about individual enzyme-catalyzed reactions. 13 C hyperpolarization methods are sensitive to flux in specific key enzymes such as lactate dehydrogenase and pyruvate dehydrogenase. We tested the hypothesis that LDH flux is increased while PDH flux is abolished in low-flow ischemia. Metabolism of HP [1- 13 C]pyruvate was studied in four groups: (1) normal perfusion pressure (NPP), 100 cm H 2 O; (2) NPP with epinephrine; (3) low-perfusion pressure (LPP), 25 cm H 2 O; (4) LPP with epinephrine. Hearts excised from male Sprague-Dawley rats were perfused (37°C) at an initial pressure of 100 cm H 2 O using standard Langendorff method with Krebs-Henseleit buffer containing 0.75% bovine serum albumin, 0.4 mM non-labeled free fatty acid, 5.5 mM [U- 13 C]glucose, 1 mM [3- 13 C]pyruvate, 0.1 mM [3- 13 C]lactate. Perfusion pressure was reduced to 25 cm H 2 O after 30 min of perfusion for ischemia group and epinephrine added at 50 th min of perfusion. HP [1- 13 C]pyruvate was injected to heart and subsequently, 13 C NMR spectra were acquired. The freeze clamped heart tissues were extracted with perchloric acid and analyzed by high-resolution 1 H and 13 C NMR (14.1 T). The 31 P NMR spectroscopy confirmed significant myocardial ischemia under LPP conditions, demonstrated by a marked increase in the [Pi]/[ATP] ratio. Surprisingly, increased LDH flux was observed in the ischemic hearts (+18.4%) while the metabolism of HP [1- 13 C]pyruvate by PDH remained unchanged as indicated by bicarbonate production. To investigate the oxidative capacity of mitochondria, epinephrine was used to stimulate hearts. As anticipated, epinephrine increased heart rate (+23%) and coronary flow (+11%) under NPP. Epinephrine increased heart rate (-60%) but did not alter coronary flow under LPP. We found that epinephrine had no effect on the rate of bicarbonate production under LPP. The study demonstrated that the conversion of pyruvate to lactate increased in ischemic hearts but the degree of pyruvate oxidation was not affected by the low-flow ischemia. Adrenergic stimulation did not increase PDH flux in these ischemic hearts. Translation to human is likely to provide new data from low-flow myocardium.

İsatinin İskemi ve Reperfüzyon Hasarı Üzerine Etkisi: İzole Kalpte Deneysel Bir Çalışma

Introduction: Isatin is an endogen indole that is found in body fluids and reported as a neuroprotective, anticonvulsant, and sedative agent. The aim of the present study was to investigate the effects of isatin on left ventricle functions before and after low-flow ischemia in the isolated rat heart. Patients and Methods: Male Wistar rats were divided into four groups. Isatin (I, 50 mg/kg intraperitoneally) was administered in the I and I-atrial natriuretic peptide (ANP) groups 20 min before the hearts were placed on the Langendorff apparatus. Serum physiologic was administered to the control (C) and ANP groups. Low-flow ischemia was applied in all groups. ANP (0.1 μM/L) was added to perfusion solution in the ANP and I + ANP groups 15 min before ischemia. Left ventricular developed pressure (LVDP) and maximum and minimum pressure changes were recorded before and after ischemia. Cyclic guanosine monophosphate (cGMP) levels were measured in the perfusate in all groups. Results: LVDP and minimum pressure change values of the I group were found to be similar to the C group at 60 min of reperfusion. The level of cGMP in the I group was similar to the C group but lower than the ANP and I + ANP groups before and after ischemia. Conclusion: The administration of isatin prior to cardiac ischemia does not significantly alter cardiac function during the reperfusion period in rat heart. The results of the present study showed that isatin may not appear to have a disturbing effect on cardiac functions after low-flow ischemia.

Low-flow ischaemia and reperfusion in rat hearts: energetic of stunning and cardioprotection of genistein.

Low-flow ischemia (LFI) is consequent to coronary disease and produces cardiac stunning during reperfusion (R). Energetic performance and mechanisms of Ca2+ handling during LFI/R are not known. Moreover, cardioprotection of the phytoestrogen genistein (Gen) remains to be demonstrated in LFI/R. The aim was to study the mechanisms of the stunning consequent to LFI/R and the effects of Gen on both sexes. Rat ventricles were perfused inside a calorimeter to measure maximal pressure development (P) and total heat rate (Ht) before and during exposition to LFI/R. The mechanisms of stunning were evaluated with selective drugs. Female hearts (FH) developed higher postischemic contractile recovery (PICR) and muscle economy (P/Ht) than males (MH). Cardioprotection was sensitive to blockade of mKATP channels, UCam and NOS. Perfusion of 20μmol/l Gen reduced PICR and P/Ht during LFI/R in FH, and dysfunction was increased by mNCX blockade with mPTP opening. However, intraperitoneal 5mg/kg Gen (Gen-ip) was cardioprotective in both sexes, and the beneficial effect of Gen-ip was blocked by 100μmol/l 5-HD. FH are more protected than MH against the LFI/R dysfunction, which involves mitochondrial Ca2+ loss; Gen-ip was more cardioprotective in MH than in FH, mainly by activation of the mKATP channels.

GW28-e1228 Eleclazine (GS-6615), a novel and selective late sodium current inhibitor, suppresses ventricular arrhythmias induced by acute global low-flow ischemia

Late sodium current (INa,L) is increased in many pathological conditions, e.g., ischemic heart. Enhanced Na,L has been shown to elicit after-depolarizations and triggered arrhythmic activity. Eleclazine (GS-6615) has the properties of reducing INa,L in atrial and ventricular tissue and minimal

Cardiac myosin binding protein C, adrenergic stimulation and cardiac contractility

Myosin binding protein C remained a perplexing although integral component of the sarcomeric thick filament until the discovery that genetic defects in its corresponding gene is a frequent cause of hypertrophic cardiomyopathy. Basic science investigation subsequently revealed that it is one of the most potent regulators of cardiac contractility. Phosphorylation of its N-terminus upon adrenergic stimulation, causes increased order in myosin heads as well as increased ATPase activity, Fmax and Ca2+-sensitivity of contraction, while its dephosphorylation upon cholinergic stimulation or during low flow ischaemia leads to changes in the sarcomeric thick filament that diminish interaction between myosin heads and actin. This dynamic flux in phosphorylation upon adrenergic stimulation is not only crucial to normal cardiac function and structure, but also vital for protection against ischaemic injury. Genetically-driven deficiency or inadequacy in cMyBPC leads to severe cardiac dysfunction and structural changes, including cardiac hypertrophy and dilation, and particularly attenuates the adaptive increase in left ventricular contractility that follows on β-adrenergic stimulation or pressure overload, resulting in decreased systolic function, and reduced cardiac output.