Cardiac Ischemia Model Research Articles

The prophylactic and therapeutic effects of cannabidiol on lung injury secondary to cardiac ischemia model in rats via PERK/NRF2/CHOP/BCL2 pathway

Background Inflammation and oxidative stress are key players in lung injury stemming from cardiac ischemia (LISCI). Cannabidiol (CBD) demonstrates tissue-protective properties through its antioxidant, anti-inflammatory, and anti-apoptotic characteristics. This study aims to assess the preventive (p-CBD) and therapeutic (t-CBD) effects of CBD on LISCI. Methods Forty male Wistar Albino rats were divided into four groups: control (CON), LISCI, p-CBD, and t-CBD. The left anterior descending coronary artery was ligated for 30 min of ischemia followed by 30 min of reperfusion. Lung tissues were then extracted for histopathological, immunohistochemical, genetic, and biochemical analyses. Results Histopathologically, marked hyperemia, increased septal tissue thickness, and inflammatory cell infiltrations were observed in the lung tissues of the LISCI group. Spectrophotometrically, total oxidant status and oxidative stress index levels were elevated, while total antioxidant status levels were decreased. Immunohistochemically, expressions of cyclooxygenase-1 (COX1), granulocyte colony-stimulating factor (GCSF), interleukin-6 (IL6) were increased. In genetic analyses, PERK and CHOP expressions were increased, whereas Nuclear factor erythroid 2-related factor 2 (NRF2) and B-cell leukemia/lymphoma 2 protein (BCL2) expressions were decreased. These parameters were alleviated by both prophylactic and therapeutic CBD treatment protocols. Conclusion In LISCI-induced damage, both endoplasmic reticulum and mitochondrial stress, along with oxidative and inflammatory markers, were triggered, resulting in lung cell damage. However, both p-CBD and t-CBD treatments effectively reversed these mechanisms, normalizing all histopathological, biochemical, and PCR parameters.

Cardiotoxic Effects Produced by Omeprazole and Methylene Blue in an Animal Model of Cardiac Ischemia and Reperfusion and Potential Implications for the Pharmacological Strategy for Vasoplegic Syndrome.

Defined as systemic hypotension caused by intense vasodilation due to the loss of systemic vascular resistance, vasoplegic syndrome (VS) is associated with elevated morbidity and mortality in humans. Although vasopressors such as norepinephrine and vasopressin are the first-choice drugs for VS treatment, several other drugs such as methylene blue (MB) can be used as adjuvant therapy including rescue therapy. To develop new pharmacological strategies to reduce the risk of VS, we investigated the effects of treatments with MB (2 mg/kg/IV), omeprazole (OME, 10 mg/kg/IV), and their combination in an animal model of cardiac ischemia-reperfusion (CIR). The ventricular arrhythmia (VA), atrioventricular block (AVB), and lethality (LET) incidence rates caused by CIR (evaluated via ECG) and serum levels of the cardiac lesion biomarkers creatine kinase-MB (CK-MB) and troponin I (TnI) in adult rats pretreated with saline solution 0.9% and submitted to CIR (SS + CIR group) were compared to those pretreated with MB (MB + CIR group), OME (OME + CIR group), or the MB + OME combination (MB + OME + CIR group). The AVB and LET incidence rates in the MB + CIR (100%), OME + CIR (100%), and MB + OME + CIR (100%) groups were significantly higher compared to the SS + CIR group (60%). The serum level of CK-MB in these groups were also significantly higher compared to the SS + CIR group, demonstrating that the treatments before CIR with MB, OME, and MB + OME produced similar effects in relation to cardiac function and the occurrence of lesions. These results demonstrate that the treatment of animals subjected to the CIR protocol with OME produced the same effects promoted by the treatment with MB, which may suggest the possibility of using OME alone or in combination with MB in medical clinics in treatment of VS.

Role of long non-coding RNA Zeb2os in protection of the injured heart

Abstract Heart failure due to ischemic heart disease is one of the leading causes of death worldwide. Terminated vasculature and cardiomyocyte apoptosis due to myocardial infarction are critical events in infarct healing. Therefore, identifying factors that can enhance cardiac repair by restoring the capillary network and inhibiting cardiomyocyte death is of great significance. Accumulating evidence implies that long non-coding RNAs (lncRNAs) are potent regulators of cardiac development and disease. Our preliminary data show the potential cardioprotective function of the long non-coding RNA Zeb2 opposite strand (Zeb2os). This lncRNA overlaps and is antisense to ZEB2 at the 5' splice site of an intron within the 5' UTR. Zeb2os was shown to prevent splicing of the intron containing an internal ribosome entry site (IRES) that increases translation of the ZEB2 transcript. In line with the known function of ZEB2, Zeb2os has pro-angiogenic and anti-apoptotic functions in different organs. However, its role in the heart still has to be determined. Our results show that Zeb2os expression is decreased in cultured cardiomyocytes subjected to H2O2-mediated oxidative stress, which correlates with increased apoptosis and reduced antioxidant defence of cardiac muscle cells. AAV6-mediated overexpression of Zeb2os in hiPSC-derived cardiomyocytes protects from H2O2-induced apoptosis, suggesting it may have cardioprotective activity, possibly through regulating Zeb2 translation. Additionally, systemic AAV9-mediated delivery of Zeb2os to the mouse heart enhanced angiogenesis by increasing the number of proliferating endothelial cells. Moreover, RNA-sequencing of Zeb2os overexpressing hearts at baseline implicated Zeb2os in the regulation of numerous genes related to response to hypoxia. In conclusion, we uncovered/identified Zeb2os as a new potential cardioprotective factor with pro-angiogenic and anti-apoptotic properties. We are currently investigating the effect of AAV9-mediated delivery of Zeb2os in a mouse model of cardiac ischemia to understand further its protective role in the injured heart, which is an essential step towards the development of improved therapies for ischemic heart disease.

Tipping the scales of understanding: An engineering approach to design and implement whole-body cardiac electrophysiology experimental models.

The study of cardiac electrophysiology is built on experimental models that span all scales, from ion channels to whole-body preparations. Novel discoveries made at each scale have contributed to our fundamental understanding of human cardiac electrophysiology, which informs clinicians as they detect, diagnose, and treat complex cardiac pathologies. This expert review describes an engineering approach to developing experimental models that is applicable across scales. The review also outlines how we applied the approach to create a set of multiscale whole-body experimental models of cardiac electrophysiology, models that are driving new insights into the response of the myocardium to acute ischemia. Specifically, we propose that researchers must address three critical requirements to develop an effective experimental model: 1) how the experimental model replicates and maintains human physiological conditions, 2) how the interventions possible with the experimental model capture human pathophysiology, and 3) what signals need to be measured, at which levels of resolution and fidelity, and what are the resulting requirements of the measurement system and the access to the organs of interest. We will discuss these requirements in the context of two examples of whole-body experimental models, a closed chest in situ model of cardiac ischemia and an isolated-heart, torso-tank preparation, both of which we have developed over decades and used to gather valuable insights from hundreds of experiments.

Rapid fall in circulating non-classical monocytes in ST elevation myocardial infarction patients correlates with cardiac injury.

Myocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. We retrospectively analyzed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical, and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand, circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (P<.05) and correlated inversely with left ventricular function (r=0.41, P=.04). The early fall in NC monocytes post-reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. In conclusion, rapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature.

Cardioprotection stimulated by resveratrol and grape products prevents lethal cardiac arrhythmias in an animal model of ischemia and reperfusion.

ABSTRACTPurposeTo evaluate the preventive cardioprotective effects of resveratrol and grape products, such as grape juice and red wine, in animal model of cardiac ischemia and reperfusion.MethodsMale Wistar rats orally pretreated for 21-days with resveratrol and grape products were anesthetized and placed on mechanical ventilation to surgically induce cardiac ischemia and reperfusion by obstruction (ischemia) followed by liberation (reperfusion) of blood circulation in left descending coronary artery. These rats were submitted to the electrocardiogram (ECG) analysis to evaluate the effects of pretreatment with resveratrol and grape products on the incidence of ventricular arrhythmias (VA), atrioventricular block (AVB) and lethality (LET) resulting from cardiac ischemia and reperfusion.ResultsIt was observed that the incidence of AVB was significantly lower in rats pretreated with resveratrol (25%), grape juice (37.5%) or red wine (12.5%) than in rats treated with saline solution (80%) or ethanol (80%). Similarly, incidence of LET was also significantly lower in rats pretreated with resveratrol (25%), grape juice (25%) or red wine (0%) than in rats treated with saline solution (62.5%) or ethanol (75%).ConclusionsThese results indicate that the cardioprotective response stimulated by resveratrol and grape products prevents the lethal cardiac arrhythmias in animal model of ischemia and reperfusion, supporting the idea that this treatment can be beneficial for prevention of severe cardiac arrhythmias in patients with ischemic heart disease.

VEGF-B electrotransfer mediated gene therapy induces cardiomyogenesis in a rat model of cardiac ischemia

Atherosclerosis induced myocardial infarction (MI) continues to be a major public health concern. Regenerative therapies that restore cardiac muscle cells are largely absent. The rate of cardiomyogenesis in adults is insufficient to compensate for MI damage. In this study, we explored the capacity of a gene therapy approach to promote cardiomyogenesis. We hypothesized that VEGF-B, critical during fetal heart development, could promote cardiomyogenesis in adult ischemic hearts. Gene electrotransfer (GET), a physical method of in vivo gene delivery, was adapted to the rat model of MI. Favorable pulsing parameters were then used for delivery of pVEGF-B and compared to a sham control in terms of infarct size, cardiomyocyte proliferation and presence of new cardiomyocytes. Ki67 immunoreactivity was used for proliferation analysis. Newly synthetized DNA was labeled with BrdU to identify new cells post-infarction. Cardiac troponin co-localization indicated proliferating and new cardiomyocytes histologically. Eight weeks post-treatment, GET pVEGF-B treated hearts had significantly smaller infarcts than the sham control group (p < 0.04). Proliferating and new cardiomyocytes were only present in the GET of pVEGF-B group, and absent in the controls. In summary, GET pVEGF-B promoted cardiomyogenesis post-MI, demonstrating for the first time direct evidence of myocardial regeneration post-infarction.

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery.

Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.

An investigation of postmortem urotensin II receptor levels in brain and kidney tissues in a rat model of cardiac ischemia

This study aimed to investigate changes in postmortem urotensin II receptor (UTR) levels in brain and kidney tissues in a rat model of cardiac ischemia. The rats were divided into two groups: a control group and a cardiac ischemia-induced group. Cardiac ischemia was created by an intraperitoneal injection of a single lethal dose of isoproterenol (ISO; 850 mg/kg). Plasma UT, blood urea nitrogen, and creatinine levels were determined 0 h postmortem. Brain and kidney UTR mRNA expression levels were determined 0, 1, 3, 6, 12, 24, 48, and 72 h postmortem. The histopathological appearance of brain and kidney tissues was also evaluated. Plasma UT and plasma creatinine levels were increased in the cardiac ischemia-induced group as compared with those in the control group (P < 0.001). Ischemia resulted in histopathological changes in brain and cerebellum tissue. The morphological evaluation revealed Purkinje cell degeneration (P = 0.037) and dark neurons (P = 0.004). The UTR expression level decreased after 1 h postmortem in the brain and after 3 h postmortem in the kidneys in the cardiac ischemia-induced group as compared with that in the control group (P < 0.001). The observed changes in UTR expression levels may be valuable in clinical practice in the field of forensic medicine. These changes may be used as a marker in postmortem evaluations of sudden death caused by ischemia-induced cardiac shock.

Cardioprotective effect of N-methylnicotinamide salt of pyruvate in experimental model of cardiac hypoxia.

Pyruvate improves contractility of normal, hypoxic, and post-ischemic myocardium. However, sodium overload is a major problem with its therapeutic application if sodium pyruvate is used. Development of alternative forms such as N-1-methylnicotinamide (MNA) pyruvate may help to overcome this problem. The aim of the study was to investigate the effect of MNA pyruvate in a murine model of cardiac ischemia. Seven month old male ApoE-/-LDLr-/- mice that develop myocardial infarction when exposed to hypoxic stress, were used in this study. Hypoxia (8% O2 in inspired air) was maintained for 8min and was followed by reoxygenation (21% O2 in inspired air). Four groups of mice were treated 10min before the hypoxic event by intravenous injection of MNA, MNA pyruvate, sodium pyruvate, and saline as control. The myocardial ischemia and damage was recorded by ECG. Four hours following the hypoxic episode serum troponin T and creatine kinase activity were measured. Significant hypernatremia was found in the sodium pyruvate group. During hypoxia, control and MNA group developed profound STU depressions on ECG while no changes were observed in MNA pyruvate and sodium pyruvate group. Creatine kinase activity and troponin T content in the mice plasma were significantly higher in the control and MNA group as compared to the MNA pyruvate and sodium pyruvate group. This study demonstrated that administration of MNA pyruvate prior to a hypoxia-induced cardiac event was cardioprotective. This intervention did not cause hypernatremia in contrast to sodium pyruvate.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

CMX-2043 Efficacy in a Rat Model of Cardiac Ischemia-Reperfusion Injury.

α-Lipoic acid (LA) has been shown to offer protection against ischemia-reperfusion injury (IRI) in multiple organ systems. N-[(R)-1,2-dithiolane-3-pentanoyl]-L-glutamyl-L-alanine (CMX-2043), a novel analogue of LA, was studied as part of a preclinical development program intended to identify safe and efficacious drug candidates for prevention or reduction in myocardial IRI. This study was designed to evaluate the efficacy of CMX-2043 in an animal model of myocardial IRI and to establish effective dosing conditions. CMX-2043 or placebo was administered at different doses, routes, and times in male Sprague-Dawley rats subjected to 30-minute left coronary artery ligation. Fluorescent microsphere injection defined the area at risk (AR). Animals were euthanized 24 hours after reperfusion, and the hearts were excised, sectioned, and stained with triphenyltetrazolium. Cytoprotective effectiveness was determined by comparing the unstained myocardial infarction zone (MI) to the ischemic AR. The reduction in the MI-AR ratio was used as the primary measure of drug efficacy relative to placebo injections. Treatment with CMX-2043 reduced myocardial IRI as measured by the MI-AR ratio and the incidence of arrhythmia. The compound was effective when administered by injection, both before and during the ischemic injury and at reperfusion. The most efficacious dose was that administered 15 minutes prior to the ischemic event and resulted in a 36% (P < .001) reduction in MI-AR ratio compared to vehicle control.

Abstract 19076: ATAC-seq Implicates Epigenome-wide CTCF Dissociation in the Progression of Cardiac Ischemia

Introduction: Heart disease is currently the most common cause of death in the United States. In 2010 alone, over 375,000 people died of coronary heart disease, of which over 120,000 were from myocardial infarction. Due to technical challenges associated with current techniques, profiling epigenome changes due to myocardial infarction has been limited. We have optimized an assay for transposase-accessible chromatin with sequencing (ATAC-seq) protocol that circumvents these challenges and works well with frozen tissue. Hypothesis: We assessed the hypothesis that ATAC-seq would enable the identification of novel chromatin remodeling and transcription factor binding events in a murine model of cardiac ischemia. Methods: Six 10 ± 2 week old C57BL/6J mice were paired by same gender, age, and litter, and mice from these pairs were selected for either mid left anterior descending artery ligation or sham surgery, both for 30 minutes. Successful ligation was confirmed by blanching of the tissue and representative electrocardiography changes. We performed our ATAC-seq protocol on left ventricular tissues, and regions of open chromatin were determined using the Hotspot algorithm at a false detection rate of 0.5%. Gene ontology was performed with the Genomic Regions Enrichment of Annotations Tool (GREAT), and motif discovery was performed with the Multiple Em for Motif Elicitation (MEME) Suite. Results: From stringent overlap criteria were obtained 159 loci that became “open” due to ischemia and 782 loci that became “closed”. Downregulation of growth and signaling processes was determined by GREAT. MEME strongly implicated the dissociation of CCCTC-binding Factor (CTCF) due to ischemia (MEME E-score = 8.10e-235; motif identification p -value = 1.88e-18; 262/782 of “closed” loci). CTCF ChIP-seq data and footprint analysis enabled the further identification of a robust set of 189 CTCF differential binding events. Conclusions: ATAC-seq was able to identify widespread chromatin remodeling due to cardiac ischemia alone. We identified a robust set of 189 gene loci from which CTCF binding is attenuated in response to this disease state. To our knowledge, this represents the first study that has examined chromatin accessibility changes in the setting of cardiac ischemia.

Beneficial properties of selenium incorporated guar gum nanoparticles against ischemia/reperfusion in cardiomyoblasts (H9c2).

Nanotechnology for the treatment and diagnosis has been emerging recently as a potential area of research and development. In the present study, selenium incorporated guar gum nanoparticles have been prepared by nanoprecipitation and characterized by transmission electron microscopy and particle size analysis. The nanoparticles were screened for antioxidant potential (metal chelation, total reducing power and hydroxyl radical scavenging activity) and were evaluated against the cell line based cardiac ischemia/reperfusion model with special emphasis on oxidative stress and mitochondrial parameters. The cell based cardiac ischemia model was employed using H9c2 cell lines. Investigations revealed that there was a significant alteration (P ≤ 0.05) in the innate antioxidant status (glutathione↓, glutathione peroxidase↓, thioredoxin reductase↓, superoxide dismutase↓, catalase↓, lipid peroxidation↑, protein carbonyl↑, xanthine oxidase↑ and caspase 3 activity↑), mitochondrial functions (reactive oxygen species generation, membrane potential, and pore opening) and calcium homeostasis (calcium ATPase and intracellular calcium overload) during both ischemia and reperfusion. For comparative evaluation, selenium, guar gum and selenium incorporated guar gum nanoparticles were evaluated for their protective properties against ischemia/reperfusion. The study reveals that selenium incorporated guar gum nanoparticles were better at protecting the cells from ischemia/reperfusion compared to selenium and guar gum nanoparticles. The potent antioxidant capability shown by the sample in in vitro assays may be the biochemical basis of its better biological activity. Further, the nanodimensions of the particle may be the additional factor responsible for its better effect.

In vivo Evans blue dye infusion allows monitoring of auto‐reperfusion in a rat model of cardiac ischemia

One major problem with animal models of cardiac ischemia is the huge variability in the degree and the actual duration of coronary occlusion, in large because of auto‐reperfusion that results in early‐onset partial‐reperfusion and the subsequent partial‐ischemia. The Evans blue dye (EBD) is a commonly used tracer for defining the boundary of ischemia in excised heart with re‐occluded coronary artery. This same dye is also commonly used in vivo for blood‐brain barrier research, and has been used safely in human subjects. In this study, we intravenously injected the rat with EBD immediately following left coronary arterial ligation in vivo, and observed its initial clear‐cut exclusion from the ischemic region. This initial exclusion was later followed by slow leakage of EBD into the ischemic region, at a pace depending on the “tightness” of the initial occlusion. Moreover, complete reperfusion of the heart resulted in the accumulation of EBD in the ischemic region, after which perfusion of the excised heart resulted in a visible clear‐cut boundary showing EBD‐filled ischemic region, which closely resembles the initial pre‐marked ischemic region. In conclusion, our refined rat model of cardiac ischemia allows for in vivo detection of early auto‐reperfusion and post hoc ex vivo detection of initial area of ischemia, and thus can effectively reduce technical variability in pre‐clinical experiments. (NSC of Taiwan)

TREATMENT OF REPERFUSION INJURY WITH RECOMBINANT ADAMTS13 IN A PORCINE MODEL OF ACUTE MYOCARDIAL INFARCTION

Background: No reflow and decreased microvascular perfusion after percutaneous coronary intervention increase morbidity and mortality in ST-elevation myocardial infarction (STEMI) patients. No reflow may be mediated by platelet vessel wall interaction that is governed by von Willebrand factor. ADAMTS13 is a metalloprotease that cleaves von Willebrand factor, thereby reducing its prohemostatic properties. There is considerable evidence that ADAMTS13 levels decrease and von Willebrand factor levels increase in STEMI patients. Recombinant ADAMTS13 has been effective in reducing cerebral infarct size in a murine model of stroke. Aims: In this study recombinant ADAMTS13 was tested as a potential treatment of no reflow in a porcine model of cardiac ischemia and reperfusion. Methods: In 23 female swine (median age 83 days, median weight 30 kg) a balloon was inflated in the circumflex coronary artery for 75 min. Fifteen minutes after reperfusion, an intracoronary bolus of either recombinant ADAMTS13 (400 U/kg, Baxter Innovations Vienna, Austria) or vehicle was given. Results: ADAMTS13 activity significantly increased in treated pigs (from median 18%, IQR 14.5-24.0 to median 324%, IQR 117.0- 384.0, P = 0.003) whereas no change was observed in the control group. Animals were sacrificed 7 days later for histopathology. There was no difference in the size of myocardial necrosis as assessed with plasma Troponin T measurements, continuous 12 leads ECG, macroscopical infarct analysis, and histopathology using phosphotungstic acid-hematoxylin staining. Microvascular obstruction as estimated by staining with anti-CD31/Hematoxylin and counting of vessels and microthrombi was similar for both groups. Conclusions: Intracoronary treatment with recombinant ADAMTS13 did not prevent formation of microthrombi and did not decrease infarct size in this porcine coronary model of ischemia and reperfusion.

Sulfaphenazole Attenuates Myocardial Cell Apoptosis Accompanied With Cardiac Ischemia–Reperfusion by Suppressing the Expression of BimEL and Noxa

We previously reported the administration of a potent cytochrome P450 inhibitor, sulfaphenazole (SPZ), to suppress oxidative stress and the extension of myocardial infarct size in a rat model of cardiac ischemia–reperfusion (I/R). The aim of this study was to investigate the effects of SPZ on the myocardial cell apoptosis induced by I/R in rats. I/R injury was evoked by ligation of the left anterior descending coronary artery for 1 h, followed by reperfusion for 3 h. TUNEL-positive nuclei were detected and nucleosomal DNA fragmentation was observed 3 h after reperfusion. The administration of SPZ largely suppressed the cardiac DNA fragmentation induced by I/R. A pan-caspase inhibitor, z-VAD-fmk, had no effect on DNA fragmentation. Caspase-3/7 was not activated 3 h after reperfusion. Decreases in the mitochondrial membrane potential and cytochrome c release from the mitochondria to cytosol were detected 3 h after reperfusion. The expression levels of BimEL and Noxa were elevated 3 h after reperfusion. These phenomena were suppressed by the administration of SPZ. Taken together, treatment with SPZ could attenuate the myocardial cell apoptosis accompanied with I/R by inhibiting the mitochondrial dysfunction due to decreases in the expression of BimEL and Noxa.

A synthetic prostacyclin agonist, ONO-1301, ameliorates ventricular remodeling after acute myocardial infarction via upregulation of HGF in rat

It has been shown that administration of angiogenic factors can augment collateral growth in ischemic tissues. We previously reported that ONO-1301, a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity, promotes production of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) from various cell types. We evaluated therapeutic efficacy of ONO-1301 in rat cardiac ischemia model. Ligation of left anterior descending coronary arteries was performed to 10-week-old Wistar rats, and the slow releasing form of ONO-1301 was administrated subcutaneously 3 days after the ligation (control group and ONO-1301 group). Hemodynamic parameters and plasma BNP levels were significantly improved by ONO-1301. Histological analysis revealed that ONO-1301 suppressed fibrotic changes in the myocardium (27.7±3.1% vs 23.3±3.6%, p<0.01). HGF and c-Met expression in the myocardium was significantly up-regulated by ONO-1301. Beneficial effects of ONO-1301 were abrogated by administration of a neutralizing anti-HGF antibody. These findings indicate that ONO-1301 has cardioprotective effects in the rat ischemic heart model via augmentation of endogenous HGF production.

Acid-Sensing Ion Channel 3, But Not Capsaicin Receptor TRPV1, Plays a Protective Role in Isoproterenol-Induced Myocardial Ischemia in Mice

Cardiac angina is the hallmark of myocardial ischemia, but the role of the cardiac sensory nerve has received relatively little attention. Recently, both acid-sensing ion channel 3 (ASIC3) and capsaicin receptor (TRPV1) have been suggested as important mediators in sensing cardiac ischemia. However, studies comparing the physiological roles of ASIC3 and TRPV1 in the neuronal-cardiac sensing circuits in vivo are lacking. Isoproterenol (1.5 mg/kg, intraperitoneally) was used to induce transient cardiac ischemia in Asic3(+/+) and Asic3(-/-) mice and a radio-telemetry system was used for electrocardiography with mice in a conscious state. Isoproterenol-induced cardiac ischemia was first demonstrated with ST-segment depression and further confirmed by hypoxia-mediated chemical reactions in cardiac tissue. Mice lacking Asic3 showed prolonged duration of ST-segment depression compared with Asic3(+/+) mice (44.3 ± 3.1 vs. 31.7 ± 2.9 min; P < 0.05). Although ischemia was transient, severe cardiac fibrosis was found in Asic3(-/-) but not in Asic3(+/+) mice littermates. In contrast, isoproterenol-injected Trpv1(+/+) and Trpv1(-/-) mice showed no difference in duration of ST-segment depression and, surprisingly, deletion of Trpv1 did not aggravate cardiac fibrosis. An isoproterenol-induced cardiac ischemia model mimicking clinical conditions of early cardiac angina was used to demonstrate that ASIC3 but not TRPV1 plays a protective role in sensing cardiac ischemia.