Perfused Heart Model Research Articles

Myocardial Protective Effect of Xuebijing Injection on Extracorporeal Membrane Oxygenation Perfused Isolated Langendorff Heart Based on the PI3K/AKT Pathway

Objective: This study aimed to investigate the myocardial protective effect of Xuebijing injection in a model of extracorporeal membrane oxygenation (ECMO) isolated heart perfusion and determine the role of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Methods: To establish an ECMO isolated heart perfusion model, Guangxi Bama miniature pigs were randomly divided into two groups: (1) a normal saline group (NS group), in which 50 mL of normal saline was added to the perfusion solution; and (2) a Xuebijing injection group (XBJI group), in which 10 mL of XBJI was added to the perfusion solution and then continuously pumped at 5 mL/h. Perfusion was maintained for 8 h. The hemodynamic changes, inflammatory response, and myocardial enzyme levels at T0, T2, T4, T6, and T8 were evaluated. The protein expression level of the PI3K/AKT pathway was analyzed by Western blot analysis and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Hematoxylin and eosin staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of T8 cardiomyopathy. Results: There was no significant difference in hemodynamics between the two groups. XBJI could reduce serum inflammatory factors and myocardial enzyme levels. XBJI also upregulated the expression of PI3K and AKT mRNA and the phosphorylation levels of PI3K and AKT proteins. The results of pathological and electron microscopy showed that XBJI effectively reduced myocardial cell and mitochondrial damage. Conclusion: XBJI can reduce myocardial inflammation and myocardial cell and mitochondrial damage in isolated heart perfusion. XBJI may play a role in the myocardial protection of ECMO isolated heart perfusion by activating the PI3K/AKT pathway.

History of the development of isolated heart perfusion experimental model and its pioneering role in understanding heart physiology.

The isolated heart perfusion model, a fundamental tool in cardiovascular research, has evolved significantly since its inception in the late 19th century. This review traces the development of the isolated heart model, from its early adaptations by pioneers such as Langendorff and Starling to modern advancements by researchers like Morgan and Neely. We discuss the various applications of the model in pharmacological testing, disease modeling, and educational settings, emphasizing its crucial role in understanding cardiac function and disease mechanisms. Recent technological enhancements, including high-resolution imaging, integration with bioengineering, and advanced genomic and proteomic analyses, have significantly broadened the capabilities of these models. Looking forward, we explore potential future developments such as the integration of precision medicine, stem cell research, and artificial intelligence, which promise to revolutionize the use of isolated heart perfusion models. This review highlights the model's crucial role in bridging experimental research and clinical applications.

CREB-binding protein and HIF-1α/β-catenin to upregulate miR-322 and alleviate myocardial ischemia-reperfusion injury.

Myocardial ischemia/reperfusion injury (MIRI) is a prevalent condition associated with numerous critical clinical conditions. miR-322 has been implicated in MIRI through poorly understood mechanisms. Our preliminary analysis indicated potential interaction of CREB-binding protein (CBP), a transcriptional coactivator and acetyltransferase, with HIF-1α/β-catenin, which might regulate miR-322 expression. We, therefore, hypothesized that CBP/HIF-1α/β-catenin/miR-322 axis might play a role in MIRI. Rat cardiomyocytes subjected to oxygen-glucose deprivation /reperfusion (OGD/R) and Langendorff perfused heart model were used to model MIRI in vitro and in vivo, respectively. We used various techniques such as CCK-8 assay, transferase dUTP nick end labeling staining, western blotting, RT-qPCR, chromatin immunoprecipitation (ChIP), dual-luciferase assay, co-immunoprecipitation (Co-IP), hematoxylin and eosin staining, and TTC staining to assess cell viability, apoptosis, and the levels of CBP, HIF-1α, β-catenin, miR-322, and acetylation. Our results indicate that OGD/R in cardiomyocytes decreased CBP/HIF-1α/β-catenin/miR-322 expression, increased cell apoptosis and cytokines, and reduced cell viability. However, overexpression of CBP or miR-322 suppressed OGD/R-induced cell injury, while knockdown of HIF-1α/β-catenin further exacerbated the damage. HIF-1α/β-catenin bound to miR-322 promoter to promote its expression, while CBP acetylated HIF-1α/β-catenin for stabilization. Overexpression of CBP attenuated MIRI in rats by acetylating HIF-1α/β-catenin to stabilize their expression, resulting in stronger binding of HIF-1α/β-catenin with the miR-322 promoter and subsequent increased miR-322 levels. Therefore, activating CBP/HIF-1α/β-catenin/miR-322 signaling may be a potential approach to treat MIRI.

Effect of Dunaliella salina on myocardial ischemia-reperfusion injury through KEAP1/NRF2 pathway activation and JAK2/STAT3 pathway inhibition

Myocardial ischemia-reperfusion injury (MIRI) is a life-threatening vascular emergency, in which oxidative stress and excessive inflammation reactions play significant roles. Dunaliella salina is rich in natural beta (β)-carotene, which is considered an antioxidant and anti-cancer compound, preventing night blindness, delaying aging, and regulating immunity. Due to its unique nutritional and functional components, D. salina has the potential to be developed as a nutritional health food. At present, it is unknown whether D. salina can rescue MIRI. The present study aimed at investigating whether D. salina has protective effect on MIRI and at exploring its potential mechanism. We developed a Langendorff perfused heart model in mice. The mice were given D. salina by gavage at a dose of 500 mg/kg for 7 days consecutively. It was found that D. salina could improve left ventricle function and reduce the rate of malignant arrhythmia and infarct size (P < 0.01). Furthermore, D. salina administration increased superoxide dismutase and decreased malondialdehyde content in myocardial tissue (P < 0.01). Importantly, real-time polymerase chain reaction and Western blot results showed that D. salina caused nuclear factor (erythroid-derived 2)-like 2 (NRF2) activation and enhanced the expression of antioxidative genes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) (P < 0.05 and P < 0.01, respectively). At the same time, the phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription 3 (STAT3) was inhibited (P < 0.05 and P < 0.01, respectively), and proinflammatory cytokines interleukin-1 beta (IL-1β) and IL-6 decreased with D. salina administration (P < 0.05). In conclusion, D. salina has a protective effect on MIRI, which may be mediated by promoting kelch-like ECH-associated protein 1 (KEAP1)/NRF2 pathway and inhibiting JAK2/STAT3 signaling pathway.

Acetate suppresses myocardial contraction via the short-chain fatty acid receptor GPR43.

The heart has high energy requirements, with an estimated 40%-60% of myocardial ATP production derived from the oxidation of fatty acids under physiological conditions. However, the effect of short-chain fatty acids on myocardial contraction remains controversial, warranting further research. The present study sought to investigate the effects and mechanisms of acetate, a short-chain fatty acid, on myocardial contraction in rat ventricular myocytes. Echocardiography and Langendorff heart perfusion were used to evaluate cardiac function. Cell shortening and calcium transient were measured in isolated cardiomyocytes. The patch-clamp method determined the action potential and L-type Ca2+ current in cardiomyocytes. Moreover, the expression of GPR43, a type of short-chain fatty acid receptors in cardiomyocytes was examined by immunofluorescent staining and Western blot. We demonstrated that acetate transiently reduced left ventricular developmental pressure in isolated Langendorff heart perfusion model, with no effect on stroke volume and cardiac output in vivo. In addition, acetate transiently and reversibly inhibited cardiomyocyte contraction and calcium transient. Acetate did not affect the action potential and L-type Ca2+ currents in cardiomyocytes. As a short-chain fatty acid receptor, GPR43 was expressed in rat cardiomyocytes. Furthermore, the GPR43 antagonist GLPG0974 prevented the acetate-induced inhibitory effect on myocardial contraction. We conclude that acetate transiently inhibits contraction via the short-chain fatty acid receptor GPR43 in cardiomyocytes.

Abstract 14127: The Role of Sodium Glucose Co-Transporter 1 in Hyperglycemia Ischemia Reperfusion Injury

Introduction: Hyperglycemia is a common finding in ACS patients in both diabetic and non-diabetic, it is considered a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury is not fully understood, whether the Sodium Glucose Co-Transporter 1(SGLT1) plays a role in increase injury, before and/or after reperfusion, remains to be elucidated. SGLT2 inhibitors clinical trials have shown significant improvements in cardiovascular outcomes in diabetic and non-diabetic, yet the mechanism is not fully understood and whether SGLT1 plays a role in infarct augmentation remains to be elucidated. Hypothesis: High glucose at reperfusion leads to excess myocardial injury and the increased injury is mediated through the activity of SGLT1. Methods: RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and Zucker diabetic rats. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose on myocardium at reperfusion. Canagliflozin a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 and SGLT2 transporter and 5nmol/L to block only the SGLT2 transposer) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results: Our data reveal that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model and it is abrogated through the administration of both mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, and through the administration of novel specific SGLT1 inhibitor, Mizagliflozin. Conclusions: We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

The cardioprotective effect of inhibiting SGLT1 in hyperglycemia ischemia reperfusion injury

Abstract Background Diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Hyperglycemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. Purpose Investigate if SGLT1 is involved in a glucotoxicity injury during IRI and whether inhibiting SGLT1 with an SGLT1 inhibitor may reduce infarct size. Method RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22mmol) on myocardium at reperfusion. Canagliflozin (CANA) a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 receptor and SGLT2 and 5nmol/L to block only the SGLT2 receptor) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results We showed that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. we demonstrate that hyperglycemia at reperfusion is injurious to myocardium with an increase of myocardial infarction. Our data reveal that glucose exacerbation of injury appears to be mediated via SGLT1. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model is abrogated through the administration of a clinically available mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, but by the SGLT2-selective concentration. Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this incre Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): The government of saudi Arabia

Protein Phosphatase 2A Improves Cardiac Functional Response to Ischemia and Sepsis.

Reversible protein phosphorylation is a posttranslational modification of regulatory proteins involved in cardiac signaling pathways. Here, we focus on the role of protein phosphatase 2A (PP2A) for cardiac gene expression and stress response using a transgenic mouse model with cardiac myocyte-specific overexpression of the catalytic subunit of PP2A (PP2A-TG). Gene and protein expression were assessed under basal conditions by gene chip analysis and Western blotting. Some cardiac genes related to the cell metabolism and to protein phosphorylation such as kinases and phosphatases were altered in PP2A-TG compared to wild type mice (WT). As cardiac stressors, a lipopolysaccharide (LPS)-induced sepsis in vivo and a global cardiac ischemia in vitro (stop-flow isolated perfused heart model) were examined. Whereas the basal cardiac function was reduced in PP2A-TG as studied by echocardiography or as studied in the isolated work-performing heart, the acute LPS- or ischemia-induced cardiac dysfunction deteriorated less in PP2A-TG compared to WT. From the data, we conclude that increased PP2A activity may influence the acute stress tolerance of cardiac myocytes.

The Pleiotropic Effects of Oxygen-Derived Free-Radical Scavengers on the Graft During Normothermic Ex Situ Heart Perfusion

<h3>Purpose</h3> The management of oxidative stress is pivotal during ESHP. The lack of sufficient antioxidant systems allow the accumulation of reactive oxygen species (ROS) which can negatively affect myocardial function. We aim to evaluate the use of oxygen-derived free-radical (OFR) scavengers on cardiac functional preservation during ESHP. <h3>Methods</h3> Using a porcine heart perfusion model, we compared the effect of different OFR scavengers. Hearts from juvenile Yorkshire pigs were perfused in working mode for 6 hours either in the control group (n=6) or adding polyethylene glycol (PEG)-catalase (10,000U/L, n=6) and PEG-superoxide dismutase (SOD) (550U/L, n=6) at the initiation of perfusion. Another group (n=6) of freshly procured heart were obtained as tissue control. Contractile function, myocardial injury and endothelial integrity were evaluated. The oxidative modification and antioxidant activity were determined in the myocardium and coronary vasculature. <h3>Results</h3> Cardiac function was better preserved in the catalase group (p<0.05, Figure A). Catalase and SOD both decreased perfusate cTnI, indicating less myocardial injury. The catalase group had lower perfusate vWF than the other two groups, suggesting less endothelial injury (p<0.05, Figure B). Compared to SOD, catalase reduced ROS production and oxidatively modified protein, indicated by protein carbonyl content in the myocardium and nitrotyrosine in the coronary vasculature (p<0.05, Figure C). The catalase and glutathione peroxidase activity decreased in all groups. However, adding catalase partially restored enzyme activity (Figure D). <h3>Conclusion</h3> The antioxidant enzyme activity decreased during <i>ex situ</i> heart perfusion. Catalase better preserved cardiac function and coronary endothelium than SOD, likely resulting from distinct oxidative stress states in the graft. Catalase and SOD have opposing effects on hydrogen peroxide detoxification, which may explain the differences observed in this study.

Entecavir competitively inhibits deoxyguanosine and deoxyadenosine phosphorylation in isolated mitochondria and the perfused rat heart

Deoxyguanosine kinase (dGK) is reported responsible for the phosphorylation of deoxyadenosine (dA) and deoxyguanosine (dG) in the mitochondrial purine salvage pathway. Antiviral nucleoside analogs known as nucleoside reverse transcriptase inhibitors (NRTIs) must be phosphorylated by host enzymes for the analog to become active. We address the possibility that NRTI purine analogs may be competitive inhibitors of dGK. From a group of such analogs, we demonstrate that entecavir (ETV) competitively inhibited the phosphorylation of dG and dA in rat mitochondria. Mitochondria from the brain, heart, kidney, and liver showed a marked preference for phosphorylation of dG over dA (10–30-fold) and ETV over dA (2.5–4-fold). We found that ETV inhibited the phosphorylation of dG with an IC50 of 15.3 ± 2.2 μM and that ETV and dG were both potent inhibitors of dA phosphorylation with IC50s of 0.034 ± 0.007 and 0.028 ± 0.006 μM, respectively. In addition, the phosphorylation of dG and ETV followed Michaelis–Menten kinetics and each competitively inhibited the phosphorylation of the other. We observed that the kinetics of dA phosphorylation were strikingly different from those of dG phosphorylation, with an exponentially lower affinity for dGK and no effect of dA on dG or ETV phosphorylation. Finally, in an isolated heart perfusion model, we demonstrated that dG, dA, and ETV were phosphorylated and dG phosphorylation was inhibited by ETV. Taken together, these data demonstrate that dGK is inhibited by ETV and that the primary role of dGK is in the phosphorylation of dG rather than dA.

Экспериментальное изучение влияния иммобилизированных субтилизинов на работоспособность изолированного сердца крысы

Introduction. Currently, a new pharmacological technology of thrombolytic therapy using immobilized subtilisins (ISs) has been introduced into clinical practice. Experimental and clinical studies have shown the high efficacy and safety of the treatment of thrombotic diseases using the drug Thrombovasim®, created on the basis of ISs. Although this drug has been widely studied as a thrombolytic, its pluripotent pharmacological properties have not been studied in detail. Aim. To study the performance of an isolated rat heart under the coronary perfusion with ISs. Materials and methods. The Langendorff’s perfused heart model was used in the study. The experiment included 50 Wistar rats. Animals were divided into 5 groups (10 animals each): group 1 – control (hearts perfused with Krebs-Henseleit solution) and 4 experimental groups – hearts perfused with ISs at 4 concentrations of 170, 340, 510 and 1020 IU/l respectively. The performance indicator of an isolated heart was assessed, reflecting the actual work of the heart muscle and expressed as the product of the heart rate and the strength of its contractions in the form of the pressure developed by the left ventricle. Results. No negative ISs effect on the performance of the isolated rat heart was found. A tendency to increase the performance in the first 5 minutes of using ISs was revealed. The achievement of the maximum and the duration of this effect depend on the ISs contents in perfusate. At a concentration of 170 IU/l, an obvious increase in the effect is found from the 10th minute, at higher concentrations – from the 5th minute. The duration of the increasing performance effect when using doses of 170–510 IU/l is maintained up to the 20th minute, when using ISs at a dose of 1020 IU/l – up to the 30th minute. The return of the performance indicators to the baseline values is observed at the 30th minute, at a dose of 340 IU/l – at the 40th minute. When perfused with a solution of ISs at concentrations of 510 and 1020 IU/l, by the 40th minute, the effect of increasing performance weakens, but the parameters still remain at higher levels as compaired to those at baseline. Conclusion. The experimental data obtained show that ISs increase the performance of the heart. This circumstance will allow expanding the use of the drug Thrombovasim® in a catastrophe of the coronary circulation.

Male and Female Rats Have Different Physiological Response to High-Fat High-Sucrose Diet but Similar Myocardial Sensitivity to Ischemia-Reperfusion Injury.

Prediabetes is a strong predictor of type 2 diabetes and its associated cardiovascular complications, but few studies explore sexual dimorphism in this context. Here, we aim to determine whether sex influences physiological response to high-fat high-sucrose diet (HFS) and myocardial tolerance to ischemia-reperfusion injury. Male and female Wistar rats were subjected to standard (CTRL) or HFS diet for 5 months. Then, ex-vivo experiments on isolated perfused heart model were performed to evaluate tolerance to ischemia-reperfusion injury. HFS diet induced fasting hyperglycemia and increased body fat percent to a similar level in both sexes. However, glucose intolerance was more pronounced in female HFS. Cholesterol was increased only in female while male displayed higher level of plasmatic leptin. We observed increased heart weight to tibia length ratio only in males, but we showed a similar decrease in tolerance to ischemia-reperfusion injury in female and male HFS compared with respective controls, characterized by impaired cardiac function, energy metabolism and coronary flow during reperfusion. In conclusion, as soon as glucose intolerance and hyperglycemia develop, we observe higher sensitivity of hearts to ischemia-reperfusion injury without difference between males and females.

B-PO01-066 FALLACIES OF AND NOVEL FINDINGS OF MAPPING INTRAMURAL ARRHYTHMOGENIC SOURCES USING ELECTROGRAM MORPHOLOGY

A unipolar ‘QS’ pattern is typically recorded adjacent to ventricular arrhythmogenic focal origin and indicative of a successful catheter ablation site. Alternatively, a unipolar ‘R’ wave infers a mapping site remote from the focal origin. We investigated unipolar electrogram (EGM) patterns generated by modifying the depth of an intramural focal source. Two models were created: an experimental and simulation model. Explanted healthy swine hearts were used in a Langendorff perfused heart model. An idealized human myocardial slab (up to 12mm thickness) was used in a simulation model with a 56-pole electrode grid represented on both endocardial and epicardial surfaces to generate unipolar EGMs. In the centre of the electrode grid, the intramural stimulation source depth varied between 1-10mm with increments of 1mm. A stimulus pacing depth of 1mm demonstrated unipolar ‘QS’ waves occupying 48% of the electrode array surface area, equating to an area of 64mm2. Distinct differences in unipolar EGM patterns and complexity were seen between epicardial and endocardial surface mapping (Figure). Endocardial maps showed at a depth of 1mm, a ‘R’ wave as the predominant unipolar pattern (54% of sites) whereas with an increasing depth of 5mm, ‘Q’ and ‘QS’ occupied >90% of sites and even up to 10mm, a ‘Q’ wave was the predominant pattern, occupying >70% of sites. On epicardial maps, a unipolar ‘QS’ pattern was rarely seen at superficial focal intramural sites (<5% of sites with a 1-2mm focal depth) whereas a ‘R’ wave was more commonly observed in 50% of sites. Furthermore, a monophasic ‘R’ wave was absent from all intramural sites >5mm, replaced largely by multi-component unipolar EGMs with increased complexity (QrS and qSS patterns). A ‘QS’ unipolar pattern lacks sensitivity for superficial foci and can be even the predominant pattern generated when mapping deep intramural foci >5mm at accessible mapping surface. Furthermore, a ‘R’ wave can be observed adjacent to superficial foci. Additionally, qSS pattern is a better indicated of deeper intramural focus at mapping surface. These findings suggest a revision is needed to electrogram morphology guided ablation of intramural arrhythmogenic foci.

Cardiac and Metabolic Impact of Functional Foods with Antioxidant Properties Based on Whey Derived Proteins Enriched with Hemp Seed Oil.

The impaired ability to feed properly, evident in oncologic, elderly, and dysphagic patients, may result in malnutrition and sarcopenia. Increasing the consumption of dietary proteins by functional foods and enriching their composition by adding beneficial nutrients may represent an adjuvant therapy. We aimed to evaluate the safety and the positive effects of a standard diet (SD) supplemented with whey-derived protein puddings (WDPP), with appropriate rheological properties, and hemp seed oil (HSO), rich in polyphenols. Rats were assigned to SD, WDPP, WDPP plus hemp seed oil (HSOP), and HSO supplemented diets for eight weeks. “Anthropometric”, metabolic, and biochemical variables, oxidative stress, tissue injury, liver histology, and cardiac susceptibility to ischemia/reperfusion were analyzed. All the supplementations did not induce significant changes in biochemical and metabolic variables, also in relation to glucose tolerance, and livers did not undergo morphological alteration and injury. An improvement of cardiac post-ischemic function in the Langendorff perfused heart model and a reduction of infarct size were observed in WDPP and HSOP groups, thanks to their antioxidant effects and the activation of Akt- and AMPK-dependent protective pathways. Data suggest that (i) functional foods enriched with WDPP and HSOP may be used to approach malnutrition and sarcopenia successfully under disabling conditions, also conferring cardioprotection, and that (ii) adequate rheological properties could positively impact dysphagia-related problems.

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

IntroductionAerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.PurposeTo investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.MethodsWe studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).ResultsAt baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.ConclusionAerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

Diabetic animal fed with high-fat diet prevents the protective effect of myocardial ischemic preconditioning effect in isolated rat heart perfusion model.

Diabetic heart (diabetes mellitus [DM]) has been shown to attenuate the beneficial effect of ischemic preconditioning (IPC) in rat heart. But the effect of IPC on diabetic rat heart that develops myopathy remains unclear. This study was designed to test the impact of IPC on diabetic cardiomyopathy (DCM) rat heart. Male Wistar rats were grouped as (a) normal, (b) DM (streptozotocin: 65 mg/kg; fed with normal diet), and (c) DCM (streptozotocin: 65 mg/kg; fed with high-fat diet). Isolated rat hearts from each group were randomly subjected to (a) normal perfusion, (b) ischemia-reperfusion (I/R), and (c) IPC procedure. At the end of the perfusion experiments, hearts were analyzed for injury, contractile function, mitochondrial activity, and oxidative stress. The results obtained from hemodynamics, cardiac injury markers, and caspase-3 activity showed that DCM rat displayed prominent I/R-associated cardiac abnormalities than DM rat heart. But the deteriorated physiological performance and cardiac injury were not recovered in both DM and DCM heart by IPC procedure. Unlike normal rat heart, IPC did not reverse mitochondrial dysfunction (determined by electron transport chain enzymes activity, ATP level, and membrane integrity, expression levels of genes like PGC-1ɑ, GSK3β, complex I, II, and V) in DCM and DM rat heart. The present study demonstrated that IPC failed to protect I/R-challenged DCM rat heart, and the underlying pathology was associated with deteriorated mitochondrial function.

Acanthopanax senticosus Protects Against Myocardial Ischemia Reperfusion Injury in Rats

The protective effects of Acanthopanax senticosus on myocardial ischemia reperfusion injury in rats was explored. A total of 60 male Sprague Dawley rats were randomly divided into the sham operated group (group C), the ischemia reperfusion group (group IR), the Acanthopanax senticosus plus blocker group (group ASS+Z), and the Acanthopanax senticosus group (group ASS). The rat myocardial ischemia reperfusion injury models were established through ligating left anterior descending branches of coronary artery in rats. TUNEL assay and Western Blot was used to detect the apoptotic index of myocardial cells and the expression levels of extracellular regulating kinase 1/2 (ERK1/2) and phosphorylation ERK1/2 (p-ERK1/2). Meanwhile, a total of 30 rats were randomly selected and divided into 5 groups; the control group (group c) was the simple ischemia reperfusion group, while groups P1, P2, P3, and P4 were given different dosage concentrations of Acanthopanax senticosus for pre-adaptation. The Langendorff isolated heart perfusion model was used to record the dynamic changes in cardiac functions and coronary artery flow in each group. The results have shown that the Acanthopanax senticosus had certain protective effects on the ischemia reperfusion injury of rat myocardium, as well as the pre-adaptive effects on the systolic functions of left ventricles after reperfusion, which would increase coronary flow and resist the arrhythmia, thereby reducing the apoptosis rates of myocardial cells and protecting the myocardium through activating the ERK1/2 pathway.

Physiologic Factors Affecting the Circulatory Persistence of Copolymer Microbubbles and Comparison of Contrast-Enhanced Effects between Copolymer Microbubbles and Sonovue

Ultrasound contrast agents have been widely used in clinical diagnosis. Knowledge of the physiologic factors affecting circulatory persistence is helpful in preparing long-lasting microbubbles (MBs) for blood perfusion and drug delivery research. In the study described here, we prepared copolymer MBs, compared their characteristics and contrast-enhanced effects with those of SonoVue and investigated the influence of external pressure, temperature, plasma components, renal microcirculation and cardiac motion on their circulatory persistence. The mean size of the copolymer MBs was 3.57 μm, larger than that of SonoVue. The copolymer MBs had longer circulatory persistence than SonoVue. At external pressures of 110 and 150 mm Hg, neither the quantity nor the morphology of the copolymer MBs changed. Further, their quantity and size were similar after incubation at 4°C and 39.4°C and when rabbit plasma and saline were compared. In vivo contrast-enhanced ultrasonography revealed a slightly larger area under the curve for the renal artery than for the renal vein. Thus, copolymer MBs exhibited good stability. However, the quantity of copolymer MBs decreased significantly after 180 s of circulation in an isolated toad heart perfusion model, indicating that cardiac motion was the main factor affecting their circulatory persistence.

Abstract 295: Cysteine 202 of Cyclophilin D is a Site of Multiple Post-translational Modifications and Plays a Role in Cardioprotection

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the binding of CypD to the PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). In a Langendorff perfused heart model, infarct size is reduced by 51% in CypD-C202S hearts compared to WT (n=4, p=0.003). Isolated cardiac mitochondria from CypD-C202S mice also have a 49% higher calcium retention capacity compared to WT (n=5, p=0.011). We hypothesized that oxidation of C202 might target CypD to the PTP and that the cardioprotection in CypD-C202S mice is due to a post-translational modification on C202. Indeed, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD-C202S to the proposed PTP component ATP synthase by 48% compared to WT (p=0.012, n=4). We previously found C202 to be S-nitrosylated in ischemic preconditioning. Cysteines can also undergo S-acylation (attachment of a fatty acid), and C202 matched a S-acylation motif. To assess S-acylation of CypD-C202 we employed a resin-assisted capture. Lysates were treated with N-ethylmaleimide to block free cysteines, and then with hydroxylamine to free cysteines from fatty acids. Subsequently, free cysteines were captured by a sepharose resin, followed by western-blot of the eluate. CypD-C202 is abundantly S-acylated, but CypD-C202S hearts exhibit less S-acylation than WT by 63% (n=6, p&lt;0.001). We investigated the importance of S-acylation of CypD-C202 in cell death in a mouse embryonic fibroblast (MEFs) model. CypD-C202S MEFs exhibit 20% less oxidative cell death than WT (n=5, p=0.003). Inhibition of S-acylation with 2-bromopalmitate attenuates oxidative cell death in the WT by 20% (n=5, p=0.018), while CypD-C202S MEFs are unaffected. Thus, S-acylation and oxidation of CypD-C202 possibly target CypD to the PTP, sensitizing it under oxidative stress and making them potent regulators of cardiac I/R injury.

MECHANISM OF ACTION OF ORBITAL ATHERECTOMY IN SEVERELY CALCIFIED CORONARY ARTERIES: INSIGHTS FROM A CADAVERIC PERFUSION MODEL

Orbital atherectomy (OA) is indicated for facilitating stent delivery in patients by reducing coronary plaque, but complete understanding of the therapeutic mechanism has not been established. We performed a mechanistic study using a cadaveric heart perfusion model to investigate the impact of OA on