RESUMEN
Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.
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Perfusión , Animales , Perfusión/métodos , Ratas , Trasplante de Corazón/métodos , Preparación de Corazón Aislado/métodosRESUMEN
The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.
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Ventrículos Cardíacos , Corazón , Animales , Corazón/fisiología , Frecuencia Cardíaca , Humanos , Preparación de Corazón Aislado/métodos , Ratones , Miocardio , Perfusión/métodosRESUMEN
AIMS: Recently, a new defibrillation modality using nanosecond pulses was shown to be effective at much lower energies than conventional 10 millisecond monophasic shocks in ex vivo experiments. Here we compare the safety factors of 300 nanosecond and 10 millisecond shocks to assess the safety of nanosecond defibrillation. METHODS AND RESULTS: The safety factor, i.e. the ratio of median effective doses (ED50) for electroporative damage and defibrillation, was assessed for nanosecond and conventional (millisecond) defibrillation shocks in Langendorff-perfused New Zealand white rabbit hearts. In order to allow for multiple shock applications in a single heart, a pair of needle electrodes was used to apply shocks of varying voltage. Propidium iodide (PI) staining at the surface of the heart showed that nanosecond shocks had a slightly lower safety factor (6.50) than millisecond shocks (8.69), p = 0.02; while PI staining cross-sections in the electrode plane showed no significant difference (5.38 for 300 ns shocks and 6.29 for 10 ms shocks, p = 0.22). CONCLUSIONS: In Langendorff-perfused rabbit hearts, nanosecond defibrillation has a similar safety factor as millisecond defibrillation, between 5 and 9, suggesting that nanosecond defibrillation can be performed safely.
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Electroporación/métodos , Corazón/fisiología , Preparación de Corazón Aislado/métodos , Animales , Cardioversión Eléctrica/métodos , Electrodos , Electrofisiología , Femenino , Masculino , Propidio , Conejos , Seguridad , Fibrilación VentricularRESUMEN
In an integrative approach, we studied cardiac effects of recently published novel H2 receptor agonists in the heart of mice that overexpress the human H2 receptor (H2-TG mice) and littermate wild type (WT) control mice and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H2 receptor agonists UR-Po563, UR-MB-158, and UR-MB-159 increased force of contraction in left atrium from H2-TG mice with pEC50 values of 8.27, 9.38, and 8.28, respectively, but not in WT mice. Likewise, UR-Po563, UR-MB-158, and UR-MB-159 increased the beating rate in right atrium from H2-TG mice with pEC50 values of 9.01, 9.24, and 7.91, respectively, but not from WT mice. These effects could be antagonized by famotidine, a H2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff-perfused hearts from H2-TG but not WT mice. Similarly, UR-Po563, UR-MB-158, or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H2-TG mice. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H2-TG mice were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H2 receptors in vitro and in vivo. We speculate that these compounds might be of some merit to treat neurologic disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients. SIGNIFICANCE STATEMENT: Recently, a new generation of histamine H2 receptor (H2R) agonists has been developed as possible treatment option for Alzheimer's disease. Here, possible cardiac (side) effects of these novel H2R agonists have been evaluated.
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Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Contracción Miocárdica/efectos de los fármacos , Receptores Histamínicos H2/metabolismo , Anciano , Animales , Relación Dosis-Respuesta a Droga , Femenino , Histamina/farmacología , Humanos , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Contracción Miocárdica/fisiologíaRESUMEN
Pharmacological postconditioning (PPC), drug intervention before or during the early minutes of reperfusion, could stimulate cardioprotection as ischemic postconditioning. In this study, we examined whether PPC with sappanone A (SA), a homoisoflavanone with potent antioxidant and anti-inflammatory activity, has a protective effect on myocardial ischemia reperfusion injury (MIRI), and explored the underlying mechanism. A MIRI model was established using the Langendorff method. After 30 min of ischemia, isolated rat hearts were treated with SA at the onset of reperfusion to stimulate PPC. The changes in myocardial infarct size, mitochondrial function, mitochondrial biogenesis, mitophagy, and mitochondrial fission and fusion were detected. The results showed that SA postconditioning decreased the myocardial infarct size, inhibited the release of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and cardiac troponin (cTnI), as well as improved cardiac function, enhanced myocardial ATP content and mitochondrial complex activity, and prevented the loss of mitochondrial membrane potential and opening of mitochondrial permeability transition pore (mPTP). Mechanistically, we found that SA was an AMP-activated protein kinase (AMPK) activator, and SA postconditioning could facilitate mitochondrial biogenesis by increasing mitochondrial DNA (mtDNA) copy number and the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). In addition, it balanced mitochondrial dynamics by decreasing fission and increasing fusion, and enhanced mitophagy in an AMPK-dependent manner. Moreover, AMPK silencing abolished the cardioprotection of SA postconditioning. Collectively, our study demonstrated that SA postconditioning ameliorated MIRI and mitochondrial dysfunction by regulation of mitochondrial quality control via activating AMPK. This finding provides a new insight into pharmacological action and clinical use of SA.
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Proteínas Quinasas Activadas por AMP/metabolismo , Poscondicionamiento Isquémico/métodos , Isoflavonas/farmacología , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Isoflavonas/uso terapéutico , Preparación de Corazón Aislado/métodos , Masculino , Mitocondrias Cardíacas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Rumex dentatus has been used traditionally for ailment of cardiovascular diseases. The aim of the present study was to assess cardiovascular effects in isolated perfused rabbit heart. Aqueous and n-butanolic fractions were assessed for their effect on perfusion pressure (PP), force of contraction (FC) and heart rate (HR) of rabbit heart using Langendorff's method. The possible mechanisms of action of extracts/fraction were assessed with and without application of different agonist/antagonist. Phytochemical, toxicity and anti-oxidant activities were also determined. Both extracts at 1mg/mL dose produced a highly significant decrease in FC and HR but PP remained unchanged. Moreover, aqueous fraction of Rumex dentatus at 0.001mg/mL dose produced a highly significant decrease in FC and HR but no significant change in PP was observed. Atropine 10-5 M did not inhibit the cardiac depressant response of both fractions. Furthermore, both fractions blocked the positive ionotropic and chronotropic effects of adrenaline and calcium chloride. Phytochemical studies have shown the presence of some phytochemicals. Acute and sub-chronic toxicity studies demonstrated that test extracts are safe and produced no significant changes in haematological and biochemical parameters. Crude extract showed significant antioxidant activity like ascorbic acid. This study revealed that this plant have good cardiac depressant effect.
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Antioxidantes/farmacología , Fármacos Cardiovasculares/farmacología , Corazón/efectos de los fármacos , Preparación de Corazón Aislado , Extractos Vegetales/farmacología , Rumex/química , Animales , Atropina/farmacología , Cloruro de Calcio/farmacología , Fármacos Cardiovasculares/efectos adversos , Epinefrina/farmacología , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Contracción Miocárdica/efectos de los fármacos , Extractos Vegetales/efectos adversos , Conejos , Ratas , Ratas Sprague-Dawley , Rumex/efectos adversosRESUMEN
BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
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Canales Iónicos Sensibles al Ácido/biosíntesis , Canales Iónicos Sensibles al Ácido/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Noqueados , Isquemia Miocárdica/terapia , Daño por Reperfusión Miocárdica/terapia , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Polimorfismo de Nucleótido Simple/fisiología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Venenos de Araña/farmacologíaRESUMEN
Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Separación Celular/instrumentación , Separación Celular/métodos , Preparación de Corazón Aislado/instrumentación , Preparación de Corazón Aislado/métodos , Miocitos Cardíacos/citología , Animales , Calcio/farmacología , Cloruro de Calcio/farmacología , Células Cultivadas , Colagenasas/química , Colagenasas/farmacología , Ratones , Miocitos Cardíacos/metabolismo , Perfusión/métodosRESUMEN
Recent studies have shown that pre and postconditioning the heart with sodium thiosulfate (STS) attenuate ischemia-reperfusion (IR) injury. However, the underlying mechanism involved in the cardioprotective signaling pathway is not fully explored. This study examined the existing link of STS mediated protection (as pre and post-conditioning agents) with PI3K, mTOR, and mPTP signaling pathways using its respective inhibitors. STS was administered to the isolated perfused rat heart through Kreb's Heinselit buffer before ischemia (precondition: SIPC) and reperfusion (postcondition: SPOC) in the presence and absence of the PI3K, mTOR, and mPTP signaling pathway inhibitors (wortmannin, rapamycin, and glibenclamide respectively). SIPC failed to improve the IR injury-induced altered cardiac hemodynamics, increased infarct size, and the release of cardiac injury markers in the presence of these inhibitors. On the other hand, the SPOC protocol effectively rendered the cardioprotection even in the PI3K/mTOR/KATP inhibitors presence. Interestingly, the SIPC's identified mode of action viz reduction in oxidative stress and the preservation of mitochondrial function were lost in the inhibitors' presence. Based on the above results, we conclude that the underlying mechanism of SIPC mediated cardioprotection works via the PI3K/mTOR/KATP signaling pathway axis activation.
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Adenosina Trifosfato/metabolismo , Precondicionamiento Isquémico Miocárdico/métodos , Daño por Reperfusión Miocárdica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tiosulfatos/administración & dosificación , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Preparación de Corazón Aislado/métodos , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Ratas , Ratas Wistar , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
BACKGROUND: Concomitant apamin-sensitive small conductance calcium-activated potassium current (IKAS) activation and sodium current inhibition induce J-wave syndrome (JWS) in rabbit hearts. Sudden death in JWS occurs predominantly in men at night when parasympathetic tone is strong. OBJECTIVE: The purpose of this study was to test the hypotheses that acetylcholine (ACh), the parasympathetic transmitter, activates IKAS and causes JWS in the presence of ajmaline. METHODS: We performed optical mapping in Langendorff-perfused rabbit hearts and whole-cell voltage clamp to determine IKAS in isolated ventricular cardiomyocytes. RESULTS: ACh (1 µM) + ajmaline (2 µM) induced J-point elevations in all (6 male and 6 female) hearts from 0.01± 0.01 to 0.31 ± 0.05 mV (P<.001), which were reduced by apamin (specific IKAS inhibitor, 100 nM) to 0.14 ± 0.02 mV (P<.001). More J-point elevation was noted in male than in female hearts (P=.037). Patch clamp studies showed that ACh significantly (P<.001) activated IKAS in isolated male but not in female ventricular myocytes (n=8). Optical mapping studies showed that ACh induced action potential duration (APD) heterogeneity, which was more significant in right than in left ventricles. Apamin in the presence of ACh prolonged both APD at the level of 25% (P<.001) and APD at the level of 80% (P<.001) and attenuated APD heterogeneity. Ajmaline further increased APD heterogeneity induced by ACh. Ventricular arrhythmias were induced in 6 of 6 male and 1 of 6 female hearts (P=.015) in the presence of ACh and ajmaline, which was significantly suppressed by apamin in the former. CONCLUSION: ACh activates ventricular IKAS. ACh and ajmaline induce JWS and facilitate the induction of ventricular arrhythmias more in male than in female ventricles.
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Acetilcolina/farmacología , Ajmalina/farmacología , Arritmias Cardíacas/tratamiento farmacológico , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Canales de Potasio Calcio-Activados/efectos de los fármacos , Canales de Sodio/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Agonistas Colinérgicos/farmacología , Modelos Animales de Enfermedad , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Preparación de Corazón Aislado/métodos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Imagen Óptica , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/metabolismo , Conejos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Sodio/efectos de los fármacos , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Heart transplantation is a life-saving therapy for end-stage organ failure. Organ deterioration during transportation limits storage to 4 hours, limiting hearts available. Approaches ameliorating organ damage could increase the number of hearts acceptable for transplantation. Prior studies show that adipose-derived stem/stromal cell secretome (ASC-S) rescues tissues from postischemic damage in vivo. This study tested whether ASC-S preserved the function of mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iCM) exposed to organ transportation and transplantation conditions. Hearts were subjected to cold University of Wisconsin (UW) cardioplegic solution ± ASC-S for 6 hours followed by analysis using the Langendorff technique. In parallel, the effects of ASC-S on the recovery of iCM from UW solution were examined when provided either during or after cold cardioplegia. Exposure of hearts and iCM to UW deteriorated contractile activity and caused cell apoptosis, worsening in iCM as a function of exposure time; these were ameliorated by augmenting with ASC-S. Silencing of superoxide dismutase 3 and catalase expression prior to secretome generation compromised the ASC-S cardiomyocyte-protective effects. In this study, a novel in vitro iCM model was developed to complement a rodent heart model in assessing efficacy of approaches to improve cardiac preservation. ASC-S displays strong cardioprotective activity on iCM either with or following cold cardioplegia. This effect is associated with ASC-S-mediated cellular clearance of reactive oxygen species. The effect of ASC-S on the temporal recovery of iCM function supports the possibility of lengthening heart storage by augmenting cardioplegic transport solution with ASC-S, expanding the pool of hearts for transplantation.
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Soluciones Cardiopléjicas/toxicidad , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Miocitos Cardíacos/metabolismo , Soluciones Preservantes de Órganos/toxicidad , Recuperación de la Función/fisiología , Adenosina/toxicidad , Alopurinol/toxicidad , Animales , Glutatión/toxicidad , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Insulina/toxicidad , Preparación de Corazón Aislado/métodos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Rafinosa/toxicidad , Recuperación de la Función/efectos de los fármacosRESUMEN
We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt)max - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.
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Corazón/fisiología , Preparación de Corazón Aislado/métodos , Adulto , Animales , Carbenoxolona/farmacología , Electrocardiografía/métodos , Acoplamiento Excitación-Contracción , Femenino , Corazón/efectos de los fármacos , Humanos , Preparación de Corazón Aislado/instrumentación , Masculino , Miocardio/metabolismo , PorcinosRESUMEN
The multi-electrode mapping method was used to analyze electrical activity of isolated rat heart under conditions of standard perfusion, pharmacological stimulation of fibrillation, and mechanical stretching of the right atrium both under normal conditions and before cardiac fibrillation. It was shown that stretching of the right atrium prevented the increase of repolarization dispersion and latency of the electrical signal in the myocardium that were observed before cardiac fibrillation.
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Fibrilación Atrial/fisiopatología , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/fisiopatología , Mecanotransducción Celular , Potenciales de Acción , Animales , Electrodos , Preparación de Corazón Aislado/métodos , Masculino , Miocardio/patología , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
Oxidative stress is the key factor of myocardial ischemia-reperfusion injury (MIRI). Anthocyanins are considered to be effective anti-oxidants. In this study, we observed the anti-MIRI effect of petunidin, one member of anthocyanins, and further explored its mechanism. In present study, anoxia/reoxygenation (A/R) models were replicated on Langendorff-perfused heart and neonatal rat primary cardiomyocytes by A/R treatment. The hemodynamic parameters of isolated hearts were monitored. The levels of oxidative stress and apoptosis in isolated heart and neonatal rat primary cardiomyocytes were evaluated. The expression levels of NADPH oxidase 2 (NOX 2), NOX 4, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax) and cytochrome c were detected by Western Blot. The results showed that petunidin could significantly improve isolated heart function, reduce oxidative stress, inhibit cardiomyocyte apoptosis, up-regulate Bcl-2 protein expression, down-regulate NOX4 and Bax expression, and reduce the level of cytoplasmic cytochrome c after A/R. However, it has no significant effect on NOX 2 protein expression, suggesting that NOX 4 may be the molecular target of petunidin. In vitro, petunidin had shown a consistent effect with that in isolated hearts. It also showed a significant inhibitory effect on reactive oxygen species (ROS) generation. However, the protective effects of petunidin on A/R injury were attenuated by over-expression of NOX 4 in neonatal rat primary cardiomyocytes. These data suggested that the protective effects of petunidin on MIRI may be achieved through targeting NOX 4, thus inhibiting the production of ROS, reducing oxidative stress, and regulating the Bcl-2 pathway to prevent cardiomyocytes apoptosis.
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Antocianinas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Hipoxia/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , NADPH Oxidasa 4/antagonistas & inhibidores , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hipoxia/metabolismo , Preparación de Corazón Aislado/métodos , Masculino , Daño por Reperfusión Miocárdica/metabolismo , NADPH Oxidasa 4/biosíntesis , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
This article presents a primed left ventricle heart perfusion method to generate physiologic aortic pressure (AoP) and perform functional assessment. Isolated hearts of male Yorkshire pigs were used to study the hemodynamic behaviors of AoPs generated in the primed left ventricle heart perfusion (n = 6) and conventional (zero-loaded left ventricle) Langendorff perfusion (n = 6). The measurement results show that left ventricular pressure generated in the primed left ventricle heart perfusion is a determinant of physiologic AoP (i.e. systolic and diastolic pressures within physiologic range). The aortic pulse pressure (systolic pressure = 124.5 ± 1.7 mm Hg, diastolic pressure = 87.8 ± 0.9 mm Hg, aortic pulse pressure = 36.7 ± 2.6 mm Hg) from the primed left ventricle heart perfusion represents close match with the in vivo physiologic data. The volume in the left ventricle remains constant throughout the primed left ventricle heart perfusion, which allows us to perform isovolumetric left ventricular pressure measurement in ex vivo heart perfusion (EVHP). Left ventricular contractility measurements (maximum and minimum rates of left ventricular pressure change) were derived for cardiac assessment. In summary, the proposed primed left ventricle heart perfusion method is able to create physiologic AoP and enables left ventricular functional assessment in EVHP in porcine hearts.
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Presión Arterial/fisiología , Preparación de Corazón Aislado , Perfusión/instrumentación , Perfusión/métodos , Función Ventricular/fisiología , Animales , Presión Sanguínea/fisiología , Corazón/fisiología , Ventrículos Cardíacos , Hemodinámica/fisiología , Preparación de Corazón Aislado/instrumentación , Preparación de Corazón Aislado/métodos , Masculino , PorcinosRESUMEN
RATIONALE: Costly proprietary panoramic multielectrode (64-256) acquisition systems are being increasingly used together with conventional electroanatomical mapping systems for persistent atrial fibrillation (PersAF) ablation. However, such approaches target alleged drivers (rotational/focal) regardless of their activation frequency dynamics. OBJECTIVES: To test the hypothesis that stable regions of higher than surrounding instantaneous frequency modulation (iFM) drive PersAF and determine whether rotational activity is specific for such regions. METHODS AND RESULTS: First, novel single-signal algorithms based on instantaneous amplitude modulation (iAM) and iFM to detect rotational-footprints without panoramic multielectrode acquisition systems were tested in 125 optical movies from 5 ex vivo Langendorff-perfused PersAF sheep hearts (sensitivity/specificity, 92.6/97.5%; accuracy, 2.5-mm) and in computer simulations. Then, 16 pigs underwent high-rate atrial pacing to develop PersAF. After a median (interquartile range [IQR]) of 4.4 (IQR, 2.5-9.9) months of high-rate atrial pacing followed by 4.1 (IQR, 2.7-5.4) months of self-sustained PersAF, pigs underwent in vivo high-density electroanatomical atrial mapping (4920 [IQR, 4435-5855] 8-second unipolar signals per map). The first 4 out of 16 pigs were used to adapt ex vivo optical proccessing of iFM/iAM to in vivo electrical signals. In the remaining 12 out of 16 pigs, regions of higher than surrounding average iFM were considered leading-drivers. Two leading-driver + rotational-footprint maps were generated 2.6 (IQR, 2.4-2.9) hours apart to test leading-driver spatiotemporal stability and guide ablation. Leading-driver regions (2.5 [IQR, 2.0-4.0] regions/map) exactly colocalized (95.7%) in the 2 maps, and their ablation terminated PersAF in 92.3% of procedures (radiofrequency until termination, 16.9 [IQR, 9.2-35.8] minutes; until nonsustainability, 20.4 [IQR, 12.8-44.0] minutes). Rotational-footprints were found at every leading-driver region, albeit most (76.8% [IQR, 70.5%-83.6%]) were located outside. Finally, the translational ability of this approach was tested in 3 PersAF redo patients. CONCLUSIONS: Both rotational-footprints and spatiotemporally stable leading-driver regions can be located using iFM/iAM algorithms without panoramic multielectrode acquisition systems. In pigs, ablation of leading-driver regions usually terminates PersAF and prevents its sustainability. Rotational activations are sensitive but not specific to such regions. Single-signal iFM/iAM algorithms could be integrated into conventional electroanatomical mapping systems to improve driver detection accuracy and reduce the cost of patient-tailored/mechanistic approaches.
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Fibrilación Atrial/fisiopatología , Fibrilación Atrial/cirugía , Mapeo del Potencial de Superficie Corporal/métodos , Ablación por Catéter/métodos , Frecuencia Cardíaca/fisiología , Imagenología Tridimensional/métodos , Potenciales de Acción/fisiología , Adulto , Anciano , Animales , Fibrilación Atrial/diagnóstico por imagen , Femenino , Humanos , Preparación de Corazón Aislado/métodos , Masculino , Persona de Mediana Edad , Ovinos , PorcinosRESUMEN
A murine line haploinsufficient in the cardiac sodium channel has been used to model human Brugada syndrome: a disease causing sudden cardiac death due to lethal ventricular arrhythmias. We explored the effects of cholinergic tone on electrophysiological parameters in wild-type and genetically modified, heterozygous, Scn5a+/- knockout mice. Scn5a+/- ventricular slices showed longer refractory periods than wild-type both at baseline and during isoprenaline challenge. Scn5a+/- hearts also showed lower conduction velocities and increased mean increase in delay than did littermate controls at baseline and blunted responses to isoprenaline challenge. Carbachol exerted limited effects but reversed the effects of isoprenaline with coapplication. Scn5a+/- mice showed a reduction in conduction reserve in that isoprenaline no longer increased conduction velocity, and this was not antagonized by muscarinic agonists.
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Síndrome de Brugada/metabolismo , Haploinsuficiencia/fisiología , Preparación de Corazón Aislado , Contracción Miocárdica/fisiología , Canal de Sodio Activado por Voltaje NAV1.5/deficiencia , Animales , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatología , Femenino , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canales de Sodio/deficiencia , Canales de Sodio/genéticaRESUMEN
p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK-/- and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK-/- mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK-/- mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK-/- mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.
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Péptidos y Proteínas de Señalización Intracelular/deficiencia , Contracción Miocárdica/fisiología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/prevención & control , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Péptidos y Proteínas de Señalización Intracelular/genética , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We maintained the function of an extracted rat heart after 24-48 h preservation in a high-pressure gaseous mixture of carbon monoxide (CO) and oxygen (O2). Here, we assessed the effects of different partial pressures of hyperbaric CO and O2 for 24-48 h at 4 °C on rat heart preservation and compared conditions including immersion in University of Wisconsin solution. Preserved hearts were transplanted into recipient rats via heterotopic cervical heart transplantation for in vivo evaluation and perfused using the Langendorff system for ex vivo evaluation. The survival rate of transplanted hearts was 100% at postoperative day 7 in the CO + O2 (PCO:PO2 = 1.5:2.0 atm) group but only 33% in the CO + O2 (PCO:PO2 = 2.0:1.5 atm) group. Langendorff system and histopathological analysis revealed that the left ventricular pressure of preserved hearts in the CO + O2 (PCO:PO2 = 1.5:2.0 atm) group was better than the CO + O2 (PCO:PO2 = 2.0:1.5 atm). We demonstrate that exposure of rat hearts to hyperbaric CO and O2 is superior to the immersion method and that partial pressure of hyperbaric CO and O2 is crucial to preservation.
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Trasplante de Corazón/métodos , Preparación de Corazón Aislado/métodos , Preservación de Órganos/métodos , Recolección de Tejidos y Órganos/métodos , Animales , Monóxido de Carbono/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Miocardio/metabolismo , Oxígeno/farmacología , Presión , Ratas , Ratas Endogámicas LewRESUMEN
In this study we used Visible Heart® methodologies featuring cyclic temperature modulation of porcine hearts in order to establish characteristic temperature responses. This isolated and perfused model is a more predictable and modifiable analog for human heart preservation and isolates the response of the cardiac tissue. We comprehensively monitored isolated porcine hearts undergoing temperature change and demonstrated optimization of isolated cardiac function under mild hypothermia. We tracked metrics of cardiac function as continuous variables during temperature changes (~ 31 to 39 °C), eliciting a well-defined reduction in metabolic demand and in heart rate modulation. Optimization of function appeared to occur around 34.7 ± 0.9 °C (n = 13). Cardiac response was further investigated in the presence of active pacing in order to assess pacing capture and the heart's functional response without a means of regulating rate. Our results may have direct clinical implications for emerging heart preservation methods prior to transplantation, as well as benefits for investigators using isolated heart models for preclinical device testing. Clinically, this porcine model is a basis for finding new ways to extend the window of viability for transplantable organs, thereby restoring or improving graft function and potentially enhancing recipient outcomes.