Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts.

Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.

Electrical remodeling of atrioventricular junction: a study on retrogradely perfused chick embryonic heart.

Atrioventricular (AV) accessory pathways (APs) provide additional electrical connections between the atria and ventricles, resulting in severe electrical disturbances. It is generally accepted that APs originate in the altered annulus fibrosus maturation in the late prenatal and perinatal period. However, current experimental methods cannot address their development in specific locations around the annulus fibrosus due to the inaccessibility of late fetal hearts for electrophysiological investigation under physiological conditions. In this study, we describe an approach for optical mapping of the retrogradely perfused chick heart in the last third of the incubation period. This system showed stability for electrophysiological measurement for several hours. This feature allowed analysis of the number and functionality of the APs separately in each clinically relevant position. Under physiological conditions, we also recorded the shortening of the AV delay with annulus fibrosus maturation and analyzed ventricular activation patterns after conduction through APs at specific locations. We observed a gradual regression of AP with an area-specific rate (left-sided APs disappeared first). The results also revealed a sudden drop in the number of active APs between ED16 and ED18. Accessory myocardial AV connections were histologically documented in all positions around the annulus fibrosus even after hatching. The fact that no electrically active AP was present at this stage highlights the necessity of electrophysiological evaluation of accessory atrioventricular connections in studying AP formation.

Cardiac effects of 5F-Cumyl-PEGACLONE.

Synthetic cannabinoids become increasingly popular as a supposedly safe and legal alternative to cannabis. In order to circumvent the German New Psychoactive Substances Law, producers of so-called herbal mixtures rapidly design new substances with structural alterations that are not covered by the law. Acting as full agonists not only at the cannabinoid receptors 1 and 2, synthetic cannabinoids might have not only desired mental but also serious physical adverse effects. However, knowledge of adverse effects of specific substances is sparse and incomplete. This also accounts for 5F-Cumyl-PEGACLONE, a synthetic cannabinoid, which has been detected regularly in Germany in recent years. By using an animal model, the isolated perfused Langendorff heart, the study at hand aimed on finding out more about possible cardiovascular adverse effects of 5F-Cumyl-PEGACLONE. Hearts of male Wistar rats, which were excised postmortem, were exposed to two different concentrations of 5F-Cumyl-PEGACLONE: 13 hearts were exposed to 50 ng/ml and 12 hearts were exposed to 100 ng/ml. Thirteen control hearts were merely exposed to an additional amount of buffer solution. Functional parameters heart rate, minimal and maximum left ventricular pressure and coronary flow were documented at pre-defined time points during and after the administration of 5F-Cumyl-PEGACLONE/additional buffer solution. Electrocardiograms (ECGs) were documented throughout the experiments and evaluated afterwards. Kruskal-Wallis analysis was performed for each functional parameter as well as for the duration of the QRS complexes and the duration of RR intervals as derived from the ECGs. Furthermore, a multivariate analysis, comprising all functional and ECG parameters, was performed. Kruskal-Wallis analysis revealed only single significant p-values for QRS duration and minimum left ventricular pressure that did not pass a Bonferroni test. The results of the multivariate approach were also comparably homogeneous, but still the model correctly recognized hearts exposed to 100 ng/ml of 5F-Cumyl-PEGACLONE more often than hearts exposed to the low concentration of 5F-Cumyl-PEGACLONE or additional buffer solution. Evaluation of the ECGs presented single cases of ST depression and QT prolongation. Though certainly not unambiguous, these findings support the assumption that 5F-Cumyl-PEGACLONE can cause severe, if not lethal, cardiac adverse effects like arrhythmias or myocardial infarctions especially if it is consumed in combination with other drugs like alcohol or if the consumer suffers from pre-existing heart diseases.

Amplification of Cardioprotective Response of Remote Ischemic Preconditioning in Rats by Quercetin: Potential Role of Activation of mTOR-dependent Autophagy and Nrf2.

OBJECTIVES: Noninvasive remote ischemic preconditioning (RIPC) is a practical, acceptable, and feasible conditioning technique reported to provide cardioprotection in myocardial ischemia-reperfusion injury (MIRI). It has been well-reported that quercetin possesses antioxidant and anti-inflammatory properties. This study investigates the modification of the cardioprotective response of RIPC by quercetin. METHODS: Adult Wistar rats were randomized into 12 groups of six animals each. MIRI was induced by subjecting the isolated hearts of Wistar rats to global ischemia for 30 min, succeeded by reperfusion of 120 min after mounting on the Langendorff PowerLab apparatus. Hind limb RIPC was applied in four alternate cycles of ischemia and reperfusion of 5 min each by tying the pressure cuff before isolation of hearts. RESULTS: MIRI was reflected by significantly increased infarct size, LDH-1, and CK-MB, TNF-α, TBARS, and decreased GSH, catalase, and hemodynamic index, and modulated Nrf2. Pretreatment of quercetin (25 and 50 mg/kg; i.p.) significantly attenuated the MIRI-induced cardiac damage and potentiated the cardioprotective response of RIPC at the low dose. Pretreatment of ketamine (10 mg/kg; i.p.), an mTOR-dependent autophagy inhibitor, significantly abolished the cardioprotective effects of quercetin and RIPC. CONCLUSIONS: The findings highlight the modification of the cardioprotective effect of RIPC by quercetin and that quercetin protects the heart against MIRI through multiple mechanisms, including mTOR-dependent activation of autophagy and Nrf-2 activation.

Ultrafast four-dimensional imaging of cardiac mechanical wave propagation with sparse optoacoustic sensing.

Propagation of electromechanical waves in excitable heart muscles follows complex spatiotemporal patterns holding the key to understanding life-threatening arrhythmias and other cardiac conditions. Accurate volumetric mapping of cardiac wave propagation is currently hampered by fast heart motion, particularly in small model organisms. Here we demonstrate that ultrafast four-dimensional imaging of cardiac mechanical wave propagation in entire beating murine heart can be accomplished by sparse optoacoustic sensing with high contrast, ∼115-µm spatial and submillisecond temporal resolution. We extract accurate dispersion and phase velocity maps of the cardiac waves and reveal vortex-like patterns associated with mechanical phase singularities that occur during arrhythmic events induced via burst ventricular electric stimulation. The newly introduced cardiac mapping approach is a bold step toward deciphering the complex mechanisms underlying cardiac arrhythmias and enabling precise therapeutic interventions.

Imatinib attenuates reperfusion injury in a rat model of acute myocardial infarction.

Following an acute myocardial infarction, reperfusion of an occluded coronary artery is often accompanied by microvascular injury, leading to worse long-term prognosis. Experimental studies have revealed the potential of tyrosine-kinase inhibitor imatinib to reduce vascular leakage in various organs. Here, we examined the potential of imatinib to attenuate microvascular injury in a rat model of myocardial reperfusion injury. Isolated male Wistar rat hearts (n = 20) in a Langendorff system and male Wistar rats (n = 37) in an in vivo model were randomly assigned to imatinib or placebo and subjected to ischaemia and reperfusion. Evans-blue/Thioflavin-S/TTC staining and Cardiac Magnetic Resonance Imaging were performed to assess the extent of reperfusion injury. Subsequently, in vivo hearts were perfused ex vivo with a vascular leakage tracer and fluorescence and electron microscopy were performed. In isolated rat hearts, imatinib reduced global infarct size, improved end-diastolic pressure, and improved rate pressure product recovery compared to placebo. In vivo, imatinib reduced no-reflow and infarct size with no difference between imatinib and placebo for global cardiac function. In addition, imatinib showed lower vascular resistance, higher coronary flow, and less microvascular leakage in the affected myocardium. At the ultrastructural level, imatinib showed higher preserved microvascular integrity compared to placebo. We provide evidence that low-dose imatinib can reduce microvascular injury and accompanying myocardial infarct size in a rat model of acute myocardial infarction. These data warrant future work to examine the potential of imatinib to reduce reperfusion injury in patients with acute myocardial infarction.

C-type natriuretic peptide induces inotropic and lusitropic effects in human 3D-engineered cardiac tissue: Implications for the regulation of cardiac function in humans.

The role of C-type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well-characterized engineered cardiac tissues (ECTs) generated from human stem cell-derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP-induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs. NEW FINDINGS: What is the central question of this study? What are the acute responses to C-type natriuretic peptide (CNP) in human-engineered cardiac tissues (ECTs) on cardiac function and how well do they translate to matched concentrations in animal ex vivo and in vivo models? What is the main finding and its importance? Acute stimulation of ECTs with CNP induced positive lusitropic and inotropic effects on cardiac contractility, which closely reflected the changes observed in rat ex vivo and in vivo cardiac models. These findings support an important role for CNP in the regulation of human cardiac function and highlight the translational value of ECTs.

Manipulation of Nitric Oxide Levels via a Modified Hydroxyethyl Starch Molecule.

INTRODUCTION: Although the improving effect of nitric oxide (NO) donors has experimentally been demonstrated in shock, there are still no NO donor medications clinically available. Thiol-nitrosothiol-hydroxyethyl starch (S-NO-HES) is a novel molecule consisting of NO coupled to a thiolated derivative of hydroxyethyl starch (HES). It was aimed to assess the ability of S-NO-HES to serve as an NO donor under a variety of in vitro simulated physiologic conditions, which might be the first step to qualify this molecule as a novel type of NO donor-fluid. METHODS: We studied the effect of temperature on NO-releasing properties of S-NO-HES in blood, at 34°C, 37°C, and 41°C. Ascorbic acid (Asc) and amylase were also tested in a medium environment. In addition, we evaluated the activity of S-NO-HES in the isolated aortic ring and Langendorff-perfused heart setup. RESULTS: The NO release property of S-NO-HES was found at any temperature. Asc led to a significant increase in the production of NO compared to S-NO-HES incubation (P < 0.05). The addition of amylase together with Asc to the medium further increased the release of NO (P < 0.05). S-NO-HES exerted significant vasodilatory effects on phenylephrine precontracted aortic rings that were dose-dependent (P < 0.01). Furthermore, S-NO-HES significantly increased the heart rate and additionally reduced the duration of the cardiac action potential, as indicated by a reduction of QTc-B values (P < 0.01). CONCLUSIONS: We demonstrated for the first time that the S-NO-HES molecule exhibited its NO-releasing effects. The effectiveness of this new NO donor to substitute NO deficiency under septic conditions or in other indications needs to be studied.

Immunohistochemical characteristics of local sites that trigger atrial arrhythmias in response to high-frequency stimulation.

AIMS: The response to high frequency stimulation (HFS) is used to locate putative sites of ganglionated plexuses (GPs), which are implicated in triggering atrial fibrillation (AF). To identify topological and immunohistochemical characteristics of presumed GP sites functionally identified by HFS. METHODS AND RESULTS: Sixty-three atrial sites were tested with HFS in four Langendorff-perfused porcine hearts. A 3.5 mm tip quadripolar ablation catheter was used to stimulate and deliver HFS to the left and right atrial epicardium, within the local atrial refractory period. Tissue samples from sites triggering atrial ectopy/AF (ET) sites and non-ET sites were stained with choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), for quantification of parasympathetic and sympathetic nerves, respectively. The average cross-sectional area (CSA) of nerves was also calculated. Histomorphometry of six ET sites (9.5%) identified by HFS evoking at least a single atrial ectopic was compared with non-ET sites. All ET sites contained ChAT-immunoreactive (ChAT-IR) and/or TH-immunoreactive nerves (TH-IR). Nerve density was greater in ET sites compared to non-ET sites (nerves/cm2: 162.3 ± 110.9 vs. 69.65 ± 72.48; P = 0.047). Overall, TH-IR nerves had a larger CSA than ChAT-IR nerves (µm2: 11 196 ± 35 141 vs. 2070 ± 5841; P < 0.0001), but in ET sites, TH-IR nerves were smaller than in non-ET sites (µm2: 6021 ± 14 586 vs. 25 254 ± 61 499; P < 0.001). CONCLUSIONS: ET sites identified by HFS contained a higher density of smaller nerves than non-ET sites. The majority of these nerves were within the atrial myocardium. This has important clinical implications for devising an effective therapeutic strategy for targeting autonomic triggers of AF.

Automation and Optimization of Rat Heart Decellularization Using a Vibrating Fluid Column.

This paper presents the validation of a software application to optimize the discoloration process in simulated hearts and to automate and determine the final moment of decellularization in rat hearts using a vibrating fluid column. The implemented algorithm specifically for the automated verification of a simulated heart's discoloration process was optimized in this study. Initially, we used a latex balloon containing enough dye to reach the opacity of a heart. The complete discoloration process corresponds to complete decellularization. The developed software automatically detects the complete discoloration of a simulated heart. Finally, the process stops automatically. Another goal was to optimize the Langendorff-type experimental apparatus, which is pressure-controlled and equipped with a vibrating fluid column that shortens the decellularization time by mechanically acting directly on cell membranes. Control experiments were performed with the designed experimental device and the vibrating liquid column using different decellularization protocols for hearts taken from rats. In this work, we used a commonly utilized solution based on sodium dodecyl sulfate. Ultraviolet spectrophotometry was used to measure the evolution of the dye concentration in the simulated hearts and, similarly, to determine the concentrations of deoxyribonucleic acid (DNA) and proteins in the rat hearts.

Changes in Left Ventricular Ejection Fraction and Oxidative Stress after Phosphodiesterase Type-5 Inhibitor Treatment in an Experimental Model of Retrograde Rat Perfusion.

Background and objectives: Taking into consideration the confirmed role of oxidative stress in ischemia/reperfusion injury and the insufficiency in knowledge regarding the phosphodiesterase 5 (PDE5)-mediated effects on the cardiovascular system, the aim of our study was to investigate the influence of two PDE5 inhibitors, tadalafil and vardenafil, with or without the addition of N(G)-nitro-L-arginine methyl ester (L-NAME), on oxidative stress markers, coronary flow and left ventricular function, both ex vivo and in vivo. Methods: This study included 74 male Wistar albino rats divided into two groups. In the first, 24 male Wistar rats were orally treated with tadalafil or vardenafil for four weeks in order to perform in vivo experiments. In the second, the hearts of 50 male Wistar albino were excised and perfused according to the Langendorff technique in order to perform ex vivo experiments. The hearts were perfused with tadalafil (10, 20, 50 and 200 nM), vardenafil (10, 20, 50 and 200 nM) and a combination of tadalafil/vardenafil and L-NAME (30 µM). The CF and oxidative stress markers, including nitrite bioaviability (NO2-), superoxide anion radical (O2-), and the index of lipid peroxidation, were measured in coronary effluent. Results: The L-arginin/NO system acts as the mediator in the tadalafil-induced effects on the cardiovascular system, while it seems that the vardenafil-induced increase in CF was not primarily induced by the NO system. Although tadalafil induced an increase in O2- in the two lowest doses, the general effects of both of the applied PDE5 inhibitors on oxidative stress were not significant. The ejection function was above 50% in both groups. Conclusions: Our results showed that both tadalafil and vardenafil improved the coronary perfusion of the myocardium and LV function by increasing the EF.

Reevaluating methods reporting practices to improve reproducibility: an analysis of methodological rigor for the Langendorff whole heart technique.

In recent decades, the scientific community has seen an increased interest in rigor and reproducibility. In 2017, concerns about methodological thoroughness and reporting practices were implicated as significant barriers to reproducibility within the preclinical cardiovascular literature, particularly in studies using animal research. The Langendorff, whole heart technique has proven to be an invaluable research tool, being modified in a myriad of ways to probe questions across the spectrum of physiological and pathophysiological functions of the heart. As a result, significant variability in the application of the Langendorff technique exists. This literature review quantifies the different methods employed in the implementation of the Langendorff technique and provides brief examples of how individual parametric differences can impact the outcomes and interpretation of studies. From 2017 to 2020, significant variability of animal models, anesthesia, cannulation time, perfusate composition, pH, and temperature demonstrate that the technique has diversified to meet new challenges and answer different scientific questions. The review also reveals which individual methods are most frequently reported, even if there is no explicit agreement upon which parameters should be reported. The analysis of methods related to the Langendorff technique suggests a framework for considering methodological approach when interpreting seemingly contradictory results, rather than concluding that results are irreproducible.

Modification of the Langendorff system of the isolated beating heart for experimental radiotherapy at a synchrotron: 4000†Gy in a heart beat.

Microbeam radiotherapy could help to cure malignant tumours which are currently still considered therapy-resistant. With an irradiation target in the thoracic cavity, the heart would be one of the most important organs at risk. To assess the acute adverse effects of microbeam irradiation in the heart, a powerful ex vivo tool was created by combining the Langendorff model of the isolated beating mammalian heart with X-Tream dosimetry. In a first pilot experiment conducted at the Biomedical and Imaging Beamline of the Australian Synchrotron, the system was tested at a microbeam peak dose approximately ten times higher than the anticipated future microbeam irradiation treatment doses. The entire heart was irradiated with a dose of 4000 Gy at a dose rate of >6000 Gy s-1, using an array of 50 µm-wide microbeams spaced at a centre-to-centre distance of 400 µm. Although temporary arrhythmias were seen, they reverted spontaneously to a stable rhythm and no cardiac arrest occurred. This amazing preservation of cardiac function is promising for future therapeutic approaches.

Development of a cardiovascular magnetic resonance-compatible large animal isolated heart model for direct comparison of beating and arrested hearts.

Cardiac motion results in image artefacts and quantification errors in many cardiovascular magnetic resonance (CMR) techniques, including microstructural assessment using diffusion tensor cardiovascular magnetic resonance (DT-CMR). Here, we develop a CMR-compatible isolated perfused porcine heart model that allows comparison of data obtained in beating and arrested states. Ten porcine hearts (8/10 for protocol optimisation) were harvested using a donor heart retrieval protocol and transported to the remote CMR facility. Langendorff perfusion in a 3D-printed chamber and perfusion circuit re-established contraction. Hearts were imaged using cine, parametric mapping and STEAM DT-CMR at cardiac phases with the minimum and maximum wall thickness. High potassium and lithium perfusates were then used to arrest the heart in a slack and contracted state, respectively. Imaging was repeated in both arrested states. After imaging, tissue was removed for subsequent histology in a location matched to the DT-CMR data using fiducial markers. Regular sustained contraction was successfully established in six out of 10 hearts, including the final five hearts. Imaging was performed in four hearts and one underwent the full protocol, including colocalised histology. The image quality was good and there was good agreement between DT-CMR data in equivalent beating and arrested states. Despite the use of autologous blood and dextran within the perfusate, T2 mapping results, DT-CMR measures and an increase in mass were consistent with development of myocardial oedema, resulting in failure to achieve a true diastolic-like state. A contiguous stack of 313 5-µm histological sections at and a 100-µm thick section showing cell morphology on 3D fluorescent confocal microscopy colocalised to DT-CMR data were obtained. A CMR-compatible isolated perfused beating heart setup for large animal hearts allows direct comparisons of beating and arrested heart data with subsequent colocalised histology, without the need for onsite preclinical facilities.

Chronic magnesium deficiency causes reversible mitochondrial permeability transition pore opening and impairs hypoxia tolerance in the rat heart.

Chronic magnesium (Mg) deficiency induces and exacerbates various cardiovascular diseases. We previously investigated the mechanisms underlying decline in cardiac function caused by chronic Mg deficiency and the effectiveness of Mg supplementation on this decline using the Langendorff-perfused isolated mouse heart model. Herein, we used the Langendorff-perfused isolated rat heart model to demonstrate the chronic Mg-deficient rats (Mg-deficient group) had lower the heart rate (HR) and left ventricular pressure (LVDP) than rats with normal Mg levels (normal group). Furthermore, decline in cardiac function due to hypoxia/reoxygenation injury was significantly greater in the Mg-deficient group than in the normal group. Experiments on mitochondrial permeability transition pore (mPTP) using isolated mitochondria revealed that mitochondrial membrane was fragile in the Mg-deficient group, implying that cardiac function decline through hypoxia/reoxygenation injury is associated with mitochondrial function. Mg supplementation for chronic Mg-deficient rats not only improved hypomagnesemia but also almost completely restored cardiac and mitochondrial functions. Therefore, proactive Mg supplementation in pathological conditions induced by Mg deficiency or for those at risk of developing hypomagnesemia may suppress the development and exacerbation of certain disease states.

Electrophysiological Safety Profile of Antiestrogenic Therapies in the Isolated Rabbit Heart.

INTRODUCTION: Hormone-mediated therapies are on the rise and are key therapies in the treatment of some of the most common cancer entities. Proarrhythmic effects have been described in patients treated with SERMs while little to none is known about the electrophysiological effects of the potentially less arrhythmogenic selective estrogen receptor degraders. METHODS: Twenty hearts of female New Zealand White rabbits were excised and retrogradely perfused employing a Langendorff setup. An electrophysiology study was performed to obtain CL-dependent action potential duration at 90% of repolarization (APD90), QT interval, effective refractory period (ERP), and post-repolarization refractoriness (PRR = ERP-APD90). After generating baseline data, the hearts were assigned to two groups and perfused with (n = 10) increasing doses of fulvestrant (1 µM and 5 µM; n = 10) or tamoxifen (2.5 µM and 7.5 µM; n = 10), and the protocol was repeated again. RESULTS: Fulvestrant led to a decrease in APD90 and QT interval and an increased PRR. The inducibility of ventricular tachycardia (VT) episodes was unaltered. Tamoxifen showed similar effects, resulting in a shortened APD90 and QT interval and no increased VT incidence in the setting of a prolonged PRR. CONCLUSION: The present study shows no acute proarrhythmic effect of tamoxifen and fulvestrant in an established whole heart model when employing clinically relevant concentrations.

Novel Labdane Diterpenes-Based Synthetic Derivatives: Identification of a Bifunctional Vasodilator That Inhibits CaV1.2 and Stimulates KCa1.1 Channels.

Sesquiterpenes such as leucodin and the labdane-type diterpene manool are natural compounds endowed with remarkably in vitro vasorelaxant and in vivo hypotensive activities. Given their structural similarity with the sesquiterpene lactone (+)-sclareolide, this molecule was selected as a scaffold to develop novel vasoactive agents. Functional, electrophysiology, and molecular dynamics studies were performed. The opening of the five-member lactone ring in the (+)-sclareolide provided a series of labdane-based small molecules, promoting a significant in vitro vasorelaxant effect. Electrophysiology data identified 7 as a CaV1.2 channel blocker and a KCa1.1 channel stimulator. These activities were also confirmed in the intact vascular tissue. The significant antagonism caused by the CaV1.2 channel agonist Bay K 8644 suggested that 7 might interact with the dihydropyridine binding site. Docking and molecular dynamic simulations provided the molecular basis of the CaV1.2 channel blockade and KCa1.1 channel stimulation produced by 7. Finally, 7 reduced coronary perfusion pressure and heart rate, while prolonging conduction and refractoriness of the atrioventricular node, likely because of its Ca2+ antagonism. Taken together, these data indicate that the labdane scaffold represents a valuable starting point for the development of new vasorelaxant agents endowed with negative chronotropic properties and targeting key pathways involved in the pathophysiology of hypertension and ischemic cardiomyopathy.

New Thiazole Acetic Acid Derivatives: A Study to Screen Cardiovascular Activity Using Isolated Rat Hearts and Blood Vessels.

Cardiovascular diseases are one of the major causes of mortalities worldwide. In the present research, new synthetic derivatives of thiazole were studied using isolated hearts and blood vessels of rats. The heart and thoracic aorta were tested with six new synthesized thiazole acetic acid derivatives (SMVA-10, SMVA-35, SMVA-40, SMVA-41, SMVA-42 and SMVA-60), and the data obtained were statistically analyzed and compared. Isolated rat hearts were used to record the changes in developed tension and heart rate, while thoracic aortas were used to measure the contractile response, before and after treatments. Analysis of the results indicated a significant (p < 0.01) increase in developed tension with the addition of SMVA-35, SMVA-40, SMVA-41 and SMVA-42, which was augmented in the presence of adrenaline without affecting the heart rate. On the other hand, acetylcholine significantly decreased the developed tension, which was significantly reversed (p < 0.01) in the presence of compounds (SMVA-35 and SMVA-60). However, in the presence of SMVA-35 and SMVA-40, acetylcholine-induced bradycardia was significantly (p < 0.01) reduced. Furthermore, only SMVA-42 induced a dose-dependent contractile response in the isolated blood vessel, which was abolished in the presence of prazosin. Therefore, it can be concluded that some of the new synthesized thiazole derivatives exhibited promising results by raising the developed tension without changing the heart rate or blood vessel function, which could be helpful in failing heart conditions. However, more research is required to fully comprehend the function, mechanism and effectiveness of the compounds.

An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping.

Cardiovascular disease is a leading cause of death and disability worldwide. Although genetically modified mouse models offer great potential for robust research in vivo, in vitro studies using isolated cardiomyocytes also provide an important approach for investigating the mechanisms underlying cardiovascular disease pathogenesis and drug actions. Currently, isolation of mouse adult cardiomyocytes often relies on aortic retrograde intubation under a stereoscopic microscope, which poses considerable technical barriers and requires extensive training. Although a simplified, Langendorff-free method has been used to isolate viable cardiomyocytes from the adult mouse heart, the system requires enzymatic digestions and continuous manual technical operation. This study established an optimized approach that allows isolation of adult mouse cardiomyocytes and epicardial activation mapping of mouse hearts using a Langendorff device. We used retrograde puncture through the abdominal aorta in vivo and enzymatic digestion on the Langendorff perfusion device to isolate adult mouse cardiomyocytes without using a microscope. The yields of isolated cardiomyocytes were amenable to patch clamp techniques. Furthermore, this approach allowed epicardial activation mapping. We used a novel, simplified method to isolate viable cardiomyocytes from adult mouse hearts and to map epicardial activation. This novel approach could be beneficial in more extensive research in the cardiac field.