Heart Preparation Research Articles

The heart, a secondary organ in the control of blood circulation.

Circulation of the blood is a fundamental physiological function traditionally ascribed to the pressure-generating function of the heart. However, over the past century the 'cardiocentric' view has been challenged by August Krogh, Ernst Starling, Arthur Guyton and others, based on haemodynamic data obtained from isolated heart preparations and organ perfusion. Their research brought forth experimental evidence and phenomenological observations supporting the concept that cardiac output occurs primarily in response to the metabolic demands of the tissues. The basic tenets of Guyton's venous return model are presented and juxtaposed with their critiques. Developmental biology of the cardiovascular system shows that the blood circulates before the heart has achieved functional integrity and that its movement is intricately connected with the metabolic demands of the tissues. Long discovered, but as yet overlooked, negative interstitial pressure may play a role in assisting the flow returning to the heart. Based on these phenomena, an alternative circulation model has been proposed in which the heart functions like a hydraulic ram and maintains a dynamic equilibrium between the arterial (centrifugal) and venous (centripetal) forces which define the blood's circular movement. In this focused review we introduce some of the salient arguments in support of the proposed circulation model. Finally, we present evidence that exercising muscle blood flow is subject to local metabolic control which upholds optimal perfusion in the face of a substantive rise in muscle vascular conductance, thus lending further support to the permissive role of the heart in the overall control of blood circulation.

Donor heart preconditioning during orthotopic transplantation

Background. To date, there are about 500 patients in the heart transplant waiting list in Ukraine. The study included 20 donors for orthotopic heart transplantation. Their average age was 32.3 years. The study evaluated the preparation of the donor heart in terms of its protection against ischemia-reperfusion injury during transplantation and the effect of donor heart preconditioning in a recipient after orthotopic heart transplantation. Тhe purpose was to evaluate the effectiveness of donor heart preconditioning for increasing myocardial resistance to ischemia-reperfusion injury and its preparation for orthotopic transplantation. Materials and methods. The work is based on the results of 20 donors who were diagnosed with brain death and authorized for the use of transplantation of human anatomical materials. Then changes in the main biochemical parameters (lactate, troponin, acid-base balance, creatinine clearance, blood glucose) and the functional state of systemic hemodynamics (blood pressure, heart rate, central venous pressure, ejection fraction, cardiac index) were analyzed at the time of donor heart explantation. Results. It was found that in 100 % of donors, norepinephrine was used for inotropic support, in 40 % — dobutamine and in 25 % — dopamine. Lactate levels were higher than normal in 75 % of cases and troponin levels were increased in all donors, indicating a possible microcirculatory dysfunction that led to a deterioration in donor preconditioning and enhanced ischemia-reperfusion injury to the transplant. According to the correlation coefficient between time and cardiac index, there was a tendency to the deterioration in the donor’s condition depending on the time of making a decision about heart transplantation before heart explantation. Conclusions. The study showed that in 100 % of donors, norepinephrine was used for inotropic support — more often than dobutamine or dopamine. Lactate levels were higher than normal in 75 % of donors and troponin levels were increased in all donors, indicating a possible microcirculatory dysfunction that led to a deterioration in donor preconditioning and increased ischemia-reperfusion injury to the transplant. According to the correlation coefficient between time indicators and cardiac index, it is noted that the longer the time of authorization for orthotopic heart transplantation, the greater the deterioration in the donor’s condition, which is expressed in a decreased myocardial contractility. Based on the correlation coefficient between time indicators and troponin, it was concluded that the growth of troponin complexes was observed with an increase in the time of making a decision for obtaining permission to transplant human anatomical materials, which indicated the onset of ischemic changes in the myocardium.

LDL electronegativity and male predominance in early ventricular tachyarrhythmias following ST-elevation myocardial infarction: evidence of sexual dimorphism associated reduction of sodium currents

Abstract Background Early ventricular tachycardia/fibrillation (VT/VF) in patients with ST-elevation myocardial infarction (STEMI) was associated with a higher morbidity and mortality rate. We sought to investigate the gender-specific risk and explore the underlying mechanisms of early VT/VF in STEMI patients. Methods We analyzed the clinical and laboratory data among 2965 consecutive STEMI patients between Jan 1, 2008 and Dec 31, 2021. Early VT/VF was defined as occurrence of spontaneous VT/VF of ≥30 seconds or requirement of immediate cardioversion/defibrillation due to hemodynamic compromise within the first 48 hours after onset of symptoms. To explore the mechanisms underlying early VT/VF, ex vivo ischemic-reperfusion experiments were conducted in 8-week-old ApoE-/- mice fed a high-fat diet for 8 weeks to promote atherogenesis. Results Of the 2965 STEMI patients studied, 256 (8.6%) experienced early VT/VF. Patients who developed early VT/VF were younger (58±13 vs. 61±13 years old, P=0.012) and had a higher proportion of males (87% vs. 83%, P=0.043). Notably, some patients with early VT/VF had significantly higher plasma levels of L5, the most electronegative subclass of low-density lipoprotein (LDL), compared to those without early VT/VF (n=19, L5: 10.14±3.09% vs. n=43, L5: 2.11±0.23%, P=0.006). In animal study, we have previously showed that male mice have higher plasma L5 concentrations than female mice. In this study, all male mice (n=4) developed VT/VF following the sham operation, whereas none of the female mice (n=3) did. Moreover, male mice had significantly slower heart conduction velocity than female mice in whole heart preparations (13.37±3.81 cm/s vs. 40.70±4.97 cm/s, P<0.001), despite having similar action potential duration (61.00±7.07 vs. 50.33±4.16 ms, P=0.999). In addition, isolated ventricular myocytes from male mice exhibited markedly lower sodium current density (-29.20±3.04 pA/pF, n=6) compared to those from female mice (-114.05±6.41 pA/pF, n=6, P<0.001). The reduced sodium current density was accompanied by lower membranous Nav1.5 protein expression (0.38±0.06 vs. 0.89±0.09 A.U., P<0.001) and higher cytosolic Nav1.5 levels (0.59±0.06 vs. 0.29±0.04 A.U., P<0.01) in male mice. Conclusions Our study demonstrates that male STEMI patients with early VT/VF have higher levels of L5, the most electronegative LDL subclass. This gender disparity in the susceptibility of early VT/VF may be caused by compromised sodium channel trafficking, likely linked to increased LDL electronegativity.

Abstract 14259: Contractile Effects of Glucagon in Atrial Preparations From Transgenic Mice and Men

Glucagon, a peptide composed of 29 amino acids, produced mainly in the pancreas can also stimulate cardiac function. Glucagon might contribute to hyperglycemia in diabetes mellitus type 2 and is thus of clinical relevance. There is controversy in the literature whether glucagon affects force and beating rate in the human atrium and which receptor is involved. Hence, we studied the effect of glucagon on atrial preparations of wild type mouse hearts (WT), from transgenic mice with cardiac overexpression of the human glucagon receptor (GR-TG) and for comparison in human right atrial preparations. We noted that glucagon exerted concentration- and time-dependent positive chronotropic effects in atrial preparations from WT and GR-TG starting at 0.1 nM glucagon and amounting to 25 % of pre-drug value at 3 nM glucagon (n=8, p<0.05). These effects were antagonized by the glucagon receptor antagonist SC203973 (1 μM), the inhibitor of cAMP dependent protein kinase H89 (1 μM), by the M-cholinoceptor agonist 1 μM carbachol, by the A1-adenosine receptor antagonist R-phenylisopropyladenosine (1 μM) and the inhibitors of hyperpolarization-activated cyclic nucleotide gated channels namely ivabradine (1 μM) or cesium chloride (2 mM) (n=5-6, p<0.05). In left atrial preparations from GR-TG but not WT, glucagon exerted starting at 1 nM a positive inotropic effect. Interestingly, in human atrial preparations glucagon at 1 μM in the additional presence of the phosphodiesterase III inhibitor cilostamide (1 μM) increased force of contraction by 34 % (n=5, p<0.05). We conclude that glucagon can exert contractile effects in mammalian hearts via GR, but they exhibit considerable regional and species differences. The physiological and therapeutic relevance of glucagon in the human heart needs to be fully elucidated.

Abstract 15483: Mitoquinone Pretreatment Mitigates Dox-induced Systolic Dysfunction in Isolated Rat Hearts

Background: Doxorubicin (DOX) is a common and effective chemotherapeutic agent that can exert cardiotoxicity by inducing oxidative stress in mitochondria, compromising mitochondrial function, and inducing apoptosis. Currently, there is no treatment that targets this mechanism. We previously found that pretreatment with higher concentrations of mitoquinone (MQ), a mitochondrial antioxidant, exerts cardioprotection against DOX in myoblast H9C2 cells. Hypothesis: We hypothesize that MQ pretreatment would preserve cardiac function against DOX in isolated rat hearts. Methods: Cardiac parameters (heart rate (HR) and left ventricular end systolic pressure (LVESP)) were monitored before and after 60 min infusion of DOX, MQ, or DOX with MQ pretreatment (10-15 min) in a Langendorff preparation of male SD rat hearts. TUNEL assay was conducted on heart tissue slides to evaluate apoptosis. The ratio between the final and initial cardiac parameters was calculated and compared among experimental groups. Results: Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, to 39±5% than lower dose DOX infusion (20 μM, n=3; 74±3%). DOX had no effects on HR. A 60-minute perfusion of higher doses of MQ (1-5 μM, n=7) unexpectedly reduced the ratios of LVESP and HR to 73±12% and 66±5%, respectively. Higher dose MQ (1-2.5 μM, n=6) pretreatment showed similar improvement of LVESP ratio (68±9%) to lower dose MQ (0.25-0.5 μM, n=6; 71±11%). TUNEL assay showed that higher dose DOX caused a higher rate of apoptosis (number of TUNEL-positive nuclei) (326.3±6.4. n=3) than lower dose (214±29.5, n=3). Pretreatment with higher dose MQ prior to DOX 25 μM infusion resulted in a notable reduction of apoptosis (60.3±5, n=6) compared to DOX alone, while lower dose pretreatment reduced apoptosis to 70.5± 0.5 (n=4). Conclusion: This study suggests that infusion of DOX into the heart acutely attenuated cardiac systolic function accompanied with higher apoptotic cell damage. Although prolonged administration of higher doses of MQ suppressed cardiac function, MQ pretreatment for 10-15 minutes mitigated DOX-induced systolic dysfunction and reduced cardiomyocyte apoptosis. In summary, MQ pretreatment may mitigate DOX-induced cardiac damage.

Visualization of cardiac thick filament dynamics in ex vivo heart preparations

RationaleCardiac muscle cells are terminally differentiated after birth and must beat continually throughout one's lifetime. This mechanical process is driven by the sliding of actin-based thin filaments along myosin-based thick filaments, organized within sarcomeres. Despite costly energetic demand, the half-life of the proteins that comprise the cardiac thick filaments is ∼10 days, with individual molecules being replaced stochastically, by unknown mechanisms. ObjectivesTo allow for the stochastic replacement of molecules, we hypothesized that the structure of thick filaments must be highly dynamic in vivo. Methods and resultsTo test this hypothesis in adult mouse hearts, we replaced a fraction of the endogenous myosin regulatory light chain (RLC), a component of thick filaments, with GFP-labeled RLC by adeno-associated viral (AAV) transduction. The RLC-GFP was properly localized to the heads of the myosin molecules within thick filaments in ex vivo heart preparations and had no effect on heart size or actin filament siding in vitro. However, the localization of the RLC-GFP molecules was highly mobile, changing its position within the sarcomere on the minute timescale, when quantified by fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy. Interestingly, RLC-GFP mobility was restricted to within the boundaries of single sarcomeres. When cardiomyocytes were lysed, the RLC-GFP remained strongly bound to myosin heavy chain, and the intact myosin molecules adopted a folded, compact configuration, when disassociated from the filaments at physiological ionic conditions. ConclusionsThese data demonstrate that the structure of the thick filament is highly dynamic in the intact heart, with a rate of molecular exchange into and out of thick filaments that is ∼1500 times faster than that required for the replacement of molecules through protein synthesis or degradation.

Endothelium-Derived Dopamine and 6-Nitrodopamine in the Cardiovascular System.

The review deals with the release of endothelium-derived dopamine and 6-nitrodopamine (6-ND) and its effects on isolated vascular tissues and isolated hearts. Basal release of both dopamine and 6-ND is present in human isolated umbilical cord vessels, human popliteal vessels, nonhuman primate vessels, and reptilia aortas. The 6-ND basal release was significantly reduced when the tissues were treated with Nω-nitro-l-arginine methyl ester and virtually abolished when the endothelium was mechanically removed. 6-Nitrodopamine is a potent vasodilator, and the mechanism of action responsible for this effect is the antagonism of dopamine D2-like receptors. As a vasodilator, 6-ND constitutes a novel mechanism by which nitric oxide modulates vascular tone. The basal release of 6-ND was substantially decreased in endothelial nitric oxide synthase knockout (eNOS-/-) mice and not altered in neuronal nitric oxide synthase knockout (nNOS-/-) mice, indicating a nonneurogenic source for 6-ND in the heart. Indeed, in rat isolated right atrium, the release of 6-ND was not affected when the atria were treated with tetrodotoxin. In the rat isolated right atrium, 6-ND is the most potent endogenous positive chronotropic agent, and in Langendorff's heart preparation, it is the most potent endogenous positive inotropic agent. The positive chronotropic and inotropic effects of 6-ND are antagonized by β1-adrenoceptor antagonists at concentrations that do not affect the effects induced by noradrenaline, adrenaline, and dopamine, indicating that blockade of the 6-ND receptor is the major modulator of heart chronotropism and inotropism. The review proposes that endothelium-derived catecholamines may constitute a major mechanism for control of vascular tone and heart functions, in contrast to the overrated role attributed to the autonomic nervous system.

Interaction of the antiarrhythmic drug Amiodarone with the sodium channel Nav1.5 depends on the extracellular pH

IntroductionAmiodarone (AMD) is a clinically used drug to treat arrhythmias with significant effect upon the cardiac sodium channel Nav1.5. AMD has a pKa of 6.56, and changes in extracellular pH (pHe) may alter its pharmacological properties. Here we explored how changes in pHe impacts the pharmacological properties of AMD upon human-Nav1.5-sodium-current (INa) and in ex vivo rat hearts. MethodsEmbryonic-human-kidney-cells (HEK293) were used to transiently express the human alpha-subunit of NaV1.5 channels and the isolated heart of Wistar rats were used. Patch-Clamp technique was deployed to study INa and for electrocardiogram (ECG) evaluation the ex vivo heart preparation in the Langendorff system was applied. ResultsThe potency of AMD upon peak INa was ∼25x higher in pHe 7.0 when compared to pHe 7.4. Voltage dependence for activation did not differ among all groups. AMD shifted the steady-state inactivation curve to more hyperpolarized potentials, with similar magnitudes for both pHes. The recovery from INa inactivation was delayed in the presence of AMD with similar profile in both pHes. Interestingly, the use-dependent properties of AMD was distinct at pHe 7.0 and 7.4. Finally, AMD was able to change the ex vivo ECG profile, however at pHe 7.0+AMD a larger increase in the RR and QRS duration and in the QT interval when compared to pHe 7.4 was found. ConclusionsThe pharmacological properties of AMD upon NaV1.5 and isolated heart preparation depends on the pHe and its use in vivo during extracellular acidosis may cause a distinct biological response in the heart tissue.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 μg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 μg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 μg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 μg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

Optical mapping of cardiac electromechanics in beating in vivo hearts

Optical mapping has been widely used in the study of cardiac electrophysiology in motion-arrested, ex vivo heart preparations. Recent developments in motion artifact mitigation techniques have made it possible to optically map beating ex vivo hearts, enabling the study of cardiac electromechanics using optical mapping. However, the ex vivo setting imposes limitations on optical mapping such as altered metabolic states, oversimplified mechanical loads, and the absence of neurohormonal regulation. In this study, we demonstrate optical electromechanical mapping in an in vivo heart preparation. Swine hearts were exposed via median sternotomy. Voltage-sensitive dye, either di-4-ANEQ(F)PTEA or di-5-ANEQ(F)PTEA, was injected into the left anterior descending artery. Fluorescence was excited by alternating green and amber light for excitation ratiometry. Cardiac motion during sinus and paced rhythm was tracked using a marker-based method. Motion tracking and excitation ratiometry successfully corrected most motion artifact in the membrane potential signal. Marker-based motion tracking also allowed simultaneous measurement of epicardial deformation. Reconstructed membrane potential and mechanical deformation measurements were validated using monophasic action potentials and sonomicrometry, respectively. Di-5-ANEQ(F)PTEA produced longer working time and higher signal/noise ratio than di-4-ANEQ(F)PTEA. In addition, we demonstrate potential applications of the new optical mapping system including electromechanical mapping during vagal nerve stimulation, fibrillation/defibrillation. and acute regional ischemia. In conclusion, although some technical limitations remain, optical mapping experiments that simultaneously image electrical and mechanical function can be conducted in beating, in vivo hearts.

Eugenol delays the onset of ouabain-induced ventricular cardiac arrhythmias in guinea pigs.

Eugenol is an aromatic compound used in the manufacture of medicines, perfumes, cosmetics and as an anaesthetic due to the ability of the drug to block the neuronal isoform of voltage-gated Na+ channels (NaV ). Some arrhythmias are associated with gain of function in the sodium current (INa ) found in cardiomyocytes, and antiarrhythmic sodium channel blockers are commonly used in the clinical practice. This study sought to elucidate the potential mechanisms of eugenol's protection in the arrhythmic model of ouabain-induced arrhythmias in guinea pig heart. Ex vivo arrhythmias were induced using 50 μM of ouabain. The antiarrhythmic properties of eugenol were evaluated in the ex vivo heart preparation and isolated ventricular cardiomyocytes. The compound's effects on cardiac sodium current and action potential using the patch-clamp technique were evaluated. In all, eugenol decreased the ex vivo cardiac arrhythmias induced by ouabain. Furthermore, eugenol showed concentration dependent effect upon peak INa , left-shifted the stationary inactivation curve and delayed the recovery from inactivation of the INa . All these aspects are considered to be antiarrhythmic. Our findings demonstrate that eugenol has antiarrhythmic activity, which may be partially explained by the ability of eugenol to change de biophysical properties of INa of cardiomyocytes.

Autonomic neuro-cardiac profile of electrical, structural and neuronal remodeling in myocardial infarction-induced heart failure.

Heart failure is a clinical syndrome typified by abnormal autonomic tone, impaired ventricular function, and increased arrhythmic vulnerability. This study aims to examine electrophysiological, structural and neuronal remodeling following myocardial infarction in a rabbit heart failure model to establish its neuro-cardiac profile. Weight-matched adult male New Zealand White rabbits (3.2±0.1kg, n=25) were randomized to have coronary ligation surgeries (HF group, n=13) or sham procedures (SHM group, n=12). Transthoracic echocardiography was performed six weeks post-operatively. On week 8, dual-innervated Langendorff-perfused heart preparations were set up for terminal experiments. Seventeen hearts (HF group, n=10) underwent ex-vivo cardiac MRI. Twenty-two hearts (HF group, n=7) were examined histologically. Electrical remodeling and abnormal autonomic profile were evident in HF rabbits with exaggerated sympathetic and attenuated vagal effect on ventricular fibrillation threshold, ventricular refractoriness and restitution curves, in addition to increased spatial restitution dispersion. Histologically, there was significant neuronal enlargement at the heart hila and conus arteriosus in HF. Structural remodeling was characterized by quantifiable myocardial scarring, enlarged left ventricles, altered ventricular geometry and impaired contractility. In an infarct-induced rabbit heart failure model, extensive structural, neuronal and electrophysiological remodeling in conjunction with abnormal autonomic profile provide substrates for ventricular arrhythmias.

KairoSight-3.0: A validated optical mapping software to characterize cardiac electrophysiology, excitation-contraction coupling, and alternans.

Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

The influence of electrode-tissue-coverage on RF lesion formation and local impedance: Insights from an ex vivo model.

The influence of power, duration and contact force (CF) on radiofrequency (RF) lesion formation is well known, whereas data on local impedance (LI) and electrode-tissue-coverage (ETC) is scarce. The objective was to investigate their effect on lesion formation in an ex vivo model. An ex vivo model was developed utilizing cross-sections of porcine heart preparations and a force-sensing, LI-measuring catheter. N=72 lesion were created systematically varying ETC (minor/full), CF (1-5g, 10-15g, 20-25g) and power (20W, 30W, 40W, 50W). In minor ETC, the distal tip of the catheter was in electric contact with the tissue, in full ETC the whole catheter tip was embedded within the tissue. Lesion size and all parameters were measured once per second (n=3320). LI correlated strongly with lesion depth (r=-0.742 for ΔLI; r=0.781 for %LI-drop). Lesions in full ETC were significantly wider and deeper compared to minor ETC (p<.001) and steam pops were more likely. Baseline LI, ΔLI, and %LI-drop were significantly higher in full ETC (p<.001). In lesions resulting in steam pops, baseline LI, and ΔLI were significantly higher. The influence of CF on lesion size was higher in minor ETC than in full ETC. ETC is a main determinant of lesion size and occurrence of steam pops. Baseline LI and LI-drop are useful surrogate parameters for real-time assessment of ETC and ΔLI correlates strongly with lesion size.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart. Atrial tissue samples from animals with AF reveal an abnormal increase in cardiomyocytes death and alterations in calcium handling proteins. High-throughput quantitative proteomics and immunoblotting analyses at different stages of AF progression identify downregulation of contractile proteins and progressive increase in atrial fibrosis. Moreover, advanced optical mapping techniques, applied to whole heart preparations during AF, demonstrate that AF-related remodeling decreases the frequency threshold for dissociation between transmembrane voltage signals and intracellular calcium transients compared to healthy controls. Single cell simulations of human atrial cardiomyocytes also confirm the experimental results. In patients, non-invasive assessment of the atrial electromechanical relationship further demonstrate that atrial electromechanical dissociation is an early prognostic indicator for acute and long-term rhythm control.

Abstract P1013: Identification Of Genetic Links Between Cardiovascular Disease And Insomnia In Drosophila

Cardiovascular disease (CVD) is the leading cause of death worldwide. CVD is associated with insomnia such that insomnia symptoms double the risk of incident CVD. However, how they are related is unknown. Two recent genome-wide association studies (GWAS) identified genetic loci significantly associated with insomnia, including one locus with five nearby genes that are associated with CVD in an independent GWAS. Therefore, we hypothesized that genetic predisposition to insomnia contributes to the development of CVD. To test this and identify causal genes at this locus, we used Drosophila melanogaster, which are well-established model systems for sleep and cardiac studies. To assess the role of these genes on cardiac physiology, we used the cardiac-specific Hand-Gal4 driver to perform genetic knockdown (KD). One-week-old Drosophila progeny were then used for semi-intact microscopic heart preparation followed by high-speed videography to assess cardiac physiology. Similarly, to assess their role in sleep, we used the neuronal-specific Elav-Gal4 driver to perform KD of these genes. Sleep and locomotor activity of one-week-old flies were monitored using the Drosophila Activity Monitoring System. We found that neuronal and cardiac-specific RNAi KD of four genes conserved in Drosophila: Lsn, ATPSynC, Bruce, and Imp, contributes to compromised sleep and cardiac performance, respectively. Cardiac-specific KD of Lsn led to significant cardiac dilation and reduced fractional shortening, a measure of cardiac performance. KD of ATPSynC led to significantly reduced fractional shortening without dilations. Furthermore, Lsn and ATPSynC KD hearts had disrupted actin-containing myofibrillar organization. Suppression of Lsn increased Pericardin deposition, indicative of fibrosis. Also, the cardiac suppression of ATPSynC and Lsn significantly shortened lifespan. Neuronal-specific KD of three genes significantly disrupted sleep. Furthermore, KD of ATPSynC and Lsn in the heart disrupted sleep in a non-cell-autonomous manner. This work provides novel insights into genetic mechanisms linking CVD and insomnia, highlighting the potential importance of these four genes in the development of both diseases.

Ergometrine stimulates histamine H2 receptors in the isolated human atrium

Ergometrine (6aR,9R)-N-((S)-1-hydroxypropan-2-yl)-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo-[4,3-fg]chinolin-9-carboxamide or lysergide acid β-ethanolamide or ergonovine) activates several types of serotonin and histamine receptors in the animal heart. We thus examined whether ergometrine can activate human serotonin 5-HT4 receptors (h5-HT4R) and/or human histamine H2 receptors (hH2R) in the heart of transgenic mice and/or in the human isolated atrium. Force of contraction or beating rates were studied in electrically stimulated left atrial or spontaneously beating right atrial preparations or spontaneously beating isolated retrogradely perfused hearts (Langendorff setup) of mice with cardiac specific overexpression of the h5-HT4R (5-HT4-TG) or of mice with cardiac specific overexpression of the hH2R (H2-TG) or in electrically stimulated human right atrial preparations obtained during cardiac surgery. Western blots to assess phospholamban (PLB) phosphorylation on serine 16 were performed. Ergometrine exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in atrial preparations starting at 0.3 µM and reaching a plateau at 10 µM in H2-TGs (n = 7). This was accompanied by an increase in PLB phosphorylation at serine 16. Ergometrine up 10 µM failed to increase force of contraction in left atrial preparations from 5-HT4-TGs (n = 5). Ten micrometer ergometrine increased the force of contraction in isolated retrogradely perfused spontaneously beating heart preparations (Langendorff setup) from H2-TG but not 5-HT4-TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergometrine at 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, and these effects were eliminated by 10 µM of the H2R antagonist cimetidine but not by 10 µM of the 5-HT4R antagonist tropisetron. Furthermore, ergometrine showed binding to human histamine H2 receptors (at 100 µM and 1 mM) using HEK cells in a recombinant expression system (pKi < 4.5, n = 3). In conclusion, we suggest that ergometrine is an agonist at cardiac human H2Rs.

Sex-specific repolarization heterogeneity in mouse left ventricle: Optical mapping combined with mathematical modeling predict the contribution of specific ionic currents.

Ventricular repolarization shows notable sex-specificity, with female sex being associated with longer QT-intervals in electrocardiography irrespective of the species studied. From a clinical point of view, women are at a greater risk for drug-induced torsade de pointes and symptomatic long-QT syndrome. Here, we present an optical mapping (OM) approach to reveal sex-specific action potential (AP) heterogeneity in a slice preparation of mouse hearts. Left ventricular epicardial repolarization in female versus male mice shows longer and, interindividually, more variable AP duration (APD), yielding a less prominent transmural APD gradient. By combining OM with mathematical modeling, we suggest a significant role of IKto,f and IKur in AP broadening in females. Other transmembrane currents, including INaL , only marginally affect basal APD. As in many cardiac pathophysiologies, increasing [Ca2+ ]i poses a risk for arrhythmia, the response of AP morphology to enhanced activation of L-type calcium channels (LTCC) was assessed in a sex-selective manner. Both APD and its variation increased significantly more in female versus male mice after pharmacological LTCC activation, which we hypothesize to be due to sex-specific INaL expression based on mathematical modeling. Altogether, we demonstrate a more delayed repolarization of LV epicardium, a leveled LV transmural APD gradient, and a more pronounced epicardial APD response to Ca2+ influx in females versus males. Mathematical modeling quantifies the relative contributions of selected ionic currents to sex-specific AP morphology under normal and pathophysiological conditions.

Diazoxide is a powerful cardioprotectant but is not feasible in a realistic infarct scenario.

Diazoxide is a powerful cardioprotective agent that activates mitochondrial ATP-dependent K-channels and stimulates mitochondrial respiration. Diazoxide reduced infarct size in isolated rodent heart preparations and upon pretreatment in juvenile pigs with coronary occlusion/reperfusion. We aimed to study the use of diazoxide in a more realistic adult pig model of reperfused acute myocardial infarction when diazoxide was administered just before reperfusion. In a first approach, we pretreated anaesthetised adult Göttingen minipigs with 7 mg kg-1 diazoxide (n = 5) or placebo (n = 5) intravenously over 10 min and subjected them to 60 min coronary occlusion and 180 min reperfusion; blood pressure was maintained by use of an aortic snare. The primary endpoint was infarct size (triphenyl tetrazolium chloride staining) as a fraction of area at risk; no-reflow area (thioflavin-S staining) was the secondary endpoint. In a second approach, diazoxide (n = 5) was given from 50 to 60 min coronary occlusion, and blood pressure was not maintained. There was a significant reduction in infarct size (22% ± 11% of area at risk with diazoxide pretreatment vs. 47% ± 11% with placebo) and area of no-reflow (14% ± 14% of infarct size with diazoxide pretreatment vs. 46% ± 20% with placebo). With diazoxide from 50 to 60 min coronary occlusion, however, there was marked hypotension, and infarct size (44% ± 7%) and area of no-reflow were not reduced (35% ± 25%). Cardioprotection by diazoxide pretreatment was confirmed in adult pigs with reperfused acute myocardial infarction but is not feasible when diazoxide is administered in a more realistic scenario before reperfusion and causes hypotension.

A novel ex-vivo isolated rabbit heart preparation to explore the cardiac effects of cervical and cardiac vagus nerve stimulation

The cardiac responses to vagus nerve stimulation (VNS) are still not fully understood, partly due to uncontrollable confounders in the in-vivo experimental condition. Therefore, an ex-vivo Langendorff-perfused rabbit heart with intact vagal innervation is proposed to study VNS in absence of cofounding anesthetic or autonomic influences. The feasibility to evoke chronotropic responses through electrical stimulation ex-vivo was studied in innervated isolated rabbit hearts (n = 6). The general nerve excitability was assessed through the ability to evoke a heart rate (HR) reduction of at least 5 bpm (physiological threshold). The excitability was quantified as the charge needed for a 10-bpm HR reduction. The results were compared to a series of in-vivo experiments rabbits (n = 5). In the ex-vivo isolated heart, the baseline HR was about 20 bpm lower than in-vivo (158 ± 11 bpm vs 181 ± 19 bpm). Overall, the nerve remained excitable for about 5 h ex-vivo. The charges required to reduce HR by 5 bpm were 9 ± 6 µC and 549 ± 370 µC, ex-vivo and in-vivo, respectively. The charges needed for a 10-bpm HR reduction, normalized to the physiological threshold were 1.78 ± 0.8 and 1.22 ± 0.1, in-vivo and ex-vivo, respectively. Overall, the viability of this ex-vivo model to study the acute cardiac effects of VNS was demonstrated.