Langendorff-perfused Guinea Pig Hearts Research Articles

Abstract 4137488: Mild elevation of [K + ] 0 reduces atrial fibrillation burden and enhances the efficacy of flecainide to suppress the arrhythmia: an experimental study.

Background: It is desirable to improve rhythm control management of atrial fibrillation (AF). Extracellular potassium ([K + ] 0 ) may affect AF generation and anti-AF efficacy of the sodium channel blockers. Methods: AF burden (i.e., percentage of time spent in AF) was determined in Langendorff-perfused guinea pig hearts in the range of [K + ] 0 of 3-6 mM in the presence and absence of flecainide (1.0 µM). Spontaneous and single premature beat-induced AF were monitored for 30 min in the presence of acetylcholine (0.3 µM). Results: Spontaneous AF episodes were consistently recorded at 3 mM [K + ] 0 but not at 4-6 mM [K + ] 0 either with or without flecainide. AF burden was progressively reduced with elevation of [K + ] 0 and anti-AF efficacy of flecainide progressively increased at the higher [K + ] 0 (Figure). * p<0.05 vs. 3 mM [K + ] 0 with no flecainide; ** p<0.05 vs. 3 mM [K + ] 0 with flecainide; # - p<0.05 vs. respective no flecainide. n=5-6 per group. Conclusion: Our findings indicate that mild elevation of [K + ] 0 significantly reduces AF burden and enhances the efficacy of flecainide to decrease AF burden.

Validation of a Demography-Based Adaptive QTc Formula using Pediatric and Adult Datasets Acquired from Humans and Guinea Pigs.

A variety of QT rate-correction (QTc) formulae have been utilized for both clinical and research purposes. However, these formulae are not universally effective, likely due to significant influences of demographic diversity on the QT-HR relationship. To address this limitation, we proposed an adaptive QTc (QTcAd) formula that adjusts to subject demographics (i.e., age). Further, we compared the efficacy and accuracy of the QTcAd formula to other widely used alternatives. Using age as a demographic parameter, we tested the QTcAd formula across diverse age groups with different heart rates (HR) in both humans and guinea pigs. Utilizing retrospective human (n=1360) and guinea pig electrocardiogram (ECG) data from in-vivo (n=55) and ex-vivo (n=66) settings, we evaluated the formula's effectiveness. Linear regression fit parameters of HR-QTc (slope and R²) were utilized for performance assessment. To evaluate the accuracy of the predicted QTc, we acquired epicardial electrical and optical voltage data from Langendorff-perfused guinea pig hearts. In both human subjects and guinea pigs, the QTcAd formula consistently outperformed other formulae across all age groups. For instance, in a 20-year-old human group (n=300), the QTcAd formula successfully nullified the inverse HR-QT relationship (R²=5.1E-09, slope=-3.5E-05), while the Bazett formula (QTcB) failed to achieve comparable effectiveness (R²= 0.20, slope=0.91). Moreover, the QTcAd formula exhibited better accuracy than the age-specific Benatar formula (QTcBe), which overcorrected QTc (1-week human QT: 263.8±14.8 ms, QTcAd: 263.8±7.3 ms, p=0.62; QTcBe: 422.5±7.3 ms, p<0.0001). The optically measured pseudo-QT interval (143±22.5 ms, n=44) was better approximated by QTcAd (180.6±17.0 ms) compared to all other formulae. Furthermore, we demonstrated that the QTcAd formula was not inferior to individual-specific QTc formulae. The demography-based QTcAd formula showed superior performance across human and guinea pig age groups, which may enhance the efficacy of QTc for cardiovascular disease diagnosis, risk stratification, and drug safety testing. Corrected QT (QTc) is a well-known ECG biomarker for cardiovascular disease risk stratification and drug safety testing. Various QT rate-correction formulae have been developed, but these formulae do not perform consistently across diverse datasets (e.g., sex, age, disease, species). We introduce a novel QTc formula (QTcAd) that adapts to demographic variability, as the parameters can be modified based on the characteristics of the study population. The formula (QTcAd = QT + (|m|*(HR-HR mean )) - includes the absolute slope (m) of the linear regression of QT and heart rate (HR) and the mean HR of the population (HR mean ) as population characteristics parametersˍUsing datasets from both pediatric and adult human subjects and an animal model, we demonstrate that the QTcAd formula is more effective at eliminating the QT-HR inverse relationship, as compared to other commonly used correction formulae.

Effects of different dosages esketamine on cardiac conduction and heterogeneity of Cx43: the epicardial mapping in guinea pigs.

BackgroundEsketamine is favored in clinical settings. Relative to other anesthetics it preserves protective airway reflexes, maintains spontaneous respiration, stabilizes hemodynamics, and alleviates neuropathic pain. This study sought to evaluate the cardiac safety of esketamine at 3 sub-anesthetic gradient concentrations.MethodsWe examined the cardiac electrophysiological effects of esketamine with infusion rates of 0.125, 0.25, and 0.5 mg·kg−1·h−1. Short-term studies were performed in ventricular myocytes using patch-clamp techniques and optically mapped Langendorff-perfused guinea-pig hearts. Long-term studies were performed using Langendorff-perfused guinea-pig hearts and electrically mapping the receipt of the infusion for 3 hours.ResultsEsketamine changed the action potential (AP) morphology of cardiomyocytes. Notably, it increased the resting membrane potential (RMP), attenuated the amplitude of action potential (APA), reduced the maximum upstroke velocity (Vmax), and shortened the action potential duration (APD) at 30% to 70%, which led to relatively prolonged monophasic action potentials (MAP) triangulation in G0.25 and G0.5. All the effects were partially eluted. Optical mapping demonstrated almost equal and heterogeneous conduction. G0.125 resulted in an increased heart rate (HR) accompanied by a shortened APD. No detectable arrhythmia was observed at the cycle lengths (CLs) used. Long-term electrical mapping demonstrated the dose-dependent deceleration of the Vmax and APA, but only prolonged the AP parameters in G0.5. Left-ventricular isochronal conduction maps revealed the conduction heterogeneities at G0.5, and conduction velocity (CV) was increased in G0.125 and G0.25. None of these effects were reversed on drug washout. Electrocardiogram (ECG) traces revealed an accelerated HR with the associated curtailment of QT intervals in G0.125; HRs were decreased in G0.25 and G0.5; the PR intervals and QRS duration differed between G0.125 and G0.25, G0.5, which elicited electrical alternans. Connexin43 (Cx43) expression were significantly decreased in G0.125, G0.25 and G0.5.ConclusionsThese data provide a basic electrophysiology for esketamine. Specifically, we found that (I) various methods of esketamine infusion had different effects on cardiac conduction at different dosages; (II) the heterogeneous expression of Cx43 is associated with spatially dispersed conduction; and (III) potential cardiac risks should be considered for high-risk patients receiving continuous esketamine infusions of high dosages.

The Investigation of Combined Na+/Ca2+ Exchanger and the L-type Ca2+- Channel Inhibition in Langendorff-Perfused Isolated Guinea Pig Hearts

Abstract Objective The sodium/calcium exchanger (NCX) and the L-type Ca2+-channel (LTCC) are nowadays considered the major transmembrane transport mechanisms that control Ca2+ homeostasis. In pathophysiological conditions the altered function of these currents may influence the Ca2+ homeostasis and cardiac contractility and thereby, may enhance the development of severe tachyarrhythmias. The blockade of NCX current has been proposed as possible approach in the prevention and/or suppression of arrhythmias; however, this mechanism is not always favourable because the inhibition of both modes of NCX may induce Ca2+-overload. The decrease of the Ca2+ level by partial LTCC inhibition may be beneficial in increasing the antiarrhythmic efficacy. Therefore, the aim of our study was to investigate the antiarrhythmic effects of combined NCX and LTCC blockade in the ex vivo guinea pig arrhythmia model. Methods We have performed Langendorff experiments in isolated guinea pig hearts. We have recorded electrocardiograms (ECG) and left ventricle pressure. We have applied 1 μM ORM-10962 (ORM), a compound that block NCX current and 30 nM nisoldipine for the inhibition of LTCC. Arrhythmias have been provoked by decreasing the activity of the sodium/potassium pump with 5 μM ouabain. Results We found that neither LTCC nor NCX blockade alone increased, while the combined inhibition of the two currents significantly delayed (p&lt;0.05) the mean time of appearance of ouabain-induced ventricular fibrillation. The heart frequency was affected by none of the drugs, only the left ventricular pressure (end-systolic and diastolic difference) was significantly elevated by ORM (p&lt;0.001). Conclusion In the Langendorff-perfused guinea pig heart, specific, combined NCX and LTCC blockade may be favourable than the inhibition of NCX or LTCC alone. However, further investigations are necessary to identify the pathological settings in which this combined cardiac drug therapy may be a potential new approach.

The conduction velocity-potassium relationship in the heart is modulated by sodium and calcium

The relationship between cardiac conduction velocity (CV) and extracellular potassium (K+) is biphasic, with modest hyperkalemia increasing CV and severe hyperkalemia slowing CV. Recent studies from our group suggest that elevating extracellular sodium (Na+) and calcium (Ca2+) can enhance CV by an extracellular pathway parallel to gap junctional coupling (GJC) called ephaptic coupling that can occur in the gap junction adjacent perinexus. However, it remains unknown whether these same interventions modulate CV as a function of K+. We hypothesize that Na+, Ca2+, and GJC can attenuate conduction slowing consequent to severe hyperkalemia. Elevating Ca2+ from 1.25 to 2.00 mM significantly narrowed perinexal width measured by transmission electron microscopy. Optically mapped, Langendorff-perfused guinea pig hearts perfused with increasing K+ revealed the expected biphasic CV-K+ relationship during perfusion with different Na+ and Ca2+ concentrations. Neither elevating Na+ nor Ca2+ alone consistently modulated the positive slope of CV-K+ or conduction slowing at 10-mM K+; however, combined Na+ and Ca2+ elevation significantly mitigated conduction slowing at 10-mM K+. Pharmacologic GJC inhibition with 30-μM carbenoxolone slowed CV without changing the shape of CV-K+ curves. A computational model of CV predicted that elevating Na+ and narrowing clefts between myocytes, as occur with perinexal narrowing, reduces the positive and negative slopes of the CV-K+ relationship but do not support a primary role of GJC or sodium channel conductance. These data demonstrate that combinatorial effects of Na+ and Ca2+ differentially modulate conduction during hyperkalemia, and enhancing determinants of ephaptic coupling may attenuate conduction changes in a variety of physiologic conditions.

Abstract 16378: Increased Extracellular Sodium and Calcium Modify the Extracellular Potassium and Cardiac Conduction Velocity Relationship in a Langendorff-perfused Guinea Pig Heart

Background: Previous studies have demonstrated a biphasic relationship between extracellular potassium (K o ) and cardiac conduction velocity (CV). With moderate hyperkalemia, CV increases in what is referred to as supernormal conduction, but further increases in K o lead to severe conduction slowing and asystole. We recently demonstrated that altering extracellular sodium (Na o ) and extracellular calcium (Ca o ) modulates CV dependence on gap junctions (GJs). We have also shown that increasing Na o and Ca o can attenuate conduction loss caused by global ischemia ischemia. The purpose of this study was to determine if increasing Na o and Ca o would alter the K o -CV relationship and preserve CV at high K o . Hypothesis: Increasing Na o and Ca o will mitigate conduction slowing and the incidence of asystole associated with severe hyperkalemia in conditions of both normal and uncoupled GJs. Methods: Langendorff-perfused guinea pig hearts were optically mapped to measure CV. Na o was set to 145 or155mM and Ca o to 1.25 or 2.0mM. K o was varied from 4.6, 6.4, 8, to 10 mM in each experiment. Perfusion order was blinded and randomized. GJs were inhibited using carbenoxolone. Results: A biphasic K o -CV relationship was observed under all conditions. Maximum CV was achieved at either 6.4 or 8.0mM K o followed by a decrease in CV with increased K o . Importantly, the degree of CV slowing in the presence of 10mM K o was significantly reduced with the 155mM Na o / 2.0mM Ca o perfusate compared to all other Na o /Ca o combinations. Carbenoxolone reduced CV across all K o , but did not alter the K o -CV relationship. With 145mM Na o / 1.25mM Ca o , all hearts became asystolic at K o =10.0mM. Increasing Na o and Ca o significantly reduced the incidence of asystole at K o =10.0mM. Conclusions: Elevating Na o and Ca o preserves CV during severe hyperkalemia with or without strong GJ coupling. Increasing Na o and Ca o significantly reduces the incidence of asystole during severe hyperkalemia. These data suggest that non-linear and combinatorial effects of sodium, calcium, and GJ uncoupling can differentially modulate cardiac conduction during hyperkalemic perfusion. These results have important implications for cardioplegic arrest and ischemic heart disease when potassium and calcium homeostasis are disrupted.

Abstract 13191: Increased Extracellular Sodium and Intercellular Cleft Separation Synergistically Prolong Repolarization in the Long-qt Syndrome Type 3

Introduction: Long-QT syndrome type 3 (LQT3) is caused by a gain-of-function mutation in the cardiac sodium channel that increases the late sodium current and prolongs repolarization. We previously suggested that narrowing the perinexus which is adjacent gap junction conceals the LQT3 phenotype by depleting extracellular sodium ([Na]) within this nanodomain and curtails the late current and repolarization. However, it is unknown if elevating bulk [Na] alone modulates action potential duration (APD) in widened perinexi to unmask LQT3. Hypothesis: Elevated [Na] and widened perinexi synergistically prolong APD in LQT3. Methods: The dependence of APD on [Na] and perinexal width was explored with a computational model and in Langendorff-perfused guinea pig hearts. The late sodium current was induced with ATXII (7nM). Perfusate [Na] changed from 145 (145Na) to 160 mM (160Na). Perinexal expansion was induced with βadp1 (1uM). APD was quantified from whole-heart optical maps. Perinexal width was quantified by transmission electron microscopy. Results: A computational model, including preferential sodium channel location at the intercalated disk, predicts that combination of elevated [Na] and widened perinexus prolongs APD greater than summing the effect of the individual interventions alone. Therefore, the combination is synergistic and not additive. Isolated heart experiments are consistent with the model. Specifically, ATXII+βadp1 significantly widens perinexal width from 27.8±4.1 to 49.7±9.3 nm and prolongs APD by 18.1±5.1ms with 600ms pacing relative to ATXII alone. In the presence of ATXII, 160Na significantly prolongs APD by 12.0±5.8ms relative to 145Na. Furthermore, the combination of both interventions is synergistic. Specifically, in the presence of ATXII, 160Na+βadp1 significantly prolongs APD more than the sum of the individual effects (49.9±7.5ms vs. 30.2±5.4ms). Conclusions: The data demonstrate that in LQT3, enhancing sodium and perinexal width concurrently prolong APD more than the individual effects alone. This synergistic effect suggests that maintaining reduced plasma sodium level can be a simple and effective method to conceal LQT3, even in the presence of perinexal expansion associated with osmotically induced stress.

Lead Identification Strategy in Astellas for Cardiovascular Liabilities Using the xCELLigence System and the Langendorff-perfused Guinea-Pig Heart Assay

Lead Identification Strategy in Astellas for Cardiovascular Liabilities Using the xCELLigence System and the Langendorff-perfused Guinea-Pig Heart Assay

Modulation of the heart's electrical properties by the anticonvulsant drug retigabine

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here.Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug.Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons.We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system.

Age-related effects of dexmedetomidine on myocardial contraction and coronary circulation in isolated guinea pig hearts

Dexmedetomidine is a selective α2 adrenergic agonist. Although dexmedetomidine is widely used for sedation and analgesia, it frequently produces hypotension and bradycardia. The present study aimed to evaluate the effects of dexmedetomidine on cardiac function and coronary circulation using Langendorff-perfused guinea pig hearts. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously monitored, and electric field stimulation (EFS) was applied to stimulate sympathetic nerve terminals. Dexmedetomidine almost completely inhibited the EFS-induced increase in LVP at all ages. The effect of dexmedetomidine on coronary artery resistance varied according to postnatal age, i.e., dexmedetomidine had little effect on CPP in young hearts (<4 weeks) but increased CPP by 10 mmHg at 4–8 weeks and by 15 mmHg at >8 weeks. The increase in CPP in adult hearts was inhibited by imiloxan, an α2B antagonist, and prazosin, an α1 antagonist. The results suggest that dexmedetomidine acts on α2 adrenergic receptors at sympathetic nerve terminals to suppress the release of norepinephrine. In addition, the findings suggest that dexmedetomidine directly affects α1 adrenoceptors and/or α2B adrenoceptors on coronary smooth muscles to increase CPP. The age-related changes in α adrenoceptor subtypes may be linked to the cardiodepressant effects of dexmedetomidine.

New in vitro model for proarrhythmia safety screening: IKs inhibition potentiates the QTc prolonging effect of IKr inhibitors in isolated guinea pig hearts

IntroductionPreclinical in vivo QT measurement as a proarrhythmia essay is expensive and not reliable enough. The aim of the present study was to develop a sensitive, cost-effective, Langendorff perfused guinea pig heart model for proarrhythmia safety screening. MethodsLow concentrations of dofetilide and cisapride (inhibitors of the rapid delayed rectifier potassium current, IKr) were tested alone and co-perfused with HMR-1556 (inhibitor of the slow delayed rectifier potassium current, IKs) in Langendorff perfused guinea pig hearts. The electrocardiographic rate corrected QT (QTc) interval, the Tpeak–Tend interval and the beat-to-beat variability and instability (BVI) of the QT interval were determined in sinus rhythm. ResultsDofetilide and HMR-1556 alone or co-perfused, prolonged the QTc interval by 20±2%, 10±1% and 55±10%, respectively. Similarly, cisapride and HMR-1556 alone or co-perfused, prolonged the QTc interval by 11±3%, 11±4% and 38±6%, respectively. Catecholamine-induced fast heart rate abolished the QTc prolonging effects of the IKr inhibitors, but augmented the QTc prolongation during IKs inhibition. None of the drug perfusions increased significantly the Tpeak–Tend interval and the sinus BVI of the QT interval. DiscussionIKs inhibition increased the QTc prolonging effect of IKr inhibitors in a super-additive (synergistic) manner, and the QTc interval was superior to other proarrhythmia biomarkers measured in sinus rhythm in isolated guinea pig hearts. The effect of catecholamines on the QTc facilitated differentiation between IKr and IKs inhibitors. Thus, QTc measurement in Langendorff perfused guinea pig hearts with pharmacologically attenuated repolarization reserve and periodic catecholamine perfusion seems to be suitable for preclinical proarrhythmia screening.

ORM-3819 promotes cardiac contractility through Ca2+ sensitization in combination with selective PDE III inhibition, a novel approach to inotropy

This study is the first pharmacological characterization of the novel chemical entity, ORM-3819 (L-6-{4-[N'-(4-Hydroxi-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one), focusing primarily on its cardiotonic effects. ORM-3819 binding to cardiac troponin C (cTnC) was confirmed by nuclear magnetic resonance spectroscopy, and a selective inhibition of the phosphodiesterase III (PDE III) isozyme (IC50=3.88±0.3nM) was revealed during in vitro enzyme assays. The Ca2+-sensitizing effect of ORM-3819 was demonstrated in vitro in permeabilized myocyte-sized preparations from left ventricles (LV) of guinea pig hearts (ΔpCa50=0.12±0.01; EC50=2.88±0.14µM). ORM-3819 increased the maximal rate of LV pressure development (+dP/dtmax) (EC50=8.9±1.7nM) and LV systolic pressure (EC50=7.63±1.74nM) in Langendorff-perfused guinea pig hearts. Intravenous administration of ORM-3819 increased LV+dP/dtmax (EC50=0.13±0.05µM/kg) and improved the rate of LV pressure decrease (−dP/dtmax); (EC50=0.03±0.02µM/kg) in healthy guinea pigs. In an in vivo dog model of myocardial stunning, ORM-3819 restored the depressed LV+dP/dtmax and improved % segmental shortening (%SS) in the ischemic area (to 18.8±3), which was reduced after the ischaemia-reperfusion insult (from 24.1±2.1 to 11.0±2.4). Our data demonstrate ORM-3819 as a potent positive inotropic agent exerting its cardiotonic effect by a cTnC-dependent Ca2+-sensitizing mechanism in combination with the selective inhibition of the PDE III isozyme. This dual mechanism of action results in the concentration-dependent augmentation of the contractile performance under control conditions and in the postischemic failing myocardium.

Ventricular cycle length irregularity affects the correlation between ventricular rate and coronary flow in isolated, Langendorff perfused guinea pig hearts

IntroductionHeart rate affects coronary flow, but the mechanism is complex. The relationship between rhythm and flow is unclear, especially in experimental settings used for determining drug actions. The present study examined whether ventricular irregularity influences coronary flow independently of heart rate. MethodsGuinea pig hearts were perfused (Langendorff mode) at constant pressure. Hypokalemic Krebs solution facilitated spontaneous development of arrhythmias. The ECG, left ventricular and perfusion pressures were recorded, and the coronary flow was measured. Beat-to-beat ventricular cycle length variability was quantified. Hearts were retrospectively allocated to arbitrary ‘Low’ or ‘High’ RR variability groups. ResultsA positive linear correlation was found between mean ventricular rate and coronary flow. The slope of the regression line was significantly greater in the ‘High’ versus ‘Low’ RR variability group, with greater coronary flow values in the ‘High’ RR variability group in the physiological heart rate range. During regular rhythm, left ventricular pressure exceeded perfusion pressure and prevented coronary perfusion at peak systole. However, ventricular irregularity significantly increased the number of beats in which left ventricular pressure remained below perfusion pressure, facilitating coronary perfusion. DiscussionIn isolated hearts, cycle length irregularity increases the slope of the positive linear correlation between mean ventricular rate and coronary flow via producing beats in which left ventricular pressure remains below perfusion pressure. This means that changes in rhythm have the capacity to influence coronary flow independently of heart rate in isolated hearts perfused at constant pressure, which should be noted in drug studies on arrhythmias performed in Langendorff hearts.

Disorders of Ca2+ handling and cell-to-cell coupling are implicated in the development of acute heart failure in intact animal hearts: An ultrastructural study

BAckground: It has been previously reported that various acute interventions causing myocardial abnormalities in Ca2+ handling and defects in intercellular coupling facilitate the occurrence of malignant arrhythmias. Objectives: To comprehensively determine the impact of such Ca2+- related disorders induced in intact animal hearts on the ultrastructure of the cardiomyocytes before occurrence and during sustaining of severe arrhythmias. Methods: Three types of acute experiments known to be accompanied by disturbances in Ca2+ handling were performed. Langendorff-perfused rat and guinea pig hearts were subjected to K+-deficient perfusion to induce ventricular fibrillation; Langendorff-perfused guinea pig heart underwent burst atrial pacing to induce atrial fibrillation; and open chest pig heart was used for intramyocardial noradrenaline infusion to induce ventricular tachycardia. Tissue samples for electron microscopy examination were obtained during basal conditions, previous occurrence and during sustaining of malignant arrhythmias. Results: The comparative findings suggest that myocardial heterogeneity of high [Ca2+]i-induced subcellular injury of the cardiomyocytes and their junctions is a common feature that precede occurrence ventricular tachycardia, ventricular fibrillation or atrial fibrillation regardless of the species and atria/ventricular-related differences in Ca2+ handling and intercellular coupling. The primary changes consisted of nonuniform sarcomere shortening, which most likely reflects cytosolic Ca2+ oscillations; disturbances in Ca2+ wave propagation; and Ca2+ overload. These disturbances were linked with defects in cardiac cell-to-cell coupling that increased the propensity of the heart to malignant arrhythmias. Moreover, Ca2+overload led to disruption of myofilaments, jeopardizing contractile function. Conclusions: The results provide a novel paradigm linking Ca2+- related arrhythmogenesis and contractility disorders that may contribute to acute heart failure.

Synergistic antiarrhythmic effect of combining inhibition of Ca²⁺-activated K⁺ (SK) channels and voltage-gated Na⁺ channels in an isolated heart model of atrial fibrillation.

Application of antiarrhythmic compounds is limited by both proarrhythmic and extracardiac toxicities, as well as incomplete antiarrhythmic efficacy. An improved antiarrhythmic potential may be obtained by combining antiarrhythmic drugs with different modes of action, and a reduction of the adverse effect profile could be an additional advantage if compound concentrations could be reduced. The purpose of this study was to test the hypothesis that combined inhibition of Ca(2+)-activated K(+) channels (SK channels) and voltage-gated Na(+) channels, in concentrations that would be subefficacious as monotherapy, may prevent atrial fibrillation (AF) and have reduced proarrhythmic potential in the ventricles. Subefficacious concentrations of ranolazine, flecainide, and lidocaine were tested alone or in combination with the SK channel blocker N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) in a Langendorff-perfused guinea pig heart model in which AF was induced after acetylcholine application and burst pacing. AF duration was reduced when both flecainide and ranolazine were combined with ICA in doses that did not reduce AF as monotherapy. At higher concentrations, both flecainide and ranolazine revealed proarrhythmic properties. A synergistic effect in AF treatment was obtained by combining low concentrations of SK and Na(+) channel blockers.

Phlorizin prevents electrically-induced ventricular tachyarrhythmia during ischemia in langendorff-perfused guinea-pig hearts.

Phlorizin is a type of flavonoids and has a peroxynitrite scavenging effect. This study aimed to elucidate the effects of phlorizin on ischemia-induced ventricular tachyarrhythmia (VT). Optical signals from the epicardial surface of the ventricle or left ventricular end diastolic pressure (LVEDP) were recorded during acute global ischemia in 42 Langendorff-perfused guinea pig hearts. Experiments were performed in the control condition and in the presence of phlorizin or N-2-mercaptopropionylglycine (2-MPG), a peroxynitrite scavenger, respectively. Mean action potential duration at 20 min of ischemia did not differ among the three interventions. Impulse conduction time-dependently slowed during 20 min of ischemia in the control. Phlorizin but not 2-MPG improved the ischemic conduction slowing at 15 and 20 min of ischemia. Programmed stimulation induced VT at 20 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin (p<0.05). LVEDP was increased during 30 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin. These results indicate that phlorizin prevents VT through the improvement of impulse conduction slowing during ischemia. Phlorizin may be more useful for ischemia-induced VT than 2-MPG.

Remifentanil Has a Minimal Direct Effect on Sinoatrial Node Pacemaker Activity in the Guinea Pig Heart

Whereas remifentanil administration is associated with severe bradycardia, it has yet to be fully investigated whether the negative chronotropic action of remifentanil is mediated by its direct action on sinoatrial (SA) node pacemaker activity in the heart versus indirect results of enhanced vagal activity. We examined the effects of remifentanil and fentanyl on the spontaneous action potentials of guinea pig SA node cells at concentrations of 5, 10, 100, and 1000 nM using the amphotericin B-perforated whole-cell patch-clamp technique. Isolated guinea pig hearts were perfused in a Langendorff mode with 5, 10, 100, and 1000 nM remifentanil. The spontaneous firing rate and diastolic depolarization rate (DDR) of the SA node action potentials were 189.1 ± 14.8 /min and 74.1 ± 2.9 mV/s (n = 8), respectively, under control conditions, and were not significantly affected by exposure to 5 nM (P = 1.0 for both spontaneous firing rate and DDR; n = 6), 10 nM (P = 0.62 for spontaneous firing rate, P = 0.99 for DDR; n = 6), or 100 nM (P = 0.23 for spontaneous firing rate, P = 0.38 for DDR; n = 6) remifentanil. However, 1000 nM remifentanil modestly but significantly decreased the spontaneous firing rate (P = 0.0087) and DDR (P = 0.0072, n = 6). Remifentanil did not affect the heart rate of isolated Langendorff-perfused guinea pig hearts at concentrations of 5 nM (P = 0.98), 10 nM (P = 0.35), or 100 nM (P = 0.24) but significantly reduced the heart rate at 1000 nM (P < 0.0001). Fentanyl did not affect the spontaneous firing rate and DDR at concentrations of 5 nM (P = 1.0 for both spontaneous firing rate and DDR) and 10 nM (P = 0.62 for spontaneous firing rate, P = 0.79 for DDR), but it significantly reduced both at 100 nM (P = 0.00038 for spontaneous firing rate, P = 0.0080 for DDR) and 1000 nM (P < 0.0001 for both spontaneous firing rate and DDR). Clinically relevant concentrations (nanomolar order concentrations) of remifentanil do not produce significant direct effects on intrinsic cardiac automaticity; thus, suggesting that remifentanil-induced bradycardia in the clinical setting is independent of its direct cardiac effects.

Abstract 114: System’s Biology Approach to Non-Invasive Integrative Analysis of Cardiac Energetics

Our aim is to propose an inovative non-invasive analysis of heart contraction energetics, to be used for diagnosis and therapeutics follow-up in human heart pathologies. The analysis is based on the combination of non-invasive 31P-NMR and IRM techniques and the analytical background of metabolic modular control analysis (MoCA). Metabolic control analysis’ based approaches have been used by us and others to overcome the complexity of intra-cellular regulations and to describe and quantify controls and regulations in complex systems. We previously demonstrated that MoCA does bring new informations on the interplay between ATP demand and supply in heart contraction energetics. In the present study, MoCA was applied to beating Langendorff perfused guinea pig heart to describe qualitatively and quantitatively the global action of various inotropic effectors effector modifying (Adrenaline) or not (Levosimendan, a calcium-sensitizer; Frank-Starling effect) cytosolic calcium transients. MoCA allows to describe the relative importance of the different routes within the heart by which the inotropic effects are transmitted. The results show that in the absence of calcium transient changes, an energetic feed-back control occurs at the expense of total energy, like in skeletal muscle, while parallel activation of both supply and demand occurs after adrenaline addition, maintaining energy homeostasis (heart energetic paradigm). In conclusion, clinical application of System’s biology to the study of contraction energetics in patients may help to the understanding of the links between molecular events in pathologies and whole organ function / dysfunction.

Molecular weight of different angiotensin II polymers directly determines: Density of endothelial membrane AT1 receptors and coronary vasoconstriction

We have shown that angiotensin II (Ang II) does not diffuse across the vessel wall, remaining intravascularly confined and acting solely on the coronary endothelial luminal membrane (CELM) receptors. A sustained intracoronary infusion of Ang II causes transient coronary vasoconstriction (desensitization) due to membrane internalization of CELM Ang II type 1 receptors (CELM-AT1R). In contrast, sustained intracoronary infusion of a non-diffusible polymer of Ang II (Ang II-Pol, 15,000kDa) causes a sustained vasoconstriction by preventing CELM-AT1R internalization. In addition, a sustained intracoronary infusion of Ang II leads to a depressed response following a secondary Ang II administration (tachyphylaxis) that is reversed by Ang II-Pol. These findings led us to hypothesize that the rate of desensitization, tachyphylaxis, and AT1R internalization were dependent on Ang II-Pol molecular weight. To test this hypothesis, we synthesized Ang II-Pols of the following molecular weights (in kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15,000. Vasoconstriction was measured following intracoronary infusion of Ang II-Pols in Langendorff-perfused guinea pig hearts at constant flow. The CELM protein fraction was extracted using the silica pellicle technique at different time points in order to determine the rate of AT1R internalization following each Ang II-Pol infusion. CELM-AT1R density was quantified by Western blot. We found that the rate of desensitization and the tachyphylaxis effect varied inversely with the molecular weight of the Ang II-Pols. Inversely proportional to the molecular weight of Ang II-Pol the CELM-AT1R density decreases over time. These results indicate that the mechanism responsible for the decreased rate of desensitization and tachyphylaxis by higher molecular weight Ang II polymers is due to reduction in the rate of CELM-AT1R internalization. These Ang II polymers would be valuable tools for studying the relationship between AT1R internalization and physiological effects.

Effect of Acute Hyperglycemia on Cardiac Conduction

Diabetes is associated with a relatively high prevalence of sudden cardiac death but the underlying mechanism is not well understood. In experimental diabetes, cardiac expression of connexin43 is reduced. Downregulation of connexin43 is linked to a slowing of cardiac conduction which increases the risk of cardiac arrhythmias and thereby sudden cardiac death. It is known that hyperglycemia causes downregulation of connexin43 but it is unknown to which extend this occurs in whole hearts. Here, we test the hypothesis that acute hyperglycemia leads to slowing of cardiac conduction through downregulation of connexin43 in whole hearts. We measured ventricular conduction velocity in Langendorff perfused guinea pig hearts using a multi-electrode array under normo- and hyperglycemic conditions (5.5 and 30mM glucose respectively) for up to 4 hrs. In addition, one group of hearts was perfused with a mannitol solution as a control for changes in osmolarity. Expression of connexin43 was quantified by western blotting. In the control group, longitudinal conduction velocity remained stable over 4 hrs (63 cm/s) while transverse conduction decreased slightly (from 27 to 25 cm/s). Similar results were obtained from both the hyperglycemia and mannitol groups suggesting that conduction was unaffected by changes in glucose concentration and osmolarity. Accordingly, no differences were observed in the protein levels of connexin43 between the groups supporting the functional results. In conclusion, acute exposure to hyperglycemia does not appear to have any significant effects on cardiac conduction and does not modulate the expression of connexin43 in guinea pig hearts.