Macrophage-Dependent Interleukin-6-Production and Inhibition of IK Contributes to Acquired QT Prolongation in Lipotoxic Guinea Pig Heart

In the heart, the delayed rectifier K current, IK, composed of the rapid (IKr) and slow (IKs) components contributes prominently to normal cardiac repolarization. In lipotoxicity, chronic elevation of pro-inflammatory cytokines may remodel IK, elevating the risk for ventricular arrythmias and sudden cardiac death. We investigated whether and how the pro-inflammatory interleukin-6 altered IK in the heart, using electrophysiology to evaluate changes in IK in adult guinea pig ventricular myocytes. We found that palmitic acid (a potent inducer of lipotoxicity), induced a rapid (~24 h) and significant increase in IL-6 in RAW264.7 cells. PA-diet fed guinea pigs displayed a severely prolonged QT interval when compared to low-fat diet fed controls. Exposure to isoproterenol induced torsade de pointes, and ventricular fibrillation in lipotoxic guinea pigs. Pre-exposure to IL-6 with the soluble IL-6 receptor produced a profound depression of IKr and IKs densities, prolonged action potential duration, and impaired mitochondrial ATP production. Only with the inhibition of IKr did a proarrhythmic phenotype of IKs depression emerge, manifested as a further prolongation of action potential duration and QT interval. Our data offer unique mechanistic insights with implications for pathological QT interval in patients and vulnerability to fatal arrhythmias.

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

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<0.05) the mean time of appearance of ouabain-induced ventricular fibrillation. The heart f equency was affected by none of the drugs, only the left ventricular pressure (end-systolic and diastolic difference) was significantly elevated by ORM (p<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.

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.

P698The KCa2 channel inhibitor AP14145, but not dofetilide or ondansetron, provides functional atrial selectivity in guinea pig hearts

Background and purpose Prolongation of cardiac action potentials is considered antiarrhythmic in the atria but can be proarrhytmic in ventricles if the current carried by Kv11.1-channels (IKr) is inhibited. The current mediated by KCa2-channels, IKCa, is considered a promising new target for treatment of atrial fibrillation. Selective inhibitors of IKr— (dofetilide) and I-KCa (AP14145) were used to compare the effects on ventricular and atrial repolarisation. Ondansetron which has been reported to be a potent blocker of both IKr and IKCa was included to examine its potential atrial antiarrhythmic properties. Methods The expression of KCa2- and Kv11.1-channels in the guinea pig heart was investigated using qPCR. Whole-cell patch clamp technique was used to investigate the effects of dofetilide, AP14145, and ondansetron on IKCa and/or IKr. The effect of dofetilide, AP14145, and ondansetron on atrial and ventricular repolarisation was investigated in isolated hearts. A novel atrial paced in vivo guinea pig model was further validated using AP14145 and dofetilide. Results AP14145 increased AERP (29 ms ex vivo and 38 ms in vivo) without prolonging QTcB both ex vivo and in vivo. In contrast, dofetilide increased QTcB (41 ms) and, to a lesser extent, AERP (16 ms) in isolated hearts and prolonged QTcB (61ms) with no effects on AERP in the in vivo guinea pig model. Ondansetron did not inhibit IKCa, but did inhibit IKr in vitro. Ondansetron prolonged ventricular (25 ms), but not atrial repolarisation ex vivo. Conclusion IKCa inhibition by AP14145 selectively increased atrial repolarisation whereas IKr inhibition by dofetilide and ondansetron increases ventricular repolarisation to a larger extent than atrial repolarisation. Data support that IKCa inhibition may be of value in treating atrial fibrillation without causing adverse effects in the ventricles. Acknowledgement/Funding Innovation Fund Denmark and Wellcome Trust

Nerol Attenuates Ouabain-Induced Arrhythmias

Nerol (C10H18O) is a monoterpene found in many essential oils, such as lemon balm and hop. In this study, we explored the contractile and electrophysiological properties of nerol and demonstrated its antiarrhythmic effects in guinea pig heart preparation. Nerol effects were evaluated on atrial and ventricular tissue contractility, electrocardiogram (ECG), voltage-dependent L-type Ca2+current (ICa,L), and ouabain-triggered arrhythmias. Overall our results revealed that by increasing concentrations of nerol (from 0.001 to 30 mM) there was a significant decrease in left atrium contractile force. This effect was completely and rapidly reversible after washing out (~ 2 min). Nerol (at 3 mM concentration) decreased the left atrium positive inotropic response evoked by adding up CaCl2in the extracellular medium. Interestingly, when using a lower concentration of nerol (30μM), it was not possible to clearly observe any significant ECG signal alterations but a small reduction of ventricular contractility was observed. In addition, 300μM nerol promoted a significant decrease on the cardiac rate and contractility. Important to note is the fact that in isolated cardiomyocytes, peak ICa,Lwas reduced by 58.9 ± 6.31% after perfusing 300μM nerol (n=7, p<0.05). Nerol, at 30 and 300μM, delayed the time of onset of ouabain-triggered arrhythmias and provoked a decrease in the diastolic tension induced by the presence of ouabain (50μM). Furthermore, nerol preincubation significantly attenuated arrhythmia severity index without changes in the positive inotropism elicited by ouabain exposure. Taken all together, we may be able to conclude that nerol primarily by reducing Ca2+influx through L-type Ca2+channel blockade lessened the severity of ouabain-triggered arrhythmias in mammalian heart.