2+ release (SCR) from the sarcoplasmic reticulum can cause delayed afterdepolarizations and triggered activity, contributing to arrhythmogenesis during β-adrenergic stimulation. Excessive beat-to-beat variability of repolarization duration (BVR) is a proarrhythmic marker. Previous research has shown that BVR is increased during intense β-adrenergic stimulation, leading to SCR.Objective: We aimed to determine ionic mechanisms controlling BVR under these conditions. potential (AP) of the single cardiac myocyte to the QT interval on the body surface. 7-9 Exaggerated BVR has been reported to be a more reliable indicator of arrhythmogenic risk than repolarization prolongation, per se, at least in several experimental ventricular tachycardia models [10][11][12] and in selected human subjects. 8,13 Although BVR has been investigated in multiple studies, the mechanisms underlying this phenomenon at the singlecell level remain to be fully elucidated. Pharmacological interventions influencing ion channels that operate during the AP plateau can markedly alter BVR. 7,14 Despite the fact that inhibition of the slowly activating delayed rectifier K + current (I Ks ) alone has minimal effects on both cellular AP duration (APD) and BVR, 14 we recently have shown that during increased Ca 2+ loading in myocytes subjected to blockade of I Ks in combination with βAR stimulation, BVR is significantly enhanced, even before the occurrence of EADs and TA. 14 In the present study, we investigated the relationship between SCR and BVR using a combined experimental and computational approach in both canine ventricular myocytes and in situ hearts subjected to βAR stimulation. We show that SCRs not only lead to I ti and DAD formation but also lead to a prolonged duration of AP via increased L-type Ca 2+ current (I CaL ), which in turn leads to increased BVR when analyzing multiple consecutive APs. Pharmacological interventions that inhibit SCR (either with reduced or with preserved systolic contraction) prevent this SCR-associated AP prolongation and reduce BVR. Methods and Results: MethodsThis investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (National Institutes of Health Publication 85-23, revised 1996). Animal handling was in accordance with the European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609/EU). Full details of methods, solutions, and interventions used are given in the onlineonly Data Supplement accompanying this article. A brief summary of the main aspects is provided. Myocyte Isolation and ElectrophysiologyCanine left ventricular (LV) myocytes were isolated as previously described. 15 Transmembrane APs were recorded at ≈37°C using highresistance (30-60 MΩ) glass microelectrodes filled with 3 mol/L KCl. Myocyte contractions were recorded with a video edge motion detector. Calcium MeasurementWe used the perforated patch-clamp technique under current-clamp or vol...

Non-technical summary Heart failure is where the heart is unable to pump sufficient blood in order to meet the requirements of the body. Symptoms of heart failure often first present during exercise. During exercise the blood levels of a hormone, noradrenaline, increase and activate receptors on the muscle cells of the heart known as β-receptors causing the heart to contract more forcefully. We show that in heart failure the response to β-receptor stimulation is reduced and this appears to be due to a failure of the β-receptor to signal correctly to downstream targets inside the cell. However, by-passing the β-receptor and directly activating one of the downstream targets, an enzyme known as adenylyl cyclase, inside the cell restores the function of the muscle cells in failing hearts. These observations provide a number of potential targets for therapies to improve the function of the heart in patients with heart failure.Abstract Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca 2+ homeostatic and molecular mechanisms causing the blunted β-adrenergic (β-AR) response in HF. We induced HF by tachypacing in sheep; intracellular Ca 2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca 2+ transient amplitude and peak L-type Ca 2+ current (I Ca-L ) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P < 0.05) whereas sarcoplasmic reticulum (SR) Ca 2+ content was unchanged. β-AR stimulation with isoprenaline (ISO) increased Ca 2+ transient amplitude, I Ca-L and SR Ca 2+ content in both cell types; however, the response of HF cells was markedly diminished (P < 0.05). Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P < 0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P < 0.05). The β-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P < 0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca 2+ transient, SR Ca 2+ content and SR Ca 2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to β-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca 2+ content and therefore failure of the systolic Ca 2+ transient to increase appropriately during β-AR stimulation.

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Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells.