Rachel C. Myles

Local β-Adrenergic Stimulation Overcomes Source-Sink Mismatch to Generate Focal Arrhythmia

Rationale: &bgr;-Adrenergic receptor stimulation produces sarcoplasmic reticulum Ca2+ overload and delayed afterdepolarizations in isolated ventricular myocytes. How delayed afterdepolarizations are synchronized to overcome the source-sink mismatch and produce focal arrhythmia in the intact heart remains unknown. Objective: To determine whether local &bgr;-adrenergic receptor stimulation produces spatiotemporal synchronization of delayed afterdepolarizations and to examine the effects of tissue geometry and cell-cell coupling on the induction of focal arrhythmia. Methods and Results: Simultaneous optical mapping of transmembrane potential and Ca2+ transients was performed in normal rabbit hearts during subepicardial injections (50 &mgr;L) of norepinephrine (NE) or control (normal Tyrodes solution). Local NE produced premature ventricular complexes (PVCs) from the injection site that were dose-dependent (low-dose [30–60 &mgr;mol/L], 0.45±0.62 PVCs per injection; high-dose [125–250 &mgr;mol/L], 1.33±1.46 PVCs per injection; P<0.0001) and were inhibited by propranolol. NE-induced PVCs exhibited abnormal voltage–Ca2+ delay at the initiation site and were inhibited by either sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibition or reduced perfusate [Ca2+], which indicates a Ca2+-mediated mechanism. NE-induced PVCs were more common at right ventricular than at left ventricular sites (1.48±1.50 versus 0.55±0.89, P<0.01), and this was unchanged after chemical ablation of endocardial Purkinje fibers, which suggests that source-sink interactions may contribute to the greater propensity to right ventricular PVCs. Partial gap junction uncoupling with carbenoxolone (25 &mgr;mol/L) increased focal activity (2.18±1.43 versus 1.33±1.46 PVCs per injection, P<0.05), which further supports source-sink balance as a critical mediator of Ca2+-induced PVCs. Conclusions: These data provide the first experimental demonstration that localized &bgr;-adrenergic receptor stimulation produces spatiotemporal synchronization of sarcoplasmic reticulum Ca2+ overload and release in the intact heart and highlight the critical nature of source-sink balance in initiating focal arrhythmias.

Optical Mapping of Sarcoplasmic Reticulum Ca2+ in the Intact Heart: Ryanodine Receptor Refractoriness During Alternans and Fibrillation

Rationale: Sarcoplasmic reticulum (SR) Ca2+ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective: To measure intra-SR free [Ca2+] ([Ca2+]SR) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results: Simultaneous optical mapping of Vm (with RH237) and [Ca2+]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca2+ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P<0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca2+ alternans, with SR Ca2+ release alternans routinely occurring without changes in diastolic [Ca2+]SR. Sensitizing RyR with caffeine (200 &mgr;mol/L) significantly reduced the pacing threshold for both SR Ca2+ and APD alternans (188±15 and 173±12 ms; P<0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca2+]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca2+ release throughout VF. Conclusions: In intact hearts, RyR refractoriness initiates SR Ca2+ release alternans that can be amplified by diastolic [Ca2+]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca2+ and APD alternans. Despite increased [Ca2+]SR during VF, SR Ca2+ release was nearly continuously refractory. This novel method provides insight into SR Ca2+ handling during cardiac alternans and arrhythmia.Rationale: Sarcoplasmic reticulum (SR) Ca2+ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective: To measure intra-SR free [Ca2+] ([Ca2+]SR) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results: Simultaneous optical mapping of Vm (with RH237) and [Ca2+]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca2+ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P <0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca2+ alternans, with SR Ca2+ release alternans routinely occurring without changes in diastolic [Ca2+]SR. Sensitizing RyR with caffeine (200 μmol/L) significantly reduced the pacing threshold for both SR Ca2+ and APD alternans (188±15 and 173±12 ms; P <0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca2+]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca2+ release throughout VF. Conclusions: In intact hearts, RyR refractoriness initiates SR Ca2+ release alternans that can be amplified by diastolic [Ca2+]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca2+ and APD alternans. Despite increased [Ca2+]SR during VF, SR Ca2+ release was nearly continuously refractory. This novel method provides insight into SR Ca2+ handling during cardiac alternans and arrhythmia. # Novelty and Significance {#article-title-40}

Effect of activation sequence on transmural patterns of repolarization and action potential duration in rabbit ventricular myocardium

Although transmural heterogeneity of action potential duration (APD) is established in single cells isolated from different tissue layers, the extent to which it produces transmural gradients of repolarization in electrotonically coupled ventricular myocardium remains controversial. The purpose of this study was to examine the relative contribution of intrinsic cellular gradients of APD and electrotonic influences to transmural repolarization in rabbit ventricular myocardium. Transmural optical mapping was performed in left ventricular wedge preparations from eight rabbits. Transmural patterns of activation, repolarization, and APD were recorded during endocardial and epicardial stimulation. Experimental results were compared with modeled data during variations in electrotonic coupling. A transmural gradient of APD was evident during endocardial stimulation, which reflected differences previously seen in isolated cells, with the longest APD at the endocardium and the shortest at the epicardium (endo: 165 ± 5 vs. epi: 147 ± 4 ms; P < 0.05). During epicardial stimulation, this gradient reversed (epi: 162 ± 4 vs. endo: 148 ± 6 ms; P < 0.05). In both activation sequences, transmural repolarization followed activation and APD shortened along the activation path such that significant transmural gradients of repolarization did not occur. This correlation between transmural activation time and APD was recapitulated in simulations and varied with changes in intercellular coupling, confirming that it is mediated by electrotonic current flow between cells. These data suggest that electrotonic influences are important in determining the transmural repolarization sequence in rabbit ventricular myocardium and that they are sufficient to overcome intrinsic differences in the electrophysiological properties of the cells across the ventricular wall.

Is Microvolt T-Wave Alternans the Answer to Risk Stratification in Heart Failure?

Despite recent advances in the prevention and treatment of cardiovascular disease, sudden cardiac death (SCD) still accounts for ≈50% of all cardiovascular deaths in developed countries, thus accounting for a significant proportion of annual death worldwide.1 Reduction of the incidence of SCD depends on identification of those at most risk. In the present review we will concentrate on the challenges of risk stratification for SCD in chronic heart failure (CHF). We evaluate the utility of microvolt T-wave alternans (MTWA) as a tool for predicting SCD and consider whether MTWA is currently a valid means of selecting which patients should, or should not, receive an implantable cardioverter-defibrillator (ICD). Until recently, attempts to prevent SCD relied on pharmacological therapy. β-Blockers,2 angiotensin-converting enzyme inhibitors,3 angiotensin receptor blockers4 and aldosterone antagonists5 modestly reduce the risk of SCD in patients with CHF and after myocardial infarction (MI), whereas antiarrhythmic therapy has largely failed.6 Despite such treatments, these patients remained at high risk until the advent of the ICD. Although ICDs have further reduced the risk of SCD, they are expensive and can be associated with significant morbidity; therefore, precisely targeting their use is crucial. Implantation of ICDs for secondary prevention is clear. Prior sustained ventricular arrhythmia confers high risk and the benefit/risk balance is clearly favorable.7 Also, the secondary prevention population is relatively small and readily identified, thus the financial costs are not insurmountable. Primary prevention ICD therapy is an entirely different scenario. Large, randomized controlled trials have shown a mortality benefit with ICDs in patients with a low left ventricular ejection fraction (LVEF) and a history of MI or CHF.6,8 However, 2 major concerns have restricted implementation of this strategy. First, although analyses have estimated an acceptable cost-effectiveness profile,9 the immediate cost of implanting devices …

The link between repolarisation alternans and ventricular arrhythmia: does the cellular phenomenon extend to the clinical problem?

T-wave alternans is considered a potentially useful clinical marker for the risk of ventricular arrhythmia in patients with heart disease. Cellular repolarisation alternans is thought to underlie T-wave alternans, and moreover, to cause re-entrant ventricular arrhythmia. This review examines the experimental and clinical evidence linking repolarisation alternans and T-wave alternans with the occurrence of ventricular arrhythmia. Repolarisation alternans, manifest as alternating changes in action potential duration, is observed in isolated ventricular cardiomyocytes and in multicellular preparations. Its underlying causes are discussed particularly with respect to the role of intracellular Ca(2+). The repolarisation alternans observed at the single cell level is compared to the alternating behaviour observed in isolated multicellular preparations including the perfused ventricular wedge and Langendorff perfused heart. The evidence concerning spatial differences in repolarisation alternans is considered, particularly the situation where adjacent regions of myocardium exhibit repolarisation alternans of different phases. This extreme behaviour, known as discordant alternans, is thought to produce marked gradients of repolarisation that can precipitate unidirectional block and re-entrant ventricular arrhythmias. Finally, the difficulties in extrapolating between experimental models of alternans and arrhythmias and the clinical manifestation are discussed. The areas where experimental evidence is weak are highlighted, and areas for future research are outlined.

Optical and electrical recordings from isolated coronary-perfused ventricular wedge preparations

The electrophysiological heterogeneity that exists across the ventricular wall in the mammalian heart has long been recognized, but remains an area that is incompletely understood. Experimental studies of the mechanisms of arrhythmogenesis in the whole heart often examine the epicardial surface in isolation and thereby disregard transmural electrophysiology. Significant heterogeneity exists in the electrophysiological properties of cardiomyocytes isolated from different layers of the ventricular wall, and given that regional heterogeneities of membrane repolarization properties can influence the electrophysiological substrate for re-entry, the diversity of cell types and characteristics spanning the ventricular wall is important in the study of arrhythmogenesis. For these reasons, coronary-perfused left ventricular wedge preparations have been developed to permit the study of transmural electrophysiology in the intact ventricle. Since the first report by Yan and Antzelevitch in 1996, electrical recordings from the transmural surface of canine wedge preparations have provided a wealth of data regarding the cellular basis for the electrocardiogram, the role of transmural heterogeneity in arrhythmogenesis, and differences in the response of the different ventricular layers to drugs and neurohormones. Use of the wedge preparation has since been expanded to other species and more recently it has also been widely used in optical mapping studies. The isolated perfused wedge preparation has become an important tool in cardiac electrophysiology. In this review, we detail the methodology involved in recording both electrical and optical signals from the coronary-perfused wedge preparation and review the advances in cardiac electrophysiology achieved through study of the wedge.

Decreased inward rectifying K+current and increased ryanodine receptor sensitivity synergistically contribute to sustained focal arrhythmia in the intact rabbit heart

Heart failure leads to dramatic electrophysiological remodelling as a result of numerous cellular and tissue‐level changes. Important cellular changes include increased sensitivity of ryanodine receptors (RyRs) to Ca2+ release and down‐regulation of the inward rectifying K+ current (IK1), both of which contribute to triggered action potentials in isolated cells. We studied the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non‐failing rabbit heart using optical mapping and pharmacological manipulation of RyRs and IK1. Neither increased RyR sensitivity or decreased IK1 alone led to significant increases in arrhythmia following local sympathetic stimulation; however, in combination, these two factors led to a significant increase in premature ventricular complexes and focal ventricular tachycardia. These results suggest synergism between increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact heart and may provide important insights into novel anti‐arrhythmic treatments in heart failure.

Alternans of action potential duration and amplitude in rabbits with left ventricular dysfunction following myocardial infarction.

T-wave alternans may predict the occurrence of ventricular arrhythmias in patients with left ventricular dysfunction and experimental work has linked discordant repolarization alternans to the induction of re-entry. The aim of this study was to examine the occurrence of transmural repolarization alternans and to investigate the link between alternans and ventricular arrhythmia in rabbits with left ventricular dysfunction following myocardial infarction. Optical mapping was used to record action potentials from the transmural surface of left ventricular wedge preparations from normal and post-infarction hearts during a progressive reduction in pacing cycle length at 30 and 37°C. Data were analyzed using custom software, including spectral analysis. There were no significant differences in baseline transmural electrophysiology between the groups. Post-infarction hearts had a lower threshold for both repolarization alternans (286 vs. 333 bpm, p<0.05) and ventricular arrhythmias (79 vs. 19%, p<0.01) during rapid pacing, which was not accounted for by increased transmural discordant alternans. In VF-prone hearts, alternans in optical action potential amplitude was observed and increased until 2:1 block occurred. The degree of optical action potential amplitude alternans (12.0 ± 7.0 vs. 1.8 ± 0.3, p<0.05), but not APD(90) alternans (1.4 ± 0.6 vs. 1.1 ± 0.1, p>0.05) was associated with VF inducibility during rapid pacing. Post-infarction hearts are more vulnerable to transmural alternans and ventricular arrhythmias at rapid rates. Alternans in optical action potential amplitude was associated with conduction block and VF. The data suggest that changes in optical action potential amplitude may underlie a mechanism for alternans-associated ventricular arrhythmia in left ventricular dysfunction.

Profile of microvolt T-wave alternans testing in 1003 patients hospitalized with heart failure.

Observational studies in selected populations have suggested that microvolt T‐wave alternans (MTWA) testing may identify patients with heart failure (HF) at risk of sudden cardiac death. The aims of this study were to investigate the utility of MTWA testing in an unselected population of patients with HF and to evaluate the clinical characteristics associated with the MTWA results.

Subepicardial Action Potential Characteristics Are a Function of Depth and Activation Sequence in Isolated Rabbit Hearts

Background—Electric excitability in the ventricular wall is influenced by cellular electrophysiology and passive electric properties of the myocardium. Action potential (AP) rise time, an indicator of myocardial excitability, is influenced by conduction pattern and distance from the epicardial surface. This study examined AP rise times and conduction velocity as the depolarizing wavefront approaches the epicardial surface. Methods and Results—Two-photon excitation of di-4-aminonaphthenyl-pyridinum-propylsulfonate was used to measure electric activity at discrete epicardial layers of isolated Langendorff-perfused rabbit hearts to a depth of 500 &mgr;m. Endo-to-epicardial wavefronts were studied during right atrial or ventricular endocardial pacing. Similar measurements were made with epi-to-endocardial, transverse, and longitudinal pacing protocols. Results were compared with data from a bidomain model of 3-dimensional (3D) electric propagation within ventricular myocardium. During right atrial and endocardial pacing, AP rise time (10%–90% of upstroke) decreased by ≈50% between 500 and 50 &mgr;m from the epicardial surface, whereas conduction velocity increased and AP duration was only slightly shorter (≈4%). These differences were not observed with other conduction patterns. The depth-dependent changes in rise time were larger at higher pacing rates. Modeling data qualitatively reproduced the behavior seen experimentally and demonstrated a parallel reduction in peak INa and electrotonic load as the wavefront approaches the epicardial surface. Conclusions—Decreased electrotonic load at the epicardial surface results in more rapid AP upstrokes and higher conduction velocities compared with the bulk myocardium. Combined effects of tissue depth and pacing rate on AP rise time reduce conduction safety and myocardial excitability within the ventricular wall.