Elena G. Tolkacheva

Arrhythmogenic Mechanisms in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca2+ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugols solution, and patch clamping in a knockin (RyR2/RyR2R4496C) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2R4496C and in 9% of 11 wild-type (P=0.03) littermates perfused with Ca2+and isoproterenol; 66% of 12 RyR2/RyR2R4496C and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (P=0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His–Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugols solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2R4496C mice. Under current clamp, single Purkinje cells from RyR2/RyR2R4496C mouse hearts generated delayed afterdepolarization–induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His–Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT.

Role of Conduction Velocity Restitution and Short-Term Memory in the Development of Action Potential Duration Alternans in Isolated Rabbit Hearts

Background— Spatially discordant alternans (SDA) has been linked to life-threatening arrhythmias. The mechanisms underlying SDA development in cardiac tissue remain unclear. Methods and Results— We investigated the role of conduction velocity (CV) restitution and short-term memory in the organization and evolution of alternans in action potential duration using high-resolution optical mapping of the epicardial surface in 8 isolated, Langendorff-perfused rabbit hearts. To assess the spatial organization of alternans, we tracked the evolution of nodal lines that separate out-of-phase regions of SDA. We measured the action potential duration heterogeneity index and maximal slope of CV restitution and estimated the effects of short-term memory by calculating time constant of action potential duration accommodation (&tgr;). We found that 2 mechanisms underlie the development of SDA in the heart, leading to 2 distinct behaviors of nodal lines. The first mechanism is based on steep CV restitution and is associated with small &tgr; and stable nodal lines. The second mechanism is associated with short-term memory (large &tgr;) and is characterized by shallow CV restitution and unstable behavior of nodal lines. The maximum slope of the CV restitution was steeper (18.16±3.34 m/s2) and &tgr; was smaller (&tgr;=4.31±0.33 stimuli) for areas with stable nodal lines than for areas with unstable nodal lines (6.32±0.96 m/s2 and &tgr;=10.3±1.84 stimuli; P<0.01). Conclusions— Our results provide new insight into the mechanisms underlying SDA formation in the rabbit heart. Specifically, our results suggest that a new mechanism associated with short-term memory underlies SDA formation in the heart, in addition to steep CV restitution.

The restitution portrait: A new method for investigating rate-dependent restitution

Introduction: Electrical restitution, relating action potential duration (APD) to diastolic interval (DI), was believed to determine the stability of heart rhythm. However, recent studies demonstrate that stability also depends on long‐term APD changes caused by memory. This study presents a new method for investigation of rate‐ and memory‐dependent aspects of restitution and for assessment of mapping models of APD.

Analysis of the Fenton-Karma model through an approximation by a one-dimensional map

The Fenton-Karma model is a simplification of complex ionic models of cardiac membrane that reproduces quantitatively many of the characteristics of heart cells; its behavior is simple enough to be understood analytically. In this paper, a map is derived that approximates the response of the Fenton-Karma model to stimulation in zero spatial dimensions. This map contains some amount of memory, describing the action potential duration as a function of the previous diastolic interval and the previous action potential duration. Results obtained from iteration of the map and numerical simulations of the Fenton-Karma model are in good agreement. In particular, the iterated map admits different types of solutions corresponding to various dynamical behavior of the cardiac cell, such as 1:1 and 2:1 patterns. (c) 2002 American Institute of Physics.

Mechanisms Underlying the Antifibrillatory Action of Hyperkalemia in Guinea Pig Hearts

Hyperkalemia increases the organization of ventricular fibrillation (VF) and may also terminate it by mechanisms that remain unclear. We previously showed that the left-to-right heterogeneity of excitation and wave fragmentation present in fibrillating guinea pig hearts is mediated by chamber-specific outward conductance differences in the inward rectifier potassium current (I(K1)). We hypothesized that hyperkalemia-mediated depolarization of the reversal potential of I(K1) (E(K1)) would reduce excitability and thereby reduce VF excitation frequencies and left-to-right heterogeneity. We induced VF in Langendroff-perfused guinea pig hearts and increased the extracellular K(+) concentration ([K(+)](o)) from control (4 mM) to 7 mM (n = 5) or 10 mM (n = 7). Optical mapping enabled spatial characterization of excitation dominant frequencies (DFs) and wavebreaks, and identification of sustained rotors (>4 cycles). During VF, hyperkalemia reduced the maximum DF of the left ventricle (LV) from 31.5 +/- 4.7 Hz (control) to 23.0 +/- 4.7 Hz (7.0 mM) or 19.5 +/- 3.6 Hz (10.0 mM; p < 0.006), the left-to-right DF gradient from 14.7 +/- 3.6 Hz (control) to 4.4 +/- 1.3 Hz (7 mM) and 3.2 +/- 1.4 Hz (10 mM), the number of DF domains, and the incidence of wavebreak in the LV and interventricular regions. During 10 mM [K(+)](o), the rotation period and core area of sustained rotors in the LV increased, and VF often terminated. Two-dimensional computer simulations mimicking experimental VF predicted that clamping E(K1) to normokalemic values during simulated hyperkalemia prevented all of the hyperkalemia-induced VF changes. During hyperkalemia, despite the shortening of the action potential duration, depolarization of E(K1) increased refractoriness, leading to a slowing of VF, which effectively superseded the influence of I(K1) conductance differences on VF organization. This reduced the left-to-right excitation gradients and heterogeneous wavebreak formation. Overall, these results provide, to our knowledge, the first direct mechanistic insight into the organization and/or termination of VF by hyperkalemia.

Restitution in mapping models with an arbitrary amount of memory

Restitution, the characteristic shortening of action potential duration (APD) with increased heart rate, has been studied extensively because of its purported link to the onset of fibrillation. Restitution is often represented in the form of mapping models where APD is a function of previous diastolic intervals (DIs) and/or APDs, A(n+1)=F(D(n),A(n),D(n-1),A(n-1),...), where A(n+1) is the APD following a DI given by D(n). The number of variables previous to D(n) determines the degree of memory in the mapping model. Recent experiments have shown that mapping models should contain at least three variables (D(n),A(n),D(n-1)) to reproduce a restitution portrait (RP) that is qualitatively similar to that seen experimentally, where the RP shows three different types of restitution curves (RCs) [dynamic, S1-S2, and constant-basic cycle length (BCL)] simultaneously. However, an interpretation of the different RCs has only been presented in detail for mapping models of one and two variables. Here we present an analysis of the different RCs in the RP for mapping models with an arbitrary amount of memory. We determine the number of variables necessary to represent the different RCs in the RP. We also present a graphical visualization of these RCs. Our analysis reveals that the dynamic and S1-S2 RCs reside on two-dimensional surfaces, and therefore provide limited information for mapping models with more than two variables. However, constant-BCL restitution is a feature of the RP that depends on higher dimensions and can possibly be used to determine a lower bound on the dimensionality of cardiac dynamics.

Interventricular heterogeneity as a substrate for arrhythmogenesis of decoupled mitochondria during ischemia in the whole heart

Myocardial ischemia results in metabolic changes, which collapse the mitochondrial network, that increase the vulnerability of the heart to ventricular fibrillation (VF). It has been demonstrated at the single cell level that uncoupling the mitochondria using carbonyl cyanide p-(tri-fluoromethoxy)phenyl-hydrazone (FCCP) at low concentrations can be cardioprotective. The aim of our study was to investigate the effect of FCCP on arrhythmogenesis during ischemia in the whole rabbit heart. We performed optical mapping of isolated rabbit hearts (n = 33) during control and 20 min of global ischemia and reperfusion, both with and without pretreatment with the mitochondrial uncoupler FCCP at 100, 50, or 30 nM. No hearts went into VF during ischemia under the control condition, with or without the electromechanical uncoupler blebbistatin. We found that pretreatment with 100 (n = 4) and 50 (n = 6) nM FCCP, with or without blebbistatin, leads to VF during ischemia in all hearts, whereas pretreatment with 30 nM of FCCP led to three out of eight hearts going into VF during ischemia. We demonstrated that 30 nM of FCCP significantly increased interventricular (but not intraventricular) action potential duration and conduction velocity heterogeneity in the heart during ischemia, thus providing the substrate for VF. We showed that wavebreaks during VF occurred between the right and left ventricular junction. We also demonstrated that no VF occurred in the heart pretreated with 10 μM glibenclamide, which is known to abolish interventricular heterogeneity. Our results indicate that low concentrations of FCCP, although cardioprotective at the single cell level, are arrhythmogenic at the whole heart level. This is due to the fact that FCCP induces different electrophysiological changes to the right and left ventricle, thus increasing interventricular heterogeneity and providing the substrate for VF.

Heart rate variability and alternans formation in the heart: The role of feedback in cardiac dynamics.

A beat-to-beat alternation in the action potential duration (APD) of myocytes, i.e. alternans, is believed to be a direct precursor of ventricular fibrillation in the whole heart. A common approach for the prediction of alternans is to construct the restitution curve, which is the nonlinear functional relationship between the APD and the preceding diastolic interval (DI). It was proposed that alternans appears when the magnitude of the slope of the restitution curve exceeds one, known as the restitution hypothesis. However, this restitution hypothesis was derived under the assumption of periodic stimulation, when there is a dependence of the DI on the immediate preceding APD (i.e. feedback). However, under physiological conditions, the heart rate exhibits substantial variations in time, known as heart rate variability (HRV), which introduces deviations from periodic stimulation in the system. In this manuscript, we investigated the role of HRV on alternans formation in isolated cardiac myocytes using numerical simulations of an ionic model of the cardiac action potential. We used this model with two different pacing protocols: a periodic pacing protocol with feedback and a protocol without feedback. We show that when HRV is incorporated in the periodic pacing protocol, it facilitated alternans formation in the isolated cell, but did not significantly change the magnitude of alternans. On the other hand, in the case of the pacing protocol without feedback, alternans formation was prevented, even in the presence of HRV.

Uncoupling the mitochondria facilitates alternans formation in the isolated rabbit heart

Alternans of action potential duration (APD) and intracellular calcium ([Ca²⁺]i) transients in the whole heart are thought to be markers of increased propensity to ventricular fibrillation during ischemia-reperfusion injuries. During ischemia, ATP production is affected and the mitochondria become uncoupled, which may affect alternans formation in the heart. The aim of our study was to investigate the role of mitochondria on the formation of APD and [Ca²⁺]i alternans in the isolated rabbit heart. We performed dual voltage and [Ca²⁺]i optical mapping of isolated rabbit hearts under control conditions, global no-flow ischemia (n = 6), and after treatment with 50 nM of the mitochondrial uncoupler FCCP (n = 6). We investigated the formation of alternans of APD, [Ca²⁺]i amplitude (CaA), and [Ca²⁺]i duration (CaD) under different rates of pacing. We found that treatment with FCCP leads to the early occurrence of APD, CaD, and CaA alternans; an increase of intraventricular APD but not CaD heterogeneity; and significant reduction in conduction velocity compared with that of control. Furthermore, we demonstrated that FCCP and global ischemia have similar effects on the prolongation of [Ca²⁺]i transients, whereas ischemia induces a significantly larger reduction of APD compared with that in FCCP treatment. In conclusion, our results demonstrate that uncoupling of mitochondria leads to an earlier occurrence of alternans in the heart. Thus, in conditions of mitochondrial stress, as seen during myocardial ischemia, uncoupled mitochondria may be responsible for the formation of both APD and [Ca²⁺]i alternans in the heart, which in turn creates a substrate for ventricular arrhythmias.

The effect of cardiac sympathetic denervation through bilateral stellate ganglionectomy on electrical properties of the heart

The role of the cardiac sympathetic nerve activity in various cardiac diseases is typically evaluated using β-adrenergic receptor antagonists. However, these antagonists induce global denervation effects not only in the cardiovascular system, but also in the brain and kidney. The objective of this study was to detect the electrophysiological property changes due to 8 days of cardiac sympathetic denervation and investigate the possible mechanisms underlying these changes using a more cardiac-specific bilateral stellate ganglionectomy (SGX) rat model. High-resolution optical mapping using a voltage-sensitive dye was performed in isolated Langendorff-perfused sham and SGX hearts, which were paced at progressively reduced basic cycle lengths under several different conditions: control, pretreatment with isoproterenol, and administration of atenolol and esmolol. Several electrophysiological parameters were recorded during periodic pacing and ventricular fibrillation (VF). Our results demonstrate that cardiac sympathetic denervation by bilateral SGX shortens action potential duration (APD) and flattens the APD restitution curve, but does not significantly affect spatial dispersion of APD. We found that, although the vulnerability of sham and SGX hearts to VF is similar, the dynamics of VF are different. The maximum dominant frequency is higher, and the spatial distribution of VF is more complex in the SGX heart, resulting in different mechanisms of VF. We demonstrated that β(1)-adrenergic receptors are downregulated in the SGX compared with sham hearts. In addition, our data suggest that the mechanism of cardiac sympathetic denervation by SGX surgery is more similar to the administration of β-blocker esmolol than atenolol.