Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.

Antiarrhythmic Hit to Lead Refinement in a Dish Using Patient-Derived iPSC Cardiomyocytes.

Ventricular cardiac arrhythmia (VA) arises in acquired or congenital heart disease. Long QT syndrome type-3 (LQT3) is a congenital form of VA caused by cardiac sodium channel (INaL) SCN5A mutations that prolongs cardiac action potential (AP) and enhances INaL current. Mexiletine inhibits INaL and shortens the QT interval in LQT3 patients. Above therapeutic doses, mexiletine prolongs the cardiac AP. We explored structure-activity relationships (SAR) for AP shortening and prolongation using dynamic medicinal chemistry and AP kinetics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using patient-derived LQT3 and healthy hiPSC-CMs, we resolved distinct SAR for AP shortening and prolongation effects in mexiletine analogues and synthesized new analogues with enhanced potency and selectivity for INaL. This resulted in compounds with decreased AP prolongation effects, increased metabolic stability, increased INaL selectivity, and decreased avidity for the potassium channel. This study highlights using hiPSC-CMs to guide medicinal chemistry and "drug development in a dish".

Atrial Fibrillation Initiated by Early Afterdepolarization-Mediated Triggered Activity during Acute Oxidative Stress: Efficacy of Late Sodium Current Blockade.

BACKGROUND: The mechanism of Atrial Fibrillation (AF) that emerges spontaneously during acute oxidative stress is poorly defined and its drug therapy remains suboptimal. We hypothesized that oxidative activation of Ca-calmodulin dependent protein kinase (CaMKII) promotes Early Afterdepolarization-(EAD)-mediated triggered AF in aged fibrotic atria that is sensitive to late Na current (INa-L) blockade. METHOD AND RESULTS: High-resolution voltage optical mapping of the Left and Right Atrial (LA & RA) epicardial surfaces along with microelectrode recordings were performed in isolated-perfused male Fisher 344 rat hearts in Langendorff setting. Aged atria (23-24 months) manifested 10-fold increase in atrial tissue fibrosis compared to young/adult (2-4 months) atria (P<0001. Spontaneous AF arose in 39 out of 41 of the aged atria but in 0 out of 12 young/adult hearts (P<001) during arterial perfusion of with 0.1 mm of hydrogen peroxide (H2O2). Optical Action Potential (AP) activation maps showed that the AF was initiated by a focal mechanism in the LA suggestive of EAD-mediated triggered activity. Cellular AP recordings with glass microelectrodes from the LA epicardial sites showing focal activity confirmed optical AP recordings that the spontaneous AF was initiated by late phase 3 EAD-mediated triggered activity. Inhibition of CaMKII activity with KN-93 (1 µM) (N=6) or its downstream target, the enhanced INa-L with GS-967 (1 µM), a specific blocker of INa-L (N=6), potently suppressed the AF and prevented its initiation when perfused 15 min prior to H2O2 (n=6). CONCLUSIONS: Increased atrial tissue fibrosis combined with acute oxidative activation of CaMK II Initiate AF by EAD-mediated triggered activity. Specific block of the INa-L with GS-967 effectively suppresses the AF. Drug therapy of oxidative AF in humans with traditional antiarrhythmic drugs remains suboptimal; suppressing INa-L offers a potential new strategy for effective suppression of oxidative human AF that remains suboptimal.

Enhanced Late Na and Ca Currents as Effective Antiarrhythmic Drug Targets.

While recent advances clarified the molecular and cellular modes of action of antiarrhythmic drugs (AADs), their link to suppression of dynamical arrhythmia mechanisms remains only partially understood. The current classifications of AADs (Classes I, III, and IV) rely on blocking peak Na, K and L-type calcium currents (ICa,L), with Class II with dominant beta receptor blocking activity and Class V including drugs with diverse classes of actions. The discovery that the calcium and redox sensor, cardiac Ca/calmodulin-dependent protein kinase II (CaMKII) enhances both the late Na (INa-L) and the late ICa,L in patients at high risk of VT/VF provided a new and a rational AAD target. Pathological rise of either or both of INa-L and late ICa,L are demonstrated to promote cellular early afterdepolarizations (EADs) and EAD-mediated triggered activity that can initiate VT/VF in remodeled hearts. Selective inhibition of the INa-L without affecting their peak transients with the highly specific prototype drug, GS-967 suppresses these EAD-mediated VT/VFs. As in the case of INa-L, selective inhibition of the late ICa,L without affecting its peak with the prototype drug, roscovitine suppressed oxidative EAD-mediated VT/VF. These findings indicate that specific blockers of the late inward currents without affecting their peaks (gating modifiers), offer a new and effective AAD class action i.e., "Class VI." The development of safe drugs with selective Class VI actions provides a rational and effective approach to treat VT/VF particularly in cardiac conditions associated with enhanced CaMKII activity such as heart failure.

Increased susceptibility of spontaneously hypertensive rats to ventricular tachyarrhythmias in early hypertension.

Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that, in early hypertension, the susceptibility to stress-induced VT/VF increases. We compared the susceptibility of 5- to 6-month-old male spontaneously hypertensive rats (SHR) and age/sex-matched normotensive rats (NR) to VT/VF during challenge with oxidative stress (H2 O2 ; 0.15 mmol l(-1) ). We found that only SHR hearts exhibited left ventricular fibrosis and hypertrophy. H2 O2 promoted VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-calcium optical mapping of Langendorff-perfused SHR hearts revealed that H2 O2 -induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except with larger EAD amplitude. During the early stages of hypertension, when challenged with oxidative stress, SHR hearts showed an increased ventricular arrhythmogenicity that stems primarily from tissue remodelling (hypertrophy, fibrosis) rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.

Stochastic pacing reveals the propensity to cardiac action potential alternans and uncovers its underlying dynamics.

KEY POINTS: Beat-to-beat alternation (alternans) of the cardiac action potential duration is known to precipitate life-threatening arrhythmias and can be driven by the kinetics of voltage-gated membrane currents or by instabilities in intracellular calcium fluxes. To prevent alternans and associated arrhythmias, suitable markers must be developed to quantify the susceptibility to alternans; previous theoretical studies showed that the eigenvalue of the alternating eigenmode represents an ideal marker of alternans. Using rabbit ventricular myocytes, we show that this eigenvalue can be estimated in practice by pacing these cells at intervals varying stochastically. We also show that stochastic pacing permits the estimation of further markers distinguishing between voltage-driven and calcium-driven alternans. Our study opens the perspective to use stochastic pacing during clinical investigations and in patients with implanted pacing devices to determine the susceptibility to, and the type of alternans, which are both important to guide preventive or therapeutic measures. ABSTRACT: Alternans of the cardiac action potential (AP) duration (APD) is a well-known arrhythmogenic mechanism. APD depends on several preceding diastolic intervals (DIs) and APDs, which complicates the prediction of alternans. Previous theoretical studies pinpointed a marker called λalt that directly quantifies how an alternating perturbation persists over successive APs. When the propensity to alternans increases, λalt decreases from 0 to -1. Our aim was to quantify λalt experimentally using stochastic pacing and to examine whether stochastic pacing allows discriminating between voltage-driven and Ca(2+) -driven alternans. APs were recorded in rabbit ventricular myocytes paced at cycle lengths (CLs) decreasing progressively and incorporating stochastic variations. Fitting APD with a function of two previous APDs and CLs permitted us to estimate λalt along with additional markers characterizing whether the dependence of APD on previous DIs or CLs is strong (typical for voltage-driven alternans) or weak (Ca(2+) -driven alternans). During the recordings, λalt gradually decreased from around 0 towards -1. Intermittent alternans appeared when λalt reached -0.8 and was followed by sustained alternans. The additional markers detected that alternans was Ca(2+) driven in control experiments and voltage driven in the presence of ryanodine. This distinction could be made even before alternans was manifest (specificity/sensitivity >80% for -0.4 > λalt  > -0.5). These observations were confirmed in a mathematical model of a rabbit ventricular myocyte. In conclusion, stochastic pacing allows the practical estimation of λalt to reveal the onset of alternans and distinguishes between voltage-driven and Ca(2+) -driven mechanisms, which is important since these two mechanisms may precipitate arrhythmias in different manners.

Molecular Basis of Hypokalemia-Induced Ventricular Fibrillation.

BACKGROUND: Hypokalemia is known to promote ventricular arrhythmias, especially in combination with class III antiarrhythmic drugs like dofetilide. Here, we evaluated the underlying molecular mechanisms. METHODS AND RESULTS: Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0-3.5 mmol/L) in the absence or presence of dofetilide (1 µmol/L). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation occurred in 50% of hearts at 2.7 mmol/L [K] in the absence of dofetilide and 3.3 mmol/L [K] in its presence. Pretreatment with the Ca-calmodulin kinase II (CaMKII) inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced ventricular tachycardia/fibrillation, as did the selective late Na current (INa) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/L) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late INa and the L-type Ca current. K current suppression by hypokalemia and dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs. CONCLUSIONS: We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late INa. Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.

Perspective: a dynamics-based classification of ventricular arrhythmias.

Despite key advances in the clinical management of life-threatening ventricular arrhythmias, culminating with the development of implantable cardioverter-defibrillators and catheter ablation techniques, pharmacologic/biologic therapeutics have lagged behind. The fundamental issue is that biological targets are molecular factors. Diseases, however, represent emergent properties at the scale of the organism that result from dynamic interactions between multiple constantly changing molecular factors. For a pharmacologic/biologic therapy to be effective, it must target the dynamic processes that underlie the disease. Here we propose a classification of ventricular arrhythmias that is based on our current understanding of the dynamics occurring at the subcellular, cellular, tissue and organism scales, which cause arrhythmias by simultaneously generating arrhythmia triggers and exacerbating tissue vulnerability. The goal is to create a framework that systematically links these key dynamic factors together with fixed factors (structural and electrophysiological heterogeneity) synergistically promoting electrical dispersion and increased arrhythmia risk to molecular factors that can serve as biological targets. We classify ventricular arrhythmias into three primary dynamic categories related generally to unstable Ca cycling, reduced repolarization, and excess repolarization, respectively. The clinical syndromes, arrhythmia mechanisms, dynamic factors and what is known about their molecular counterparts are discussed. Based on this framework, we propose a computational-experimental strategy for exploring the links between molecular factors, fixed factors and dynamic factors that underlie life-threatening ventricular arrhythmias. The ultimate objective is to facilitate drug development by creating an in silico platform to evaluate and predict comprehensively how molecular interventions affect not only a single targeted arrhythmia, but all primary arrhythmia dynamics categories as well as normal cardiac excitation-contraction coupling.

Cardiac late Na⁺ current: proarrhythmic effects, roles in long QT syndromes, and pathological relationship to CaMKII and oxidative stress.

Myocyte sodium channel current that persists throughout the plateau of the cardiac action potential is referred to as late sodium current (I(Na-L)). The magnitude of I(Na-L) is normally small, but can increase significantly in common acute and chronic pathological settings as a result of inherited and/or acquired Na(+) channelopathies that alter channel opening and closing (ie, gating), location (trafficking), or anchoring and interactions with cytoskeletal proteins. An increase in I(Na-L) reduces repolarization reserve in atrial and ventricular myocytes and prolongs the action potential duration and the QT interval. An enhanced I(Na-L) is a cause of long QT syndrome 3. I(Na-L) may be a cause of afterdepolarizations, triggered arrhythmias, and spontaneous diastolic depolarization-induced automaticity. In addition, enhancement of I(Na-L) increases both the temporal and the spatial dispersion of repolarization in the myocardium and may lead to spatially discordant action potential duration alternans, wavebreak, and reentrant arrhythmias. Positive feedback loops between increases in I(Na-L) and the activity of Ca(2+)/calmodulin-dependent protein kinase II appear to contribute to the genesis of arrhythmias and to certain abnormalities of the ischemic heart. In this review, we discuss some of the more relevant experimental results, clinical findings, and insights from cellular and animal models that highlight the role of I(Na-L) in the genesis of arrhythmias, long QT syndromes, and intracellular Ca(2+) homeostasis.

Selective inhibition of late sodium current suppresses ventricular tachycardia and fibrillation in intact rat hearts.

BACKGROUND: Enhanced late inward Na current (INa-L) modulates action potential duration (APD) and plays a key role in the genesis of early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) and triggered activity. OBJECTIVE: The purpose of this study was to define the influence of selective block of INa-L on EAD- and DAD-mediated triggered ventricular tachycardia (VT) and ventricular fibrillation (VF) in intact hearts using (GS967), a selective and potent (IC50 = 0.13 ± 0.01 µM) blocker of INa-L. METHODS: VT/VF were induced either by local aconitine injection (50 µg) in the left ventricular muscle of adult (3-4 months) male rats (N = 21) or by arterial perfusion of 0.1 mM hydrogen peroxide (H2O2) in aged male rats (24-26 months, N = 16). The left ventricular epicardial surface of the isolated-perfused hearts was optically mapped using fluorescent voltage-sensitive dye, and microelectrode recordings of action potentials were made adjacent to the aconitine injection site. The suppressive and preventive effects of GS967 (1 µM) against EAD/DAD-mediated VT/VF were then determined. RESULTS: Aconitine induced VT in all 13 hearts studied. Activation map (N = 6) showed that the VT was initiated by a focal activity arising from the aconitine injection site (cycle length [CL] 84 ± 12) that degenerated to VF (CL 52 ± 8 ms) within a few seconds. VF was maintained by multifocal activity with occasional incomplete reentrant wavefronts. Administration of GS967 suppressed the VT/VF in 10 of 13 hearts (P < .001). Preexposure to GS967 for 15 minutes before aconitine injection prevented initiation of VT/VF in 5 of 8 additional hearts (P < .02). VF reoccurred within 10 minutes on washout of GS967. Microelectrode recordings (N = 7) showed that VT/VF was initiated by EAD- and DAD-mediated triggered activity at CL of 86 ± 14 ms (NS from VT CL) that preceded the VF. GS967 shortened APD, flattened the slope of the dynamic APD restitution curve, and reduced APD dispersion from 42 ± 12 ms to 8 ± 3 ms (P < .01). H2O2 perfusion in eight fibrotic aged hearts promoted EAD-mediated focal VT/VF, which was suppressed by GS967 in five hearts (P < .02). CONCLUSION: The selective INa-L blocker GS967 effectively suppresses and prevents aconitine and oxidative stress-induced EADs, DADs, and focal VT/VF. Suppression of EADs, DADs, and reduction of APD dispersion make GS967 a potentially useful antiarrhythmic drug in conditions of enhanced INa-L.

Cardiac fibrosis as a determinant of ventricular tachyarrhythmias.

Animal and emerging clinical studies have demonstrated that increased ventricular fibrosis in a setting of reduced repolarization reserve promotes early afterdepolarizations (EADs) and triggered activity that can initiate ventricular tachycardia and ventricular fibrillation (VT/VF). Increased ventricular fibrosis plays a key facilitatory role in allowing oxidative and metabolic stress-induced EADs to manifest as triggered activity causing VT/VF. The lack of such an arrhythmogenic effect by the same stressors in normal non-fibrotic hearts highlights the importance of fibrosis in the initiation of VT/VF. These findings suggest that antifibrotic therapy combined with therapy designed to increase ventricular repolarization reserve may act synergistically to reduce the risk of sudden cardiac death.

Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to ß-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.

Early afterdepolarizations in cardiac myocytes: beyond reduced repolarization reserve.

Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets.

Bifurcation theory and cardiac arrhythmias.

In this paper we review two types of dynamic behaviors defined by the bifurcation theory that are found to be particularly useful in describing two forms of cardiac electrical instabilities that are of considerable importance in cardiac arrhythmogenesis. The first is action potential duration (APD) alternans with an underlying dynamics consistent with the period doubling bifurcation theory. This form of electrical instability could lead to spatially discordant APD alternans leading to wavebreak and reentrant form of tachyarrhythmias. Factors that modulate the APD alternans are discussed. The second form of bifurcation of importance to cardiac arrhythmogenesis is the Hopf-homoclinic bifurcation that adequately describes the dynamics of the onset of early afterdepolarization (EAD)-mediated triggered activity (Hopf) that may cause ventricular tachycardia and ventricular fibrillation (VT/VF respectively). The self-termination of the triggered activity is compatible with the homoclinic bifurcation. Ionic and intracellular calcium dynamics underlying these dynamics are discussed using available experimental and simulation data. The dynamic analysis provides novel insights into the mechanisms of VT/VF, a major cause of sudden cardiac death in the US.

Oxidative stress, fibrosis, and early afterdepolarization-mediated cardiac arrhythmias.

Animal and clinical studies have demonstrated that oxidative stress, a common pathophysiological factor in cardiac disease, reduces repolarization reserve by enhancing the L-type calcium current, the late Na, and the Na-Ca exchanger, promoting early afterdepolarizations (EADs) that can initiate ventricular tachycardia and ventricular fibrillation (VT/VF) in structurally remodeled hearts. Increased ventricular fibrosis plays a key facilitatory role in allowing oxidative-stress induced EADs to manifest as triggered activity and VT/VF, since normal non-fibrotic hearts are resistant to arrhythmias when challenged with similar or higher levels of oxidative stress. The findings imply that antifibrotic therapy, in addition to therapies designed to suppress EAD formation at the cellular level, may be synergistic in reducing the risk of sudden cardiac death.

Dynamics of early afterdepolarization-mediated triggered activity in cardiac monolayers.

Early afterdepolarizations (EADs) are voltage oscillations that occur during the repolarizing phase of the cardiac action potential and cause cardiac arrhythmias in a variety of clinical settings. EADs occur in the setting of reduced repolarization reserve and increased inward-over-outward currents, which intuitively explains the repolarization delay but does not mechanistically explain the time-dependent voltage oscillations that are characteristic of EADs. In a recent theoretical study, we identified a dual Hopf-homoclinic bifurcation as a dynamical mechanism that causes voltage oscillations during EADs, depending on the amplitude and kinetics of the L-type Ca(2+) channel (LTCC) current relative to the repolarizing K(+) currents. Here we demonstrate this mechanism experimentally. We show that cardiac monolayers exposed to the LTCC agonists BayK8644 and isoproterenol produce EAD bursts that are suppressed by the LTCC blocker nitrendipine but not by the Na(+) current blocker tetrodoxin, depletion of intracellular Ca(2+) stores with thapsigargin and caffeine, or buffering of intracellular Ca(2+) with BAPTA-AM. These EAD bursts exhibited a key dynamical signature of the dual Hopf-homoclinic bifurcation mechanism, namely, a gradual slowing in the frequency of oscillations before burst termination. A detailed cardiac action potential model reproduced the experimental observations, and identified intracellular Na(+) accumulation as the likely mechanism for terminating EAD bursts. Our findings in cardiac monolayers provide direct support for the Hopf-homoclinic bifurcation mechanism of EAD-mediated triggered activity, and raise the possibility that this mechanism may also contribute to EAD formation in clinical settings such as long QT syndromes, heart failure, and increased sympathetic output.

Enhanced sensitivity of aged fibrotic hearts to angiotensin II- and hypokalemia-induced early afterdepolarization-mediated ventricular arrhythmias.

Unlike young hearts, aged hearts are highly susceptible to early afterdepolarization (EAD)-mediated ventricular fibrillation (VF). This differential may result from age-related structural remodeling (fibrosis) or electrical remodeling of ventricular myocytes or both. We used optical mapping and microelectrode recordings in Langendorff-perfused hearts and patch-clamp recordings in isolated ventricular myocytes from aged (24-26 mo) and young (3-4 mo) rats to assess susceptibility to EADs and VF during either oxidative stress with ANG II (2 µM) or ionic stress with hypokalemia (2.7 mM). ANG II caused EAD-mediated VF in 16 of 19 aged hearts (83%) after 32 ± 7 min but in 0 of 9 young hearts (0%). ANG II-mediated VF was suppressed with KN-93 (Ca(2+)/calmodulin-dependent kinase inhibitor) and the reducing agent N-acetylcysteine. Hypokalemia caused EAD-mediated VF in 11 of 11 aged hearts (100%) after 7.4 ± 0.4 min. In 14 young hearts, however, VF did not occur in 6 hearts (43%) or was delayed in onset (31 ± 22 min, P < 0.05) in 8 hearts (57%). In patch-clamped myocytes, ANG II and hypokalemia (n = 6) induced EADs and triggered activity in both age groups (P = not significant) at a cycle length of >0.5 s. When myocytes of either age group were coupled to a virtual fibroblast using the dynamic patch-clamp technique, EADs arose in both groups at a cycle length of <0.5 s. Aged ventricles had significantly greater fibrosis and reduced connexin43 gap junction density compared with young hearts. The lack of differential age-related sensitivity at the single cell level in EAD susceptibility indicates that increased ventricular fibrosis in the aged heart plays a key role in increasing vulnerability to VF induced by oxidative and ionic stress.