Sandeep V. Pandit

A Novel Form of Short QT Syndrome (SQT3) Is Caused by a Mutation in the KCNJ2 Gene

Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (IKr) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (IKs) channel. Here we identify a new variant, “SQT3”, which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (IK1) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward IK1 than the wild-type (P<0.05) at potentials between −75 mV and −45 mV, with the peak current being shifted in the former with respect to the latter (WT, −75 mV; D172N, −65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward IK1 greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients’ consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias.

Mechanisms of Wave Fractionation at Boundaries of High-Frequency Excitation in the Posterior Left Atrium of the Isolated Sheep Heart During Atrial Fibrillation

Background— High-frequency fractionated electrograms recorded during atrial fibrillation (AF) in the posterior left atrium (PLA) and elsewhere are being used as target sites for catheter ablation. We tested the hypothesis that highly periodic electric waves emerging from AF sources at or near the PLA give rise to the most fractionated activity in adjacent locations. Methods and Results— Sustained AF was induced in 8 isolated sheep hearts (0.5 &mgr;mol/L acetylcholine). Endocardial videoimaging (DI-4-ANEPPS) and electric mapping of the PLA enabled spatial characterization of dominant frequencies (DFs) and a regularity index (ratio of DF to total power). Regularity index showed that fractionation was lowest within the area with the maximal DF (DFmax domain; 0.19±0.02) and highest within a band of ≈3 mm (0.16±0.02; P=0.047) at boundaries with lower-frequency domains. The numbers of spatiotemporal periodic episodes (25.9±2.3) and rotors per experiment (1.9±0.7) were also highest within the DFmax domain. Most commonly, breakthrough waves at the PLA traveled toward the rest of the atria (76.8±8.1% outward versus 23.2±8.1% inward; P<0.01). In both experiments and simulations with an atrial ionic model, fractionation at DFmax boundaries was associated with increased beat-to-beat variability of conduction velocity and directionality with wavebreak formation. Conclusions— During stable AF, the PLA harbors regular, fast, and highly organized activity; the outer limit of the DFmax domain is the area where the most propagation pattern variability and fractionated activity occur. These new concepts introduce a new perspective in the clinical use of high-frequency fractionated electrograms to localize sources of AF precisely at the PLA and elsewhere.

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.

Rotors and the Dynamics of Cardiac Fibrillation

The objective of this article is to present a broad review of the role of cardiac electric rotors and their accompanying spiral waves in the mechanism of cardiac fibrillation. At the outset, we present a brief historical overview regarding reentry and then discuss the basic concepts and terminologies pertaining to rotors and their initiation. Thereafter, the intrinsic properties of rotors and spiral waves, including phase singularities, wavefront curvature, and dominant frequency maps, are discussed. The implications of rotor dynamics for the spatiotemporal organization of fibrillation, independent of the species being studied, are described next. The knowledge gained regarding the role of cardiac structure in the initiation or maintenance of rotors and the ionic bases of spiral waves in the past 2 decades, as well as the significance for drug therapy, is reviewed subsequently. We conclude by examining recent evidence suggesting that rotors are critical in sustaining both atrial and ventricular fibrillation in the human heart and its implications for treatment with radiofrequency ablation.

Human Atrial Action Potential and Ca2+ Model: Sinus Rhythm and Chronic Atrial Fibrillation

Rationale: Understanding atrial fibrillation (AF) requires integrated understanding of ionic currents and Ca2+ transport in remodeled human atrium, but appropriate models are limited. Objective: To study AF, we developed a new human atrial action potential (AP) model, derived from atrial experimental results and our human ventricular myocyte model. Methods and Results: Atria versus ventricles have lower IK1, resulting in more depolarized resting membrane potential (≈7 mV). We used higher Ito,fast density in atrium, removed Ito,slow, and included an atrial-specific IKur. INCX and INaK densities were reduced in atrial versus ventricular myocytes according to experimental results. SERCA function was altered to reproduce human atrial myocyte Ca2+ transients. To simulate chronic AF, we reduced ICaL, Ito, IKur and SERCA, and increased IK1,IKs and INCX. We also investigated the link between Kv1.5 channelopathy, [Ca2+]i, and AF. The sinus rhythm model showed a typical human atrial AP morphology. Consistent with experiments, the model showed shorter APs and reduced AP duration shortening at increasing pacing frequencies in AF or when ICaL was partially blocked, suggesting a crucial role of Ca2+ and Na+ in this effect. This also explained blunted Ca2+ transient and rate-adaptation of [Ca2+]i and [Na+]i in chronic AF. Moreover, increasing [Na+]i and altered INaK and INCX causes rate-dependent atrial AP shortening. Blocking IKur to mimic Kv1.5 loss-of-function increased [Ca2+]i and caused early afterdepolarizations under adrenergic stress, as observed experimentally. Conclusions: Our study provides a novel tool and insights into ionic bases of atrioventricular AP differences, and shows how Na+ and Ca2+ homeostases critically mediate abnormal repolarization in AF.Rationale: Understanding atrial fibrillation (AF) requires integrated understanding of ionic currents and Ca2+ transport in remodeled human atrium, but appropriate models are limited. Objective: To study AF, we developed a new human atrial action potential (AP) model, derived from atrial experimental results and our human ventricular myocyte model. Methods and Results: Atria versus ventricles have lower IK1, resulting in more depolarized resting membrane potential (≈7 mV). We used higher Ito,fast density in atrium, removed Ito,slow, and included an atrial-specific IKur. INCX and INaK densities were reduced in atrial versus ventricular myocytes according to experimental results. SERCA function was altered to reproduce human atrial myocyte Ca2+ transients. To simulate chronic AF, we reduced ICaL, Ito, IKur and SERCA, and increased IK1,IKs and INCX. We also investigated the link between Kv1.5 channelopathy, [Ca2+]i, and AF. The sinus rhythm model showed a typical human atrial AP morphology. Consistent with experiments, the model showed shorter APs and reduced AP duration shortening at increasing pacing frequencies in AF or when ICaL was partially blocked, suggesting a crucial role of Ca2+ and Na+ in this effect. This also explained blunted Ca2+ transient and rate-adaptation of [Ca2+]i and [Na+]i in chronic AF. Moreover, increasing [Na+]i and altered INaK and INCX causes rate-dependent atrial AP shortening. Blocking IKur to mimic Kv1.5 loss-of-function increased [Ca2+]i and caused early afterdepolarizations under adrenergic stress, as observed experimentally. Conclusions: Our study provides a novel tool and insights into ionic bases of atrioventricular AP differences, and shows how Na+ and Ca2+ homeostases critically mediate abnormal repolarization in AF. # Novelty and Significance {#article-title-67}

Bone Marrow Adipose Tissue Is an Endocrine Organ that Contributes to Increased Circulating Adiponectin during Caloric Restriction

The adipocyte-derived hormone adiponectin promotes metabolic and cardiovascular health. Circulating adiponectin increases in lean states such as caloric restriction (CR), but the reasons for this paradox remain unclear. Unlike white adipose tissue (WAT), bone marrow adipose tissue (MAT) increases during CR, and both MAT and serum adiponectin increase in many other clinical conditions. Thus, we investigated whether MAT contributes to circulating adiponectin. We find that adiponectin secretion is greater from MAT than WAT. Notably, specific inhibition of MAT formation in mice results in decreased circulating adiponectin during CR despite unaltered adiponectin expression in WAT. Inhibiting MAT formation also alters skeletal muscle adaptation to CR, suggesting that MAT exerts systemic effects. Finally, we reveal that both MAT and serum adiponectin increase during cancer therapy in humans. These observations identify MAT as an endocrine organ that contributes significantly to increased serum adiponectin during CR and perhaps in other adverse states.

Unique Kir2.x Properties Determine Regional and Species Differences in the Cardiac Inward Rectifier K+ Current

The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (IK1). In addition to differences in current density, atrial and ventricular IK1 have differences in outward current profiles and in extracellular potassium ([K+]o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular IK1 in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than −30 mV (I≈0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, IK1 rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, IK1 did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1±0.1 pS in 15 cells, which corresponded with IK1 in sheep atria (9.9±0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K+]o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial IK1 showed an increase in outward currents in 10 mmol/L [K+]o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward IK1 currents rectified completely and increased in high [K+]o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K+]o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of IK1 in the heart.

Human Atrial Action Potential and Ca2+ Model

Rationale: Understanding atrial fibrillation (AF) requires integrated understanding of ionic currents and Ca2+ transport in remodeled human atrium, but appropriate models are limited. Objective: To study AF, we developed a new human atrial action potential (AP) model, derived from atrial experimental results and our human ventricular myocyte model. Methods and Results: Atria versus ventricles have lower IK1, resulting in more depolarized resting membrane potential (≈7 mV). We used higher Ito,fast density in atrium, removed Ito,slow, and included an atrial-specific IKur. INCX and INaK densities were reduced in atrial versus ventricular myocytes according to experimental results. SERCA function was altered to reproduce human atrial myocyte Ca2+ transients. To simulate chronic AF, we reduced ICaL, Ito, IKur and SERCA, and increased IK1,IKs and INCX. We also investigated the link between Kv1.5 channelopathy, [Ca2+]i, and AF. The sinus rhythm model showed a typical human atrial AP morphology. Consistent with experiments, the model showed shorter APs and reduced AP duration shortening at increasing pacing frequencies in AF or when ICaL was partially blocked, suggesting a crucial role of Ca2+ and Na+ in this effect. This also explained blunted Ca2+ transient and rate-adaptation of [Ca2+]i and [Na+]i in chronic AF. Moreover, increasing [Na+]i and altered INaK and INCX causes rate-dependent atrial AP shortening. Blocking IKur to mimic Kv1.5 loss-of-function increased [Ca2+]i and caused early afterdepolarizations under adrenergic stress, as observed experimentally. Conclusions: Our study provides a novel tool and insights into ionic bases of atrioventricular AP differences, and shows how Na+ and Ca2+ homeostases critically mediate abnormal repolarization in AF.Rationale: Understanding atrial fibrillation (AF) requires integrated understanding of ionic currents and Ca2+ transport in remodeled human atrium, but appropriate models are limited. Objective: To study AF, we developed a new human atrial action potential (AP) model, derived from atrial experimental results and our human ventricular myocyte model. Methods and Results: Atria versus ventricles have lower IK1, resulting in more depolarized resting membrane potential (≈7 mV). We used higher Ito,fast density in atrium, removed Ito,slow, and included an atrial-specific IKur. INCX and INaK densities were reduced in atrial versus ventricular myocytes according to experimental results. SERCA function was altered to reproduce human atrial myocyte Ca2+ transients. To simulate chronic AF, we reduced ICaL, Ito, IKur and SERCA, and increased IK1,IKs and INCX. We also investigated the link between Kv1.5 channelopathy, [Ca2+]i, and AF. The sinus rhythm model showed a typical human atrial AP morphology. Consistent with experiments, the model showed shorter APs and reduced AP duration shortening at increasing pacing frequencies in AF or when ICaL was partially blocked, suggesting a crucial role of Ca2+ and Na+ in this effect. This also explained blunted Ca2+ transient and rate-adaptation of [Ca2+]i and [Na+]i in chronic AF. Moreover, increasing [Na+]i and altered INaK and INCX causes rate-dependent atrial AP shortening. Blocking IKur to mimic Kv1.5 loss-of-function increased [Ca2+]i and caused early afterdepolarizations under adrenergic stress, as observed experimentally. Conclusions: Our study provides a novel tool and insights into ionic bases of atrioventricular AP differences, and shows how Na+ and Ca2+ homeostases critically mediate abnormal repolarization in AF. # Novelty and Significance {#article-title-67}

Up‐regulation of the inward rectifier K+ current (IK1) in the mouse heart accelerates and stabilizes rotors

Previous studies have suggested an important role for the inward rectifier K+ current (IK1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1–green fluorescent protein (GFP) fusion protein in a cardiac‐specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff‐perfused TG hearts were able to sustain stable VT/VF for 350 ± 1181 s at a very high dominant frequency (DF) of 44.6 ± 4.3 Hz. In contrast, tachyarrhythmias in wild‐type hearts (WT) were short‐lived (3 ± 9 s), and the DF was 26.3 ± 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 μm Ba2+, suggesting an important role for IK1. Moreover, by increasing IK1 density in a two‐dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (≈45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in IK1, compared to other K+ currents. In combination, these results provide the first direct evidence that IK1 up‐regulation in the mouse heart is a substrate for stable and very fast rotors.

KCNJ2 mutation in short QT syndrome 3 results in atrial fibrillation and ventricular proarrhythmia

We describe a mutation (E299V) in KCNJ2, the gene that encodes the strong inward rectifier K+ channel protein (Kir2.1), in an 11-y-old boy. The unique short QT syndrome type-3 phenotype is associated with an extremely abbreviated QT interval (200 ms) on ECG and paroxysmal atrial fibrillation. Genetic screening identified an A896T substitution in a highly conserved region of KCNJ2 that resulted in a de novo mutation E299V. Whole-cell patch-clamp experiments showed that E299V presents an abnormally large outward IK1 at potentials above −55 mV (P < 0.001 versus wild type) due to a lack of inward rectification. Coexpression of wild-type and mutant channels to mimic the heterozygous condition still resulted in a large outward current. Coimmunoprecipitation and kinetic analysis showed that E299V and wild-type isoforms may heteromerize and that their interaction impairs function. The homomeric assembly of E299V mutant proteins actually results in gain of function. Computer simulations of ventricular excitation and propagation using both the homozygous and heterozygous conditions at three different levels of integration (single cell, 2D, and 3D) accurately reproduced the electrocardiographic phenotype of the proband, including an exceedingly short QT interval with merging of the QRS and the T wave, absence of ST segment, and peaked T waves. Numerical experiments predict that, in addition to the short QT interval, absence of inward rectification in the E299V mutation should result in atrial fibrillation. In addition, as predicted by simulations using a geometrically accurate three-dimensional ventricular model that included the His–Purkinje network, a slight reduction in ventricular excitability via 20% reduction of the sodium current should increase vulnerability to life-threatening ventricular tachyarrhythmia.