Robert S. Kass

Proposed Diagnostic Criteria for the Brugada Syndrome Consensus Report

Asyndrome characterized by ST-segment elevation in right precordial leads (V1 to V3) that is unrelated to ischemia, electrolyte disturbances, or obvious structural heart disease was reported as early as 1953,1 but was first described as a distinct clinical entity associated with a high risk of sudden cardiac death in 1992.2–4⇓⇓ The Brugada syndrome is a familial disease that displays an autosomal dominant mode of transmission, with incomplete penetrance and an incidence ranging between 5 and 66 per 10 000. In regions of Southeast Asia where it is endemic, the clinical presentation of Brugada syndrome is distinguished by a male predominance (8:1 ratio of male:female) and the appearance of arrhythmic events at an average age of 40 years (range: 1 to 77 years).2,5⇓ Although a number of candidate genes are considered plausible, thus far the syndrome has been linked only to mutations in SCN5A , the gene encoding for the α subunit of the sodium channel.6 A number of ambiguities exist concerning the diagnosis of Brugada syndrome. The electrocardiographic signature of the syndrome is dynamic and often concealed, but can be unmasked by potent sodium channel blockers such as flecainide, ajmaline, and procainamide,7 although the specificity of this effect for uncovering patients at risk for sudden death has been an issue of concern. A recent report by Remme et al8 has shown that the number of idiopathic ventricular fibrillation patients diagnosed as having Brugada syndrome is a sensitive function of the diagnostic criteria applied. What are the proper diagnostic criteria for identifying Brugada syndrome? A definitive answer to this question has been out of reach and is the reason for the establishment of a special Arrhythmia Working Group of the European Society of Cardiology that met from August 31 to …

Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists.

We have investigated the mechanisms of blockade of calcium channel current by the dihydropyridines, e.g. nisoldipine, nitrendipine, and nicardipine. Membrane current was recorded in isolated calf Purkinje fibers using a two-microelectrode voltage-clamp technique, and voltage protocols were designed to identify voltage- and use-dependent block by these compounds systematically. Our results show that calcium channel blockade by dihydropyridine derivatives is strongly modulated by membrane potential. Block is more pronounced when current is measured from depolarized holding potentials, but in contrast to verapamil, this voltage-dependent block occurs in the absence of repetitive depolarizations. Use-dependent block by dihydropyridines is observed at pulse frequencies greater than 1 Hz. Our results suggest that dihydropyridines bind preferentially to the inactivated state of the calcium channel, and that the development of usedependent block is related to the ionization constants of the compounds. Furthermore, binding is approximately one thousand times stronger to inactivated channels than to resting channels. This state-dependent difference in binding affinities may account for the previously reported contrast between electrophysiological and binding data for these compounds.

Long QT syndrome: from channels to cardiac arrhythmias

Long QT syndrome, a rare genetic disorder associated with life-threatening arrhythmias, has provided a wealth of information about fundamental mechanisms underlying human cardiac electrophysiology that has come about because of truly collaborative interactions between clinical and basic scientists. Our understanding of the mechanisms that control the critical plateau and repolarization phases of the human ventricular action potential has been raised to new levels through these studies, which have clarified the manner in which both potassium and sodium channels regulate this critical period of electrical activity.

Dependence of the GABAA receptor gating kinetics on the alpha‐subunit isoform: implications for structure‐function relations and synaptic transmission.

1. To examine the dependence of gamma‐aminobutyric acid (GABAA) receptor gating on the alpha‐subunit isoform, we studied the kinetics of GABA‐gated currents (IGABA) of receptors that differed in the alpha‐subunit subtype, alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S. cDNAs encoding rat brain subunits were co‐expressed heterologously in HEK‐293 cells and the resultant receptors studied with the whole‐cell patch clamp technique and rapidly applied GABA pulses (5‐10 s). 2. IGABA of both receptors showed a loosely similar dependence on GABA concentration over a wide range (1‐5000 microM). Generally, IGABA manifested activation reaching an early current peak, subsequent slower spontaneous desensitization, and deactivation of open channels at pulse termination. Lowering GABA concentrations reduced peak currents and slowed activation and desensitization kinetics. 3. The presence of alpha 3 altered the peak IGABA concentration‐response relationship by shifting the fitted Hill equation to tenfold greater GABA concentrations (GABA concentration at half amplitude: alpha 1, 7 microM; and alpha 3, 75 microM) without affecting Hill coefficients (alpha 1, 1.6; alpha 3, 1.5). These findings indicate a reduction in the apparent activating site affinity and are consistent with previous reports. 4. To investigate differences in gating, we normalized for apparent activating site affinities by analysing the time course of macroscopic gating at equi‐activating GABA concentrations. The presence of alpha 3 slowed activation fourfold (time to current peak (means +/‐ S.E.M.): alpha 1, 1.2 +/‐ 0.06 s (2 microM); alpha 3, 4.7 +/‐ 0.5 s (20 microM)), desensitization nearly twofold (reciprocal of time to 80% decay: alpha 1, 2.5 +/‐ 0.48 s‐1 (100 microM); alpha 3, 1.5 +/‐ 0.15 s‐1 (1000 microM)) and deactivation threefold (monoexponential decay time constant: alpha 1, 0.22 +/‐ 0.026 s (2 microM); alpha 3, 0.68 +/‐ 0.1 s (20 microM)). 5. To gain an insight into the gating mechanisms underlying macroscopic desensitization, we extended a previous gating model of GABAA receptor single‐channel activity to include a desensitization pathway. Such a mechanism reproduced empirical alpha 1 beta 2 gamma 2S activation, desensitization and deactivation kinetics. 6. To identify molecular transitions underlying the gating differences between alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S receptors, we explored parameter alterations of the alpha 1 beta 2 gamma 2S gating model that provided an accounting of alpha 3 beta 2 gamma 2S empirical responses. Remarkably, alteration of rates and rate constants involved in ligand binding alone allowed reproduction of alpha 3 beta 2 gamma 2S activation, desensitization and deactivation. 7. These results indicate that substitution of the alpha 3 subunit variant in an alpha 1 beta 2 gamma 2S receptor alters transition rates involved in ligand binding that underlie changes in apparent activating site affinity and macroscopic current gating. Furthermore, they argue strongly that the structural determinants of these functional features reside on the alpha‐subunit.

A Novel Channelopathy in Pulmonary Arterial Hypertension

BACKGROUND Pulmonary arterial hypertension is a devastating disease with high mortality. Familial cases of pulmonary arterial hypertension are usually characterized by autosomal dominant transmission with reduced penetrance, and some familial cases have unknown genetic causes. METHODS We studied a family in which multiple members had pulmonary arterial hypertension without identifiable mutations in any of the genes known to be associated with the disease, including BMPR2, ALK1, ENG, SMAD9, and CAV1. Three family members were studied with whole-exome sequencing. Additional patients with familial or idiopathic pulmonary arterial hypertension were screened for the mutations in the gene that was identified on whole-exome sequencing. All variants were expressed in COS-7 cells, and channel function was studied by means of patch-clamp analysis. RESULTS We identified a novel heterozygous missense variant c.608 G→A (G203D) in KCNK3 (the gene encoding potassium channel subfamily K, member 3) as a disease-causing candidate gene in the family. Five additional heterozygous missense variants in KCNK3 were independently identified in 92 unrelated patients with familial pulmonary arterial hypertension and 230 patients with idiopathic pulmonary arterial hypertension. We used in silico bioinformatic tools to predict that all six novel variants would be damaging. Electrophysiological studies of the channel indicated that all these missense mutations resulted in loss of function, and the reduction in the potassium-channel current was remedied by the application of the phospholipase inhibitor ONO-RS-082. CONCLUSIONS Our study identified the association of a novel gene, KCNK3, with familial and idiopathic pulmonary arterial hypertension. Mutations in this gene produced reduced potassium-channel current, which was successfully remedied by pharmacologic manipulation. (Funded by the National Institutes of Health.)

Characterization of Sodium Channel α- and β-Subunits in Rat and Mouse Cardiac Myocytes

Background—Sodium channels isolated from mammalian brain are composed of α-, β1-, and β2-subunits. The composition of sodium channels in cardiac muscle, however, has not been defined, and disagreement exists over which β-subunits are expressed in the myocytes. Some investigators have demonstrated β1 expression in heart. Others have not detected any auxiliary subunits. On the basis of Northern blot analysis of total RNA, β2 expression has been thought to be exclusive to neurons and absent from cardiac muscle. Methods and Results—The goal of this study was to define the subunit composition of cardiac sodium channels in myocytes. We show that cardiac sodium channels are composed of α-, β1-, and β2-subunits. Nav1.5 and Nav1.1 are expressed in myocytes and are associated with β1- and β2-subunits. Immunocytochemical localization of Nav1.1, β1, and β2 in adult heart sections showed that these subunits are expressed at the Z lines, as shown previously for Nav1.5. Coexpression of Nav1.5 with β2 in transfected cell...

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers.

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.

Cardiac Voltage-Gated Sodium Channel Nav1.5 Is Regulated by Nedd4-2 Mediated Ubiquitination

Nav1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Nav1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Nav1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Nav1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Nav1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Nav1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Nav1.5 and Nedd4-2. Nav1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Nav1.5. Consistent with the functional findings, localization at the cell periphery of Nav1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Nav1.5, suggesting that Nedd4-2 is a specific regulator of Nav1.5. These results demonstrate that Nav1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Nav1.5 by decreasing the channel density at the cell surface.

A Computational Model to Predict the Effects of Class I Anti-Arrhythmic Drugs on Ventricular Rhythms

Two- and three-dimensional models of cardiac excitability based on sodium channel kinetics can predict the adverse effects of class I anti-arrhythmic drugs. Crowdsourcing the Heart for Drug Screening The old way: Consult a specialist to answer your question. The new way: Consult a crowd of generalists who in the aggregate can come up with a better answer. The old way—testing drugs on single cardiac cells in vitro—has not worked well for screening out potential anti-arrhythmia agents that can occasionally block conduction in the heart or exacerbate arrhythmia, serious problems that cause sudden death in treated patients. Instead, Moreno et al. have called on the crowd by building a model of heart tissue that includes many cardiac cells and their interactions. When anti-arrhythmia drugs are “applied” to the model’s beating heart tissue—but not when they are applied to the single cardiac cells that make up the model—the drugs that cause side effects, and the concentrations at which they do so, are revealed, results that the authors were able to validate experimentally. The model starts with the detailed kinetics of the heart’s sodium channels, first in the context of a single cell, then in two- and three-dimensional cardiac tissue. The authors compared the action of lidocaine, a class 1B anti-arrhythmic drug not known to cause conduction block, and flecainide, a prototypical class 1C drug that carries a warning from the Food and Drug Administration. In the modeled analyses of single cardiac cells, both drugs slowed excitability at concentrations that matched those used in patients, but the cells retained the ability to generate action potentials. But when the model incorporated coupled groups of cells, the behavior of the drugs diverged. Lidocaine lowered excitability without causing block, but at the higher concentrations (used clinically), flecainide caused serious conduction block when heart rates reached 160 beats per minute. Experiments in rabbit heart confirmed the results of the model. In scaled-up, 500 by 500 groups of cells, the authors’ model could also successfully predict the tendency of flecainide, but not lidocaine, to make the heart extra sensitive to heartbeats occurring too early or too late, an effect that causes even more severe arrhythmias in patients when they take anti-arrhythmia drugs. Again, experiments in rabbit hearts replicated the model’s predictions, as did simulations of anatomically accurate human hearts derived from magnetic resonance imaging images. The ability of this sophisticated model of living cardiac tissue to replicate the clinical adverse effects of lidocaine and flecainide is promising, but it will be necessary to validate its performance with other drugs to understand how to deploy it most effectively. Ideally, such models will be useful for screening out potential arrhythmic drugs that promote conduction block or exacerbate arrhythmias. Such a view of how drugs affect the collective activity of cardiac cells should help in these situations in which the cure proves more deadly than the disease. A long-sought, and thus far elusive, goal has been to develop drugs to manage diseases of excitability. One such disease that affects millions each year is cardiac arrhythmia, which occurs when electrical impulses in the heart become disordered, sometimes causing sudden death. Pharmacological management of cardiac arrhythmia has failed because it is not possible to predict how drugs that target cardiac ion channels, and have intrinsically complex dynamic interactions with ion channels, will alter the emergent electrical behavior generated in the heart. Here, we applied a computational model, which was informed and validated by experimental data, that defined key measurable parameters necessary to simulate the interaction kinetics of the anti-arrhythmic drugs flecainide and lidocaine with cardiac sodium channels. We then used the model to predict the effects of these drugs on normal human ventricular cellular and tissue electrical activity in the setting of a common arrhythmia trigger, spontaneous ventricular ectopy. The model forecasts the clinically relevant concentrations at which flecainide and lidocaine exacerbate, rather than ameliorate, arrhythmia. Experiments in rabbit hearts and simulations in human ventricles based on magnetic resonance images validated the model predictions. This computational framework initiates the first steps toward development of a virtual drug-screening system that models drug-channel interactions and predicts the effects of drugs on emergent electrical activity in the heart.