Augustus O. Grant

Inherited Arrhythmias A National Heart, Lung, and Blood Institute and Office of Rare Diseases Workshop Consensus Report About the Diagnosis, Phenotyping, Molecular Mechanisms, and Therapeutic Approaches for Primary Cardiomyopathies of Gene Mutations Affecting Ion Channel Function

The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop (September 14 to 15, 2006, in Bethesda, Md) to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. These included the following: (1) Na+ channelopathies; (2) arrhythmias due to K+ channel mutations; and (3) arrhythmias due to other inherited arrhythmogenic mechanisms. Another major goal was to provide recommendations to support, enable, or facilitate research to improve future diagnosis and management of inherited arrhythmias. Classifications of electric heart diseases have proved to be exceedingly complex and in many respects contradictory. A new contemporary and rigorous classification of arrhythmogenic cardiomyopathies is proposed. This consensus report provides an important framework and overview to this increasingly heterogeneous group of primary cardiac membrane channel diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular biology and novel therapeutic approaches in cardiology, as well as the introduction of many recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy in consensus with a recent American Heart Association Scientific Statement.

Role of Mahaim fibers in cardiac arrhythmias in man.

Twelve patients with evidence of Mahaim fibers are reported, six with nodoventricular (NV) fibers and six with fasciculoventricular (FV) fibers. All patients with NV fibers had left bundle branch block morphology, and a sustained reentrant tachycardia with this morphology was proved in each case. In three of the six, ventriculoatrial dissociation occurred during tachycardia. We postulate that the mechanism of this tachycardia is a macroreentry circuit using the NV fiber for the antegrade limb and the His-Purkinje system with a portion of the atrioventricular node for the retrograde limb. ECGs of patients with FV fibers were varied, suggesting a functional relation to the right or left side of the septum. No direct relationship of FV fibers to observed arrhythmias could be found.

Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (DeltaK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and DeltaK1500 mutant channel. The mutation shifted V(1/2)h( infinity ) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h( infinity ) was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the DeltaK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, -1 and -2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the DeltaK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.

The electrophysiologic basis and management of symptomatic recurrent tachycardia in patients with ebstein's anomaly of the tricuspid valve

Twenty-two patients with Ebsteins anomaly were evaluated because of recurrent tachycardia. A total of 30 accessory pathways were present in 21 of the 22 patients. Twenty-six accessory pathways were of the atrioventricular (A-V) type while four were Mahaim fibers. Multiple accessory pathways were present in eight patients. Twenty-five of the 26 accessory A-V pathways were right-sided, either in the posterior septum (12 pathways) or the posterolateral free wall (13 pathways); one patient with corrected transposition of the great arteries had a left-sided accessory A-V pathway in a lateral free wall location. Patients with accessory A-V pathways had a long minimal ventriculoatrial (V-A) conduction time during reciprocating tachycardia (192 +/- 47 ms) and usually showed a persistent complete or incomplete right bundle branch block morphology. At surgery, preexcitation was invariably localized to the atrialized ventricle. The long V-A conduction time during reciprocating tachycardia appeared to consist of late activation of the local ventricle in the region of the accessory pathway with a further delay occurring before excitation of adjacent atrium presumably due to conduction over the accessory pathway. Accessory A-V pathways were successfully sectioned with no deaths in 13 of 15 patients. On the basis of these data, certain electrocardiographic findings encountered in the study of patients with recurrent tachycardia should point to the possibility of associated Ebsteins anomaly: morphology of the surface electrocardiogram suggesting preexcitation of the right posterior septum or right posterolateral free wall as well as the combination during reciprocating tachycardia of a long V-A interval and right bundle branch block.

Antiarrhythmic drug action. Blockade of the inward sodium current.

THE mechanisms of action of antiarrhythmic drugs were discussed in these reviews over a decade ago (Rosen and Hoffman, 1973). Since then, important new concepts of the blocking action of drugs have been proposed and tested experimentally. Several attempts at a quantitative voltage clamp analysis of the blocking action of antiarrhythmic drugs have been made. The mechanism of the blocking action of local anesthetics has been studied in nerves to the limit of current electrical techniques—that of gating currents (Cahalan, 1980; Yeh, 1982). The volume of data dictates that we restrict the studies covered. We shall focus on the mechanism of the blockade of the sodium current. We neglect discussion of many otherwise important studies which do not have this focus. Knowledge of the number, kinetics, and relative importance of the individual pacemaker currents is sufficiently incomplete as to limit a discussion on the drug action on the individual currents. Blockade of membrane sodium conductance (GNa) is probably a major mechanism of action of antiarrhythmic drugs. To obtain a quantitative analysis of the kinetic and steady state effects of drugs on the sodium conductance, it is necessary to measure the sodium current, INa, in a stable system with the temperature and extracellular milieu that may be obtained in both normal and diseased tissues in vivo. The strategies that have been exploited for the study of INa in heart muscle include: (1) indirect estimation from Vmax of action potentials, (2) direct measurement of macroscopic sodium currents in multicellular and isolated cell preparations under voltage clamp, and (3) direct recording of unitary sodium conductance using the extracellular patch clamp technique. All these techniques use measurement of electrical properties to assess the binding kinetics of drugs to their receptor site(s). The quantitative precision of these techniques may vary. However, it is important to establish their ability to produce qualitatively similar results.

Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

Optical measurements of transmembrane potential changes during electric field stimulation of ventricular cells.

We evaluated transmembrane potential changes at the ends of isolated rabbit ventricular myocytes during defibrillation-strength shocks given in the cellular refractory period. The myocytes were stimulated (S1 pulse) to produce an action potential. Then a constant-field shock (S2 pulse) with an electric field of 20 or 40 V/cm was given at an S1-S2 interval of 50 msec. The cells were stained with potentiometric dye (di-4-ANEPPS), and the cell end facing the S2 anode or cathode was illuminated with a laser while the fluorescence was recorded. During S2, the cell end facing the S2 cathode became more positive intracellularly, whereas the cell end facing the S2 anode became more negative intracellularly. The S2-induced transmembrane potential change at the cell end (delta Vm) was determined relative to the amplitude of the S1-induced action potential (APA) in each recording (i.e., delta Vm/APA). In Tyrodes solution containing 4.5 mM potassium, delta Vm/APA for 40-V/cm S2 was 1.36 +/- 0.34 at the cell end facing the S2 cathode and -1.65 +/- 0.61 at the cell end facing the S2 anode (n = 9). For the 20-V/cm S2, delta Vm/APA was 0.61 +/- 0.33 at the cell end facing the S2 cathode and -0.71 +/- 0.33 at the cell end facing the S2 anode (n = 6). The delta Vm/APA was not significantly influenced by 20 mM diacetyl monoxime. These results indicate that large delta Vm values occurred at the ends of the cells during S2. The calculated values of delta Vm, assuming a nominal APA of 130 mV, were 177 and -214 mV for the 40-V/cm S2 and 79 and -93 mV for the 20-V/cm S2. The delta Vm was correlated with cell size (r > or = 0.95) and agreed with values predicted by the S2 electric field strength multiplied by half of the cell length to within 27%. When the potassium concentration was increased to 20 mM, delta Vm/APA for 40 V/cm S2 increased 85% and 67% at the cell ends facing the S2 cathode and anode, respectively (n = 9, p < 0.005 versus 4.5 mM potassium), consistent with reduced APA. Thus, with normal or elevated extracellular potassium, transmembrane potential changes at the ends of cells during defibrillation-type stimulation are large enough to produce activation or recovery of voltage-dependent ion channels and may produce the effects responsible for defibrillation.

Proarrhythmic response to sodium channel blockade. Theoretical model and numerical experiments.

BackgroundThe use of flecainide and encainide was terminated in the Cardiac Arrhythmia Suppression Trial because of an excess of sudden cardiac deaths in the active treatment group. Such events might arise from reentrant rhythms initiated by premature stimulation in the presence of anisotropic sodium channel availability. Drugs that bind to sodium channels increase the functional dispersion of refractoriness by slowing (a result of the drug-unbinding process) the transition from an inexcitable state to an excitable state. It is interesting that encainide and flecainide unbind slowly (15–20 seconds), whereas lidocaine and moricizine unbind rapidly (0.2–1.3 seconds). Methods and ResultsWith a computer representation of a cable with Beeler-Reuter membrane properties, we found a small (6 msec) vulnerable window that occurred 338 msec after the last drive stimulus. Premature stimuli falling within the vulnerable window resulted in unidirectional block and reentrant activation. In the presence of a slowly unbinding drug, the window was delayed an additional 341 msec, and its duration was extended to 38 msec. The delay (antiarrhythmic effect) before the onset of the vulnerable window and its duration (proarrhythmic effect) were both dependent on the sodium channel availability and the recovery process. Both effects were also prolonged when sodium channel availability was reduced by membrane depolarization. Defining the proarrhythmic potential as the duration of the vulnerable window, we found that hypothetical use-dependent class I drugs have a greater proarrhythmic potential than non-use-dependent drugs. ConclusionsThe antiarrhythmic and proarrhythmic properties of pure sodium channel antagonists are both dependent on sodium channel availability. Consequently, the price for increased antiarrhythmic efficacy (suppressed premature ventricular contractions) is an increased proarrhythmic vulnerability to unsuppressed premature ventricular contractions.

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.

Use of the Esophageal Lead in the Diagnosis of Mechanisms of Reciprocating Supraventricular Tachycardia

Recent studies have emphasized the role of concealed accessory pathways in reciprocating supraventricular tachycardia. Diagnosis has generally required multicatheter electrophysiologic study. We recorded esophageal electrograms during study in 16 patients with reciprocating tachycardia due to reentry using an accessory alriovenlricular pathway, and in 12 patients with reciprocating tachycardia due to reentry in the AV node. The interval from onset of ventricular depolarization to earliest atrial activation (V‐AMIN), ear‐liest atrial activity on the esophageal lead (V‐AESO).and high right atrium (V‐HRA) was measured. No patient with RT due to an accessory atrioventricular pathway had a V‐AMIN or V‐AESO less than 70 ms, or a V‐HRA less than 95 ms. In contrast, 11 of 12 patients with reentry in the AV node had V‐Aggo intervals less than 70 ms. Esophageol recording during reciprocating tachycardia provides a simple screening procedure available to all practicing physicians to exclude the diagnosis of accessory atrioventricular pathways in the genesis of paroxysmal supraventricular tachycardia.