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RationaleThe antianginal ranolazine blocks the human ether-a-go-go-related gene-based current IKr at therapeutic concentrations and causes QT interval prolongation. Thus, ranolazine is contraindicated for patients with preexisting long-QT and those with repolarization abnormalities. However, with its preferential targeting of late INa (INaL), patients with disease resulting from increased INaL from inherited defects (eg, long-QT syndrome type 3 or disease-induced electric remodeling (eg, ischemic heart failure) might be exactly the ones to benefit most from the presumed antiarrhythmic properties of ranolazine.ObjectiveWe developed a computational model to predict if therapeutic effects of pharmacological targeting of INaL by ranolazine prevailed over the off-target block of IKr in the setting of inherited long-QT syndrome type 3 and heart failure.Methods and resultsWe developed computational models describing the kinetics and the interaction of ranolazine with cardiac Na(+) channels in the setting of normal physiology, long-QT syndrome type 3-linked ΔKPQ mutation, and heart failure. We then simulated clinically relevant concentrations of ranolazine and predicted the combined effects of Na(+) channel and IKr blockade by both the parent compound ranolazine and its active metabolites, which have shown potent blocking effects in the therapeutically relevant range. Our simulations suggest that ranolazine is effective at normalizing arrhythmia triggers in bradycardia-dependent arrhythmias in long-QT syndrome type 3 as well tachyarrhythmogenic triggers arising from heart failure-induced remodeling.ConclusionsOur model predictions suggest that acute targeting of INaL with ranolazine may be an effective therapeutic strategy in diverse arrhythmia-provoking situations that arise from a common pathway of increased pathological INaL.

Long QT syndrome (LQTS) type 3 (LQT3), typified by the DeltaKPQ mutation (LQT3 mutation in which amino acid residues 1505 to 1507 [KPQ] are deleted), is caused by increased sodium entry during the action potential plateau resulting from mutation-altered inactivation of the Na(v)1.5 channel. Although rare, LQT3 is the most lethal of common LQTS variants. Here we tested the hypothesis that cellular electrical dysfunction, caused not only by action potential prolongation but also by mutation-altered Na(+) entry, distinguishes LQT3 from other LQTS variants and may contribute to its distinct lethality. We compared cellular electrical activity in myocytes isolated from mice heterozygous for the DeltaKPQ mutation (DeltaKPQ) and myocytes from wild-type littermates. Current-clamp pause protocols induced rate-dependent spontaneous diastolic activity (delayed after depolarizations) in 6 of 7 DeltaKPQ, but no wild-type, myocytes (n=11) tested. Voltage-clamp pause protocols that independently control depolarization duration and interpulse interval identified a distinct contribution of both depolarization duration and mutant Na(+) channel activity to the generation of Ca(i)(2+)-dependent diastolic transient inward current. This was found at rates and depolarization durations relevant both to the mouse model and to LQT3 patients. Flecainide, which preferentially inhibits mutation-altered late Na(+) current and is used to treat LQT3 patients, suppresses transient inward current formation in voltage-clamped DeltaKPQ myocytes. Our results demonstrate a marked contribution of mutation-altered Na(+) entry to the incidence of pause-dependent spontaneous diastolic activity in DeltaKPQ myocytes and suggest that altered Na(+) entry may contribute to the elevated lethality of LQT3 versus other LQTS variants.

Na(v)1.5, 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 Na(v)1.5 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 Na(v)1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na(v)1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na(v)1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na(v)1.5 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 Na(v)1.5 and Nedd4-2. Na(v)1.5 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 Na(v)1.5. Consistent with the functional findings, localization at the cell periphery of Na(v)1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na(v)1.5, suggesting that Nedd4-2 is a specific regulator of Na(v)1.5. These results demonstrate that Na(v)1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na(v)1.5 by decreasing the channel density at the cell surface.