Eleonora Grandi

A Novel Computational Model of the Human Ventricular Action Potential and Ca Transient

We have developed a detailed mathematical model for Ca handling and ionic currents in the human ventricular myocyte. Our aims were to: (1) simulate basic excitation-contraction coupling phenomena; (2) use realistic repolarizing K current densities; (3) reach steady-state. The model relies on the framework of the rabbit myocyte model previously developed by our group, with subsarcolemmal and junctional compartments where ion channels sense higher [Ca] vs. bulk cytosol. Ion channels and transporters have been modeled on the basis of the most recent experimental data from human ventricular myocytes. Rapidly and slowly inactivating components of I(to) have been formulated to differentiate between endocardial and epicardial myocytes. Transmural gradients of Ca handling proteins and Na pump were also simulated. The model has been validated against a wide set of experimental data including action potential duration (APD) adaptation and restitution, frequency-dependent increase in Ca transient peak and [Na](i). Interestingly, Na accumulation at fast heart rate is a major determinant of APD shortening, via outward shifts in Na pump and Na-Ca exchange currents. We investigated the effects of blocking K currents on APD and repolarization reserve: I(Ks) block does not affect the former and slightly reduces the latter; I(K1) blockade modestly increases APD and more strongly reduces repolarization reserve; I(Kr) blockers significantly prolong APD, an effect exacerbated as pacing frequency is decreased, in good agreement with experimental results in human myocytes. We conclude that this model provides a useful framework to explore excitation-contraction coupling mechanisms and repolarization abnormalities at the single myocyte level.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late INa Augmentation Leading to Cellular Na and Ca Overload

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

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}

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}

Calcium/calmodulin-dependent kinase II regulation of cardiac ion channels.

Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase expressed abundantly in the heart. CaMKII targets numerous proteins involved in excitation-contraction coupling and excitability, and its activation may simultaneously contribute to heart failure and cardiac arrhythmias. In this review, we summarize the modulatory effects of CaMKII on cardiac ion channel function and expression and illustrate potential implications in the onset of arrhythmias via a computer model.

Ca/Calmodulin Kinase II Differentially Modulates Potassium Currents

Background—Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression. Methods and Results—We examine the influence of overexpressing cytoplasmic CaMKII&dgr;C, both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKII&dgr;C-transgenic mice, on transient outward potassium current (Ito), and inward rectifying current (IK1). Acute and chronic CaMKII overexpression increases Ito,slow amplitude and expression of the underlying channel protein KV1.4. Chronic but not acute CaMKII overexpression causes downregulation of Ito,fast, as well as KV4.2 and KChIP2, suggesting that KV1.4 expression responds faster and oppositely to KV4.2 on CaMKII activation. These amplitude changes were not reversed by CaMKII inhibition, consistent with CaMKII-dependent regulation of channel expression and/or trafficking. CaMKII (acute and chronic) greatly accelerated recovery from inactivation for both Ito components, but these effects were acutely reversed by AIP (CaMKII inhibitor), suggesting that CaMKII activity directly accelerates Ito recovery. Expression levels of IK1 and Kir2.1 mRNA were downregulated by CaMKII overexpression. CaMKII acutely increased IK1, based on inhibition by AIP (in both models). CaMKII overexpression in mouse prolonged APD (consistent with reduced Ito,fast and IK1), whereas CaMKII overexpression in rabbit shortened APD (consistent with enhanced IK1 and Ito,slow and faster Ito recovery). Computational models allowed discrimination of contributions of different channel effects on APD. Conclusion—CaMKII has both acute regulatory effects and chronic expression level effects on Ito and IK1 with complex consequences on APD.

Late Sodium Current Contributes to the Reverse Rate-Dependent Effect of IKr Inhibition on Ventricular Repolarization

Background— The reverse rate dependence (RRD) of actions of IKr-blocking drugs to increase the action potential duration (APD) and beat-to-beat variability of repolarization (BVR) of APD is proarrhythmic. We determined whether inhibition of endogenous, physiological late Na+ current (late INa) attenuates the RRD and proarrhythmic effect of IKr inhibition. Methods and Results— Duration of the monophasic APD (MAPD) was measured from female rabbit hearts paced at cycle lengths from 400 to 2000 milliseconds, and BVR was calculated. In the absence of a drug, duration of monophasic action potential at 90% completion of repolarization (MAPD90) and BVR increased as the cycle length was increased from 400 to 2000 milliseconds (n=36 and 26; P<0.01). Both E-4031 (20 nmol/L) and d-sotalol (10 &mgr;mol/L) increased MAPD90 and BVR at all stimulation rates, and the increase was greater at slower than at faster pacing rates (n=19, 11, 12 and 7, respectively; P<0.01). Tetrodotoxin (1 &mgr;mol/L) and ranolazine significantly attenuated the RRD of MAPD90, reduced BVR (P<0.01), and abolished torsade de pointes in hearts treated with either 20 nmol/L E-4031 or 10 &mgr;mol/L d-sotalol. Endogenous late INa in cardiomyocytes stimulated at cycle lengths from 500 to 4000 milliseconds was greater at slower than at faster stimulation rates, and rapidly decreased during the first several beats at faster but not at slower rates (n=8; P<0.01). In a computational model, simulated RRD of APD caused by E-4031 and d-sotalol was attenuated when late INa was inhibited. Conclusion— Endogenous late INa contributes to the RRD of IKr inhibitor–induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.

Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.

Ranolazine for Congenital and Acquired Late INa-Linked Arrhythmias In Silico Pharmacological Screening

Rationale: The 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. Objective: We 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 Results: We 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 &Dgr;KPQ 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. Conclusions: Our 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.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late I Na Augmentation Leading to Cellular Na and Ca Overload

Rationale: In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective: We tested whether CaMKII&dgr; is required for ROS-dependent late INa regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results: 40 &mgr;mol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 &mgr;mol/L), the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKII&dgr;−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKII&dgr;−/− myocytes (versus WT). CaMKII&dgr;−/− myocytes developed significantly less H2O2-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKII&dgr; in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 &mgr;mol/L KN93 or 0.1 &mgr;mol/L AIP [autocamtide 2–related inhibitory peptide]). Conclusions: Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKII&dgr; enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.