Simulation and Mechanistic Investigation of the Arrhythmogenic Role of the Late Sodium Current in Human Heart Failure

Heart failure constitutes a major public health problem worldwide. The electrophysiological remodeling of failing hearts sets the stage for malignant arrhythmias, in which the role of the late Na+ current (INaL) is relevant and is currently under investigation. In this study we examined the role of INaL in the electrophysiological phenotype of ventricular myocytes, and its proarrhythmic effects in the failing heart. A model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of INaL. A sensitivity analysis of the model was performed and simulations of the pathological electrical activity of the cell were conducted. The proposed model for the human INaL and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. The sensitivity analysis of the modulation of electrophysiological parameters of myocytes from failing hearts due to ion channels remodeling, revealed a role for INaL in the prolongation of action potential duration (APD), triangulation of the shape of the AP, and changes in Ca2+ transient. A mechanistic investigation of intracellular Na+ accumulation and APD shortening with increasing frequency of stimulation of failing myocytes revealed a role for the Na+/K+ pump, the Na+/Ca2+ exchanger and INaL. The results of the simulations also showed that in failing myocytes, the enhancement of INaL increased the reverse rate-dependent APD prolongation and the probability of initiating early afterdepolarizations. The electrophysiological remodeling of failing hearts and especially the enhancement of the INaL prolong APD and alter Ca2+ transient facilitating the development of early afterdepolarizations. An enhanced INaL appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of [Ca2+]i homeostasis of failing myocytes

Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIδC can be reversed by inhibition of late Na+ current

Transgenic (TG) Ca2+/calmodulin-dependent protein kinase II (CaMKII) δC mice develop systolic heart failure (HF). CaMKII regulates intracellular Ca2+ handling proteins as well as sarcolemmal Na+ channels. We hypothesized that CaMKII also contributes to diastolic dysfunction and arrhythmias via augmentation of the late Na+ current (late INa) in early HF (8-week-old TG mice). Echocardiography revealed severe diastolic dysfunction in addition to decreased systolic ejection fraction. Premature arrhythmogenic contractions (PACs) in isolated isometrically twitching papillary muscles only occurred in TG preparations (5 vs. 0, P < 0.05) which could be completely terminated when treated with the late INa inhibitor ranolazine (Ran, 5 μmol/L). Force–frequency relationships revealed significantly reduced twitch force amplitudes in TG papillary muscles. Most importantly, diastolic tension increased with raising frequencies to a greater extent in TG papillary muscles compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm2; P < 0.05). Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm2 (at 10 Hz; P < 0.05) without negative inotropic effects. Mechanistically, the late INa was markedly elevated in myocytes isolated from TG mice and could be completely reversed by Ran. In conclusion, our results show for the first time that TG CaMKIIδC overexpression induces diastolic dysfunction and arrhythmogenic triggers possibly via an enhanced late INa. Inhibition of elevated late INa had beneficial effects on arrhythmias as well as diastolic function in papillary muscles from CaMKIIδC TG mice. Thus, late INa inhibition appears to be a promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is found to be increased

Differences in MEF2 and NFAT Transcriptional Pathways According to Human Heart Failure Aetiology

BACKGROUND:Ca(2+) handling machinery modulates the activation of cardiac transcription pathways involved in heart failure (HF). The present study investigated the effect of HF aetiology on Ca(+2) handling proteins and NFAT1, MEF2C and GATA4 (transcription factors) in the same cardiac tissue. METHODOLOGY AND PRINCIPAL FINDINGS:A total of 83 hearts from ischemic (ICM, n = 43) and dilated (DCM, n = 31) patients undergoing heart transplantation and controls (CNT, n = 9) were analyzed by western blotting. Subcellular distribution was analyzed by fluorescence and electron microscopy. When we compared Ca(+2) handling proteins according to HF aetiology, ICM showed higher levels of calmodulin (24%, p<0.01), calcineurin (26%, p<0.01) and Ca(2+)/Calmodulin-dependent kinase II (CaMKIIδ(b) nuclear isoform 62%, p<0.001) than the CNT group. However, these proteins in DCM did not significantly increase. Furthermore, ICM showed a significant elevation in MEF2C (33%, p<0.01), and GATA4 (49%, p<0.05); also NFAT1 (66%, p<0.001) was increased, producing the resultant translocation of this transcriptional factor into the nuclei. These results were supported by fluorescence and electron microscopy analysis. Whereas, DCM only had a significant increase in GATA4 (52%, p<0.05). Correlations between NFAT1 and MEF2C in both groups (ICM r = 0.38 and DCM r = 0.59, p<0.05 and p<0.01, respectively) were found; only ICM showed a correlation between GATA4 and NFAT1 (r = 0.37, p<0.05). CONCLUSIONS/SIGNIFICANCE:This study shows an increase of Ca(2+) handling machinery synthesis and their cardiac transcription pathways in HF, being more markedly increased in ICM. Furthermore, there is a significant association between MEF2, NFAT1 and GATA4. These proteins could be therapeutic targets to improve myocardial function

Regulatory Effect of Connexin 43 on Basal Ca2+ Signaling in Rat Ventricular Myocytes

Background: It has been found that gap junction-associated intracellular Ca 2+ [Ca 2+]i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca 2+ signaling, in particular the basal [Ca 2+] i activities, is unclear. Methods and Results: Global and local Ca 2+ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca 2+ transients and local Ca 2+ sparks in monolayer NRVMs and Ca 2+ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP3 butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca 2+ signal and LY uptake by gap uncouplers, whereas blockade of IP 3 receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca 2+ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibod

Characterization of Multiple Ion Channels in Cultured Human Cardiac Fibroblasts

Background: Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. Methods and Findings: A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 μM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR). Conclusions: These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. © 2009 Li et al.published_or_final_versio

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200–400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca2+ influx via voltage-gated L-type Ca2+ channels activates Ca2+ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca2+]i transients in hESC-CMs required Ca2+ entry through L-type Ca2+ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca2+ or treatment with diltiazem, an L-type channel inhibitor. Ca2+ release from the SR also contributes to the [Ca2+]i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca2+ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca2+ currents (ICa) and corresponding whole-cell [Ca2+]i transients in hESC-CMs and hFVMs, and the amplitude of both ICa and the [Ca2+]i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca2+]i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca2+ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca2+ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca2+ release by the ICa during EC coupling develops early in human cardiomyocytes

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca2+) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca2+ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects

Modeling CICR in rat ventricular myocytes: voltage clamp studies

<p>Abstract</p> <p>Background</p> <p>The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect <it>Ca</it>²⁺loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR <it>Ca</it>²⁺release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic <it>Ca</it>²⁺concentration ([<it>Ca</it>²⁺]<it>_myo</it>).</p> <p>Methods</p> <p>The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed <it>Ca</it>²⁺channels (trigger-channel and release-channel). It releases <it>Ca</it>²⁺flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[<it>Ca</it>²⁺]<it>_myo</it>.</p> <p>Results</p> <p>Our model reproduces measured VC data published by several laboratories, and generates graded <it>Ca</it>²⁺release at high <it>Ca</it>²⁺gain in a homeostatically-controlled environment where [<it>Ca</it>²⁺]<it>_myo</it>is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR <it>Ca</it>²⁺release, its activation by trigger <it>Ca</it>²⁺, and its refractory characteristics mediated by the luminal SR <it>Ca</it>²⁺sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence <it>Ca</it>²⁺homeostasis.</p> <p>Conclusions</p> <p>We examine the role of the above <it>Ca</it>²⁺regulating mechanisms in handling various types of induced disturbances in <it>Ca</it>²⁺levels by quantifying cellular <it>Ca</it>²⁺balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.</p

Electrophysiological and Structural Remodeling in Heart Failure Modulate Arrhythmogenesis. 1D Simulation Study

Background: Heart failure is a final common pathway or descriptor for various cardiac pathologies. It is associated with sudden cardiac death, which is frequently caused by ventricular arrhythmias. Electrophysiological remodeling, intercellular uncoupling, fibrosis and autonomic imbalance have been identified as major arrhythmogenic factors in heart failure etiology and progression. Objective: In this study we investigate in silico the role of electrophysiological and structural heart failure remodeling on the modulation of key elements of the arrhythmogenic substrate, i.e., electrophysiological gradients and abnormal impulse propagation. Methods: Two different mathematical models of the human ventricular action potential were used to formulate models of the failing ventricular myocyte. This provided the basis for simulations of the electrical activity within a transmural ventricular strand. Our main goal was to elucidate the roles of electrophysiological and structural remodeling in setting the stage for malignant life-threatening arrhythmias. Results: Simulation results illustrate how the presence of M cells and heterogeneous electrophysiological remodeling in the human failing ventricle modulate the dispersion of action potential duration and repolarization time. Specifically, selective heterogeneous remodeling of expression levels for the Na+ /Ca2+ exchanger and SERCA pump decrease these heterogeneities. In contrast, fibroblast proliferation and cellular uncoupling both strongly increase repolarization heterogeneities. Conduction velocity and the safety factor for conduction are also reduced by the progressive structural remodeling during heart failure. Conclusion: An extensive literature now establishes that in human ventricle, as heart failure progresses, gradients for repolarization are changed significantly by protein specific electrophysiological remodeling (either homogeneous or heterogeneous). Our simulations illustrate and provide new insights into this. Furthermore, enhanced fibrosis in failing hearts, as well as reduced intercellular coupling, combine to increase electrophysiological gradients and reduce electrical propagation. In combination these changes set the stage for arrhythmias.This work was partially supported by (i) the "VI Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica" from the Ministerio de Economia y Competitividad of Spain (grant number TIN2012-37546-C03-01) and the European Commission (European Regional Development Funds - ERDF - FEDER), (ii) the Direccion General de Politica Cientifica de la Generalitat Valenciana (grant number GV/2013/119), and (iii) Programa Prometeo (PROMETEO/2012/030) de la Conselleria d'Educacio Formacio I Ocupacio, Generalitat Valenciana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Gómez García, JF.; Cardona, K.; Romero Pérez, L.; Ferrero De Loma-Osorio, JM.; Trénor Gomis, BA. (2014). Electrophysiological and Structural Remodeling in Heart Failure Modulate Arrhythmogenesis. 1D Simulation Study. PLoS ONE. 9(9). https://doi.org/10.1371/journal.pone.0106602S9