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Review

Physiological roles of the transient outward current Ito in normal and diseased hearts

Jonathan M.Cordeiro1, Kirstine Calloe2, Roozbeh Aschar-Sobbi3, Kyoung-Han Kim4, Adam Korogyi3, Dona Occhipinti1, Peter H. Backx3, Brian K. Panama1,*
1
Department of Experimental Cardiology, Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, New York, 13501 USA
2
Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Dyrlægevej 100, 1870 Frederiksberg C, Denmark
3
Departments of Physiology and Medicine, University Health Network, University of Toronto. 1 King’s College Circle, Toronto, Ontario, M5S 1A8 Canada
4
Program in Developmental & Stem Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada
DOI: 10.2741/S454 Volume 8 Issue 1, pp.143-159
Published: 01 January 2016
*Corresponding Author(s):  
Brian K. Panama
E-mail:  
panamab@mmrl.edu
Abstract

The Ca2+-independent transient outward K+ current (Ito) plays a critical role in underlying phase 1 of repolarization of the cardiac action potential and, as a result, is central to modulating excitation-contraction coupling and propensity for arrhythmia. Additionally, Ito and its molecular constituents are consistently reduced in cardiac hypertrophy and heart failure. In this review, we discuss the physiological role of Ito as well as the molecular basis of this current in human and canine hearts, in which Ito has been thoroughly studied. In particular, we discuss the role of Ito in the action potential and the mechanisms by which Ito modulates excitation-contraction coupling. We also describe the effects of mutations in the subunits constituting the Ito channel as well as the role of Ito in the failing myocardium. Finally, we review pharmacological modulation of Ito and discuss the evidence supporting the hypothesis that restoration of Ito in the setting of heart failure may be therapeutically beneficial by enhancing excitation-contraction coupling and cardiac function.

Key words

Arrhythmia, Calcium, Heart Failure, Potassium Channel, Review

2. Introduction

The Ca2+-independent transient outward K+ current (Ito), also referred to as A-type current in neuronal tissue, plays a critical role in shaping the action potential (AP). Ito is observed in atrial and ventricular myocytes as well as Purkinje fibers of mammals including human (1, 2), dog (3, 4), feline (5), ferret (6), rabbit (7), rat (8, 9) and mouse (10). In larger mammals,Ito underlies the early phase of repolarization of the cardiac action potential (AP), and growing evidence suggests that Ito plays a key role in excitation-contraction (EC) coupling (11, 12), as well as in arrhythmogenesis (13). In addition, Ito and its molecular constituents are consistently reduced in cardiac hypertrophy and heart failure (1, 14).This review will highlight the physiological role of Ito as well as the molecular basis of this current in human and canine hearts. We will also discuss the role of Ito in the normal and failing myocardium, as well as under various pathophysiological conditions, such as the Brugada syndrome. Finally, we will discuss pharmaceutical modulation of Ito and how restoration of Ito in the setting of heart failure may be therapeutically beneficial.

3. The action potential and role of Ito

The AP in cardiac muscle is critical for initiating and coordinating contraction of the working myocardium (15) while also minimizing arrhythmias via its relatively long refractory period (16, 17). The AP is generated by highly coordinate changes in different ionic conductances. In the human (or canine) heart, the AP is divided into five phases, 0 through 4 (Figure 1). In ventricular myocytes or Purkinje fibers, the initial depolarization (upstroke) to approximately +35 mV (Phase 0) is generated by Na+ influx (INa), from coordinated opening of voltage-gated sodium channels (18) (Figure 1). The brief repolarization (Phase 1), or “notch,” following the upstroke is due primarily to a Ca2+-independent (fast) transient outward K+ current (Ito) (8, 19). Ito can be divided into two separate currents: Ito, f (fast) and Ito, s (slow), based upon their recovery times from inactivation. In addition, Ca2+-activated Cl-currents have also been thought to contribute to the Phase 1 repolarization (20, 21). The plateau (Phase 2) of the AP arises from a delicate balance between K+ efflux through voltage-gated potassium channels and Ca2+ influx through voltage-gated calcium channels (ICa). In the late repolarization phase (Phase 3), the balance of ionic currents is shifted in favor of repolarization by the progressive increase in slow delayed rectifier potassium channels (IKs) due to slow activation as well as in the rapid delayed rectifier potassium channels (IKr) which has a unique rapid recovery from inactivation coupled with slow deactivation as the membrane potential becomes more negative. In the late phase 3, the K+ conductance increases further due to the unblocking of inwardly rectifying potassium channels (IK1), which are required to stabilize the resting membrane potential following full repolarization (Phase 4) (22). Marked differences in the shape and duration of the cardiac AP have been described in different regions of the mammalian heart, reflecting differences in the underlying ionic currents (7, 8, 23, 24). In humans and canines, the ventricular epicardium (Epi) tissue has a more prominent Ito compared to endocardial (Endo) tissue (7, 25, 26) (Figure 1). This transmural gradient of Ito results in the classical spike-and-dome morphology of Epi and midmyocardial (Mid) APs that is absent in Endo cells. In humans and canines, the Epi Ito is predominantly Ito, f whereas Ito, s is predominantly expressed in the Endo (1, 27). Besides transmural differences in phase 1 repolarization, there are marked differences in AP duration (APD), with long APs in Endo and Mid, whereas the APD is short in Epi (28).

Figure 1. A cartoon depicting APs (Panel A) and underlying ionic currents (Panel B) from a canine left ventricular Endo and Epi cell. The time courses of ionic currents are partly based on recordings from previous publications (87,122-124).

4. Physiological role of Ito

4.1. AP waveform and the initiation of Ca2+ transients

Although much attention has focused on the electrical distinctions between the diverse ventricular cells types, it is clear the mechanical properties also vary across the ventricular wall (11, 12, 29). Coordinated contraction of the ventricle is necessary for the efficient ejection of blood to the body. For synchronization of ventricular contraction to occur one would anticipate that all areas of muscle are electrically excited at the same time (30, 31), however this is not the case. In dog, as in human, the electrical impulse distributed through the His-Purkinje system excites predominantly Endo. The wave of excitation must then traverse the ventricular muscle to excite the Mid and Epi layers. Although the Epi is the last region to be excited, it is the first region to repolarize. This observation is reflected by the positive T wave in the canine wedge model electrocardiogram (28). Studies have shown that the transmural conduction time is about 30-40 ms in the canine left ventricle (28). In spite of this difference in transmural conduction, all parts of the ventricle contract in unison. These observations suggest there are regional differences in cardiac excitation-contraction coupling must compensate for the inherent electrical time delays. Differences in AP waveform and duration, which are associated with variations in Ca2+ transient and Ca2+ homeostasis between Epi, Mid and Endo cells, are hypothesized to be important contributing factors for the appropriate synchronization of ventricular contraction (12) (Figure 2).

Figure 2. Panel A: Representative APs (top traces) and corresponding normalized Ca2+ transients (bottom traces) recorded from an Epi, Endo and Mid cell. Myocytes were paced at a cycle length of 2 s. The corresponding Ca2+ transients (lower trace) show that Endo cells have a slower time-to-peak as well as a slower decay of the Ca2+ transient. The tick marks on the Ca2+ transient traces denote the peak. Panel B: Representative APs (top) and corresponding unloaded cell shortening (bottom) recorded from and Epi, Endo and Mid cells. Myocytes were paced at a cycle length of 2 s. AP recorded from both Epi and Mid cells exhibit a rapid phase 1 repolarization and prominent spike and dome configuration compared to Endo cells. The latency to onset of contraction was relatively short in Epi and Mid cells and time-to-peak was longest in Endo cells. Modified with permission from (29).

The Ca2+ transient is a dynamic process governed by Ca2+ influx and release as well as intracellular Ca2+ extrusion and re-uptake. Depolarization of the cardiac membrane results in influx of Ca2+ and subsequent release of Ca2+ from the SR, the process of EC coupling. There are two sources of Ca2+ that contribute to this rise in intracellular Ca2+: A) influx of Ca2+ across the sarcolemma via various pathways (32) and B) a release of Ca2+ from the sarcoplasmic reticulum (SR) (33-35). The classic model of excitation-contraction suggests that Ca2+ enters the cardiac cell via L-type Ca2+ channels and triggers a larger release of Ca2+ from the SR, a process referred to as “Ca2+-induced Ca2+ release” (32, 36). Although the main source of trigger calcium is from L-type Ca2+ channels (37), activation of T-type Ca2+ current (38), and the Na+-Ca2+ exchange operating in ‘reverse-mode’ (39, 40) are capable of initiating SR Ca2+ release to cause a contraction. The small, local increases in intracellular Ca2+ from ventricular cells (Ca2+ sparks; reviewed by (41)) are the result of brief, spontaneous openings from clusters of ryanodine receptors (RyRs) and are believed to be the elementary event underlying EC coupling in cardiac muscle. Ca2+ transients in cardiomyocytes represent the spatial and temporal summation of many Ca2+ sparks.

Consistent with the role of AP profile on Ca2+ release and contraction properties, regional differences in AP waveform and duration can dramatically affect the Ca2+ transient and mechanical shortening. The bulk of Ca2+ entry through ICaL occurs during phase 1 and the plateau phase of the AP, where the membrane potential is in the optimal range for Ca2+ entry via ICaL The magnitude of ICaL is equivalent across the wall in the canine left ventricle when measured using a square wave voltage clamp pulse (Figure 3A) (29, 42, 43). However, differences in AP morphology alter the size and kinetics ICaL across the ventricular wall. Increasing the early rate of (phase 1) repolarization increases the driving force, which results in a large peak ICaL amplitude, a faster time to peak of ICaL, as well as greater total charge (Figure 3B) (11, 29, 44). These observations suggest that an AP waveform with a spike-and-dome morphology is a more efficient releaser of SR Ca2+. Conversely, an AP with a small phase 1 repolarization, such as in Endo, results in a smaller ICaL, desynchronized SR Ca2+ release and reduced efficiency of EC coupling. This results in a slower velocity of cell shortening (Figure 3C). Nevertheless, variations in Ito and the size of the phase 1 repolarization across the ventricular wall contribute to differences in electrical and mechanical characteristics of the three cell types. The implications relative to the mechanical function of the heart under normal and diseased condition are numerous and will be described in the sections to follow.

Figure 3. Panel A: Representative traces showing ICa recorded from a canine Mid ventricular myocyte. Ca2+ currents were recorded during a 300 ms step depolarization from –40 to +50 mV in 10 mV increments. Current-voltage relationship for ICa is shown on right side of Panel A. Panel B: Representative ICaL current traces (defined as the nicardipine-sensitive difference current) recorded from a cell in response to an Endo (left side) or Epi (right side) AP waveform. To eliminate the effects of INa, the AP was modified so that -50 mV was the start of the waveform (top of figure). Application of the Epi- waveform produced a larger peak current and greater charge entry compared to the Endo- waveform. Panel C: Endo and Epi AP waveforms and corresponding cell shortening (bottom traces) recorded from a cell in response to endocardial and Epi AP waveforms (top). Five pre-pulses were applied to the cell to maintain uniform SR Ca2+ content. Application of the Epi waveform caused a faster time-to-peak and greater cell shortening. The tick marks on the cell shortening traces denote the peak. Modified with permission from (29).

4.2. The decline of Ca2+ transients

The duration of myocyte contraction is limited by the inactivation of ICaL coupled with Ca2+ removal from the cytosol through several pathways, leading to a reduction in the cytosolic free Ca2+, dissociation of Ca2+ from the contractile machinery and relaxation of the myocardium (45). The dominant pathway for removal of Ca2+ from the cytosol is through the activity of the SR Ca2+-ATPase (SERCA), which derives the energy from ATP to actively pump Ca2+ into the lumen of the SR. The contribution of SERCA in the removal of Ca2+ from the cytosol varies between 70-92% depending on the species examined. The large contribution of SERCA to Ca2+ cycling ensures that the SR is replenished with Ca2+ and the cell is ready for the next round of EC coupling. In addition to SERCA, Ca2+ is also removed from the cytosol into the extracellular matrix through the activity of the Na+-Ca2+ exchanger (NCX) which depending on the species, can contribute up to 30% to Ca2+ efflux (46). Evidently, the fraction of Ca2+ efflux by NCX is approximately equal to the amount of Ca2+ that enters the cell through the activity of ICaL (47). In addition to playing a role in removal of Ca2+, it has been suggested that Ca2+ influx through the reverse mode of the NCX may directly act as a trigger for Ca2+ release from the SR. The relative contribution of Ca2+ influx through NCX towards EC coupling remains to be determined.

When differences in the AP waveform and duration are eliminated by application of a square wave under voltage clamp conditions, some of these differences in cell shortening persists, suggesting there are intrinsic differences in E-C coupling between the three cell types. Therefore, factors other than AP morphology can contribute to the mechanical distinctions. These variations may be due to differences in intracellular Ca2+ buffering or expression of Ca2+ handling proteins. In canine ventricular myocardium, it has been shown that the expression of SERCA is greater in the Epi versus Endo (Figure 4). The greater SERCA2a expression in Epi results in a faster decline of the Ca2+ transient (48).

Figure 4. Representative recording of ionic currents (upper panel) and cell shortening (lower panel) from an Endo, Mid and Epi myocardial cell recorded under voltage clamp conditions. The voltage clamp protocol is shown at the top. Five pre-pulses were applied to maintain a constant SR load. Following application of a 300 ms test-pulse to +20 mV, a large Ito can be observed in the Epi and Mid cells (upper traces). The tick marks on the cell shortening traces denote the peak. Representative recording of corresponding confocal xt line scans (panel B) from an Endo, Mid and Epi cell recorded under voltage clamp. The voltage clamp protocol is shown at the top of the figure. Five pre-pulses were applied to the myocytes to maintain a constant SR load. Following application of a 300 ms test-pulse to +20 mV, a large Ito can be observed in the epi and mid cell (panel A). The corresponding line scan recordings (panel B) and time course of F/Fo (panel C) for Epi and Mid cells show similar kinetics. In contrast, the line scan recorded from the Endo cell has a much slower decay compared to the other two cell types. Modified with permission from (29, 125).

5. Molecular components and regulation of Ito

5.1. Molecular composition of cardiac Ito

Ito, f currents are generated by channels consisting of voltage-gated α-pore-forming Kv4 subunits. Assembly of four Kv α-subunits into a tetrameric structure forms functional Ito, f channels (49). In human and canine hearts, the majority of studies have shown that Kv4.3 is the predominant α-subunit of Ito, f (50, 51). Heterologous expression of Kv4 α subunits show rapidly activating, inactivating and recovering Ito, f-like outward K+ currents (9), which are sensitive to heteropodatoxins (HpTx) (52), and phrixotoxin (PaTxs) similar to native Ito, f. Several transgenic mouse studies have provided further evidence regarding molecular identity of Ito, f. Heterologously expressed Kv4 channels do not recapitulate entirely the kinetics of native cardiac Ito, and Kv4 channels have been shown to assemble with various ancillary β-subunits in cardiac tissue (Figure 5). The most prominent, K+ Channel Interaction Protein 2 (KChIP2) is one of four KChIPs (KChIP1–4) that are members of the neuronal calcium sensor subfamily of Ca2+ binding proteins characterized by four Ca2+-binding EF-hand motifs (53). It is believed that only KChIP2 is specifically expressed in the heart (54), and KChIP2 co-immunoprecipitates with both Kv4.2 and Kv4.3 (55).

Figure 5. Disease causing mutations in Kv4 and interacting subunits as well as putative drug binding sites. Heteropoda venatoria toxins selectively inhibit Kv4 channels by stabilization of the closed state by binding to the S3B to S4 linker segment (108). NS5806 has been suggested to modulate the interaction between the hydrophobic groove on KChIP and Kv4 as well as to affect calcium sensitivity of KChIP (118). Loss-of-function mutations in Kv4 have been linked to spinocerebellar ataxia (SCA) (indicated by blue dots). SCA19 is associated with T352P, M373I and S390N (92). SCA22 is associated with an in-frame 3-nucleotide deletion F227del, V338E, G345V and T377M (126). Gain-of-function mutations (marked by reddots) have been linked to sudden unexpected death, V392I and G600R (86), the Brugada syndrome (BrS), L450F and G600R (85) and early onset atrial fibrillation, A545P (87). P450L (marked by red dot with blue outline) has been associated with both BrS and SCA (93). P450L results in a gain of function of Kv4.3. In the accessory subunit KCNE3, R99H resulting in increased Kv4 currents has been linked to BrS (90). Likewise, mutations in KCNE5, Y81H, D92E+E93X, resulting in increased Kv4 currents has been linked to BrS (73).

KChIP2 increases Kv4.3 current density by facilitating trafficking, slowing inactivation and accelerating recovery kinetics (10, 56). Co-expressed with KChIP2, Kv4-encoded currents in heterologous systems are almost identical to native Ito, f in rodent cardiomyocytes (57). Interestingly, mice lacking KChIP2 have no Ito, f, (10, 58)suggesting an additional role of KChIP2 in facilitating expression of Kv4 channels by stabilizing Kv4 proteins in their formation (59, 60). Several studies suggest that Kv4.3 mRNA levels are uniform throughout the canine left ventricle and the gradient in Ito expression is due to a gradient in KChIP2, as KChIP2 mRNA is more abundant in Epi-and Mid than in Endo (2, 27, 61, 62). Interestingly, the time constant (t) of decay was faster in Epi and Mid than in Endo (6, 63). As KChIP2 slows Kv4.3. current decay (64), this may suggest that Endo Ito is not merely Kv4.3. in absence of KChIP2 and indicates that other K+ subunits contribute to Endo Ito in human and canine hearts.

Besides KChIP2, several other proteins expressed in ventricular tissue have been shown to interact with Kv4.3. Similar to KChIP2, dipeptidyl-peptidases (DPP6 and DPP10) can facilitate Kv4.3 trafficking and accelerate recovery from inactivation; however, in contrast to KChIP2, the DPPs accelerate Kv4.3 inactivation (65) and increase single channel conductance of Kv4.2 (66). DPP6 expression in the heart has been demonstrated both at mRNA and protein level (67). Whether DDP10 is expressed in cardiomyocytes is at the present unclear, though DPP10 mRNA has been detected in atrial tissue (68). Very little is known about the nature of the interaction between Kv4 and DPP, it has been suggested to involve the transmembrane regions of both proteins (69).

Several members of the KCNE β-subunit family have been demonstrated to modulate Kv4 density and kinetics. Loss of KCNE2 in mice leads to a reduction in ventricular Ito, f (70).whereas KCNE3 inhibits Kv4 (71). A recent study demonstrated that KCNE4 expression is localized in the transverse tubules of murine cardiac myocytes, where it co-assembles with Kv4.2 regulating Ito in a “KChIP2-dependent manner” (72). KCNE5 modulates Kv4 channels in the heterologous system and a mutation in KCNE5 resulting in less inhibition of Kv4.3 current have been linked to the Brugada syndrome (BrS) (73). Interestingly, the KCNE5 expression is elevated in failing human hearts (74). Other subunits have been proposed to modulate Ito, f but the physiological importance may be uncertain.

Several studies have demonstrated that Ito in human and canine is not simply mediated by Kv4.3 channel but that other α subunits may be involved. The presence of Kv1.4 and Kv1.5 has been noted in the ventricle of both human and canine ventricle (3, 14). These α-subunit are expressed at lower levels compared to Kv4.3 but their presence have been consistently measured by numerous investigators. Kv1.4 gives rise to a slowly recovering Ito, s and is likely responsible for the slow phase of Ito recovery in dog ventricle (27). The presence of a TEA-sensitive Ito conducted through Kv3.4. was noted in dog ventricular and Purkinje tissue (75). The presence of Kv3.4 message was verified in human tissue by Gaborit et al. (51). In that same study, the authors noted the presence of Kv4.2 message, an α-subunit thought to only be present only in rodents (51). Interestingly, a more recent publication noted a rare genetic mutation of the KCND2 gene in a patient with J-wave syndrome supporting a functional role for Kv4.2 in human ventricle (76). These observations suggest that Ito, in human ventricle may also consist of Kv1.4, Kv1.5, Kv3.4 and Kv4.2, but the precise subunit composition remains to be elucidated.

5.2. Molecular regulation of Ito

Ito and its molecular constituents are highly regulated by multiple transcription factors, both constituently and in response to pro-hypertrophic stimulation, as well as stimulation of adrenergic pathways. With regards to constitutive regulation, Iroquois-class homeo-domain protein 5 (Irx5), which is expressed transmurally as a gradient from Endo-to-Epi (large to small), is a repressor of Kv4.2 expression in the mouse heart and Irx5 has been suggested to establish the Ito, f Epi-to-Endo gradient in murine hearts (77). In addition, Jia et al. found that GATA and FOG2 transcription factors can drive expression of the Kv4.2. promoter (78). The same group also found that c-Jun NH(2)-terminal kinases play a role in mediating the α-adrenergic receptor.-induced reduction in KChIP2, and the MEK-ERK pathway mediates the protein kinase C-mediated down-regulation. In mouse, Ito was found to negatively regulate by the calcineurin/nuclear factor activated T-cell system (NFAT) (79).This mechanism is intriguing since calcineurin/NFAT is activated in heart failure (HF) (80), which coincides with decreases in Ito. However, in rat neonatal cardiomyocytes calcineurin/NFAT increases Ito via up-regulation of Kv4.2 (81). Adding to this complexity, we found that ubiquitous transcription factor nuclear factor kappaB mediates α-adrenergic receptor-induced reductions in KChIP2 expression (82). Further studies are warranted to more fully understand the complicated transcriptional regulation of Ito subunits by adrenergic stimulation in the context of heart disease.

6. Pathophysiological conditions and the role of Ito and its pharmacology

The transient outward potassium current Ito has been linked to several pathophysiological conditions and inherited syndromes. In the cardiac field, Ito has been suggested to play a role in the BrS or J-wave syndromes, atrial fibrillation (AF) as well as in hypertrophy and HF. Since Ito is altered in multiple diseases, its potential as a pharmacological target is also highly relevant.

6.1. Inherited Ito linked channelopathies

The Brugada syndrome was first described as a new clinical entity in 1992 (83) and is associated with nocturnal polymorphic ventricular tachycardia and sudden death, predominantly in young males. BrS is characterized by a ST-segment elevation in the right precordial leads, occurring in the absence of structural heart disease. The majority of BrS cases have been linked to loss-of function mutations in cardiac INa ((84) for a recent review); however, several gain-of-function mutations in Kv4.3. have been linked to BrS (L450F and G600R (85)) or sudden unexpected death (SUD, V392I and G600R (86). Kv4.3. gain of function has also been linked to atrial fibrillation (AF, A545P (87)). All these known gain-of-function mutations in Kv4.3. are located to the C-terminus of Kv4.3. (Figure 5). The C-terminus harbors several sites involved in post-translational modifications (reviewed by (88)), and may be involved in stabilizing the interaction between KChIP2 and Kv4 subunits (89). Furthermore, mutations in the accessory subunits KCNE3 (90) and KCNE5 (91) have been linked to BrS. Both KCNE3 and KCNE5 normally exert an inhibitory effect on Kv4.3. to suppress Ito (71, 90) and mutations in these subunits remove this inhibitory effect resulting in higher Ito densities.

It is important to note that not all BrS patients harboring mutations in Kv4.3. exhibit documented AF (85, 86), and conversely, a Kv4.3. AF patient did not show a BrS ECG phenotype when administered a flecanide challenge (87), suggesting that the expression and/or regulation of other cardiac currents may influence the phenotype. In addition, the Kv4.3. protein is expressed in many different tissue types including neuronal tissue. Loss-of-function mutation in the Kv4.3.α-subunit has been associated with inherited spinocerebellar ataxia (SCA), a progressive disease associated with atrophy of the cerebellum causing ataxia of gait and limbs as well as eye movement and speech impediments. SCA has been linked to loss-of-function and impaired trafficking mutations in Kv4.3 (92). Interestingly, P450L has been associated with both BrS and SCA (93). This puzzling observation has not been resolved, but electrophysiological studies revealed P450L results in a gain of function of Kv4.3. For the other loss-of-function mutations (Figure 5), no cardiac phenotype has been reported.

6.2. Alterations in heart failure

Besides the inherited syndromes due to mutations in Ito subunits, changes in Ito expression has repeatedly been linked to a number of cardiac pathologies, including hypertrophy and HF.

During the development of HF, a cascade of events occurs to cardiac tissue that ultimately results in a reduced cardiac output. These changes are multiple and complicated but include alterations in ion channel currents, Ca2+ cycling proteins and ultrastructure changes. The changes that occur, predispose the failing heart to ventricular tachyarrhythmias and sudden cardiac death triggered by early afterdepolarizations (EADs) or delayed afterdepolarizations (DADs). Typically, studies have focused on defects in intracellular Ca2+ cycling and the subsequent remodeling of these processes. Indeed, altered Ca2+ handling during heart failure has been linked to changes in Ca2+ handling proteins such as a decrease in ICaL density (94) and increased NCX (95, 96).

Down regulation of many repolarizing potassium currents during heart failure is well documented (for review see (97,98)). Ito, f is reduced in both human and animal models of HF (1, 99-101), concomitant with a decrease in phase 1 repolarization and an increase in AP duration (102).The early repolarization phase of the ventricular AP determines the electrochemical driving force for Ca2+ influx through L type Ca2+ channels. Therefore, cell types with a large Ito and spike and dome morphology have a significantly larger peak ICaL as well as greater total charge compared to a cell type where Ito is absent (29, 44). However, what happens under pathophysiological conditions when Ito is altered? How does this affect the magnitude and kinetics of ICaL?

A smaller Ito results in a significant reduction in peak ICaL as well as total charge when compared with a cell type where Ito is prominent (Figure 3B) (12, 29). The reduction in ICaL will affect Ca2+ transients (12) and ultimately contraction (Figure 3C). These observations were verified in a study by Cooper et al., (103) who applied previously recorded failing human hearts APs to healthy ventricular rat myocytes using AP voltage clamp techniques. The results demonstrated that applying HF-like AP waveforms to healthy cardiomyocytes lead to a reduced ICaL and a reduced Ca2+ transient compared to when a human non-failing AP waveform was applied to the same cell. The loss of Ito, f also results in a loss of the transmural gradient in phase 1 repolarization resulting in Epi and Endo tissue having a similar AP morphology (Figure 6). As the early repolarization phase is important to synchronize Ca2+ transients and cell shortening across the ventricular wall, a loss of Ito will ultimately affect contraction and worsen energy expenditure in the failing heart. This indicates that restoration of the early repolarization may be beneficial to patients in heart failure. However, it is important to note that in the failing heart, changes in AP waveform and duration due to loss of Ito and other ionic currents should not be considered separately from changes in EC coupling.

Figure 6.Ventricular tachypacing-induced heart failure-induced reduction in epicardial AP notch and ECG J wave is reversed by NS5806. Left Panel: Representative LV wedge isolated from a normal heart showing Epi and Endo AP as well as corresponding transmural ECG. A pronounced spike-and-dome AP is apparent in the Epi recording. Middle Panel: Representative LV wedge isolated from a 2-week HF heart showing a decrease in Phase 1 repolarization, loss of spike-and-dome morphology and J wave. Right panel: 10 µM NS5806 restores AP notch and J wave toward control. Basic cycle length = 2000 ms. Modified with permission from (120).

6.3. Pharmacology

Kv4 currents are selectively inhibited by several spider toxins containing three disulfide bonds that form an “inhibitory cysteine knot,” including the Heteropoda venatoria toxins (104) HpTX2 (105) and HpTX3 (6), the Phrixotrichus auratus toxins PaTx1 and PaTx2 (106) and the Theraphosa leblondi toxins TLx1-3 (107).These toxins exert their function by binding to the extracellular linker region between transmembrane segments S3B and S4 (105, 108)and rather than occluding the channel pore, these toxins work by interfering with the channel’s gating mechanism and stabilizes the closed state of the channel protein (108,109). Recently, Kv4 channels have been shown to be blocked by the AmmTX3 of the α-KTX15 family of scorpion toxins, principally in the presence of the auxiliary subunits DPP6 and DPP10 (110).

Kv4.3/Ito, f is blocked by 4-aminopyridine (4-AP) in millimolar range concentrations (111). However, 4-AP also blocks Kv1 channels/Ito, s in micromolar concentrations, which limits the use of 4-AP to discriminate between Ito, f and Ito, s. Interestingly the underlying mechanisms are different. Ito, s 4-AP is blocked in the open-state whereas 4-AP binds to Ito, f in the closed state (112), which results in complex recovery properties of Ito(112). Ito, f is blocked by several sodium channel blockers, including flecainide (113) and quinidine (114) and several calcium channel blockers, including nifedipine (115). Since a large phase 1 repolarization and Ito is thought to play a central role in the BrS, Ito blockers could be of therapeutic value in BrS. In line with this, hydroquinidine could prevent VT/VF induction in a group of asymptomatic BrS patients with inducible arrhythmias (116) as well as reduced the occurrence of arrhythmias in BrS patients that had suffered several ICD shocks (117). However, quinidine is associated with frequent gastrointestinal side-effects (84) and the development of better Ito antagonists could be beneficial for treating BrS patients.

We have recently added an Ito, f activator, NS5806, to this list of Ito affecting compounds. NS5806 increases Ito, f peak currents and slows current decay (27). This resulted in enhanced phase 1 repolarization in canine ventricular wedge preparations (62). NS5806 has been suggested to modulate the interaction between the hydrophobic groove on KChIP2 and Kv4 as well as to affect Ca2+ sensitivity of KChIP2 (118); however, in the absence of KChIP2 there is still substantial effect of NS5806 on Kv4.3. currents (119) suggesting additional binding sites for NS5806 on the Kv4 subunits.

7. Ito as therapeutic target for heart failure?

Several lines of evidence suggest that restoring Ito in the setting of heart failure could be beneficial: 1) Application of an Ito, f activator will result in restoration of the spike-and-dome morphology (120) resulting in an immediate increase in Ca2+ influx into the cell during the course of an AP; 2) by restoring Ito, f, the transmural gradient in phase 1 repolarization would also be restored which would results in a better coordination of the calcium transients and transmural contraction. This could potentially decrease the energy expenditure and improve the contractile function of the heart, and 3) on the long term basis, enhancement of Ito, f will maintain SR load since a greater amount of Ca2+ will enter the cell during each AP. We have recently demonstrated that application of the Ito activator NS5806, resulted in restoration in the spike and dome morphology of the AP in failing hearts (Figure 6). Despite the clear evidence for the potential benefit of modulation of Ito, f in heart failure, these studies are still lacking in animal models with classical spike-dome AP morphology, which would be more applicable to humans. Novel Ito, f agonists, such as NS5806, and the greater technological potential for viral gene transfer (121) will greatly enhance the prospects of exploring this hypothesis in the near future.

8. Summary

Because of its prominent role in cardiac excitability, Ito is one of the most studied K+ currents. Ito plays a role Brugada syndrome and is altered in a number of cardiomyopathies including heart failure. In addition, it is clear that the Ito mediated spike-and-dome morphology is essential for normal repolarization and can have important effects on EC coupling in canine and human hearts. The spike and dome-morphology results in a significant increase in peak ICaL as well as total charge when compared to a cell type where Ito is reduced or absent. In the setting of heart failure, where Ito is known to be reduced, this reduction in Ito results in reduced Ca2+ influx into the cell via ICaL. Augmentation of Ito in heart failure may have a beneficial effect on cardiac output since application of an Ito activator will result in restoration of the spike-and-dome morphology resulting in an immediate increase in Ca2+ influx into the cell during the course of an AP. Furthermore, enhancement of Ito may re-establish the transmural gradient of phase 1 repolarization and improve the synchronization of contraction which will minimize the metabolic stress of the failing heart. On a long term basis, increasing Ito may help maintain SR load since a greater amount of Ca2+ will enter the cell during each AP. Further studies are warranted to examine this hypothesis.

9. Acknowledgements

JM. Cordeiro, K. Calloe and R. Aschar-Sobbi contributed equally to this work. The authors are grateful to Judy Hefferon for assistance with Figure formatting. This study was supported by the Masons of New York State, Florida, Massachusetts and Connecticut, Danish National Research Foundation Centre for Cardiac Arrhythmia (KC) in addition to grants from Heart and Stroke Foundation (H&SF) of Ontario Canada (T6485) and Canadian Institutes for Health Research (MOP185773) to PHB. PHB is a Career Investigator with the H&SF of Ontario.

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Jonathan M.Cordeiro, Kirstine Calloe, Roozbeh Aschar-Sobbi, Kyoung-Han Kim, Adam Korogyi, Dona Occhipinti, Peter H. Backx, Brian K. Panama. Physiological roles of the transient outward current Ito in normal and diseased hearts. Frontiers in Bioscience-Scholar. 2016. 8(1); 143-159.