PHYSIOLOGICAL CONSEQUENCES OF TRANSIENT OUTWARD K⁺ CURRENT ACTIVATION DURING HEART FAILURE IN THE CANINE LEFT VENTRICLE

Abstract

Background

Remodeling of ion channel expression is well established in heart failure (HF). We determined the extent to which I_to is reduced in tachypacing-induced HF and assessed the ability of an I_to activator (NS5806) to recover this current.

Method and Results

Whole-cell patch clamp was used to record I_to in epicardial (Epi) ventricular myocytes. Epi- and endocardial action potentials were recorded from left ventricular wedge preparations. Right ventricular tachypacing-induced heart failure reduced I_to density in Epi myocytes (Control=22.13±1.9 pA/pF vs 16.12±1.4 after 2-weeks and 10.69±1.4 pA/pF after 5-weeks, +50 mV). Current decay as well as recovery of I_to from inactivation progressively slowed with development of heart failure. Reduction of I_to density was paralleled by a reduction in phase 1 magnitude, epicardial action potential notch and J wave amplitude recorded from coronary-perfused left ventricular wedge preparations. NS5806 increased I_to (at +50 mV) from 16.12±1.4 to 23.85±2.1 pA/pF (p<0.05) at 2 weeks and from 10.69±1.4 to 14.35±1.9 pA/pF (p<0.05) in 5 weeks tachypaced dogs. NS5806 increased both fast and slow phases of I_to recovery in 2 and 5-week HF cells and restored the action potential notch and J wave in wedge preparations from HF dogs.

Conclusions

The I_to agonist NS5806 increases the rate of recovery and density of I_to, thus reversing the HF-induced reduction in these parameters. In wedge preparations from HF dogs, NS5806 restored the spike-and-dome morphology of the Epi action potential providing proof of principal that some aspects of electrical remodelling during HF can be pharmacologically reversed.

INTRODUCTION

Congestive heart failure (HF) is one of the most common causes of death and disability in United States affecting about 2.5 million in this country alone. Despite advances in pharmacological therapy, it is estimated that the mortality rate approaches 50% after 5 years. Associated with heart failure are a number of other problems such as cardiac arrhythmias occurring in both the atria and ventricle [1].

Causes of HF are numerous but include ischemic heart disease, myocardial infarction, hypertension and valvular disease. During the development of HF, the heart undergoes structural, functional and electrophysiological remodeling that ultimately results in a reduced cardiac output. Increased sympathetic tone initially compensates for the reduced cardiac output by maintaining blood pressure and perfusion. However, this added stress leads to increased metabolic demands. Current treatment involve administration of agents that reduce afterload (such as angiotensin converting enzyme inhibitors, diuretics or beta-blockers) thereby reducing the workload of the heart.

Altered intracellular Ca²⁺ handling appears to play a central role during the progression of heart failure as well as in the development of cardiac arrhythmias. In a process known as calcium induced calcium release (CICR), Ca²⁺ influx through L type calcium channels (I_Ca) initiates a much greater Ca²⁺ release from the sarcoplasmic reticulum resulting in cell contraction. Typically, studies have focused on defects in intracellular Ca²⁺ cycling and the subsequent remodeling of these processes during HF. Indeed, altered Ca²⁺ handling during heart failure has been linked to changes in Ca²⁺ handling proteins. The expression of several proteins involved in Ca²⁺ regulation is reduced, including a lower expression of the SR Calcium ATPase (SERCA). Interestingly, other proteins are upregulated such as the sodium-calcium exchanger (NCX) resulting in a greater proportion of Ca²⁺ ions being pumped out of the cell [2], whereby the intracellular and SR Ca²⁺ content is reduced. The decrease in SR Ca²⁺ loading results in reduced Ca²⁺ transients, a reduced synchronization in the release of Ca²⁺ from the SR and impaired contraction.

Down regulation of many repolarizing potassium currents during HF is well documented (for review see [3,4]) resulting in a prolongation of the action potential duration (APD) as well as a decrease in phase 1 repolarization, presumably due to a decrease in I_to. In canine failing hearts, this loss in I_to may be due to loss of either K_v4.3 and/or KChIP2 proteins [5,6]. Action potential waveform can profoundly affect size and kinetics of I_Ca. For example, the presence of a phase 1 repolarization can increase both peak and total charge of I_Ca during the course of an action potential [7–9]. Therefore in pathophysiological conditions where I_to is reduced, such as HF, loss of I_to may contribute to decreased calcium influx, CICR and contraction.

The present study evaluates the effect of an I_to activator (NS5806) in tachypacing induced HF in canine heart. Results of our study show that there is a progressive loss in the magnitude of I_to as well as a change in kinetics of the current following prolonged ventricular tachypacing. Application of NS5806 increased I_to density toward normal levels, resulting in restoration of AP morphology in both single and multicellular preparations. Results have been previously presented in abstract form.

METHODS

Rapid-Pacing Induced Heart Failure

We used a well characterized rapid-pacing induced heart failure model which results in congestive HF model [10]. Adult mongrel dogs of either sex were used for the study. This investigation conforms to the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (The Eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC 2011)) and was approved by the Institutional Animal Care and Use Committee.

Dogs were premedicated with 0.1 mg/kg hydromorphone and 0.02 mg/kg acepromazine. Anesthesia was induced by intravenous administration of 12 mg/kg thiopental and maintained by isoflurane (1–1.5%) inhalation. Pacemaker generators (modified Medtronic) were implanted in a subcutaneous pocket in the left cervical region. An active fixation bipolar pacing lead was positioned in the interventricular septum of the right ventricle with the aid of fluoroscopy and transesophageal echocardiography. After recovery (1 day), the dogs were paced at 220 bpm for a period of either 2 or 5 weeks. Pulse rates were monitored daily and a 12 lead ECG was recorded weekly to ensure proper pacing. HF was verified by measurement of LV ejection fraction, fractional shortening, end-systolic volume, and end diastolic volume using echocardiography and by measurement of BNP before and at the end of the 2-week (9 dogs) or 5 week (8 dogs) pacing periods.

Ventricular Wedge Preparations

The animals were anticoagulated with heparin and anesthetized with pentobarbital (30–35 mg/kg, i.v.). The chest was open via a left thoracotomy, the heart excised, placed in a cardioplegic solution (4° C-Tyrode's solution with 12 mM [K⁺]_o). Transmural wedges with dimensions of up to 3 × 2 × 1.5 cm (left ventricular wedge) were dissected from the antero-apical aspects of the canine left ventricle as previously described [11]. During the cannulation procedure the preparations were initially arterially perfused with cardioplegic solution through a distal diagonal branch of the left anterior descending coronary artery. Subsequently, the wedges were placed in a tissue bath and perfused with Tyrode's solution of the following composition (mM): 129 NaCl, 4 KCl, 0.9 NaH₂PO₄, 20 NaHCO₃, 1.8 CaCl₂, 0.5 MgSO₄, 5.5 glucose, buffered with 95% O₂ and 5% CO₂ (37±0.5° C). The perfusate was delivered at a constant pressure (45–50 mmHg). A transmural ECG was recorded using two Ag/AgCl half cells placed at ~1 cm. from the Epi (+) and Endo (−) surfaces of the preparation and along the same axis as the transmembrane recordings. Action potentials were simultaneously recorded from the epicardial surface (Epi) and from subendocardial regions or endocardial surface (Endo) using floating microelectrodes. Pacing was applied to the endocardial surface (BCL= 2 s). All amplified signals were digitized and analyzed using Spike 2 for Windows (Cambridge Electronic Design [CED], Cambridge, UK).

Isolation of adult myocytes

Myocytes from Epi regions were prepared from canine hearts using techniques previously described [12–14]. A wedge consisting of the left ventricular free wall was cannulated and perfused with nominally Ca²⁺-free Tyrode’s solution containing 0.1% BSA for about 5 minutes. The wedge preparations were then subjected to enzyme digestion with the nominally Ca²⁺-free solution supplemented with 0.5 mg/ml collagenase (Type II, Worthington), 0.1 mg/ml protease (Type XIV, Sigma) and 1 mg/ml BSA for 8–12 minutes. After perfusion, thin slices of tissue from the Epi (<2 mm from the epicardial surface) were shaved from the wedge using a dermatome. The tissue slices were then placed in a beaker, minced and incubated in fresh buffer containing 0.5 mg/ml collagenase, 1 mg/ml BSA and agitated. The supernatant was filtered, centrifuged at 200 rpm for 2 minutes and the pellet containing the myocytes was stored in 0.5 mM Ca²⁺ HEPES buffer at room temperature.

Solutions

The nominally Ca²⁺-free dissecting buffer contained (mM): NaCl 129, KCl 5.4, MgSO₄ 2.0, NaH₂PO₄ 0.9, glucose 5.5, NaHCO₃ 20 and was bubbled with 95% O₂/5% CO₂. Ventricular cells were superfused with HEPES buffer (mM): NaCl 126, KCl 5.4, MgCl₂ 1.0, CaCl₂ 2.0, HEPES 10, glucose 11, pH=7.4 with NaOH. For I_to recordings 300 µM CdCl₂ was present in the extracellular solution to block I_Ca. The pipette solution consisted of (mM): K-aspartate 125, KCl 10, MgCl₂ 1, EGTA 5, MgATP 5, HEPES 10, NaCl 10, pH=7.2 with KOH.

Electrophysiology

I_to recordings from myocytes were performed as previously described [15] and all myocyte experiments were performed at 36°C. Voltage-clamp and conventional recordings were made using a MultiClamp 700A amplifier and MultiClamp Commander (Axon Instruments). Patch pipettes were fabricated from borosilicate glass capillaries (1.5 mm O.D., Fisher Scientific, Pittsburg, PA). Pipettes were pulled using a gravity puller (Narishige Corp) and the resistance ranged from 0.9–3 MΩ when filled with the internal solution. Cell capacitance was measured by applying −5 mV voltage steps. Electronic compensation of series resistance to 60–70% was applied. All analog signals were acquired at 10–25 kHz, filtered at 4–6 kHz, digitized with a Digidata 1322 converter (Axon Instruments) and stored using pClamp9 software.

Statistics

Pooled data are presented as Mean±SEM. Statistical analysis was performed using an ANOVA test followed by a Student-Newman-Keuls test or Student t-test, as appropriate, using SigmaStat software. Statistical analysis of RNA and protein expression was performed with a repeated measures ANOVA followed by a Tukey’s post test. p<0.05 was considered statistically significant.

RESULTS

Right ventricular tachypacing induced progressive development of HF as assessed by hemodynamic parameters measured using echocardiography following 2 and 5 weeks of tachypacing (Figure 1A). LV end systolic volume (LVESV) increased from 8.6±1.4 to 19.2±3.0 ml at 2 weeks and from 18.2±4.0 to 48.6±9.4 ml at 5 weeks and end diastolic volume (LVEDV) increased from 22.8±3.1 to 29.8±4.0 ml at 2 weeks and from 41.4±7.6 to 70.5±13.0 ml. Left ventricular ejection fraction (LVEF) decreased from 63.2±2.0 to 35.9±2.5 % at 2 weeks and from 59.0±4.0 to 36.3±4.5% at 5 weeks.

Figure 1.

Panel A: Summary of left ventricular hemodynamic changes in response to rapid pacing. Hemodynamic parameters were collected before pacing and at the end of the 2-week (9 dogs) or 5 week (8 dogs) pacing periods. EF=ejection fraction, IDD=internal diameter in diastole, IDS=internal diameter in systole, EDV=end diastolic volume, ESV=end systolic volume, FS=fractional shortening, FWD=front wheel drive, FWS=wall thickness in systole Panel B: Representative recordings of action potentials recorded from a Normal and 5-week HF Epi myocyte. Each panel shows 5 consecutive APs paced at a cycle length of 1 second. HF Epi cells show a drastically reduced phase 1 repolarization and longer AP duration.

As an initial basis of comparison, action potentials (AP) were recorded in Epi cells isolated from hearts subjected to either 2 or 5-weeks of rapid pacing. Representative APs from normal and 5 week paced Epi cells paced at a basic cycle length of 1 s are shown in Figure 1B. AP recordings from normal Epi cells show a prominent spike-and-dome configuration. In contrast, APs recorded from Epi cells isolated from a HF dog showed loss of the spike and dome configuration and smaller phase 1 repolarization, suggesting loss of I_to. This was accompanied by a prolongation of the AP duration suggesting down regulation of other repolarizing K⁺ currents.

The density of I_to was examined in myocytes isolated from Epi in control animals and dogs tachypaced for 2 and 5 weeks. Cd²⁺ (300 µM) was present in the extracellular solution to block the calcium current (I_CaL). Following a brief step to −50 mV to discharge sodium channels, voltage steps from −40 to +50 mV applied to Epi cells elicited fast activating and rapidly inactivating I_to currents. Representative currents recorded from normal and 5-week Epi are shown in Figure 2A–B. The mean current-voltage (I-V) relation of peak I_to showed a progressive reduction in I_to density in HF Epi cells compared to controls (Figure 2C). Analysis of the decay of I_to (tau) revealed a progressive slowing of current inactivation with 2 to 5 weeks of tachypacing (Figure 2D).

Figure 2.

Representative I_to traces recorded from a normal Epi (Panel A) and 5-week HF Epi cell (Panel B). The voltage clamp protocol is shown at the top of the figure and Cd²⁺ was present to block I_CaL. Mean I-V relation for peak I_to from normal, 2-week HF and 5-week HF Epi cells (Panel C). The time constants of decay (Tau) were obtained by fitting a single exponential equation to the decaying phase of the currents and plotted as a function of voltage (Panel D). A time-dependent reduction in the density of I_to as well as a slowing of current decay was observed with prolonged pacing.

Changes in steady state gating parameters may contribute to the difference in current density between normal and HF cells. Steady state inactivation of I_to was evaluated using a prepulse-test pulse voltage clamp protocol (top of Figure 3) in presence of Cd²⁺. Peak current following a 2 s prepulse was normalized to the maximum current and plotted as a function of the prepulse voltage to obtain the availability of the channels. A Boltzmann function was then fitted to the data. In normal Epi cells, the mid-inactivation voltage was −47.4±0.31 mV (Figure 3C). In 2-week and 5-week HF Epi cells, mid-inactivation voltage was −49.9±0.45 mV for 2-week and −46.8±0.48 mV for 5-week Epi cells (Figure 3C).

Figure 3.

Representative traces recorded from a normal Epi (Panel A) and 5-week HF Epi (Panel B) showing voltage dependence of inactivation of I_to. Boltzmann curves showing mid-inactivation voltages for normal, 2-week HF and 5-week HF Epi (Panel C).

We next determined if I_to recovery from inactivation was altered following rapid pacing. Representative traces from a normal Epi (Figure 4A) and 5-week HF Epi (Figure 4B) are shown. Reactivation of I_to at −80 mV for normal and HF Epi cells showed a fast and a slow phase of recovery as follows: i) τ1= 28.4±2.54 ms and τ2=177.5±7.82 ms for normal Epi cells, ii) τ1=56.6 ± 5.92 ms and τ2=337.8±28.9 ms for 2-week and iii) τ1=73.4±3.52 ms and τ2=546.3±38.6 ms for 5-week Epi HFcells (Figure 4C). These results demonstrate that HF causes a significant time dependent slowing in both the fast and slow phase of recovery of I_to in Epi cells.

Figure 4.

Representative traces recorded from a normal (Panel A) and 5-week HF Epi cell (Panel B) showing recovery of I_to. Cd²⁺ was present to block I_CaL. Mean data showing the recovery time-course of I_to recorded from normal, 2-week HF and 5-week HF Epi cells (Panel C).

The results thus far demonstrate a reduction in the density of I_to as well as a slowing in the recovery of the current with progressive development of HF. In previous studies, we have identified a selective I_to agonist (NS5806). Although this compound also produces a minor inhibition of I_Na and I_Ca with no effect on other K⁺ currents [11], we utilized its I_to agonist effect to restore this current in Epi cells isolated from HF hearts. In the first series of experiments the effect of NS5806 on I_to density in HF Epi cells was examined. Representative I_to recordings for Epi cells isolated from 5 week tachypaced dogs in the absence and presence of 10 µM NS5806 are shown in Figure 5A–B. An increase in I_to peak current density was observed as well as a slowing in the decay of the current. Analysis of the current-voltage (I-V) relation of peak I_to showed that NS5806 increased current density in both 2-week (Figure 5C) and 5-week HF Epi cells (Figure 5D). On a percentage basis, we found that NS5806 increased I_to (at +50 mV) by 180% in normal Epi cells, 148% in 2 weeks and 134% in 5 weeks tachypaced dogs. Analysis of the decay of I_to (tau) showed that NS5806 slowed current decay in HF Epi cells (Figure 5E–F), similar to previously described results in normal cells [12].

Figure 5.

Representative I_to traces recorded from a 5-week HF Epi cell under control conditions (Panel A) and after 10 µM NS5806 (Panel B). Cd²⁺ was present to block I_CaL. Mean I-V relation for peak I_to from 2-week (Panel C) and 5-week (Panel D) HF Epi cells in absence and presence of 10 µM NS5806. Mean I-V relation for peak I_to from normal Epi cells are also illustrated for comparison. The time constants of decay (Tau) were obtained by fitting a single exponential equation to the decaying phase of the currents and plotted as a function of voltage for 2-week (Panel E) and 5-week (Panel F) HF Epi cells. Tau values from normal Epi cells are illustrated for comparison. NS5805 increased I_to peak current density and slowed current decay in HF Epi cells paced for 2 or 5 weeks. *Statistically significant from normal Epi cells, p<0.05; #Statistically significant from HF Epi cells, p<0.05.

Steady state inactivation of I_to was next evaluated in HF cells. In the absence of drug, mid-inactivation voltage was −49.9±0.45 mV in 2 week HF Epi cells and −46.8±0.48 mV in 5 week HF Epi cells (Figure 6). Application of NS5806 (10 µM) caused a significant negative shift in the mid-inactivation potential in both HF Epi cells with mid-inactivation voltages of −54.3±0.36 mV at 2 weeks (Figure 6C) and −52.1±0.46 mV at 5 weeks (Figure 6D). This shift in mid-inactivation could not account for the increase in current density observed in the presence of NS5806.

Figure 6.

Representative traces recorded from a 5-week HF Epi cell under control conditions (Panel A) and after 10 µM NS5806 (Panel B) showing voltage dependence of inactivation of I_to. Boltzmann curves showing mid-inactivation voltages for 2-week (Panel C) and 5-week (Panel D) HF Epi in absence and presence of NS5806.

We next examined the difference in time dependent recovery from inactivation of I_to in HF Epi cells in the absence and presence of 10 µM NS5806 (Figure 7A–B). Application of NS5806 significantly increased both the fast and the slow phase of recovery of I_to in both 2 and 5 weeks HF Epi cells. In 2 week HF Epi cells, the time constants of recovery were: τ1=56.6±5.92 ms and τ2=337.8±28.9 ms in the absence of drug and τ1=31.1±4.28 ms and τ2=149.9±7.98 ms after 10 µM NS5806 (Figure 7C). In 5 week HF Epi cells recovered with τ1=73.4±3.52 ms and τ2=546.3±38.6 ms in the absence of drug and τ1= 51.2±4.22 ms and τ2=257.5±16.83 ms after drug (Figure 7D).

Figure 7.

Representative traces recorded from a 5-week HF Epi cell under control conditions (Panel A) and after 10 µM NS5806 (Panel B) showing recovery of I_to. Cd²⁺ was present to block I_CaL. The recovery time-course of I_to recorded from 2-week HF Epi (Panel C) and 5-week Epi cells (Panel D) in the absence and presence of drug.

The results demonstrate that NS5806 can restore I_to in Epi cells isolated from HF hearts. We next determined the effect of NS5806 in multicellular preparations. APs and corresponding pseudo-ECG were simultaneously recorded in Epi and Endo layers from a canine left ventricular wedge preparation (Figure 8). In a control wedge preparation, the Epi layer of the wedge exhibited a pronounced phase 1 repolarization with minimal phase 1 repolarization present in Endo layer (left panel). The resulting transmural voltage gradient inscribed a distinct J wave in the ECG. In wedge preparations isolated form dogs tachypaced for a period of 2-week, phase 1 and the epicardial action potential notch were dramatically reduced, as was the J wave in the ECG (middle panel). Application of 10 µM NS5806 restored the spike-and-dome morphology in the Epi recording (phase 1 repolarization increased from 3% of the amplitude of phase 0 to 20.5%, n=5 wedges) with negligible effect in the Endo recording and no changes in APD₉₀. The dramatic increased in the Epi notch was accompanied by a comparable augmentation of the J wave amplitude of the ECG.

Figure 8.

Ventricular tachypacing-induced heart failure-induced reduction in epicardial action potential 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 pseudo-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 action potential notch and J wave toward control. Basic cycle length = 2000 ms.

DISCUSSION

The results of our study demonstrate that rapid-pacing induced heart failure causes a prolongation of the cardiac action potential and a marked reduction of phase 1 repolarization in both single and multicellular preparations. Patch clamp analysis of I_to showed a time-dependent reduction in current density as well as a slowing of current decay in failing hearts. A progressive slowing in recovery from inactivation was also observed. The I_to activator NS5806 increased I_to density toward normal in HF cells and restored early repolarization in ventricular wedge preparations from HF hearts.

In the normal canine ventricle, the majority of transient outward K⁺ channels are comprised of K_V4.3 channels with lower levels of K_V1.4 [6,16]. The electrophysiological properties of K_V4.3 channels are modulated by several β-subunits including KChIP2, which increase peak K_V4.3 current density, accelerate recovery from inactivation, and slow the decay (τ) of the current [17]. The reduction in I_to coupled with the change in current kinetics during HF suggests that one or more of the molecular correlates of I_to is altered. Previous studies have found that both K_V4.3 and KChIP2 are reduced following rapid pacing induced HF in canine hearts[5]. In contrast, a reduction in K_V4.3 but no change in KChIP2 was observed in another study[6]. Interestingly, the expression of K_V1.4 proteins has been shown to be increased in failing canine hearts [6]. The fact that K_V1.4 typically displays very slow recovery from inactivation as well as a slow current decay [18], suggests the possibility that the slower τ values of I_to decay as well as the slowing of the time dependent recovery observed in the present study may be due to a relatively larger contribution of K_V1.4 to I_to in failing hearts. In addition, I_to may be affected by elevated levels of angiotensin II, a known modulator of I_to [19].

In rodent models of HF it has been shown that loss of I_to may be directly involved in the hypertrophic response [20]. Reducing I_to prolongs APDs, resulting in larger Ca²⁺ influx which activates the hypertrophic factor calcineurin [20] whereas increasing I_to slows the progression of HF [21–23]. In larger mammals I_to is not directly involved in the phase 3 repolarization and the increased APD is likely due to down-regulation of currents other than I_to, thus the mechanisms of the potential beneficial effect of increasing I_to is fundamentally different in smaller and larger mammals (see review by Sah et al. [24]).

Application of NS5806 resulted in a substantial increase in I_to density in isolated HF Epi cells. In 2 weeks HF Epi cells, I_to density in the presence of NS5806 was comparable to normal Epi cells (Figures 2 and 5). After 5 weeks, I_to density was further reduced in Epi HF cells and while NS5806 increased I_to density, the effect was smaller than for 2 week. The slower recovery and reduced ability of NS5806 to enhance I_to after 5 weeks may suggest a larger contribution of K_V1.4 to the remaining I_to. Some studies have demonstrated that a reduction in KChIP2 occurs during HF, suggesting that this is responsible for the reduced I_to density. However, in the absence of KChIP2, I_Kv4.3 has a faster decay [25] but in the present study we observed a slowing in tau. Furthermore, our previous work has demonstrated that NS5806 slows I_to current decay only in the presence KChIP2 [17]. This suggests that the reduction of I_to in HF is not merely due to a reduction in KChIP2 levels.

In LV wedge preparations from failing hearts, phase 1 repolarization of Epi APs was decreased, presumably due to a loss in I_to. Moreover, APD (and the corresponding ECG) was prolonged in Endo and Epi compared to normal canine hearts. Application of NS5806 restored the phase 1 repolarization in Epi, and normalized the pseudo-ECG by restoring the J-wave. AP morphology in Endo recordings was not affected by application of NS5806, consistent with very little expression of I_to in the endocardium [12]. In both Epi and Endo, the APD remained prolonged, which was reflected on the ECG as an increased QT interval compared to normal, suggesting that factors other than a decrease in I_to are responsible for the effects on APD. The early repolarization phase determines the electrochemical driving force for Ca²⁺ influx during the early plateau phase. Cell types with a large I_to exhibit a prominent early repolarization and have a significantly larger peak I_CaL as well as greater total Ca²⁺ influx compared to a cell type where I_to is absent [7–9]. Since the size of the Ca²⁺ transient is proportional to Ca²⁺ influx via I_CaL, a large influx translates into greater SR Ca²⁺ release as well as increased cell shortening. Furthermore, in the presence of a prominent phase 1 repolarization, the SR Ca²⁺ release exhibits more temporal synchronization and spatial synchronization on single cell level [7,8], which in turn increases contraction as well as accelerates time to peak contraction [7]. In the normal canine heart, Epi and M cells have a prominent phase 1 repolarization compared to Endo, reflecting the transmural gradient in I_to density. Since the normal activation pattern of the ventricle is from Endo to Epi, the differences in phase 1 repolarization may serve to synchronize contraction across the ventricle and may improve the contractile efficiency [26]. These results suggests that in HF cells, the reduction of I_to and loss of phase 1 repolarization will affect L-type Ca²⁺ currents and reduce the synchronization of the calcium transient as well as disrupt the transmural coordination of contraction.

In theory, the augmentation of I_to in the setting of heart failure should have a beneficial effect on cardiac output for several reasons: Application of an I_to 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 action potential. Since an AP with spike and dome morphology is a more efficient releaser of Ca²⁺ form the SR, this may result in a better synchronization of the calcium transients in single cells [8,27]. Furthermore, restoration of transmural gradient of phase 1 repolarization may improve synchronization of contraction across the ventricular wall. On the long term basis, enhancement of I_to will maintain SR load since a greater amount of Ca²⁺ will enter the cell during each action potential. The improved contractile efficiency may outweigh the risk of increasing metabolic workload of the failing heart.

In summary, we demonstrate a time dependent reduction in the magnitude of I_to as well as a slowing in the recovery from inactivation in response to rapid pacing-induced HF. Application of NS5806 increased I_to density in Epi ventricular myocytes as well as phase 1 repolarization in multicellular preparations. Our results suggest that restoration of I_to may be a novel approach to the treatment of HF or other pathophysiological conditions (such as diabetic cardiomyopathy) where I_to is known to be reduced.

Highlights.

• -Heart failure (HF) reduced phase 1 repolarization in both single and multicellular preparations.
• -I_to current density was reduced in HF Epi cells compared to normal Epi cells.
• -The I_to activator NS5806 increased I_to density toward normal in HF cells
• -NS5806 restored phase 1 repolarization in ventricular wedge preparations from failing hearts.
• -Restoration of I_to may be a novel approach to the treatment of HF.