Ionic Mechanism of Action Potential Prolongation in Ventricular Myocytes From Dogs With Pacing-Induced Heart Failure
Abstract Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097±73 milliseconds [failing hearts, n=30] versus 842±56 milliseconds [control hearts, n=25]; P<.05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca2+ current and whole-cell Na+ current did not differ in cells from failing and from control hearts, but significant differences in K+ currents were observed. The density of the inward rectifier K+ current (IK1) was reduced in cells from failing hearts at test potentials below −90 mV (at −150 mV, −19.1±2.2 pA/pF [failing hearts, n=18] versus −32.2±5.1 pA/pF [control hearts, n=15]; P<.05). The small outward current component of IK1 was also reduced in cells from failing hearts (at −60 mV, 1.7±0.2 pA/pF [failing hearts] versus 2.5±0.2 pA/pF [control hearts]; P<.05). The peak of the Ca2+-independent transient outward current (Ito) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0±0.9 pA/pF [failing hearts, n=20] versus 20.4±3.2 pA/pF [control hearts, n=15]; P<.001), while the steady state component was unchanged. There were no significant differences in Ito kinetics or single-channel conductance. A reduction in the number of functional Ito channels was demonstrated by nonstationary fluctuation analysis (0.4±0.03 channels per square micrometer [failing hearts, n=5] versus 1.2±0.1 channels per square micrometer [control hearts, n=3]; P<.001). Pharmacological reduction of Ito by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp–release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of Ito in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.
Sudden cardiac death, often the result of ventricular arrhythmias, accounts for up to 50% of deaths in patients with heart failure.1 Despite intensive investigation, the underlying pathogenesis of ventricular arrhythmias in heart failure is poorly understood.2 Myocardial cells and tissue isolated from failing animal and human hearts consistently reveal abnormalities in repolarization, independent of the mechanism of heart failure.3 4 5 6 7 Prolongation of the APD can predispose to dispersion of repolarization, leading to nonexcitable gap reentry.8 Long action potentials also favor the development of afterdepolarizations, which in turn can induce triggered arrhythmias.9
The membrane current abnormalities that underlie action potential prolongation in failing human myocytes have recently been described.6 Studies involving isolated myocytes from failing human hearts highlight the importance of K+ current downregulation in the action potential prolongation observed in heart failure. According to these studies, Ito is markedly reduced in cardiac myocytes isolated from patients with heart failure.6 Other studies have suggested a reduction in Ito from failing human myocytes limited to cells isolated from the subendocardium.10 Nonetheless, even this regional reduction in current density could predispose to cardiac arrhythmias.
Measurements of ionic current in human cellular studies rely on a single time point in the course of disease, usually terminal heart failure of several years’ duration. The etiology of the myocardial failure, its duration, and medical treatment are varied and uncontrolled. In this regard, an animal model of heart failure characterized by malignant ventricular arrhythmias and sudden death would facilitate the investigation of the changes in the cardiac electrical properties.
The canine pacing-induced tachycardia model11 reliably reproduces the hemodynamic changes seen in human heart failure.12 13 14 15 We characterized the changes in the action potential in dogs with pacing-induced heart failure compared with nonpaced control dogs. The ionic currents underlying these changes in action potentials and the mechanism for the reduction in Ito density were investigated. In addition, we tested the idea that a reduction in Ito density suffices to produce the observed changes in action potential shape and duration.
Materials and Methods
Canine Pacing-Induced Tachycardia Heart Failure Model
Thirteen adult mongrel dogs of either sex underwent sterile surgical preparations for hemodynamic measurements and pacemaker implantation as previously described.15 The dogs (20 to 30 kg) were sedated with sodium thiamylol, intubated, and anesthetized with 1% to 2% halothane. Oxygen saturation and expired CO2 were maintained within the physiological range throughout the procedure by continuous volume ventilation. A left lateral thoracotomy was performed, and two right ventricular free wall pacing wires were placed. A subcutaneous pocket was fashioned for the placement of a VVI pacemaker (Activitrax or Spectrax, Medtronics). A rapid pacing mode was maintained by permanently attaching a magnet to the posterior surface of the pacemaker pulse generator. Animals were allowed to recover fully from surgery for 1 to 2 days, after which pacing was initiated at 240 bpm for 3 to 4 weeks. Terminal heart failure for the purpose of this and previous studies is defined by symptoms of lethargy, loss of appetite, dyspnea, and/or ascites.12 15 Prior work from our laboratory reveals that dogs subjected to this pacing protocol consistently develop an elevated left ventricular end-diastolic pressure (>25 mm Hg), depressed dP/dtmax (<1700 mm Hg/s), and a delayed time constant of relaxation (>45 milliseconds). Hemodynamics recorded in an unselected subset of 6 of the failing dogs revealed a left ventricular end-diastolic pressure of 36±2.8 mm Hg, dP/dtmax of 1187±239 mm Hg/s, and τ value of 69.7±9.4 milliseconds, consistent with severe myocardial failure. The hearts were harvested at terminal heart failure with the animals anesthetized as described above. The chest was opened by a left lateral thoracotomy, and the chest cavity was flooded with ice-cold saline. The great vessels were banded, and the coronary arteries were perfused retrogradely with cold cardioplegic solution containing (mmol/L) NaCl 110, KCl 16, MgCl2 16, NaHCO3 10, and CaCl2 1, pH 7.4, until diastolic cardiac arrest. The heart was quickly excised and submerged in cold cardioplegic solution. The time from tissue harvest to start of cell isolation was <15 minutes. To obtain normal control cells, euthanasia was performed in 16 adult mongrel dogs (20 to 30 kg) in the same manner but without prior pacing.
Isolation of Ventricular Myocytes
Ventricular myocytes were isolated as previously described with minor modifications.16 The territory perfused by the LAD from the aortic root to the apex was excised. This myocardial segment was then perfused via the LAD at a rate of 15 mL/min with the following solutions: 30 minutes with a nominally Ca2+-free modified Tyrode’s solution containing (mmol/L) NaCl 138, KCl 4, MgCl2 1, glucose 10, NaH2PO4 0.33, and HEPES 10, pH 7.3, followed by 40 minutes in the same solution with collagenase (type I, 178 U/mL, Worthington Biochemical Corp) and protease (type XIV, 0.12 mg/mL, Sigma Chemical Co). The enzyme solution (80 mL) was recirculated. The myocardial segment was then washed for 15 minutes with modified Tyrode’s solution containing 200 μmol/L Ca2+. All solutions were oxygenated with 100% O2 and warmed to 37°C. To control for the previously described transmyocardial variability of the currents,10 17 all cells were isolated from the central third of the myocardial wall, excluding endocardial and epicardial layers. At the end of perfusion, epicardial and subepicardial layers were dissected and discarded, and small chunks of well-digested midmyocardial tissue were taken from the area of the left ventricular anterior free wall between the LAD and the first diagonal branch. Ventricular cells were then mechanically disaggregated, filtered through a nylon mesh, and stored at room temperature in Tyrode’s solution containing 2.0 mmol/L Ca2+ until electrophysiological study. Only Ca2+-tolerant cells with clear cross striations and without spontaneous contraction or significant granulation (≈20% to 40% of those isolated in both control and failing hearts) were selected for experiments.
The experimental conditions were chosen to be comparable to those used in previous studies of human ventricular myocytes.6 18 For action potential and total membrane current recordings, the external solution contained (mmol/L) NaCl 138, KCl 4, CaCl2 2.0, MgCl2 1, glucose 10, NaH2PO4 0.33, and HEPES 10, pH 7.3. For the measurement of whole-cell K+ currents, CdCl2 (0.3 mmol/L) was added to block Ca2+ currents. For the measurement of whole-cell Ca2+ currents, the external solution contained (mmol/L) N-methyl-d-glucamine 140, CsCl 5, CaCl2 2.0, MgCl2 0.5, glucose 10, HEPES 10, and 4-AP 5, pH 7.4. 4-AP was prepared fresh before each use. Single-channel K+ currents were recorded in the cell-attached configuration in a depolarizing bath solution containing (mmol/L) KCl 140, NaCl 10, MgCl2 1, glucose 10, HEPES 10, and EGTA 0.1, pH 7.4. The pipette solution for action potential recordings contained (mmol/L) KCl 140, NaCl 5, MgCl2 1, HEPES 10, EGTA 2, and Mg-ATP 4, pH 7.4. For recording whole-cell K+ currents, the patch-clamp electrodes contained (mmol/L) potassium glutamate 120, KCl 10, MgCl2 2, HEPES 10, EGTA 5, Mg-ATP 2, QX 314 (ALMONE Laboratories) 5, pH 7.2. The pipette solution used for single-channel recording of Ito was identical to the external solution used for action potential recordings. For single-channel recordings of IK1, patch-clamp electrodes contained (mmol/L) KCl 140, EDTA 0.1, and HEPES 10, pH 7.4. For whole-cell Ca2+ current recordings, patch-clamp electrodes contained (mmol/L) CsCl 125, TEA-Cl 20, HEPES 10, EGTA 10, and Mg-ATP 4, pH 7.3. Whole-cell Na+ currents were recorded in symmetrical 5 mmol/L Na+; the external solution contained (mmol/L) NaCl 5, MgCl2 2, CsCl 5, TEA-Cl 125, and HEPES 20, pH 7.4 with TEAOH, and the pipette solution contained (mmol/L) NaCl 5, CsF 145, and HEPES 10, pH 7.2 with CsOH. Whole-cell Na+ currents were recorded in symmetrical 5 mmol/L Na+; the external solution contained (mmol/L) NaCl 5, MgCl2 2, CsCl 5, TEA-Cl 125, and HEPES 20, pH 7.4 with TEAOH, and the pipette solution contained (mmol/L) NaCl 5, CsF 145, and HEPES 10, pH 7.2 with CsOH. All chemicals were purchased from Sigma unless otherwise stated.
Electrophysiological Recording Techniques
Both the whole-cell and cell-attached configurations of the patch-clamp technique were used.19 Myocytes were transferred to the stage of an inverted microscope and superfused with external solution at a rate of 1 to 2 mL/min. Action potential recordings were performed at 37°C. All other experiments were performed at room temperature (22°C to 23°C). Patch electrodes were pulled from borosilicate glass and had 2- to 5-MΩ tip resistances for whole-cell recordings and 12- to 15-MΩ tip resistances for single-channel recordings. Cell-attached patches were formed with seal resistances of 20 to 100 GΩ. Currents were recorded using an Axopatch 200A patch-clamp amplifier (Axon Instruments) interfaced with a personal computer. Voltage or current control and data collection were performed using custom-written software. Cell capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 20-mV depolarizing test pulse from a holding potential of −80 mV. Series resistance was then compensated as much as possible without ringing, typically 60% to 80%. Given the average series resistance of our electrodes, the maximal uncompensated voltage error was <7 mV for the largest currents studied here. Whole-cell currents were low-pass–filtered at 2 kHz and digitized at 5 to 10 kHz via a TL-1 or Digidata 1200 A/D (Axon Instruments) interface for off-line analysis. The repetition interval was 5 seconds, unless otherwise specified. The currents recorded for nonstationary fluctuation analysis were filtered at 5 kHz. For cell-attached single-channel currents, records were filtered at 1 kHz and sampled at 5 kHz with a repetition interval of 5 seconds. Action potentials were recorded using an Axoclamp 2A voltage-clamp amplifier and sampled at 0.2 kHz.
The data were analyzed using custom-written software. Leakage and capacity currents were subtracted from unitary current records by fitting a smooth template, containing a sum of exponentials, to null traces. Single-channel current amplitudes were determined by amplitude histograms fitted by multiple gaussian distributions (IK1) or by averaging the amplitudes of well-resolved openings (Ito). Leak-subtracted current records were idealized using a half-height criterion,20 and idealized records were used to construct ensemble-averaged currents. The number of channels in a patch was estimated from the maximal stacking of the unitary events in long runs (>200 sweeps). The peak open probability was determined from the ensemble-averaged current using the single-channel current amplitude and the estimated number of channels in the patch.
Nonstationary fluctuation analysis was used to estimate the number (N) of functional Ito channels in the membrane. For a homogeneous population of channels gating independently, the mean macroscopic current (I) is defined as follows: I=N·i·po. The macroscopic current variance (ςI2) is defined as follows: ςI2=N·i2·po·[1−po], where i is the single channel current amplitude and po represents the open probability of the channel.21 22 Variance was calculated by subtracting pairs of sequential current records to obtain the difference current, squaring, and dividing by 2. The variance was then averaged over all the records collected.23 Provided that I and ς2 are determined for a range of open probabilities, i and N can be estimated by a plot of variance versus mean current fit by the following parabolic function: ςI2=i·I−I2/N.24 25
The currents plotted in the current-voltage relations were determined from non–leak-subtracted records, with the exception of Na+ current (Fig 3A⇓) and IK1 (Fig 4B⇓). For Na+ current, capacity transients and leak currents were subtracted using the P/4 method. In the case of IK1, a linear leak component defined by a line connecting the origin and the current at −80 mV was subtracted from the net current values at each potential to facilitate the analysis of the small outward component of IK1.
Pooled data are presented as mean±SEM unless otherwise stated. Statistical comparisons were made using Student’s t test or ANOVA, with P<.05 considered to be significant. A Wilcoxon signed-rank test was used to compare the APD before and during the phase-1 current injection.
The surface area of the myocytes was estimated by the cell capacitance. The capacitance was comparable in cells isolated from failing (142±4.4 pF [n=91]) and control (146±4.4 pF [n=87]) hearts (P=NS). The resting membrane potential of cells isolated from control and failing ventricles was nearly identical (−82 mV; see Table 1⇓), suggesting that cells from failing hearts were not systematically damaged by the cell isolation procedure.
Action Potential Changes in Heart Failure
Action potentials recorded in cardiac myocytes from failing hearts show significant changes in shape and duration compared with control hearts. The action potential features from 7 dogs with pacing-induced heart failure and 6 control dogs are summarized in Table 1⇑. Control cells typically have a prominent notch in phase 1, which is markedly attenuated in the cells from failing hearts (Fig 1⇓). The depth of the notch measured from the peak of the overshoot to the voltage minimum before the plateau was significantly decreased in cells from failing hearts. The diminutive notch is followed by a significantly elevated maximum plateau potential in cells from failing hearts. APD50 and APD90 were significantly prolonged in cells from failing hearts (Fig 1⇓). The average duration at 90% repolarization, under our recording conditions, was >1 second in cells from failing hearts. The resting membrane potential did not differ between the two groups.
We next examined the ionic currents that underlie the heart failure–associated alterations in the action potential. Although the prolongation of the APD may be the result of changes in a number of currents active during the plateau phase, the reduction in the size of the notch in phase 1 suggests a decrease in Ito.6 17
L-Type Ca2+ Current
Robust L-type Ca2+ currents were present in both ventricular myocytes from failing and normal canine hearts (Fig 2A⇓). There was no difference in the peak L-type Ca2+ current density (at +5 mV, −2.7±0.6 pA/pF [failing hearts] versus −2.7±0.5 pA/pF [control hearts]; P=NS). The current-voltage relation in the absence of β-adrenergic stimulation was not altered in cells isolated from failing hearts compared with those from control hearts (Fig 2B⇓, circles), although the response to isoproterenol (1 μmol/L) was blunted in myocytes from failing ventricles (Fig 2B⇓, triangles). The mean peak L-type Ca2+ current density at +5 mV was increased 74% in myocytes isolated from failing hearts compared with 111% in those from control hearts (P=NS). The tendency toward reduced responsiveness to β-adrenergic stimulation is consistent with previous data in myocytes isolated from patients with heart failure26 and may reflect β-receptor downregulation and/or distal changes in β-adrenergic signal transduction, consistent with the severity of heart failure produced in this animal model.
Data from other investigators27 concluded that Na+ current is not altered in failing human myocytes. We observed no significant difference in Na+ current measured in cells isolated from the failing canine ventricle compared with control cells. The currents activated at test potentials above −50 mV and peaked at ≈−25 mV (Fig 3A⇓). The peak current density did not differ between the two groups (−12±7 pA/pF [control hearts, n=5] versus −15±4 pA/pF [failing hearts, n=3], Fig 3B⇓). Steady state inactivation was similar in both groups, with a half-inactivation midpoint and slope factor of −73±0.6 and 3.8±0.3 mV, respectively, in cells isolated from failing ventricles and −71±0.6 and 4.4±0.3 mV, respectively, in control cells (P=NS). The rate and voltage dependence of whole-cell current decay were similar in both groups, and the currents were almost completely inhibited by 20 μmol/L tetrodotoxin (data not shown).
It does not appear that changes in depolarizing currents mediate the action potential prolongation in canine pacing-induced tachycardia. We next examined changes in K+ currents.
IK1 is important in maintaining the resting membrane potential and in shaping the contour of the action potential. Fig 4A⇓ illustrates representative whole-cell currents in myocytes from failing and control hearts. The density of IK1 in this preparation is similar to that reported in human ventricular myocytes.6 Cells from failing hearts exhibit a reduction in the current density at negative potentials, which is confirmed by the pooled steady state current-voltage relations. The curves diverge significantly between test potentials of −90 to −150 mV (P<.05, Fig 4B⇓). The relevant component of IK1 for repolarization is the outward current “hump” observed at potentials positive to the K+ reversal potential. The peak outward component of IK1 at −60 mV was significantly decreased in cells from failing hearts (1.7±0.2 pA/pF) compared with control cells (2.5±0.2 pA/pF, P<.05) (Fig 4B⇓, inset).
Fig 5A⇓ shows representative single-channel IK1 records from cells isolated from failing and control hearts. Channel activity appears with voltage steps negative to the K+ reversal potential (≈0 mV under these ionic conditions). The open duration of the channels decreases monotonically with more hyperpolarized voltage steps; subconductance current levels were observed in both groups. Representative single-channel current-voltage relations demonstrate no difference in the IK1 single-channel conductance of cells from failing and control hearts (Fig 5B⇓). The average slope conductance for four control and three failing cells was 28.6±1.4 and 30.8±0.9 pS, respectively (P=NS). Thus, the difference in IK1 whole-cell current density reflects either a decrease in open probability, a modest reduction in channel number, or a change in the frequency of substate openings.
The attenuation of the prominent notch in phase 1 of the action potential of myocytes from failing hearts suggested a reduction in Ito. Canine ventricular myocytes exhibit two components of transient outward current: a Ca2+-independent 4-AP–sensitive current and a Ca2+-dependent current.28 29 We focused on the former, which has previously been shown to be decreased in failing human ventricular myocytes,6 10 and refer to it simply as Ito. The Ca2+-dependent component was suppressed in the present study by the inclusion of 5 mmol/L EGTA in the intracellular solutions.
Upon depolarization, control myocytes exhibit a rapidly activating Ito, which then decays to a nonzero steady state current level (Fig 6A⇓, left). Cells isolated from failing canine hearts exhibit a dramatic decrease in this current (Fig 6A⇓, right). Panels B and C of Fig 6⇓ show the current-voltage relations of the peak and the steady state current components for 20 cells from failing hearts and for 15 control myocytes. Peak current density measured at +80 mV was significantly reduced in the failing myocytes (7.0±0.9 pA/pF [failing hearts] versus 20.4±3.2 pA/pF [control hearts], P<.001). In contrast to the peak current, the steady state component was not significantly altered (4.6±0.9 pA/pF [failing hearts] versus 3.2±0.6 pA/pF [control hearts], P=NS).
The reduction in whole-cell Ito could reflect a shift in the voltage dependence of activation and/or inactivation. Alternatively, maximal Ito conductance may be reduced. To distinguish between the possibilities, we characterized the kinetics of Ito in more detail. In the myocytes from both failing and control hearts, Ito activated at test potentials of ≥−10 mV. The voltage dependence of activation was determined by measurement of tail currents at −20 mV. Cells from failing hearts and control myocytes had similar activation curves (Fig 7A⇓). The half-maximal activation voltages and slope factors were 4.2±0.2 and 8.2±0.4 mV, respectively, for failing myocytes and 7.1±0.3 and 11.6±0.4 mV, respectively, for control myocytes (P=NS). Other gating properties of Ito were also unchanged in cells from failing hearts. The voltage dependence of steady state inactivation, assessed using a standard two-pulse protocol, did not differ between the two groups (half-inactivation voltages and slope factors of −33±0.4 and 5.5±0.3 mV, respectively, for failing cells versus −37±0.2 and 4.7±0.2 mV, respectively, for control cells). These steady state gating parameters are comparable to those reported previously for cardiac ventricular Ito in several species.17 28 30 The time course of inactivation was determined by single-exponential fits to the whole-cell current decay over a range of test voltages from +30 to +80 mV. Pacing tachycardia–induced heart failure did not affect the time course of inactivation of Ito (Fig 7C⇓). The rates of current decay in cells from failing hearts and in control cells overlapped at all potentials. The recovery kinetics at −100 mV were determined using a standard two-pulse protocol. Repriming in cells from failing hearts and in control cells is biexponential; neither the time constants nor the relative amplitudes of the components differed significantly between the groups (Fig 7D⇓).
By exclusion, the reduction in macroscopic Ito density in myocytes from failing hearts appears to reflect an underlying decrease in maximal Ito conductance at the whole-cell level. This could be due to alterations in channel number, single-channel conductance, or open probability. Therefore, we further examined the mechanism of the reduction in Ito density using single-channel recording. Fig 8A⇓ shows representative single-channel current records at various test potentials (+40 to +80 mV) from a holding potential of −80 mV. The channels tend to open at the beginning of the voltage step and rapidly inactivate; openings late in the voltage steps are rare. This single-channel gating pattern was similar in the myocytes from failing hearts and in the control myocytes and was reflected in the similar decay rate of the ensemble-averaged currents (Fig 8B⇓). The whole-cell (WC) current decay at +80 mV was faithfully reproduced by the ensemble-averaged (EA) current (τEA, 55 milliseconds [failing hearts] and 50 milliseconds [control hearts]; τWC, 34 milliseconds [failing hearts] and 34 milliseconds [control hearts]). The ensemble averages, corrected for channel number and represented as open probabilities, show the same peak opening probability in myocytes from failing hearts and in control myocytes (Fig 8B⇓). The single-channel conductance of Ito in myocytes from failing and control hearts is nearly identical (Fig 8C⇓). Differences between the conductance measured in the present study and in other studies30 31 32 33 34 reflect differences in recording conditions, species, or tissue of origin. Previous recordings were from ferret,30 mouse,31 and rat34 ventricle and rabbit atrium32 ; these are the first single-channel Ito recordings from canine ventricular myocytes.
The absence of changes in single-channel current or unitary open probability suggest, by exclusion, that the number of functional Ito channels is reduced. As a more direct means of estimating number of functional channels in myocytes from failing and control hearts, we used nonstationary fluctuation analysis of whole-cell Ito. Fig 9⇓ shows plots of variance as a function of mean current in a normal cell (left) and in a cell isolated from a failing ventricle (right). The peak of the parabolic fit yields the number of functional channels, which is obviously higher in the control cell. Pooled data from three control and three cells from failing hearts are summarized in Table 2⇓, illustrating that channel density was significantly reduced in myocytes from failing hearts.
Can the Reduction of Ito Account for the Prolongation of Action Potentials?
If the reduction in Ito that we observed is sufficient to produce prolongation of the action potential of myocytes from failing hearts, pharmacological inhibition of Ito in a control cell should result in action potential prolongation. Blockers of K+ channels are not specific, but low concentrations of 4-AP preferentially inhibit Ito. Peak Ito elicited by a voltage step to +60 mV is >50% blocked by 2 mmol/L 4-AP and almost completely eliminated by 5 mmol/L, with a half-blocking concentration (IC50) of 1.1 mmol/L (Fig 10A⇓). Peak Ito is inhibited over the entire range of test voltages, with more pronounced block at positive potentials (Fig 10B⇓ and 10C⇓, right). The records also confirm that there is little effect on the maintained component of Ito. To determine whether the drug was having an unanticipated effect on currents other than Ito, we examined the effect of 4-AP on total membrane current over a wide range of voltages. Fig 10C⇓ shows the result of such experiments in three control cells and three myocytes from failing hearts. 4-AP had no effect on total membrane current over the range of test potentials from −150 to +20 mV but did reduce outward current at more positive voltages, consistent with a predominant effect on Ito.
We then examined the effect of 4-AP on action potentials. Fig 11A⇓ illustrates the action potential prolongation by 4-AP, with attenuation of the phase-1 notch and no change in the resting membrane potential. These changes mimic faithfully the action potential contour in failing myocytes. Fig 11B⇓ summarizes the effects of 4-AP on six control cells. The amplitude of the notch was consistently reduced, and APD50 was significantly increased. In the absence of a substantial effect of 4-AP on other membrane currents, the present findings support the notion that the reduced Ito in failing myocytes is important in the prolongation of repolarization.
If inhibiting Ito in normal cells can reproduce the failing action potential phenotype, it is logical to predict that an action potential from a failing ventricular myocyte may be normalized by simply reconstituting the electrical effect of Ito. We addressed this question by injecting a brief repolarizing current pulse during phase 1 of the action potential in myocytes from failing hearts to mimic the effect of Ito. If Ito is essential for setting the plateau potential and indirectly influencing the trajectory of the remainder of the action potential, artificially restoring the notch should shorten the APD. In Fig 11C⇑, a 250-pA repolarizing current pulse delivered for 8 milliseconds during phase 1 was sufficient to shorten the duration of the action potential. This pulse represents an underestimate of the current contribution of Ito during this phase of the action potential. Nonetheless, in 9 of 10 cells in which the protocol was attempted, APD50 and APD90 were shortened (P<.02). Thus, APD in cells from failing hearts could be effectively abbreviated by brief injections of repolarizing current during phase 1 of the action potential.
Action potential prolongation is a prominent feature of cardiac myocytes isolated from pacing tachycardia–induced heart failure in the dog. The changes include prolongation of both APD50 and APD90 and a dramatic attenuation of the notch in phase 1 without a significant change in the resting membrane potential. There is a concomitant reduction in Ito density with a significant reduction in channel number estimated from nonstationary fluctuation analysis. We also observed a reduction in IK1, which may be involved in the terminal phase of repolarization. Our findings are consistent with reports of heart failure–induced action potential lengthening in other species35 36 as well as those described in human cells isolated from failing hearts explanted at the time of cardiac transplantation.6 16
Significance of the Canine Pacing-Induced Heart Failure Model
Congestive heart failure induced by rapid pacing in the dog exhibits many mechanical and neurohumoral derangements reminiscent of human heart failure. After 3 to 4 weeks of rapid ventricular pacing, the animals exhibit marked contractile depression, chronic chamber remodeling, and blunted β-adrenergic responsiveness.12 Neurohumoral activation is evident as a substantial rise in plasma and myocardial norepinephrine levels.37 Biochemical abnormalities in the β-adrenergic receptor–G protein–adenylyl cyclase pathway resemble those in human heart failure.38 39 Therefore, this model appears to reproduce the major mechanical, biochemical, and clinical features of human heart failure. As an important first step in determining the mechanism of repolarization abnormalities that develop with heart failure and their relation to ventricular tachyarrhythmias and sudden cardiac death, we have characterized the cellular electrophysiology of this model. As we sought to compare our results directly with those obtained in human myocytes, the experimental conditions were designed to mimic those of Beuckelmann et al6 and Näbauer et al.18
Canine ventricular myocytes exhibit two components of the transient outward current28 29 : the Ca2+-independent transient outward component (referred to here as Ito) and a Ca2+-dependent component (sometimes referred to as Ito2). The Ca2+-dependent component of Ito is not active under the present experimental conditions, given the presence of 5 mmol/L EGTA in the intracellular solutions. The density of Ca2+-independent Ito was reduced by 66% in cells from failing hearts compared with cells from control hearts (at +80 mV). The voltage dependence of activation and inactivation curves did not differ in ventricular myocytes from failing hearts compared with those from nonfailing control hearts. The kinetics of whole-cell current decay could be fit with a single exponential, and the rates of decay did not change with the induction of heart failure. Recovery of peak Ito at a physiologically relevant voltage was biexponential and did not differ in cells from failing hearts and cells from control hearts. The absence of changes in single-channel conductance and open probability left only a reduction in channel number as an explanation for the reduction in the current density in cells from failing hearts; this interpretation was confirmed by nonstationary fluctuation analysis. The mechanism of the reduction in functional channel number is still under investigation, but the unchanged electrophysiological properties of the residual current remaining in cells from failing hearts argues against a posttranslational mechanism. Alternatively, a reduction in transcription (or translation) of Ito genes could reduce channel number. At present, these mechanisms remain speculative; indeed, the prospects for elucidating these mechanisms are rendered somewhat daunting, given the uncertain identity of the molecular components of Ito.
Comparison With Human and Other Animal Data
Our data are quite consistent with those reported in similarly isolated human ventricular myocytes.6 18 However, other laboratories have not found a significant difference in the current density of Ito in cells from normal and failing human hearts.10 The cause of this discrepancy is not known, but several possibilities exist. An important difference may be the method of cell isolation. We used a coronary perfusion technique similar to that described by Beuckelmann et al16 rather than a chunk method and observed a viable cell yield of 20% to 40% compared with a considerably lower yield when the chunk method is used. The use of a coronary artery for perfusion may limit cell damage during isolation because of superior perfusion and oxygenation of the tissue. In addition, we restricted our sampling to the midmyocardium, a region of the myocardial wall with a large Ito density. It is possible that Ito in these “M” cells17 40 has a fundamentally different response to myocardial failure than either the endocardial or epicardial cells studied by Wettwer et al.10 Species differences may also have significant effects on the Ito density and its change in heart failure. A complicating issue in all human studies, which is circumvented in the present study, is the variability in the etiology, stage, and prior treatment of the explanted hearts from which cells were isolated.
The transmural variability in Ito density has been well documented.10 17 41 Since our main goal was to compare electrical properties in the presence and absence of heart failure, we isolated cells from only one region of the heart, the midmyocardial layer (see “Materials and Methods”). We did not address possible regional changes or alterations in the transmyocardial Ito gradient that may occur in heart failure.
Other K+ Currents
IK1 may also contribute to repolarization of ventricular myocytes, at least at near-diastolic potentials. We observed a 40% reduction (at −150 mV) in the steady state current density of IK1 in ventricular myocytes from failing canine hearts compared with nonfailing control cells. However, the reduction at very negative potentials will not influence the action potential profile. Outward current through IK1 is observed at voltages positive to the K+ reversal potential and is likely to affect the terminal phase of the action potential. The small but significant reduction in the outward IK1 component observed in myocytes from failing hearts (Fig 4B⇑, inset) could account for some of the prolongation of APD90 but is unlikely to figure in the equally prominent abbreviation of APD50.
The delayed rectifier K+ current (rapidly and slowly activating components) has recently been described in canine ventricular myocytes. However, this current is present at a much lower density (0.4 to 0.9 pA/pF) in midmyocardial cells than is Ito.42 The change in the action potential contour in the present study suggested a downregulation of Ito. The delayed rectifier K+ current was never resolved under our experimental conditions; if present, it would form part of the “maintained component” of Ito, which was not altered in cells from failing hearts. Nevertheless, the fact that these currents were not specifically examined in the present study does not exclude the possibility that they may influence repolarization, particularly under more physiological conditions (37°C, no cell dialysis) than those used.
Significance of Reduced Ito in Heart Failure for Alterations in APD
If the major change in cells from failing canine ventricles is a reduction in Ito, the obvious question is whether this change in Ito suffices to explain the observed changes in the action potential profile and duration. The main features of the action potential of ventricular myocytes isolated from failing hearts could indeed be reproduced by exposing myocytes from normal hearts to 5 mmol/L 4-AP. Under our recording conditions, this concentration of 4-AP affects only Ito. In fact, 4-AP prolongs the action potential more dramatically in myocytes from failing hearts than in normal myocytes (data not shown), implying that myocytes from the failing heart are even more dependent on the residual Ito for repolarization. We further demonstrated the importance of Ito in ventricular repolarization by restoring the action potential notch in myocardial cells from failing hearts. Abbreviation of the APD was effected by artificially recreating the phase-1 notch with a repolarizing current pulse delivered during the first 8 milliseconds of the action potential. These data suggest that Ito, despite its transient nature, is important in determining the shape and duration of the ventricular action potential.
Physiological and Clinical Implications
Ito is prominent in normal canine ventricular myocardium, but its physiological role has not been fully defined. The prominent notch found in myocytes that have been isolated from the midmyocardium and the initial level of the plateau of the action potential have been attributed to Ito.6 17 18 The importance of Ito in the genesis of cardiac arrhythmias is suggested by its reduction in arrhythmogenic substrates such as heart failure (Reference 6 and the present study), myocardial infarction,43 hypertrophy,44 and anoxia.45 Nevertheless, much remains to be learned regarding the mechanistic links between the downregulation of Ito and the pathogenesis of ventricular arrhythmias.
Selected Abbreviations and Acronyms
|APD||=||action potential duration|
|APD50, APD90||=||APD at 50% and 90% repolarization|
|IK1||=||inward rectifier K+ current|
|Ito||=||Ca2+-independent 4-AP–sensitive transient outward current|
|LAD||=||left anterior descending coronary artery|
This study was supported by grants HL P50 52307 (SCOR in Sudden Cardiac Death) and RO1 HL-47511 (Dr Kass) from the National Institutes of Health; by an American Heart Association, Maryland Affiliate, Inc, Grant-in-Aid (Dr Tomaselli); and by fellowships from the National Institutes of Health T32 HL-07227 (Dr Pak), the Medical Research Council of Canada (Dr Chiamvimonvat), the American Heart Association, Maryland Affiliate, Inc (Dr Nuss), and the Deutsche Forschungsgemeinschaft (Dr Kääb). We are grateful to Richard S. Tunin for his expert technical assistance.
- Received May 24, 1995.
- Accepted October 17, 1995.
- © 1996 American Heart Association, Inc.
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