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Cardiovascular Research 2002 54(1):42-50; doi:10.1016/S0008-6363(02)00236-5
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Repolarization abnormalities and their arrhythmogenic consequences in porcine tachycardia-induced cardiomyopathy

Dominique Lacroixa,*, Pascale Gluaisb, Christelle Marquiéa, Christine D'Hoinnea, Monique Adamantidisb and Michèle Bastideb

aDepartment of Cardiology, University of Lille, Lille, France
bDepartment of Pharmacology, University of Lille, Lille, France

* Corresponding author. Present address: Cardiologie A, Hôpital Cardiologique de Lille, Boulevard du Pr Leclercq, F-59037 Lille (Cedex) France. Tel.: +33-320-445-038; fax: +33-320-446-898 dlacroix{at}chru_lille.fr

Received 12 September 2001; accepted 27 December 2001


   Abstract
Top
 Abstract
1 Methods
 2 Results
 3 Discussion
 Acknowledgments
 References
 
Objectives: Action potential prolongation related to the alteration of several membrane currents is constantly reported in heart failure (HF) but reports about its role in arrhythmogenesis are sparse. Our aim was to determine, by analogy with long QT syndromes, whether prolonged repolarization is associated with increased dispersion or linked to bradycardia-dependent ventricular arrhythmias in pacing-induced cardiomyopathy. Methods: QT intervals, action potentials and transmural activation-to-recovery intervals (ARIs) along with whole-cell delayed rectifier (IK) and transient outward (Ito1) K+ currents were recorded in left ventricle from pigs with HF and controls. HF was obtained after 14 days of rapid pacing at 250 ms. Results: Repolarization was delayed as indexed by corrected QT intervals (13.7% increase, P<0.01) or ARIs (252±4 to 340±7 ms, P<0.01). ARIs were uniformly prolonged with disappearance of the transmural gradient, spatial dispersion of repolarization decreased by 50% (P<0.05). Ito1 density was reduced in HF from 1.35±0.1 to 0.57±0.04 pA/pF subepicardially, from 1.05±0.19 to 0.55±0.08 pA/pF midmyocardially and from 1.04±0.1 to 0.48±0.04 pA/pF subendocardially. IK density was significantly decreased in HF pigs vs. controls: subepicardially from 0.46±0.04 to 0.22±0.02 pA/pF; midmyocardially from 0.46±0.05 to 0.25±0.03 pA/pF; and subendocardially from 0.49±0.04 to 0.20±0.04 pA/pF following depolarization at +50 mV. Electrocardiogram (ECG) monitoring at the time of death did not disclose any polymorphic ventricular tachyarrhythmia. Conclusion: Despite a profound alteration in K+ currents, repolarization is uniformly prolonged in this model with no proclivity to develop bradycardia-dependent arrhythmias.

KEYWORDS Heart failure; Ion channels; Mapping; Ventricular arrhythmias

This article is referred to in the editorial by R. Coronel (pages 11–12) in this issue.

Electrical remodeling characterized by isolated action potential prolongation has been documented in patients with heart failure as well as in experimental models [1]. Among the available models, tachycardia-induced cardiomyopathy has been extensively studied especially in the dog [2,3]. In this model, prolongation of repolarization has been mainly ascribed to a downregulation of the K+ channels carrying the transient outward current Ito [4]. Available evidence demonstrates a downregulation at the transcriptional level with a decrease in mRNA encoding the corresponding Kv 4.3 channels [5]. A decrease in the inwardly rectifying K+ current IK1 [4], a reduced activity of the Na+/K+ pump current and an enhanced activity of the Na+/Ca2+ exchanger have also been shown [1]. However, reports linking this remodeling to arrhythmogenesis remain scarce [6,7]. Simulation studies suggest a marginal role of the decrease in Ito and the utmost importance of the delayed rectifier K+ current IK in failing myocytes [8]. Both components of this latter (IKr and IKs) have been found downregulated in a rabbit model of pacing-induced cardiomyopathy [9] but not in the dog model. This observation is of paramount importance, and at least two hypotheses have been formulated about the consequences of a decrease of this primary repolarizing current [10]. First, prolonged action potential duration may promote the occurrence of bradycardia-dependent early afterdepolarizations (EADs) which in turn can initiate triggered activity. In addition, variation in action potential duration may create dispersion of repolarization with non uniform recovery from inexcitability favoring functional reentry. Second, the larger intracellular Ca2+ load indirectly induced by a prolonged repolarization enhances the role of the Na+/Ca2+ exchanger in its normal mode generating an inward current which is thought to be an important potential mechanism of delayed afterdepolarizations (DADs). Therefore our purpose was to seek a downregulation of K+ currents with a focus on a possible decrease of IK and to explore its arrhythmogenic consequences in a large animal model of heart failure.


   1 Methods
Top
 Abstract
 1 Methods
2 Results
 3 Discussion
 Acknowledgments
 References
 
Following Chow et al. [11], experiments were performed on 13-week-old female hybrid pigs weighing 35–38 kg. All procedures for animal care and experimentation followed the NIH guidelines of the Guide for the Care and Use of Laboratory Animals, and were approved by an institutional committee.

1.1 Tachycardia-induced cardiomyopathy
Under deep sedation (subcutaneous ketamin hydrochloride 20 mg kg–1, nalbuphin 0.5 mg kg–1 and midazolam 0.25 mg kg–1) and after local anaesthesia with lidocaine a venous access to the right jugular vein was prepared surgically. A unipolar pacing electrode with active fixation (Medtronic, USA) was inserted into the right atrial appendage under fluoroscopic guidance and connected to a modified pulse generator (courtesy of Medtronic France) which was implanted subcutaneoulsy in the neck. Atrial pacing at 250 ms was activated 24 h later for 14 days. A body surface electrocardiogram (ECG) was obtained every 3 days to verify atrial capture and 1:1 atrioventricular conduction. Post-operatively, antibiotics (cefamandole 1.5 g/day) were injected for 3 days. Control animals underwent the same procedure but the pulse generator was not turned on. Animals were examined on a daily basis, oedema of the paws indicated right ventricular failure; symptoms of low output included asthenia, inability to stand up, anorexia, vomiting, mottling of the skin. Tachypnea, cyanosis and lung crepitations suggested pulmonary congestion.

The pacemaker implantation was carried out in 45 pigs; 27 were paced to induce heart failure the remaining 18 were unpaced as controls. Six paced animals (22%) died during the 2nd week of pacing, five from congestive heart failure the last one suddenly. Seven control animals and seven pigs with heart failure were sacrificed for hemodynamic and mapping experiments. Four failing animals were used for purpose of prolonged ECG monitoring. The remaining 10 animals with heart failure as well as 11 controls were sacrificed for electrophysiological experiments. Volumes of pericardial, pleural and abdominal fluids were measured at autopsy.

In the last nine failing pigs echocardiography was performed before sacrifice after light sedation (midazolam 0.25 mg kg–1) and compared with a preceding one obtained at baseline. A complete M-mode and two-dimensional examination was performed with the animal lying on its left side with a Vingmed Sound CFM750 (courtesy of General Electric France) equipped with a 3.25 MHz probe. These examinations were done following the standards developed by the American Society for Echocardiography. Measurements were recorded together with a lead II ECG at a speed of 25 mm s–1 with a fiber-optic recorder and generated in triplicate. These paper recordings were reassessed by a second independent observer then averaged. M-mode study of the left ventricle was obtained from a long-axis left parasternal view with the probe in the fifth intercostal space. Care was taken to obtain simultaneous recordings of the interventricular septum and of the left posterior free wall in a section of the left ventricle below the maximal deflection of the mitral valve leaflets, and to direct the ultrasound beam perpendicular to the posterior wall.

1.2 Isolated perfused porcine heart preparations
Under anaesthesia (IV sodium thiopentone 10 mg kg–1 and nalbuphin 1 mg kg–1) the thorax was opened by a midsternal incision and a small 16-gauge catheter was inserted directly into the left ventricle (Portex, UK) to record pressures via a high fidelity transducer (Harvard Apparatus, USA). Then the heart was removed and connected to a Langendorff non-working recirculating perfusion system with a modified Tyrode's solution, the details of this procedure have been published previously [12].

Sixteen plunge needle electrodes were inserted in the free wall of the left ventricle (between the papillary muscles). These electrodes contained six unipolar electrodes, each pole separated by 1.5 mm. Electrodes were inserted through a flexible plate containing 16 holes, this grid consisted of a 4x4 square arrangement with a distance of 7.5 mm between rows and columns. The grid was centered on the basal third of the ventricle with the first electrode row parallel to the mitral annulus and 1 cm apart. The sinoatrial nodal area was crushed so allowing bipolar atrial pacing at 1000 ms. After termination of the experiment, each electrode was carefully removed and replaced by a rigid polyamid thread. A solution of paraformaldehyde was infused through the coronary arteries then the area of interest was removed, frozen and cut in 5 mm slices parallel to the mitral annulus. The outline of these slices was drawn to show the insertion and direction of each plunge electrode. The most proximal contacts were located {approx}0.5 mm from the epicardial surface. Usually the most distal poles did not reach the endocardial surface being distant from 1.5 to 3 mm.

1.3 Data acquisition and cardiac mapping
The mapping system and mapping methodology have been described elsewhere [12]. Activation–recovery interval (ARI) was chosen as index of local repolarization duration and measured on each of the 96 electrograms during atrial pacing at 1000 ms. It was defined as the interval between the time of minimum first derivative (dV/dtmin) in the activation complex (RS wave) to the maximum positive slope (dV/dtmax) in the T wave of each of the unipolar electrograms. Iso-ARI areas were derived from ARIs values and drawn at 10-ms intervals. Mean ARI (mARI) of a given layer was the arithmethic mean of the 16-ARI values obtained within a same beat, the dispersion index (DI) was expressed as the coefficient of variation.

1.4 Electrophysiological experiments
Isolation of single myocytes from the myocardial wedge related to the marginal branch of the circumflex artery was cannulated on a Langendorff perfusion apparatus. All solutions used during the cell dissociation procedure were oxygenated and kept at 37 °C. The wedge was first perfused with K+-enriched Tyrode's solution then with a Ca2+-free Tyrode's solution containing 0.83 mg/ml collagenase B, 0.13 mg/ml protease XIV and 0.44 mg/ml bovine serum albumin for 20 min [13]. Then the heart was rinsed with Kraftbrühe solution. Great care was taken to cut off very thin and superficial (<0.5 mm) samples of subepicardial and subendocardial muscle owing to the reported thinness of these layers [14]. Small parts were dissected from the central third of the ventricular wall. These fragments were minced in Kraftbrühe solution. After gentle agitation, the resulting myocytes suspension was filtered and stored at room temperature in Tyrode's solution (pH 7.35).

An aliquot of the cell suspension was placed in a recording chamber on the stage of an inverted microscope and perfused with the following solution: in mmol/l, NaCl, 135; KCl, 4; NaH2PO4, 0.8; CaCl2, 1; MgCl2, 1; glucose, 10; HEPES, 10; pH 7.4. The whole-cell configuration of the patch-clamp technique was employed at room temperature (20 °C) to evaluate the K+ currents by using a patch-clamp amplifier (RK 400, Biologic, France) and pCLAMP software (Axon Instruments, USA). Only Ca2+-tolerant cells with clear cross striations and without contraction were selected for experiments. Currents were low-pass filtered at 3 kHz and digitized at 3.33 kHz (Ito recordings) or 333.3 Hz (IK recordings) (Labmaster TL-1, Scientific Solutions, USA). Borosilicate glass pipettes had a resistance of 1.4–1.7 M{Omega} when filled with the intracellular solution (in mmol/l: KCl, 130; MgCl2, 2; K2-ATP, 3; EGTA, 10; HEPES, 10; creatine phosphate, 5; pH 7.3). Cell capacitance was calculated by integrating the area under a capacitive transient elicited by a 5-mV hyperpolarizing pulse from a holding potential of –50 mV. For comparison of currents, amplitudes were normalized to the cell capacitance which was 138±7 pF and 128±4 pF in failing and control myocytes, respectively (P=ns). Myocytes were superfused with a low-Na+ solution including N-methyl-D-glucamine (NMDG) (in mmol/l: NaCl, 54; KCl, 4; NMDG, 86; CaCl2, 1; MgCl2, 1; glucose, 10; HEPES, 10; pH 7.4).

In parallel, action potentials were recorded with standard glass microelectrode technique in strips of left ventricular epicardial muscle and from free running strands of Purkinje tissue dissected from the endocardium in the area of the posterior papillary muscle with ventricular muscle attached at both extremities. Preparations were mounted in a tissue bath and superfused continuously with Tyrode's solution (in mmol/l: NaCl, 118.2; KCl, 4; CaCl2, 1.8; MgCl2, 1; NaH2PO4, 1.8; NaHCO3, 25; glucose, 11; pH 7.35±0.05). The solution was gassed with carbogen (O2 95%–CO2 5%) at 36.5±0.5 °C. During stabilization the preparation was paced at 2000 ms (epicardial muscle) or 500 ms (Purkinje tissue) for 30 min then at 1250 ms for 2 h. Others methodological details are identical to those described in rabbit Purkinje studies from our laboratory [15].

1.5 ECG monitoring
In six animals, an automatic implantable electrocardiographic monitor (Reveal PlusTM, courtesy of Medtronic Inc.) was inserted subcutaneously in the precordial area at the time of the implantation of the pulse generator. Care was taken to choose a location allowing the recording of discernable P, R and T waves with appropriate relative amplitudes according to the policy of the manufacturer. Four of them survived the initial pacing period of 14 days, then pacing was suspended periodically for 3–5 days with the Reveal Plus device being activated. These monitoring phases alternating with pacing phases were repeated until spontaneous death occurred.

1.6 Statistical analysis
Group values are presented as mean±S.E.M. Measurements between groups were compared by ANOVA followed by appropriate post test or by Student's t-test according to the case. Welch's alternate t-test was applied when variances were not homogenous. Comparisons were two-tailed and statistical analyses were made using the INSTAT and PRISM software packages (GraphPad, USA).


   2 Results
Top
 Abstract
 1 Methods
 2 Results
3 Discussion
 Acknowledgments
 References
 
2.1 Characteristics of heart failure in surviving animals
Apart from six paced animals who died, severe symptoms of low output developed in 11 out of the 27 paced animals, frank pulmonary congestion also occurred in three of these 11 pigs. In the remaining 10 animals symptoms were moderate. Necropsy revealed pleural effusion (<100 ml), pericardial effusion (<20 ml), or ascites (<200 ml) in three, five and eight failing pigs, respectively. Control animals were asymptomatic and no effusion was detected at autopsy.

The heart weight-to-body weight ratio was higher in failing animals (7.6±0.48 g kg–1, n=7) compared to controls (6.4±0.26 g kg–1, n=7, P<0.05). Table 1 summarizes hemodynamic data: left ventricular end diastolic pressure and relaxation constants were increased and dP/dtmax was reduced in failing animals compared to controls. Table 2 gives the results of the echocardiographic measurements obtained in the last nine failing pigs at baseline and before sacrifice. Left ventricular ejection fraction fell from 67±2.5 to 34±3.5%, P<0.001, the end diastolic diameter of the left ventricle significantly increased by 5 mm and the septum as well as the posterior free wall thinned by 1.8 and 1.2 mm, respectively.


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  		Table 1 Hemodynamic findings in control and failing pigs

 

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  		Table 2 Left ventricular echocardiographic measurements in nine failing pigs

 
2.2 QT interval prolongation
In the failing pigs the corrected QT interval (Bazett's formula) was significantly prolonged by 49 ms (13.7%) after 14 days of pacing but the mean cycle length was decreased by 71 ms (13.3%). In contrast these variables did not change in control pigs (Table 3).


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  		Table 3 Electrocardiographic findings in control and failing pigs

 
In support of these results, epicardial action potentials were obtained in both groups confirming a lengthening of repolarization with a marked decrease in the phase 1 notch in failing hearts (examples are given in Fig. 1). The action potential duration at 90% of repolarization (APD90) was 402±20 ms (n=7) in fibers from failing compared to 320±23 ms (n=6) in fibers from control hearts (P<0.05) indicating an increase of 82 ms (or 26%). Other parameters are detailed in Table 4, but action potential amplitude, resting membrane potential and maximum upstroke velocity were unaltered.


Figure 1
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  		Fig. 1 Representative action potentials recorded from epicardial left ventricular muscle. Action potentials from failing animals are longer and the phase 1 is blunted. Pacing cycle length is 1250 ms.

 

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  		Table 4 Action potential characteristics of epicardial muscle in control and failing pigs

 
2.3 Transmural repolarization mapping
In all groups of animals, repolarization patterns were well organized as evidenced by ARI mapping. In failing animals, ARIs were uniformly longer and DIs computed from ARI values were lower. Composite data indicating the distribution of mARI and DI across the ventricular wall are summarized in Fig. 2. In control animals the mean ARI progressively increased from the subepicardial layers toward the endocardium from 246±4 ms to a maximum of 257±3 ms. This gradient of 11 ms was highly significant (ANOVA, P<0.0001). In failing pigs, mARIs were systematically longer at any depth within the ventricular wall, and the transmural gradient from epicardium to endocardium was no longer observed. Overall, the mARI was 340±7 ms in failing animals (mean from all layers) compared to 252±4 ms in controls (i.e. 88 ms longer or 35%). DIs were significantly lower in failing experiments compared to controls except in the subepicardial layer. Overall this index was 2.6%±0.5% in failing pigs compared to 5.2%±0.8% in controls.


Figure 2
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  		Fig. 2 Mean ARI (mARI) and dispersion of repolarization (DI) plotted as a function of the depth of the electrodes. Electrograms from 96 unipolar electrodes in 16 plunge needles are obtained in controls ({blacksquare}, n=7) and failing hearts (bullet, n=6). Levels from 1 to 6 represent contacts separated by 1.5 mm from subepicardial muscle (Epi=level 1) toward subendocardium (Endo=level 6). Heart failure increases repolarization duration but decreases dispersion at all levels except subepicardially. * P<0.05 vs. controls, ** P<0.01 vs. controls.

 
2.4 Transient outward current (Ito1)
We focused on the Ca2+-independent 4-aminopyridine-sensitive component of Ito and suppressed its Ca2+-dependent component by inclusion of 10 mmol/l EGTA in the intracellular solution. Whole-cell currents were elicited by application of 380-ms depolarizing voltage steps between –30 and +70 mV in increments of 10 mV from a holding potential of –80 mV. Ito1 current was measured in solution including nifedipine 5 µmol/l or Co2+ 4 mmol/l for blockade of ICa-L. INa was blocked by tetrodotoxin 40 µmol/l or by a 30 ms pre-pulse from –80 to –50 mV. Ito1 was measured as the difference between the peak outward current and the sustained current at the end of the depolarizing pulse. Representative current records obtained from subepicardium, midmyocardium and subendocardium in failing and control experiments are shown in Fig. 3A.


Figure 3
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  		Fig. 3 Heart failure reduces the peak Ito1current in subepicardial, midmyocardial and subendocardial myocytes. In (A), representative examples of currents in different types of myocytes from control (left) and failing hearts (right) from a holding potential of –80 mV in response to voltage steps of 380 ms from –30 to +70 mV in 10 mV increments. In (B), left: peak current–voltage relations for subepicardial ({blacksquare}, n=11), midmyocardial ([medium shade square], n=6) and subendocardial cells ({square}, n=11) from control hearts. Peak Ito1 is stronger in subepicardial than subendocardial myocytes above +40 mV (* P<0.05). In (B), right: peak current–voltage relations for subepicardial (bullet, n=8), midmyocardial ([medium shade circle], n=7) and subendocardial cells ({circ}, n=8) from failing hearts.

 
Fig. 3B showed the mean current density–voltage relationships of Ito1 obtained from control and failing myocytes. A significant current density gradient was evidenced between epicardial and endocardial layers in control animals for potentials from +50 to +70 mV. The current density values at +50 mV in 11 control subepicardial cells were significantly higher than in 11 control subendocardial cells: 1.35±0.10 pA/pF vs. 1.04±0.10 pA/pF, respectively (P<0.05). Midmyocardial cells (n=6) displayed values similar to those obtained subendocardially (1.05±0.19 pA/pF).

In failing pigs this gradient disappeared, the current density–voltage relationships obtained from subepicardial and subendocardial myocytes were closely superimposed. Moreover the current density was significantly reduced in all layers: 0.57±0.04 pA/pF in eight subepicardial cells (P<0.001), 0.55±0.08 pA/pF in seven midmyocardial cells (P<0.05) and 0.48±0.04 pA/pF in eight subendocardial cells (P<0.001) at +50 mV.

2.5 Delayed rectifier current (IK)
Total IK was activated by applying voltage steps for 3000 ms from a holding potential of –40 mV to a range of depolarizing levels between –30 and +50 mV in increments of 10 mV [16]. IK was measured as tail current on repolarization at –40 mV in solution including Co2+ 4 mmol/l for blockade of ICa-L and tetrodotoxin 40 µmol/l for blockade of INa. The null tail current observed when E4031 (5 µmol/l; a specific IKr blocker) was applied suggested that in our experimental conditions the IKs component was not detected (n=6 myocytes, data not shown) therefore we considered IKr as the main component of IK in our preparations. The IK tail current amplitude was estimated as the difference of peak and steady-state current. Representative tracings obtained in control and failing myocytes are illustrated in Fig. 4A and B.


Figure 4
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  		Fig. 4 Heart failure reduces the delayed rectifier tail current IK in subepicardial, midmyocardial and subendocardial myocytes. Representative families of currents recorded by applying depolarizing potentials from a holding potential of –40 mV to levels of –10, +10, +30 and +50 mV for 3000 ms are shown in control myocytes (panel A) and myocytes from failing hearts (panel B). Tail currents are reduced in the three types of cells compared to controls. In (C), average tail current–voltage relations plotted for subepicardial failing (bullet, n=7), and control myocytes ({blacksquare}, n=9) (left panel), for midmyocardial failing ([medium shade circle], n=6) and control myocytes ([medium shade square], n=7) (middle panel) and subendocardial failing ({circ}, n=7) and control myocytes ({square}, n=6) (right panel). * P<0.05 vs. controls, ** P<0.01 vs. controls, *** P<0.001 vs. controls.

 
The mean tail current–voltage relationships normalized by cell capacitance are plotted as a function of test potentials in Fig. 4C. In failing myocytes from subepicardium, midmyocardium and subendocardium the tail currents on repolarization of IK were smaller than those obtained in control cells. In subepicardial myocytes, the current density was significantly reduced in cells from failing hearts from +10 mV to more positive potentials (at +50 mV, 0.22±0.02 pA/pF, n=7, vs. 0.46±0.04 pA/pF, n=9, P<0.001). In midmyocardial myocytes, the current density was also significantly lower in cells from failing hearts from +10 mV to more positive potentials (at +50 mV, 0.25±0.03 pA/pF, n=6, vs. 0.46±0.05 pA/pF, n=7, P<0.01). In subendocardial myocytes as well, the current density was significantly reduced in cells from failing hearts from +20 mV to more positive potentials (at +50 mV, 0.20±0.04 pA/pF, n=7, vs. 0.49±0.04 pA/pF, n=6, P<0.001).

2.6 Arrhythmogenesis
Prolonged ECG monitoring was obtained in four pigs. From these experiments the cumulative duration of monitoring of native rhythms was 38 days. Extreme bradycardia or asystole was the terminal event in every animal. Spontaneous cycle lengths >1000 ms were commonly observed in the hours preceding death. A single episode of monomorphic ventricular tachycardia was recorded 2 h before death in an animal later found asystolic (Fig. 5). The automatic detection criteria for the device was the occurrence of at least 16 consecutive beats with a cycle length <333 ms, therefore short runs of non sustained ventricular tachycardia as well as premature beats or slower tachycardia episodes were not captured. Moreover, this automatic detection precluded any arrhythmia recording during the pacing phases.


Figure 5
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  		Fig. 5 ECGs recorded at the time of death in three pigs with heart failure after cessation of pacing (panels A, B and C). In all cases the terminal event is electromechanical dissociation with severe sinus or ventricular bradycardia (cycle lengths are 1400, 1450 and 900 ms in A, B and C, respectively). In panel (A), whereas sinus tachycardia is recorded 150 min before death (cycle length 275 ms) monomorphic ventricular tachycardia (cycle length 240 ms) occurs 120 min before the final event.

 
Despite pacing at long cycle lengths (i.e. from 1000 to 5000 ms) EADs were not obtained in free running strands of Purkinje fibers from failing hearts. This was observed in corroboration of the lack of bradycardia-dependent polymorphic ventricular tachycardia. After addition of nor-epinephrine 1 µmol/l and atenolol 1 µmol/l resulting in {alpha} adrenergic stimulation, EADs were observed neither in fibers from failing myocardium nor in fibers from control animals.


   3 Discussion
Top
 Abstract
 1 Methods
 2 Results
 3 Discussion
Acknowledgments
 References
 
Electrical remodeling occurred in failing pigs after 14 days of atrial pacing. QTc interval increased by 49 ms (or 13.7%) as well as action potential duration or ARI measured in vitro. These latter indicated delays in repolarization duration greater than those observed in vivo mainly due to longer cycle lengths compared to the spontaneous heart rate of failing animals, the absence of mechanical load being a second possible factor. Intramural mapping in the left ventricular free wall showed a uniform prolongation of ARIs with a disappearance of the transmural gradient and a decrease in the dispersion of repolarization. As expected, Ito1 density was reduced in failing animals and found at a same low level in subepicardial, midmyocardial and subendocardial muscle; therefore the transmural gradient of the current also disappeared. This reduction in Ito1 density was associated to a substantial reduction in IK density, the reduction reaching the same magnitude in the different types of cells considered. Despite this profound alteration in K+ currents with the occurrence of bradycardia episodes, prolonged ECG monitoring at the time of death did not disclose any proclivity to develop polymorphic ventricular tachyarrhythmias.

3.1 Repolarization abnormalities in the intact heart
Prolongation of repolarization was much more pronounced in this porcine model compared to that reported in others species with tachycardia-induced cardiomyopathy. Pak et al. [7] reported a 8.3% increase in the corrected QT in a dog study, Tsuji et al. [9] a 6.8% increase in this parameter in their rabbit study.

In control hearts a transmural gradient of repolarization was found with a 11 ms difference, a value lower than that reported in the dog with similar methodology [17]. As detailed in the methods, true superficial layers are thin, it is likely that the true subendocardial layer was not explored by the distal contacts of the plunge electrodes. Furthermore, the use of multicontact unipolar electrodes is known to reduce the physiological electrical gradient because of shunts in the extracellular currents [14]. Nevertheless unipolar ARI is recognized as a reasonable approximation of action potential duration. In line with previous studies [17,18] this recording technique has been shown to be able to measure regional heterogeneity following exposure to Class III agents [12].

In the present study prolongation of repolarization was uniform across the ventricular wall and dispersion was reduced. Regional differences in repolarization duration have been rarely studied in the failing or hypertrophied heart [7,19] and mapping studies with appropriate spatial resolution are lacking. Actually, measurements done at sites separated by several centimeters are inadequate to disclose steep gradients. Strong repolarization gradients indeed, may provide refractory barriers around which reentry can occur. In the isolated canine heart, the induction of ventricular arrhythmia was observed when steep repolarization gradients (<10 s m–1) were generated between adjacent areas [20,21]. Our results do not indicate the occurrence of such abnormalities in the failing heart. Conversely, dispersion was reduced and the transmural gradient disappeared. This latter finding is in accordance with published studies in the hypertrophied heart [19].

3.2 Ito1density
This current has been suggested to contribute significantly to regional electrophysiological heterogeneity in myocardial cells and tissue of several animal species and to cause electrical gradients across the ventricular wall. A reduction in this current density has been constantly reported in this animal model as well as in patients with heart failure [4,22]. Consistent with these reports is our finding of a reduced density in all types of cells studied. We also denoted a disapperance of its gradient between subepicardial and subendocardial failing myocytes that may be advanced to explain the concomitant disappearance of the ARI gradient across the ventricular wall.

Data about regional or transmural differences in its reduction during heart failure are conflicting in human hearts. When cells from explanted failing and nonfailing donor hearts were compared, Wettwer et al. [23] reported that Ito1 was not different in subepicardial cells; however, it was larger in subendocardial cells from nonfailing hearts. Conversely, according to other investigators [24] the density of Ito1 was found significantly smaller in failing hearts in subepicardial cells but not different in subendocardial myocytes.

This current, however, is expressed not only in a cell type-specific but also in a species-specific fashion and subject to developmental changes. Its contribution to early repolarization appears to be moderate in normal immature porcine myocardium. Compared to the adult dog or to mature human myocardium the current density measured at room temperature in the present report was at least five to seven times lower [4,22,24]. Even when compared with 12-week-old dogs the current was found smaller [25].

3.3 IKdensity
This current has not been studied in human heart failure, although some authors did not relate any decrease in the mRNA of HERG, the gene encoding the rapid component of the current [5]. More recently, Tsuji et al. showed in tachycardia-induced cardiomyopathy in the rabbit a downregulation of both components of IK that predominates on its slow component [9]. The present report is in line with this finding in the same model but in large mammals. Given our experimental conditions it was not possible to make a separate analysis of IKr an IKs because this latter was not detected. This is a limitation that justifies further attempts to characterize IKs in the pig.

Additionally, our comparative analysis in different types of cells was in agreement with the absence of regional differences in the prolongation of repolarization duration in the intact heart. Specifically we did not show a different decrease in total IK density in midmyocardial myocytes whereas these cells are believed in the dog to possess a less dense IKs than cells from other layers [26]. This characteristic of midmyocardial cells however, cannot be necessarily transposed to porcine myocardium given the fact that some investigators did not find typical M cells in immature pigs [27].

3.4 Arrhythmogenic consequences
In accordance with studies describing automaticity or DADs but not EADs in failing human myocardium [6], EADs were not induced in Purkinje fibers. This result conflicts with a study that demonstrated a propensity to EADs in canine midmyocardial myocytes [28]. This discrepancy should be interpreted with caution because sensitivity to class III agents is lower in porcine Purkinje fibers compared to dog or rabbit fibers and EADs have not been described yet in this species [29]. Our results obtained in vivo confirm the arrhythmogenicity of the model. Our monitoring methodology however, is not without limitations: in two cases the final event occurred at night without witness, the possibility of post mortem recording of bradycardia cannot be ruled out in the absence of blood pressure monitoring. In addition, a single lead was available on the implantable monitor to classify the tachycardia as monomorphic. Despite these technical limitations, the main difference with syndromes of acquired or congenital repolarization prolongation (LQTS) came from the absence of recording of bradycardia-dependent polymorphic ventricular tachyarrhythmias a fact that can be linked to the lack of increased dispersion of repolarization. Theoretically, the absence of such spontaneous events should not preclude a greater risk upon exposure of the heart to IKr-reducing agents. Nevertheless, in this regard it is important to note that a placebo-controlled study was conducted with the IKr blocker dofetilide in 1518 patients with heart failure with no impact of the drug on mortality [30]. Clearly the failing process alters several important ionic currents in addition to K+ currents that were not explored in our study especially those involved in calcium homeostasis [1]. Therefore, electrical remodeling in porcine heart failure does not fully mimick LQTS.

Time for primary review 26 days.


   Acknowledgments
Top
 Abstract
 1 Methods
 2 Results
 3 Discussion
 Acknowledgments
References
 
The authors wish to thank Cécile de Monclin-Laiter and Alain Lasquellec from Medtronic France for their continuing technical support throughout this study.

Work supported by a Grant from CH&U Lille


   References
Top
 Abstract
 1 Methods
 2 Results
 3 Discussion
 Acknowledgments
 References
 

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