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Department of Cardiology, University of Heidelberg, 69115 Heidelberg, Germany
Submitted 5 November 2003 ; accepted in final form 10 May 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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regression of hypertrophy; mapping; torsade de pointes
In the present study, the CAVB model was used to address the following questions: Is CsA relevant in the prevention and/or regression of biventricular hypertrophy in dogs? Because morphological remodeling in the CAVB model is accompanied by electrical remodeling, how are potential effects of CsA on morphology related to electrophysiology? Finally, if changes in morphology and/or electrophysiology occur, is inducibility of PVT affected? Respective information should help determine whether hypertrophy represents an epiphenomenon, the cause of electrophysiological changes, or the anatomic substrate for PVT.
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METHODS |
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Model preparation.
In 45 foxhounds weighing 29.5 ± 1.5 kg, general anesthesia was induced with pentobarbital sodium (0.5 mg/kg) and maintained by halothane (
1.0%). Complete AV block was achieved by radio-frequency ablation of the AV node (17). Dogs were studied acutely (AAVB) or allowed to recover for 6 or 12 wk (CAVB). This related to dogs with or without CsA treatment. CsA (Sandimmune, Novartis) was administered orally at 10–20 mg·kg–1·day–1 in an effort to achieve plasma levels of 400–800 µmol/l. To exclude direct CsA effects on electrophysiology and to allow for an analysis of CsA effects on prevention and regression of hypertrophy, the following control (CTL) and treatment groups were formed: 1) AAVB, no treatment (CTL-AAVB, n = 6), 2) AAVB, permanent CsA treatment, starting 6 wk before AV node ablation (CsA-AAVB, n = 3), 3) CAVB for 6 wk, no treatment (CTL-CAVB-6W, n = 11), 4) CAVB for 6 wk, permanent CsA treatment, starting on the day of AV node ablation (CsA-CAVB-6W, n = 7), 5) CAVB for 12 wk, no treatment (CTL-CAVB-12W, n = 12), and 6) CAVB for 12 wk, permanent CsA treatment from week 7 to 12 (CsA-CAVB-12W, n = 6).
Echocardiographic analysis. Transthoracic echocardiographic analyses were performed before AV node ablation and every 2 wk thereafter with the dogs in the conscious state. A 2.5-MHz imaging transducer (Toshiba Sonolayer SSH-260 A) was used to determine LV end-diastolic diameter and thickness of the septum and posterior wall.
Recording techniques. To allow for bipolar recording and stimulation at 240 intramural sites at depths of 1, 4, 7, and 10 mm, 60 plunge needles (12 mm long) containing 4 bipolar electrodes each (interpolar distance 0.5 mm, interelectrode distance 2.5 mm) were used. Electrodes were connected to a 256-channel computerized multiplexer mapping system developed at the University of Limburg (Limburg, The Netherlands) (1). Thus, apart from measuring local refractory periods with the extrastimulus technique, reconstruction of tridimensional activation maps from local activation times was also possible (17).
Study protocol. Electrophysiological studies were performed 2 h, 42 ± 2 days, or 84 ± 1 days after AV node ablation. The dogs were reanesthetized, and the heart was exposed through an extended midsternal approach. After pericardectomy, all 60 needles were inserted into both ventricles in 5 transverse sections from base to apex (Fig. 1), as described elsewhere (17). The chest cavity was closed, and body temperature was adjusted to 38°C with a heating lamp. For 20 min, surface ECG leads I, II, and III and all intracardiac electrograms were continuously recorded to capture any spontaneously occurring arrhythmia. To determine local effective refractory periods (ERPs), 24 ± 10 electrode sites within the right ventricle (RV) and 32 ± 12 electrode sites within the LV were randomly selected for stimulation. At each site, stimulation thresholds and ERPs were measured at a basic cycle length of 800 ms. After eight basic stimuli (S1) at twice diastolic threshold amplitude, an extrastimulus (S2) was introduced, decreasing the S1-S2 coupling interval in steps of 10 ms. ERP was defined as the maximum S1-S2 interval that failed to evoke a propagated ventricular response. Dispersion of ERP was defined as the difference between the longest and the shortest ERP found at any site. All measurements were repeated after intravenous administration of 0.34 µmol/kg almokalant.
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200 beats/min. If no TdP occurred with the first dose of almokalant within 20 min, a second dose was given. Drug effects were observed for 20 min thereafter. Morphological measurements. After the experiment, the heart was excised. To determine ventricular weight, the ventricles were removed from the atria and the RV was separated from the LV; the septum was taken as part of the LV. The thickness of the anterior LV wall, the RV wall, and the septum 1 cm below the AV ring was determined for each heart (Fig. 2). Measurements were adjusted for body weight (heart weight/body weight).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Electrophysiological studies were performed in 9 dogs with AAVB (6 CTL and 3 CsA), 12 dogs with CAVB for 6 wk (6 CTL and 6 CsA), and 13 dogs with CAVB for 12 wk (7 CTL and 6 CsA).
With respect to morphological parameters (wall thickness and heart weight), there was no significant difference between CTL-CAVB-6W and CTL-CAVB-12W dogs or between CTL-AAVB and CsA-AAVB dogs.
Effects of CsA on development of ventricular hypertrophy. CAVB led to biventricular hypertrophy, indicated by a significant difference in heart weight-body weight index (HBWI) between CTL-AAVB and CTL-CAVB-6W dogs. CsA treatment did not totally prevent, but considerably attenuated, hypertrophy (Fig. 3). Accordingly, HBWI was significantly higher in CsA-CAVB-6W than in AAVB dogs but significantly lower than in CTL-CAVB-6W dogs. CsA treatment resulted in a 26.6% decrease in HBWI. The CsA effect was similarly reflected by total heart weight, LV weight, and RV weight. Compared with AAVB, CAVB led to a significant increase in wall thickness. Again, CsA treatment significantly reduced the increase in the thickness of the septal wall as well as the LV and RV walls. As a result, wall thickness was not significantly different between CTL-AAVB and CsA-CAVB-6W dogs. Data are summarized in Table 1. Given the operator dependence and the difficulties in obtaining echocardiographic data from conscious dogs, respective values must be interpreted with caution. Still, with respect to the time course of hypertrophy development, it appeared that LV end-diastolic diameter increased most 1–2 wk after AV node ablation, whereas the increase in wall thickness was most prominent 2–4 wk after AV node ablation (Table 2). CTL-CAVB-6W dogs had significantly thicker septal and posterior LV walls and larger end-diastolic LV diameters than CTL-AAVB dogs. In CsA-CAVB-6W dogs, the thickness of the septal and posterior LV wall was comparable to that in CTL-AAVB dogs. However, end-diastolic LV diameters were increased as in CTL-CAVB-6W dogs (Table 3). CAVB and/or CsA treatment did not seem to affect cardiac function.
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To discern whether CsA treatment exerts its effect on TdP inducibility primarily through trigger elimination or through substrate modification, the number of premature ventricular contractions (PVCs) occurring within the first 6 min after application of almokalant was compared for the various subgroups. Not revealing any significant difference, this comparison was limited by the relatively small number of dogs per group and by considerable interindividual variability in the incidence of PVCs (5.5 ± 3.9, 9.9 ± 2.9, 8.8 ± 3.7, 4.4 ± 3.0, 3.0 ± 1.8, 3.3 ± 1.6, and 6.4 ± 2.1 PVCs/min for AAVB, inducible CTL-CAVB-6W, noninducible CTL-CAVB-6W, inducible CTL-CAVB-12W, noninducible CTL-CAVB-12W, CsA-CAVB-6W, and CsA-CAVB-12W, respectively, P = not significant). Nevertheless, the findings are at least compatible with the hypothesis that the antiarrhythmic effects of CsA are based on substrate modification, rather than on trigger elimination.
ECG and activation pattern of PVTs. All seven PVTs occurred spontaneously after drug application. Four episodes were initiated by a short-long-short sequence. Nonsustained episodes (6 of 7) lasted for 6–60 beats. One six-beat run was too short for a meaningful analysis of ECG morphology. All other episodes revealed characteristic changes in the QRS axis and amplitude closely resembling TdP in patients. A representative electrocardiographic example is given in Fig. 4A, and respective three-dimensional activation patterns of selected beats are presented in Fig. 4, B-F.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Calcineurin inhibition and morphology. Data on the effects of calcineurin inhibition on hypertrophy are inconsistent, and whether calcineurin plays a key role in the development of hypertrophy is the subject of an ongoing debate (19). In vitro and some in vivo studies in transgenic mice and in pressure-overloaded rodents have demonstrated that inhibition of calcineurin prevents or at least attenuates hypertrophy (7–9, 11, 18, 20). Other studies, however, indicate that CsA and FK-506 are ineffective in preventing hypertrophy (8, 12, 25) and may even be deleterious (7–9, 25). With respect to regression of hypertrophy, there are also conflicting data (7, 15, 18). Some investigators demonstrated that CsA administration in hypertensive rats partially reverses hypertrophy and fibrosis in a dose-dependent manner (7, 18). In contrast, using the same animal model, Sakata et al. (15) found that FK-506 attenuated the initial stage of hypertrophy without affecting fibrosis but failed to prevent cardiac remodeling and to induce regression in the advanced stage of hypertrophy. Species differences, variations in the methodology and the models used, and the timing, dose, and route of CsA administration may account for these controversial results. It is well known that there are marked differences in physiology and drug metabolism between certain species (2). Recent data also suggest that the signal transduction pathway may vary depending on the duration and/or the type of intervention (i.e., volume or pressure overload) inducing hypertrophy. Thus calcineurin may only be of relevance for a certain phenotype of hypertrophy (10, 15 19). Our results would then suggest that the calcineurin pathway is involved in the development of volume overload-induced hypertrophy in dogs. CAVB results in biventricular hypertrophy accompanied by electrical remodeling (23, 24). In our study, CsA attenuated the development of hypertrophy and partially reversed established hypertrophy without increasing mortality. HBWI was significantly lower in CsA-treated dogs with CAVB than in nontreated CAVB dogs but higher than in AAVB dogs. The fact that CsA did attenuate, but did not completely block, hypertrophy could have been a matter of dose and/or timing of administration. It could, however, also indicate that not only a single pathway is responsible for the overall hypertrophic response. Instead, an interaction with other signaling pathways, such as the MAPK pathways, seems to be likely.
Calcineurin inhibition and mortality.
Whether hypertrophy is salutary or detrimental is a matter of debate. In mice subjected to pressure overload, Meguro et al. (9) found a 7.2-fold higher mortality due to heart failure in the CsA-treated group, supposedly due to the lack of an adequate hypertrophic response. In the present study, none of the animals developed heart failure. One could speculate that an attenuated hypertrophic response might still suffice to compensate for the hemodynamic situation. Increased mortality associated with CsA treatment in pressure-overloaded rats in the study by Zhang et al. (25) was attributed to the general toxicity of systemic CsA. Consistent with the findings of other groups (4, 15, 18), we could not find increased mortality rates in CsA-treated animals. On the contrary, mortality was higher in untreated CAVB dogs dying prematurely from sudden death. The incidence of severe drug-related complications was very low (1 of 13 dogs), possibly because of the short-term drug application (6 wk). Furthermore, as with the beneficial effects of CsA, detrimental effects may also depend on drug metabolism. Thus controversial findings might again be explained by differences in the model, the species, and the drug dose.
Calcineurin inhibition and changes in electrophysiology. Hypertrophy is associated with prolonged repolarization parameters and a predisposition to ventricular arrhythmias (22–24). Thus regression of hypertrophy might resolve electrophysiological abnormalities and the susceptibility to ventricular arrhythmias. Rials et al. (13) provided convincing evidence, in vitro and in vivo, that angiotensin-converting enzyme inhibition induces regression of hypertrophy, normalizes action potential duration, and decreases VF inducibility in different hypertrophy models (13). In our study, CAVB also resulted in prolongation of mean ERPs. The fact that we could not demonstrate a significant increase in the dispersion of ERP is in contrast with other studies (13, 23) and probably points to a weakness of the methodology applied. ERP measurements with the extrastimulus technique do not allow us to perceive dynamic changes and are very time consuming. This could have accounted for different time windows for data acquisition, a factor of potential relevance with respect to the stability of the preparation, temperature, and drug levels. Furthermore, because of a mismatch between needle length and wall thickness, longer endocardial ERPs may have been missed in some cases, thereby falsely suggesting a minor degree of dispersion. Although it would have been reassuring to reproduce previous findings on the dispersion of repolarization (13, 23), even with ERP measurements, this was certainly not the focus of the present study. Fundamental effects of CAVB, namely, biventricular hypertrophy, ERP prolongation, and PVT inducibility, could be confirmed. Thus assessment of the effects of CsA relied on these parameters. CsA did not seem to affect electrical remodeling. Despite a significant ERP prolongation, CsA-treated CAVB dogs were no longer susceptible to almokalant-induced PVTs but produced single PVCs as frequently as untreated dogs. This latter finding is compatible with the idea that the initial beat of a PVT is due to prolongation of repolarization and early afterdepolarizations (EADs). The lack of PVT inducibility in CsA-treated CAVB dogs with significantly reduced hypertrophy suggests that hypertrophy provides the substrate for continuation of the arrhythmia.
Recently, Schoenmakers et al. (16) demonstrated that CAVB dogs may develop dofetilide-induced TdP, even after 2 wk of complete AV block (CAVB2). The authors concluded that ventricular hypertrophy in the CAVB dog is not a prerequisite for electrical remodeling or drug-induced TdP. However, they also found that CAVB2 dogs exhibit longer Q-T intervals, LV monophasic action potential durations, RV monophasic action potentials, and ventricular ERPs than control AAVB dogs, indicating the presence of electrophysiological changes associated with the development of hypertrophy. Electrophysiological changes associated with hypertrophy could relate to local heterogeneities in conduction and/or refractoriness or to alterations of electrical coupling. It is quite conceivable that respective changes could precede morphological changes evident at a macroscopic level.
Another study in the CAVB model found that hypertrophy is associated with an increase in the dispersion of activation recovery intervals and that PVTs are exclusively due to reentry (6). Because dispersion of refractoriness as a prerequisite for functional conduction block is the hallmark of reentry, both observations are compatible with the idea that hypertrophy by increasing dispersion facilitates reentry. Kozhevnikov et al. (6) analyzed 14 episodes of PVT, and their typical example shows a 9-beat run based on macroreentrant circuits. We did, indeed, also observe a six-beat PVT with focal initiation but transition to this type of macroreentrant activation, at least indicating that the methodology applied was able to trace respective activation patterns. However, the majority of episodes, which consisted of up to 60 beats and closely resembled TdP in patients, clearly exhibited a centrifugal spread of activation and were, thus, termed "focal." This does not necessarily imply a certain mechanism with respect to the focus, which could as well be based on microreentry. However, such a focal activation pattern is clearly different from the more-or-less circular spread of activation observed in one of our experiments and by others (6). The prevalence of activation pattern during PVTs in the CAVB model and the basis of long-lasting, TdP-like runs need to be determined.
Furthermore, the fundamental question on the issue of focal versus reentrant activity during TdP is the relevance of hypertrophy. Our data and present knowledge only allow for speculation and for the creation of hypotheses that need to be confirmed. The concept of reentry as the basis of TdP does not explain the unique ECG morphology of the arrhythmia or clinical characteristics, such as spontaneous termination, mode of induction, or etiology (e.g., drug induced and congenital). It is also surprising that one rarely sees transition of TdP to a monomorphic tachycardia, which is usually based on reentry. Our hypothesis would be different: The initial beat of a TdP is generally accepted to result from EADs and triggered activity. That, however, is a bradycardia-dependant process and could, therefore, not account for subsequent beats of the tachycardia. If, however, subtle changes in local electrophysiology (uncoupling, local conduction delay, and local dispersion of refractoriness) associated with hypertrophy would result in tachycardia-dependant entrance block to certain regions, these regions would experience long reactivation intervals and could, thus, develop bradycardia-dependent EADs during TdP. This would explain the need for a continuous shift in the site of earliest activation. This would also explain the ECG appearance, its similarity from patient to patient, and the tendency toward spontaneous termination. QT prolongation alone might not be sufficient to create TdP; otherwise this arrhythmia should be easily inducible in every patient or in multiple experimental settings. Instead, one would need QT prolongation to provoke EADs, as well as local electrophysiological changes (e.g., those associated with hypertrophy) to provide the basis for local entrance block.
Limitations of the study.
Electrical and ventricular remodeling processes due to volume-overload hypertrophy start within 24 h after induction of AV block (21, 23, 24). Sufficient blood levels of CsA required 2–3 days of treatment. Thus pretreatment of the dogs with CsA might have resulted in a more pronounced effect on hypertrophy.
The development of hypertrophy could have been influenced by the blood pressure of the dogs, which was not measured in this study. Furthermore, cyclosporin-induced changes in blood pressure could have contributed to the development and/or regression of hypertrophy.
The resolution with our electrode array allowed for 1.0- to 1.5-cm gaps between individual needles. Furthermore, a significant part of the interventricular septum was not covered. This might not have been sufficient to resolve small functional reentrant circuits. However, even with small circuits, the activation pattern at a distance from the circuit should remain somehow circular, except one would postulate a microreentrant circuit being surrounded by functional conduction block with only limited exit.
Apart from the limitations of multiple ERP measurements already discussed, the lack of septal sites might have also contributed to the inability to detect significant dispersion. However, functional conduction block as a prerequisite for reentry requires local refractory gradients. Thus the relevance of ERP differences between septal and other sites at a certain distance is at least questionable.
For refractory measurements, a train of eight basic beats preceded application of an extra beat. With that, steady-state conditions were probably not reached. However, the emphasis of our study was on the comparison of normal and hypertrophied hearts, and the given limitation applies to both.
Our data on the role of CsA treatment for the occurrence of PVCs are limited by the small number of observations. Thus these findings have to be interpreted with caution.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. D. Schreiner and K. Kelemen contributed equally to this study. 
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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leads to cardiac hypertrophy and dilated cardiomyopathy by calcineurin-dependent and -independent pathways. Proc Natl Acad Sci USA 95: 1393–1398, 1998.
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