4.3. A Novel Mechanism Behind Flecainide Proarrhythmia
4.3.1. APD xx Prolongation and Increased APD xx Dispersion (ΔAPD xx ) with Flecainide in the Murine Heart
Despite some similarities between the latter observations and ours, particularly with epicardial APs that initially showed a prolongation with Flecainide followed by an abbreviation, major and crucial differences exist between their results and ours on several scales. The comparison between their results and ours should be done with caution: starting from the different animal models used (canine vs. murine hearts), the experimental tools to characterize the electrophysiology in the tissue (extracellular electrodes at a few sites on the epicardium vs. optical mapping using 10,000 recording sites and a spatial resolution of 100μm), the high concentrations of Flecainide that are of little practical significance in a clinical setting631, 632 (15μM vs. 1μM; therapeutic range of Flecainide [0.5-‐2μM]lxxix) and the adopted pacing protocol to reveal the heterogeneity in the epicardium (S1-‐S2 protocol vs. steady state pacing at a fixed BCL). The impacts of these differences are tackled in a more detailed manner in the next subsections (4.3.1 and 4.3.2).
The concentrations of drugs Krishnan and Antzelevitch used in their in-‐vitro studies were very high compared to ours411. The trend in pharmacological interventions is to use as high concentrations as possible, until an effect is observed633, 634. Although these pharmacological studies are of great scientific importance, serious questions should be raised concerning the ones that use tens or hundreds of mM of drug to study their channel blocking action, when the therapeutic range lies more than 4-‐5 orders of magnitude lower452. We therefore attempted to use clinically valid concentrations, to obtain a more realistic correlation with what could possibly be clinically observed.
4.3.1. APDxx Prolongation and Increased APDxx Dispersion (ΔAPDxx) with Flecainide in the Murine Heart
Flecainide has been reported to cause APD90 prolongation in mammalian ventricular myocytes479,
635, as well as human, guinea pig, and canine atria636, 637. However it was also shown to cause APD90 shortening in canine Purkinje fibers635. In our measurements, APD prolongation was observed at all
lxxix Manual of Laboratory & Diagnostic Tests. 7th edition. Lippincott Williams & Wilkins.
repolarization levels (APD25, APD50 and APD75) with Flecainide (Figure 30, Figure 32, Figure 42), and most prominently in APD25 measurements (Figure 34, Figure 43) in all substrates (WT, mdx, and ΔKPQ). This suggests that with an increasingly shallower upstroke, the amplitudes and kinetics of sequential ionic currents could be modified, causing shifts in the phases of the epicardial AP with respect to voltage and time. In reality, with the strong use-‐dependence of Flecainide, APD prolongation was not expected. Since our experiments were conducted using only one frequency of stimulation, carried at steady state, we cannot comment on how the rate of APD prolongation would change, if different frequencies were involved, neither can we speculate on the restitution behavior of the APD in the presence of Flecainide. Although we have observed an APD prolongation in all sinus measurements (both WT and mdx) with Flecainide compared to the starting control conditions, this finding is not reliable because the measured APDs are at different locations on the restitution curve (due to the varying diastolic interval between initial control conditions (t0) and after Flecainide was introduced into the system). At control conditions, the sinus rate was no less than 8-‐8.3Hz (i.e. 480-‐500 bpmlxxx). After ten minutes of Flecainide exposure, the sinus rate dropped to a value between 4.5-‐5.8Hz (270-‐350 bpm) in almost all preparations. Hence, we cannot confirm whether the observed APD prolongation is secondary to pure Flecainide effects or due to the final lower heart rates.
The observation that Flecainide causes an actual prolongation of the APD doesn’t correlate well with the fact that this drug has a strong use-‐dependence block479. Indeed, Flecainide is unique among AADs, where use-‐dependence block of NaV1.5 causes a prolongation of the APD and not retraction499, 638. This might suggest that Flecainide blocks other channels than just NaV1.5. Follmer et al. showed that Class Ic agents have a markedly different ionic profile among NaV1.5 blockers:
Flecainide has the potential to block the rapid component of the delayed K-‐rectifier current (IKr) using concentrations as low as 2.1μM in cat ventricular myocytes639. Although the concentration used in the latter experiments is at least twice as large as the one used with our murine hearts, we cannot eliminate the possibility that blocking K+ channels could be involved in the observed APD prolongation with Flecainide.
In a previous study done by Starmer et al., they showed that prolonging refractoriness by blocking NaV1.5 alone increases the likelihood of unidirectional block in the presence of premature excitations in an otherwise normal tissue492. Follmer et al. showed that the interaction of Flecainide with K+ channels closely correlated with channel activation, suggesting an open-‐state blocking effect639. As the result of the two previous findings, Follmer et al. argue that the antiarrhythmic efficacy of Flecainide resides in its ability to act on both NaV1.5 and K+ channels, by prolonging refractoriness in the setting of decreased excitability, without the need to solely reduce INa,f in order to achieve the same degree of refractoriness639.
APD prolongation is of different magnitude for different levels of repolarization, with Flecainide. For higher repolarization levels for which the APDs were originally defined (i.e. the closer temporally is the repolarization level to the upstroke), the more prominent is the percentage increase in prolongation observed between t0 and t10, in either WT or mdx (Figure 34). Both APD50 and APD75 showed no directional bias, in such a way that the percentage increase didn’t vary whether the APs were picked up along the longitudinal or transversal directions. To illustrate, in the WT APD50,Long prolonged by ~65% and APD50,Trans by ~60%. Although the percentage increases in the mdx were slightly higher (but not statistically significant) compared to the WT, no directional preferences were detected; APD50, Long and APD50,Trans prolonged by ~70% and ~69% respectively. APD75 and APD90 in both WT and mdx prolonged overall by ~40% and ~30% respectively in both directions.
lxxx In cardiology, the heart rate is measured in bpm, i.e. beats per minute.
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The dynamics of APD25 were dramatically different than other APDs. This parameter appeared to be the most susceptible to Flecainide, acquiring both direction-‐ and substrate-‐dependences (Figure 34) as the treatment progressed. In the WT, the prolongation of APD25 peaked along the longitudinal direction with an increase exceeding ~190%, vs. ~170% in the transversal direction. The mdx heart, with lower available NaV1.5 channels, showed significantly lower percentages of APD25 increase between t0 and t10 upon exposure to Flecainide, with ~155% and ~110% in the longitudinal and transversal directions, respectively. These data emphasize three important findings:
• As APD25 encloses the time interval from AP takeoff till the end of phase 1, it represents the interval during which the dynamical processes the most affected by functional NaV1.5 take place. In other words, this correlates well with the fact that under control conditions, only APD25 showed a significant difference between WT and mdx, whereas other APDs were not statistically different (Figure 30, Figure 32). In our simplistic assumptions, the major difference in the plasma membrane ionic composition between WT and mdx is the absence of NaV1.5 on the LM in the mutated heart. Hence, if subsequent ionic currents were perturbed in the mdx compared to the WT in the earliest phases of repolarization (in the absence of Flecainide), they should stem from the differences in NaV1.5 availability between the two substrates (Figure 19).
• Flecainide enhanced the appearance of directional bias only in APD25, whether in the WT alone (by comparing longitudinal to transversal directions), in the mdx alone or when comparing the two substrates. The lower percentage increases in APD25,mdx (compared to APD25,WT) further underline that the lack of NaV1.5 on the LM means lower available channels to modulate. Henceforth, the total change induced by Flecainide in either direction is expected to be reduced in the mdx (as it was indeed observed in the statistics in Figure 34).
• The increased susceptibility of APD25 to Flecainide (compared to APD50, APD75 or APD90) highlights that in our experiments, whether [1μM] Flecainide also blocks K+ channels, the main functional effect of the drug is localized to high enough phases of the AP, where repolarizing K+ channels play practically no role.
Because of the fact that APD25 incorporates other AP processes beyond the upstroke phase, its sensitivity to directional influences is less pronounced than the one observed for the maximum upstroke velocity (dF/dt)max (Figure 25), where the decay in (dF/dt)max was similar between WT and mdx along the longitudinal direction, but more pronounced in the WT along the transversal direction (compatible with the differences in NaV1.5 cellular distribution between the two substrates). Hypothetically speaking, if APD25 were to represent the phase of the AP where NaV1.5 is the only functional channel, then the behavior of APD25 and (dF/dt)max with Flecainide at any time point of the treatment with Flecainide should exactly match. Since the latter statement is precisely not true, as Flecainide exposure proceeded, the coupling between ADP25 and (dF/dt)max became increasingly ambiguous, indicating a decoupling between the two parameters (Figure 35). This could suggest that as the upstroke velocity becomes progressively weaker, under the influence of the drug, the ability to NaV1.5 to orchestrate the sequential activation of the consecutive channels becomes increasingly more limited (Figure 35b). At t10, further deterioration of the upstroke would hinder the capacity of NaV1.5 to even coordinate the earliest phases of the AP (particularly the ones involving Ito,1 and ICa,L) after the upstroke; a mechanism that could promote electrical instability in the tissue.
The spatial distribution of the above macroscopic processes couldn’t have been elucidated using traditional microelectrodes positioned at few sites of the epicardium. The strength of the observed findings relies in the methodology used to dissect both spatially and temporally the effects related to the propagating electrical activity across the entire epicardium. Furthermore, without optical
mapping, the increase in spatial dispersion of repolarization (SDR) couldn’t have been described with the precision adopted in this work. Flecainide not only induced a differential prolongation in the APD values, but also in the spatial organization of these APDs across the epicardium, where WT and mdx hearts both showed a pronounced increase in their SDR at different AP repolarization (Figure 33). Under control conditions (i.e. when no Flecainide was introduced), APD25 was not only significantly prolonged in the mdx, but also the dispersion of the APD25 (i.e. ΔAPD25) was significantly widened in the mdx (ΔAPD25,WT =8.4±1.7ms, ΔAPD25,mdx =13.1±4.0ms, p-‐value<0.01).
Although ΔAPD25,mdx was higher on average (29.3±7.1ms) than ΔAPD25,WT (25.6±6.6ms), bistability erupted in at least 70% of the WT hearts (n=10) at t10 and in 0% of mdx heart (n=7). The emergence of bistability in the APD50 profile in the WT heart at t10 led to the significance increase in dispersion (Figure 33). Hence, we believe that the ΔAPD50,WT and ΔAPD75,WT widening at t10 is a consequence of bistability, rather the causative agent behind it.
To further illustrate why this point could be crucial in our understanding of symmetry breaking in APD, the same analysis of APD prolongation and dispersion was carried out in the ΔKPQ model treated with Flecainide [1μM] and their WT control, over a protocol that lasted for 5min (due to the premature loss of steady state conditions with the ΔKPQ model beyond 5min of treatment).
Although the spatial organization of APDs in the ΔKPQ heart doesn’t show the same sharp transition observed previouslylxxxi, the heterogeneous distribution of APD25 and APD50 at t5 indicates the presence of steep gradients between the distinct zones observed on the epicardial surface (Figure 39a and b). Although ΔAPD50,WT was significantly higher in the WT at t5 compared to ΔAPD50,ΔKPQ, symmetry breaking emerged in the ΔKPQ and not the WT (the difference in APD50 values is not significantly different between the two groups, Figure 42). We believe that since we are principally targeting NaV1.5 with Flecainide, the mechanism by which modulation of NaV1.5 occurs is capable of influencing the subsequent events leading to symmetry breaking. We hypothesize that in the presence of Flecainide, neither APD25 prolongation nor widening of ΔAPD25 alone may evoke symmetry breaking in a well-‐coupled tissue. However, cumulatively, whenever the global change in ΔAPD25 with Flecainide exceeds the overall prolongation in APD25, the substrate becomes more susceptible to symmetry breaking (indicated by the statistics in Figure 43). The latter statement is by far just a hypothesis based on an observation from our experiments, further investigations using computer models of the mouse heart will be required to find out, whether symmetry breaking with Flecainide could occur under the pre-‐requisites set above.
The concept of heterogeneous repolarization, as a facilitator of arrhythmogenic activity, has been recognized for more than four decades407-‐409. When transmembrane repolarization at some sites of the cardiac tissue outlasts repolarization at an adjacent site, local current will be expected to flow in proportion to the voltage gradient between the two sites411. If the current is of sufficient magnitude, it can reexcite the earlier repolarizing site by bringing the cells’ potential to threshold triggering reentry264. The prominent increase in SDR observed in our murine hearts is likely to be arrhythmogenic because the dispersion in repolarization occurs over very short distances264 (approximately, the apico-‐basal length of the murine heart is ~6mm and the width of the LV free wall in our field of view ~3mm), creating a very steep repolarization gradient (in the example in Figure 28a, the gradient can exceed 20ms.mm-‐1 across the borderline from the zone with the prolonged APDs to the zone of the abbreviated ones). It’s currently believed that the steepness of the repolarization gradient, rather than the total magnitude of the dispersion in a cardiac tissue, determines the arrhythmogenic susceptibility of the substrate involved263, 264.
lxxxi i.e. in the WT control group of the mdx mice.
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