4.3. A Novel Mechanism Behind Flecainide Proarrhythmia
4.3.3. Hypothetical Mechanisms Behind Bistability and Arrhythmogenesis The current experimental outcomes with Flecainide motivated a search for a possible mechanism
This symbiosis between slowed conduction and heterogeneous repolarization would be of a particular importance in pathological diseases of the heart483, where structural heterogeneity can amplify the predisposition of cardiac patients to drug-‐induced arrhythmias485, 486, in concordance with the outcomes of CAST I474. In a normal epicardium, bistability is expected to give rise to reentrant patterns in the presence of premature stimuli497. These data support the hypothesis that electrophysiological heterogeneity upon exposure to Flecainide could play a critical role in the proarrhythmic effects of the drug411. Given that arrhythmias are multicellular292, it becomes legitimate to question the efficacy and applicability of antiarrhythmic drugs as a primary mean of patients’ management, when their outcomes were established based on isolated “single” cell measurements643, 644.
4.3.3. Hypothetical Mechanisms Behind Bistability and Arrhythmogenesis The current experimental outcomes with Flecainide motivated a search for a possible mechanism that could potentially account for the observed bistability in healthy tissue. Cardiac excitation involves local regenerative processes at the individual cell level and transmission of this transient dynamic process from one cell to another via intercellular connections through the flow of depolarizing charges. However, spread of excitation from one point to another in the cardiac tissue occurs exclusively when a critical “amount” of cells (source) is simultaneously excited, as to generate a depolarizing current sufficient to overcome the sink provided by the quiescent tissue in order to bring downstream cells to threshold645. Therefore, several cells below this critical mass cannot entrain its neighbors to acquire the same characteristics. Given that myocytes in an intact tissue are well coupled to their neighbors, how do these cells synchronously develop two almost opposing dynamics in such a small sized heart?
We hypothesize about the generic mechanism that could have lead to the bistability observed in our experiments. We start by considering two observed properties of bistability: First, bistability is not dependent on anatomy or structure and its spatial configuration, whenever it appears, was not conserved across preparations (i.e. among the WT preps). Second, WT hearts that eventually showed a steep repolarization gradient with Flecainide and the mdx hearts that failed to do so, shared similar APD distributions at initial conditions (i.e. in the absence of the drug). Henceforth, we are reluctant to associate bistability to differences in the ionic machinery of individual cells, which was shown by Szentadrassy et al. to account for apico-‐basal differences in APD in the canine ventricle646. We, therefore, make the assumption that all cells of the LV free wall epicardium are homogeneously equipped with the same ionic channels. The first possibility assumes that each and every individual cell has the capacity to either acquire a long or a short APD. The presence of two adjacent zones with a dominating APD on each side suggests that cells, when present in a tightly coupled mesh, have the potential to help their neighboring cells deciding on which APD to display, creating finally two zones (they could be hypothetically more than just two) with each zone sharing similar electrophysiological characteristics. Another possibility assumes the presence of “leader cells” (that form a “critical mass” as explained earlier), which pick the initial conditions (i.e. a long or a short APD) and force the remaining mass of cells in the mesh to follow their lead. The emphasis in either hypothetical behavior is on these tight electrotonic interactions that overturn the cells in a multistable system to acquire one particular electrophysiology over another. Since we are not aware of the intrinsic dynamics occurring at the level of one cell, we cannot predict which possibility is more dominant.
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From an electrophysiological perspective, we hypothesize that Flecainide-‐induced bistability in the murine epicardium could originate from competing currents (one outward repolarizing and another inward depolarizing) acting between the end of phase 0 (i.e. the upstroke) and before the phase 2 of the AP (i.e. the plateau) gains momentum. The reason for this assumption is simple: bistability was prominent after 25% AP repolarization, and wasn’t detectable prior to this level (Figure 28, Figure 31). The two competing currents are Ito,1 and ICa,L. The idea behind the “competing currents”
hypothesis is similar to the elegantly presented work by Weiss et al. in Heart Rhythm (2010)308, which provided a consistent explanation for the dynamics of afterdepolarizations at the end of the plateau phase, using a rabbit heart numerical model.
The bidirectional influence of membrane voltage and Ca2+ on each other is well documented in the literature647, 648. In a murine or rat ventricular myocyte, Ca2+ homeostasis is strictly controlled via uptake in the sarcoplasmic reticulum or SR (more than 95% of the cytoplasmic Ca2+ available for mechanical contraction is taken up by the SR, the remaining 5% through plasma channels)113. We consider first the dynamics of Ca2+ entry into the cell and its intricate modulation by the Ito,1 current.
Although the INa,f -‐dependent rapid depolarization activates ICa,L, the amplitude of ICa,L doesn’t reach its maximum at the peak and consequently doesn’t contribute substantially to the upstroke of the AP. Bers explains that this phenomenon is partly due to the fact that the activation of ICa,L is not just voltage-‐dependent but also acquires some intrinsic time scale of activation, leading to a trade-‐off in the co-‐existence of relatively high channel conductance (gCa) and low driving force (Embr -‐ ECa)peak, with ICa,L being the product of these two components649. In reality, at the AP overshoot, the membrane voltage is ~30-‐40mV, which is relatively close to the reversal potential of Ca2+ (ERev,Ca), which is measured under physiological conditions to be ~50-‐60mVlxxxii, so the driving force of Ca2+ is low by the end of phase 0. As the NaV1.5 channels inactivate and the Ito,1 has already activated, phase 1 lowers the membrane potential, increasing the driving force of Ca2+ into the cell. In conclusion, ICa,L activates over two phases: A rapid increase in conductance followed by an increase in the driving force, with Ito,1 directly modulating Ca2+ influx649. Inactivation of ICa,L is both voltage and Ca2+
dependentlxxxiii, with the latter being a far more predominant component of inactivationlxxxiv, hence ICa,L inactivation is extremely slow113.
On the other hand, Ito,1 is not a voltage dependent current only, but also acquires outward rectification properties107, which means that the amplitude of this current is strongly dependent on the amplitude and velocity of the preceding upstroke, in other words on INa,f. The sizeable amplitude of this current in rat and mouse ventricular cells abbreviates the APD and reduces the plateau phase substantially113, 650. A decrease in INa,f in ventricular cells is expected to lower the intensity of Ito,1, henceforth decrease the early repolarization phase. This implies that the plateau will start at higher membrane voltages than usual, an effect which might decrease the driving force of Ca2+ into the cell, potentially altering ICa,L peak in amplitude and time. The intricate balance in time (and within a limited range of membrane voltages) between Ito,1 and ICa,L peaks has the potential to tip the intrinsic dynamics of the cells into one stable trajectory or another. In the case of the observed APD bistability in our measurements, one stable state is APD prolongation, the other APD abbreviation.
lxxxii ERev,Ca, the reversal potential of Ca2+ in physiological experiments is measured to be ~50-‐60mV, in contrast to its thermodynamical equilibrium potential (ECa), which is ~120-‐125mV. Substantial amount of Ca2+ flows across the L-‐type channels between ERev,Ca and ECa. But in a real cell at ERev,Ca the inward ICa,L is counterbalanced by the outward K-‐current (Ito,1), so the electrophysiologically important ICa,L becomes negligible at the peak, despite some influx of Ca2+ (Reference: J Physiol (2000) 523:57-‐66).
lxxxiii Refer to Introduction section 1.1.4 on Ca2+ inactivation.
lxxxiv Since the predominant factor in Ca2+ inactivation is metabolic, this process is considered relatively slow compared to inactivation of other currents which are completely voltage or time dependent (like INa and Ito,1).
Henceforth we hypothesize that by lowering INa,f with Flecainide and continuously reducing its rate of increase as the treatment progresses, Ito,1 activation and inactivation processes are also altered. A plateau phase is sustainable whenever repolarizing currents (K+ currents) are not fully activated and depolarizing currents still didn’t inactivate (ICa,L). Following the upstroke phase, if Ito,1 peaks early enough, it will lower the membrane voltage and allow for the increase in Ca2+ driving force. Ito,1 inactivates very quickly, allowing ICa,L to peak at a later stage as Ca2+ rush into the cell, which would ultimately keep the plateau phase at higher voltages. As Flecainide also affects IKr, the current would be further weakened to counteract Ca2+ influx and the plateau is substantially increased. If on the other hand, Ito,1 activation is delayed enough to peak at a later stage, phase 1 is prolonged and Ca2+
influx could face a fierce competing opposing current as it’s trying to reach its maximum. This would obliterate the plateau and bring the cell into resting potential, abbreviating the APD.
This hypothesis on competing currents following the upstroke has to await further experiments and numerical simulations to be approved as valid or disapproved. One way to elucidate the role of Ito,1 would be to disable it by introducing 4-‐aminopyridine (4-‐Ap) into the protocol, which could provide important and specific insights on the role played by Ito,1 in creating this bistability in the epicardium. We expect that the presence of this antagonist molecule may potentially alter the responsiveness of the epicardium to Flecainidelxxxv or even prevent bistability from occurring altogether. Alternatively, hypercalcemia may favor one stable state over another, however it can alter other Ca2+ dependent mechanisms in the cell, which could render the final results harder to interpret. Therefore, the detailed explanations given in this section should only be regarded as one potential mechanism explaining the emergence of bistability in the murine heart.
Although our study was limited to steady state pacing at a fixed BCL (100ms), we hypothesize that a premature excitation occurring in the epicardium would give rise to drug-‐induced arrhythmias that are characterized by a reentrant mechanism. The mechanism by which reentry is initiated would depend on the specific location of the ectopic beat with respect to the different zone of AP morphology. APD prolongation goes hand in hand with repolarization abnormalities, hence is capable of influencing conduction of the propagating impulse. Fibers with prolonged APD will elicit a slowing of conduction with increased entrainment rate, so that each consecutive AP arises prior to the completion of repolarization of the preceding beat. The frequency at which conduction block might ensue will be relatively lower in the case of the prolonged APD, than the one associated with a normal APD318. If the ectopic beat arises in the area governed by shorter APDs (APDShort), the electrical wave would spread across the tissue, until it hits the borders separating the two adjacent zones. Crossing to the prolonged APD zone (APDProlong) will depend on the excitatory state of the cells in that region: if theses cells had sufficient time to recover from the previous excitation, the wave will propagate. Otherwise, conduction block will occur. In this setting, either the electrical wave dies out or persists somewhere else until the cells in the APDProlong zone regain excitability. In this case, circus movements are highly probable and reentry occurs. If however the ectopic beat would arise in APDProlong zone, the activation would spread directly in the APDShort zone, exciting the cells in that region. Interestingly, for reexcitation to occur, sustenance of propagation would then depend on the electrotonic interactions between the cells on either side of the borders. It has been suggested that in tissues where depolarization is normally Na-‐mediated, the contribution of Ca2+ in maintaining AP propagation increases significantly in the context of severely depressed membrane excitability (i.e. <20% INa availability)585. In the case of Flecainide induced heterogeneity, it is the prominent plateau at some sites (APDProlong) but not others (APDShort) that could generate local
lxxxv The effects due to antagonizing Ito,1 using 4-‐AP were described by Krishnan and Antzelevitch in epicardial layers in the presence of Flecainide. Ito,1 blocking was reported to prevent AP abbreviation in epicardial cells (Reference: Circulation (1993) 87:562-‐72).
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circuit currents411, by acting as a reservoir of depolarizing charges (Ca2+), which would increase the electrotonic driving force in the abbreviated AP cells, consequently, the depolarizing current to the downstream tissue585, bringing membrane voltage to threshold and initiating depolarization. Kléber and Rudy described this membrane-‐switch from Na+ to Ca2+ -‐supported propagation as an excellent example of the “intimate interaction” between passive network properties and the excitatory membrane currents during conduction in the cardiac tissue254. Whether this switch in membrane conduction is sufficiently strong to maintain a self-‐perpetuating reentrant mechanism in the heart in the context of bistability is yet to be investigated.