3.3. Expatiated Pathophysiological Heterogeneity in ΔKPQ
3.3.1. Atypical Activation Spread in ΔKPQ with Flecainide
In the next set of experiments, ΔKPQ murine hearts were subjected to the same treatment protocol that was followed for the WT and mdx heart (section 3.2). Over a period of 5min, the mutated hearts (n=4) and their WT counterparts (n=4) were perfused with Flecainide [1μM]. The same experimental conditions used previously were reproduced in the treatment of ΔKPQ hearts. The exposure to the drug was limited to 5min only (half the exposure time in the previous sets of experiments), due to difficulty in entraining the mutated hearts into a 10Hz steady state pacing beyond 5min. From the previous experiments, the increased sensitivity of the WT group to Flecainide compared to a substrate with lower availability of NaV1.5 was a major finding. This sensitivity was translated into a symmetry breaking effect that lead to the coexistence of two adjacent stable zones entrained by the same frequency. As the outcomes of Flecainide exposure are yet to be clarified, activation, functional heterogeneity profile and drug proarrhythmia are investigated next, since a NaV1.5 blocker might turn to be completely ineffective or overly effective in interacting with the mutant channel.
3.3.1. Atypical Activation Spread in ΔKPQ with Flecainide
The ΔKPQ mutation destabilizes inactivation, as previously explained. Wang et al. measured INa from a whole cell voltage clamp using ΔKPQ mutated cardiac cells and reported that the steady state activation curve was slightly (~6mV) shifted towards depolarizing voltages compared to the WT395. In addition, the fractional amplitude of the fast component of the INa,f was actually greater in the ΔKPQ cells than the WT. The recovery from inactivation kinetics was also considerably faster for the mutated cells: in the WT the largest fraction (77%) of channels seem to have recovered from inactivation with a time constant of τWT =6.7ms vs. 59% in ΔKPQ at τΔKPQ =2.1ms395. Based on these cellular results, and assuming that the resting membrane potential (Vrmp) in both substrates is unaltered, we do not expect the maximum upstroke velocity (dF/dt)max to be modified in ΔKPQ. As we have seen in the previous WT and mdx study, (dF/dt)max may represent an index of NaV1.5 availability in some conditions, however may not necessarily give an accurate prediction of the CV in the medium.
In Figure 36a, the optical APs (OAP) were measured from the LV free wall of the WT and ΔKPQ.
Prior to normalization, the OAPs were compared for amplitude and FC% (as previously described).
The APΔKPQ is superimposed over the dotted WT representation (same as the APWT, left) for a direct comparison. On the right, the slightly shallower upstroke is followed by a hampered notch, which precedes a plateau phase at more positive voltages. Unlike the APmdx (Figure 19, left), where the early prolongation phases are compensated with strong enough repolarization currents leading to an almost normal termination of the AP, the APΔKPQ is characterized by a continuous non-‐
compensated prolongation and a considerably delayed repolarization. In that regard, the APΔKPQ appears comprehensively prolonged compared to the APWT in all phases (Figure 36a, right).
Expatiated Pathophysiological Heterogeneity in ΔKPQ 103
After 5min exposure to Flecainide (t5), qualitative differences between the two maps reveal an atypical pattern of activation in the mutated heart. Meanwhile, the WT map maintains a global anisotropic propagation with slower components compared to t0 (at t5: Vmax,WT = 0.62±0.05m.s-‐1 and Vmin,WT = 0.21±0.03m.s-‐1). As the position of the pacing electrode remains unchanged (Figure 45), the wave clearly emanates from the tip of the electrode where the pacing current is introduced into the tissue. However it acquires a preferential shift towards the base where conduction is clearly faster (the first isochrone is located towards the aortic side), maintains an elliptical shape until the convex ends along the major axis of the ellipses reach the right and left boundaries of the heart (within the red colored area of the map), then resumes propagation towards the apex in quasi-‐planar waves.
From the movie of the measurement (Figure 44 and Figure 45), an artifact due to electrode position change was ruled out. This pattern appears to be generic for all ΔKPQ preps treated with Flecainide at t5 (n=4) in this study. The cause of such activation is unknown. Hence, it’s tempting to speculate that the mechanism behind the deviation from global anisotropy (to partial anisotropy) could possibly be due to a source-‐sink mismatch. A point stimulation, unlike a line stimulation, creates an elliptical pattern of activation founded on fiber orientation; principally due to “facilitation” of spread in the direction parallel to the fiber’s long axis and increased “discontinuities” in the direction perpendicular to it254. In addition, planar waves have the propensity to travel faster than convex ones572, due to more efficient distribution of depolarizing charges as the wave front crosses the medium573. Since the depolarizing current was introduced into the epicardium from a point stimulation, it will give rise to ellipses with convex wave fronts. For unknown reasons, the upper half (from LV free wall center to base) of the heart appears to facilitate conduction and becomes fully activated within 15ms of pulse induction (Vmax,ΔKPQ=0.44±0.04m.s-‐1). The remaining half (center to the apex) requires an additional 15ms to fully activate (Vmin,ΔKPQ=0.11±0.02m.s-‐1). In consequence, even if conduction block is not yet present here, double this time was still needed to reach the apex.
As the elliptical activation terminates in the upper half of the heart, a quasi-‐planar wave front is formed, more capable of surpassing the electrical load across the remaining epicardium to the apex than the originally expected convex wave.
The difference in CV between the two groups as Flecainice proceeds is shown in Figure 37. Bar graphs for Vmax (N=8, per group) and Vmin (N=8, per group) indicate a decrease in velocities in both substrates with an overall change (between t0 and t5) of 32% and 26% in Vmax for WT and ΔKPQ respectively; 40% and 39% in Vmin for WT and ΔKPQ respectively. It’s noteworthy to emphasize that at this time point of the current protocol, the values reached in the WT group are consistent with what was reported in the previous section for the control group of the mdx (during the 10min protocol), where at t5, Vmax,WT and Vmin,WT decreased by 33% and 39% respectively (Figure 22). At control conditions, the velocities recorded for the ΔKPQ substrate are globally 15-‐18% lower than the ones of the WT. The difference at that stage is significant between the two groups with Vmax,WT = 0.78±0.10m.s-‐1, Vmax,ΔKPQ = 0.66±0.06m.s-‐1 (p-‐value<0.05); and Vmin,WT = 0.37±0.08m.s-‐1, Vmin,ΔKPQ = 0.31±0.03m.s-‐1 (p-‐value<0.05). As NaV1.5 blocking proceeds, both velocities in the two substrates start to decrease, until t5 where the difference in velocities between the groups vanishes. The percentage decrease Vmax is slightly higher for the WT group than it is for the ΔKPQ, but Vmin seems to be similarly affected in both groups at t5. In the 10min protocol used to compare mdx and WT, patchy high velocity (or supernormal conduction) zones erupt at t10 in the WT group along the longitudinal direction. In the current results, the protocol was stopped at 5min, without an area of supernormal conduction being detected in either group.
In Figure 38, (dF/dt)max is calculated at each pixel for each of the maps presented in Figure 36. This approach is necessary to identify how NaV1.5 blocking alters (dF/dt)max in the ΔKPQ substrate and whether the fluctuations detected in CV are reproducible for (dF/dt)max .
Expatiated Pathophysiological Heterogeneity in ΔKPQ 105
In a similar fashion to CV, WT and ΔKPQ (dF/dt)max dispersion maps show a decrease in all directions with Flecainide (Figure 38a). The maps’ corresponding histograms are shown in Figure 40c. In the leftmost panel, the WT upstroke velocities have a bimodal distribution with two peaks at
~0.31 and ~0.44 ms-‐1, suggestive of the presence of longitudinal and transversal clusters. The ΔKPQ map also shows a semi-‐bimodal configuration at control conditions, with the majority of values lingering in the lower range compared to WT peaking at ~0.28ms-‐1 and a tail of higher values in the vicinity of 0.36ms-‐1. With Flecainide (at t5), histograms shift to the left indicating lower range of values in the two substrates: the WT maintains the bimodal distribution with the highest frequency around 0.24ms-‐1 and a smaller peak towards 0.35ms-‐1; whereas the ΔKPQ histograms concentrate to the left with a high peak around 0.1ms-‐1 and another smaller one around 0.16ms-‐1.
On average, the shallower upstrokes in the mutated substrate (upstroke = 0.43±0.03 a.u.ms-‐1) do not reveal a significant difference to their WT counterpart at t0 (0.47±0.10 a.u.ms-‐1) with p-‐value = 0.34 (Figure 38b). The sharper decrease in the ΔKPQ heart with Flecainide alters this difference in favor of considerably larger values in the WT with ~71% decrease in ΔKPQ in the longitudinal direction (WT: 0.31±0.08 a.u.ms-‐1; ΔKPQ: 0.15±0.04 a.u.ms-‐1; p-‐value<0.05), and ~60% decrease in ΔKPQ in the transversal direction (WT: 0.25±0.08 a.u.ms-‐1; ΔKPQ: 0.14±0.04 a.u.ms-‐1; p-‐value<0.05).
The decrease in the WT upstrokes, unlike the ΔKPQ substrate, doesn’t show directional differences, in such a way that the total change in (dF/dt)max is almost similar in the longitudinal (~36%) and transversal (~38%) directions.
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Figure 37. Bar graphs of Vmax and Vmin for WT (N=8) and ΔKPQ (N=8) at t0 and different time points of Flecainide [1μM]
perfusion. a. Decrease in Vmax is consistent for both groups. At t5, a maximum decrease of
~32% was recorded in the WT group vs.
~26% in the ΔKPQ (To note: a similar value was recorded in the previous set of experiments in the mdx control group (N=22) at t5 with a decrease of 33%, Figure 22).
Despite a larger percentage decrease in the WT, both groups have almost identical velocities at t5 (Vmax,WT = 0.53 ± 0.12m.s-‐1 ; Vmax,ΔKPQ = 0.49 ± 0.11m.s-‐1). The initially larger velocity in the WT group at t0 accounts for the larger decline (Vmax,WT = 0.78 ± 0.10m.s-‐1, Vmax,ΔKPQ = 0.66 ± 0.06m.s-‐1, p-‐value < 0.05). b.
A gradual decrease in Vmin with Flecainide.
A total of ~41% and ~39% decrease in Vmin was recorded for WT and ΔKPQ at t5 respectively. Starting with Vmin,WT = 0.37±0.08 m.s-‐1 and Vmin,ΔKPQ = 0.31 ± 0.03m.s-‐1 (p-‐
value<0.05), the difference between the two groups becomes statistically insignificant at 5min of Flecainide perfusion (Vmin,WT = 0.22±0.03 m.s-‐1 and Vmin,ΔKPQ = 0.19±0.03m.s-‐1).
Annotations in figure: * (p-‐value<0.05), n.s.
not significant.
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Figure 38. Dispersion maps showing at one pixel resolution the changes in (dF/dt)max with Flecainide [1μM] in WT (N=8) and ΔKPQ (N=8) and their respective bar graphs. a. Anisotropic (dF/dt)max distribution with a consistent decrease under NaV1.5 blocking. At t0, higher values are more widely spread in the WT than the ΔKPQ. However (dF/dt)max distribution remains anisotropic. At t5, the ΔKPQ substrate reveals an area of higher upstroke velocities proximal to the base and towards the right side, whereas the area towards the apex indicates dramatically diminished (dF/dt)max, as observed in the activation map. The WT map at t5 reveals slower upstrokes, with an unremarkable distribution. Look-‐up-‐table: (dF/dt)max [0 0.8]. Scale bar =1mm. Ao = Aorta, Ap = Apex, R = Right, L =Left, P = Pacing, PE
= Pacing Electrode. (b,c). Bar graphs showing the gradual change in (dF/dt)max in longitudinal (b) and transversal (c) directions. At t0, the shallower upstroke in ΔKPQ is on average not significantly different from its WT counterpart. The sharper decrease in the ΔKPQ upstroke velocity (d) in both directions longitudinal and transversal leads to a deviation in the values between the two groups at t5 (see text). d. Overall decrease in (dF/dt)max between t0 and t5 exceeds 70.8±4.8% in ΔKPQ (vs. 35.8±11.2% in WT) longitudinally and 59.8±7.1% transversely in ΔKPQ (vs. 38.1±9.4% in WT).
Annotations in figure: # (p-‐value<0.0001), *** (p-‐value<0.001), ** (p-‐value<0.01), * (p<0.05), n.s. not significant.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 107
A decrease in (dF/dt)max was reported for the WT group with the 10min Flecainide protocol in the previous section, where (dF/dt)max maximally dropped by ~52% on average in either direction (Figure 27c). Meanwhile, it’s noteworthy to recall that the mdx substrate showed a significantly smaller drop in (dF/dt)max at t10 compared to its WT counterpart particularly along the transverse direction (with a total decrease of ~39%). Contrastingly, ΔKPQ achieves a significantly larger decrease in (dF/dt)max compared to the WT group (at t5), which could indicate that the change in upstroke velocities (including the transversal ones) with Flecainide is possibly substrate dependent.
Although, both CV and (dF/dt)max decreased with Flecainide in the WT and ΔKPQ, their characteristic differences do not match at control conditions, and their response to Flecainide further consolidates this contrast. While Vmax and Vmin are significantly larger in the WT group at t0 compared to ΔKPQ, their corresponding (dF/dt)max are almost similar (no significant difference).
Similarly, the effects at t5 are reversed: the velocities become almost comparable between the two groups in either direction, whereas their corresponding upstroke velocities considerably differ.
Indeed in these two groups, CV and (dF/dt)max appear to behave characteristically different with or without Flecainide.