Dr Wilde, could you please begin by describing the strongest key evidence in support of the depolarisation hypothesis?

Transcription

1 Welcome to the Journal of Molecular and Cellular Cardiology Podcast on the Pathophysiological Mechanism of the Brugada Syndrome. My name is from the Department of Pharmacology at the University of California in Davis and I will be moderating this podcast. Joining me are the two participants, Dr Charles Antzelevitch, Executive Director and Director of Research at the Masonic Medical Research Laboratory and Professor of Pharmacology at the Upstate Medical University at Syracuse; and Dr Arthur Wilde, Professor and Chair in the Department of Cardiology in the Academic Medical Centre in Amsterdam in the Netherlands. I d like to begin by briefly introducing the topic of discussion today. The current views on the pathophysiological mechanism of the Brugada Syndrome: Brugada Syndrome is characterised by specific ST segment changes in the right precordial ECG leads, because patients with Brugada Syndrome are at increased risk of arrhythmia and sudden cardiac death, it is very important to understand the underlying electrical mechanism that gives rise to Brugada type ST segment elevation on the ECG. Understanding the mechanism will enable better risk stratification, timely treatment and prevention of sudden death. At present the pathophysiological mechanism of Brugada Syndrome is a topic of debate among researchers including our participants, Doctors Antzelevitch and Wilde. Although the participants agree that Brugada Syndrome originates in the right ventricle and on the association between the Brugada type ECG an increased susceptibility to arrhythmia, they do not agree on the likely underlying pathophysiological mechanism. Dr Antzelevitch and his colleagues have conducted many experiments in his laboratory that support the repolarisation hypothesis. This hypothesis relies on transmural dispersion of repolarisation between the right ventricular outflow tract in the endocardium and epicardium. The significant dispersion in cellular level action potentials is expected to give rise to large repolarisation gradients that will manifest as ST segment elevation on the ECGs. In contrast Dr Wilde has compiled evidence supporting the depolarisation hypothesis that relies on right ventricular conduction slowing and involvement of mild structural abnormalities. ST segment elevation is expected to result from severe delays in conduction in the right ventricular outflow tract compared to other areas of the right ventricle thereby creating a large gradient of depolarisation that is observed on the ECG as ST segment elevation. Dr Wilde, could you please begin by describing the strongest key evidence in support of the depolarisation hypothesis? Arthur Wilde

2 Thank you, Dr Clancy. Before doing so, may I introduce, Pieter Postema, who is also on my side of the ocean and on the phone too, so he might intervene as well. We think the strongest evidence in favour of the depolarisation theory is the fact that in any study you do, particularly in the clinical studies, there is always substantial conduction delay in the right ventricle in particular and in particular in the upper part of the right ventricle. This is already clear on the ECG, which most often shows right ventricular conduction delay, either represented by a right bundle branch block or by deep S-waves in the inferior leads that represent delayed conduction of the right ventricular outflow tract. There is recent evidence that is not actually in the manuscript, because it was not officially in the public domain yet, but also that if you measure at the epicardial side of the right ventricular outflow tract in patients that you really get conduction delays. The conduction delay there is up to milliseconds and that is what you need in order to explain the ECG by depolarisation disorder. That evidence is not in the paper that we published together, but I think that is in strong favour of the depolarisation hypothesis. If you allow me the argument against the repolarisation hypothesis is that we always struggle with the fact that if you have well coupled areas within the myocardium next to each other, how can there exist such big differences in the potential level? We never understood why you can have in a very thin right ventricular wall, which in principle contains well-coupled cells, very short exit potential, so exit potential with a deep plateau and normal action potential at the endocardial side. We think or we are afraid that a lot of the evidence that comes from the elegant experiments of Dr Antzelevitch relates to the experimental setup of the wedge preparation which might not be a good model to show whether or not transmural differences can exist. If you try to study this in the impact chart, so without the cut edges of the wedge preparation you never observe these huge transmural differences. A similar type of argument holds against the M cells, but we are now discussion the transmural inhomogeneity in repolarisation. Thank you, Dr Wilde. Dr Antzelevitch, if you could please start by describing the strongest key evidence in support of the repolarisation hypothesis and then please feel free to comment on the evidence for the depolarisation hypothesis that was mentioned by Dr Wilde. Charles Antzelevitch Let me start out by addressing the comments to the depolarisation hypothesis. The issue of right bundle branch block was indeed part of the initial definition of the Brugada Syndrome when first presented by Josep and Pedro Brugada, but it s been clear that many if not most Brugada syndrome patients do not meet the criteria for RBBB.

3 With respect to major conduction delays, there is a diversity of findings in various studies. The only study that I know of that actually measured epicardial and endocardial action potential simultaneously in patients with Brugada Syndrome was the study by Kurita and co-workers, which showed that there was no major conduction delay, but rather repolarisation differences that accounted for the electrocardiographic manifestations of the Brugada Syndrome. As far as finding of major repolarisation differences in the wedge preparations, this clearly has nothing to do with the cut edges of the wedge preparations, because if you measure this inside the wall in very thick coronary-perfused preparations you can see the same distinctions, so it s not a matter of coupling. Besides the major electrophysiogical substrate in BrS occurs on the epicardial surface of the right ventricular wedge, which has not cut surface. We find that with sodium channel mutations or block, we see minimal conduction delay with a 50% reduction in sodium channel current, as one would expect in theory, but that this can lead to very pronounced repolarisation differences. Let me move to the key evidence in support of the repolarisation hypothesis. I think it s important to point out at the outset that there are numerous experimental models of Brugada Syndrome that have been created, supporting the repolarisation hypothesis. To our knowledge there are none that recapitulate the arrhythmic and electrocardiographic manifestations of Brugada Syndrome based on depolarisation hypotheses. It s not merely that these models supporting the depolarisation hypothesis do not exist, but that all attempts to create Brugada Syndrome models mimicking the genetic defects responsible for the syndrome have indeed resulted in experimental models exhibiting characteristics consistent with the repolarisation hypothesis. We now recognise mutations in 8 channel genes as being responsible for Brugada Syndrome. These include mutation that cause a loss of function in sodium and calcium channel current as well as the a gain of function in transient outward channel current. Experimental models that mimic these using sodium and calcium channel blockers or agents that augment the transient outward current recapitulate all of the electrocardiographic and arrhythmic manifestations of Brugada via repolarisation hypothesis. Even when we use sodium channel blockade to reduce sodium channel to 50% of normal, thus mimicking haploinsufficiency, depolarisation factors contribute little to the manifestation of the Brugada phenotype. Not only are the electrocardiographic and arrhythmic manifestations for Brugada Syndrome recreated in these models, but the repolarisation mechanisms that are operative give rise to both the substrate and the trigger in the form of Phase 2 re-entry. The repolarisation hypothesis, by the way, also accounts for the fact that Brugada Syndrome is a right ventricular disease. The depolarisation hypothesis accounts for none of these.

4 The only means by which we re able to mimic the depolarisation hypothesis in our models is using ischemia and the electrocardiographic and functional characteristics are clearly different. What we generally see is an apparent ST segment elevation, which is actually a prolongation of the R-waves due to delayed conduction. The other point to be made is conduction under these conditions is exquisitely sensitive to increases in rate, so we encounter conduction block very readily as we progress to faster rates. I think it is important to point out that the principal electrocardiographic and arrhythmic characteristics of Brugada Syndrome are similar whether we examine a patient with an SCN5A mutation causing a loss of function of sodium channel current or a patient that has a KCNE3 mutation causing a gain of function in Ito. It is difficult indeed to explain, if these two have major differences in substrate. If one is due to the a depolarisation defect and the other due to a repolarisation defect, why are the electrocardiographic and arrhythmic manifestations so similar. Then there are repeated observations that quinidine via its actions to inhibit the transient outward current is effective in suppressing the electrocardiographic and arrhythmic manifestations of Brugada Syndrome. If these were indeed due to the depolarisation hypothesis, quinidine, via its actions to inhibit the sodium channel, would be expected to aggravate the Brugada phenotype; this does not happen. Finally, I would point out that the only in vivo model of Brugada Syndrome in existence is a heterozygous murine knockout model of SCN5A. This model, which recreates the haploinsufficiency often observed in the clinic, was very recently characterised and published by Martin and co-workers. The results, published just a couple of weeks ago, conclude that an accentuated transmural repolarisation gradient underlie arrhythmogenicity in this model. Thus, there is compelling evidence, both experimental and clinical for the repolarisation hypothesis. Thank you, Dr Antzelevitch. Following up on several of the points that you made, are we to conclude that the depolarisation and repolarisation hypotheses are mutually exclusive or is there room for both of these mechanisms to explain varied sources of the Brugada Syndrome? Dr Wilde, maybe you could begin by describing specific situations where you believe the depolarisation hypothesis is the key mechanism of observed Brugada Syndrome. Arthur Wilde

5 Can I first comment on a few issues? The issues raised by Charlie Antzelevitch are first of all the right bundle branch block. Of course, it s clear that not every patient has a clear right bundle branch on the ECG, but the vast majority of patients have a deep S-wave in the inferior leads and all the patients exposed to flecainide or asmalin or class 1 blocking agent lead to deep S-waves in the inferior leads, and the deep S-wave is a manifestation of right ventricular conduction delay, so that is in itself and if you only have the very end of the right ventricle conduction delay, you don t see a right bundle branch block, but you do see the deep S-wave in the inferior leads. The reason we are sure that the right ventricular outflow tract is based on the vectorcardiograms that showed that there is a rightwards exit deviation, so this is not the left ventricle delay, but it s a right ventricle delay and in particular a right particular outflow tract delay. It is agreed that that is not sufficient to have to account for the ST elevation, because you need much more delay than visible within the QRS complex. The one study Charlie was referring to that measured epicardial conduction delay was the study where, if I m right, they put the catheter in the right coronary artery and you don t measure the whole right ventricular outflow tract. You measure very locally and it is the whole area that should be covered by electrodes before you can really exclude conduction delay as the mechanism in action there. The third issue the different experimental models that are in use to mimic Brugada Syndrome, the majority of them are the wedge like preparations. It is not an impact part in the majority of them. The final issue relates on rate. We do believe and we indicated that in the manuscript that a significant number of patients with Brugada Syndrome do have more severe ST-segment elevation with increased heart rates. This was not recognised in the initial studies and we always thought that the rate dependency was in the opposite direction, but I think it s very clear now that a significant and I would say substantial, not all, but a substantial number of patients with Brugada Syndrome get a worsening of the ST-elevation during increased heart rate. The issue of Quinidine we have discussed that many times, it s the most troublesome to me because at first glance you would indeed expect that the sodium channel blocking effect should make it worse. If you think about the reason of the conduction delay then it becomes very speculative, in reality the right ventricular outflow tract it divides from AV-nodal tissue. There is an AV-nodal tissue ring that should fade out of the right ventricular outflow tract and if some tissue of the AV nodes persist in the area of the right ventricular outflow tract you would expect Quinidine to improve conduction in that part of the heart. Quinidine is known to improve conduction in the AV node and it will do so in remnants of the AV node in the right ventricular outflow tract, so our current fault is that that might be part of the reason. It s an anatomical substrate remnants of the AV-nodal area in the right ventricular outflow tract that are sensitive

6 to Quinidine like the AV nodes are sensitive to Quinidine, and that might explain the effects of Quinidine, and you might try to improve, as Charlie stated, the ST-elevation then. I have to go back to the question that is when the repolarisation hypothesis is key in the explanation of the ST-elevation. Is that right, Colleen? Right. Arthur Wilde I think it s in the majority of cases, at least those cases with sodium channel mutation, that the main gene we know of I am still not that confident that the calcium channel mutations relate to Brugada Syndrome patients are very similar to the sodium channel mutations. I think we need some genotype/phenotype information there and I have not seen the data. The EKGs that were shown for calcium dependant Brugada Syndrome are quite similar although conduction delays is evident there and that is already what we showed, because there is a difference between conduction intervals between SCN5A positive patients and SCN5A negative patients. About the other rare patients with single mutation and the gene encoding one of the ITO components and the other ones relate to reduced sodium currents. That s in a similar direction as the sodium channel mutations themselves, and the ITO one might be one in favour of the repolarisation theory. I think in the vast majority of cases the depolarisation is the issue, and the study that I briefly refer to from Thailand from Nademanee. I had hoped it would have appeared now already in the literature. He showed me the data; he showed the data on the Heart Rhythm Society last year in Boston. There is really a pronounced conduction delay in an area of the right ventricular epicardial side. It adds up to milliseconds and very local conduction delay and if you ablate that area, so make a substrate ablation within that area, what you see is that you see is that the ST-elevation in the end disappears; it s more severe in the early phase after ablation because of the entry current, but it fades away in the weeks after the ablation and the arrhythmias are gone. You can say that the ablation destroys the whole right ventricular outflow tract transmurally, so you also destroy the transmural gradient and as long as we have no pathoanatomical specimens of these patients that have undergone this ablation procedure, we will no know, but it s at least compatible with local epicardial conduction delay as well and I don t think the one clinical study, that Charlie refers to, excludes that possibility at all.

7 Thank you, Dr Wilde. Given the comments that you ve just made I think I d like to ask the next question, which naturally stems from those comments. The mechanism of conduction slowing that you describe in the JMCC article relies on delayed depolarisation in a specific structure in the right ventricular outflow tract in the right ventricle and the source sync relationship within the closed loop circuit. Could you talk about how conduction delay mechanisms would be expected to extend to ST-segment elevation in the full atria and in the ventricles that are observed during challenge tests with drugs to block depolarising currents? Arthur Wilde I don t think are able to see ST-elevation in the atria on the EKG, so with permission I would like to skip that question and in the left side I must admit I have no clear explanation, but you do not see it often. It s rare that you see ST-elevation you see it in the lateral leads, so in AVL and lead one and it s rare. You never see it in the left lateral leads V5, V6 which are closer to the epicardial side of the left ventricle and you don t see it there, but you do see it in lead one and the AVL, and I don t know what you see there. In a very rare case you see it only in those leads and not in the right epicardial lead. I have no clue to explain that. Okay thank you, and now a final question for Dr Antzelevitch, one thing that I ve always wondered about is that pharmacological models of the Brugada Syndrome, how exactly to those experimental models relate to inherited and acquired forms of Brugada Syndrome? Let me be more specific. When drugs are used to mimic Brugada Syndrome in experimental preparations they act by dramatically reducing depolarising sodium and calcium currents, which tips the balance of currents to allow for a potassium currents to dominate and cause abbreviated action potential in some cells and coved dome morphologies in others, but many of the inherited Brugada linked mutations cause only modest reductions and peak sodium currents, and the clinically relevant doses of this sodium channel blockers would be expected to reduce current less than would be needed for premature repolarisation. Can you help to reconcile the potential for a common mechanism observed in experimental studies and in the clinically observed pathology? Charles Antzelevitch Fascinating question Colleen, but before I get to it can I just address some of the comments that Arthur made, which I found very interesting. The fact that RVOT derives from AV-nodal and that quinidine

8 exerts an atropine like effect is interesting, but I don t believe that the RVOT or any part of the ventricular myocardium has been shown to have muscarinic receptors, so that would be an issue to probe further. I am at a loss to comment on Nademanaee s data because I haven t see it. I would point out that when we create a mark dispersion of repolarisation and one introduces a premature beat, you encounter major conduction delays, as a consequence of the repolarisation defect. The question is what comes first the chicken or the egg? Is repolarisation heterogeneity contributing to the marked delay in conduction or is the marked delay in conduction contributing directly to the sequela associated with the Brugada Syndrome. Now to address your question, we know that in some cases we find SCN5A mutations and they cause a fairly mild reduction sodium channel current. In fact, Arthur s group recently published one on SCN1B and SCN1Bb. We found similar mutations in SCN1B and we expressed these and also found that heterozygously expressed they produce only approximately a 20% reduction in sodium channel current, which in our view is insufficient to produce the Brugada phenotype. From experimental models we know that we need at least 40 to 50% reduction of sodium channel current in order to create the repolarisation heterogeneity, so we looked to see whether SCN1B can affect other channels and based on the work of Isabelle Deschenes and Gordon Tomaselli s group, we looked at Ito. We co-expressed SCN1B with KCND3 (Kv4.3) and found that the mutant channel produced a marked gain of function in Ito. It turns out that the sodium channel inhibition was actually a very minor component of the expression of the Brugada phenotype and the gain of function in Ito, was largely responsible. We need to consider all of these factors including the entire haplotype when we examine mechanisms. This I think is part of the explanation for why we can have Brugada Syndrome when we see fairly mild reduction in sodium channel currents. The other point that I would like to bring up is that we have been very busy looking at calcium channels in the ventricular myocardium, and find a high yield of calcium channel mutations. I am referring to the L- Type calcium channel, comprised of the α1, β2 and α2delta subunits. We find mutations in these channels associated with Brugada Syndrome. They are not associated with any type of major conduction problem. The yield is about 12-14%. Our yield with sodium channels is 18%, so it is almost equivalent. Here again, when we mimic this using calcium channel blockers we observe only repolarisation abnormalities in the wedge preparations as being responsible for the Brugada phenotype. No conduction impairment is observed, once again in support of the repolarisation hypothesis. Dr Wilde, do you care to comment?

9 Arthur Wilde I made the comment before; I indicated that as to the calcium channel mutations I am not sure why that would work out in the same. It is more difficult to reconcile the depolarisation hypothesis; that is sure. It could affect the cells, when there are AV-nodal cells in that right ventricular outflow tract it could still have an effect there that gets you into this direction. The main issue remains that the conduction relay data are to me quite convincing. It is a pity that we have no access, both of us right at this moment or in this manuscript on the Nademanee data, because they are really quite convincing. We should have a careful look, so I have never seen a calcium channel mutation related to Brugada Syndrome patients. We have checked it in the early days but we never found it, so I have no possibility to compare the EKGs of these patients. We are easy to group things that at a later stage appear not to be the same thing. This could be the case here. Thank you. Do either of you have any further comments that you would like to make before we end this podcast? Pieter Postema Can I drop in for a moment about the mutual exclusivity of the depolarisation and the repolarisation hypothesis? Two thing that didn t skip the discussion yet is; one is structural abnormalities, which might be related to Brugada Syndrome as well, which have been shown in the [unclear] of a Brugada Syndrome patient and that has also been shown in studies into histopathological examinations by biopsies from the right ventricle, which were actually not reproduced by another group, so that is still open for discussion, but as for strong depolarisation changes or conduction slowing will shorten exponentials in the latest activated parts. Actually you will get shortening of repolarisation when there are strong depolarisation changes present. That is something that has been used from our side to back up the depolarisation hypothesis while including repolarisation changes also on the cell level. Charles Antzelevitch I would just add as a final comment that I have no question that slowed conduction and mild structural defects that Pieter points out; in some Brugada cases, particularly those involving the sodium channel loss of function, contribute to the Brugada phenotype. Our view however is that this is a relatively minor

10 contribution. In order to get major conduction delays in the heart you need to drop sodium current by much more than 50%. There are associations with structural disease. We know that the Brugada phenotype is in some cases associated with arrhythmogenic right ventricular cardiomyopathy, as described by Corrado and coworkers. Recent studies from Mario Delmar s group show that a Plakophilin-2 mutation can disrupt the desmosome, causing major conduction delays, but it also reduces the expression of sodium channel current. It is interesting to speculate that it is the reduction of sodium channel current that is responsible for the Brugada phenotype via repolarisation heterogeneity and that the desmosomal disruption underlies the major conduction problems. It is very likely that there are other overlap syndromes in which common genetic variations may exist and lead to a combination of repolarisation and depolarisation defects, but our view is that these are the exception rather than the rule. Excellent. I think that is a great way to end this podcast. I would like to than the participants for your insightful comments and for your participation in JMCC Podcast. To read more about the pathophysiological mechanisms of Brugada Syndrome please visit the JMCC Homepage to obtain the point/counterpoint article authored by today's guests and their colleagues. Thank you very much