a very exciting session on the basic mechanisms and effects of pulse field ablation. You're welcome to send in, we can use the structured app for questions or you guys can come up here to the mic and ask questions at the end. We have a star lineup and I'd love to get started so we can spend more time discussing the topics. We'd like to first invite Dr. Koss to discuss mechanisms of cell injury by electroporation in cardiac and non-cardiac tissue. Hello, thank you very much. I have to click on start, I hope. All right, thanks very much again for the introduction, for the invitation. While this is coming up, I've already been introduced. So I've been asked to talk about the mechanisms of cell death. And by now, you've all heard quite several times during the conference that pulse field ablation equals electroporation. We know that electroporation is caused by exposure of cells to electric fields. And what these electric fields do is they induce a large transmembrane voltage on the cell and of course, that can cause depolarization, but this transmembrane voltage is so large that it does other things to the cells. I've seen already quite a few slides showing pores in the lipid domain, but we found that this is not the only mechanism of injury to this membrane. And there are several other mechanisms that can happen at the same time as the lipid pores formation. So there is lipid oxidation and protein denaturation, so the ion channels and other stuff. So we have to think about electroporation as a sequence of events that starts when we apply the pulses, and then goes on for quite some time. And after a certain period of time, the cells will either recover, and we will call this reversible electroporation, or they will die. And that is, of course, what is called irreversible electroporation. We did a large review of papers dealing with different kinds of injury to cell after electroporation. And I have to say that this is a bit of a chicken and egg problem, because some of these mechanisms are, let's see, there's no pointer here. Some of these mechanisms are like ATP depletion. They can lead to membrane damage, and membrane damage can also lead to ATP depletion. I think it's fine. Well, we're doing okay. Now you can use the pointer. Okay. So now, with the help, it's... I'm sorry. It's fine. We can leave it like this. No, we lost the screen. Well, now, as I said, there are different mechanisms that are going on. At the same time, it's probably not going to be possible to separate them, one from the other, really specifically. Now, we do have to say that there is quite a big, large dearth of information, especially in the longer pulse durations. As you can see here, there are only a couple of studies in this area where using longer pulses, but most of this stuff was done using nanosecond pulses. Now, let's transition from this cell injury, the injury to the cell membrane, to what's going on with cell death. And this cell death is also a process, as I mentioned earlier. And in this study, we can see very nicely in in vitro 3D tissue matrix collagen experiments, where we can see that the lesion formation is really increasing a lot from three hours after the pulsation up to 24 hours after the pulses. And there are also some difference between waveforms. Now, in our lab, we did a study that looked at the dynamics in a different kind of way, so using flow cytometry. And I'll walk you through this slide because it's quite a lot going on. So what's interesting in this picture is that we have this large part of cells at a certain, after exposure to fields, that they are the normal cells. They are alive, they have a very relatively low fluorescence as visualized by the flow cytometry using propidium iodide. Then we have this compartment of immediately dead cells that have a very large fluorescence. And these are cells that have died immediately. And you can see also a small part of these cells are also in the blue control group. So they're also normally every kind of culture we have, there'll be a couple of dead cells. But we also see a large spike in the fluorescent in the treatment group where the cells have died. What's interesting though is this middle part. So these are cells that have an intermediate fluorescence with PI. So these are cells that are clearly distressed. There's something going on with their membrane because normally these cells don't fluoresce if you add this fluorescent dye. So we looked specifically, the next graph I'll show is looking specifically only at this compartment, okay? But, and there is a lot of data points here. We are looking at this at different time points and using very different amplitudes of the electric fields. And we can see that there is a time dynamic to this evolution, to this injury of this cell, that in this delayed cell that, and some of these cells of course can then recover, but we can see that after a certain duration of time, we have a large percentage of cells in this compartment, and then this reduces after a longer period of time. That means that either these cells at a later time stage have died, so they don't show up anymore, or they have recovered, so they again don't show up anymore. And additionally, what we can see is there's also some difference between cell lines. So we look at the Chinese hamster ovary cells or these cardiomyoblasts. You can clearly see different time courses of this mechanism after post-field ablation. Now, I've heard several times the mention of lethal electric field threshold. Why is this important? So the lethal electric field threshold is actually a very useful concept, but we have to keep in mind that this is a simplification. So what we, after post-field ablation, every time we get some cells that survive and some cells that die, and this is generally a gradual transition. So when we introduce the lethal electric field, this is a sharp transition, but it helps us wrap our minds around what is going on in tissue. And we did a study that was done in an ex vivo model with pig hearts, applying pulses to the pig hearts, first using a metronic post-field ablation protocol, but then we also did the same experiments and with 100 microsecond post-field protocol. That was in order to find how similar or how different it is to other tissues. And other tissues have been targeted with electroporation much more extensively prior to cardiac becoming the main star of this show. And the way we go about figuring out the lethal electric field threshold is, of course, you have experiments. These are lesions in pigs visualized by TTC staining. And we compare these lesions to numerical models that will show us the distribution of the electric field. And, of course, we threshold this electric field with a certain electric field threshold and looking at all the time at the best electric field that is matching the experimental results in the best way. We cannot really compare these studies because these are done using very different, they're all used 100 microsecond pulses, but that's basically all they have in common. They have different tissues, they have different ways of evaluating the outcome, and they also looked at the cell death at different time points. So this is, but nevertheless, if you would want to compare them, I have put together this nice slide. And what we can see is that the cardiac tissue, even though it has a little bit lower thresholds than most other tissues, it's actually the skeletal muscle that using the same kind of approach, exactly the same as in this study, has an even lower threshold. We've also done estimations of lethal electric field threshold in vivo. So that is visualized using late gadolinium enhanced MRI imaging. We did this study with multiple different number of pulse trains ranging from one to up to 16. And on these pulse trains, of course, for every different lesion, we estimated the lethal electric field threshold, and then we fitted these curves to this function that we found already in an earlier publication. And when we combine this with the numerical model of the electric field around the catheter that was used in the study, we can also then estimate how deep we can get by just increasing the number of pulse trains. And you can see a very large plateau effect after a certain number of pulse trains. And that's one of the reasons why we don't want to increase the number of pulse trains to infinity. First of all, we'd get diminishing returns and no other, not much to gain, but we do have much to lose. That's because every kind of energy delivery to tissue also comes together with temperature rise. And we have to keep in mind that every time nearest the catheter, there is a quite significant temperature rise. But of course, we want to keep the mechanism of cell death non-thermal. And now to wrap up, I think we've shown that the cell death is a very dynamic process that is definitely not instantaneous and that it takes some time. And also it's important to keep in mind that when we do any kind of treatment in tissue that we're exposing the cells to very different electric field strengths based on the distance from our treating electrodes. And also it's important to keep in mind that different tissues can have different dynamics of cell death. And of course, what's then maybe the differential effect for the treatment can come from is that different tissues can have different capacity for healing. So with this, I'd like to thank you very much for your attention. Thank you. Thank you very much. Very nice thing. So just a question. So is it correct if I understand you right, even though the pictures show these zones, reversible, irreversible, and no, it's a continuum. So some things in each zone will be reversible and irreversible. Exactly. So what we see here is a model from the same study where we show that these zones are sort of overlapping. There may be nested one in the other, but they're always most of them present every time we apply the treatment. What do we know about this reversible zone? Are they normal cells coming back? Are they different? Are they better senescent cells, more like juvenile cells? What do we know about that? Are they the same as normal? So I'm now going a bit outside the scope of this presentation, but we found that you can, in vitro cardiomyocytes, sometimes you can pace them and they respond to pacing even after a minute, even if they are not immediately able to pace them. So they can come back and then behave normally. I cannot really discuss if they have changed this kind of, any kind of morphology or other things, because we don't, I don't think we've looked at that in such a long time period. Great. Thank you. Thank you very much. Thank you. Thank you. Thank you, Dr. Koss. Our next topic is tissue and animal models of cardiac injury and nearby organ damage with pulse field ablation by Dr. Meisels from Sheba Medical Center. Welcome. I'll download the presentation. Just click on yours. Did that. No, I'm trying to swipe it. I think it got switched on the type of view. Let me have them reset. Sorry about that. That's OK. So good afternoon, everybody. I'm Leonid Meisels. I'm from the Royal Melbourne Hospital and Chiba Medical Centre. I'd like to thank you, the committee, very much for the opportunity and invitation to talk in the conference. And when the presentation is up, I'll be able to talk about PFA models. I've got no disclosures and my research is completely independent of the industry, which I think is good for being objective about the advantages but also disadvantages of the system. I know. There it is. Thank you. All right. So why do we need models for PFA? I think the answer is evident in this sort of graph. All these protocols were evaluated preclinically, in vitro, in vivo. And still, during the clinical trial, there were iterations of clinical improvement, clinical adjustment of the protocol to achieve better results. And this is quite unanimous across all the big systems that were introduced clinically. And when we consider so many parameters that affect PFA, it's not surprising that we do need good enough models to incorporate all these different parameters to allow us better predict the clinical outcomes of PFA. And, for instance, when we see... This is a sentence from a good publication. And we do need to ask ourselves, can PFA actually affect the esophagus? Can PFA cause phrenic nerve paralysis? Is PFA actually cardiac-specific? So that's why we need models. So, for instance, esophageal injury. One example, Mara Koshola took human esophageal smooth muscle cells and human-induced pluripotent stem cell-derived cardiomyocytes, compared the irreversibility threshold of the two using very detailed protocols and viability staining, showing that cardiac cells are two to three times more susceptible to killing than esophageal cells. So, on the one hand, these are very good models in vitro because they can specifically address one or several questions that are highly controlled. But the disadvantage of these kind of models is that they do not reproduce the clinical scenario very well, for which we need large animal models. So, for instance, this publication by Kaz Nevin, open chest swine model stem PFA applicator directly delivering electroporation to the esophagus and demonstrated intraepithelial vesicles, some threaded muscle degeneration, some late fibrosis, but no otherwise significant sequela long-term. And similar findings were shown in a rabbit open chest model of the esophagus. A bit more contemporary, clinically relevant models would be the esophageal deviation models. So, with a balloon, the esophagus is deviated closer to endocardial structures where ablation is performed. This is a more clinical scenario. So, in these kind of models, we can see overall either non or minimal esophageal damage chronically, but recently there's increasing evidence that acutely we can actually see various degrees of non-transmural damage that is healed over the course of a couple of weeks. Now, what about blood vessels? So, this work by Elad Maor. So, PFA delivery directly to the arteries did not result in any distortion of the anatomy or the extracellular matrix, but nevertheless it did destroy the whole medial muscular layer of the artery. And some epicardial, endocardial, and intracoronary models, including from Professor Asif Adam's group, they all show different degrees of coronary spasm, some degrees of chronic imprint, including intimal hyperplasia and medial fibrosis. And we also need to remember that these models do not account for the presence of coronary artery disease or tendency for coronary spasm or delayed vascular spasm, which are all clinically relevant scenarios. PFA can definitely affect the blood pool. Several models demonstrating ex vivo and in vivo that increasing amount of pulses or reduced tissue contact are both associated with hemolysis. Potentially some differences between different PFA systems as well, or some of the differences might be explained by the different experimental setup. There are quite a lot of models of injury to nerves, but this one is pretty comprehensive, demonstrating or using accelerometers to measure diaphragmatic contractions. So it's a very nice model that demonstrated, that you can actually damage or stun the phrenic nerve, and that this effect is voltage threshold related and proximity related. And in this model, by the end of the experiment, all phrenic function recovered. There was no chronic histological evidence of damage. But nevertheless, it can definitely affect the phrenic nerve. So from all these studies, I think we can say that electroporation, not only PFA, but electroporation can obviously affect any tissue, probably can kill any cell you want. It only depends on the protocol and how we do it. And also PFA, as PFA and clinical PFA, has a variable thermal footprint, which sometimes might not be negligible. And taking this together and using this famous quote from the House of God, I wonder if there is a strong enough tool and a strong enough arm, can we actually damage tissues that we currently think that we cannot damage? In my opinion, we might. Different models are used for PFA optimization as well. So several models of epicardial and endocardial swine preparations that overall taught us that contact is important to generate adequate lesions. Contact force is probably also important to an extent. This is a matter of ongoing study now. Small animal models can be easily used to study and compare different protocols. So in this work from our group, Eyal Heller demonstrated reduced muscle stimulation with high frequency by phasic pulses, and he also compared different protocols and different tissue effects. This work nicely evaluated the differences between unipolar and bipolar pulse delivery while confirming the cumulative effect of additional pulses on lesion dimensions, also suggesting a trend for deeper lesions with unipolar delivery. But just as Boer mentioned earlier, so if we increase the pulse number four times, we don't actually increase the lesion depth by four times. It's not a linear effect, and it has a plateau. So we cannot increase pulse number indefinitely expecting that this will simply increase lesion depth. There are several models available to assess lesion dynamics as well. So in this work from our group as well, we used human induced pluripotent stem cell derived cardiomyocytes and loaded them with voltage sensitive dyes, and then we were able to assess functional electrical activity in these cells and functional lesion dimensions. So we can see here different recovery dynamics over time and some differences between different protocols. And this model also demonstrated the importance of delayed recovery. So when assessed at 24 hours post-PFA, we could notice further recovery of the lesion periphery, but also we could see in some of the cultures that this conduction barrier actually was breached. And this is very important because we need to be able to predict this and then eliminate this if we're looking into clinical meaning of such treatments. Again, recent publication that Boer also mentioned by Damian Niklavchich, a very comprehensive model where they were able to characterize the MRI imprint of PFA lesions and also correlate them with histology. They also show this sort of biphasic recovery trend. Also nicely showed correlation between current of injury and additional parameters to lesion depth and transmurality. Quite a few models there to show that PFA penetrates scar tissue and probably does so better than RF. And finally, there is a growing interest in the predictability of long-term PFA outcomes. So this work from Tomas and Anthony Vora's group used a 3D printed scaffold that was applied epicardially to record EGMs after lesion deployment. And they were able to characterize the changes in EGMs in different areas of the lesion and also to correlate these parameters with long-term lesion durability. Another report from Atul Verma's group assessed a novel PFA catheter that has optical recording abilities that records tissue birefringence, which is reduced following PFA. And they were able to demonstrate a cutoff that predicts transmural chronic lesions. And finally, this work from Damian's group as well assessed components of the high frequency and low frequency bipolar and unipolar EGMs that were also predictive of lesion transmurality and chronicity. So all these models, they have some limitations, some issues, but it is very promising that we will be able to better predict PFA durability and chronic effects. So to summarize, and we didn't actually touch computational models, but they are extremely important in the study of PFA, and they complement all tissue models that we saw here. Modeling is critical for the study of PFA, in my opinion. Each model has specific advantages and disadvantages. In vitro models have the advantage of being controlled. They can address specific questions mechanistically, but they do not represent the clinical setup as well, for which we're using large animal models. But these are not perfect as well, because, for instance, swine anatomy and physiology is still different from human. And again, we see that when we try to implement our knowledge from the in vivo to clinical practice. In my opinion, it's very important to maybe consider developing some set of standardized, protocolized data that we would like to require from every new system and every new protocol that are being published, including the presumed field potential and how it affects the tissue, and also that the companies start disclosing their protocols so we can actually assess them in vitro and preclinically much better to understand also the safety concerns or implications that these systems might have. And I think, yeah, we definitely need a better chronic endpoint prediction. And with that, I'd like to thank you all for listening, and I'm happy to take any questions. Thank you so much. That was quite eye-opening. My question is, if you crank up the threshold on a particular system, are you likely to see damage to the esophagus, phrenic nerve, and the vessels linearly? So would you see all three affected, or do you still think it's questionable? So if you had setting A and setting B, and setting A showed evidence of phrenic nerve injury, and setting A is stronger than setting B, would you also see more vessel injury and esophageal injury? That's a great question. Thank you. I'm not sure. I'm not sure. I don't think—I think they're different. Phrenic nerve, I think, is a bit different than other tissues because—or I would say myelinated nerve, because electroporation per se would much less likely, in my opinion, affect myelinated nerve chronically. It can definitely acutely affect it, but these nerves are more likely to recover as long as the epineurium is intact, whereas electroporation as itself can kill any other cell type. But I guess that if we crank up the voltage, if we crank up the durations or other parameters, or we stack pulses, I think we can definitely see irreversible damage potentially to the esophagus, maybe phrenic nerve. And also, again, as we said, that there is thermal imprint of different systems, and we might inadvertently reach to significant thermal damage. So it will not be electroporation-related, but it will be PFA procedure-related. Just one other question. You know, you kind of mentioned this, but you know, Eilat Mao's rabbit carotid study, it's like eye-opener for the whole field at that time. Nothing happens to the artery, there's a scaffold. But so much early work, we never really saw spasm. You know, why is that? Because, you know, some of the models used, for instance, the swine, is actually a spasm model for study of early calcium blockers and nitric oxide. Why was it so late an entrant into our thinking? Again, a great question, and I really thought about it myself, and I don't know, because the animals, maybe it was the different model, maybe the rat and swine are not the same, because they had zero consequences on the animals. The animals were fine, and then when they were sacrificed chronically, they saw normal structure, endothelium recovered, and the medial layer did not. But yeah, if there was significant spasm, the animals should have been stroked, but they weren't. Maybe different animal. Thank you very much. Thank you. Our next talk is on different PFA protocols and delivery tools to maximize myocardial injury and minimize extracardiac tissue injury by Dr. Kohlke. Hello, everyone. Thank you so much for the opportunity to present. Thank you, Dr. Mehta and Dr. Asurabhatam. It is indeed an honor to share stage with some amazing pioneers in the field, and I'm a clinical cardiac electrophysiologist, so I'll try to provide some background to the clinicians here about different PFA protocols and how they apply to our ablation techniques. So we'll go over some energy delivery parameters for PFA and some of the effect of the different tools we have available in our arsenal at present, and we'll cover some of the safety profile of PFA techniques. And at the end, I would like to urge you all to frame the right questions. Is PFA truly non-thermal? And there's a difference in saying that the thermal mechanism isn't the reason for cell death versus saying there's no thermal effect at all. Is myocardial selectivity sufficient? And then a lot of our clinical trials at this point have focused on atrial fibrillation, and then that mainly involves ablation of myocardium, atrial myocardial sleeves and the pulmonary veins. But beyond that, we know there is some benefit of ablating a neural tissue as well in AFib. So is myocardial selectivity sufficient, or do we need to have techniques where we increase ablation of these non-myocardial cells as well? And does one-size-fit-all? So at present, the way we are using these ablation modalities is we have different vendors with their catheters and then their profile set, and then we're essentially pushing a button and delivering energy. Is that, is an ablation on a first-time paroxysmal PVI in a healthy atrium the same as someone in persistent AFib and hypertrophic cardiomyopathy? So we'll try to cover some of these aspects. And my prior speakers have covered this in detail, but just a quick review of when we apply electric field to tissue, it is essentially a non-thermal effect created by delivery of direct current electrical energy, and then this creates pores within the lipid bilayer. And if there's sufficient energy delivered, these pores are irreversible, leading to alteration in the cellular homeostasis. And in the initial days of the field, reversible electroporation was used for delivery of chemotherapy agents, for example, in cancer patients. For our purposes, we are mostly interested in irreversible electroporation and destruction of the cell via apoptosis with intracellular calcium influx. And if the energy is increased even further, that can lead to more necrosis and thermal damage as well. And the expectation of PFA safety in the early days comes from a schematic like this, where we know as we increase the dose of any energy, there's more tissue response. So with radiofrequency, you increase the wattage, you increase the temperature at the contact site, the tissue temperature, and the time of delivery, and more contact force that leads to more tissue response. And the hope is with pulse field ablation that we can achieve the same tissue response and we have a wider safety margin. So if we keep increasing the parameters, sort of like the question you asked, is there a point at which we can deliver more energy and destroy the tissue of interest and still not get adverse effects? So the factors governing myocardial selectivity are that the cardiomyocytes require substantial energy for usual activities. And when we create these pores, the insufficient energy hinders the rapid correction of the lipid bilayer. And then we have this Schwan equation, which is the induced transmembrane voltage is governed by the electric field and then the radius of the cell and the angle of the electric field to the cell. So keeping everything else constant, larger cells will have, by definition, more induced transmembrane velocity or voltage. And for that reason, myocardial cells are more susceptible to the same field as opposed to nerve cell as well as smooth muscle cells. Beyond the properties of the myocardial cells themselves, we can vary the parameters of delivery. So some of this is intuitive. So if you increase the energy, increase the voltage of application, and reduce the distance from the application to the tissue, you get more effect. And the shape of the waveform can cause significant changes as well. So there are many different kinds of waveforms that have been studied. For the purpose of clinical application, we mostly deal with square waveforms as opposed to sinusoidal. So speaking of square waveforms, you can have these monophasic pulses where a pulse is delivered and then there's a relatively longer delay before another pulse is delivered. This can be done with alternating monophasic with changing polarity as well. Biphasic pulse will have a shorter delay between a delivery and a packet is defined as multiple pulses that constitute one packet and then a longer delay before the next packet is delivered. So what we know is that with monophasic delivery, we do get stronger treatment effect. There's a greater electric field, but comes at a cost of increased muscle contraction and barotrauma and microbubble formation. And we'll get to some of this when we get to the clinical section. And with biphasic, there is less muscle contraction, but again, we get a bigger treatment effect. So there's a trade-off. And fundamental frequency comes into play for biphasic waveforms and can significantly alter treatment effect. So higher frequencies which have shorter individual pulse durations can reduce muscle contraction, but again, progressively smaller treatment effects. And pulse width is the duration for which this is delivered. We know more of the pulse width will create more cell damage. And this curve is similar to those of you performing devices, our strength duration curve. So we have asymptotic values on either side. So beyond a certain point, increasing the pulse width and increasing the electric field does not create more damage. So we do reach a threshold. And as these curves go higher here, we end up towards thermal injury, which can be almost as similar to what we see with the radiofrequency ablation. So this is where I think there's a lot of interest in most of us to see, does the effect of delivery tools change how we create these lesions? And there are a slew of catheters and technologies available. And then a lot of new catheters being presented at the session this time. But I'll just focus on some of the concepts of what governs the electric field generated. So the first thing I would like to talk about is catheter contact. And when PFA was first proposed to the clinical application for cardiac ablation, it was marketed as a contact-less approach. So contact is not important. But we know and we've seen this in preclinical studies and in clinical studies that contact certainly does improve the lesion delivery. And maybe contact isn't as important as what we see with the radiofrequency, but it certainly is an important factor. Beyond that, the electrode size and the surface area. So the smallest surface area of contact leads to a more dense electric field and more cell damage. The orientation of the catheters are the poles as well. So if you have a multipolar catheter, it leads to more of an outward electric field as opposed to a single short catheter that leads to an inward electric field at the point of delivery. So those are generally better, the outward electric fields, for creating lesions at the place where you want it with less collateral damage. And you can change, some of these have different configurations in terms of unipolar or bipolar delivery. So that is governed by the return electrodes. You can have both the poles, anode and cathode, on the delivery catheter. Or you can have one situation as unipolar where the return electrode is the back of the patient, for example. So wider electric field will lead to more stimulation beyond the cardiac tissue, so you can have more skeletal muscle stimulation in that situation. So coming to safety of PFA, there are a few concerns that have been raised with clinical studies. And I'd like to talk about each of them individually. Gas bubble formation and how many of you have done PFA cases and have seen gas bubbles or bubbles during ablation? So a lot of you. And then we all have seen this and it is not clear as to, so we know why it happens, it's mostly because of hydrolysis of the blood pool. But it's not clear what factors change this because part of the issue that we are dealing with now is there is this enigma about what are we actually delivering. And we don't know if we change parameters catheter to catheter, is there less electrolysis with one versus the other. But there can be clinical consequences. So we've seen up to three to nine percent asymptomatic thromboembolic events in these patients. And long term studies up to a year of follow-up have shown no significant neurological damage over time. But then just seeing that is quite concerning because at this point we are ablating mostly inside the left atrium. And even in their microemboli, we need to figure out, we need to do better as a community to figure out what is causing this. There have been some preclinical studies showing anodal delivery versus cathodal delivery can change gas bubble formation, as well as alternating current delivery can help with reducing gas bubbles. But there's no consensus as to one protocol that can minimize this significantly. With phrenic nerve injury, the reassuring thing is the manifest study, which had over 17,000 patients studied, did have a low event rate of permanent phrenic nerve damage. We do see some phrenic nerve paresis, and that does recover over time. But then there have been some recent reports in 2024, up to 64% stunning with SVC isolation performed with the pentaspline catheter. Now this is concerning, and again the reasons this may happen is the proximity of the ablation catheter to the phrenic nerve. If we get to a point where we do epicardial ablations with PFA catheters, then certainly this number can go up because of the shorter distance to the phrenic nerve. But this is not a non-issue. It is good to see that the event rates are low, but it's something that we need to keep following. We spoke a little bit about coronary spasm in the prior talk, and the event rates were again low and manifest. But a lot of us have seen or heard about these cases happening, and some of the studies have shown responsiveness to pretreatment with nitroglycerin. That being said, it is still a large unknown as to why we did not see a lot of this in our preclinical studies, and why we are seeing a lot of this now. Some folks have said that because radiofrequency leads to more of an occlusive kind of injury leading to PCI, spasm may be a little better than that. But again, this is not something that we can say clearly. The other concerning thing is long-term follow-up does show that some of these patients may have a predisposition to long-term atherosclerotic cardiovascular disease. So it is, even if the spasm recovers, these patients may not be out of risk as we follow them. And then RBC hemolysis, and this was an interesting one because again, a lot of the preclinical studies showed that the thresholds for RBC damage was much higher than what we expected for myocardial damage. Yet, we are seeing the effect of hemolysis, and it is dose dependent. The more ablation lesions we deliver, we see hemolysis more often, and hydration has been effective. And generally, for PVI, when we limit it to standard pulmonary vein isolation without additional lesions, the incidence of hemolysis has been quite low. And the biggest take-home point with this is we have seen a significant decrease, and the incidence of AE fistulas and PB stenosis have practically gone. And this is probably the biggest take-home. As was mentioned earlier, the lack of clinical AE fistulas does not mean we're not causing esophageal injury. Studies have shown serial rise in esophageal temperatures during ablation and follow-up. These do get better over time, and endoscopic ultrasound studies have shown some changes in the luminal membrane as well. And PB stenosis has been studied in detail in the preclinical models and clinically, and the outcomes are reassuring. And I mentioned this earlier about cardiac selectivity not being enough, and there are cases again in AFib where we want to target those atrial ganglionated plexi, and certain other conditions where there's hypervagotonia, and we are trying to ablate the nerve cells. So is PFA appropriate for that kind of ablation? There are certain conditions where we want to ablate the conduction tissue. We can have reentrant circuits in the ventricle and bundle branch, reentrant ventricular tachycardia. We can have complex structures such as papillary muscle, which is dense muscle, and then Purkinje fibers interspersed within, and that certainly will change the threshold of delivery. So how do we account for these? And this is some of our work looking at the effect of PFA on the ganglia cells. So the way we determine adequate lesion during the procedure, and these are post-scene models, the way we determine lesions was by extracardiac vagal stimulation. So we perform vagal stimulation at baseline and get a vagal response, and we ablate until that response goes away. So we know we are causing denervation. With radiofrequency, this has been, this is a well-established technique, and then a lot of centers have done cardio-neural ablation using that technique. When we did this with PFA, and then we saw that with the standard delivery that was useful for pulmonary vein isolation, we were not getting ganglia denervation with that. As we cranked up the settings, we delivered more voltage and more pulses. We saw that we were getting vagal denervation, but that came at the cost of hemorrhage and necrosis. So certainly more tissue destruction at the point of getting nerve damage. Now it's important to remember that these lesions were delivered endocardially. So again, if we deliver endocardially, certainly the thresholds would be different, but this is the case in these experiments. And then looking at the cellular changes with staining for ganglia, we certainly did see vacuolization and nuclear pycnosis consistent with damage of the nerve cells. But again, those were at significantly higher energy deliveries. And in chronic models as well, we saw some evidence of fibrosis, and at four weeks when we brought these pigs back for testing the vagus nerve denervation, it was, it remained denervated at the one-month mark. So it is reassuring. So like was mentioned earlier, we have a tool that can ablate any tissue given the right settings, but at what cost are we doing that? So I'd like to summarize by saying that PFA offers promise, and there's definitely some safety advantages over traditional ablation techniques. And early studies and trials have been reassuring, and they've demonstrated encouraging results. But there's a lot that we still need to learn about. We need to have discussions regarding individualized PFA delivery, because what may work for one patient will not work for another patient. As we change our targets from atrial to ventricular tissue to other than myocardial cells, we need to be able to titrate the parameters. And this is indeed the need of the hour. So a lot of research still to come. Thank you all for your attention. Thank you, sir. In the interest of time, we'll move on to the last speaker, Future Technology and Applications of Electroporation and EP, by Professor Miklovic from University of Leblon. Thank you very much. Well, with a little time left and with almost everything said, I want to first thank for the invitation, but then complain about the title, because how can you tell the future? I mean, I don't have the crystal ball, so the easiest way is to look at it. If anybody can do it, you can do it, don't you? Yeah, thanks. So the second best way I know is you look at the history, but there's no history virtually. If we look back five years ago, there was almost nothing about PFA. So a year ago, we wrote a review paper, and you know, there's a lot of things in that review that has been already outdated when the paper was out. What I want to say, though, is that in June, when it was published, there was about 50,000 patients treated. I think June this year, it's going to be half a million. That's how fast it goes. So with that said, I think we have enough evidence that PFA is safe, even with imperfect system, even in, let me say, hands of not fully educated EPs. So it's still safe, yeah? I know you will chase me down the hall for what I said, but I take my chances, I take my risks. So I think in a study that we did, and I think this is really about the ventricles, because the atrium, the PVI, is really something that is going on. You already see at this meeting a very strong interest in going into the ventricles. I think the ventricles are probably one of the next targets. So I'm focusing now on the future, which is obvious, at least to me. We, but also others, have shown that with respect to PFA lesion, it seems that the scar, at least the young scar produced in a model, in a swine model, is not really preventing us from being able to ablate beyond. But let me remind you, this is a young myocardial infarction. This is not an old one. So we need to see whether this is going to play as we want. One of the things that I would like to say is that when we were determining the lesion depth, both in myocardium healthy part of the tissue, but also in the infarcted part, the lesion depth was the same. We couldn't, I mean, you can't tell, there's no statistically significant difference. Of course, because the wall is thinner where you have the scar, the transpirality was achieved earlier. So I'm just showing a couple of early papers, and there's more and more coming out. So I think we're going to soon learn about that. But let's make a step back. I'm coming from a country where leukodontics is coming from. So a step back, which he does to score, is something what we do. So what was fascinating for the early adopters and people who were used to do RF was really the disappearance of the signals. Electrograms almost immediately disappearing. So let me just take you back to what Bohr said at the beginning. Within seconds, we see, because of the increased permeability, because of electroporation, the exchange of ions across the membrane. What does that do in a cardiomyocyte? In a cardiomyocyte, we have a very nicely orchestrated action potential, increasing intracellular calcium, and then, of course, followed by the contraction. Now you can only imagine what happens when you do the electroporation. You mess up completely this mechanism. There is no control of it. So let me show you. We've done a study. The paper is under preparation. What we did is we actually took adult rat cardiomyocytes, isolated them, and then we have a system which allows us to follow the action potential, so the transmembrane voltage, the intracellular calcium, and, of course, also measure sarcomere shortening, the contraction, at the same time. We did that. We pace, of course, the cells and measure this, and then we deliver different waveforms, so the monophasic, the short nano, and the biphasic, and we observe what happens. We can achieve electroporation and similar effect with all these different waveforms. So there's no magic about the waveform with respect to the electroporation. Now this is what you see, and this is how it happens. After a minute, though, when taking a rest, you can get, again, the pacing. So you disrupt, with electroporation, this mechanism of contraction, but then, after a minute, it recovers. Not always, depending on how much you do actually electroporate. But if you have it after a minute, if the cardiomyocytes can be paced, you will be able to follow that for additional 20 minutes. So they will not die out in the next 5, 10 minutes. On the contrary, even those cardiomyocytes that you cannot pace at one minute, they may recover within 5 to 10 minutes, and then they stay. So we have a certain dynamics that is in place, and that we should be aware of it. And I think we know that. So that's one part, ventricles and what electroporation does, and why we are losing electrograms. I agree with Leonid. He said that there is a chance that electrograms are carrying the information that is useful. And I think we are working, many of us are working on that. Now, I would like to make another step back. The old literature tells us a lot, and many things are based on early work. So in this paper, published in 2000, that's not that old, at least not for me. Defibrillation was tested as a hypothesis being a mechanism for successful defibrillation. Now, in the discussion, what they say, the success or failure of defibrillation therapy has usually been attributed to one or both of the following mechanisms. Success in extinguishing ongoing fibrillatory activity, and failure to reinitiate a new arrhythmia. And the authors conclude that their data suggests that electroporation may be actively involved in both of these processes. Now based on that, and knowing one more thing, we all know in which, let me say, in which ballpark are the pulse parameters for defibrillators. And this is, of course, in the range of milliseconds, several milliseconds. And this is again, this is even older. This is from 1980s. The success is achieved reliably if you use five millisecond pulses. But back in 1980, you could not produce one microsecond pulses or 200 nanosecond pulses. So now, if we have electroporation, and we know electroporation is involved in defibrillation, then you could be, it should be possible to defibrillate with a 10 microsecond pulse or a nanosecond pulse. And Nick Dunn actually did the experiment at Mayo. And yes, I mean, this is not really defibrillation, but you can terminate the atrial arrhythmia successfully with a single pulse of 10 or 20 microseconds. And Nick here can give you more detail on that if you're interested. So what else is done in the field of medical use of electroporation? Well, of course, PFA, the big, big, big star. Tumor treatment, which I personally was working, and my team, for 20 years. Drug delivery, but also irreversible electroporation. What about gene therapy? Now, this is what we usually are told. We can choose where we're going to work, whether it's going to be non-thermal irreversible or non-thermal irreversible or reversible. So if we, at one point, decide we want to explore this, rather than focusing on irreversible, maybe we can focus on reversible. And if we focus on reversible, then we can introduce genes. Now, I'm not talking about cardiomyocytes being able to be paced and so on, but really surviving the electroporation, and at the same time, delivering genes. So people have been working on gene therapy with better or no success. But quite often, any method that has been used so far had shortages in success. And sometimes, of course, it was really driving to uncontrolled hyperproliferation and fatal arrhythmias. So a possible solution might be reversible electroporation, especially because we will have all these generators. We will have all these catheters, and we will learn much more about electroporation. So I would say the next thing will be, of course, introducing genes. A colleague of ours, Lea Rims, she just started a starting grant, ERC. This is a very prestigious European grant. It's called Reincarnation, and that's her job. So if you're interested in that part, feel free to contact her or me, and thank you very much. That was a really fantastic review, Damian. Just a couple of questions. One of the things that Dr. Koulgi brought up was some of the effects, including like gas formation, was different from the anode than the cathode. And you know, pacing history from Mauer and all, they had shown this, like less trauma to tissue from anodal versus cathodal stimulation. If contact is equal for pulse field, is there a fundamental difference between cathode and anode as the point of delivery? This will depend on the duration of the pulse. So if the pulse is long, and if we talk about long, five milliseconds is a long pulse. The damage around the anode will be different than the anode damage around the cathode, because we have a pH production changes around the cathode and the anode. We have extreme changes very close to the electrodes. If we now shorten the pulses, we will lose this, because the pH changes will be very short. Because this is, I mean, the chemical reactions are related to the charge. So current multiplied with the time. The current is the same if you have the same voltage. The time is much longer, 100 microseconds with respect to 10 milliseconds. It's huge. So that is one of the things. And I think that's maybe one difference. And just one more question for you. You know, this focus on reversible electroporation, you said it's so important when we want the cells to survive. But is that a homogenous zone, like, you know, do some cells survive more than others? Some are going to be unhealthy. So even if it was irreversible. Because the irreversible side is, you know, dead or alive. But here, you know, that spectrum, what do we know about that? Yeah. It's very interesting. So I would say there's a lot of work that has been done in vitro on gene transfer. And you have very successful, I would say, protocols. So there's a device from Lonza, which is called the nucleofactor, where, you know, for any type of cell and cargo plasmid you want to introduce, you look up their table, and you put, enter which program, and pretty much you get a good result. When you move to in vivo, it's not so easy. Because we have two things that we don't control well. One is the distribution of plasmid DNA after being injected. We don't know where it goes. People just assume you inject and it's everywhere. No, it's not. And second, this need to be the plasmid and the reversible extirpation, which is just a narrow band around, you know, irreversible and nothing, have to be co-localized. Yeah. And so it's, I think we are working on that, obviously. But it's not, it's a continuum, like you alluded before. So there's more cells, less cells, even less cells. And, you know, like when you talk about the future, we also have to talk about PFA outside the heart. And a big, big focus there for, you know, deodenum, for bladder, is regeneration of cells. So idea there is they're not doing anything biological, but just the cells that come back are... That's really the big, big difference between ablating in the cardiac tissue and anywhere else. So you probably know much better than I, that cardiac muscle does not regenerate. Yeah. So after ischemic... Yes, for sure cardiac muscle, but you know, the other cells that are there along with the cardiac muscle. So the macrophages, resident macrophages. So anyway, all the other tissues are there to regenerate. So the skin, the, you know, endothelium, the epithelium, the muscle that we, you know. So that is something that is always regenerating. So it really depends whether you want to, if you just get rid of the cells, they will, the new will repopulate the place, unless you have thermal damage and you damage the extracellular matrix and the microcirculation. But if we are talking about gene delivery, these cells which are there have to survive. So that's different. So resurfacing is maybe easier than doing actually the gene delivery. Thank you very much. And thank you everyone for a really amazing set of talks. Thank you.

pulse field ablation

electroporation

cardiac tissues

non-cardiac tissues

reversible electroporation

irreversible electroporation

PFA models

safety and efficacy

gene therapy

ventricular ablation