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EP Fellows Curriculum: Molecular Mechanisms Underl ...
EP Fellows Curriculum: Molecular Mechanisms Underl ...
EP Fellows Curriculum: Molecular Mechanisms Underlying Atrial Fibrillation - Implications for New Gene Based Therapies
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Okay, good morning, everyone. Thanks again for joining us. It's a pleasure this morning to introduce one of my senior colleagues at Northwestern, Dr. Rishi Arora. Dr. Arora is Professor of Medicine and Cardiology at Northwestern, where he also serves as the Director of Experimental EP Research for us. He's done significant amount of both molecular and genetic research in AFib, particularly as it relates to oxidative stress. So he's gonna speak to us this morning about some of his work. The title of his talk is Molecular Mechanisms Underlying AFib Implications for New Gene-Based Therapies. So I'd like to thank him for being here and taking the time out of his schedule to do this, and I will turn it over to him. Thanks, Rishi. Yeah, thanks for the introduction, Nishant. Appreciate everybody being tuned in at this early hour. Okay, so I will skim through some of the things because I believe most of the audience is people that are very clinically attuned. So I would try to skim through some of the clinical work that most of you are familiar with. For example, I think most of you are familiar with age of population and its disease burden and critical impact. Suffice it to say, AF is the most common organ disorder. It affects not more than three, but most people feel more than five to six million people in this country. I think most of you know the statistics in terms of the numbers of hospitalized patients, percentage of patients with heart failure, et cetera, that have high percentage of strokes, lots of symptoms, and when it comes to electrophysiologists, huge economic impact. So suffice it to say that the successful treatment of AF is an important challenge in cardiovascular medicine. Again, I think most of you are also familiar with the existing therapeutic approaches to treat AF, both drugs as well as ablated procedures, which obviously have been around for two decades now, and that there's a certain domain procedure. Unfortunately, there are limitations to current therapies and therapeutic approaches. Drugs are less than 50% efficacious and can cause life-threatening arrhythmias. Ablation, as most of you know, has about 70 to 75% efficacy in paroxysmal AFib, but even there, it often requires repeat procedures. Ablation success in a more advanced stage of persistent AFib or AFib in the setting of structural heart disease is less than 50%, even at good centers, especially in long-standing persistent AF, that's quality here. And unfortunately, progressive improvements in ablation capacity, energy sources, et cetera, over the last 10 to 15 years have not led to a significant increase in ablation success. So this begets the larger question, why are current treatments so ineffective? And I would say most people in the field would agree that one of the major reasons for this suboptimal efficacy of ablation and other treatments is that current drugs and ablation procedures are not targeted at chemolective mechanisms that we like to take care of. So having said that, my lab has been studying a whole host of both up- and downstream elective mechanisms for a number of years. But this talk today, vis-a-vis the translational component of what we do, namely gene therapy, I was going to focus on three main aspects of our work, namely the autonomic nervous system, our work on pro-fibrotic signaling, and more recently, in oxidative stress or oxidative issues. Okay, so let's start with the autonomic nervous system. The autonomic nervous system, as several of you know, has been thought for many years now to play a role in both the genesis and the maintenance of AFib. The classical teaching has been that adrenergic AF occurs in the setting of increased activity or stress in patients with spectral heart disease, for example, post-MI, whereas the classic description of vagal AF is that it's more common during sleep or at rest in gender patients with normal hearts. However, we also know, and from studies done about 15 years ago, 20 years ago now, that the vagus nerve is probably the dominant autonomic within the creation of AF substrate. And we also know that during ablation of the pulmonary veins, vagal responses such as bradycardia, asystole, and hypertension are frequently noted. In fact, while the early studies in the ablation era showed that if you religiously ablate these regions in the atria that demonstrate vagal responses during radiofrequency, you can actually increase success of the AF ablation. But that was early, and then since then, this line of ablation has not been pursued. That being said, there are groups, again, as several of you may know, that are trying to elicit vagal responses at different regions of the atrium and they're trying to target those. This is called GP ablation, but more about that later. So I haven't told you what I've just did about the background as to the role of the autonomic nervous system in AF. The precise contribution of the autonomic nervous system to the electrophysiological substrate for AF is not known still. And therefore, over the last several years, we chose to systematically assess the functional and structural characteristics of sympathetic and thyrosympathetic nerves in the atria. We chose to do this in normal animals, as well as in dogs that have structural demodulants that can do the heart failure, and dogs that have electrical demodulants that can do the rapid atrial pacing. And the reason we chose both these models is because, as you know, AF is a disease of electrical and structural demodeling, and consequently, if you really are to understand the disease state well, you have to be able to understand it in these two complementary models. So this is just some of our very early work where we showed that you just do immunostaining. We and Frank Chang's group pretty much showed this data at the same time, that nerve trunks in the atria have normally, if in a very peculiar location, they are located epicardially, they are located around blood vessels, certainly a predilection for more nerve trunks in the posterior left atrium. Obviously, there are a lot of nerve trunks in the fat pads, but about 30% of the nerve trunks that we saw were actually outside the fat pads. And so these are nerve trunks which are composed of sympathetic and thyrosympathetic nerve fibers. But obviously, these fibers, these brown fibers and thyrosympathetic fibers arise in these ganglion cells, these thyrosympathetic cells, postganglionic thyrosympathetic cells. So all these fibers that I'm showing you here actually arise from, as you can see over here, from these GP cells, and then these little blue fibers are postganglionic sympathetic fibers. So as you can see, blue and brown are co-localized, or sympathetic and thyrosympathetic fibers tend to co-localize. So clearly, the two systems travel together anatomically, and obviously, that's one reason why they functionally often appear to act together vis-a-vis genesis of atrial and ventricular arrhythmias. We also showed back in the day when we were still starting our work that a large number of the vagal fibers that supply the posterior left atrium of the pulmonary veins are carried by the ligament of Marshall. This was Joseph Obadi's work in our lab when he was here, and he painstakingly did these dissections and found that a lot of the nerves that travel in the ligament of Marshall, ultimately are the ones that then end up supplying the posterior left atrium of the pulmonary veins. In a related study, we also wanted to understand the functional significance of these nerves, and so we performed extensive, very detailed, electrophysiological studies using high-density plaques, where we looked at multiple refractive mirrors in the pulmonary veins and the related left atrium, and found that vagal adhesion refractive mirror shortening actually correlates very closely, we found, with the spatial distribution of anionic current called IKAC-H. Obviously, this was the correlation here with the protein subunits of IKAC-H, namely KiO 3.1 and 3.4. And in addition to that, what we discovered was that the distribution of IKAC-H also appeared to affect electrophysiological responsiveness. So as you can see over here, IKAC-H, which is the pink stain or the brown stain, is very homogeneous in the left atrium appendage and very heterogeneous in the posterior left atrium, and that's one of the reasons we felt that refractive mirror responsiveness in response to vagal stimulation was more homogeneous in the appendage and more heterogeneous in the posterior left atrium. So having studied the normal atrium, we then went on to look at top-numbered modeling in a canine model of congestive heart failure. This is a canine model where we performed rapid ventricular pacing at 240 beats a minute for three to four weeks. This ventricular tachypacing caused a progressive increase in atrial and left ventricular size and a decrease in left ventricular systolic function, and ultimately reduced our sense of fibrosis both in the atrium and the pulmonary veins. So this model, we discovered, was to do a significant neural remodeling of the posterior left atrium in heart failure. So again, these are normal nerve bundles, mostly brown, which is sympathetic, but there's a spattering of blue, which is, sorry, brown, which is parasympathetic, but there's a splattering of blue in there, which is sympathetic. And what you can see over here, these other three panels over here are sections taken from heart failure dogs. As you can see over here, these nerve clumps are much, much larger, both the ones with these GP neurons and those without. So this is just a quantification, but just about every aspect of nerve bundle size and parasympathetic and sympathetic nerve number and whatever else we could measure, we looked at everything with upper heart failure. On the physiological side, even though parasympathetic nerves appear to be increased, we found that, strangely, parasympathetic were back to theory. So again, what's the canonical thinking in the ventricle? Sympathetic signaling goes up and parasympathetic signaling goes down. That's the canonical teaching in the ventricle. Clearly, as I'm showing you over here, there's very much an excess of parasympathetic nerves in the atrium and heart failure. So something is very different in the heart failure atrium as compared to the ventricle. But interestingly, even though parasympathetic nerves were significantly increased in this part, we found that refractive responsiveness in response to parasympathetic manipulation was decreased. So interestingly, when we looked at M2 receptors using radio ligand binding studies, there was no significant difference. So what I'm really showing you is that the nerves go up significantly in number, but the receptors don't change in number. So if the nerves go up and the receptors don't change, you would expect a very dramatic parasympathetic effect. But interestingly, when you do vagal stimulation or you give atropine, parasympathetic responsiveness is blunted in heart failure. And the reason we found that was the case ultimately is because there's an increase in acetylcholine esterase at the end. It's a compensation that basically chews up all the acetylcholine that's secreted by these nerves because without this, I think it would permanently be inactive all the time. So it's a very sensible, intelligent, compensatory response by the body. And so when we gave an acetylcholine esterase inhibitor, basically isostigmine, we found that we would completely restore vagal stimulation as well as the applied carbonyl directly to the atria. Even without acetylcholine esterase inhibition, we found that the responsiveness was very good. Again, I'm going to show you that this is not a vagal release problem. The problem will be near resolved is that the acetylcholine esterase increases. But despite this compensation by an increase in acetylcholine esterase, interestingly, we found that when we gave atropine, even without physostigmine in these animals, we found a significant decrease in AM duration. And that actually did not get any better with double blockade. Clearly goes to show you that even in the presence of increased acetylcholine esterase as a compensation, the increased vagal nerve that we see in this model clearly are contributing to the maintenance of it. But clearly, the autonomies played a key role. And in a subsequent study, we also looked at the role of the autonomic nervous system in the creation of AF electrograms. Again, as several of you know, there's a considerable, there's a fair amount of literature that supports the fact that that the physiological substrate for AF may be reflected in AF electrograms. So in this study, we gave atropine as well as double blockade in dogs that have been just one failure. We wanted to obviously understand the electrophysiological significance of the anatomic data that I just showed you on the previous slides. And what we found was, again, as you can see over here, clearly there's an organization that occurs in the presence of both atropine and then especially double blockade. And then another study, related study, what we wanted to do was correlate these AF electrogram characteristics with the distribution of autonomic nerve points and nerve fibers. And so what we found was that change in AF complexity that occurs with autonomic blockade actually correlates very nicely with the amount of nerve ridge fat. Again, as I said, the majority of these large nerve points are located in the fat, even if 30% or not. So this is Shannon's entropy. And the change in Shannon's entropy that you see with atropine or with autonomic blockade actually correlates with the amount of nerve ridge fat. And this is another example where AF organization with autonomic blockade is greatest over a region of high nerve concentration. So again, the encircled regions have lots of nerve fronts, as you can see over here. And when we give autonomic blockade, again, just look at the regions. So this is dominant frequency before and after double blockade the regions that show the greatest organization are also the ones that appear to have a larger number. So clearly there's a spatial correlation between these remodeled autonomic nerves and the electrical logical changes that occur on top of those nerves as reflected in the changes that you saw in these AF electrographs. Again, as I said, we also wanted to see what happens in a canine model of rapid atrial facing because that's where a lot of the remodeling is reflected. And so this model, very quickly what this model entails is we perform rapid atrial facing at 600 beats a minute for weeks to months. And as you'll see over here, and our very own Anna Penninger was actually a joint first author of the study. It's a fairly recent study where what we found was that in a rapid atrial facing model, and we've known about the sympathetics of this model for a long time, that there's an increase in sympathetic nerves, but no one has had vigorously looked at parasympathetic nerves on this model. And no one had done a detailed electrophysiology anatomic nerve correlation study either on this model. Even though we obviously knew that in this model nerve tend to increase, especially sympathetic ones. So when we looked at this from soup to nuts in terms of bundle size, parasympathetic nerves, sympathetic nerves, ganglion cells, the whole idea we found was that clearly you can see over here sort of akin to what I showed you in the heart failure model. The bundle size has tended to increase. Normal nerve bundle size, this is obviously a larger nerve bundle. And then we also found these so-called megabundles after rapid atrial facing where these bundles, some of them actually were so large that you actually could even view them under a 5X microscope. Massive, massive neural hypertrophy. A lot of it is quantified over here. We also then looked at the individual components of these nerve trunks. And as you can see over here, both sympathetic and parasympathetic nerve fibers go up. This was sort of known from before, at least at the myocardial level. And then we obviously looked at this at the level of the individual bundles themselves, but obviously the myocardium is supplied ultimately by these same nerve trunks. But just focus your attention. Parasympathetic nerves, this was a totally new finding, go up very, very dramatically in this model. Okay, so having said that, just look over here. When you have an increase in these nerve trunks, it's not unreasonable to presume that since these nerve bundles are the ones that ultimately distribute nerve fibers to the myocardium, that myocardial nerve fiber number is also going to go up. And that's exactly what happens. As compared to the normal dog, as you can see in the myocardium now, the number of parasympathetic and sympathetic nerves goes up very nicely. We also found that when we actually looked at just the spatial inhomogeneity or homogeneity of parasympathetic and sympathetic nerve, interestingly, this is the posterior left atrium, left atrial free wall, and left atrial appendage. Even though sympathetic fibers were fairly homogeneously distributed, parasympathetic nerve fibers tended to be more heterogeneously distributed in the posterior left atrium as compared to the free wall and as compared to the left atrial appendage. You can kind of blink a little bit of that from here. It's hard to tell, I suppose. But again, the number of nerve fibers in these fairly evenly spaced panels in the appendage is about the same. Just look at the arrows, whereas the number of arrows in the number of fibers that you can see in these fairly evenly spaced panels tends to be more heterogeneous in the posterior left atrium. Ultimately, obviously, as I said, we're electrophysiologists. No matter how good or beautiful these nerves look, we want to see what effect they have in electrophysiology. And so what we found just looking at baseline of the AF electrogram was that electrograms were more organized in the left atrial appendage, where these parasympathetic nerves, as I said, were more homogeneous than the posterior left atrium. And none of you are foreigners to this. If you've been to the EP lab and have done any kind of mapping of the left atrium, again, shown over here, a posterior left atrium electrogram as compared to an appendage electrogram. You see this all the time. Electrograms in the posterior left atrium and the rest of the free wall tend to be more chaotic and more disorganized as compared to what you tend to see in the appendage. And we think this is because of nerve distribution at least in part. That's just a quantification of above and below, but this is really the money slide over here that basically tells you that AF electrograms tend to be more organized in regions where nerve fibers, especially parasympathetic nerve fibers are more homogeneously distributed. Obviously, the proof of the pudding lies in eating it. So if you're going to, you have to disrupt the system to really make sure that the correlation that we found in the previous slide is real. And so this is a disruption slide where we gave atropine. And the response to atropine we discovered was more homogeneous than the left atrial appendage again. So again, the money slide over here, I know it's a complicated figure, but just focus your attention on this. These are high density flags located placed on the atrium. Baseline map in the left atrial appendage and the posterior left atrium. We gave atropine and then this is a delta map where you're subtracting the baseline from the atropine pixel. And as you can see over here, if we just look at the delta map for dominant frequency, the delta map is far more heterogeneous or patchy for the endoposterial left atrium as compared to the left atrial appendage. Same thing happens with organizational index. Again, this just goes to show you that clearly the autonomic innervation is playing a role, the parasympathetic innervation because in the presence of parasympathetic blockade, the response is much more heterogeneous in the posterior left atrium spatially as compared to the appendage, which is actually consistent with the nerve innervation pattern that we just saw. Okay, and we did not see this with sympathetic double autonomic blockade. And so what could be a potential mechanism for what might be going on over here? So this was a very interesting finding when we found was that nerve growth factor, which the literature has known for many years now, is a major upstream factor that's responsible for nerve sprouting in the atrium and the ventricle. So we know from previous studies and our own studies reflected the same thing, that the amount of nerve growth factors goes up very significantly in the rapidly changing atrium. That's not new. But interestingly, when we looked at this in a regional fashion, number one, we found interestingly that there was in a strange way, even though, yes, NGF goes up at the back path, it also goes up at the myopartium. And so we began to scratch the head that we thought about what might be going on over here as to what the source of this NGF might be. And then if you look at this in a regional way, again, what really surprised us was that the amount of nerve growth factors in the atrium was actually highest in the left atrial appendage. So this led us to believe that nerve growth factors is probably coming from the myopartium. The hypothesis was that it's been secreted by atrial myocytes in response to rapid atrial stimulation. So obviously, this is a rapid atrial spacing model. But in AFib, our feeling was that the rapidly stimulated myocytes produced nerve growth factor. So we took an atrial cell line called an HL1 cell line and we did the same thing with atrial myocytes as well, the canine atrial myocytes. But when you paste these HL1 cells, as you can see, are getting faster and faster, the amount of NGF that's secreted goes up. So clearly, this is a pacing dependent phenomenon, a stimulation or frequency dependent phenomenon. And what was even more interesting was when we did variable pacing, what we mean by that is you can do the same six-hertz pacing regularly or irregularly. When we paste the HL1 cell, you can do it regularly or irregularly. When we pace more irregularly, you produce less NGF. And this led us to believe that one of the reasons you're probably seeing more NGF secretion of the appendage is because the appendage that I showed you earlier is more regular in its electrograms, right? We know that from our own experiences on the GP lab and what have you, where, so we believe that based on this data, that one of the reasons we have greater secretion of nerve growth factor in the appendage is because myocytes in the electrical appendage tend to be more regularly stimulated as compared to myocytes in the other regions of the atrium. So this does become a bit of a chicken and an egg story as to what happens first, but this is our working model where what we're proposing is that nerve growth factor is secreted by rapidly stimulating apomyocytes. What I didn't show you was that in addition to this huge hypertrophy of the ganglion and plexi and the nerve trunks in the GPs, we also see a very significant increase in stellate ganglia and hypertrophy. That's not new. Peng Chen's group has shown that before, but we found the same thing. But our working model over here is that NGF is secreted by rapidly stimulating apomyocytes. This leads to rectigrade NGF transport to the GPs and the stellate ganglia, leading to neuronal hypertrophy, which in turn then stimulates nerve sprouting in both the atria. Again, this is basically NGF being secreted by different parts of the atrium. More we think of the appendix, the regions we talked about. Regardless, we know that nerve growth factor can travel rapidly up nerves. That's something that's been described in the literature before. So we believe that's exactly what's happening over here. It's produced in the atria. It goes up to the GPs and the stellate ganglia. These tend to hypertrophy in response to stimulation. And once these hypertrophy, you have this diffuse increase in sympathetic and parasympathetic nerves throughout the atria, which is exactly what we found. Also tells you that the appendage may not be an innocent player in what we do in the atria. So to summarize as far as the talk goes, pronounced remodeling of the parasympathetic, to a lesser extent, the sympathetic nervous system, again, produced by structural and electrical remodeling. Neural remodeling is more pronounced in the posterior left atrium compared to the left atrium. The clinical implication of this is that the parasympathetic and sympathetic is the innervation of the atria. The viable therapy is targeted AF, both in the setting of structural and electrical remodeling of the atrium. So then the question that arises then is, how do you selectively target parasympathetic signaling in the atria? So this now is a segue into our gene therapy talk. Again, the reason I took my time getting to it is because, remember, first and foremost, the ability for us to know what we do in my lab, we're really an AF mechanism lab, and the gene therapy work is the translational component of our work. So without understanding the mechanistic piece, I think the point was going into gene therapy right away. So this is the autonomic signaling cascade. If you had to target parasympathetic signaling, you could do it a couple different ways. You could target the M2 receptor, but that's not easy to do. As you may know, remember from med school biology, there are five types of M2 receptors, and unfortunately, there's a very large, very high sequence homology between these receptors, and so it's very hard to come up with selective M2 receptors. Just very, very difficult, pharmacologically. But at the same time, we know from other people's studies in the last several years that if you wanted to target a G-protein coupled receptor, you could do it much more selectively by targeting the interface between the GPCR, in this case, the M2 receptor, and the downstream G-alpha G-protein. So we know that the docking of the GPCR with the G-alpha protein, in this case, G-alpha IMO, is an area that's evolutionarily very, very conserved, and so if you wanted to target any GPCR signal, you could do it by going after the interface of the G-protein coupled receptor with its protein of G-alpha G-protein. And so that's what we, and again, this is something that's been described several years ago by a group out of Vanderbilt, where people have shown that G-protein inhibitors with peptides that target the C-terminus of the G-alpha subunit, which is the one that interacts with the G-protein coupled receptor, can selectively inhibit downstream GPCR signals. And so this is just one example, but you can do this with other G-protein coupled receptors as well. People have done this with endothelium, for example, G-alpha Q, G-alpha 11, 12, a lot of different receptors that have been targeted by this method. So our hypothesis over here was that targeted disruption of G-alpha IMO signaling in the posterior left atrium, the G-alpha IMO is downstream of muscarinic signaling, so our hypothesis was that targeted disruption of G-alpha IMO signaling in the posterior left atrium using C-terminal inhibitory peptides would lead to selective vagal dilation of the left atrium and a consequent decrease in vagal IF. So what we've done initially, what we did was we put in a mini gene, a small gene with a very small insert that expresses these G-alpha IMO inhibitory peptides. And then we looked at the fact that there is an IF-induced ability of 48 to 70 from our exact third injection of gene. And so what we found over here is the posterior left atrium is where the gene was injected. And this is just delta ERP in response to vagal stimulation. Vagal stimulation, as expected, in the presence of scrambled or dummy peptide, it's very strong. It's like a normal atrium. 40, 50 milliseconds refractive period shortening. If you just give the one gene expressing the one peptide G-alpha I2 over here, you will find that there's about a 50% abrogation of vagal dilation refractive period shortening. If you give both the peptides in mini gene form, you're almost completely a ton vagal dilation refractive period shortening. What's even more interesting over here is that even though we injected the gene only in the posterior left atrium, and not unexpectedly you see effects of the posterior left atrium, but interestingly you see an effect also in areas that are remote from that region of injection. So the plum remains. They're not too far, but they are because we're not injecting on top of the plum remains. And yet you see a response in the plum remains and even more surprisingly in the appendage, which as electrophysiologists know is really quite distant from the posterior left atrium. And again, we see a very powerful effect in the left atrium as well. So clearly just injecting the posterior left atrium is affecting vagal induced refractive period shortening of the whole left atrium. And this was obviously reflected in a decrease in vagal induced AF, both in the number of episodes as well as the duration as well as the number of episodes that you induce. And why do we think we get this remote effect in the whole atrium? We think the reason for that is the brown stain over here is a flag stain. So basically the gene that you inject has an epitope on it, a flag epitope. And so if the gene gets expressed, then you should be able to see the flag epitope as well, which is basically brown. And so as you can see over here, this is a normal myocardium uninjected. There's no brown over here. This is a thick brown stain. This is a lot of flag peptide in this myocardium. These are myocardial myocytes. But interestingly, as you can see over here, there's also a good amount of the flag and the nerve fronts. These are nerve cells and these are GP neurons. So clearly with the electrophoresis atrium of these low energies, a gene is taken up not just by the myocytes, but also by the neurons. And I know the word on the street is that when you do high energy electrophoration, that it's very selective for tissue. And that's one of the reasons people believe that it might be a better energy source for the future in terms of collateral damage and so on. But I can tell you in our own hands, if we do low energy electrophoration, you clearly are getting gene into multiple tissue types. So that's just our observation. And then obviously, since the goal would be to go longer term with this gene-based approach, we have also performed longer term expressions of G-alpha-ionoid peptides in a chronic AF model. This is ongoing work in the lab. And what we found is that there's delayed onset of AF after injection of these many genes expressing G-alpha-ionoid peptides. So to do this, we have to be able to clone the insert of interest into a plasmid that can express longer. So we have a plasmid that's called polyubiquitin C plasmid. It's a polyubiquitin C promoter. And it's a Pol II promoter that several studies have shown over the years actually expresses much longer. It's not silenced as easily as the promoter that I showed you in the previous slide, which is a CMV promoter. And so when we've used this promoter, we've actually had very good success in terms of long term expression. But this is just some of our earlier data that goes to show that if you inject this gene of interest, if you don't inject this gene, within about a week or so of pacing, as Dr. Kettinger will tell you, these dogs begin to go into AFib that begins to sustain more than 30 minutes at a time. But if you inject the G-alpha-ionoid peptides together, as you can see in this particular series of three or four animals, you very significantly delay the time to onset of AFib. So to summarize this part of the talk, these results demonstrate the feasibility of a targeted mini-gene-based approach in achieving G-alpha-ionoid inhibition of the posterior left atrium, with a resulting change in vagal responsiveness in the entire left atrium. Non-ablated methods as a result of this data, we believe, can be successfully employed to attain selective parasympathetic inhibition of the left atrium, and appropriately selected ablation sites, we believe, can lead to downstream effects that lead to favorable modification of AF substrate. Okay. Quickly shifting gears now to the next part of the talk, fibrosis. Atrial fibrosis has been thought to be, for years now, to be one of the most important factors in the formation of AF substrate. Atrial fibrosis has been observed in biopsies from patients with AF, and people with risk factors for AF, which is advanced age, valvular disease, dominant triglyceride myopathy, and the list goes on and on. In the heart, we know that TGF-beta-1 is a very important signaling molecule that increases atrial fibrosis, as evidenced by over-expression and knock-out models. We know that serum TGF-beta levels increase in patients with AF. Early clinical studies, and we also know that when you cardioverte people with AF, you have a significant decline in TGF-beta levels. And so this is, I mean, again, fibrosis is complex. There are multiple signaling pathways that are involved in it, but we know that the TGF-beta is an important one, and so this was one that we decided to target, and the reason I'm showing you this slide is because we know that TGF-beta signals by one of two important mechanisms of the canonical pathway, which is a SMAD-related pathway where, by a phosphorylation of Fas-SMAD2 and 3, you turn on probiotic genes in the nucleus, which is a SMAD-transporter to the nucleus where it turns on these transcription factors that ultimately lead to collagen synthesis, but there's an alternative pathway as well, it's ACK1, alternate pathway, which via the MAP-MAP kinase pathway, it also turned on the same transcription factors in fibrosis. So our hypothesis over here was a decreasing TGF-beta saline in a canine posterior lactatum of a 1008 Mark-Bayer-induced fibrosis, heterogeneous conduction, and resulting AFM. How did we accomplish this? We injected a plasmid that expresses a dominant negative type 2 TGF-beta receptor, so the classic TGF-beta receptor, like all receptors, has to signal to the inside of the cell by some mechanism, and so what you're doing in this particular approach is you're overexpressing this pipated receptor that cannot phosphorylate downstream. So basically, it lacks an intracellular kinase domain, and so as a result of that, it's not able to signal, and if you overexpress enough of this, hopefully you'll be able to basically competitively decrease the native normal TGF-beta receptor, and as a result, stop downstream signaling, or at least decrease it. So that was the goal, to competitively inhibit the normal TGF-beta signaling by creating a defective TGF-beta receptor, and so again, this was performed in the canine heart failure model, as I have shown you before. This is rapidly increasing at 40 beats a minute, but the reason I'm showing this to you again is because this model, as I said earlier, produces fibrosis, and this fibrosis leads to an increase in conduction heterogeneity as measured by something called conduction homogeneity index, and this will come up again. That's why I mentioned it here. So this is how we performed a gene injection, transgenes out of the control of the same promoter that I mentioned earlier, the UBC promoter. So basically, the TGF-beta delta-negative R2 receptor was flown into a UBC backbone. Controlled plasmid, in this case, was a laxative-expressor plasmid. We performed electroporation, so you do a gene injection. It's a sub-epicardial gene injection. You raise these little blebs over the epicardium, and then you perform electroporation. These are electroporation electrodes. After gene injection, you perform, a few days later, we started ventricular tachycasing at 240 beats a minute for three weeks, and then we performed a terminal study to assess conduction and fibrosis, et cetera. So just quickly to the results, what we found was that the dominant-negative receptor attenuates conduction and homogeneity in the heart, sphincter, and posterior left atrium. So again, in controlled animals receiving the laxative plasmid from baseline as compared to three weeks of ventricular tachycasing, there's a very significant increase in conduction and homogeneity. However, in animals that got the active gene, as you can see over here, at different cycle lengths, pacing cycle lengths, when you go from baseline to post-ventricular tachycasing, there's no significant increase in conduction and homogeneity. It's just an example over here. But interestingly, we found that not only do you improve conduction characteristics, but you also positively affect the APD restitution slope. So we looked at MAPs in these atria, and using a monophasic action potential probe, and we found that APD restitution was actually improved in these dogs. So we think it's a dual mechanism. Obviously, ultimately, what are we interested in? We're interested in AFib, and with the dominant-negative receptor, there's a very significant decrease in AFib. Again, we're electrophysiologists. Obviously, all aspects of electrophysiology interest us, and so the electrograms are always near and dear to our heart. As you can see over here, interestingly, this came as a bit of a surprise to us, but if you think about it, it doesn't have to be. If you take away a little bit of the fibrosis, kind of the thinking today is, well, if fibrosis is going to cause more chaotic or more disorganized AF electrograms. Well, in our case, we found that electrograms were more disorganized in the presence of lots of fibrosin, which is three weeks of epithelial attack increasing, and then when you took away some of the fibrosis, obviously, you can't remove it completely. You find that electrograms tend to get faster, a little bit more disorganized. So we think what happens is that, yes, electrograms do tend to disorganize electrograms early, when you have a little bit of interstitial fibrosis, but once you get lots of fibrosis, eventually, and we've seen these atria. Several of us have. When you have somebody with structural heart disease, what you see in these atria, or think of the post ablative atrium, or the post base atrium, or a heart failure atrium, you see these slower, sick-looking, chronically low-amplitude type electrograms in the atria. We think that's what chronic severe fibrosis does to you in the long term. Limited fibrosis, yes, there's a lot of disorganized activity, but once fibrosis becomes severe, you slow down and basically organize the AF electrogram. So why did all this happen? Why did we see these improvements in AF and conduction? Well, the reason was the tissue level and improvement in the amount of fibrosis. So again, this is a control animal. Lots of blue or fibrosis you can see over here. This is a dog that received a dominant negative receptor. As you can see over here, there's very little fibrosis, much less. So it doesn't go away completely, but there's a lot less. That's quadrified over here in multiple animals. Again, with gene therapy, one has to be careful that you get gene cargo into as many regions as possible. Again, basic electrophysiological concept. If your gene expression is patchy, potentially you can always be prorythmic for reasons that most electrophysiologists are familiar with. And so in our hands, we find that with the approach that I showed you, the electrophoresis approach, the sequential injected electrophoresis approach, gene expression is moderately homogeneous. The green is a gene. It's not completely, but moderately homogeneous. This is the uninjected region. That's a V5 tag. And in terms of the signaling pathway that I mentioned to you there too, we were able to get this improvement in fibrosis and downstream electrophysiology, not only by affecting the canonical SMAP pathway, but also by affecting your MAP kinase phospho-ERK pathway. Last part of the talk, oxidative stress. Oxidative stress is thought to be an important contributor to both electrical overmodeling and structural overmodeling in AF. Oxidative stress is thought to affect cardiac ion channels and excitation contraction of the coupling, at least in part, by increased CAFK2 signaling. Unfortunately, most data that supports the role of oxidative stress in AF is indirect. Extrapolated findings of inflammation and oxidative data damage in its black faecal tissue. And the precise mechanisms by which oxidative stress creates a vulnerable substrate for AF in the intact atrium are not known, and neither is it known whether AF, whether oxidative stress affects the emergence of AF triggers in intact food remains. So the next few slides are just going to summarize data from the last four or five years of work in the lab, in this space, but really it's a two-part hypothesis. Number one, in heart failure, we believe that there's a preferential increase in oxidative stress in downstream CAFK2 signaling in the intact posterior left atrium, which creates a vulnerable substrate for triggered activity and re-entry. And the second part of the hypothesis is that oxidative stress contributes to both the initiation and the maintenance of electrical overmodeling in AF. So this is some of our data in a heart failure model, which is the first part of the hypothesis where we think an increase in CAFK2 signaling by an increase in loss leads to conditions for both triggered activity and re-entry. Again, in this heart failure K9 model, very significant increase in superoxide generation. It appears to be preferential in the posterior left atrium, even though it does go up in the appendage too. So this increase in superoxide then actually leads to an increase in protein oxidation as measured by an assay for carboxylation. If we just focus over here, for example, you're looking at just the total number of bands over here in the dark, and you can see it's clearly carboxylation is very significantly increasing in the setting of oxidation, right? Quantified, which is protein oxidation. And again, we found that it was preferential. There was more protein oxidation in the posterior left atrium compared to the appendage. This increase in oxidation is accompanied by an increase in CAMK phosphorylation for, well, two things happen. There's an increase in oxidation of CAMK2, which is a master signaling molecule that exists in both of the atrium and the ventricle. There's an oxidized form of CAMK2 that goes up in the atrium in response to this increase in oxidative stress that I showed you. And this increase in oxidative stress then is accompanied by an increase in expression of CAMK phosphorylated RYR2. And this particular site of phosphorylation of RYR2 that several of our fellows have been familiar with is something that has been described before, initially by Andy Marks and then by Zonda-Warrens as being a site that actually makes the ryanine receptor leaky. This is one of the famous calciquestion mutations. Sites of calc... One of the famous mutations of the calciquestion mouse, which leads to CPVT. But we know also from Zonda-Warrens and other people's work, standard health work, that the same phosphorylation of RYR2 can also lead to leakiness of ryanine receptors in the setting of AFib. So anyway, bottom line is when you have an increase in morosity of an increase in oxidation of CAMK2, which then leads to an increase in CAMK phosphorylation of RYR2 at this very interesting site. And then at the physiological level, we had shown together with Andy Wasserstrom's group a couple of years ago that when you actually... So there's something called triggered calcium wave. It is a very unique calcium release phenomenon that we see in atrial myocytes. Not in ventricular myocytes. We believe that because T-tubules are pretty poorly developed in the atrium as compared to the ventricle, when you pace myocytes faster and faster with the calcium transient, at fast atrial pacing, you will not see this in normal ventricles. You begin to see these calcium waves. We call them triggered calcium waves. There's a very atrial-specific calcium waves. But the important point for this particular slide is that in heart failure, we found that there's a very significant increase in these triggered calcium waves. So the one reason we believe that happens is because there is a very significant breakdown of T-tubules that happens in heart failure. So in heart failure, you begin to see these triggered calcium waves at much slower cycles. That's quantified over here. So again, to put everything together, increase in oxidized CAMK2, which leads to an increase in CAMK2 phosphorylation of RYR2. And our belief is that this increase in CAMK2 phosphorylation of RYR2 is one of the reasons you begin to get this excessive calcium release in the heart failure atrium. So as I said before, there's T-tubule breakdown, but we think this is another major mechanism by which you might be getting more, this Ross-induced increase in CAMK2 phosphorylation of RYR2 at this 2A14 site. We think that may be one mechanism whereby you might be getting more triggered calcium waves in heart failure. So again, the proof of the pudding, putting all those lies in eating it. So what we wanted to do was then see whether these triggered calcium waves are actually Ross-sensitive. So in the presence of phytotempoverate, which are Ross inhibitors, as you can see, these triggered calcium waves tend to get abundant. So again, that I think is very significant proof of the fact that these triggered calcium waves are indeed likely, at least partially, caused by an increase in oxidized CAMK2. Obviously, with large animal studies, you know, as I said, with science, it's always about doing, the sufficiency experiment is always large. Yes, it's easy to show that there is, if you take away something, that Ross is contributing to these calcium waves, but if you want to show that Ross is sufficient, then you have to have to go the other way and increase the Ross and see if you bring back these calcium waves. And that's a little bit harder to do at a chronic model. It's easier, in a large animal model, easier than on a mouse, but you can actually compensate for that by doing computational studies, computational modeling studies, and that's what we did over here, where in a computational model that one of our collaborators created with us, what we did was we basically married a couple of different models that already were in existence. There is a model that you can use, actually, to increase CAMK2 that comes out of Tom Hunt's lab at Ohio State. There's a model that you can use. This is by a model of my collaborator, Johannes Schipperol, where you can actually cause formation of triggered calcium waves in the atrium. And so what Johannes did in this particular case was he basically married these two models so that now in the presence of an increase in CAMK and use Ross, the question is, can you then bring on triggered calcium waves? And sure enough, as you can see over here, low concentrations of oxidative stress, nothing much happens, but as you pace, as you get more and more Ross into the atrium, the same cycle of pacing as you can see over here, you go from these sparks to these waves that begin to form, and then you see these very real calcium waves, large calcium waves, micro-calcium waves, along the lines of the calcium waves that I've shown you experimentally. So clearly, as you increase Ross in this model, instead of getting increased CAMK2, you can actually lead to an increase in triggered calcium waves. In the same model, we had found, we also found that there's a preferential increase in CAMK phosphorylation in the sodium channel, NMV1.5. Unlike NMV1.5, the CAMK, unlike native NMV1.5, what we found was that this CAMK phosphorylated NMV1.5. So again, this was a work that came out of Peter Mohler's lab where he showed that CAMK phosphorylation of NMV1.5 at this site, so NMV1.5 is the most important cardiac subunit of the sodium channel, center of sodium channel, and CAMK phosphorylation at the NMV1.5 site, we know from Mohler group's findings, can actually make the sodium current, can actually change the sodium current so that it actually, in their studies, they find that with the CAMK phosphorylation of NMV1.5 at this S571 site, there's a very significant increase in late sodium current. That's their finding, but we were curious to see whether the same mutation, so there are really two groups that have done this, or three groups that have done this in a very systematic way. Don Berg's group has actually looked at CAMK phosphorylation of NMV1.5. These identified a couple of mutations that lead to late sodium current, but for a variety of different reasons, we're especially interested in this mutation from Peter Mohler's lab. So we obtained his antibody, and what we really wanted to see was what happens to the sodium channel per se, just histologically. And so what we found was that there was an increase in CAMK phosphorylation of NMV1.5 at this site, in this Hartwigian model. But interestingly, what we found was, when we looked at CAMK phosphorylation, CAMK phosphorylation at NMV1.5, very surprising. So you're expected to always have sodium in two main compartments of the cell, at the intercalated disk, and then also at the lateral membrane. Those are the two main compartments where NMV1.5, where sodium current is thought to exist. And again, this is what you're expected to see in controlled biosites, but in Hartwigia, surprisingly, unlike native NMV1.5, it doesn't exist in both compartments. What you see over here is for this CAMK phosphorylated channel, it only exists in one compartment, which is the intercalated disk. And we know that this is a very important finding, that the ramifications of this finding are huge, because we know that absence of NMV1.5 at the lateral membrane will decrease your peak sodium current by about 40 to 50%. I mean, it's a mass effect. We know that you need sodium current in both compartments. And so if you take the NMV1.5 away from one compartment, we know that peak sodium current will go down. And so based on this, the implication of the work, from our perspective, was that an increase in this defective variant of NMV1.5, this CAMK phosphorylated variant, likely contributes to slow conduction of the Hartwigia phosphorylated channel. Now, why does this happen? Again, there's some very elegant biology that has been done by a couple of different groups in this area. Let's see why. So, you have two different pools of sodium current. Suffice it to say, we believe that the reason sodium channel exists in different pools is because different binding partners take it there, different parts of the cell membrane. And so, ANCHOR-NG is one of the key proteins, we know, binding partners, that has been known to bind to the NMV1.5, the CAMK-phosphorylated NMV1.5, we know that's Molo's group. And so that's one of the first things we want to look at. And then we looked at the amount of ANCHOR-NG, we found that the amount of ANCHOR-NG actually goes down very nicely in Hartwigia. So this actually then makes a lot of sense. And this is, again, fluorescence, as you can see over here, even though there's a lot of cadherin at the endoclinic test, there's not much ANCHOR-NG. So, if you have a decrease in the main binding partner that takes your NMV1.5 to the lateral cell membrane, it goes to reason, stands to reason, that that may be one of the reasons why not enough NMV1.5 is being transported to the lateral membrane. And so, again, this just is a mechanism, is a likely mechanism whereby we think there is an insufficient amount of, or actually a negligible amount of CAMK-phosphorylated NMV1.5 because its main binding partner is missing in Hartwigia. So what happens as a result of that is that there's patchy dropout of CAMK-phosphorylated NMV1.5. Again, this is just, again, if you were to do this in intact parts, you have to put it all in the context of anomalous conduction and conduction velocity. But suffice it to say that because there's patchy dropout of NMV1, CAMK-phosphorylated NMV1.5, again, as I said, it barely exists at the lateral membrane. It only exists at the integrated disk. So as it is, there's a decrease in conduction velocity. You can definitely see that in the Hartwigia model. But in addition to that, because this knockout is patchy, so interestingly, even the little bit of, in the interest of time, I don't want to belabor this, but even the CAMK-phosphorylated NMV1.5 that is left at the integrated disk, which we know is not done in a patchy way, maybe I did not do justice to this. Anyway, what I want to say is that because of this decrease in anchorage, what happens is that even the CAMK-phosphorylated NMV1.5 that exists at the integrated disk, that misspoke accuracy actually takes the NMV1.5 to the integrated disk and not to the lateral membrane. So it's the early hour. And so what we find is that because of a decrease in anchorage, there's a decrease in CAMK-phosphorylated NMV1.5 in a patchy fashion also at the integrated disk. Anyway, to make a long story short, there's a very heterogeneous distribution of CAMK-NMV1.5 because of this decrease in anchorage, which is reflected in more heterogeneous conduction of the posterior ligament. So we again created another computational model with the help of Ignatius Sheparov. But what we wanted to see was, in the presence of fibrosis alone, which people have for years said is enough to give you inhomogeneous conduction, is that indeed the case? So what we found was that inhomogeneous low conduction as created by this CAMK-phosphorylated NMV1.5 actually interacts with regions of fibrosis to exacerbate substrate for reentry. So if you just have fibrosis along with the atrium, as you can see over here, these holes are basically regions of fibrosis. That's an S2 beat. As you can see over here, yes, fibrosis alone is circulatory, but the reentry extinguishes pretty quickly. Now, on top of this, if you alter conditions so that you have fibrosis, and on top of that, you have now decreased conduction velocity in the surrounding atrium, as you can see over here, now see the difference. You've got a double-bomb, you've got fibrosis, and you've got the slowing conduction, one mechanism for which we think this is increasing CAMK-phosphorylated NMV1.5. Now, as you can see over here, we saw this multiple times, different iterations of fibrosis plus the slowing of conduction. Now, as you can see, the reentry tends to perpetuate itself. And so we saw this with a variety of different dissimilations, again, fibrosis alone. It's possible, but hard to get sustained reentry. Now, if you add some slowing of conduction to the surrounding minor atrium, different iterations of fibrosis, as you can see, you can sustain the reentry. And these are some of the electrograms that we also think are pretty useful. And so this is, again, basically showing that if you have inhomogeneous slow conduction, juxtaposed regions of fibrosis that exacerbate conditions for AF. This is a model that we've put together. So our low mechanism was a CAMK-phosphorylation R0AO2, which we think leads to triggered calcined wave, and therefore to triggered activity. Whereas there, by a CAMK-phosphorylation of NMV1.5, again, upstream to the downstream of oxygenated stress, we think what happens is that there's a patchy droplet of NMV1.5 at the integrated disk, which leads to inhomogeneous conduction. So again, very quickly now, last couple of minutes, this is the gene therapy part of the oxidative stress. Oxidative stress can be reached both via decision and the mechanism of electrical remodeling in AF, which is the second part of the hypothesis. So the rapid neutral pacing model, lately we've found that NOX2-generated oxidative stress tends to get increased, kind of what we saw in the bacteria model, but then we also found that the sources of oxidative stress are, well, in this case, we found that NOX2 was a big source, and mitochondrial growth was the second source. This is the buddy slide, and it was joined first off with Xinyu on this paper. What we found was that when you actually perform targeted expression of NOX2-HRT in the intact atrium, again, these are, just focus your attention to the right side of the panel. As you can see over here, if you just pace normal dogs, and now we're looking at more than eight hours of AFib, if you pace normal dogs, after about four or five weeks of pacing, they'll develop eight hours of AFib, and then they'll stay in it thereafter. But in animals that received NOX2-SHRT, as you can see over here, there were six of them. These animals developed AFib for that entire period, and we've actually followed a couple of these animals up to six or seven months, and while they were going to flutter, but they do not go into AFib. Possible mechanism is that we think there's prolongation of refractive hemorrhage in these atria. This is just for the presence of NOX2. ERPs tend to be longer. And then at the cellular level, we believe the reason you have this attenuation of ERP shortens because there is an iron channel called constitutive reactive IKACH. And so, canonical thinking, we know for years, people have known from standard health data that L-type calcium channel, a decrease in L-type calcium channel is shown over here. So this is a normal atrium. This is rapidly pacing. There's a decrease in L-type calcium channel, which for years has been thought to be responsible for refractive hemorrhage shortening, but we also know from Stan's group and other people's group that there's an increase in IK1 that occurs in these atria, and there's an increase in this constitutive reactive IKACH channel. So we looked at all three to see which of these would be the most RAS sensitive, and interestingly, L-type calcium channel did not appear to respond very much to RAS inhibition, neither did the IK1, but the constitutive reactive IKACH was very, very sensitive to RAS attrition. And so we believe that the reason, and we actually saw this, when we actually performed targeted expression of nonspecific RNA in these atria, we were actually able to uptend this upregulation of the constitutive reactive IKACH channel. So we think that that's a mean or so mechanism by which this nonspecific RNA would lead to a prolongation of refractive periods and a decrease in AFib. Summarized, this is the end of the talk. Increased parasympathetic nervous clouding contributes to formation of a vulnerable AF substrate, both in the presence and absence of spectral heart disease. Pro-fibromyalgia TGF-beta signaling in the posterior left atrium underlies adverse atrial hemorrhagic heart failure. Oxidative injury creates substrate for frigate activity and re-entering the pump remains in heart failure. And non-sterogenerated oxidative injury underlies the genesis of both maintenance of electrical and hemorrhagic atria. So the clinical implications of this are the gene-based therapies, whether we over-express G-alpha-iodoenobutyric peptides, go after TGF-beta signaling, or go after oxidative stress, may allow for the successful targeting of electrical abstracts for remodeling in AF, and that future optimization of gene-based therapy, gene therapy approaches, may translate into an effective therapy for AF. Thank you for your attention. Just wanted to thank the people in my lab, my collaborators, as well as funding sources, and thank you for your attention. That was great, Rishi. You know, obviously very thought-provoking stuff. Maybe I can just ask a couple questions. We're a little over here. So Alex asked a question which we've discussed before, but because the autonomics probably play a very important role, do you think that this new attempt at pulse field ablation will have an Achilles' heel if it doesn't target that? Or as you kind of alluded to in your statement, do you think it's not as selective as people are saying? Yeah, I think the latter. I think the latter. I really do think electrophoresia, at least in our end, is fairly agnostic in terms of cell type. So as long as you get nerve fibers and yet you don't get... So the question is, does it spare certain cell types or does it spare certain structures because they're far-reaching? I think the answer is the latter. I do think in its field, it does get its... Whichever field you like to put it in, I do think it gets most cell types under its field, but I think its added distance effects are the beneficial ones and that you don't get the collateral damage that you would expect. I think that's what's going on this time. I don't know for sure, but at least if I had to surmise what might be happening in terms of what the benefits of PFA might be, it'd be collateral damage. And I would say, I do think you get effect on neurons and I think the reason you do is because the nerve fibers are right there. They're actually in your field of electroporation. So I think whatever's in your field, you will get whatever's a little bit further away, you probably will not. I would say that's my take on it. Now, that being said, when we've done electroporation, we found that we actually do see a far-field effect. I don't know how big it is, but we do see that if you have electrodes in close contact with myocardium, you don't need great contact with myocardium to get, and that's what the studies I believe are saying also. You don't need great contact with myocardium to create the electric field. So I don't know what the perfect answer is. So I think you do have some far-field effect of PFA, but I think it's a limited far-field effect, which is one of the reasons you don't get collateral damage. But I think within your site, but within the region that's actually getting electroporated I do think you get more sometimes. I don't know if that makes sense. Okay. Well, I mean, we'll probably get more information as more of us get our hands on those catheters, but in the interest of time, I'll just ask one more because our audience here is mostly the fellows. So as a clinical electrophysiologist, it seems that our recent attempts at anatomic-based or even electrogram-based ablation for AFib have not really moved the needle in terms of success rates over the last few years. So if you had to give your opinion to the upcoming trainees, where would you say that we should focus our efforts since it doesn't seem like we're doing much in terms of adding to our ablation strategies? Yeah, that's a great question. So I would say my answer to that would be that you're right. There is this dismay in the field that electrogram-based approaches have failed us. And unfortunately, anatomic approaches have their limitations for the reasons that we talked about. But I do believe that one of the reasons electrogram-based approaches have failed is because we were targeting a phenomenon, namely rotational activity that may or may not really reflect true substrate. So what I mean by that is we can see, for example, in some of our own studies, clearly underlying AF substrate, for example, what I showed you with the autonomic data and fibrosis and what have you, does affect AF electrograms. So I think if you're going to target, if you're going to do a mechanism-guided AF ablation, I do think you can target electrograms, but then you should target electrograms based on whether they're really reflecting true pathophysiological AF substrate instead of going after a phenomenon that may or may not be reflecting that substrate. So I would say, for example, if you wanted to target the autonomics, one of the mechanisms, one of the ways by which you could do that potentially from our own data is you could use Atropine or you could do, as some other groups are doing, is do vagal nerve stimulation. But if you were to use Atropine and then go after regions or electrograms organized most in response to Atropine and then ablate those, I would say that would be a better way to do mechanism-guided or electrogram-guided ablation than to, again, focus your efforts in finding something that looks like a re-entrant phenomenon and then chasing that instead of going after the root cause mechanism of that. I'm not saying rotors don't exist, but I also feel that there are many mechanisms that undermine these rotors, and ultimately, if you're really going to get success, it has to be after going after the root cause of fundamental mechanisms. So I do think there's a lot you can still do in terms of using electrograms to better guide ablation, especially in persistence, primarily other than, for sure, wall isolation and expanded ablation, where there's little else we can offer those patients right now. I think in those patients, we have to seriously think about, about developing approaches to target electrograms that, again, as I said, are truly, true mechanism-guided. And as you know, my lab has been working on a couple of those lately. So I think there is still a lot that can be done in that area. I just think that we started in the right direction, but then we quickly, I think, went down the rabbit hole and decided to go after the electrical phenomenon instead of going after the root cause.
Video Summary
Dr. Rishi Arora's video presentation on atrial fibrillation (AFib) discusses the molecular mechanisms underlying the condition and highlights potential gene-based therapies for targeted treatment. He begins by explaining that AFib is a common heart rhythm disorder and that current treatments are not focused on the underlying causes of the disease.<br /><br />Dr. Arora focuses on the autonomic nervous system's role in AFib and the remodeling that occurs in the atrial nerves. His research demonstrates that parasympathetic and sympathetic nerves play a significant role in initiating and maintaining AFib.<br /><br />Fibrosis, characterized by scar tissue accumulation, is also discussed as an important factor in AFib. Dr. Arora's research aims to understand the molecular pathways involved in fibrosis and develop gene-based therapies to target these pathways.<br /><br />One potential therapy mentioned is disrupting the signaling protein G-alpha-i2, which reduces parasympathetic signaling and AFib. Dr. Arora also explores the role of nerve growth factor in atrial remodeling and its potential as a therapeutic target.<br /><br />The talk emphasizes the importance of understanding the molecular mechanisms of AFib and the potential for gene-based therapies to provide more effective and targeted treatments.<br /><br />The other video transcript focuses on the role of TGF-beta-1, oxidative stress, and electrophoretic ablation in treating AFib. Dr. Arora explains that TGF-beta-1 is a signaling molecule that increases fibrosis and that targeting its signaling can improve heart conduction. He also discusses the role of oxidative stress in AFib and its contribution to electrical and structural remodeling. Dr. Arora suggests that targeting TGF-beta signaling and oxidative stress through gene-based therapies could be effective in treating AFib.<br /><br />He further explores the use of electroporation, a technique that uses electrical currents to deliver genes, as a method to overexpress or knock out specific genes involved in AFib. The limitations of current ablation strategies are also mentioned, and Dr. Arora suggests targeting electrograms that reflect the true underlying AFib substrate.<br /><br />Overall, Dr. Arora emphasizes the importance of understanding the underlying mechanisms of AFib to develop more effective treatment approaches.
Keywords
Atrial fibrillation
AFib
Molecular mechanisms
Gene-based therapies
Autonomic nervous system
Atrial nerves
Fibrosis
G-alpha-i2
Nerve growth factor
TGF-beta-1
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