Well, it's my pleasure to welcome you to San Diego and Heart Rhythm 2025, the 46th annual meeting of the Heart Rhythm Society. So if you have not already done so, please download the app from your preferred app store. This is how you can participate in live question and answers during the sessions. But in addition, also please feel free to raise your hand. I think that will be handier to just ask a question, speak up, and then the presenter can repeat the question, and that will facilitate the discussion. Please know that visual reproduction of Heart Rhythm 2025, either by video or still photography, is still strictly prohibited. Well, these are the formalities. And welcome to the session on inherited and ventricular arrhythmias. It's a pleasure to invite Georgia Bertoli, and she will present on the anatomy of the nucleus and then damage and deformation in PKP2 arrhythmogenic cardiomyopathy. Okay, and then it's just, okay, perfect. Yeah, thank you so much. Okay, good afternoon, everyone. It's a pleasure for me to be here today. Thanks for the organizer. And so, the main topic of interest of our laboratory is the study of placophilin-2-arithmogenic cardiomyopathy, also called PKP2-ACM. It's an inheritable heart muscle disease that may result in arrhythmia, heart failure, and sudden cardiac death. It's characterized by progressive fibrosis and fibrofat infiltration of the ventricles, most often of right ventricular predominance. And pathogenic variants in placophilin-2 are responsible for about 50% of cases of arhythmogenic cardiomyopathy. My colleagues in 2022 published this paper that was done in collaboration with the Laboratory of Alicia Lumby in Copenhagen. And we were very lucky because we were able to assess to heart biopsies of patients with placophilin-2-arhythmogenic cardiomyopathy. And in these slides, you are seeing two electromicroscopy images. Here, you are seeing cardiomyocytes of an healthy person. And especially, we are looking at the nucleus. As you can observe, the nucleus presents a really nice, smooth surface. When we look into the cardiomyocytes of placophilin-2-arhythmogenic cardiomyopathy patients, we instead observe nuclei that were presenting a lot of ruffling of the nuclear envelope. And we were able to observe a nuclear envelope impairment. Therefore, we decided to investigate better what was happening at the nuclear level when there was a deficit of placophilin-2. And we did that utilizing our mouse model of PKP deficiency, PKP-2 knockout. That is a cardiomyocyte-specific and tamoxifen-induced mouse model. So the mice develop normally, and then around three, six months, we inject tamoxifen, and we silence the expression of placophilin-2 only in myocytes. We do that in male and female mice, and the mice are monitored for our function and survival by means of neocardiogram, EKGs, and also, post-euthanasia, we perform fibrosis evaluation. Here, what we really wanted to do was to explore the effect of the absence of placophilin-2 on the myocytes. And we wanted to study the nuclei and sub-nuclear structure. So we knew we really need a lot of power of resolution. And therefore, we decided to apply expansion microscopy. So here in these slides, you are seeing two cardiomyocytes, stained both for desmin to show the Z-line. And you are seeing that the myocytes in the upper panel is a not-expanded myocytes. You can observe that it's around 100 micrometer lens, while the second myocyte is always stained for desmin, but is acquired instead of with a 63x lens, with a 10x lens. And you can observe that the structure is really similar, but this time, this myocyte is around 600 micrometer lens. So the beauty of this is that, if I look at this myocyte then, with a 63x lens, you can see not only that there is a nice preservation of the Z-line, but also that you are increasing your power of resolution. So we then combined expansion microscopy with structural illumination, another super-resolution technique, to look at the interaction between desmin filament and the nucleus in three dimension and in the nanometric range. We know that desmin filament is extremely important in maintaining the nuclear shape and position, and this is a movie, you will see a movie, of desmin filament approaching to the nuclear envelope stained for Lamin-B in a controlled myocytes. I think you can observe that the desmin filaments are very organized, and they are all approaching the nucleus perpendicular. And also you see that the nucleus is very elongated, presents very few invagination, and when then we looked at all these lights, one on top of the other, we can observe that there is a really nice wrapping of desmin filaments to maintain the nucleus in place. We then repeat the same exercise for a PKP2 knockout myocyte, and here you will see, already from the very beginning of the movie, that the nucleus looks different, that there are a lot of profound invagination in the nuclear envelope, and that the desmin filaments are not reaching only perpendicular to the nucleus, but they are reaching with many different angles, especially in this moment, you are seeing the desmin filaments are even parallel to the nucleus sometimes. And it looks like the desmin is applying different forces to the nuclear envelope. Also the nuclei were looking with a weirder shape, and we also quantify they were looking bigger. The reconstruction of the PKP2 knockout looks definitely way different from the control. You see that definitely there is a disorganization of desmin filaments, and the nucleus also has a disruption of the shape. So in the course of these years, some of the results were acquired with this technique, and over the course of my project, and some of the results that I'm listing here were acquired by my colleagues in precedent studies. We basically, I don't have the time to show you everything today, but we basically observed that in PKP2 knockout, there were larger nuclei of altered morphology, there was a weakening of the nuclear membrane barrier, there was increased DNA damage, and increased ROS production, and also we were able to look at nucleoli inside the nuclei, and we observed that they were looking larger, and with weirder shape. So I'm listing all this data here, because then we realized that those data were already reported in literature, and they are reported as a sign of premature aging. So we wondered if in our PKP2 knockout, we were observing a premature aging phenotype. We therefore compare the proteome of a PKP2 knockout, published by our group, that at the time of the experiments were around three, four months old, with the proteome of an aging mouse, that was published in literature. In this graph, you are observing that there is a comparison between the proteins that are differently regulated between mice of six versus 30 months of age, and control versus PKP2 knockout. As you can observe, the majority of the protein are located into the second and third quadrant, and so they are both or up or down regulated in PKP2 knockout, and in the aged mouse. Few of them were going to go into the different direction. We made a correlation coefficient, we did a correlation analysis, and we got a piece of coefficients of 0.7, so it really looks like that PKP2 knockout strongly correlate with the aging of the mice. So we started to search for sign of aging, sign of senescence that are well common, established in literature, and we started searching for senescence-associated secretory phenotype, also called SASP. Here you're looking at a differential transcriptomic analysis of enriched myocyte region of PKP2 knockout versus control. Everything that is on the right side of the volcano plot, it's upregulated in PKP2 knockout, everything that is on the left is downregulated. Especially the red dots are the ones that are significantly upregulated in PKP2 knockout. And you see many of the SASP were upregulated in knockout versus control, and as I said, both in the left and in the right ventricle. As I said, we tried to select enriched myocyte regions, but when we saw a lot of this secretory phenotype, we also thought that it may be that we are also picking up non-myocytes that are infiltrating in the ecosystem of the myocytes. For that reasons, we decided to look also at the non-myocytes of PKP2 knockout hearts. And here, this is an immunostaining for myocytes, for non-myocytes that were isolated from PKP2 knockout hearts. They are staying for alpha-smooth muscle acne ingrain, so you see many of them are myofibroblast. And this is not surprising, this does not surprise us, because of course, there is a lot of fibrosis in our hearts of PKP2 knockout, so it makes sense that we found a lot of myofibroblast. But what I want to show you, especially today, is that we look at the nuclei, state for DAPI, of this population of non-myocytes, and we found, I hope you can see them, a lot of compacted, rounded heterochromatin inside the nuclei, inside of a homogeneous distribution of the DAPI. And these nodes of heterochromatin are described in literature as senescence-associated heterochromatin foci, also known as SAF. And these are well-described sign of senescence, and we observed many of them in the non-myocytes of PKP2 knockout hearts. Similarly, we stain for P21, P21 is all marks of senescence, it's a protein that, when it's expressed, cause the cell cycle arrest, so the cells stop divides, so they become senescent, and many of those nuclei, both in myofibroblast and also in our non-myocytes, were positive for P21. So what we think is that our PKP2 knockout myocytes are communicating with the non-myocytes, and non-myocytes, vice versa, are producing SASP, pro-inflammatory molecules that are affecting the myocytes, creating a positive feedback loop. And in conclusion, so we observe sign of premature aging and senescence in both myocytes and non-myocytes in absence of placofilin-2, and therefore we think that it may be interesting to look into the senescence in neurodegenerative disease pharmacology to search for insights in the treatment of PKP2 arythmogenic cardiomyopathy. And with that, I would like to thank all my lab, especially my mentor, Mario Delmar, and my colleagues, former colleagues, the Microscopic Core in NYU, and also the DeLazaro Lab in NYU as well. And thank you for the attention, of course. All right, are there any questions? Yes, please. Mhm. Yes. Yes. Could you repeat the question, please? Well, yes. I'll try. So, basically, the question was, let me know if I understood correctly, that our idea is that there is a mechanotransaction between the disruption of the connection between placofilin 2 and desmine and intercalated this that then connects to the impairment of the nuclear envelope. Right? Is that the question? Okay. Yes. We think that that's exactly what's happening. So our idea is that, of course, we know that desmine is interactive at the intercalated disk with placofilin 2. And when placofilin 2 is missing, there is a disruption of the desmosomal structure. And so that may relate with a disorganization of desmine that then affects the... And desmine is super important in maintaining the nuclear shape. So this could actually affect the nuclear shape. Damaging the container can also damage then the chromatin that is inside the nucleus and creating so a prescription of reprogramming in our myocytes. And then now we are also thinking that this prescription of reprogramming correlates with the myocytes communicating with the non-myocytes that are also then affecting vice versa the myocytes. Yes. That's our idea. So I think we have to move on to the next presentation, but maybe you can find each other after the session. Yes. Yeah. All right. Thank you. Thank you. Okay. So the next presenter is Luigi Anastasia, and he will talk about lethal autoimmune channelopathies and NFE1.5 autoantibodies and their role in sudden cardiac death. Okay, thank you very much, and thank you for the opportunity to drive you through this journey about this, what we try to define as lethal autoimmune channelopathies, and as you will see, these NAV1.5 autoantibodies that we discovered probably will redefine the way you will see sudden cardiac death. So in the last few years, I had the opportunity to work with Carlo Pappone and Giuseppe Cicconte to try to understand what's behind Bugatti syndrome. Of course, they defined very well this arythmogenic substrate, but we really wanted to understand what is the mechanism of this genetic disorder that is associated with an increased risk of cardiac sudden death. So over the years, this disease has been associated to a mutation of the SCN5A, which is encoding the NAV1.5 channel, but if you really think about it, I mean, even if we account all the papers that have been written on this topic, we can only cover about 25% of the patients. So the other 75%, they don't have a mutation that is known, so why do they have this disease? Of course, one of the landmarks of this disease is the ST elevation, that it can be either spontaneous or it can be elicited, for instance, by injecting a sodium channel blocker like Ajmelin. So this is actually an important thing. So no matter what, even if you don't have a mutation in the channel, this, at least right now, is one of the gold standard to see. So you inject a sodium channel blocker. So the sodium channel must play a role in this disease. And we all have been working on this, thinking about the cardiac channelopathy, so we focus on the heart. But what if the critical piece lies somewhere else behind the heart? And that's how we started to look into this, especially because in the last few years, there was a lot of attention about autoimmunity and arrhythmias, and of course, autoantibodies that have been found in many diseases. We saw them in atrial fibrillation, we saw them in long QT and short QT, but nothing really was coming out on Brugada. So we actually conducted a very large study where we did a multi-omic study on about 600 individuals, about 300 Brugada patients, 300 controls, we tested negative for Ashmolen. And one thing that was always coming out was this activation of the immune system that you could see both in transcriptomics and proteomics. And we confirmed also this inflammatory cytokines when we analyzed the blood with Analyzer. So we started to think that maybe there was some autoimmune in this disease. And I have to thank Andrea and Adriana in my lab, because they really pushed this forward. So again, we are thinking about the sodium channel that can be impaired, either because there's a mutation or maybe there is something that is blocking it, and we are thinking that maybe an antibody is blocking it. And Ashmolen, or another channel blocker, is actually combining the effect, and that's why we see the ST elevation when we do this drug. So how did we test that? Unfortunately, there was no ELISA, so we had to sort of like come up with an idea. And so what we did, we overexpressed NAV1.5 in X cells, and we used the protein extract as a probe to look for the antibodies. And I don't want to bore you. I mean, you can read it in the paper that we just published about the details. But basically, what we found was that we could see a specific binding of antibodies against the NAV1.5 in Brugada patients. And this was independent of having a spontaneous type 1 mutation or not mutation in the sodium channel. And of course, we did a lot of samples, and the results were quite stunning, and we were impressed. And so we think that maybe one day we can develop an non-invasive test to look for these patients simply by a blood test. And of course, we had to prove that this was specific. We did it in vitro and in vivo on a heart specimen. We did immunoprecipitation. I don't have time to go into the details, but I think some of the experiments were quite interesting. So these antibodies, they're not just the biomarker, but they are functionally impairing the channel. And we proved that because we took over-expressing the channel, and when we incubated either with the control blood or the plasma coming from the Brugada patient, we saw a stunning reduction of 41% in the sodium current. But I think that actually the best experiment was suggested by one of the reviewers that asked, what happens if you inject them into a mouse? And Mark in my lab was really smart to do a very simple experiment that I hope somebody else will try to repeat. So he injected 200 microliters of the plasma of Brugada patients and controls, and he just recorded an ECG. So when we injected the control plasma, nothing happens. We followed the mice for 45 minutes. But what was impressive was, and we were really shocked about this, when we injected blood from Brugada patients, within a few minutes, we started to see some electrical instability. And within five minutes, the typical pattern that you see in humans started to appear. And then arrhythmias were coming, and these mice died within a few minutes. We were really, really, really scared about what we found, that basically you could transfer the disease by just the blood transfusion. So we wanted to prove that actually this was due to the antibodies. So we removed the antibodies from the blood of the patients using G-protein coated beads. And so we injected the same plasma from a patient with or without the antibodies. And if you inject it without the antibodies, nothing happens, like in the control cells. So we wanted to understand where these antibodies bind. So we sent out several samples to this company in Germany that is specialized in epitope mapping. And we found the exact sequence, and of course more than one, where these antibodies bind. So now we are synthesizing peptides, and we want to develop an ELISA to hopefully try to find a way to detect it in the blood of the patients. So in the last few minutes, if I have time, I want to tell you a story that really was not expected. As this movie, I don't know if some of you saw this movie a few years ago on Netflix, it was called Brain on Fire. This woman was misdiagnosed with a psychiatric illness, and what it turns out is that she had cancer, and the autoimmune system was producing antibodies that were cross-reacting also with the brain. What if something like this happens also in the heart? And actually, if you look at the literature, there was a recent paper that metastatic cancer patients that die within the first six months, up to 20% they die of sudden cardiac death. And there's no way that this is justified by just simply the cardiotoxicity of the drugs that they're taking. So what is known in the literature is that most metastatic cancer, especially colon cancer, breast cancer, and prostate cancer, they overexpress the sodium channel to make it more metastatic. And they have this neonatal isoform that is only seven amino acids different from the one that we have in the heart. And we found one report of out antibodies that women with breast cancer are generating against the tumor, against the sodium channel. So we tested whether the blood of about 40 women that had metastatic breast cancer was able to cross-react with the cardiac sodium channel. And we were shocked that that was true. So they tested positive, and so they cross-reacted. So basically what is happening here, that the body of these patients are trying to fight the cancer, but they are generating antibodies that can't cross-react as a side effect to the heart. So what happens if we inject the blood of a metastatic breast cancer woman in a wild type mouse with a completely normal heart? We inject it in the tail, and we see exactly the same thing that we saw with Brugada patients. So we see an ST elevation, and we start to see arrhythmia, and the mice die. And when we look at the heart, we injected the blood in the tail, and we found antibodies, human antibodies, bound on the heart of these mice. And when we remove the antibodies from the blood, nothing happens, exactly like with the controls. And it's the same when we did the electrophysiological study. So to finish up this presentation, of course this is the beginning of the story, but we really think that maybe Brugada might just be a heart manifestation of something bigger that are these lethal, as we define it, autoimmune, lethal antibodies that the body can generate also against something else that is not with the heart. So the heart might be even normal, but these antibodies might bind to the heart and induce sudden cardiac death. So we need to find a threshold and see if these are dangerous or not. And of course, if you are doing a chemotherapy and your heart is already under pressure, maybe you are at higher risk of sudden cardiac death. So I'd like to thank, in the last slide, my lab, everything that worked with us, and of course Giuseppe and Carlo that really supported us over the years. Thank you very much. So we have time for a few questions. That's a good question. We, I don't expect it because, oh yeah, so they asked me would you expect a large amount of patients with cancer to have an ST elevation, right? Well, we don't, we don't see this in Brugada patients. Only a very small fraction have an ST elevation unless you provoke it with a sodium channel blocker. And actually you see them overnight. So we have patients that have a completely normal ECG during the day. You put an alter at four in the morning, they have a typical type one pattern. We are starting to follow these patients, these cancer patients with an alter and we started to see some alteration overnight but of course we just started. We just got approved so we need to study it better. But with, you know, at least in the mice we see exactly the same phenotype. That's a good question. The question is why do we expect the male predominance? We don't know. What I can tell you is that people that have a mutation in the sodium channel, it seems they have a little less antibodies than people that don't have the mutation. But male and female, we didn't see any difference in the amount. But we need a quantitative test that we don't have right now. Well, yeah, so the question is, how do we explain these results as compared to the polygenic risk score that has been developed for Brugada syndrome? Well, right now we cannot really explain this difference. Well, what we publish is that people that have mutation in STN5A have a worse phenotype. They have a worse substrate when they map the substrate. So we think that it can be a combination of these two effects. We're not saying that it's just an autoimmune disease. Of course, it's a genetic disease. But we don't know at this stage because we cannot really measure the amount until we have an ELISA. We cannot be sure about the amount of antibodies that this patient has. So right now we can only say yes or no, but we cannot say exactly how much they have. It can be, but as I said, when we injected the blood of the patient in a normal mouse with a normal heart, we saw the ST elevation and we saw the arrhythmias. And these mice, they didn't have any previous disease. And we don't see, we absolutely don't see it with the normal controls. And we even inject ijmelin and nothing compared to what we see here. And when we remove the antibodies, now we are trying to fish out only the antibodies against the sodium channel with the peptide. We still don't see any effect anymore. Thank you. So the next presenter will be Molly O'Reilly from the Amsterdam University, and she will talk on redefining cholinergic polymorphic ventricular tachycardia as a neurocardiac disorder. Okay. Hi, everyone. Good afternoon. It's a real pleasure to be presenting here today. My name is Molly O'Reilly. I'm a postdoc at the Amsterdam UMC. And today I've gone for quite a daring title, but I'm hoping by the end of my talk, I'll have convinced you of its accuracy. So redefining CPVT as a neurocardiac disorder. So CPVT stands for catecholaminergic polymorphic ventricular tachycardia, and it's a condition that's characterized by exercise or emotion-induced sudden cardiac death in very young and otherwise healthy patients. In most cases, it's caused by mutations in ryanodine receptor 2, which has a really important role in intracellular calcium homeostasis. In cardiomyocytes, mutations lead to a leaky transporter, which leads to a leakage of calcium into the intracellular space. This leads to delayed after depolarizations, and this leads to arrhythmias. Now, traditionally, it's been thought of as being a purely cardiac condition, but I suspect that there are other factors involved. So we know that ryanodine receptor 2 is also expressed in the neurons of the central nervous system, such as in the brain. Patients often present with clinical signs of autonomic dysfunction, things such as syncopes and seizures. And the mainstay treatment approaches are beta blockers or a stilectomy, both of which are targeting the interaction between the nervous system and the heart. So all of this leads me to hypothesize that there is an unrecognized or rather underappreciated dysfunction of the autonomic nervous system, which may be contributing to arrhythmia. But why do we care about dysfunctional neurons? Well, that is, of course, because the heart is under constant control of the autonomic nervous system, which is broadly split into two divisions. So there is the parasympathetic nervous system, which predominates at rest and keeps the heart rate low. Then there is the sympathetic nervous system, which takes over during exercise or emotion, and it sends projections from the structure known as the stellate ganglia directly to the heart in order to increase the heart rate. So because in CPVT, the arrhythmias typically occur during sympathetic activity, I'm really interested in the stellate ganglia structure. And the questions I've been addressing in my project is whether ryanodeme receptor 2 is also expressed in these peripheral cardiac modulating neurons and whether mutations in ryanodeme receptor 2 lead to any functional or structural alterations of the nervous system. So for this project, I've been taking or isolating stellate ganglia neurons from mice. And here you can see the location of the stellate ganglia structure, located next to the first and second rib alongside the longus coli muscles. So using enzymes, you can isolate individual neurons. And you can see here what these look like when they're freshly isolated. And then after several days in culture, when they really regain their structural architecture and regrow those neurites and dendrites. So after I've cultured them for several days, I then perform immunocytochemistry to look for my ion channel of interest. So here you can see immunostaining in three different channels. We have in blue, DAPI, the nuclear marker. In red, we have beta-3-tubulin, a general neuronal marker. And in green, we have ryanodeme receptor 2. And I think it's very clear from these images that indeed, ryanodeme receptor 2 is expressed in these peripheral cardiac modulating neurons. So the next question is whether mutations lead to any functional alterations. And for this, we turn to a CPVT mice model. So I chose to work with the mutation R2474S. This is a really classic, well-characterized CPVT model. So I've been isolating stellate ganglion neurons, and I've been investigating their function. Of course, the first thing I investigated was calcium homeostasis. So what you can see here is some live cell imaging of freshly isolated stellate ganglion neurons loaded with a calcium-sensitive dye, Fura Red, and then exposed to caffeine to cause release of all of the calcium from the intracellular stores. So what you can do is you can actually quantify that reduction in fluorescence, and that tells you about how much calcium was stored in the intracellular stores and released upon caffeine exposure. When I compared my mutants and my wild types, I found a significantly reduced reduction in fluorescence in the CPVT neurons. So what that tells us is that there is less calcium stored in those intracellular stores ready to be released upon caffeine exposure. So what we think is going on here is that the mutant ryanodeme receptor 2 channel is leaking calcium into the intracellular space, just like we know occurs in cardiomyocytes. So what would be the consequence of that? Well, we know from neuroscience studies that if there is leakage of calcium in the intracellular space of a neuron, then it will extend more neurites from the cell body. So the next thing I investigated was whether all of this leads to increased neurite outgrowth. To do that, I isolated my stellate ganglion neurons, and I put them into culture for seven days, and then I stained them for DAPI and beta-3-tubulin. I then quantified outgrowth, neurite outgrowth, by looking at the beta-tubulin positive area normalized to DAPI. When I did this, I found a significant increase in the outgrowth of neurites from these mutant neurons. So this is really exciting. We now have both functional and structural alterations of stellate ganglion neurons as a result of ryanodeme receptor 2 mutations. The next question is whether all of this leads to increased cardiac innervation, because I told you that these stellate ganglion neurons project directly to the myocardium. So to answer this question, I've been taking mid-ventricular cryosections from my CPVT hearts, and I've been staining those for the sympathetic neuron marker tyrosine hydroxylase. When I compared the TH positive area in these hearts, I did find a significant increase in my CPVT myocardium. So we now know that CPVT hearts are sympathetically hyper-innovated. There are more sympathetic neurons. And not only that, I also observed an increase in innervation heterogeneity. So when you compare the most and least innovated areas of these myocardial slices, there was a much greater spread in the data in the CPVT hearts. And we know that innervation heterogeneity is a pro-arrhythmic factor. So the final question in this project is whether all of this leads to increased neurotransmitter concentrations, because of course neurotransmitters are the final functional link between the nervous system and the heart. So before I show you the data, I'm just going to try and explain how neurotransmitters in the heart work. So when a sympathetic neuron is stimulated, it will release norepinephrine. This norepinephrine binds to beta receptors on the cardiomyocyte membrane, triggering intercellular cascades that leads to contraction. Most of that norepinephrine is taken back up into the sympathetic neuron and repackaged into presynaptic vesicles. However, if there is an excess of norepinephrine, then this is actually taken up by the cardiomyocytes and converted into its metabolite normetinephrine, and that's then secreted into the bloodstream. So I took my ventricular tissue of my CPVT heart, and I sent this off for neurotransmitter quantification, and what we found was slightly lower levels of norepinephrine, elevated levels of normetinephrine, and a significant increase in the normetinephrine to norepinephrine ratio. What this tells us is that there is an excess spillover of norepinephrine in the cardiac interstitial space, and a significant increase in extra-neuronal norepinephrine turnover. So I just want to finish off now with my newly proposed mechanism of arrhythmogenesis. So in a wild-type scenario, a sympathetic neuron is activated, it releases norepinephrine, this binds to beta receptors on the cardiomyocyte membrane, this triggers intercellular cascades, which leads to the overload of calcium in the SR, and this is the triggering for reanidine receptor 2 to open. The current prevailing dogma in CPVT is that everything is identical, other than that mutant reanidine receptor 2 channel, which causes a leakage of calcium. So what I'm now adding to this mechanism, based on the data that I presented to you today, is the neuronal component. So we can now also say that the hearts of these mice are sympathetically hyper-innovated. So when sympathetic activity occurs during exercise or emotion, there will be an enhanced release of norepinephrine, enhanced activation of beta receptors, this will lead to enhanced intracellular cascades, and a more enhanced flooding of calcium into the SR, and of course this is a potent trigger for reanidine receptor 2 to leak calcium and lead to arrhythmias. And this really nicely explains why stelectomies or beta blockers are the treatment approaches that are effective for this condition, as they would target both of these aspects. So we can now conclude by stating that stelate ganglia abundantly express reanidine receptor 2, mutations lead to functional differences in the way of altered calcium homeostasis, this is likely responsible for the innovation differences in the way of sympathetic hyper-innovation, and importantly, all of this leads to increased extra-neuronal norepinephrine turnover. So I hope I've managed to convince you today that CPVT is indeed a neurocardiac disorder. And just to finish with the translational outlook here, we can now state that autonomic nervous system alterations are indeed a component of CPVT, and thus provide a novel avenue for risk stratification and treatment design. We can incorporate assessments of autonomic function for improved risk stratification, we could potentially use normetanephrine as a novel blood biomarker, and we could stabilize ReR2 in the stelate ganglia as a novel treatment approach. And if you'd like to read more about this, this paper and project is now on bioRxiv. And that just leaves me to thank my colleagues in my lab for supporting me in this project, especially my supervisor Caroline Remy, I'd like to thank my funders, and of course I'd like to thank you all for listening, so thank you. Thank you for your presentation, I'll take advantage and ask the first question. So a few days ago I saw a poster where pharmacological blockade of the ryanodine receptor was antiarrhythmic in patients with structural heart disease, would it be via a similar mechanism? Yeah, absolutely. I think stabilizing the stelate ganglia would have all kinds of consequences for the innervation of the heart in numerous different conditions, but of course we don't know what stabilizing ryanodine receptor 2 would do in a cardiomyophyte, that could affect things like neurotrophic factors and other things that feed backwards to the stelate ganglia. So potentially, but I can't confirm. Yes, so the question is in my eye growth assays, whether I have applied flecainide or dantrolene and seen an effect. I have, but I'm not going to go into too much detail because it's very preliminary at the moment. So watch this space for that result, maybe. Carol told me not to tell. So for saying that there are autonomic, oh sorry, the question is whether, yes, so the question is whether there is a human objective here. So ideally we'd love to get some human hearts and confirm the hyperinnovation, but what we want to do is test CPVT patients for their blood biomarkers. Also looking at the metabolites of these neurotransmitters and then that will give us a very good indication of whether there are alterations in innovation. We would also like to do MIBT imaging too. Yeah, so the question is whether I have any data on MPY, and unfortunately I don't. The neurotransmitter quantification service I used did not offer that, but we would love to look into it. I can tell you we also see differences in epinephrine as well, so we see lower concentrations of epinephrine in the hearts of these lower, which might tie into the bradycardia that is sometimes observed in these patients also, but that's highly speculative on my part. No, I did not measure PNMT. No, so we haven't tried that yet, but I think that would be really interesting as well. Yeah, so for all of the neurotransmitter quantification, there were just mice, anesthetized, and the heart's taken out. So there was no stress involved, that's just baseline levels. Alright, thank you. Then we move on to the next presentation. We'll be by Laivi Low. Dr. Low, I'm presenting on behalf. Oh, presenting on behalf, and what's your name? Xinhui Liu, Dr. Liu. Dr. Liu, this is Dr. Liu who will be presenting on the crosstalk between neuromodulation and anti-inflammation. This is your presentation. Thank you. Okay, thank you for the introduction. My name is Xinhui Liu, and I'm from Taipei Veterans General Hospital in Taiwan, and I'm here on behalf of my mentor, Professor Dr. Liu Lai Low. And on the topic we're going to discuss today on the association between the low-level tragus nerve stimulation and also the cholinergic anti-inflammatory pathways. So this is our disclosures. So to begin with, what are the roles of the low-level tragus stimulation? So this was first reported in a canine study by the Oklahoma group, where they found nerve bundles in the auricular branch of the tragus nerve containing the acetylcholine esterase. And they tried to use the rapid atrial pacing, and they found a decreased AERP and a decreased AF window of vulnerability, whereas if these are ARP plus ALTS, cause a linear return of those parameters, as you can see from the figures here. So clinically, there is also another clinical trial called the TREAT-AF trial, where they tested the transcutaneous electrical vagus nerve stimulation to see if it suppresses AF. And they found that the chronic intermittent ALTS had an 85% lower AF burden at six months. So most importantly, they also found that the TNF alpha was also significantly decreased at 23% in the active group. So in this study, they highlighted the role of inflammation in AF pathogenesis and possibly the possibility of a neural circuit regulating immunity through the cholinergic anti-inflammatory pathway, the CAIP. So what is a CAIP? So we think that the CAIP is a missing link in the neuroimmunomodulation that controls the cytokines. As you can see from the figures here, the efferent signals from the vagal nerve, which could be controlled by the cholinergic brain network, and it further inhibits the cytokine production via pathways dependent on the alpha-7 subunit of the acetylcholine receptor on macrophages. And it further inhibits the release of TNF or interleukin-1 and other cytokines. As we dive deeper into the mechanism, we already know that the cholinergic signals derived from the vagus nerve stimulation inhibit the release of cytokines by transducing a signal cell that inhibits the nuclear activity of the NF-kappa-B. And as a result, the alpha-7 subunit of the acetylcholine receptor ligation activates the JAK2-STAT3 pathway, which initiates a signal transduction that negatively regulates the NF-kappa-B binding to the DNA. So this led to an anti-inflammatory response. So in one of our previous studies, we had similar results, and we tried to focus on obstructive sleep apnea models and tried to interfere it with renal denervation. So in our study, we found that the obstructive apnea model caused an atrial immune cell infiltration, and furthermore, it reduced the presentation of the alpha-7 acetylcholine receptor and also reduced the phosphorylation of the JAK2-STAT3 and also increased the interleukin-1, 6, and TNF-alpha and finally activated the NF-kappa-B. So in our study, we found that there's a link between the alpha-7 subunit of the acetyl receptor and the JAK-STAT pathway. So this finding was also similar under a proof-of-concept study in humans where they tried to use the LLTS to treat ischemic reperfusion in semi-patients. So they found that the ventricular arrhythmias were significantly attenuated by the acute LLTS in the first 24 hours. The eryangeal curve for the CK-MB and the myoglobin over the 72 hours were smaller in the acute LLTS group, and finally, the inflammatory markers were decreased by the LLTS. So in our study, we hypothesized that the chronic LLTS attenuates inflammation, improves cardiac function, and reduces ventricular arrhythmogenesis in the rabbit model of ischemic cardiomyopathy. So in our methods, we prepared 18 male New Zealand white rabbits and ran dynes into DSHAM control, heart failure, and heart failure resync in the LLTS group. And all rabbits underwent general anesthesia under the final experiment. As for the heart failure model, we used a post-MI heart failure model, and it was induced by the LED ligation in both the heart failure and the heart failure LLTS groups, and we monitored for eight weeks until the heart failure maturation. As for the final experiments, cardiac EP study was done, and at the two times and ten times diastolic pacing threshold, inflammatory markers, trichostain, western blot analysis were also done. As for the creation of the LLTS model, we tried to use electrical clips placed at the left tragus of the rabbits, and we used simulation at a frequency of 20 hertz with a pulse duration of 0.2 milliseconds, currently with a current ranging from two to four microamples for each session. And in the LLTS groups, all the rabbits underwent a daily 30-minute LLTS at an 80% below the threshold for sinus rates lowering for at least one month. As a result, for the echocardiogram, we found that there was a deterioration of the LVEF, and it was observed in the heart failure group, and it was less significant in the heart failure LLTS group. As for the inflammation marker, compared to the sham control group, the pro-inflammatory cytokines such as the interleukin-6 and TNF-alpha were increased in the heart failure group, but both markers were decreased in the heart failure LLTS group. Compared to the sham control group, the anti-inflammatory markers such as the interleukin-10 decreased in the heart failure group and decreased in the heart failure LLTS group. As for the cardiac EP study, we found that the VERP was significantly prolonged in the heart failure group and moderately prolonged in the heart failure LLTS group compared to the sham control group. We also did a ventricular inducibility test, and we found the inducibility was greater in the heart failure group than in sham control and the heart failure LLTS group. And most importantly, there was no differences between the sham control and the heart failure LLTS group. So the cardiac EP study indicated that electrical remodeling in the heart failure can be prevented by an LLTS. And next is the trichostain for the myocardial fibrosis. We found that there was a significant increase in the LV and RV fibrosis in the heart failure group compared to the sham control and heart failure LLTS groups respectively. So these findings indicated that the structural remodeling in the heart failure can be prevented by LLTS. And next is the Western blot analysis for ventricular ion channels, and we found there was an altered protein expression of all of these cardiac ion channels, and they were found from the heart failure group and were normalized in the heart failure LLTS group. So this indicated that there could be a calcium channel dysregulation in the heart failure and can be prevented by LLTS. And next is the cell cellocytes that revealed expression of the phosphorylation of JAK1 and STAT1 were increased in the heart failure group but decreased in the heart failure LLTS group, whereas the phosphorylation of the JAK2 and STAT3 were increased after the LLTS. So this indicated that the elevation of the phosphorylation of JAK2 and STAT3 pathway suggested activation by ligation of alpha-7 acetylcholine receptor, and this suggested that the LLTS increased the anti-inflammatory response through the CAIP. So as a conclusion, the heart failure causes a significant inflammation response with ventricular electrical and restructural remodeling, leading to the risk of ventricular arrhythmias and sudden cardiac death. The JAK2-STAT3 pathway is inactivated by ligation of the alpha-7 acetylcholine receptor, suggesting that the LLTS increased the anti-inflammatory response through the CAIP. And next is the LLTS, it may mitigate the heart failure by reverse modeling and reduce ventricular arrhythmias and inflammation with the CAIP playing an important role. And then finally, these findings suggest that the LLTS may benefit extend beyond neuromodulation and involve the CAIP activation. And I would like to acknowledge the members from our team, and Professor Chen and Dr. Lo and Dr. Liu is the original presenter today. And thank you for your attention. Are there any questions? Yes, please. Okay, so the question was whether the mechanism was through local or through another mechanism through the spleen. So we're still working on it, but we think there could be another off-target mechanism pathway that we're still looking at. But as you said, it could possibly be both, both from the inflammatory pathway and possibly another off-target focusing on the ion channels. I have one question. So what was the effect on heart rate? Maybe I missed it. Is there an effect on heart rate? Oh, so the heart rate, because the vagus nerve stimulation, supposedly it's a parasympathetic stimulation and it would slower the heart. So we do not want to cause bradycardia. So that's not our goal. Our goal is just to have a low-level stimulation instead of a high-level stimulation. If it was a high-level stimulation, it would cause bradycardia, and the sham control will not be the exact condition. That was indeed what I was aiming at. But it wasn't different, heart rate? Did you check? Oh, so there's an 80% threshold. We don't want to be below the 80% of a baseline during rest. So was heart rate lower or not different? Oh, no difference, no difference. Any other questions? Well, infraction size, because this model is quite stable in our lab, so I think the majority is almost the same, and we ligate almost at the same location. So I think because it's done by the same member from our team, so I think in general it's the same. Yeah, so that is what we're still working at, because we're afraid there could be other off-target pathways, so that's what we're trying to work at. Thank you. And then I welcome the last speaker of this session, Justin Brilliant, who will talk on circadian pattern of ventricular arrhythmias in gene-edited porcine to baboon cardiac xenotransplantation. Please. Hi everyone. Thank you so much for coming. My name is Justin Brilliant. I'm a cardiology fellow at the University of Maryland Medical Center and had the pleasure of presenting our research on the circadian patterns of ventricular arrhythmias in gene edited porcine to baboon cardiac xenotransplants. So as an introduction, cardiac arrhythmias are a leading cause of cardiovascular death. It has actually been long accepted as well that life-threatening cardiac arrhythmias, particularly VT and VF, can occur and are more likely to occur in the morning after waking. And circadian rhythms profoundly influence various physiological processes including cardiac function, electrophysiological parameters, and arrhythmia timing. And also and furthermore that may play a critical role in the timing and manifestation of arrhythmias. So therefore, you know, one thing to keep in mind is heart transplants is the most effective treatment for end-stage heart failure for patients with terminal or advanced cardiac failure, offering the best chance for high quality of life. However, the supply of donor hearts is much smaller than the demand leading to long wait lists, high wait list mortality, and a reliance on temporary mechanical circulatory support for patients deteriorating while on the transplant list. And so gene edited cardiac xenotransplantation has emerged as a promising approach to alleviate the chronic shortage of donor hearts for those patients with end-stage heart failure on the transplant list. And so University of Maryland, we had the fortune of performing the first two successful porcine to human heart transplants, the first one being January of 2022 with a recipient surviving for 60 days, and the second patient receiving a transplant September 2023, ultimately surviving for 40 days. However, there is limited human and animal data really even on the mechanism of arrhythmias and even circadian patterns of arrhythmias in these gene edited xenotransplants. And so given this limited physiologic knowledge, we assess the circadian distribution of ventricular arrhythmias in porcine hearts, transplant it into baboon recipients, and hopefully this can help improve our understanding of the proarrhythmic risk in future human cardiac xenotransplant cases. And so for our methods, we evaluated gene edited porcine to baboon xenotransplants where pigs actually had genetic editing performed where certain carbohydrate antigens were removed from the pig hearts and also some anti-inflammatory epitopes were removed as well. And these were transplanted into baboons in 15 animals from 2017 to 2022. And all of this was performed by the Revivacor company. And at the time of transplant, they were surgically placed intrathoracic epicardial leads, bipolar ECG electrodes. One of the ECG leads being placed epicardially in the right atrial appendage and the ground electrode being placed in the left ventricular apex. And right here is a kind of an image of the telemetry machine that's placed in the pre-peritoneal area right below the subxiphoid process of these baboons. And with these surgically placed epicardial ECG electrodes, we had the availability of continuous single lead ECG telemetry as exemplified right here, this being normal sinus rhythm. And we had continuous measurements for the first 30 days postoperatively and thereafter with intermittent use of this telemetry for battery preservation. And finally, arrhythmia analysis for arrhythmic episodes were performed with specialized software for identification, but followed by full human visual beat by beat review. And so when we looked at the postoperative course for these 15 baboons and looking for particular arrhythmias, we were able to find 226 days worth, about 5,432.9 hours of total intermittent ECG telemetry data with automatic and additional visual beat to beat review of these arrhythmias. And some of these arrhythmias that we found included non-sustained monomorphic VT, non-sustained polymorphic VT, sustained monomorphic VT, and sustained polymorphic VT slash VF. And the documented animal survival for these 15 animals ranged from two days to 264 days with a mean of 82 day survival duration. And the total time of monitoring versus animal survival was 20% during these experiments. And ventricular arrhythmias were observed in 13 out of 15 of these cardiac xenotransplanted baboons, 87% of our sample size. And here are some, before going into our data, here are some examples of what these arrhythmias looked like on the single ECG lead that was epicardially placed in these pig-to-baboon xenotransplants. So this is an example of non-sustained monomorphic ventricular tachycardia. This is an example of non-sustained polymorphic VT. This is an example of the onset and offset of monomorphic VT. This is an example of sustained polymorphic VT. And this is an example of ventricular fibrillation. And all these examples, again, are through single lead ECG telemetry with each row representing 10 seconds. And so when we've looked at the data overall for total ventricular arrhythmias, we found a sizable amount of total arrhythmic episodes of 1,313. And when we looked at these total arrhythmic episodes hourly over the 24-hour day, we kind of noticed this bimodal distribution starting to occur, particularly from 10 a.m. here, starting from 10 a.m. all the way to 12 a.m. here, versus 12 a.m. to 10 a.m. on the left side of this graph. And we saw that there were 1,012 episodes that occurred from 10 a.m. to 12 a.m., 77% total, compared to 301 episodes, or 23% total, occurring from 12 a.m. to 10 a.m. And so we call the 10 a.m. to 12 a.m. the daytime distribution, and 12 a.m. to 10 a.m. the nighttime distribution. And when looking at total episodes per hour, about 72.3 episodes per hour versus 30.1 episodes per hour, daytime versus nighttime. And when looking at the physiologic significance of this, breaking down these total amount of arrhythmias into the kind of ventricular arrhythmias that we saw, when we look at non-sustained monomorphic VT, again we see that trend still kind of emerging here from 10 a.m. to 12 p.m. versus 12 a.m. to 10 a.m. And for non-sustained monomorphic VT, total episodes were almost 1,200, 77% in this daytime distribution, 23% in nighttime. And the numeric trend here, 65.1 per hour versus 27.4 per hour, more than double increase per hour here at that rate. And then for non-sustained polymorphic VT, number of total episodes was decreased. Again, we didn't have as much of a trend here when looking at 10 a.m. to 12 a.m. versus 12 a.m. to 10 a.m., but when we looked at other distribution here from 2 p.m. to 11 p.m. versus 11 p.m. to 2 p.m., we saw more of a trend here, 6.7 per hour versus 2.2 per hour, a little over three times increase at that rate. And then finally, we wanted to look at sustained VT and VF episodes, since these would carry the greatest physiologic significance in post-transplant survival and, again, pro-arrhythmic risk. And when looking at sustained monomorphic VT, we had about 23 total episodes, 21 episodes in this daytime distribution versus two episodes from 12 a.m. to 10 a.m. and a p-value approaching significance of 0.05. And with sustained polymorphic VT slash VF, only around 11 episodes, not as many that we see here in our graph, but when we looked at the distribution rather from 2 p.m. to 11 p.m. versus 11 p.m. to 2 p.m., we did see some kind of trend start to emerge here. So in conclusion, there's a strong trend towards four ventricular arrhythmias to occur more commonly during the late morning slash early afternoon to the midnight time frame, rather than at night or the early morning hours. The circadian pattern in animal studies suggests potential effects of circulating endogenous or exogenous factors and or effects of the residual ventricular nervous system in a setting of decentralization post-operatively after heart transplants. And understanding the circadian patterns of ventricular arrhythmias really may allow us to improve the management of these potential life-threatening arrhythmias and xenotransplants, particularly for the human population moving forward. And I just want to send a warm thank you to Dr. Dickfeld, my mentor during this experiment and the study. Also Dr. Griffith and the cardiac surgery team at the University of Maryland. And Dr. Muhyiddin and his research team, who's really carried research in xenotransplant forward over the past 10-20 years at University of Maryland. So I really appreciate all your attention. Thank you. Very nice talk. The population is excellent. When you show the circadian rhythm, is it associated with any changes in sympathetic or parasympathetic tone? Did you notice changes in heart rate? Because the heart's supposed to be denervated at this time. Or you actually notice re-nervation and it's already present, change autonomic nerve stimulation. So the question was, were we able to see differences in parasympathetic versus sympathetic inputs post-operatively in these experiments, even in the setting of decentralization. So certainly we know that there is variability in the heart rates that we saw. We didn't particularly look at faster heart rates versus slower heart rates to really know if there was greater parasympathetic versus sympathetic tone that correlated with the circadian rhythm. But we do know that the heart does vary in response to exogenous and endogenous stressors in these experiments. But that's something that we can certainly look for, especially looking at endogenous mediators in the bloodstream that may be an explanation for the circadian rhythm that we're seeing in the experiment. For more information visit www.FEMA.gov So the question is, did we look at, you know, sympathetic mediators in the bloodstream that correlated with these circadian rhythms in these experiments? So the first one, unfortunately, we did not correlate that, you know, so we don't have particular biomarkers that would explain, you know, this distribution, this bimodal distribution in these experiments. We're still continuing to do these experiments at University of Maryland, so I think that's going to be one of our future questions in terms of the mechanism behind our findings. And then as for the two humans that were transplanted at University of Maryland, they actually were not on beta-blockers post-operatively. They were actually largely dependent on inotrypstil and vasopressors. You know, one of the interesting things that we're still trying to look at is sizing for these pig hearts and human recipients, and, you know, they still remain inotryp-dependent, vasopressor-dependent, because the stroke volume may be low, because those pig hearts are a lot smaller in humans. So there are some confounders where we're not able to put on as much, you know, GDMT or beta-blockers in those patients because of sizing issues and other inflammatory markers that we need to discover or evaluate further, but so, yeah, it's a good question, I think. So I think we have to close the session for the sake of time. I do would like to ask the speakers to come forward and fill out an evaluation via the QR code here. And all the others, thank you for attending the session.

heart rhythms

genetics

neurocardiac interaction

CPVT

LLTS

ventricular arrhythmias

xenotransplants

beta-blockers

stellate gangliectomy

cholinergic anti-inflammatory pathway