All right, good morning, everyone. My name is Peter Hanna. I'm from UCLA. I'm joined by my co-chair, Nick Moisea, from Ohio State University. And it's my pleasure to get things started with our session this morning. We have four excellent speakers talking on the diverse roles of inflammation and arrhythmogenesis. Along with each of the talks, we'll have a Q&A session. So when the QR code pops up on the screen, go ahead and scan it and enter in your questions or come up to the mic to ask. At this point, we'll get started with our first speaker, Dr. Giovanni Pareto from Milan. Thank you. Thank you very much. Dear moderators, dear colleagues, my name is Giovanni Pareto from Sarafella Hospital, Milan. And it is my pleasure to give this talk today. My background is as a clinical and interventional electrophysiologist. I work at the Referral Center for Ventricular Tachycardia Ablation. And we also have a multidisciplinary unit entirely dedicated to myocarditis and inflammatory heart disease with more than 600 patients with biopsy-proven disease in active follow-up at our center. So let's start with an overview. The concept of myocardial inflammation actually is really relevant because it doesn't apply only to the classic setting of acute viral or autoimmune myocarditis, but actually displays relevant overlaps with genetic dilated cardiomyopathy, but also primary arrhythmogenic cardiomyopathy. It has been recently defined as the hot-face presentation of some patient with desmosomal gene variance in particular. To make things simple, we can consider that we have any kind of exogenous noxa. It can be infectious or noninfectious. And it can be, I don't know why it doesn't work, okay. It can be a classic lymphocytic myocarditis or a special form of myocarditis, for example, the granulomatous ones. In the event you have a complete healing, you can have restitution integrum without scar, but more commonly, you have a non-ischemic scar as a result of myocardial inflammation. Of course, you can have also chronicization to a chronic active myocarditis, and in the end, the end-stage form is chronic dilated inflammatory cardiomyopathy. And it is easy to understand that the risk of heart failure increases as far as we have higher degrees of left ventricular dilation and dysfunction. But what is relevant is that the arrhythmic risk may be high even in patient with normal or near-normal ejection fracture at normal volumes. That means that we have an increased arrhythmic risk depending on myocardial inflammation and subsequent fibrosis. The pathophysiology is quite complex. We don't have time to discuss about all the possible mechanism of arrhythmias. We have a supraventricular arrhythmias, bradyarrhythmias, and ventricular arrhythmias. Today, we will mainly focus on ventricular arrhythmias and two main mechanisms. One is the non-ischemic scar, which is the final result of myocardial inflammation, and the other one is the impaired connection between cells, which can cause arrhythmias. So starting from myocardial inflammation, we know that inflammasome is the cornerstone of inflammatory cascade activation in myocarditis. This has been proven both in biopsy and autoptic cases, and the degrees of activation of inflammasome is somehow proportional to the degree of heart failure, NIA class, but also left ventricular systolic dysfunction and failure to recover, for example, differently from classic fulminant myocarditis. Even in the context of chronic inflammatory dilated cardiomyopathy, the inflammasome is activated. Here is an example of a calcineurin transgenic mice, in which we have a spontaneous sterile inflammation, which depends on interleukin-1 cascade, and accordingly can be partly reverted by interleukin-1 axis blockade by means of anakinra, for example. You can see that the degree of myocardial inflammation and also the degree of fractional shortening impairment may be reverted by the use of this drug. But more focusing on arrhythmias, and in particular, arrhythmogenic cardiomyopathy, we have a beautiful paper, just for an example, by Celco and Safis, in which, again, the activation of inflammasome and FKB cascade in particular, and again, partly reversible by the use of a specific antagonist, has been proven associated with improved substrate in a number of models. In vitro, so with a JAP-mutated ventricular neonatal cultures. In vivo, in models of mice with double homozygous desmoglian nocaine mutation, and even in human-derived iPSC with inpatients from arrhythmogenic cardiomyopathy. So again, we have this functional in the mechanic part, which can be reverted by the use of inhibitor, but look at the fibrosis, for example, the degree of fibrosis using pre-treating mice with an FKB molecule can lead to lower degrees of fibrosis. And consistently, lower degrees of pro-arrhythmogenic substrate, for example, the so-called late potential or epsilon wave on your surface ECG, which is a classical feature of arrhythmogenic cardiomyopathy. Also, we have significant changes in the landscape of cytokines and chemokines, and in particular, interleukin-1, interleukin-6, by the targeting of an FKB pathway. And this actually has clear clinical correlates, because this is our experience in humans with myocarditis. We observe that the features of ventricular arrhythmias are different in patients with active myocarditis, where there is a prevalence of irregular and polymorphic ventricular arrhythmias, as in contrast with the post-inflammatory stage, in which we have stable re-entry circuits and regular and monomorphic tachycardias. So even in clinical setting, we can target the ventricular tachycardias by using immunosuppressive agents, steroids, as a thioprene, and a number of even biological agents nowadays, to achieve relevant improvement in arrhythmic outcomes late after treatment termination, so after 12 months of treatment. The second part of my talk is about the intercellular junction, which is another hotspot for the pathophysiology of arrhythmias in myocarditis. Intercalated disks are complex molecular structures, which provide the electrical and mechanical coupling between cardiac myocytes. And it is very important, looking at this example of transmission electron microscopy, that the space, the intercellular cleft width between consecutive myocytes, is kept very narrow, because this provides the haptic coupling between myocytes. If we have disruption of this structure, we have an increased trend towards arrhythmogenicity. This has been demonstrated, for example, in a mice model of arrhythmogenic cardiomyopathy, again, the small-gland mutation. Look at how enlarged intercellular cleft width we have in these cases, which has been associated with conduction anisotropy and prevalence of averse spontaneous or induced ventricular tachycardias as compared to healthy controls. Similar effects can be obtained by pharmacological modulation of the connection between connexin-43 at the gap junctions and voltage-gated sodium channels, and with exponential effect if combined together. Even in the world of autoimmune diseases, we observed an association between anti-intercalated disks autoantibodies directed against antigens inside the junctions and prevalence of ventricular arrhythmias in humans with myocarditis of autoimmune nature. So the idea here is that whatever the background, genetic in some cases, this immune may be toxic, we have some changes in structure or function of intercalated disks that finally lead to arrhythmias, not only ventricular arrhythmias. And this is basically the results of my recent PhD thesis, which has been just published in JAK Clinical Electrophysiology. And here the concept is that we can have a number of patients, humans, with variable overlap between genetic and autoimmune intercalated disk abnormalities. The common finding is that an increase in intercellular space width, in particular above 40 nanometers in our experience, was associated with an increased risk of either spontaneous or induced arrhythmias, both at baseline and during follow-up. We attempted to transfer our model in a preclinical platform, in particular, BALB-C mice with experimental autoimmune myocarditis, to observe an equivalent cutoff, because the value of ECV, intercellular space width, was comparable in human and mice. But in mice with arrhythmic features, we observed a significantly increase, above 40 frequently, of intercellular cleft width. Here are just some examples. This is a normal mouse, normal clefts, no arrhythmias, never spontaneous or induced, and normal propagation on microelectrode arrays. Whereas in cases with increased spaces between consecutive myocytes, we have evidence of either spontaneous or induced arrhythmias, atrial and ventricular, and abnormal electrical substrate and re-entry circuits, even proven by multi-electrode arrays. The next step of this project would be in collaboration with the myoclinic Jacksonville, with Dr. Leslie Cooper and Delisa Fairweather, who is an expert in sex-related differences, aiming to further characterize the relationship between interleukin-1 pathway and inflammation in general, and intercalated disruption with regard with sex-related differences. Here we have some changes. First of all, we will implant mice with loop recorders to provide continuous monitoring, which is better as compared to 30-minute CCG recording. We will use an Akira to target interleukin-1 pathway to observe differences in inflammation and arrhythmias, and we will explore differences in females and males. Among future direction, we have to consider that the pharmacological targeting of arrhythmias in inflammatory cardiomyopathies does not only rely on anti-inflammatory drugs, but maybe on some glue-like molecules trying to connect and tighten the space between consecutive myocytes, the ECW. Even if today we have a dimensional filter, we have a limitation on drug delivery because the 3-kilodalton is the dimensional limit for the junctions. So the conclusion for my talk today is that arrhythmogenesis in inflammatory cardiomyopathy is quite complex. It is often multifactorial. Today I've discussed only two of the main mechanisms. The targeting of myocardial inflammation is feasible and promising to target arrhythmias in this setting, both in the preclinical and clinical settings. Intercalated disks may play a critical role in arrhythmogenesis and may constitute a future target for pharmacological treatment of ventricular arrhythmias and arrhythmias in general associated with inflammatory disease. And of course, personalized medicine requires a strict and bidirectional interplay between the basic and clinical research. Thank you very much for your attention. Hello, my name is Sam Dudley. I'm from the University of Minnesota. If I understood your data correctly, inflammation can cause arrhythmogenesis in the absence of structural disease. Is that correct? Yeah. Does the inflammation always have to be in the heart or can it be somewhere else? Yeah, this is a very relevant question. Thank you. Of course, we have also a model of systemic inflammatory diseases. A number of models have been provided also for the COVID-19 pathophysiology, but even some systemic autoimmune diseases, for example, systemic sclerosis and so on, have a specific model for activating interlocking one pathway and also alternative pathway throughout the body, so not only in the heart. The point is that we don't frequently observe the structural heart disease in terms of left ventricular dilation and dysfunction. But if you collect biopsy from this patient, we will find some cell signs of structural diseases, sometimes fibrosis, sometimes enlarged myocytes and edema. So is the mediator of all of this IL-1? Why did you choose anakinra? Yeah, we used anakinra because that was our main target in the clinical setting. We have anakinra readily available for many of our patients with systemic or just organ-specific myocarditis. Of course, there are also alternative targets, for example, tocilizumab for interlocking six. It is more expensive and we have easy availability of anakinra even for the preclinical setting. So it's a good starting point to have a parallel design to compare the clinical and preclinical setting in this scenario. But it is not the only drug, of course. Oh, yeah, hi. You didn't say anything about age, I don't think. So I wondered about chronic sterile inflammation and if you comment on age. If you've looked at the impact of aging or frailty? Yeah, this is a relevant point. We didn't have considered that so far. But starting from the clinical setting, usually inflammatory heart disease are common in young patients, not in the elderly. And even in the so-called hot phase genetic cardiomyopathy, think of hot phases of classic arrhythmogenic cardiomyopathy, it is frequently observed in very, very young patients because the end stage of a disease is just fibrosis without evidence of overt inflammation. So it's, okay, I've got it. Thank you. Welcome. Maybe just one quick last question. You mentioned biopsy, MRI. Can you comment on the utility of cardiac PET in these patients? Yeah, of course. The PET is currently approved only for the diagnosis of sarcoidosis, which is, by the way, one form of inflammatory heart disease. But in our experience, it is a good way to replace MRI in patients with arrhythmic myocarditis, especially in the presence of instable arrhythmias or frequent patterns because it is difficult to have a good ECG gating from MRI, so you have artifacts and interference. And of course, in cardiac divide carriers, because even if you have an ICD, which is permissive for MRI, you have relevant artifacts impairing the evaluation of T1 and T2 mapping. In that setting, PET is a good alternative, but it has good correlates with myocardial inflammation only in truly acute myocarditis, not in the chronic stage, which is not sensitive. All right. Thank you. Thank you. And now I'd like to invite Dr. Sharon George from Pittsburgh. All right, good morning, everyone. I would first like to thank all the organizers for this opportunity to present my research. And next, I would like to thank all of you for being here super early in the morning, all in the name of science. So today, we're going to talk about mechanisms of arrhythmia after cancer chemotherapy. And yes, I'm going to talk about doxorubicin. But even with a model such as this, there's always something new to learn. And we'll talk a little bit more about that at the end of this presentation. So doxorubicin is an anti-cancer chemotherapeutic agent with well-established cardiotoxic effects. Today, we're going to focus on a relatively less known aspect of doxorubicin cardiotoxicity. We're going to look at how it affects cardiac EP. A lot of what we know about dox and cardiac EP is based on case reports, small clinical trials, and very few research that is based on acute studies with very large or high doses of doxorubicin. So here, we're going to discuss more chronic in vivo mouse models and human donor heart data. So watch out for the silhouettes at the top left corner of your screen to look for if the data is from mouse or the human heart models. All right. So dox and cardiac EP, what do we know? We previously showed that doxorubicin alters the ECG in a sex-specific manner. So shown here are ECG parameters recorded from female and male hearts of mice treated with doxorubicin or controlled. So controlled in black, 20 mgs per kilogram dox is in red, and 30 mgs per kilogram is in blue. And when we treated them with doxorubicin, we see significant changes only in female hearts. Particularly, we saw a shortening of P wave duration. And more importantly, we saw increased QT, QTC, and RR intervals in these mice. There were no significant differences observed in males. So to explore these sex differences a little further, we performed triple parametric optical mapping, where we looked at transmembrane potential, intracellular calcium, and metabolic parameters simultaneously from the same field of view. We used a chronic dox model here. So we treated the mice with 5 mgs per kilogram doxorubicin weekly for six weeks. So that brings our cumulative dose up to 30 mgs per kilogram, which is the high dose from the previous slide. And at the end of six weeks, we performed optical mapping. And then we measured several different parameters of cardiac function. I will not go through all of them. If you are interested, I would refer you to our paper on triple parametric mapping and a more recent paper on BioRxiv on quadruple parametric optical mapping for more information. But what I will point out is this right here. So as we saw sex-specific QT prolongation, we also saw sex-specific APD prolongation, which was preferentially observed only in female hearts, not in males. In males, we do see an increase in calcium rise time. However, none of the electrical parameters were altered. So the next question we asked was, is this mouse-specific or does this translate to humans? And do we also see sex-specific effects in humans? To do that, we used a human donor heart model. So these are donor hearts that don't get used in transplantation. We generate organotypic cardiac slices at 400 micron thickness from the left ventricle. We then can culture these slices, which we did for 24 hours with doxorubicin or without doxorubicin for control in this case. And from these slices, we optically mapped them and we measured multiple parameters. We will go over two specific parameters that we measured. First one is APD, just as we did in the mice. We have four different groups here. So you have young and old, male and female. 50 years of age was used as a cutoff for young and old. I'm sorry to anyone I offended so early in the morning, but I assure you it's only for the purpose of ease of explanation. Anyway, so at baseline, there were no significant differences observed in APD between any of the four groups. But when we added doxorubicin, now you start to see differences. So in the male hearts, you have control APD in black and doxorubicin in blue, and there were no significant differences at any of the doses that we tested. When you look at the female hearts, you see a different story. You see APD prolongation, and this was observed as early as five or as low as five micromolar. So this is a dose response that we did for doxorubicin with increasing doses on the x-axis and percent change in APD on the y-axis, percent change from each heart's own control. And we saw APD prolongation in females, not in males. When you look at the older group, now you start to see effects in both males and females. We also looked at conduction velocity in the transverse direction of propagation. And yet again, at baseline, without any doxorubicin, there were no significant differences between the groups. When we added doxorubicin, now we see a dose-dependent decrease in conduction velocity. However, the sex specificity in the younger group was no longer observed. So we see conduction slowing in both males and females, as well as in the older male category. So the next question we asked was, what causes these effects? So Amber Mills, who is a brilliant postdoc in my lab, did some gene expression analysis to look at how different major ion channels in the heart are expressed in the four categories. So the four groups here being young female, old female, young male, and old male. So let's take a look at this one by one. So sodium channels are all in red, which means they were increased after dox treatment. Now this is contradictory to what I just showed you with the conduction velocity data. But keep in mind, these are gene expressions. It may or may not correlate with channel expression, channel function, channel localization, and many other parameters that can contribute to conduction velocity. So we're still working on this. But let's move on to the calcium channels and potassium channels. And let's see if we can find an explanation for what causes a sex-specific APD prolongation that we observe in females. So when you look at calcium channels, now you start seeing differences between the group. They are primarily increased in the first three groups, whereas we see no change or decrease in the old female group. Now potassium channels are even more different between the groups. And let's take a look at this one by one. So in the first group, which is your young females. Now this is a group where we did see APD prolongation. You see that there's a decrease in potassium channel genes that contribute to the early phase of repolarization. And this effect was not observed in any of the other groups, except for old males, where we also did see slight prolongation. So early phase potassium channels reduced in young females, where we saw APD prolongation. In the old female and the young male group, they were pretty much identical. You do see increase in some ion channels that contribute to IKR and IKS, and even IK1. However, these effects were preserved across the groups, across the four different groups. They do not explain the sex differences. And lastly, in the old male group as well, we see reduction in KCND3, which contributes to ITO. So early phase of repolarization seems to be a very important part of Doxorubicin's effects on APD. So to further look at this hypothesis that the early phase of repolarization is important, we went back to the action potential and performed a repolarization analysis. Now this work is very new. It's being done by a new grad student in my lab, Erin O'Neill. What she did was she looked at action potential duration at different phases of repolarization. So we looked at 20, 50, and 80. And what that tells you is, for example, if APD20 is affected, it tells you which currents could be the contributor to that effect on action potential. So what we have here is repolarization at 20, 50, and 80% and action potential durations on the y-axis. We also did this at varying Doxorubicin doses. You see that at the lower doses of Dox, you don't see any changes in action potential, just as we previously saw. At 5 micromolar, now you start seeing effects. And this effect was observed as early as 20% of repolarization. And this APD prolongation that you saw, 20%, was maintained throughout the rest of the action potential at both 5 and 10 micromolar Doxorubicin. So this further confirms the fact that the effect of Doxorubicin is very significant in the early phases of repolarization. And the ion channels that contribute to repolarization at this point may be underlying the observed APD prolongation. So the next question was, what causes these ion channels to be modulated? And the thing with a multifactorial problem like Doxorubicin cardiotoxicity is that it could be numerous factors all working together. But for the sake of time and for synthesis and inflammation session, let's look at some cytokines and chemokines. Doxorubicin did alter several pro- and anti-inflammatory cytokines, but I want to focus on these two which were preferentially suppressed in the young female group which showed the APD prolongation. So the first one is CCL19, which is involved in migration of immune cells. I want to point out TMP1 here, though. So TMP1 is an anti-inflammatory, sometimes pro-inflammatory cytokine. And it's known for modulating the expression of MMPs or matrix metalloproteinases. Now these proteins which are primarily known for their effect on ECM and their degradation has also been very recently been shown to alter the function, expression, and trafficking of ion channels such as the L-type calcium channel and potassium channels. So this is where our research is focused right now on the role of TMP1 in cardiotoxicity and particularly sex differences. So what do we know? We already knew that dox causes cardiotoxicity, it causes electrical remodeling. We confirmed that in our model. What's new? We showed that there are sex-specific differences, particularly when it comes to APD and QT prolongation, which was observed only in female mice. And the APD prolongation was observed in the early phases of action potential. And this correlated with sex-specific modulation of potassium channels that contributed to this early phase. And TMP1 or suppression of TMP1 could play a critical role in the modulation of these ion channel expression and function. With that, I would like to thank all the people involved. This was a very long project, so I would like to particularly acknowledge all the students who contributed to this work. I would like to thank our collaborators and funding sources, and I would be happy to take any questions. So the most common cause of QT prolongation in cardiomyopathy is not ion channel alterations. It's diastolic calcium release from the sarcoplasmic reticulum, usually because of oxidation or phosphorylation of the receptor. So did you look at that at all? So disrupted calcium homeostasis is a well-known effect of doxorubicin, and as I said, it could be several different factors that are contributing to this QT prolongation. In terms of what we looked at with calcium, we looked at... So we performed optical mapping. We looked at calcium transient durations. We looked at rise time. We did not look at... Well, you won't see diastolic calcium release by doing that. Yeah. So we did not look at any of those. And doxorubicin is known to cause oxidative stress, which is known to cause this problem. So second of all, most doxorubicin toxicity is late. It can be years after the problem, and it's either ascribed to topoisomerases or mitochondrial dysfunction or both, or occasionally ion metabolism issues. So how do you think your studies that are acute are relevant to this problem? So you're right. Most of the cardiotoxicity occurs decades later, but there is also acute cardiotoxicity. And our model, particularly with a human heart, is a super acute model. Well, it's 24 hours, so it's still longer in duration compared to what we previously know. With the human data, that's where we are at right now, unless we get tissue from patients who underwent chemotherapy decades earlier. But our mouse model, which is why we are using a more chronic approach, so we're using six weeks of treatment in the mouse model. And I know that's not decades, but in mouse life, that is multiple years. So that's why we do both mouse and human comparison. Sharon, really nice work. Thank you. Did you collect any samples for structural studies? Did you do any histology, particularly looking at perivascular edema? So we have the tissue collected. We have not looked at the structure yet. So yeah, all the tissue is frozen and safely kept. Yeah. Thank you. Great. Thank you. I'll introduce our next speaker, Dr. Jin Lee from Bern, who's going to speak with us about autoantibodies in arrhythmogenesis. So first, I would like to thank the organizing committee for this invitation. It's a great pleasure to speak today about cardiac autoantibodies and arrhythmias. So let's start with a little bit some background. We all know the heart is composed of different cardiac cells. And at the cardiomyocyte level, we have action potentials that result from the activation of different cardiac ion channels. Now, disruption at any level, time point of this perfectly balanced system may prompt automated activities, trigger early or delayed after depolarizations, ultimately leading to cardiac arrhythmias. And usual suspects, we all know them, it's gene variants, structural heart diseases, inflammation, and so on. But how about autoantibodies? Could autoantibodies be a cause of cardiac arrhythmias? Congenital heart block, or neonatal lupus, is the classic example of an autoantibody-induced cardiac arrhythmia. Pregnant individuals with anti-rho-SSA antibodies may pass on those anti-rho-SSA autoantibodies through the placenta to the neonate, and where it inhibits the calcium channel at the AV node, thus causing an AV block. So interestingly, in adults, anti-rho-SSA antibodies have been associated with prolonged QT interval. It's believed to inhibit the HERC channel, and thus causing a prolongation of the cardiac repressation. Now, how about other types of cardiac arrhythmias, such as ventricular fibrillation, or at the atrial level? So to answer that question, we performed a non-antibody screening. We have in select patients. We primarily focused on autoantibodies, IgG. Autoantibodies focus targeting the extracellular sites of cardiac ion channels. And once we have identified a potential target, we then purified them to perform functional studies. With patch cams, we can characterize the electrophysiological effects of those autoantibodies. And then we have experimental animal models to validate our results. And that's how we then close the loop with a potential new autoantibody biomarker, but also a potential new therapeutic target that can be beneficial to the patient. So the story begins what has been more than 10 years ago now. We started with the first autoantibody screening in patients with dilated cardiomyopathy. And in 6% of patients, we detected autoantibodies targeting casein Q1 auto channels. And interestingly is that patients with casein Q1 autoantibodies presented a significant shortening of QT interval. What is casein Q1? It's the voltage-gated potassium channel. It plays a role in cardiomyopatization, as we've heard about just before. The current across the channel is the IKS slow, delayed, rectified potassium current. So next, we purified that casein Q1 autoantibodies. And when true cells stably expressing human IKS channels were exposed to casein Q1 autoantibodies, we could measure a significant increase in IKS current, which matches our clinical observation of QT shortening. Next, we also performed an experimental autoimmune animal model, where New Zealand white rabbits were immunized against casein Q1 channel peptide in order for them to produce circulating casein Q1 autoantibodies, just as in our patients. And we recorded in cardiomyocytes from casein Q1 immunized rabbits a significant shortening of action potential duration. And the rabbits also presented a shorter QT interval. So we had here identified an autoantibody that leads to a gain of function of a channel and shorter QT. Now, how about ventricular fibrillation? Why is that relevant? Well, about 5% to 10% of sudden cardiac arrest remains unexplained despite the workup. And so we screened patients with idiopathic ventricular fibrillation, and we detected autoantibodies targeting CAV1.2. As you all know, CAV1.2 is the voltage-gated calcium channel. The L-type calcium current is the current across the channel. It plays a role in electromechanical coupling and is responsible for the plateau phase of the action potential. So next, we isolated that autoantibody from patient plasma samples, and we incubated IPS-derived ventricular cardiomyocytes with CAV1.2 autoantibodies. And we could show, as shown here, we can see that there is a significant inhibition of the L-type calcium channel, the calcium current. And it's not shown here, but the APD was also shortened, so both are potentially leading to arrhythmias. Now, there is a particular subtype of VF that is called short-coupled VF, where VF is initiated by triggering PVCs with a short coupling interval. And despite many, several research, up until now, there has been no convincing evidence for a disease-causing gene variant, so we were speculating about an autoimmune etiology for short-coupled VF. So we screened patients and detected circulating autoantibodies targeting TRACK1 channels. What is TRACK1? It's a stretch-activated, two-pole domain-portation channel. Interestingly, it's highly expressed in the right ventricle outflow tract and predominates in the cardium. So these are all areas with increased stretch activities. So next, we purified TRACK1 autoantibodies and treated hexa-cell-stably expressing TRACK1 channels, and so we could observe a significant activation of TRACK1 channels, and thus adding another piece to the puzzle as far as adults are concerned. Now, how about infants? We've all heard about sudden infant death syndrome, SIDS, or CURB-DEF. A perplexing diagnosis with no clear underlying biological substrate. So we were wondering whether autoantibodies could be a potential cause of SIDS. So we had samples from SIDS cases, and strikingly, we detected in 85% of SIDS cases TRPV2 autoantibodies. What is TRPV2? It's a stretch-activated calcium permeable non-selective cation channel. Not much is known about TRPV2 channels, but it has been found to play well in the intercalated disc, and we know that it is expressed in cardiomyocytes and fibroblasts. So we not only detected maybe potentially the very first biomarker for SIDS, but we also could demonstrate that it is a functional autoantibody. As you can see here, upon patch-cam studies in HEK cells, TRPV2 autoantibodies induced an inhibition of TRPV2 occurrence. And when iPS-derived cardiomyocytes were co-cultured with iPS-derived cardiac fibroblasts, upon mere recordings, we could observe a significant shortening of EFP, extracellular field potentials. So there is potentially a role of electroctonic coupling between cardiomyocytes and fibroblasts. Next, we needed to validate our results to establish a causal relationship, so we also demonstrated this in an animal model where female BAPC mice were immunized against TRPV2 channels, so they had circulating TRPV2 autoantibodies. They were then mated with healthy BAPC mice, and after an uneventful gestational period, cubs were born and followed up for a month. And as you can see here on the Kaplan-Meier survivor curve, there was a significant increase in postnatal mortality, thus confirming our clinical observation. So now we've talked a lot about the ventricular arrhythmias. How about supraventricular arrhythmias? For example, AF, the most common type of arrhythmia. So because cervical comorbidities can promote AF, we selectively chose a screening of autoantibodies in patients with so-called idiopathic AF, and we compared them with age and sex-matched healthy controls, and we also had a third group of patients with here called pre-AF, meaning that we have the blood samples before actually idiopathic AF was then diagnosed later on. So we performed the autoantibody screening, and we detected out of hundreds of potential targets, only one single autoantibody stands out, it's the autoantibody targeting KF3.4. And why is that such a remarkable finding? Well, KF3.4 is the, it's exclusively expressed in the HRA, it's the Inwardly Rectifying Potassium Childhood Subunit 3.4. In the heart, it forms a heterotetramer with the 3.1 subunit, and underlies the acetylcholinegated KCH channel. So when activated, it's supposed to shorten APD, ERP, and provides the optimal substrate for AF. So we purified KF3.4 20 weeks out of patient samples, and in iPS-derived atrial cardiomyocytes induced a significant increase in KCH. And we also performed an animal study to demonstrate the causal relationship. So we had mice immunized against KF3.4, so it developed circulating KF3.4 autoantibodies. On EKG, they were all presented normal sinus rhythm, but upon in vivo electrophysiological studies, the birth pacing protocol induced 80% of KF3.4 immunized mice atrial fibrillation, compared to only 28% in sham immunized mice. So there was a nearly threefold increased susceptibility to AF, and thus confirming our clinical observation in patients. So here's the, in low view, the summary of all the results up until now. We have different cardiac autoantibodies targeting different cardiac ion channels leading to several types of cardiac arrhythmias. So with this, I would like to thank all my collaborators, and thank you for attention, and I'll be happy to answer any questions. Thank you. of understanding the idiopathic nature of these arrhythmias. Could you explain the possible, whether it's serologic studies of working that up for these sorts of patients, or even possible future directions for treatments for these patients? Could you go into a little bit of depth of what you've seen in your practice and other literature? Yeah, so our studies started with patient screening. So we detected those pro-ephemogenics O2 antibodies. So they can be used not only as a biomarker, prognostic, or diagnostic, as in cases of AFib or SIDS. And then next, of course, there is a therapeutic part where there are potential new therapeutic targets. One possible approach would be immunoabsorption therapy, where we move circulating pro-ephemogenic O2 antibodies. Or we can also neutralize those pathogenic O2 antibodies any kind of way. Thank you. How does the body know which antibody to produce? So we did a microarray, peptide microarray assays, so there were hundreds of different extracellular epitopes we were screening for and then we did analysis to see which one stands out statistically significant in that particular patient population. We always had control samples to match. Thank you. Thank you. And now I'd like to introduce Dr. Budadip Don from the University of Las Vegas. Good morning. I'd like to start by thanking the organizers for the invitation. There is a disclosure which has nothing to do with this talk. So the company is related to neurologic and stem cells. It has no relation with this talk. T-cells are a pretty broad subset of different subsets of cells in the lymphoid system. They come from the bone marrow, go to thymus after that. They develop TCR alpha beta, and then they differentiate into many different subsets of cells, as you may know, Th1, Th2, Treg, and they belong to two broad categories, CD4 and CD8. When they are activated, they are able to secrete a set of cytokines. Those could be pro-inflammatory or anti-inflammatory, and those things can have direct relation with arrhythmogenesis. Not until recently it was known that T-cells are actually present in normal heart, and they participate in immune homeostasis. These are from mouse heart, and as you can see, T-cells constitute about 3% of all the leukocytes in the heart. These are the total non-myocytes in mice heart. T-cells comprise only 0.3% of those cells. Data from humans, there is a difference about the distribution of immune cells. In the atria, it's about 10%, ventricles, 5%. So it is consistent with the fact that T-cells are more implicated in the genesis of atrial arrhythmias. There are eight different subgroups of lymphoid cells in the heart. When it comes to arrhythmias, there are two different types of broad categories. One that promote arrhythmia on the left, one that are sort of anti-arrhythmic on the right, that CD4 positive regulatory T-cells tregs. And it is hypothesized that the balance between pro-inflammatory Th1 and Th17 cells and the treg cells are important for arrhythmogenesis. Only about the last 15 years or so, we have come to know that T-cells can cause arrhythmias, both in the atria and in the ventricle. So why is this balance important between Th1, Th17, and treg cells? It directly relates to what they do. And on the left are all the bad stuff that can be arrhythmogenic with the cytokines, TNF-alpha, IFN-gamma, IL-21, IL-22, recruiting more neutrophils to the heart, also increasing the matrix activating MMPs and activating myofibroblasts that would cause myocardial fibrosis and also electrical heterogeneity. On the right, the treg cells are predominantly pro- they suppress pro-inflammatory activities in the heart, reduce fibrosis, and also they promote tissue repair. If we take an example of a cytokine, why is cytokine, pro-inflammatory ones, bad for arrhythmogenesis? If you take TNF-alpha as a protagonist pro-inflammatory cytokine, it can activate pathways that would lead to fibroblast activation that would lead to increased alpha-smooth muscle actin, also changes in the extracellular matrix, and decreased connexin-43 expression. Overall, this would lead to increased atrial fibrosis, and that would cause arrhythmogenic substrate. Now, if you open this up to a combination of multiple cytokines, multiple cells as well, because T cells do not act in isolation, so they act with macrophages, and that really broadens the factors that are pro-inflammatory and that cause changes in the myocardium that are too numerous, and they act in concert to make different changes in the action potential duration. They slow conduction. They cause electrical conduction heterogeneity. Broadly, they lead to electrical remodeling and structural remodeling that can provide the arrhythmogenic substrate, both in the atrium and in the ventricle. Now, if we add another layer of arrhythmogenesis possibilities to that, then you will see the triggered tachyarrhythmia possibilities through changes in DADs and EADs, and those don't have to be local. There are local factors that can directly impact the ion channels and that can directly impact the QT duration. There are also systemic factors through autonomic nervous system that can impact the neural input into the heart, for example, through stellate ganglion, and there is some emerging evidence that that could be one way that these cells are playing a role in arrhythmogenesis. So one of the first studies came only about 15 years ago where right atrial appendage tissue in patients with atrial fibrillation were shown to contain more CD45-positive cells, which are inflammatory. So this drew attention to the fact that T cells in the atria are associated with atrial fibrillation. Since then, there have been a number of studies that have pointed out the fact that T cells either in the atrial tissue or in the peripheral blood and different subsets of T cells could be CD3-positive cells or CD4-positive CD28-null T cells. They are increased, and they are not only increased, they also have correlation with clinical outcomes. In one study, in fact, there was increased CV mortality in patients with increased number of CD4-positive CD28-null cells. So the outcome data actually indicate that there could be more than association between T cell subsets and atrial fibrillation. In another study, in fact, senescent CD8-positive T cells were shown to not only correlate with the predicted AF prevalence and also AF recurrence after ablation. These data are actually quite striking. The senescent T cells were identified by either an increased expression of CD57 or a decreased expression of CD27 or CD28. Now when a T cell loses CD28, the cell becomes resistant to apoptosis and it becomes more pro-inflammatory. So that could have potentially, but these are associations still, could have potentially a relation with AF prevalence and recurrence. And the authors concluded that these T cells could be a potential biomarker for atrial fibrillation risk. There was a larger study analysis of data from two studies, CHS and MESA, a total of 3,700 patients with blood samples that were collected between 1998 and 2002 and analyzed around 2016 to 2018. And follow-up duration was 15 years, mean of 8 years. So it was expected that the T cell, different subgroups of T cells, will correlate with the incidence of atrial fibrillation during the follow-up. However, the pre-specified cutoff P values were not met. From this right side, you may think these are significant numbers. However, for the secondary analysis to be positive, the P value needed to be less than .0015, which they did not meet. There was a suggestion there was a trend, but no specific T cell subset was identified to be associated with atrial fibrillation incidence. Moving on to ventricular arrhythmias, there are many basic science studies which show the cytokines, including TNF, TH17, that can cause ventricular arrhythmia in the setting of myocardial infarction, where T cells do move to the myocardium. Now, this one was from stellate ganglion, left stellate ganglion, in patients who had resistant either PCVT or long QT syndrome. The stellate ganglion in patients with CPVT on the left, one patient, the HNE stain in the top panels showed increased leukocyte infiltration. At the bottom, they were positive for CD3. The middle one is from long QT syndrome patient, and this is control. That indicates that T cells are in the myocardium, in the stellate ganglion, in close proximity to stellate ganglion cells, which may potentially induce ganglionitis, causing increased autonomic disturbance in the heart that may lead to arrhythmias. It's a basic science study which looked at the ability of interleukin-17 to induce ventricular arrhythmias by activating MAP kinase pathways. So there is a clear relation with pro-inflammatory cytokines, such as TNF, IL-6, interleukin-17, to be able to induce and perpetuate ventricular arrhythmias. I believe this is the final slide. Many of you know immune checkpoint inhibitors are associated with myocarditis, sometimes fatal in many cases. This has increased in number in the last decade or so. They block PD-1 and CTLA-4, and that can promote arrhythmia in itself. In these two, the same person had heart block and also sustained ventricular tachycardia, and there were increased number of CD3-positive cells and CD8-positive cells in the myocardium. So myocardial infiltration by T cells plays a role in this type of myocarditis, and T cell-mediated direct cytotoxicity is a possible mechanism for this. So in conclusion, many different types of T cell populations are present in the myocardium, both in health and disease, and their mechanisms of how they cause arrhythmia are being investigated at this time. Clinical studies also suggest that T cell subpopulations, different subpopulations such as CD4-positive, CD28-null, they have a distinct role in the pathogenesis of different types of arrhythmias. T cells do contribute to the pathogenesis of atrial fibrillation by multiple mechanisms. It's not just one thing that leads to multiple different changes in atrial tissue that can cause to a fib. T cells also play an important role in ventricular arrhythmia. I did not touch upon the acute MI setting where they play a role as well, and they also are critical in arrhythmogenesis in myocarditis. I'll stop here. Thank you for your attention. APPLAUSE Thank you for a nice talk. T cells are part of the adaptive immune response. Correct. So what is the antigen? Or is it not required that T cells actually go to the heart? Because T cells are extremely rare in the heart, even in inflammatory conditions. Except, of course, maybe checkpoint inhibitors. So why do T cells go to the heart? There would have to be a cardiac antigen, right? Because otherwise you're not going there. So what is the antigen? Those receptors could be CXCR4 that can play a role in this, because even in subclinical cardiac injury, there could be expression of SDF1 and or CXCR4. We believe that's the primary reason for T cell homing into the heart. But the precise reason why they go there is unknown. Sometimes it is determined by the systemic factors. So that suggests that the antigen-presenting cells are calling them there, which would be the macrophages, which are well known to cause it, right? That's a possibility, yes. Hi there. I'm Nikki Posnack from Children's National. So I have kind of a basic, naive question. Do you have a sense of the timing for T cell infiltration and these pro-inflammatory responses? And I'm thinking more about kind of acute stress or post-operative inflammation and how some of this may play a role in post-operative arrhythmias, et cetera. There has been clear correlation between T cell infiltration and post-operative arterial fibrillation. But the time course when they disappear, because these are from human tissues, the time course studies are not possible to do. Thank you, Dr. Dhan. And with that, we'll conclude the session. I'd like to thank our speakers for fantastic talks and the audience for their attention. Enjoy the rest of the conference.

inflammation

arrhythmogenesis

myocardial

cardiac electrophysiology

autoantibodies

T-cells

arrhythmias

immune mechanisms

therapeutic targets

cytokine modulation