All right, good morning, everybody. All right, thank you. Good morning, everybody. It's my pleasure to welcome you to San Diego and Heart Rhythm 2025. This is the 46th annual meeting of the Heart Rhythm Society. If you have not already done so, you can download the app. However, if you ask questions through the app, it will not come through in any known way. So, download the app because that's what they told me to tell you to do. Otherwise, if you have questions, can the speaker please repeat the question before answering it? Also, speakers, at the end of the session, can you please come up, scan a QR code and take a very brief survey? And with that introduction, you can hear me okay? Okay. I think one of the red buttons is up and one is down. All right, while we're working with AV technicalities, I would like to introduce our very first speaker, Dr. Yazaki, who will be talking about the restoration of ventricular electrical synchrony via CBIN1 gene therapy in a canine model of ischemic cardiomyopathy. Thank you for your questions. I came from University of Utah, so today I'll talk about the restoration of the ventricular electrical synchrony via CBIN1 gene therapy in a canine ischemic heart failure model. As most of you know, this heart failure prevalence is steadily increasing over time, and as of now, over 6 million American people suffer from heart failure. And the therapeutic option for the heart failure has been changed. And in the 1990s, a standard medication for the heart failure has been developed, including ACE inhibitors, ARVs, beta blockades, and MRIs. And in the 2000s, cardiac implantable devices have been developed for the advanced heart failure, and including cardiac resynchronization therapy. And in the very recent three, there's a new drugs, and new medications has been demonstrated to be effective for the heart failure patients, including ARNI and SJRT2 inhibitors. But the problem is, heart disease remains a leading cause of death in the U.S. after 2021 and 2022, and we may need another option for the heart failure treatment. Gene therapy could be the one for the treatment. And before jumping into the gene therapy, let me talk a little bit about the t-tubular structures networks in the cardiomyocyte. This is a schematic image of the cardiomyocyte, and as you can see, the t-tubular is a specialized structure to the skeletal muscle and heart muscle. Those extensions penetrate to the membrane and they're creating a broad network in the cardiomyocyte. And then, in a healthy subject, as you can see the left side panel, t-tubules are well-organized, as you can see in the comforter imaging image. And looking at the left side, like cartoons, these like banana-shaped protein, and so cardiac bridging integrator, one so-called CB1, and create a dyad between the L-type calcium channel and the Y-anogen receptor, which facilitates the calcium handling machinery, leading to efficient excitation-contraction coupling. But once heart failure developed, the t-tubular structure get disorganized, as you can see in the left panel comforter imaging, and the CB1 level get decreased, leading to inefficient EC coupling. So to investigate the level of the CB1 in maintaining a t-tubular structure, we developed a heterozygous and a homozygous CB1 non-I-mass model. And as you can see in the top panel, in the Y-type mass, had a well-folded membrane in the t-tubule, but in a heterozygous or homozygous and a non-I-mass model, which developed a heart failure, and then lose micro force, and the t-tubule got round-shaped. And so, you can see that CB1 has a crucial role in maintaining a t-tubular structure. So we started investigating the efficacy of the CB1 exogenous gene therapy, and using a transverse aortic constriction mouse model, and a pacing-induced cardiomyopathy porcine model, which are like a non-ischemic heart failure model. And we demonstrate that CB1 gene therapy improves LVF and survival rate in those models. And very recently, we also demonstrated this efficacy in using ischemic heart failure canines as well. And this is a timeline of our study. And the first, we ligated the LAD, creating a broad anterior infarction in the left ventricle. And 10 to 16 weeks after the ligation, we injected either the CB1 or GFP as a control, and into the mouse, directly into the mouse caudium. And eight to, five to 80 weeks after the injection, we had a terminal study. And as a consequence, CB1 gene therapy groups, LVF got increased by 5%, but while the GFP group, LVF got worsened by 8%. So the CB1 gene therapy could improve the ventricular remodeling. And another thing we may be concerned with is like LV synchrony. And there are LV synchrony mainly divided into electrical and mechanical one. And the electrical one was represented as an acute situation, and in case these, and the mechanical one is presented as a peak strain difference on ultrasound. But once heart failure progressed, and the LV synchrony is supposed to be worsened. So our hypothesis is like, of this study, is a CB1 gene therapy could improve the LV, electrical and mechanical synchrony in ischemic heart failure canines model. So this is our study timelines. And 30 canines were included, and all LV were ligated. And after creating our heart failure model, eight to 16 weeks after the ligation, we injected either GFP or CB1. And the five to 13 weeks after the injection, we had a terminal study. And at the two time points on the injection of the terminal points, we got every total activation time, every TAT, and the cure situation, co-active QT interval on EKG, and the peak strain time difference on the strain echocardiography. So every TAT was acquired with the NOGA-XP system, which is an electronical mapping system, which allow us to see the LV geometry, as well as on the activation and the voltage map. We got over the 100 points for each animals, and we calculated every TAT by means of the time difference between the earliest and the latest activation. And as you can see on the light graph, in the CB1 group, LV TAT are significantly reduced as compared to the GFP group. And also the cure situation showed a significant decrease in the CB1 gene therapy group as compared to the GFP group. But unfortunately, the co-active QT interval didn't show a significant improvement in the CB1 group as compared to the GFP. And regarding the mechanical synchrony, and the peak strain time differences also show the significant shortening in the CB1 gene therapy group in both radial strain and the longitudinal strains, as you can see in this light side graph. So the possible mechanism for the LV synchronization after the gene therapy will be the wider fibrosis burden. In the previous study, we demonstrated the fibrosis burden was significantly decreased in the CB1 group. And this decreases and contributed to the smoother propagation of the electrocortical impulses. And another reason would be LV endodiastric volume. And also in the previous study, we demonstrated the CB1 groups showed a significant reduction in the LVEDV. And that goes to compact average geometry and less world stress, and creating a uniform depolarization and contraction. So in conclusion, CB1 gene therapy shortens every varic ventricular total occupational time and cure situation, as well as a reduced peak strain time difference on autism. And I appreciate a lot of contribution from the collaborators, like this study is funded in a lot of foundation. Thank you for your attention. Okay, we have time for a few questions. I can hear without my headphones. Does anybody have any questions? In this case, we didn't look at the sodium channel or sodium chitosan exchangers, yeah, so unfortunately we didn't look at that. There is another question. Yes. What do you know about the molecular signaling that's driving these changes? Driving exchanger. No, the molecular signaling, the molecular pathway. Molecular pathway, so like, yes, a CB1 gene therapy, oh no, in my thought like a CB1 gene therapy could promote that, sorry, can you break down your question, like, sorry. CB1 is sucking up the T2 building, it's making them tighter, it's getting things synchronized, but then how does that become the end result that you're looking for? Okay, so yeah, we're we didn't like suggest like a direct pathway from the CB1 to these every synchronization and so like it that's why it's like we propose like that's like a possible mechanism like those every synchronization and the one is a regular fibrosis and mediated by the fibrosis and another the other one it will be like every EDV decrease so like we didn't like a proposal those like a direct relationship between the CB1 to the such a sodium channel or calcium exchanger or like a gap junction or any kind of sauce so yeah as of now we didn't have any like data like demonstrating don't like direct the relationship between the CB1 and those like consequences okay thank you very much our next speaker is dr. Liu who will be talking about unlocking sodium glucose co-transporter to inhibitors potential reducing arrhythmias and ion channel remodeling and cardio renal syndrome Okay, thank you for the introduction. My name is Xinhui Liu, and I'm from Taipei Veterans General Hospital in Taiwan. And today, it is my honor here today to represent my topic on unlocking SGLT2 inhibitors potential and reducing arrhythmias and ion channel remodeling in cardio renal syndrome. And I have no disclosures. And to begin with, we're going to discuss a little bit about cardio renal syndrome. And as we know that the chronic kidney disease, which is the CKD, will exhibit an elevated risk to develop CV diseases such as CAD, heart failure, or arrhythmias. And on the other hand, heart failure contributes to congestion, hyperpigmentation, declining kidney function, and promoting the CKD. So there is this vicious cycle between the heart and the kidney, which causes fatal risk factors, including terminal arrhythmias. So what exactly are terminal arrhythmias? As you can see from this figure on the left side, there are different etiologies that may lead to terminal arrhythmias in cardio renal syndrome. And patients are usually at high risks of terminal arrhythmias with cardiac etiology such as tachyarrhythmias, bradyarrhythmias, MI, and heart failure. However, in CRS, they include longstanding pathological anomalies that may increase arrhythmias, including these fatal conditions such as systole, ventricular arrhythmias, and sudden cardiac death. So how do we reduce the risk in CRS patients? So there are various pharmaceuticals methods, which include managing risk factors such as blood pressure control, glucose control, lipid profile control, and also it is proven that with ACEI and ARB, it has improved the CV outcomes and also reduced kidney events. And as for the SGLT2 inhibitors, it's also proven with improved CV outcomes and decreased CKD progression. And finally, there are MRAs such as fenurenone that also have improved CV outcomes and also reduced the CKD progression. And as to all of these various clinical trials for SGLT2 inhibitors, we know that they previously focus on DM and heart failure and CKDs. And these clinical trials, they demonstrate risk reductions, including improved LEVF, and then reduced mortality and hospitalization for heart failure, and also reduced CKD progression. But however, the population for post-MI, half-path, and arrhythmias, they're still limited, and these trials, some are just very new. So in one of the trials, the DAPA-AF trial, which was conducted recently, and they included 4,744 patients with HF-REF, and they applied the DAPA-Glyphosine for these patients. And most importantly, as we already know, they decreased the risk of worsening heart failure and death, and also improved symptoms in patients with and without AF. But most importantly is that the DAPA-Glyphosine, it did not reduce the risk of new onset AFs. So this raised the question of whether the SGLT2 inhibitor is actually beneficial for these patients. So in terms of the mechanism, so how does the SGLT2 inhibitor reduce risks of AF? So you can break down into three different aspects, and one is the metabolic aspects, which we already know, which could reduce blood sugar, BP, and body weight, and it reduces epicardio-adipose tissue, and reduce the pro-inflammatory factors and inhibit fibrosis. As for the hemodynamic aspects, it improves the LV diastolic function and reduces LV filling pressure and hemodynamic stress, which may reduce AF triggers or ventricular arrhythmia triggers. And finally is the off-target effects on the cardiomyocytes. The SGLT2 inhibitors have proven that they may regulate the cardiomyocytes' ion channels and potentially prevent the onset of arrhythmias. So this is a figure of the off-target effects of SGLT2 for arrhythmias, and this is the mechanism for the cardiac ion channels among the arrhythmias. And it is proven that SGLT2 inhibitors, it affects the sodium-hydrogen exchanger, which could impact the sodium-calcium exchanger here, as you can see. And furthermore, it also increases the expression of the SERCA protein ion channels, which improves contractility. And there are also other trials or investigations that investigated thopaglifosin, which also reduced the vulture-dependent L-type calcium channels and also reduced the sodium-calcium exchanger. And finally, the empaglifosin was also proven with improved CKA expression. And so in our study, we noticed that previous study, they just emphasized on the benefits of SGLT2 in heart failure or CKD alone and with limited investigations on the cardiac EP properties. So we aimed to examine the cardiac EP properties and cardiac reverse modeling of SGLT2 inhibitors and CKD rabbits with heart failure. So as for our methods, we included 18 New Zealand white rabbits, and we randomized it into three different groups, the SHAM, CKD group, and the CKD-DAPA group. And all of them underwent baseline blood tests, ECG, and echocardiogram. As for the CKD model, we chose the FIT6 nephrectomy as our CKD model. And after four weeks, we used a blood test to confirm a CKD. And in the CKD-DAPA group, they were given oral thopaglifosin for four weeks. And then finally, echocardiogram was confirmed for heart failure. And finally, all these groups underwent another final blood test, ECG, echocardiogram, cardiac EP study, and also the immunochemistry stain in western blot analysis. And as for the experimental procedure, as mentioned before, all the baseline datas, and also we included the histology, which we tried to identify collagen fiber through Mason's trichostain, sympathetic neurons through the tyrosine hydroxylase stain, and also the western blot analysis, where we try to identify these calcium voltage-gated L-type channel, the SERCA expression, the rhenodine receptors, and also the sodium calcium exchanger. And this is just one of the examples of the cardiac EP study, where we tried to induce the ventricular arrhythmias. And as a result, for the baseline characteristics, among the three groups, the CKD and the CKD-DAPA group were confirmed with renal failure, and in the CKD-DAPA group were also confirmed with a decreased LVEF. And as for the cardiac EP study, we tried to pace at two times and ten times pacing threshold at all the cardiac chambers, and we found that the ERP of all cardiac chambers in the CKD-DAPA group were significantly shorter than those in the CKD group, respectively. And as for the arrhythmic inducibility test, the atrial and the ventricular arrhythmia inducibility test in the CKD-DAPA group were significantly shorter than those in the CKD group, respectively. As for the examples of inducible—these are the examples of inducible arrhythmias, including AF and ventricular arrhythmias in the CKD group. And next is the cardiac collagen fiber, where we used the Mason's trichostame. And as you can see from the blue areas, these are the collagen deposition in the myocardium, and we tried to measure the areas where the CKD group had a significantly larger area of collagen fiber than the SHAM and the CKD-DAPA group. And next is the cardiac sympathetic neurons, where we used the tyroxine hydroxylase stain, and we also measured the areas of the sympathetic innervation, and we found that the CKD group had a significantly larger area of sympathetic neurons than the SHAM and the CKD-DAPA group. And then finally is the cardiac ion channels, where we try to identify all of these cardiac ion channels, and we found that in the CKD-DAPA group, in all of the cardiac chambers had a significantly decreased the L-type calcium channels, as you can see from the figure here, the expressions were decreased and compared with those in the CKD group. And as for the SIRCA expression, the CKD-DAPA group also had an increased expression and compared with those in the CKD group. So as a result, in our conclusion, is that we found that the CRS harbors arythmogenic myocardial substrate with cardiac remodeling, and the CRS rabbits demonstrated increased collagen deposition and sympathetic innervation, and also the SGLT2 inhibitors reversed the cardiac remodeling, reduced inducible arrhythmias and improved cardiac EP properties in CRS rabbits, suggesting a potential to lower arrhythmia risk in CRS patients. So thank you for your attention. So, have you looked, to tie the first and second talk together, have you looked to see if, for example, protein clustering precedes the retention of all the proteins you looked at, all the calcium handling proteins, or have you looked at any of the structure, or just the total expression of your proteins? Just the total expression of the proteins, so we haven't looked that deep into the clustering. Okay. Thank you. Yeah. All right. Any other questions? Great. Thank you very much. All right. Our next speaker is Dr. Chelko, who will be talking about the therapeutic effects of anti-CD14 antibody and arrhythmogenic cardiomyopathy. Thank you for, HRS, y'all can all hear me, I don't have my, for the opportunity to disseminate this work. Let's get started. The relevant disclosures for today, it's just a material transfer agreement with Implicit Bioscience. So, arrhythmogenic cardiomyopathy, the prevalence is rather low, although it is often misdiagnosed as myocarditis. Clinical characteristics are ventricular dysfunction, wall motion abnormalities, ECG repolarization, depolarization abnormalities, and pathological characteristics, cell death, fibromyalgia hallmark characteristics is myocardial inflammation. Our work and others have demonstrated that desmodermal disruption, beta-catenin suppression, and GSKB activation contributes to ACM pathogenesis. In 2019, our lab showed NF-kappa-B mediated transcription drives both intrinsic and extrinsic mechanisms of myocardial inflammation. I'll probably be using those words quite a bit intrinsic. What I mean is that the cardiomyocytes themselves, and then extrinsic is infiltrating immune cells. And then in a follow-up study, we reported last year that persistent NF-kappa-B activation in cardiomyocytes is driving ventricular arrhythmias in a mouse model of ACM, and that inhibition prevents disease progression. Yet, as you can all imagine, prolonged NF-kappa-B inhibition will most likely not muster a clinical trial given that it will most likely suppress the innate immune response. So therefore, we decided to look upstream of NF-kappa-B. So in 2016, under mentorship of Dan Judge, I generated a knock-in desmoglan-2 mouse model of ACM, which robustly recapitulates disease features seen in patients as early as eight weeks of age. These initial studies, I've been investigating NF-kappa-B mediated transcription of inflammatory cytokines and chemokines, as well as the immune cell populations that are contributing to myocardial inflammation and their temporal invasion. We found that CCR2-positive macrophages are an early and persistent driver of myocardial inflammation. We additionally uncovered a key inflammatory molecule, CD14, that was upregulated in DST mutant mice, as well as two different IPS cardiomyocyte lines harboring two of the desmosomal mutations linked to ARBD. In the setting of sterile immune activation, CD14 is activated by DANTs that are released from dying or dead cells, triggering immune activation. So using our previously generated RNA-seq panel, we assessed both the differential expression of CD14 and the genes associated with downstream CD14 signaling. The majority of these genes were increased in DST2 mutant mice compared to wild-type controls, where macrophages, monocytes, and neutrophils were the predominant immune cells with the largest CD14 activation signature. On the right are the UMAPs displaying myeloid cells that were sub-clustered by the major genes expressed by these immune cells. For macrophage 1, as you can see, both CD14 expression, as well as CXCL2, which is a secreted chemokine for monocytes, macrophages, and neutrophils. And for our monocyte 2 population, particularly CCL2, which is the chemokine ligand for CCR2. Given our results in mice, we performed RNA-seq on left ventricular specimens from donor controls in patients with ACM, and we found that the CD14 expression to be the highest in myeloid cells, which additionally corresponded with higher levels of N-kappa-B predicted by Dorothy Green regulatory network. So collectively, given our prior works, the timeline of disease phenotypes in our mice, as well as the infiltrating immune cells in DST2 mutant mice, along with our current findings presented here, supports the possibility of CD14 as a therapeutic in ACM. We divided, we had four groups, wild-type controls and DST2 mutant mice that received either isotype control, which is mouse anti-IDG, or mouse anti-CD14. We performed ECHOs, 8-lead ECGs, before and after treatment, and in a separate set of mice, we also did PET imaging for CCR2-positive cells. So prior to study initiation, there was no significant difference in cardiac function between cohorts that were noted. However, at study endpoint, we observed recovery in both left and right ventricular function in CD14-treated DST2 mutant mice. This was additionally observed in decrease in ventricular ectopy in mice that were treated with anti-CD14 antibody, which was accompanied by reduced depolarization and repolarization abnormalities, which can be seen in the signal average ECGs. During functional assessment, hearts were extracted, and downstream proteomic transcriptomic and immunohistochemistry analyses were performed. We showed a substantial or almost near-complete loss of myocardial fibrosis in anti-CD14-treated mutant mice. So to further elucidate the impact of anti-CD14 on myeloid populations, we performed single cell RNA sequencing and sorted immune cell populations using our previously generated library. Subclustering revealed 14 distinct myeloid populations, and to determine which populations actually are relevant to human disease in ACM patients, our human ACM signature was overlaid onto our mouse UMAP space, and which showed a substantial overlay of macrophages one and monocytes one and two populations. So these cell composition plots were constructed from our global UMAP myeloid populations and revealed that a collective proportion of these three myeloid populations were reduced in CD14-treated DST2 mutant mice. Heat maps and top reactome pathways were generated from the top 25 differentially expressed myeloid genes, and we observed a variety of genes that were downregulated in CD14-treated mice. However, what I found the most interesting were the genes that were actually upregulated following anti-CD14 treatment, particularly APOE, which is known to polarize macrophages from a pro-inflammatory to a pro-reparative phenotype, and RGAP24, which is a known deactivator of NFkappaB. So these results further suggest the anti-inflammatory effect of anti-CD14 antibody treatment. Additionally, we performed multiplex cytokine arrays and found that anti-CD14 treatment substantially decreased myocardial levels of several primordial inflammatory cytokines such as NL1β, C-reactive protein complement component 5, and potent neutrophil chemokines such as CXCL2 and 19, and fibroadipokines such as osteopotent and periostin. We also used an IPS cardiomyocyte culture system harboring a pathogenic PKB2 variant, and we transfected these cells with an NFkappaB luciferase reporter. Cells were then cultured with either the NFkappaB inhibitor that we used in 2019, Bay 11, or with anti-CD14. I'd first like to note that without any exogenous stimuli, PKB2 cells are already demonstrating significant rise in NFkappaB luciferase. The addition of exogenous TNF-alpha, interferon-gamma, and olucone-beta both drove NFkappaB luciferase activity in isogenic controls, as well as PKB2 cardiomyocytes. As anticipated, NFkappaB inhibition substantially reduced NFkappaB luciferase activity, and lastly, reduced NFkappaB was observed in a dose-dependent manner in isogenic and PKB2 cardiomyocytes. So, our work implicated infiltrating CCR2 macrophages in both ACM mice and patients' left ventricular myocardial specimens from patients, suggesting that decreasing CCR2-positive macrophages in their abundance may reduce the disease severity. So therefore, a separate cohort of mice, we assessed the impact of CD4 inhibition on CCR-positive macrophages in the hearts, and as you can see, there was a significant reduction in mice treated with anti-CD14. So in summary, CD14 is a biomarker of inflammatory diseases and has been implicated in numerous cardiovascular diseases. However, its role in arrhythmogenic cardiomyopathy has been underexplored. Here we demonstrate that CD14 is activated in ACM, and its inhibition is both cardioprotective, such as reducing myocardial inflammation, fibrosis, ventriculactomy, and cardiac function. Additionally, anti-CD14 causes a favorable shift in the cellular and transcriptomic landscapes of ACM, favoring a more pro-reparative or pro-resolving phenotype. And it's also sufficient to attenuate disease pathogenesis in an animal model of ACM. Our findings indicate targeting CD14 reduces CCR2-positive macrophages, both their activation and infiltration. It prevents myocardial inflammation, fibrotic remodeling, and thus preserving cardiac function. I'd like to thank my collaborators, Dr. Staffitz and Levine at Harvard and Wash U, respectively, for their expertise and their experiments performed in their respective labs. I'd like to thank all of our funding organizations and also Implicit Bioscience for their invaluable contribution and their protocol design. Lastly, I'd like to thank my lab members for their continuously hard work and their courage to return to campus after the tragic and vile cowardice act that occurred on campus last week. Thank you for your time. Questions? Not I have one. Yeah, go ahead. Just... No, no, I'm the next one. All right, I have a quick question then for you. What's special about weeks one through four? Is CD low in weeks one through four or does this somehow have to do with some other work in ACM of it takes a while for gap junctions to remodel within the body and... So you want the real answer or... I'll take whatever you're willing to share with us. FSU is notorious for the very hard acook in trying to get mice treated at three weeks of age, which is what I wanted to... Sorry, when we weaned them from the mice from their dams, but I'm working on that as well. I want to treat earlier, but I also want to do what we call our progression protocol starting at 16 weeks and going to 24. That way it's similar to like a patient that can't anti-CD4 treatment, keep a patient alive long enough to get a heart transplant. If the disease is so full, you know, is this can at least delay further progression. Is there any downside from this therapy or could it be like a longevity enhancement? Currently, I don't know. I mean, we saved all of our livers. I'm sure that's probably going to be a reviewer's comment regarding the toxicity, but currently no mice were like affected. They were all, you know, running around the cage all the time. It's just once a week treatment, but they seem to tolerate it very, very well. Yeah, I'm just highlighting it. I can't really talk much about it, but all right. Thank you very much. All right, thank you. Okay, our next speaker is Dr. Venkatesan on immunomodulatory effects of transcutaneous vagus nerve stimulation and heart failure with preserved ejection fraction. Hello, I'm Tamil Indian Venkatesan. I'm from Mokulagamma University of Health Science Center. Do you hear me? Okay. Thanks for the organization for accepting our abstract to present here. Here, I'm going to talk about how vagal nerve stimulation modulates immune response and improves heart function in heart failure with preserved edema fractured condition. Many population studies and registries worldwide have suggested that approximately 35 to 70 percent of heart failure cases have heart failure with preserved edema fraction. Developing an effective therapy for FF is remain challenging due to the limited understanding of its pathophysiology and disease heterogeneity. However, many numerous and experimental clinical studies have shown that systemic inflammation play a central role in FF progression. This schematic diagram highlights how systemic inflammation contributes to cardiac stiffness and diastolic dysfunction. So, the cholinergic anti-inflammatory pathway is a mechanism of nervous system, specifically vagus nerve to regulate immune system and controls inflammation. Upon VNS stimulation, the released acetylcholine interact with immune cells, mainly macrophages and suppress pro-inflammatory cytokine production. Historically, vagal nerve stimulation was performed using surgically implanted electrical devices. However, vagal nerve stimulation can also achieve by non-invasive ways. In our study, we targeted the tragus region of the air to stimulate auricular branch of the vagus nerve. Since FF is associated with systemic inflammation and VNS exerts anti-inflammatory activity, we hypothesize that chronic intermittent VNS may suppress inflammation and reverse diastolic dysfunction in outfielder erection fraction. So, in our study, we utilized a mouse model of FF. To induce FF, we fed the mice with IFAD and LNA for five weeks, and then we performed ECG and echocardiography to confirm the FF phenotype, and after five weeks, we randomized the mice into sham and VNS treatment. VNS was treated for four weeks, daily for 20 minutes in anesthetized mice. We used isofluron 2% to anesthetize the mice. For sham treatment, we just put the electrical clip to the same region, tragus region, but we didn't stimulate under anesthetized condition. What we noticed here, FF mice showed increased systolic blood pressure, increased E by E prime, which is an indicator of diastolic dysfunction, and also we noticed that FF mice showed increased heart rate due to hypertrophy and increased lung weight is due to pulmonary congestion, and also we noticed that FF mice showed increased fibrosis in left ventricular area. However, VNS treatment is reverse all these FF-associated phenotypes. We also noticed that there was no significant difference in adjacent fraction among all the three groups. To determine whether VNS induces immune modulation in our tissues, we mainly assess the cardiac resistant macrophages by flow cytometry. Here, what we noticed here, FF mice showed increased number of CCR2 macrophages in FF condition. However, VNS treatment markedly decreased this CCR2 macrophage numbers in our tissues. So, to understand where these cells come from, we performed lineage tracing study to specifically label the CCR2 macrophages. We crossed MS4A3 CRE. This CRE is specific for granulocyte, monocyte, progenitor cells, and TD tomato red for tracing the cells. These mice crossed with CCR2 knockout mice. The resulting heterozygous mice produces CCR2 cells positive for tomato red, this enabling us tracing the CCR2 positive cells. So, in confocal imaging, we noticed that there is an increased number of TD tomato positive red cells. In flow cytometry analysis of TD tomato positive cells, we noticed that there is an increased number of CCR2 positive macrophages in FF heart tissues. But, no notable difference in TLF positive and MS3 positive between the groups. We also noticed that when we knock out CCR2, it improves heart function in FF mouse model. It is evident from decreased E by E prime compared to the wild type, and also decreased heart rate, lung rate, and also decreased fibrosis in left reticular area. CCR2 knockout also reduced the expression of inflammatory marker genes, such as TNF-alpha, IL-6, IL-1-beta, SPP-1. It also reduced the expression of fibrotic genes like TGF-beta-1, collagen-3A1, 4A1, actol-3, and post-strain. We next performed single-cell RNA sequencing to analyze the target gene expression. Here, we isolated cells from heart tissue, and immune cells are sorted out based on the CD45. And then we analyzed, we performed cluster analysis. In the cluster analysis, we noticed that there were 12 different immune cell populations, among which cardiac-resistant macrophages were further sub-clustered into four different unique subsets. In consistent with previous flow cytometry results, here also we noticed that CCR2 macrophage numbers are significantly increased in FF heart tissues, but this was a small decrease by VNS treatment. Interestingly, what we noticed here from single-cell analysis, TVNS treatment significantly increased the insulin-like growth factor 1 in heart tissues, mainly in TLF and MSC-positive macrophage phenotypes. So to investigate the role of IGF-1 in TBNS-induced cardioprotective effect, we knock out the IGF-1 macrophage specifically, and in this mice, we noticed that knockout of IGF-1 macrophage specifically reversed the TBNS-induced cardioprotective effect, evidenced from increased E by E prime, heart rate, lung rate, as well as fibrosis. IGF-1 knockout also reversed the expression of inflammatory marker genes as well as fibrotic marker genes. So to summarize our study, FF is associated with increased accumulation of CCR2 macrophages in the heart tissues. Genetic knockout of CCR2 knockout improves cardiac function in FF mouse model. VNS treatment has showed increased expression of IGF-1 in TLF and MSC-positive macrophage phenotypes. However, this macrophage-specific deletion reversed the VNS effect in FF mouse model. First of all, I would like to thank my boss, Dr. Stavrakis, for giving an opportunity to work on this wonderful project. I also thank my colleagues and collaborators who were involved in our study. Finally, I thank the funding agency, NIH, American Heart Association, and Oklahoma Translation Research. Thank you all. So, the question is, the question, do you want to repeat in the, can I repeat your question? How do you know that it's due to vagus stimulation versus the restriction of the animal effectively? We use ECG, we use EGC. We have a sham, we have a sham group where we put the electrodes on, but we don't apply. We didn't stimulate, we, yeah. So, it's not the restriction. So. Yeah. So, here. There is an impulse difference in ECG. You see that we're slowing the heart rate. Slowing, yes. You see here? You see the RR interval is increasing, so we're slowing the heart rate. We're engaging heart rate now. Yes, stand it on, please. Okay, just so everybody else can follow along. They're asking, how do you know it's working? One of the responses is that there's a sham group, so they have been restricted. The second response is that you can see the vagus stimulation is slowing the RR interval. Other questions? Thank you all. All right, thank you very much. Please find your favorite speaker and have them fill out the forms on how this session goes. It's really important. This informs how Heart Rhythm will conduct these types of abstract presentations henceforth. All right. Thank you very much. Our last presentation is from Dr. Boink on AAV-HCN4T mediated biological pacing as a potential life-saving therapy for congenital complete heart block. Enjoy. Thank you. Let's see. I'd like to start by thanking the organizers for selecting this abstract for an oral presentation. I'm excited to share with you our progress on HCN4 gene therapy and related strategies in the domain of biological pacing where we aim now to develop a novel therapy for congenital complete heart block. Why is this important? Well, congenital complete heart block is actually the most severe cardiac arrhythmia in utero and in neonates. It's a rare disease that occurs in 1 in 15,000 to 1 in 20,000 live birds, but it is a disease with a severe phenotype that can progress into heart failure, multiple organ failure. What you can see here on the ultrasound, I don't know if you can see my pointer. Now you can, right? You cannot. Oh, sorry. Yeah, yeah. There we go. And now it works, right? So you see here an ultrasound of a newborn with H. fetalis. And so this is really a severe condition that has a mortality of 20%. So it would be great if we can develop something that can actually save these patients because currently the treatment is centered around immunomodulation, so with corticosteroids because this condition is typically caused by maternal autoantibodies. But yeah, that immunomodulation really doesn't work because by the time the AV node is destroyed, there's really no way you can get that right again. And then the other line of treatment is better are genetic agonists that can be used to increase heart rates, but there is just a subset of those patients that progressively fail to respond on that, progressively develop slower heart rates. And then because you can also not implant an electronic pacemaker in utero, there's really nothing we can do. And so how about developing a biological pacemaker for this problem? So that is what we've been doing over the past two decades, I can say. And so there's a variety of different strategies being explored in the domain of gene therapy, largely centering on pacemaker ion channels, so the so-called HCN channels that I will talk a lot about today. And on the other side of the spectrum, there has been explorations of transcription factor gene therapy, and in particular TBX18 takes a prominent place there. And I just only very shortly dive into TBX18, and we presented it last year, but here's just a brief update. And that is, we took notice that the TBX18 studies, although they had the pacemaker phenotype in the original adenoviral gene therapy, and also mRNA studies, this phenotype slowly declined over time. So we said, well, if you want to treat a chronic condition, it makes sense to overexpress with an AAV. And to our surprise, what happens when you provide, in this case, mice with CMV-driven TBX18 overexpression, over time, only four weeks of follow-up, fibrosis starts to develop, and really the entire injection site progresses into transmural fibrosis, and basically all the cells are dead. So, well, how can this be a biological pacemaker? So we then figured, okay, maybe because these transcription factors are highly dose-dependent, maybe we should lower the dose of expression, and we did so by inserting an upstream open reading frame between the promoter and the transgene, and thereby could effectively lower transgene expression levels. And so this took care of the toxicity, because we did not see any toxicity. It maintained transcriptional activity, so the important genes that are, for example, down-regulated primarily, are effectively down-regulated by this, what we call, dose-titrated TBX18 vector, yet the outcome is not the pacemaker cell. You see there is membrane depolarization, you have these abnormal oscillations, so really abnormal automaticity, and there is no induction of the funny current. So yeah, basically it didn't work in our hands, but we continued to use it also into our intact animal studies, just as a matter of control, to also compare then our HCN2 approach with. So that's what we did. So a little further background on these HCN channels, so most of you will be familiar with them. They're hyperpolarization activated, meaning that they open upon completion of the action potential, thereby giving rise to slow diastolic depolarization. They're cyclic nucleotide-gated. You can see here the cyclic AMP binding domain, and upon binding, channel kinetics are accelerated and increased, and thereby inward current is also increased and heart rate is increased. We studied this a long time ago already. This is HCN4 overexpression from a lentiviral vector in neonatal red cardiomyocytes, and these cells are already spontaneously active based on spontaneous calcium releases, but when you then overexpress HCN4, you actually see that you get this nice pacemaker-like action potential morphology with slow diastolic depolarization, and in this culture model provided for really reliable pacing. This has also been shown by others, for example here in human-induced pluripotent stem cell-derived cardiomyocytes, and this is just one example because many have shown it by now. And how about using this in vivo? Well, actually, this has been done also a long time ago. This was from the Medtronic group, but this has only been a preliminary study, so they did a few docs but never gotten to do a full series and never gotten to a full publication. One thing they did that was also appealing to us is that they worked with a truncated version of the HCN4 channel. So basically, just after the cyclic AMP binding domain, the rest of the nucleotide sequence was removed, leaving a channel of up to 2,200 base pairs, and this was appealing for us because it allowed us to also include the GFP marker to perform single-cell patch clamp studies in mice. So that was basically our first step, so that's shown here. Two weeks of follow-up, AAV6-mediated HCN4T overexpression, and what you can see is that there is a nice inward current generated, having the kinetics of HCN4, and actually, to our surprise, this already gave rise to very potent pacemaker phenotype in single cells. So as you can see here, 10 out of 10 of these HCN4 GFP-positive cells were spontaneously active. So we basically have an action potential morphology that is somewhat in the middle between a ventricular cardiomyocyte and a pacemaker cell because, as you can see, it has this notched action potential but also a phase 4 diastolic depolarization. So the other phenotype that was detected is the membrane depolarization, as was also to be expected from HCN4 overexpression. So then, taking this one step further, testing it in animals with complete AAV blocks. So here we leverage the RET complete AAV block model that was previously developed by Hale Cho and colleagues, and it's a nice model because we use, and they used, female RETs of six months of age, and they actually very well tolerate AAV blocks, so, yeah, giving you the opportunity to then study gene therapeutic interventions that can hopefully further increase RET. And so, basically here, we tested a total of four groups, three are shown here, so saline-injected control animals, animals that were injected with this dose titrated TBX18, without GFP in this case, and HCN2, also without GFP, and what you can see is that at baseline not much happens because here you see basically AAV block combined with a rhythm that is in line with a junctional escape rhythm, but then upon overexpression of HCN2 combined with isoprothenol, and this is important because actually this is part of the conventional treatment of CCHB patients, actually we see very reliable pacemaker activity with a extreme access, so in line with pacing from the injection site in the LV apex. And so here you see the aggregate data of this, and also the fourth group, because we also tested the combination of HCN2 together with TBX18, so this dose titrated TBX18, and actually you see here once more that TBX18, yeah, doesn't do anything in terms of baseline pacing, but also it cannot be used to augment HCN2-mediated pacemaker activity. So enthusiastic by this HCN2 outcome, we then asked the question, well how about some longer follow-up and comparing HCN2 to HCN4, this was also a long-standing question in the field but never had been studied by a full rigorous head-to-head comparative study. So this is what we did here, now again injecting the vectors one week after the implementation of AAVBlock, and then following these animals for a total of four weeks with weekly EKG testing combined with isopteranol testing, and so this is the result. So at baseline, again, these saline-injected or HCN2-injected animals do not show much other than just an intrinsic rhythm. But in case of HCN4-injected animals, every now and then we would see a baseline rhythm that was already compatible with pacing from the injection site, although the rate was fairly similar as compared to the intrinsic rhythm. And when you look at the aggregate data, you see this back, so average heart rate over this four-week follow-up at baseline was not really different between HCN4 or control animals, but what you see is that there is already some pacing occurring in HCN4, and this appears to be more prominent as compared to HCN2. Well, then the question, of course, what happens with the isopteranol challenge? Well, again, very reliable pacing in both HCN2 as well as HCN4, and this is more or less stable over the four-week follow-up, and what is intriguing is that it actually appears to be more stable in case of HCN4 as compared to HCN2, and also HCN2 has a tendency to develop slightly faster heart rates, which is compatible with the faster kinetics of HCN2 as compared to HCN4. So, yeah, then the next step was obvious, so how about testing this in a pig? And so, basically, we did pretty much the same, so again, a complete AV block, now we did monthly testing with isopteranol, and we used this RAMP protocol of isopteranol and explored the rhythms that developed. And what you can see is pretty much the same as what happened in the rats, so in case of baseline rhythm recordings, they were fairly similar between HCN4 and control animals, but upon the challenge with isopteranol, the HCN4-injected animals, again, reliably developed a pacemaker rhythm from the injection site, and here you see the aggregate data of that, so you see that the heart rates are faster in HCN4-injected animals, and they are compatible with ectopic pacing developing upon increasing isopteranol, and this was the case for the four-week follow-up time point, as well as the eight-week follow-up time point. So, yeah, that brings me to my conclusions, we're excited about this, HCN4 appears to generate spontaneous pacemaker activity in ventricular cardiomyocytes, this generates robust and long-term stable biological pacing in rats and pigs with complete heart block, in particular, in the setting of isopteranol stimulation, which is very relevant for the treatment of patients with CCHB, and therefore, we really aim to develop this further towards a gene therapy, potentially life-saving gene therapy, for severe cases of in-utero CCHB. And with that, I'd like to thank you for your attention, I'd like to thank my co-authors that are in Amsterdam, in our spin-off company, Pacingure, we closely work together with the team in Utrecht, where we do our large animal testing, and we work together with a bunch of different companies, including Nascent Science, a Korean company, Zero, that did the rat AV block studies, and Revity was involved in viral vector production, and these are consultants in terms of gene therapy pricing and translational development with regard to regulatory intelligence. So with that, I'd like to thank you for your attention, and I'm happy to take questions. So, once upon a time, some of this data I think you generated, the way to get this to work was to inject it into the existing production system, and use that machine, and it looks like you've got part of what you need, and I'm wondering what your thoughts are about really constructing the complete case picture, how many genes, what you want it to do, and how you get this to a point of automation. Yeah, yeah, that's a great question. What I can tell you is that the importance of injecting into the bundle brain system, although previously confirmed with the Adeno, we did not, let's say, reproduce in our pigs, but this can have multiple causes, and it is difficult to precisely inject in that region. Another sort of common realization was, among all the different strategies that we've been testing, is that in case of the Adenoviral gene therapies, you have a combination of local tissue inflammation, and then the ion channels on top that provide it for robust pacing eventually. Now with the AAV, we're basically one step back in that respect, because the ion channels are apparently less potent without the local tissue inflammation being present. So, I don't think that injecting HM4 into the proximal conduction system would have a very dramatic outcome. I think it's still not enough, as you're saying. Yeah, I'm intrigued, and we'll continue to try to mix and match, see if we can, for example, do dual gene transfer approaches similar to what we've done in the past, and see if we can get it to become more potent, absolutely. But I think also it's realistic to say that it's probably going to be relatively challenging, and so therefore we're really aiming to push this now as a first-in-line therapy, because I think that the combination of better genetic stimulation and one ion channel really gives a potent outcome for now that I think will be difficult to reproduce with a dual gene therapy, but we'll try anyway. So, the first question was about the duration. So, yeah, we have now only tested up to eight weeks. The pigs were eight weeks follow-up after injection. We did not go beyond that yet, but I think that the data are very suggestive that this is a stable effect, also because, actually, expression comes up relatively early. Already, after two or three days after injection, you already get expression that then slowly increases, you know, more or less up until the point of four weeks after injection, and thereafter, it is more or less stable. So, yeah, from that perspective, we anticipate that this is the anticipated stable outcome. Then, with regard to dose titration, yeah, that's an obvious next thing to do. We haven't done it yet. What we do see is that... So, with viral vectors, typically, you go in steps of a log scale. So, you would go a log scale lower, and we actually did this in injection volume in a GFP vector just by looking. So, if we inject 200 microliters, which is what we did here, we do three times 200 microliters, and with the GFP vector separate experiment, we did 200 microliters and 20 microliters, and what we saw there is that it really gave rise to a dramatically smaller size of the injected area and really became less reliable. So, I think that we are more or less on the lower limit here, although we really don't know how many pacemaker cells you would need. So, I think at some point, we will have to also do a functional study to make this step, but we just didn't do it yet. But I should also say that we're also working on novel capsid variants that, yeah, with the aim of lowering overall dose. So, that would, in our view, be the ideal setting where we do this dose titration. So, we'll do it, but it takes us some extra time to get there. Thank you very much. All right, I'd like to thank all the speakers one final time, and if you're a speaker, please come take the survey and collect the feedback on how this session went. Thank you all.

Heart Rhythm Society

cardiac therapies

gene therapy

ischemic cardiomyopathy

SGLT2 inhibitors

arrhythmogenic cardiomyopathy

vagus nerve stimulation

AAV-HCN4T gene therapy

biological pacemaker

heart diseases