Good morning, and thanks for being a hearty and interested audience. I'm David Van Wagener, and co-chair is Anna Feniger, and we're going to have an interesting session about cellular signaling pathways in AF. And our first speaker is Xun Ai from The Ohio State University, and she's going to be talking about MAP kinases and AF. Xun? Thank you. Thank you so much. And thank you for the organizer to give me the chance to share and publish the data. And unfortunately, I have nothing to disclose. So yeah, today I'm going to share our recent findings. So I was told to talk about MAP kinase. MAP kinase has different members, the ERK, that's considered the survival signaling, and the P38, a kind of a detrimental signaling, involve a lot of cell deaths. And also junk is another target involved. Those three things really involve the disease development. So the junk is a very interesting target that we really try to focus on, because that can be activated pretty quickly in responding to acute stressor, and then also be activated in endocrinic stress condition, but they involve different isoforms. So in the heart, junk 1, junk 2, that's the predominant isoform, and then junk 3, very small amount. If someone attended the session yesterday, I mentioned specifically about this. And then the endocrine conditions. So the, for example, aging is a very important risk factor, independent risk factor for AFib. So therefore, we spent quite some time to understand how does the junk activation that involve aging associate AFib initiation, as well as the sustain. Then we found in both the age of the human, and as well as the age of the rabbit, and then we found activated junk, but not P38. And we also see slightly increase of the ERK. So then this is the situation that lead to increase the AFib inducibility. It's not a spontaneous AFib, but that's the AFib inducibility pacing. And then we further go into detail, we IP the protein, the junk 2 specific protein, because the antibody we have, that's really no isoform specificity. So therefore, we pull down the junk 2, and we see the junk 2 activity, enzyme activity significant increase, but junk 1 is kind of reduced, but not significant yet. So then we use the junk 3 specific inhibitor, we'd be able to eliminate that aging associated AFib inducibility. So then we use a transgenic model, this overexpressed activated, constituted reactivated MKK7, which the predominant upstream regulator to predominantly activated junk 2, that's our, we have some data supported this claim, and also other literature suggests so. And we found that this activated junk 2 increased the AFib inducibility in this model. So then aging is one of the risk factor for AFib. Then binge alcohol is another one we know that associated with a holiday heart syndrome. So this model we found in the human heart, those were lucky, we got those binge exposed to heart, and then they had accidents, and we had a heart, and then those AFib inducibility clearly increased, and then they're associated with the activated junk. Then use an animal model, we did the same drill, so we pulled down junk 2 and assessed the enzyme activity that's also specifically increased, but not junk 1. So then we try to always use this analogy to see the junk 2, but not junk 1 associated with the AFib inducibility. And then we further to prove the junk 2 activation in the heart that lead to AFib inducibility, we use a very specific, cardiac specific animal model, and that overexpressing inactivated junk 2. So that's the dominant connective protein, and to competitively inhibit endogenous junk 2, and you can see here is the proof with the H3 tag, and then we be able to successfully inhibit the junk 2, inhibit the AFib inducibility by the alcohol exposure. So then we think about it, so the alcohol, that's in the alcohol research well known, so increase the inflammatory cytokines. So inflammation that involve with a lot of alcohol cause the organ injury. So then in our case, so the aging, actually we did assessment, it seems that's also the case. So aging was the chronic inflammation. So then there is one very important reason cause of this chronic inflammation, people accumulating data suggest, so the GI dysfunction that may link to the chronic inflammatory status. So in this case, we did the GI epithelial cell barrier permeability assay, and then we found using the dye, the fluorescent dye approach, and then we found increase the permeability in those aging animal. And this increase the permeability clearly associated with the increase in inflammatory cytokines, and also including the bacterial endotoxin. So those cytokines increased, and then we try to use animal model to prove that cytokine, the permeability increase in the GI tract, and that associated with the increase the inflammatory cytokine. So we use a model, the animal model, that's a feeding with the chemical DSS. So then in that model, we do seven days and feeding, and then we measure the AFib inducibility as well as the GI permeability. We see that very well correlated. And then we also take another method to mimic the clinical situation. Because in the clinical, the patient with the GI problem, especially with the IBD patient, if they have increased the GI inflammation, that's associated with increased the GI permeability. So that's the flare-up lead to increase the AFib prevalence. And then the remission in those patients, that also lead to reduce the AFib risk. So in those animal model, we see pretty well correlation in between that. And this leaky gut, that associated with the AFib inducibility, that's very well correlated with cardiac junk two, junk activation. In this case, it's the junk activation using the antiphosphate, the junk antibody detected. And then the recovery remission state, that's R2, that suppress the junk activation. So that's the correlation. Then we want to see whether in this model, whether or not that the junk two really plays a role here. And then we use the junk two inhibitor, specific junk two inhibitor I mentioned earlier, that we'd be able to eliminate that AFib inducibility. And in addition, with that dominant junk two overexpress the animal model, again, we see that suppress the AFib inducibility. So then here, also proof with reduced the junk two, cardiac junk two activation. So then that's kind of together, as I hope I convinced you, so junk two activation in the heart that link to inflammation associated the AFib. So then we further. So if we use that drug, probably mass C, because that's changed the osmolarity of the GI environment. And then now we use a very GI epithelial cell specific occludon knockdown model. In this, occludon is a very important anchor protein to maintain the integrity for the GI tight junction barrier. So in that 50% knockdown, we see that's increased permeability associated with increased the hydrogenality of the action potential and the calcium signal in the heart. And that translates into the AFib inducibility that clearly see here. And then in the use of the junk two specific inhibitor also pretty effectively inhibited that AFib inducibility. So together, I hope the data show you is activate junk two due to increase the GI cause the inflammatory cytokine or the other endotoxin that might contribute to. So further proof, we measure the circulating cytokines and as well as the endotoxin LPS. So we see that's a pretty well correlation in that. And then that lead to two models that all increase the cytokines as well as the LPS. So then we want to see whether or not that affected the junk two activation. So that was a very interesting data was we found that TFR is one of the pro-inflammatory cytokine be able to activate junk. And then the IL-117 is another cytokine also can activate junk. And then the LPS is obviously the same. However, that's our surprise, IL-1 beta, we didn't see change with the junk activation. And then the IL-6 doesn't. So then we want to see, because of this IL-1 beta is a pretty interesting to feel the Dr. Nali and as well as Dr. Debrow, they already showed the NRPS3 signaling associated with, I will show you more data later. So in this way, we see all those activated junk, then we use the junk two inhibitor be able to suppress it, that mean that junk activation is really specific associated with those cytokines. And then as we know, the junk activation were already published in the past. So then the activated junk that lead to SR calcium and mishandling, that more specifically lead to the ROR channel dysfunction, and that lead to diastolic SR calcium leak. And therefore, we did the confocal calcium imaging measurement and we show here with the TNF-alpha, clearly that increased diastolic SR calcium leak, that's the tetra-sensitive SR leak protocol we used. And they use this specific TNF-alpha receptor blocker, we'll be able to block it, suggest this leak is really due to TNF-alpha. But on the other hand, you can see the junk two inhibitor be able to eliminate it. And then the IL-17 has the same thing, and LPS also can cause it. But you can see here, the IL-1 beta also can cause it. And basically here, we use even the IL-1 beta receptor blocker, we'd be able to block it, suggest that's really due to the IL-1 beta. But everything here shows junk two specific inhibitor be able to eliminate. So then kind of gave us idea, and here, I just want to tell you, we have a lot more story here. Hopefully, next year, I can share with you guys. And then we further did the calcium trigger activities, and it clearly shows increased the calcium waves. And then Jaime de San Diego did this patch clamp, and we show that's the increase, the social increase, the DAD, delayed after depolarization. And then the junk two inhibitor be able to eliminate it. And then Xiaoping in Isabel's lab, and recently, we did additional data, IL-17 also be able to increase the trigger activity significantly, and the junk two inhibitor effectively inhibit. So so far, I hope I convinced you the overall. Junk two activation, that's really the response by this number of stressors. And then the inflammation could be one of those cytokines, it could be one of them. And then the inflammatory cytokines are complicated. So we just show you individual one, but you think about it, put them together, and then that's as the whole, that as the end point, activate junk two, that lead to those trigger activity, and make this junk two could be very effective therapeutic target. So I would like to thank all my lab members, as someone said here, and then also other previous members, and also my collaborators, I really thank you guys. I'm ready for question. Thanks. This is open for questions. You mean the junk isoforms? P38. Oh, P38. Right, right, right. In our case, we just did the phospho P38. We didn't see changes, but clearly we did, right, right, right, I know, I know you had a pretty beautiful data that shows the different isoforms. We didn't discriminate the isoform, but in our case, it's just general phospho P38, yeah. You beautifully showed the role for junk in cardiomyocytes, but junk too is important in other cell types, like macrophages and in other diseases. Do you think there's a role for junk too in cardiac macrophages as well in this? Absolutely. There's definitely something happening, and whether or not the cardiac junk or the cytokines that involve or interact with those immunocells, definitely that's possible. We tried to have some collaboration with Dr. Prabhu, and he indeed found the macrophage has increased the junk activation, yeah. Thank you. Thank you very much. Our next speaker is Raelynn Mo from UMass, and the title of this presentation is Calcineurin and Fatsignaling in Atrial Fibrillation. Good morning, everyone. Thank you for having me here. It's my pleasure to share my most recent research with you. To start with, I would like to quickly introduce myself. So I was born in China, and after college, I went to Australia for my PhD, and there I started to learn arrhythmias. Since then, I was obsessed with the reason disorders, so in 2019, after my PhD, I went to United States and doing my postdoc training with Dr. Donahue. So first year in Massachusetts was pretty tough. I was overwhelmed by the cold weather, so I made a wish that 2020 should be better, and guess what happened in 2020? The pandemic. But something good did happen, because we have time to think. During quarantine, Kevin says, how about we look at calcineurin in atrial fibrillation? Previously, we did research on calcium-related kinase, COMK2, so maybe this time we should jump to another end, looking at this calcium-related phosphatase. So after quarantine, we're rushing back to the lab, I did preliminary data, and yes, we got the grant. So something good did happen, and here, my calcineurin story officially begins. So what is calcineurin, why it is bad for AFib? During atrial fibrillation, the heart is beating quite fast, and that would induce sustained increases of calcine. So this calcineurin comes inside of the cell, and then finds its friend, calmodulin, and these two together can easily bind and activate calcineurin. So calcineurin as a phosphatase, the main job is just difference fibrillation. So it will find the substrate, and difference fibrillate it, that will cause translocation of the substrate from the cytoplasm into the nucleus. Shown in here is a classic substrate of calcineurin, that NFAT will travel inside of the nucleus, and where it triggers downstream gene expression related to hypertrophy, inflammation, or pathosis, those factors for structural remodeling. Other calcineurin-related pathways are listed over here. Basically, calcineurin can difference fibrillate ion channels and change behavior of the channels and eventually contribute electrical remodeling. So calcineurin is bad. How could we stop it? We brought a virus from Dr. Mocantin, who is doing research about calcineurin, NFAT, and cardiac hypertrophy. So this virus, adkane, basically is the adenoviral vector carrier transgene of cane, which is an endogenous inhibitor of calcineurin, has a high potency, but less side effects. We validated this virus in the lab in vitro as well as in vivo. So looks like we're pretty much ready for our study. We have a good hypothesis that calcineurin inhibition would reduce the remodeling in AFib. We had good inhibitors to fulfill this goal, and then we have our pig atrial fibrillation and HF model. So we have three groups of pigs, cane, placebo, and AFPC. AFPC group received adkane on day one, just like the cane group, and pacing adjustment every day to match the AFib burden of the placebo group. The other two groups, cane and placebo, we just did the regular two seconds on and two seconds off pacing throughout the time course. On day 14, which is the end point, we did in vivo EB study, and then we collected tissues for further investigation. So let's look at the results. We're pretty excited to see calcineurin inhibition improve the cardiac rhythm. The sinus rhythm percentage in cane group was higher than the placebo, and we also have positive results from the APD90 measurement. As you can see, in cane group, the APD90 pronounced it as well as the AFPC group. Then after that, we tested the indicators of conduction velocity. So we found also very positive results from the cane group. We can see it keep a nice expression level of cognacin 43, as well as low level of interstitial fibrosis. But we noticed the beneficial effects from the AFPC group was lost. So that may tell us AF burden is very important to decide how much beneficial effects you can get from this calcineurin inhibition. And then we decided to take a look at structural remodeling. As I mentioned, we set up the AF burden in the AFPC group as similar as the placebo, which we think that is a true player to stress out the atrial tissue and cause remodeling. So here we have positive results. We can see the apoptosis signals were substantially suppressed in the AFPC group. And this positive survival response is consistent with our data from the echo. So the cardiac function was improved in AFPC group. However, we have some concerns. So first of all, we noticed the anti-inflammatory effects that by calcineurin inhibition was lost in AFPC. In cane group, we marked the infiltration by calcineurin CD3 and CD45. That was largely reduced, but it's gone in the AFPC. And then the big surprise we got is from the hypertrophy. So we measured the cellular size on sections, and we found out, surprisingly, our calcineurin inhibition enhanced rather than reduced cardiac hypertrophy. And this micro-level observation was confirmed by the echo data. So our chamber was truly dilated. So what happened? I wish I could tell you today, but unfortunately, no. So far, I had no luck for this answer. But I want to share with you today all the interesting stuff we found from sequencing. So we did R-seq and found out these pacemaker-specific genes popping on our list. So that indicating maybe the ectopic activity in the firing cardiac conduction system was suppressed by calcineurin inhibition in our AFPC group. Also from sequencing, we noticed the genes of the potassium channels are downregulated in AFPC group. That may explain the longer APD we've seen before. And together with that, so far, we have patch count data for the seven-day AFAB model, which is just half of the time cost. And we can see the calcineurin inhibition increased the L-type calcium currents. That also could contribute to the longer repolarization. And then, again, from the sequencing, we can see the downregulation of HDAC4 in our case. And to check that, I did a WESTERN and found out those found from a relation level and also the total level of HDAC4 reduced in AFPC. And according to the literature published, that may explain why the anti-apoptosis effects in the AFPC group. So to stop here, what I'm sharing with you today is about calcineurin inhibition showed benefits in AFAB. We found out it maintained sinus rhythm. We observed prolongation of action potential duration. And we found that it preserved the cardiac function, reduced apoptosis. Based on the gene expression, it suggested reduction of automaticity. But it also brings up some concerns, like the most significant one, so the cardiac hypertrophy increased by calcineurin inhibition. At the end, I would like to thank our team, so especially our lab, my mentor, Dr. Donoghue, and the lovely faculty. Also, these people from Gene Therapy Center, Dr. Gallucci helped me with the inflammation. And also, my collaborator, Yi from Broad Institute, helped me with R-seq data. And thank you, everyone. Thank you for your time. Please come forward with any questions. While people are thinking about that, I just had a comment about your calcium current measurements. So you showed that the diameter of the myocytes has increased. Did you measure the capacitance? Basically, you normalize the current to the surface area, so picoamps per picofarad. You just showed an absolute current value. Oh, so about the patch globe? Yes. Well, I'm afraid I couldn't answer that question, so because... We can talk about it afterwards. We've normalized it. Yes. Okay. Maybe I have a broader question for you. Calcineurin inhibitors are used, you know, in clinical practice pretty widely for immunosuppression. Do you know, is there any human data that is concerning for cardiac hypertrophy or that is encouraging for an effect on AFib in patients who are using these drugs? Well, you know, I've been searching for literature to support my results recently and I noticed only one publication, not from the people, but not from human, I mean, but from the transgenetic mice model. They found out that the calcineurin inhibition enhanced cardiac hypertrophy rather than reduce it in the myosin heavy chain mutant model. So from there, they showed a clear enlargement of the chamber and then, very interesting, they treat the transgenetic mice together with the L-type calcineurin inhibitor together with calcineurin inhibitor and guess what happened? The enlargement reduced. So I don't know what, well, I still, I'm gonna try hard for my study, but this may give us some clues that maybe casein is still playing something over there that I don't know. So yeah, that could be a breakthrough. And also, I do know, I've been thinking, so in our case, the pacing-induced hypertrophy may have lots of mechanisms down there and then the most, like, predominant one may be different from the pressure overload hypertrophy that has been published for the calcineurin inhibition in terms of the anti-hypertrophic effects. So yeah, I still have lots of work to do, so I will keep you updated. Thank you very much. Thank you. Okay, our next talk will be from Dr. Natel, JAK-STAT Signaling and AF. Thanks very much, David and Anna. A wonderful opportunity to be here to speak. So I'm going to talk about the relatively little information that's available about the JAK-STAT pathway in AF. You've already heard a lot about JAK2 from Junai and it seems to be important in structural remodeling. So as you know, JAK is Janus kinase and STAT stands for signal transducer and activator of transcription. The system is inhibited by ligands that interact with various receptors and cause phosphorylation of JAK. It can also, as Junai showed you, be activated by various stressors. So when JAK gets phosphorylated, among the things it does is to phosphorylate STAT, which then displaces to the nucleus and causes changes in gene transcription. And the JAK-STAT pathway is known to be a very important signaling system in cardiovascular remodeling. So a lot of the work that I'm going to present you is from a study done by Yolanda Chan in my lab a number of years ago. She studied left atrial remodeling, specifically structural remodeling, in two models. Dogs with ventricular tachycardia myopathy after two-week ventricular tachypasing and mice with left ventricular dysfunction three weeks post MI. First, Yolanda looked at PDGF expression in fibroblasts from the canine heart failure model. All of these graphs show gene expression, mRNA levels, as a function of time of ventricular tachypasing. Typically fibrosis becomes significant at one in two weeks, and you can see there's a an increase in the expression of PDGF-A, PDGF-C, and PDGF-D. So multiple components of the platelet-derived growth factor system are upregulated in this heart failure model of atrial fibrillation. She then looked at the receptors for PDGF. She found no transcriptional alterations of any significance, but she did find upregulation of PDGFR-beta protein, indicating post-transcriptional regulation of left atrial PDGFR-beta, as well as transcriptional upregulation that I showed you in the last slide of the left atrial PDGF genes. She also looked at JAK and found upregulation of JAK2 gene expression at one in two weeks of ventricular tachypasing. She looked at phosphorylated STAT3, which is a result of JAK activation of STAT. You can see that PSTAT3 levels were upregulated. These slides show atrial and ventricular levels in control dogs, as well as in heart failure dogs. This is at two weeks. You can see significant upregulation of PSTAT3 expression, some upregulation of total STAT3, but a relative increase in STAT phosphorylation based on the ratio of PSTAT3 to total. So upregulation of JAK2 and increased phosphorylation of STAT3 in heart failure atrial fibroblast from dogs. Yolanda then looked at the hypothesis that PDGF is responsible for some of these changes. She studied PDGFR-alpha and beta expression and noticed an increase in both of them in the left atrium with infusion with in vitro exposure to PDGF. I think she used PDGFD here. She also found upregulation of JAK2 and STAT3 and increased phosphorylation of STAT3. She then looked at the secretion of collagen and fibronectin into the supernatant of fibroblast exposed to PDGF and noted upregulation of collagen 1 secretion into the supernatant as well as fibronectin 1. So in vitro PDGF regulates PDGF receptors JAK2 and PSTAT3 in fibroblasts and in vitro PDGF administration to fibroblasts increases secretion of collagen 1 and fibronectin. She then looked at in vitro inhibition of JAK2 or PDGF receptor. She used these selective small molecule blockers of JAK2 and PDGF receptors and found that either of them reduced PDGF expression as well as PSTAT3 phosphorylation. She also noted that collagen 1 secretion was sorry this is collagen 1 secretion was decreased and cell proliferation was reduced based on the cell number of fibroblasts after 24 hours of exposure. In vitro inhibition of left atrial fibroblast JAK2 or PDGFR prevents PDGF induced upregulation of STAT3 collagen and fibroblast proliferation. She then looked at an in vitro STAT3 inhibitor at in vitro STAT3 inhibition with this selective inhibitor S3I and she noted that inhibition of STAT3 activation at various concentrations of S3I suppressed STAT3 mRNA expression, reduced collagen 1 and collagen 3 expression, reduced PSTAT phosphorylation and reduced collagen release into the supernatant. In vitro STAT3 inhibition prevents PDGF induced upregulation of STAT3 collagen, STAT3 phosphorylation and collagen secretion and fibroblasts. Yolanda then went on to look at the effects of in vivo STAT3 in a rat MI model. These rats develop clear left atrial fibrosis after three weeks post MI. This is suppressed by in vivo treatment with the STAT3 inhibitor S3I and P wave duration which is increased by myocardial infarction is significantly reduced by STAT3 inhibition. As well there's left atrial size changes, left atrial dimension, systole and diastole are increased in the MI model and this increase is prevented by in vivo STAT3 inhibition. So having shown that the JAK STAT system seems to be involved in fibrotic changes in atrial fibrillation associated with left ventricular dysfunction, we wondered about what activates the STAT system in AF and I want to share with you some recent work from Xander Verenz's group looking at a mouse post-operative AF model. They took mice subjected to thoracic surgery involving a biatrial pericardiectomy and aortic cross clamping for 20 seconds to mimic what happens in cardiac surgery in man and they compared these results to results in a sham group that had dissection down to this I think this should be thoracic wall but no further dissection. 72 hours later they did electrophysiological study and divided the mice into the AF inducible group which was the TAF group standing for thoracotomy and atrial fibrillation, compared this to the non inducible group the TSR for thoracotomy sinus rhythm group and compared these to a sham group. So they did single cell sequencing and looked specifically at the number of macrophages and noted an increase in the number of macrophages in the atrial macrophages in the thoracic atrial fibrillation group and this is quantified here. They also looked at the specific subtypes of macrophages and noted an increase in the number or the percentage of macrophages that had pro-inflammatory or mixed phenotypes. They noted that mice subjected to this thoracotomy developed an increase in AF incidence and then they use clodrinate liposomes to deplete macrophages and noticed that this significantly reduced paroxysmal AF incidence as well as duration with the changes in duration being qualitatively apparent but not statistically significant. So the macrophage population increased in post-op AF atria. There was an increase in the proportion of inflammatory macrophages and macrophage depletion prevents post-op AF in this model. They then looked specifically at IL-6 receptor signaling and developed a double knockout mouse that specifically targeted IL-6 receptors in macrophages and they noted that while the post-operative AF incidence was increased in thoracotomy mice, this was restored to almost normal with the macrophage specific knockout of IL-6 receptors. They also noted a decrease, an increase in PSTAT3 phosphorylation in the group that developed AF with suppression in their macrophage knockout group. So macrophage specific knockout of IL-6 receptors suppresses post-operative AF and PSTAT3 phosphorylation is a candidate downstream signal for IL-6 activation. They then looked at the role of PSTAT3 signaling in this post-op AF model using daily injection of PSTAT3 inhibitors including the S3I which we had used in our rat model. They noted suppression of post-operative AF by S3 inhibition. They then went on to look more specifically at cardiomyocyte selective STAT3 knockdown via AAV9 and noted that knockdown of STAT3 suppresses AF by reducing the incidence as well as almost significantly reducing the duration. So macrophage mediated IL-6 activation of STAT3 is a key signaling mechanism in post-operative AF. So in conclusion, in a canine model of AF due to structural remodeling there's up regulation of PDGF, PDGF receptors, JAK2 and PSTAT3. In vitro inhibition of any of these PDGF receptors, JAK or STAT3 prevents downstream effects including fibroblast activation in canine fibroblasts, canine left atrial fibroblasts. In vivo STAT3 inhibition prevents atrial structural remodeling. And finally macrophage mediated IL-6 signaling appears to be a key mediator of post-operative AF with its action mediated by STAT3 signaling. Thank you very much. This talk is open for discussion. Maybe while you're all thinking about these beautiful data, can I ask a question about the PDGF itself? Where does it come from in this model? I'm not sure I understand your question. Okay, are you asking if it comes from cardiomyocytes or fibroblasts? I think it's up regulated in fibroblasts but it may as well come from cardiomyocytes. There's lots of evidence and yours is supporting it that the macrophages are important. You didn't do any subtyping of the macrophages? Well Xander did, well not further than what I showed you. Okay, thanks, very interesting. And the last presentation for the day for this session will be from Dr. Guy Salanna who is at the University of Pittsburgh. He unfortunately is not was not able to be present so his presentation will be pre-recorded. I present our work on Wnt canonical signaling and atrial fibrillation. I regret that I'm not able to be here in person, but please don't hesitate to send me some questions or comments. My background, I'm relaxant to a hormone of pregnancy that has pleiotropic actions on the cardiovascular system in both men and women and is currently the topic of multiple clinical trials. And age rats are shown to be an excellent model of atrial fibrillation and relaxant treatment of age rats blocks sustained atrial fibrillation, reverses fibrosis, reduces inflammation and macrophage infiltration. Relaxant treatment increases the expression of NAV15, Connexin 43, beta-catenin and Wnt1. Incubation of isolated myocytes and fibroblasts with relaxant for 24 hours upregulates Wnt1 and suppresses endogenous Dikhoff peptide to trigger genomic modifications. And finally, I will show some data on RNA-seq analysis of eight and young ventricles with and without. It's a 53 amino acid hormone discovered in the early 1980s, at which time it de-orphanized a receptor, a GPCR receptor called RXFP1. It is essential in pregnancy as it increases cardiac output, systemic arterial compliance and suppresses postpartum cardiomyopathy. Its suppression of fibrosis of the uterus is a particularly important action. Relaxant knockout male and female mice develop multi-organ fibrosis. A great deal is known of its actions in multiple organs and I refer those interested to nature reviews as far back as 2009. Or strain of rats has been extensively studied for its linear aging. As can be shown here, the young animals have a low levels of fibrosis which increases as a function of age in the ventricles. The young animals following a birth space revert right back to sinus rhythm. In the aged animals, either you see a natural tachyarrhythmia or a sustained fibrillation. Ion channels remodeling in AF has been shown to occur through a decrease in L-type calcium current, decrease in sodium current and fibrosis increases gradually with age and AF. Nine-months-old or 24-month-old rats were implanted with all that many pumps to deliver either the vehicule or relaxant at 0.4 mg per kilo per day, the vehicle control being sodium acetate. At the end of a treatment of two weeks, the hearts were isolated in a Langendorff perfusion apparatus for dual optical mapping of voltage and calcium. And electrical stimulation on the right atrium or the left atrium or the LV were applied to describe and identify the arrhythmia vulnerability of these animals. The hearts were also collected for RT-PCR, histology, immunofluorescence and RNA-seq. Atrial action potentials from control animals showed that a premature impulse triggered either a transient arrhythmia or a sustained AF that essentially lasted for the duration of the experiment. In relaxant-treated animals, the typical result is that the premature pulse either captured and the animal goes back into sinus rhythm or simply failed to capture. The arrhythmia profile shown here expresses that relaxant-treated animals had much fewer arrhythmias than the controls. To elucidate the mechanism whereby relaxant protects the heart from atrial fibrillation, we measured conduction velocity restitution on the right atrium and the left atrium as well as conduction velocity as a function of cycle length. As you can see here, aged rats had considerably low conduction velocity compared to young rats which are approximately at one meter per second. At this conduction velocity at 250 millisecond cycle length also is dropped precipitously to close to 0.3 or at 150 millisecond cycle length. Relaxant therapy increased the conduction velocity markedly, particularly at fast heart rates or low cycle length. The collagen 1-to-tissue ratio decreased markedly and the mRNA levels of TGF-beta, collagen 1, and collagen 3 were reduced. Two sections of 7 micrometer thickness were dual-labeled with a beta-catenin and a conexin-43 antibody. In animals who did not receive relaxant treatment, you can see a diffused pattern of beta-catenin labeling. Relaxant-treated animals had marked increases in beta-catenin at the intercalated disk that merged and localized with the localization of conexin-43. The summary data is shown here where relaxant had a considerable effect in increasing beta-catenin expression as well as conexin-43. A similar experiment as was shown before on the atrium, these are ventricular tissue sections labeled with beta-catenin and conexin-43 antibody. In the 9-months-old adult, there is a considerable amount of beta-catenin labeling, which is much reduced in the 24-months-old aged animal and is reversed by relaxant treatment. The merging of the beta-catenin and conexin-43 shows a high degree of spatial correlation as shown here through the Pearson's correlation coefficient. Suction velocity caused by relaxant can be partly attributed to the increase in conexin-43 at the intercalated disk, but that isn't the only mechanism. If you take rat atrial myocytes and incubate them with relaxant for 10 minutes, well, not much happens, but 24 hours later, you have a marked increase in NAV1-5 expression summarized here, and patch clamping of these cells shows a marked increase in peak INA as a result of relaxant treatment for 24 hours. This is not only a rodent effect. Human iPSCMs were incubated for 48 hours with relaxant, resulted in a two-fold increase in the upregulation of the sodium current. In ventricular tissue sections from an aged rat, labeled with WNT1 antibodies, levels of WNT1 are quite low. After a two-week treatment of relaxant, there's a marked increase in WNT1 expression. DKK does somewhat the opposite. Tissue samples from ventricles of control and relaxant-treated rats shows a marked decrease in DKK expression. In isolated cells labeled with DKK and DAPI, relaxant also shows a marked decrease in DKK1, and this is the summary from 18 myocytes or more between control and DKK. Myocytes treated with NAV1-5 in the presence of exogenous DKK of 3.6 nanomolar show little expression of NAV1-5. WNT1 has a dramatic elevation of NAV1-5, and the combination of WNT1, exogenous WNT1 plus DKK shows that DKK suppresses the effects of WNT1, and statistics are shown here for a minimum of 18 cells per group. Ventricular rat myocytes were labeled with DAPI and beta-catenin, and in control conditions fewer than 10% of the nuclei contain beta-catenin. After incubation with 10 nanomolar relaxant for 24 hours, there was a considerable translocation of beta-catenin into the nucleus, and 40 to 60% of the nuclei contain beta-catenin, which is an important step to explain the relaxant-dependent genomic modification of the cells. RT-PCR of atrial muscle shows a decrease in TGF-beta-1. Both collagen 1 and 3 are reduced, metalloproteinases 2 and 9 are reduced, and alpha-smooth muscle actin is reduced by 30%, suggesting a decrease in myofibroblast. WNT3A is reduced, and WNT1 is increased by 80% along with beta-catenin. Relaxant is known to modulate collagen synthesis in cardiac fibroblasts, and in this figure we find that DKK, exogenously added, suppresses partially the effects of relaxant on collagen. As shown here on top, cardiac fibroblasts are incubated, then labeled with collagen 1 and DAPI, TGF-beta-1 for 24 hours increases collagen levels. Relaxant alone does little. Relaxant plus TGF-beta-1 inhibits and suppresses collagen synthesis. In these panels, DKK is exogenously added at 3.6 nanomolar. DKK plus or minus TGF-beta-1 has little change. Relaxant and DKK has little effect, but DKK relaxant plus TGF-beta-1 shows here that the level of collagen suppression that was observed in the absence of DKK is less effective. So we've looked at the overall picture. What about specific common markers others have found to be significant to predict severity of outcomes in patients with atrial fibrillation, heart failure, or inflammatory diseases? Again, there's a dramatic difference between males and females. Relaxant only affects transcription in males in red markers. Again, a thing to notice here is that in males, there's little increase of these transcripts due to aging. Light blue relaxant only affects transcription in the females. Here the genes suppressed by relaxant show an increase in aging, highlighting again the activation seen in females and not males. In dark blue, these are a few transcripts that RLX affects similarly in males and females. IL-1-beta pro-inflammatory reduces cardiac contractility. It's involved in hypertrophy and arrhythmia. IL-6 pro-inflammatory increases CRP, hypertrophy, increased risk of stroke. And TNF-alpha, LV dilation pro-inflammatory and heart failure progression, LV dysfunction and remodeling. NPB, brain atriotic peptide, indicative of hemodynamic wall stress, risk factor for AF and stroke. And GDF-15, growth differentiation factor, increased circulation in heart failure and may signal inflammatory stress, predictor of heart failure, re-hospitalization, and all-cause mortality. Relaxant treatment of aged animals has a very large impact on inflammatory cytokines and chemokines, 10 chemokines, making it a comprehensive anti-inflammatory. Macrophage infiltration was found to have marked sexual differences being considerably higher in females than in males, even at young adults at nine months. That would be those values versus here. At 24 months, there was a further increase in macrophage infiltration in females and in males. And relaxant treatment substantially reduced macrophage infiltration in females. Here we show a schematic to describe the interplay between relaxant and Wnt signaling. On the left, relaxant interacts with RXFP1, which is a GPCR, resulting into cyclic AMP elevation, pKa, again, Krebs activation. It also interacts with the receptor tyrosine kinase, which has an increase in PI3K, and gene transcription, which increases INOS expression, elevation of nitric oxide, which inhibits the NLRP inflammasome. What we have added to this pathway is that Wnt1 elevation occurs and is transported outside the cell and via an autocrine pathway interacts with the LRP56 core receptor and the frizzled receptor to alter GSK3 beta destruction complex and allows beta-catenin elevation, which is translocated into the nucleus and changes transcription. And now this transcription has a consequence of increasing NAV15 expression and connexin 43 expression. Connexin 43 and beta-catenin is translocated to the intercalated disk in NAV15 to T-tubules and intercalated disk locations. Two ventricular tissue sections have been labeled with a beta-catenin antibody, ANDAPI. One is from a rat that has undergone normal healthy aging. The other is from a rat that has been treated with Riloxin for two weeks. You can see a dramatic difference in the structure and the cellular hypertrophy of the heart, indicating considerable remodeling by Riloxin. I would like to acknowledge all the people who did the work, in particular Beth Gaber-Sweber, who's been in my lab tech for over 10 years, and Guillermo Romero, with whom I bounce his new ideas on a daily basis. Great. This represents the end of the session. If you have any questions for Dr. Salama, please don't hesitate to contact him via email or text messages through Igor, apparently. Thank you all for being here.

atrial fibrillation

cellular signaling

MAP kinases

calcineurin

JAK-STAT pathway

inflammation

cardiac remodeling

fibrosis

therapeutic targets