Good afternoon, everyone. Welcome to the session on the environmental effects of the environment on cardiac electrophysiology, arrhythmia, and disease. We have an interesting set of talks lined up, which I'm really excited to introduce. But I think we're going to first start off with our chair, who needs no introduction. Would you like to say a few words, and then you can? Yeah. Anyway, this is a very exciting session, so thank everyone for coming. We switched the orders around so that I could just give a brief overview of the entire field and the terminologies, and I think that will set up well for our exciting three speakers that are going to be coming up after this. And I'd like to add that I'm stepping in. My name is Faisal Saeed. I'm a cardiac electrophysiologist at UNC, and I'm stepping in for Wayne Cassio, who is the head of the EPA, and who I know personally, and he is unfortunately not allowed to attend this session because of restrictions with travel and attendance from the government. So it's my privilege to step in on his behalf. Okay, so I want to thank the organizer and Ellie again for putting together a very important session and a very important topic, and the title of the talk is going to be Linking Environmental Pollutants to Cardiovascular Disease, and my only disclosure is that I'm an interventional cardiologist and a vascular biologist, so there might be some bias where I call it CVD. It's mostly ischemic heart disease, but we are going to talk about arrhythmias and conduction disease as well. But I wanted to sort of set the stage and just kind of define what we call a pollutant, or what are the pollutants that actually affect human health. There are some established new and emerging types of environmental threats, and then we'll talk about how we consider linking these pollutants to cardiovascular disease, and I'm going to use air pollution as an example. And then there are some other concepts like lifetime cumulative risk of exposure, susceptible, vulnerable populations, and then we'll end with some challenges and future needs. So pollutum is really a convenient term to collect all the different types of pollution that could actually affect human health, and it's part of the exposome that we consider in defining human health. And it goes all the way from air pollution, toxic metal with heavy metals in the water, in the soil, petroleum-based chemicals and plastics, and some of the toxic chemicals that come out of that, including PFAS, which are these forever chemicals. Wildfires are increasing as well, increasing structural burn, which is very different from air pollution as well in terms of the types of chemicals and toxicants that gets introduced in there. And when we think about the global burden of disease caused by these pollutants, the Lancet Commission and WHO estimate that there are actually about 9 million deaths, excess death that occur every year from global pollution issue. And that can range anywhere from 9 to 12, depending on how you define the pollution. But out of those, about 60 percent actually are cardiovascular disease, and that includes ischemic heart disease, stroke, and hypersensitive heart disease as well. And when you think about what are the risk factors that increase your chance of mortality as well as cardiovascular risk, air pollution and tobacco use are among the top risk factors that leads to cardiovascular death. And about total pollution is actually estimated to contribute to about 20 percent of all cardiovascular disease and 25 percent of all ischemic heart disease. And among those, air pollution has the highest contribution with about 6 million, more than 6 million deaths attributed to air pollution. And this is only expected to increase with worsening population-weighted exposure as well as increasing population density. But this is also likely an underestimate because a lot of this estimation comes from what we call sort of zone one pollutants. And these are well-characterized health effects with well-studied pollutants. So there's causal evidence that those pollution or pollutants are actually causing health risk. And these estimates are derived only based on these type of zone one pollutants. And the zone two, zone three are still something that we have not yet been able to integrate into our health estimates. And so what are these emerging pollution and threats? There are increasing wildfire, as I said, which comes with a lot of different types of pollution and chemical exposure. There's also vaping and electronic cigarette, which is affecting our youth and exposing them to different types of chemicals and also coming with high nicotine. So it becomes, you know, people become very dependent as well. Microplastics, you've probably read about them. They're really everywhere. And then chewing a gum, sucking a baby bottle, and exposure to a lot of these and, you know, they're found in our brains and vasculature. So what are their effect on our health, as well as many other types of emerging threats? So how do we link these pollutants to cardiovascular disease? And I think I sort of think of it in three big pillars. There's epidemiology and mechanism intervention. And they help to establish causality, construct a dose response relationship where we can actually start to think about what is too much. And discover mechanism linking individual cardiovascular response to specific environmental conditions or pollutants. So let's talk about epidemiology. I'm going to use air pollution as an example. And, you know, it's an observation that people had from a not, it's not a very recent one. Even back in 1600, there was a Sir John Evelyn who wrote a report to Charles II, the king, said brewers, dyers, line burners, salt and soap boilers are making London resemble Mount Etna, Stromboli, or suburbs of hell. And there's fulginous, filthy vapor corrupting the lungs, and one half of them who perish in the city die of physical or pulmonic distempers. And in fact, the mortality was very high in London, higher than the rest of the country at the time. He proposed some, at the time, a very advanced set of interventions which were never really implemented. It wasn't really until 20th century when there was these catastrophic atmospheric events and sort of inversion events where bad air was trapped. The Great London Smog in 1952 was an example where there were excess of 14,000 deaths from that event. And then in U.S., there was Donora, which was, they had a mill town with Carnegie Steel, and there was basically an extreme event leading to half the city becoming affected with health outcomes. New York City had this event. And this basically led people to say, we've got to do something. 1950, Truman, Harry Truman, had to convene the first National Air Pollution Conference. And then subsequently in 1963, Clean Air Act comes in, and in 1970, it becomes implemented. EPA is established in 1970. And then they start setting some regulations and guidelines and standards, and they were based on some limited set of data. We had some time series studies, small longitudinal studies, and some human experiments and animal toxicology studies. And they started with sort of the 75, 50, which are much higher than what our current regulations are, and then they started with total particles, but then they started to refine this as well, which helped to refine the regulation. So now they're looking at different sizes, PM2.5 and PM10. It wasn't really until this landmark study in 1982, 1990s, actually, they started to look at prospective core studies and found that there was a causal association linking mortality and cardiovascular disease. And multiple core studies followed, helping us to establish some of this dose exposure response relationship. And when we look at these different levels, they basically found that with active smoking, passive smoking, and air pollution, there wasn't a linear relationship, but a very steep curve at the low end of this exposure, suggesting that much of this sort of marginal risk exists in that low exposure range. And so that helped to guide some of the global burden estimation, but also helped to refine our current guidelines and standards, now at nine micrograms per millicube. And so these type of work really helped to establish that dose response curve. Similar data exists for AFib as well, but not as extensive as ischemic disease. So the mechanism helps to establish plausibility, and then associates some of these different types or constituents with a mechanism. And this is human exposure studies that were done. It's hard to do these these days, because now I think some of the risks are very established. But people, they would put them in the chamber, expose them to high levels of diesel exhaust for an hour, put them on a treadmill, and basically they saw that there was increased inflammation, increased platelet viscosity, as well as ST changes during these type of stress tests, along with animal studies in rabbits and mice showing that there's increased atherosclerosis, as well as in population level, there were calcium scans that showed increased that. So there's basically multiple pathways that are proposed at this point. Long inflammation, systemic oxidative stress, direct entry into circulation, causing damage to the distal organs as well. I think relevant to arrhythmia is the neurohormonal activation that causes increased sympathetic and parasympathetic, as well as autonomic imbalance as well. And I think the key is also associated with many known risk factors, including diabetes, obesity, and hypertension, which can also lead to these risks as well. And potentially affecting channel dysfunction as well, and conduction through calcium channels and ion channels. So the last is the intervention part. And I think there's some natural experiments that were done. So the Clean Air Act helped to really lower the emission to 80%, 98% even for lead. And basically based on that, we've seen adjusted, there is a reduction of cardiovascular mortality. And in year 2020, 230,000 deaths were avoided based on this as well, with high benefit. And I think that means we still need to do these intervention trials, and these are ongoing as well. But some of these policy interventions take a long time, and they miss these local hotspots where vulnerable populations live. And using masks and filtration are sort of, especially for a vulnerable population, can help to see if there's immediate sort of ways to avoid health effects as well. And at this point, basically the clinical implication is that now AHA, ACC, ESC all consider air pollution to be a major modifier risk factor for cardiovascular disease. Some of the last few points I want to make is that cumulative exposure is basically across lifetime. So babies in utero who get affected basically have a much higher lifetime risk of developing cardiovascular disease. And they start off in this steep slope. And our goal is to basically identify them, get them intervene, get intervene, and try to get them to this low lifetime exposure curve as soon as possible. And then we discussed this briefly, but it's important to identify the susceptible and vulnerable population so that we can target our intervention better. And so with that, I just want to introduce our just concept of the three speakers. We have different types of emerging threats and pollutants that are going to, our speaker is going to discuss in terms of their association with disease as well as their potential mechanism. And these are the type of work we need to be able to really advance the field forward. So in summary, there's a large burden of cardiovascular disease from environmental pollutants. There's further research that's needed for linking these emerging pollutants to cardiovascular disease. And we need to better measure so that we can actually intervene. And early intervention is important. And also identifying susceptible and vulnerable population is important for pollutant exposure. So with that, I want to thank my team and my collaborators. Thank you very much. Okay. So just in the interest of time, I suggest we move to our first speaker, who is Nikki Posnack from the Children's National Hospital, talking to us about the effects of endocrine disrupting chemicals and cardiac electromechanical function. Okay, so thank you again to Dr. Grandi for putting together this topic and bringing it to heart rhythm, and hopefully we can continue to expand going forward. So today I've been asked to speak about the effects of endocrine-disrupting chemicals on cardiac function, and endocrine-disrupting chemicals can be a whole host of different types of chemicals that basically interfere with hormone homeostasis. They may act like hormone mimics to turn on or turn off different biological processes. And today I'm mostly going to focus on chemicals that are found in plastic materials. So as everyone here knows, plastic products and plastic devices are truly ubiquitous in our environment. If we were to take a sampling of individuals in the audience today, roughly 93 to 98 percent of us would have plastic chemicals floating through our body at any given time. And so there's increasing public health concerns about our chronic, continuous daily exposure to chemicals that are used in plastic materials, particularly food and beverage containers. And then, as was kind of mentioned by the previous speaker, these plastic materials don't disappear. They break down into smaller and smaller pieces, termed microplastics, which are now pervasive in both our food and water supply. And so it's estimated that the average person ingests approximately five grams of plastic per week, or roughly the weight of a credit card. So clearly there's a public health concern about our bodies being inundated with these chemicals, unbeknownst to us, over decades. My lab is also really interested in one or two different types of chemicals that are also found in medical devices. So to be clear, these are found in consumer products, but I work at Children's National, so we're really interested in chemicals that are found in plastic medical devices. So today I'm just going to talk about one of those, DEHP, or di-2-ethylhexyl phthalate, which is a liquid plasticizer that's commonly mixed with harder materials like PVC to make flexible plastic products. So DEHP has been found in blood bags, blood storage bags, and tubing circuits for nearly 75 years, which is pretty amazing. And so essentially what it is is it's a plasticizer. It makes things very flexible, but it's not covalently bound to the PVC matrix, and so it's very prone to leaching or migration when fluids are running through tubing or things are being stored in plastic products. And it can make up to 40 to 80% of the final weight of that product. So the reason that we've used DEHP in blood bags for nearly 75 years is that as these chemicals leach or migrate into blood bags, they actually, they're very lipophilic, and studies have shown that they can intercalate into lipid membranes, particularly in red blood cells, where they cause a stabilizing effect. They allow these cells to be more fluid. And so to date, replacement products to try to remove phthalates or DEHP from blood bags have largely resulted in inferior products. So this leaching becomes exacerbated over time. If you're not in the blood banking field, blood can be stored for up to 42 days, and this DEHP concentration can reach about a half a millimolar concentration. So DEHP is considered an endocrine disruptor. Again, it can interact with hormone receptors. It's considered antiandrogenic. It can also interact with thyroid receptors. And so it's very much a bioactive chemical. So if we look at the public health scene, individuals that have higher daily exposures to phthalates have about a 40% increased risk of cardiovascular and all-cause mortality. And then if we look not just at a public health perspective, but look at our clinical population, it's estimated that intensive care patients, particularly neonates, can be exposed to levels that are nearly 1,000-fold higher than what are considered safe. And again, as alluded to by others, there have also been some additional studies coming out recently that microplastics also contribute to these cardiovascular events where they've actually found microplastic particles in atherosclerotic plaques. So clearly there's this kind of public health concern about what these chemicals may be doing and then even more so a concern about our vulnerable patient populations. So since I work at Children's National, one of the goals or a couple of the goals of our lab have been to quantify exposure in pediatric patients. I have a cardiac electrophysiology background. I'm very interested in cardiac surgery patients. As you can imagine, cardiopulmonary bypass circuits have a lot of plastic. And these patients are being exposed to blood products during their surgical procedure. We're also looking at the impact or association to postoperative outcomes using animal models to look at direct effects. And then along the way, hoping to identify some mitigation strategies. So I'm just going to kind of dabble in some of the work that we're doing, not really focus on one specific experiment. One part of our lab is looking at phthalate chemical exposure during cardiac surgery. To date, we've collected about 800 longitudinal pediatric patient samples. So if you want to collaborate, please feel free to talk to me. To date, we've ran mass spec studies looking at phthalate exposure in about 122 of those patients. And here I'm just showing you that we're dividing our patients up into groups. Those that undergo surgery without bypass, those with bypass who have a crystalloid prime in the circuit, and those that undergo bypass with a blood prime. And I won't step through all of this, but if you look at the red box, what you can see is that pediatric patients that undergo cardiopulmonary bypass with blood priming in their circuit have a much higher exposure to these DHP chemicals, largely because it's leaking from the circuit and they're getting transfused blood products. These concentrations are, they do go down over time. They're not persistent chemicals, but we still have elevated exposure 24 to 48 hours after surgery. So next we wanted to look at what these chemicals might be doing to pediatric patients. And there's a little bit of data in the literature that look at phthalate chemical exposures in animal models. And what's been reported is that these chemicals are largely cardiodepressive, so they lead to cardiac arrest and hypotension. And so for this first set of studies, what we did was we tried to limit our patient population to a very narrow cohort. We looked only at patients that were less than one year old, because we didn't want to have age as a factor, and we looked at patients who all received cardiopulmonary bypass with red blood cell priming to try to look at the postoperative complications that might result from those being exposed to higher phthalate concentrations. And so this data essentially shows that those individuals who are experiencing postoperative outcomes related to arrhythmias, heart block, low cardiac output syndrome, and hypotension are all shown in blue, have statistically higher phthalate concentrations. Okay, so patients are heterogeneous, as are their surgeries. And so another component of the lab is to look at what are the direct effects of these chemical exposures on cardiac physiology. And so I'm just going to show an example of that data here. So in our lab, we use a number of different animal models. Here is an example of a rat model, where we've exposed these rat hearts to DHP concentrations that mimic clinical concentrations, actually about eightfold less than what are found in blood bags. And what you can see is that with increasing time, we start to see a decline in heart rate and a lengthening of the PR interval. Sorry, my pointer's here. So we have a decrease in atrial rate, ventricular rate. We have this increase in PR interval time, or the time that takes a signal to propagate from the atria to the ventricles, which eventually leads to heart block, which, again, has been observed in our patient population. We also use optical mapping studies. So an example of some of that data is just shown here, where you can see in control animals electrical propagation from the atria down to the ventricle, shown in red, as compared to the delay following DHP exposure. And the timing of these results are anywhere from 15 to 90 minutes. It gets worse with time, which coincides with the timing of a cardiopulmonary bypass procedure or cardiac surgery. So I kind of maybe misled you, because my topic was assigned to me on endocrine disruptors. And I do think endocrine disruption is incredibly important. But I think in the cardiac cell, I think the idea that these chemicals are lipophilic is possibly more important. So if we look at molecular simulation studies, DHP is very lipophilic, and it's known to embed into membranes. I mentioned that as a RBC, a red blood cell stabilizing. It can offer red blood cell stabilizing components. And so what we see, we've actually started doing some of these studies in human-induced pluripotent stem cell-derived cardiomyocytes. But what we see is that when we treat cells with DEHP, we do get an increase in membrane fluidity, which suggests that these chemicals are embedding into cardiomyocyte membranes as well. And then if we look at the literature in other cell types, so here's an example of hepatocytes. What you can see is that that membrane intercalation decreases gap junction coupling in other cell types. And the degree of that gap junction uncoupling correlates with the structure of the phthalate in particular. Here I'm showing DEHP and the length of the carbon side chains. And so I do think that some of these chemicals may be causing some adverse effects, surely through disrupting membrane organization. So again, we're kind of interested in cardiac phenotypes. So here's an example of some of our data looking at conduction velocity measurements in human-induced pluripotent stem cell cardiomyocytes. We're doing some time course studies as well as dose response studies where we see a decrease in conduction velocity, again with time, which kind of suggests that these chemicals may be intercalating into membranes. And then we've done a set, a preliminary set of fluorescence recovery studies. So the way that these work is that we have a monolayer of cardiomyocytes that are loaded with a very low molecular weight dye, so that's what that green color is. And then essentially what we can do is we can photobleach a cell and look at the time it takes for that cell to recover its fluorescence. And so if that myocyte is really well coupled and has a ton of gap junctions, that fluorescence recovery should be shorter in time. And what we see is in control cells versus DEHP treated cells or even cells treated with heptanol, a known gap junction uncoupler, that that recovery is delayed. So not only is our lab interested in kind of identifying what the problems are, but our hope is that we can start to identify some mitigation strategies. So this is kind of the third prong of our approach, where we're starting to look at how DEHP accumulates over time. We've coupled with some perfusionists and cardiac surgery to look and see whether cell washing can reduce these extracellular contaminants in blood products. And then looking at different types of biomaterials that may have less DEHP exposure, with the caveat that eventually we have to convince the blood banking field that adopting alternatives might be better as long as we can maintain that red blood cell integrity and length of storage time. So if there's some really smart biomaterial scientists in this lab that want to help kind of address this problem, I think that would be an excellent collaboration. Okay, so just to conclude, I don't want to get fired from Children's National, so I want to make sure I emphasize that plastics are absolutely necessary, right? We can't do any of these clinical procedures without sterile materials. This helps us to store blood, transport blood. It helps all the procedures that we do. But I think we should really start thinking about exposure from a public health perspective as well as a clinical environment, because we do have vulnerable patients who are exposed to very high concentrations of these adverse chemicals. I think they may act through endocrine disruption as well as affecting membrane biology. These effects are time dependent. We're trying to look at some additional mitigation strategies. And I would encourage others who are interested in this topic to help as we pursue other areas of interest like chronic daily exposures to environmental chemicals. With that, I'll thank my team, and I'll also give a shout out to Devin Guarelli. She's one of the PhD students in the lab who got a front cover picture highlighting her article as well as others in the lab who help with our studies. So thank you. Thank you. That was perfectly on time. And we'll take any questions. We have time for maybe two or three questions. If there are no in-person questions, we do have an online question. The question is, are there any known suppliers that are committed to DEHP-free tubing or bags? Is this working? Yeah. Okay. So there are some alternative tubing circuits that especially have bioactive lining that helps to reduce leaching. And bags, there is a bag of interest that we're investigating. The problem with those bags is that there's an increased risk of hemolysis, so red blood cells tend to lyse when they don't have this stabilizing solution, and so you can't store blood for the same length of time in those alternative bags. So it's a very interesting research. I'm curious about the membrane biology. Do you think they have preference in targeting the ID region of membrane versus other part of a membrane? Have you looked at them, or perhaps also transverse t-tubule membrane, et cetera? No, I'll ask you to help with that. No, we haven't. No, we just started this work. Thank you. One last question from, maybe in terms of DEHP, I think, is this something that, how do you measure it? Is it easy to measure? Is it something that other centers could also implement to measure? Can you just measure in the plasma as well, if you were to take a blood draw from a baby? That's exactly what we do. Can you actually measure it? That's what you do? Yeah, that's exactly what we do. So we have a very smart colleague who was in the thank you slides, Angelo De Alessandro's lab. They measure it through mass spec techniques. So it's not as simple as getting an assay to do it right in the lab, but... I have a question as well. So how applicable... These are great results that you demonstrated in babies and neonates. How applicable are your findings to the adult population? I think that's an excellent question. We have children at Children's National. So all of the samples that I've measured so far have just been in pediatric patients. But I do know there's maybe a handful, four to five studies, that have looked at exposure in adult populations following bypass and following ECMO support. So I do think the levels that we're getting, concentrations are about the same. To my knowledge, I don't know of any studies that are looking at post-operative outcomes though. So I can't correlate that data quite yet. Thank you, Dr. Busnak. That was an excellent talk. Next, we have Dr. Alex Carl from University of Louisville. His title is Effects of Airborne Toxicants on Autonomic Function and Erythmogenesis. Well, first off, I'd like to thank the organizers, Dr. Grandi, for inviting me. I'm sorry that my past surrogate mentor, Wayne Cassio, couldn't be here for whatever political reasons. But today, I'm going to do a little bit of a bait and switch here, unintentionally. I'm going to talk about the impacts of inhaled toxicants on cardiac function, especially arrhythmogenesis, and autonomic function, but specifically focus on my work with e-cigarettes. So if you haven't noticed, vaping or use of electronic cigarettes has become exceptionally popular over the past 15 years or so. And this has been shown in young adults and school-aged children especially. So over the past six years, the frequency or prevalence of use among young adults ages 19 to 30 has increased to roughly 25% within the past year. And school-age children, especially high schoolers, peaked in prevalence of use within the past 30 days in 2020 at 25%. But a lot of questions remain about what the actual impacts of e-cigarette exposure are. So first off, a little primer on e-cigarettes if you're not that familiar with them. They started off as the Ciga-like first generation devices but then sort of evolved into these vape pens and mod devices that allow adjusting voltage, wattage, and temperature. And then more recently, the sleek and more discreet Juul devices that look like a thumb drive sort of overtook the market in 2018. They had a new nicotine formulation that was much more tolerable and palatable at a high concentration of about 5%. And historically, the concentrations were about 1.2 to 2.4% before this new salt formulation was introduced on the market. Since Juul, disposable pod mods have been introduced and it's basically the same premise but you throw away the battery when you're done using it. And these have been especially popular among kids. So all e-cigarettes contain e-liquids. They aerosolize them by heating them and within each e-cigarette usually is nicotine with solvents, propylene glycol, and vegetable glycerin or PG and VG. In addition, there is usually a cocktail of flavorants mixed in along with additives to enhance palatability. And in heating these, a number of constituents of concern are produced, including particulates. So this is applicable to particulate matter research that's been done previously. But also aldehydes and volatile organic compounds and some metals as well. The question remains what the adverse impacts are on the heart, although some research over the past five to 10 years has shown evidence of fibrosis in the heart in mice exposed as well as impaired vascular function. A number of questions still remain. As well, included in this, what is the role of ingredients on the adverse effects? And as well, what are the roles of byproducts in these effects? So there's a lot of precedent in research looking at the effects of individual inhaled toxicants on the heart. And first off, it's understood that with smoking, or at least estimated with smoking, that acrylin, acetaldehyde, and formaldehyde contribute to the vast majority of the adverse cardiovascular effects. And these aldehydes are also found in e-cigarette aerosols. Meanwhile, animal studies, in vivo studies, have shown that acrylin exposure induces autonomic imbalance and bradyarrhythmias, consistent with irritant effects. In addition, particulate matter, or PM2.5, especially ambient particulate matter but also particles just carbon-based, have been shown to induce autonomic imbalance and arrhythmia and it's in a dose-dependent manner. So the lower the dose, if you observe an effect on autonomic balance, it usually manifests as sympathetic activation. In the early experiments and in occupational settings with the high concentrations, usually the effects manifest as sympathetic activation and tacky arrhythmias. So meanwhile, within e-cigarettes, nicotine is a critical component and it's a known sympathomimetic that has been demonstrated to evoke arrhythmias and increase risk for sudden cardiac death. But vaping itself has recently been associated, at least temporally, with sudden cardiac arrest in at least three studies, one of which included six patients that incurred sudden cardiac arrest. So the hypothesis of my research that predates these publications was that e-cigarettes disrupt cardiac electrophysiology through autonomic imbalance and chemical constituents. And our seminal research published in Nature Communications showed that acute exposure at roughly 54 puffs in mice that had radio telemeters implanted in them altered electrophysiology. Specifically, we looked at the effects of nicotine-free solvent aerosols, be it 100% propylene glycol or vegetable glycerin or a 50-50 mix of the two, and also compared to the effects of commercial flavor aerosols. In this case, it was classic tobacco from Blue or Magnificent Menthol at 2.4% nicotine. We also compared to mainstream cigarette smoke with both ultralight and full-flavor cigarettes. And we found that the solvents alone induced a clear and dramatic bradycardia in mice. I'm not gonna show you that data in full, but we did see that even when this bradycardia resolved, there was a lag in the resolution of PR prolongation and QT and JT prolongation in the solvent-exposed mice. But in addition, we observed small, subtle, but significant increases in PVCs, or ventricular premature beats. So specifically, propylene glycol and Magnificent Menthol aerosol delivered in a human-like puffing pattern significantly increased VPBs. And not shown here also, we observed an instance of what we believe to be AV reentrant tachycardia in one mouse for a total of 12 minutes with a heart rate at 850 beats per minute. So we followed up this study with a more modern e-cigarette, the Juul, and we wanted to see if menthol differentially affected arrhythmias compared to just the tobacco flavor. And we did find that with a 270-puff regimen, that there was a significant increase in the frequency of VPBs, only with the menthol flavor yet again. We also controlled for the inclusion of nicotine in the solvents and found that this did not produce a significant increase in arrhythmias. In addition, we found that total nicotine equivalents in urine were highest with the Juul menthol exposure, and the nicotine concentration in urine, or the total nicotine equivalents, including their metabolites, correlated with ventricular premature beat arrhythmia frequency. We then followed up this study with a longer-term exposure involving 180 puffs per day. But on the first day of that exposure, we evaluated the responses in heart rate variability as well as catecholamines in the urine and found that SDNN heart rate variability was depressed by exposure, indicating sympathetic activation. Epinephrine was elevated, also indicating sympathetic activation, and ventricular premature beats were increased, including an episode, a brief episode of ventricular tachycardia. We followed this study with a small-scale pilot and administered animals atenolol in their drinking water to find that delivery of this beta-1 adrenergic blocker prevented the induction of arrhythmias by Juul menthol. So to follow this study up, we're really curious, is it something other than the menthol flavor itself, perhaps, in the Juul and the Blue products, or is it the cooling agent menthol itself that is producing these effects? We were also really curious because disposable pods had kind of taken the market by storm, especially among youth, with ice flavors. So these ice flavors don't actually have really menthol in them, usually. They often have synthetic coolants, WS3 or WS23, at alarmingly high concentrations, up to 45 milligrams per milliliter. And these are very potent coolants that stimulate a TRPM8 receptor, similar to menthol. So to address this, we delivered aerosols involving either vehicle with PGBG plus 2.5% nicotine, or vehicle plus these various coolants, and compared responses in heart rate. We observed, similar to our previous studies, that there is some bradycardia during the puffing session, but there's this kind of biphasic response where there's an increase in heart rate during the washout. And this continues throughout the exposure session with increasing concentrations, but it's especially prominent with the inclusion of these coolants. In addition, we saw that STNN heart rate variability was also reduced. So this exposure increased arrhythmias, in addition, and we were curious if maybe the changes in heart rate and also changes in QT interval might relate to the arrhythmias. We found correlations, interestingly, in the QTC interval in these mice, and that inspired us to investigate further in inducible pluripotent stem cell cardiomyocytes. So we delivered coolants to these HIPSCCMs, both with and without norepinephrine co-treated. And first off, we delivered the norepinephrine to simulate the effects of nicotine, because the HIPSCCMs are not innervated. And we found, not surprisingly, norepinephrine increased beat rate and shortened field potential duration, which is an analog to QT. FPD and QT were inversely correlated. Sorry, FPD and beat rate were inversely correlated, and we corrected for that with a new QT, sorry, FPD correction formula. So we used this FPDC formula to evaluate the effects on repolarization independent of changes in beat rate. We found that the coolants themselves did not actually directly affect beat rate or field potential duration, but when norepinephrine was included, they actually slowed beat rate and accordingly prolonged field potential duration. So this was not particularly surprising that field potential duration would be prolonged in concordance with the slowing of beat rate, but when we applied the FPDC formula, we found that there was actually shortening in FPDC, suggesting accelerated repolarization directly by the inclusion of either WS3 or menthol. So this indicates direct impacts on these HIPSCCMs, both on chronotropy and repolarization. As I mentioned earlier, we did a more long-term exposure study involving 180 puffs per day over the course of 20 days. We found transient effects on heart rate, but consistent elevation in blood pressure, up to 20 millimeters of mercury. And then even after stopping the e-cigarettes, three weeks thereafter, we found a clear bradycardia in these animals with no exposures, as well as an increase in heart rate variability, suggesting parasympathetic dominance. We then examined QT interval and found that there was a prolonged QTC applying our formula, which accurately adjusts for QT according to RR interval. Finally, we evaluated the phosphoproteome in the hearts of these mice, and clearly there's a lot of detail in here. One interesting observation we made was that PITX2 was diminished in expression, but collectively in terms of pathway analyses, we found both protein expression and phosphorylation changed consistent with dilated cardiomyopathy, adrenergic signaling, and arrhythmia. So in conclusion, these studies indicate that e-cigarettes exert cardiotoxicity partly dependent upon their ingredients and activation of the sympathetic nervous system. The ingredients of greatest concern presently that we found are coolants and nicotine and potentially menthol as well. And the effects on heart rate, heart rate variability and QT interval may predict the arrhythmia induction. Long-term e-cigarette use could induce persistent electrical remodeling or autonomic dysregulation, and if these findings translate to humans, limits to these constituents need to be considered by regulatory agencies. So with that, I'd like to thank all the people who will help me conduct this research. Shown here, Anand Ramalingam, he has just published his findings in Jaha that I just presented to you today, and he is looking for a faculty position, so if anybody's looking to hire, let me know. Thank you. Thank you, that was a great series of experimental presentations. There was an online question. Has there been any investigation into the use of cannabis vaporization devices? Yes, I know there's some folks, and I believe even at your institution, investigating the impacts of cannabis, be it THC or some of the synthetic analogs, Delta-9, Delta-8, Delta-10, and I myself have not ventured into that territory, but Matt Springer at UCSF has demonstrated that cannabis either puffed as a combusted aerosol or vaped or ingested impairs, I think it's flow-mediated dilation, vascular function in rats. So there's definitely more research coming out with cannabis, too, and vaping. Yeah, we have just very briefly one more question, and then we'll move on, so if you'd like to. Hi, Kellen Rimmer, Amsterdam. Very interesting. Did you look also at changes in innervation in these hearts after long-term exposure? We're on that right now. The histopath is yet to be performed in one of our studies, and I didn't have time to present it, but we have exposed also pregnant dams to e-cigarette aerosols, and so we're kind of chasing after that specifically because there are more persistent effects in those mice in terms of basal arrhythmias. But no, we haven't yet looked. Thank you for the suggestion. Thank you very much. Thank you. So the final speaker will be Holly Shields from Manchester, UK, and thank you for being here. Okay, so I'd also like to start by thanking the organizers and the chairs for having me here. Let me just see if I can get this started. Okay. And I've been asked to speak about the effects of air pollution on ion channel dysfunction and arrhythmias, and I feel a bit like a fish out of water here at a mammalian conference speaking about arrhythmias. Most of my work has been actually in the aquatic environment, but since that's also been on polycyclic aromatic hydrocarbons, I feel like maybe it's okay that I'm a fish out of water here. So I'm going to tell you about some of the work we've done looking at the specific cardiotoxicity of the polycyclic aromatic hydrocarbon phenanthrene. So we know air pollution is a complex mixture of gases, liquids, and particles, but you may be less aware that it's also high in polycyclic aromatic hydrocarbons. These are compounds that contain two or more fused benzene rings. Here is our compound of interest. This is phenanthrene, which is made of three benzene rings. This means it's a low molecular weight, PAH, and that's quite important because it means that it has the weight when you get combustion of fossil fuels, it can bind to both the surface of the particulate matter, or the PM, but it also exists in large quantities in the gas phase. You can see that here. This is looking at phenanthrene in the, let me see if I can get my pointer. Oh, okay, that's even easier. Okay, so phenanthrene here in the kind of teal color is a percentage of all the other PAHs, and you can see it's the most abundant in the gas phase, but it's also the most abundant in the low molecular weight PAHs adhered to the surface of PM, meaning that as you breathe into the lungs, it's a high route of exposure. Phenanthrene's also really present in the food we eat and the water we drink. It is taken up by plants. It's deposited onto the soils, and of course, it's highly lipophilic, so it also accumulates in the food chain, and these multiple, these high levels and these multiple exposure routes are really concerning because it's directly cardiotoxic. What I'm going to show you today is that it inhibits cardiac ion channels. It inhibits the ion channels of the phase naught, phase one, phase two, and phase three. Interestingly and importantly, it doesn't seem to inhibit phase four ion channels, so IK1, in all studies we've conducted across all vertebrate and invertebrate species, seems to be resistant as is the ligand-gated channels. So that also, to my mind, means that these effects on these other ion channels are specific. We're not just looking at some kind of baseline toxicity. These are specific ion channel effects. And so I think the take-home message, I'll start with that, is that this direct toxicity has to be considered alongside other aspects of toxicity of air pollution in terms of contributing to arrhythmias, ones that we're probably more familiar with, like oxidative stress, immunomodulation, and also imbalance in the autonomic system. So I'm gonna tell you some work we've done. This is really quite straightforward stuff. This is our starting point, which was looking at the ex vivo exposure of mouse hearts and cardiomyocytes to acute doses of finanthrin. What you can see here on the left-hand side, we've got in black our control, and in gray our time-match control. And here we can see what happens to the mouse ECG after 15 minutes of finanthrin exposure. You can see prolongation of the RR interval and prolongation of the PR interval. Mean data is shown here, both the time-match control and the exposure dose, and you can see that we get this consistent bradycardia, a corrected prolonged PR interval. So we know that finanthrin is probably affecting conduction. We looked at this, oops, sorry, going the wrong way. Using multi-electrode arrays on the epicardial surface of the left ventricle, these isolated hearts, and you can see under control conditions, the earliest state of activation is in red, and it progresses to blue. And in the presence of finanthrin, you can see that the scale here is different. Conduction velocity is slowed. You can also see you get greater heterogeneity in the activation map, and here's the mean data. So we know that one of the key ion channels that underlies conduction velocity is the sodium channel. So we patch clamped these myocytes and looked at the impact on sodium current. Here you can see an inward sodium current in black, and you can see the inhibition of the current in the presence of finanthrin. That's at 30 micromolar, but here's the dose response across a range of concentrations, and you can see significant inhibition of the sodium current even at one micromolar finanthrin. So we've got sodium channel block, we've got slowed conductance, and we've got bradycardia, which we know are prorythmic. So what happens to arrhythmia susceptibility? So here we can see trained VCGs. This is under a control heart in the presence of a rapid pacing protocol, and here's the rapid pacing, and we look for tachycardia. Under the control conditions, we rarely see any tachycardia, but in the presence of finanthrin, you can see significant tachycardia. Most of the time, these are monophasic tachycardia, and we get about 10% in our control hearts, and that goes up to about 55% in our finanthrin-exposed hearts. And not only do we see an increase in the presence of tachycardia, but if we also score the type of tachycardia, we get a more severe type in the presence of finanthrin. So we went on in the mouse to show that finanthrin also blocks phase one repolarization, well, actually, the entire repolarization, and also the calcium current, but then it also prolongs the action potential duration. But because repolarization in the mouse heart is so significantly different from the human heart, I thought I'd take the last few minutes to switch gears a little bit and talk about what we know about the effects of finanthrin on the HERB channel. And here, we're gonna move from mouse into a heterologous expression system using hex cells. This is work done in collaboration with my colleague Jules Hancox in Bristol, where he has stably transfected the HERB channel, or transiently transfected mutant HERB channels, and then looked at the impacts of finanthrin. The data I'm showing you here is when he's co-expressed the alpha-1a and alpha-1b HERB channel in these hex cells. This co-expression's important because that's thought to underlie the native mammalian HERB channel isoform distribution. And you can see here these lovely clear tail currents in this expression system under control conditions, and you can see the inhibition of that tail current with 10-micromolar finanthrin. Here, you can see the fractional block. And here, we can see some mean data looking at the fractional block in the EC50, or the IC50, pardon me, across a range of different organisms. So here is the data I just showed you for the expression system for the HERB1a and HERB1b, and we've got an IC50 of about 1.8 micromolar. When we only express HERB1a, that significantly alters the EC50. If we look in a couple of native myocytes, first of all, the zebrafish, which express HERB, we can see the IC50's around 3.3 micromolar, and we've just started some preliminary work with human iPSCs, and it's coming out around 8 micromolar. So how does finanthrin inhibit the HERB channel? Well, together with Jules, we explored this using a combination of site-directed mutagenesis, but also some computational docking studies onto the cryo-EMM structure. Here, the Horg-Perler is shown, kind of as this green ribbon. You've got the potassium spheres transmitting, coming through the selectivity filter, but what I want to draw your attention to is here's finanthrin bonding. What they showed is that it binds to a hydrophobic pocket just outside the pore region, but when it binds, it restricts iron flow through the pore. This is a drug called halifantrin. It was an anti-malarial drug that was taken off the market, and that's because it actually was blocking in the canonical bonding site of the HERB channel, and you can see the structural resemblance of finanthrin and halifantrin. It's interesting, because this compound was pulled because of arrhythmia risk. The EC50 for halifantrin was around 0.2 micromolar, and the therapeutic plasma concentration was around two micromolar. If we think about what we know from finanthrin, we know that the EC50 is probably between two and eight micromolar. We're still trying to work this out, but if you look at human plasma, either in polluted areas, in smokers, in patients with heart failure who also smoke, it can range between low nanomolar to high nanomolar, but upwards of three micromolar, especially in occupationally dangerous things like paving workers and coat plant workers. So what about, so that's all quite concerning, but what about unhealthy hearts? So these are healthy mouse hearts. Well, what we've actually done recently is try to look to see what happens in an aged mouse heart, and we can see that we now are seeing both bradycardia, slow conduction, and increased arrhythmia sensitivity, both ventricular and atrial, or supraventricular, at far lower doses, so at below three micromolar. So to summarize, I told you that acute finanthrin exposure is proarrhythmic in healthy hearts, and I just showed you that the effects can happen in aged hearts at even lower dosages. It impacts both the polarizing and repolarizing ion currents, and therefore direct inhibition of cardiac ion channels should sit alongside other considerations like oxidative stress, immunomodulation, and autonomic imbalance as a trigger for cardiovascular dysfunction in acute poor air quality. I'll end by thanking the fantastic PhD students, Sana and Ellie, whose work I've just showed, Luigi's one of my collaborators in Manchester, Jules Hancock's from Bristol, and my colleagues at Moscow State University. Thank you. Thank you so much, and really elegant work. So I'm going to combine an online question with a related question, because I think you presented some really interesting data on how often it's present in a human sample population that you had. And presumably, those were patients with heart disease, who presumably came from an environment that this is present. Yeah, that was a study from Greece, where they've looked at a population of both people in urban areas, people, smokers. It was kind of a range of populations. And that was the parent compound phenanthroin. They also looked at all the metabolites as well. And so is there any data on what the environmental levels are like, and what happens once you're exposed to it? Does it gradually build up over time, or does your body clear it, and if so, how? And then also, are the exposures similar to PM 2.5, and are they compounding effects? Yeah, those are all great questions. I think the concentration in air ranges, so it can be extremely high. If you look at waste-burning areas or waste-recycling areas, it's outrageously high. But it's high in urban air. But when it's inhaled, it goes through phase 1, phase 2 metabolism. Some of the metabolites, it's not a strong activator of the aerohydrocarbon receptor, but its metabolites do show some cardiotoxicity and also some impacts on iron channels. But it's also highly lipophilic, so it can accumulate to higher levels than I'm talked in the serum in fat. And that's been shown in animal studies, especially in the wild during PAH exposures or oil spills. And so is there any data that once you have an acute exposure, how long does it take for your body to get clear of that? Yeah, so I think one of the key metabolites is 1-hydroxyphenanthrene. And you can see that if you've had a, it's, I think it's a couple of days. And then you see the levels cleared from the metabolites. Thank you. That's right. in the urine. Question on the floor. Hi, Steve Polzing from Virginia Tech. Thank you for a really interesting talk. I failed at my quick PubMed and Google search in the back, so I came up to the microphone to just ask. Are you aware of, or can you point me to any literature on acute phenanthrene toxicity and how it's treated clinically? I don't know. Oh, sorry. No, no worries, I appreciate it. I don't think so. I don't know if it is treated clinically. I think there's no regulation right now on phenanthrene concentrations in air. In fact, PAHs are barely regulated or monitored. When they are, we usually monitor benzoylpyrine, which is not directly cardiotoxic. It does activate downstream mechanisms through aerohydrocarbon receptors, but we use that as a proxy for phenanthrene concentrations. So it's very, we don't really know how much, unless you're specifically measuring it. It's not part of routine measurements. So correlating it with what's happening with patients coming to the hospitals is usually done through PM2.5, because we know what's on the surface. Got it. Okay, thanks. And I also failed at my Google search on how to actually reduce the levels in your immediate environment. And although we know that air filters will work for PM2.5, I didn't find any interventions that will work for this particular exposure. Do you know of anything that people can do to reduce their exposure? I mean, filters do work, and there are filters that have, that are particularly good at taking out PAHs. There are some that actually are in process right now, so people are trying to work on it, and they're hoping to be able to use them either in people with vulnerable populations, people who already have a cardiovascular distress. But I don't think they're on the market yet. But I think general air filters do help somewhat. You. Yeah, I apologize. This isn't really a question, more like an observation, but great talk, by the way. I couldn't help but think about, you mentioned smokers having elevated phenanthrene. I mean, obviously there are a lot of different constituents in cigarette smoke, but one thing that is common among e-cigarette users and smokers is that often they co-use. So if you have one to three micromolar phenanthrene in circulation, and you're delivering nicotine through another very rapid way that doesn't involve all the other combusted stuff, I could see a synergistic effect where otherwise it maybe wouldn't be observed with smoking given the carbon monoxide and all the other things that might be somewhat protective-ish. Yeah, undoubtedly. At any rate, yeah, I don't know how tobacco funding is in England, but if it's there, we might look into that. Thank you. I'm sorry, I think we're out of time, so I'm gonna have you ask a question in person. So thank you very much, everyone, for coming to this session, and I think this is an important area that we still need to pursue and continue, and I hope to see events like this in the future events as well. Thank you so much for coming.

cardiac electrophysiology

air pollution

endocrine-disrupting chemicals

DEHP

e-cigarettes

arrhythmias

cardiovascular disease

polycyclic aromatic hydrocarbons

autonomic imbalance

cardiotoxic effects