Good morning, good morning everybody. It is my pleasure to welcome you today to our session entitled Sex-Specific Cardiac Electrophysiology and Why It Matters in the Clinic. So we will be chairing the session today, Dr. Eleonora Grandi from UC Davis and myself, Beatriz Treanor from Polytechnical University of Valencia. We will have four speakers today, three of them are going to be here present and one of them is going to be online. So we have the pre-recorded session, but you will be able to ask also questions and we will manage to answer them. Okay, then we will start with the first talk by Susan Howlett and the talk is Sex Differences in Preclinical Models of Cardiac Health, Aging and Disease. Hello and thanks so much for inviting me to give this talk. I apologize in advance for my voice, I've got a laryngitis thing going on and I hope that it's going to last. So I'm going to talk today about sex differences in preclinical models of cardiac health, aging and disease. So we all know that this is a percent of the population getting different or getting cardiovascular diseases and we know very well that there are male-female differences in different cardiovascular diseases and how they manifest themselves. But the major risk for developing cardiovascular disease is advanced age in people. And despite this, most animal studies have used young adult and until fairly recently largely male mice and other models to model cardiovascular disease. And for many years, my laboratory has been interested in looking at older animals as models for how the heart changes as a function of age and then for how disease expression may reflect in those models. And so the work that I've done, a lot of it has been done on isolated ventricular myocytes and I've been interested in both aging and in male-female differences in myocyte function. And we know there are receptors for sex steroid hormones including estrogens and androgens in cardiomyocytes from mice and from people. And we also know that estrogens and androgen levels decline with age and this occurs at the same time as heart disease incidence increases. And so we wondered in some experiments whether or not these might be connected. So we know that estradiol levels decline with age in mice and this is just some work from our group. And these are a few young and old mice, a young mouse would be like a three to six month old mouse and an older mouse would be around two years of age. And that's about roughly a 75, 70-year-old human. We can also model low estrogen states in female mice by doing an overectomy and this is showing uterine atrophy. We can actually weigh these and quantify the degree of atrophy. And so in work that was published a long time ago now, we have looked at male-female differences in cardiomyocyte function, calcium handling, and ionic currents. And this is all published, I've listed the papers there. But just to point out one key male-female difference that we saw with respect to aging, I'll get my pointer. When we looked at male mice, this is applying caffeine here in a voltage-clamp myocyte to release calcium from the SR stores or the sarcoplasmic reticulum stores. And we see that both the SR calcium content or the size of this wave is very similar in young and aged male mice. They do have other differences, including reduced calcium currents that I won't discuss, but they show no effect on SR calcium handling. By contrast, if we look at young adult females and males, you can see that there's a much larger release of calcium when we apply caffeine, suggesting that the internal stores become overloaded. We can mimic this finding in over-actimized animals, and so we can recapitulate some of the things we see just as a function of age in an over-actimized model. And so why is this important? Well, the fact that the SR calcium content increases with age in females leads to spontaneous calcium release from the sarcoplasmic reticulum, for example, which we're showing here. And this can trigger arrhythmias, clearly those mediated by delayed after depolarizations, and under some circumstances, potentially, those mediated by early after depolarizations as well. So we know, in addition to estradiol levels declining with age in females, we know that testosterone levels decline with age in the males. And I draw your attention to this heterogeneity here in testosterone levels in the young adult male animals. There is also some variation in the old adult males, but if we look, we've shown here that testosterone declines, serum testosterone declines with age in male mice. But I'd like to draw your attention, this is data of serum testosterone levels from aging male mice, and you can see that the mean is way up here being driven by a number of potentially alpha males in this group with really high levels of testosterone, whereas most of the older mice have very low levels of testosterone. These are just naturally aging mice, C57 black six. And I think it's very interesting in the context of people thinking that female mice are so much more variable than male mice, given these variations in hormone levels. We see a very similar pattern in young adult male mice. So serum testosterone levels are highly variable in older male mice, and I'm not showing it here, but also in younger mice. So in order to remove outliers, shall we say, we gonadectomized the mice, and we then looked at older animals and older animals who've had a gonadectomy. And when we did some cardiac electrophysiology on these ventricular myocytes, we saw that action potential duration was greatly prolonged in the gonadectomized mice, and you can see here that this is significant at APD70 and 90 as well, not shown. So we know that gonadectomy increases the action potential duration. We looked at the underlying currents, and there's some evidence from other work that there may be impacts on potassium currents, but there's been a lot of interest in the late sodium current and its role in prolonging action potential duration. So what we did was we actually measured the late sodium current here in voltage clamp conditions in cells from these mice, and we found that there was a large increase in the size of the late inward sodium current in cells from gonadectomized males. We looked at abnormal electrical recording from these myocytes, and we found activity characteristic of early after depolarizations, which you would expect to see in these really prolonged action potential, cells with prolonged action potentials. We also saw activity with characteristics of delayed after depolarizations or spontaneous activity, and these incidents were much higher in cells from gonadectomized mice than from controls. We showed that the non-selective late inward sodium current blocker, ranolazine, blocked this prolongation action potential, but we then were interested in NAV1.8, which is the late inward sodium current. We used a selective blocker of this at only 30 nanomolar, and we found that treating the cells with this selective NAV1.8 blocker actually normalized action potential duration in the cells from the gonadectomized mice. Similarly, we did look at late inward sodium current, and we were able to show that there was a large and statistically significant decrease in the magnitude of this late inward sodium current carried by NAV1.8 in cells from the gonadectomized male mice. Likewise, we couldn't measure any abnormal electrical activity. So EADs and DADs were just, they were not present. And so then we wondered, we did some Western blot, we also did some qPCR, but suffice to say this is a Western blot for NAV1.8, and we were able to show that NAV1.8 expression was significantly and quite markedly increased in the hearts, the ventricles from mice with this aging mice with gonadectomy. NAV1.5 levels were similar, so there was no difference there. Now this also had implications for in vivo electrophysiology. Here we're showing some sham and gonadectomized recordings of the ECG, and what we found was that there was an increase in the QT interval in vivo in mice with gonadectomy. And this is actually just showing that we quantified that, it's statistically different. We also quantified arrhythmias that we could record on these in vivo recordings, and we found that there were many more arrhythmias in the GDX hearts when compared to sham controls. So just to, in terms of where we're going with some of this, we've been looking at chronic treatment with an androgen receptor antagonist, and we've used flutamide. So we've treated here, this is just showing some teaser data from a project that we're kind of finishing up, but we looked at the androgen receptor antagonist flutamide to see what impact, if any, it might have on cardiac electrophysiology, and also on ventricular function with echocardiography. I've got a whole other project where we're looking at that in low testosterone states as well. But what you can see here, so this is interesting, these are baselines, so this is at baseline when we implant the pellet, and this is going into a mouse at about 20 or 21 months of age. And so we implant the pellet, and you can see here, first in the control or placebo pellet, you can see that QTC actually increases, and this is potentially due to just the endogenous decline in testosterone levels that we see on average in male mice. And it really seems to take off around 18 or 20 months of age in those animals. So we see a significant decline here in the control group, but you see a much larger and statistically significant decline, I'm calling it a decline, lengthening of the interval in the flutamide treated group. So this is an interesting observation that suggests that treating, for example, men who may have cancer with a androgen receptor antagonist may have impacts on electrical function in the heart. And so flutamide then had some similar effects as gonadectomy. So just to conclude, age and ovarectomy seem to promote SR calcium loading, and this may trigger arrhythmias in females through these mechanisms. This doesn't happen in males. Arrhythmia increases the action potential duration in older male mice. This happens through an increase in the late inward sodium current, and this is mediated by an increase in NAV1.8 sodium channels or SCN10A. And the increase in action potential duration is clinically potentially significant as it promotes abnormal electrical activity in cells and increases QT duration and arrhythmias in vivo. And finally, there's newly emerging NAV1.8 sodium channel blockers that are being used for the treatment of pain, and these may be potentially of interest to treat arrhythmias in older men, particularly if there's—with low testosterone. And so I'd just like to thank you for listening and collaborators, especially Peter Nichol, Shubham Banga, and others identified here, and Rob Rose, who's here, who was a co-author on the paper, as well as other colleagues. And I look forward to questions. Thank you. Thank you for this interesting data. Please, go ahead. Peter? Hello. Peter Kohl, Freiburg. Thank you very much for sharing this data with us. A question that always is on my mind, we feed most of our mice with soy-based diets that are high in phytoestrogens. Do you know the levels in your animals? Do you avoid soy in the diet? Because I've heard that it is about 10 orders of magnitude above normal estrogen levels in male mice if you use standard chow. Is that a concern, or is that not really important? It's actually a really prescient question. I know about these soy-based diets. We did feed the mice a soy-based diet, sort of traditional laboratory chow. But we have measured estradiol levels. Now estradiol levels, that's a real dicey thing. I don't know how well we're measuring them, but we can see changes in response to overectomy and things that you expect. We see low levels in serum from males and high levels in serum from young females. What we do see is we do see an increase in estradiol levels in aging male mice. But we think that we haven't measured the phytoestrogens specifically linked to diet, but we do think that what's happening is they have fat, and they get some aromatization. So you end up with higher estradiol levels. So it is interesting that these levels increase in males and decrease in females in the serum. So that's for sure a potential contributor to what we see. I mean, I couldn't say no. So anyway, thanks for the question. It's just a conceptual question from my point of view, because I think Leslie Leinwand showed really impressive effects on cardiovascular function. And so what I conceptually don't understand is why wouldn't one take that out of the equation and go for a casein-based diet in sex-related difference studies? Yeah. I think that's fair. Thanks. Thanks. Katja Odening, University of Bern. Really interesting data, particularly that you looked a lot on the orchiectomy rather than the overectomy that we are usually doing. So I've been working a lot with rabbit models, with overectomy, and then hormone treatment. So I find it very interesting that also the orchiectomy actually does that much of an effect, particularly on the late sodium. But we do know that there are pronounced sex differences also in the L-type calcium that may also contribute to your EAD, DAD formation. So have you looked a bit broader and checked whether other channels are also changed? Yeah, we did measure the calcium current, and we actually found, interestingly, that peak calcium current is decreased in cells from GDX mice. So we don't think that's contributing to what we're seeing with respect to the prolongation of action potential duration. So they're smaller. That's interesting. Okay, thank you very much. You're welcome. Thank you. Our next speaker is Jason Bayer, and he's going to present computational models of sex-specific cardiac electrophysiology. No, I said to finish the presentation before, so it got to this screen, perfect, cool, great. Well thanks, it is an honor to be here. I think it's a really important topic and I'm going to give you the computational side of this session. And so, we started with the question when I got into this was, why should we model the sex differences? Well, we wanted to find a question first. Going through the literature was quite interesting. We found that men, in several studies, were more likely to develop a cardiac arrhythmia than women. However, we also found studies that show that women were more likely to have the higher risk of death, though, from the actual arrhythmia. And one of the questions we asked was, why? Why is this the case? So going from a bigger picture, what influences the electrophysiology of the heart? Well, we've seen hormones in the past talk. There's also differences in the heart size, so we can see between the right and the left, there's differences in the heart size and the shape, and also aging. But the question is, do these impact arrhythmia initiation and or maintenance? So we can study this theoretically directly in humans, but this is dangerous and potentially deadly any time we're dealing with lethal arrhythmias. Bone or hearts is another option, however, they're usually for transplantation and not for experimentation. Animal hearts is also feasible. They're expensive, time-consuming, but it terminates the animal, and as we've seen before, linking it to the human data is also sometimes a challenge. So an alternative that is safe and cost-effective is computational modeling, which is multiscale. We can have cellular models, tissue models, and ventricular models. And in these models, then, we can use simulation software and supercomputing to simulate the electrophysiology of the heart, and particularly, we want to study arrhythmias. So we can study arrhythmias in the atrium as well as the ventricles, and we can do this from the single cell to the organ level. This is pretty common. This is the typical approach, and this wasn't done just yesterday. This actually started back in the 1960s. So single cells were developed in the 80s, we went to tissue, and the past decade or two, we're really working at the full bifentricular and four-chamber hearts. Though, going back and looking at this history line, sex-specific differences really weren't included in this pipeline until about 2012. And here's a paper by Colleen Clancy. And followed looking at really in the larger scale models, this didn't really start occurring until the early 2020s. Here's an example, too, by Ellen Kuhl's group. So building upon this previous work, to answer that original question that I showed you, we said, well, maybe we need to develop new sex-specific models specifically for studying ventricular arrhythmia initiation and also the maintenance. So we applied for funding to do this initiative called the France Berkeley Fund. And from the U.S. side, Eleonora Grani's group here, and her students, Roshni and Haibo, and their expertise in single cell was then combined with our expertise at the tissue level from our group in Bordeaux, France. And so a lot of the work that you're going to see that is done here was mainly by the students here, Roshni, Haibo, and also Vladimir. So we wanted to start first at the single cell level. So circulating hormones can impact the ion channels behavior and structure. It can change the autonomic tone. And all this can influence the electrophysiological characteristics of the heart. So starting at the cell level, particularly the modified calcium channels, as well as the potassium channels. So starting with the single cell model, we can then modify the potassium channel currents of this cell model to generate action potentials that are specific to male and also females. So several groups have already done the hard work by going through the sparse literature and developing the data to fit the models to. But what we need to know is that typically this generates an action potential that's longer in the females than the male. And likewise, we can do this for calcium channel currents. So we can take the single cell model and generate calcium transients that are sex specific. So we see a less calcium transient amplitude in the female versus the male. But then there's also an interesting component at the tissue level, which is the dispersion of repolarization. And interestingly, in the past year, there was a study out of Maastricht and Imperial College where they use ECGI to collect activation maps on the surfaces of the ventricles. I won't go too much into the details because the next talk is going to be very interesting and go into all the aspects of this. But what we need to know is from this study, we can collect activation maps as well as repolarization maps from the ventricles. So we can fit our models to these between male and female patients. So using a pipeline that is existing from Natalia Trajanova's group, we can take image based models. So basically, we can take CT or MRI. We can segment the ventricles of these models, so basically the myocardium. And then we can mesh them to generate computational grids that we can solve the electrophysiology on. And it does a very good job, as you can see here, of collecting the different size and shapes between the male and the female hearts. So inside these models then, we can fit this to the clinical data, the repolarization differences. And you don't need to know the equations. But what we can do is change the diffusion of the electrical waves traveling in the heart in different directions to match the clinical data, as well as we can go back to the single cell model and change spatially within the heart the different ion channel currents to generate repolarization differences seen from the ECGI data. So with this pipeline then, we wanted to go back and ask the question, do sex differences in spatial repolarization impact arrhythmia initiation and or maintenance? So what you see here in this plot here is a bullseye plot of repolarization time. And this is from 16 males and 6 males. So what you see here is the apex, which is in center, is plotted here. And the base around the perimeter of this bullseye plot is plotted here. So we can see that between the male and the females, the repolarization times are elevated. And also, the difference between the base and the apex when plotted between the male and females have an interesting trend where it's positive and negative differences in the females, but predominantly positive differences within the males. So what does all this mean? Then we can apply the models now to this data saying, OK, well, let's look at when the difference in the repolarization times is quite steep in the males. So we take the male model. We apply an arrhythmia initiation protocol by pacing the apex, which is seen black here in the ECGI. And then the arrhythmia that follows it is in red. And in the male heart, we have a very short-lived arrhythmia. But with a very similar repolarization time difference in the female heart, we get a long-lasting arrhythmia. And this is just one case, but we clearly see in this initial study there's already differences. So what did we do? I told you they were healthy patients. So let's start looking at the steeper gradients outside of these bounds, because this is what is known in the literature to generate arrhythmogenesis. So since we have models, we can explore the parameter space much, much bigger. So we can change the differences in repolarization times from the apex to base, as well as from the base to apex. So we have a natural and an inverted gradient. And we can do this for a much larger parameter space than the patient data that we had. And we see this on the right. We have differences in the repolarization time on the x-axis. And then we have the repolarization time in the apex on the y-axis. And so when you see a triangle, that means an arrhythmia occurred from the arrhythmia protocol that I applied. And the darker it is means the longer that it lasted. And interestingly, when we paste from the apex for this natural gradient, we get several arrhythmias. But, as I told you before, the repolarization time in the females is higher than males. So we actually start to see less arrhythmias in the females along that parameter space. And also, if we were to normalize with distance and shape, this actually would shift further to the right as well. So this might explain the initial question of, why are males more vulnerable to arrhythmias? Just the initiation aspect. Well, that's because we can have less differences in repolarization time, but get more arrhythmias. And as a proof of concept, just applying these inverted gradients, too, which we saw on the females but not in the males, the same trend occurs, but we would predominantly mostly see them in just the females if it was in the data that we had. But these arrhythmias can occur when we paste from the RVOT in the opposite direction. But however, going to the arrhythmia maintenance and the duration side of things, we didn't really find a trend between the different durations between the models. So what does that mean? Our next steps, we would like to expand this parameter space to look at more different aspects, and particularly apply it to different types of geometries as well. We would like to analyze the repolarization differences and the arrhythmia complexity with respect to heart size and shape. We really think that might be one of the missing components of the difference we saw from the arrhythmia durations and the severity of the arrhythmias in females. And we'd also like to include more cases, such as long QT syndrome, fibrosis, and hearts with and without scar. So going back to the initial question, did we answer it? As I said, I think these models with the repolarization differences help to explain why the initiation is different between males and females. However, we have not yet really put our finger on what is causing differences in arrhythmia maintenance that may be causing the women to have higher mortality rates. But in all cases, we need more data, and we can determine when and why this happens. So this is really the next step of where we are moving with this project. And so acknowledgments, this work was done by multi-groups across Europe and the U.S. And with that said, I will take any questions that you may have. Hello, thank you for doing the topic. I see in my clinic, a lot of the females are perimenopausal or postmenopausal that come in with arrhythmias that 20-year-olds are now getting, like the SVTs. Do you see any disparities or changes in the models that you do based off of hormones between premenopausal, perimenopausal, postmenopausal women? This is a great question, because this is the next part of the project. So what you're seeing now is a single timestamp. It's kind of a first pass of saying, what are we trying to study? The differences in hormones with age, for example, or timing, we are going to include that, right? But the data right now, it would be nice, you know, talking to people like you, we can get these data and maybe start integrating them into the models, because this is what I really think is important, because we have seen that aging and hormones and all of these things where you study the arrhythmia is very important, and the time that you study it is really important. So I think we can do this, but we haven't done it yet. I feel like a lot of women just totally get brushed off as far as, you know, oh, you're just stressed out, and they're not really getting diagnosed with the arrhythmias first off. The other one is that when you first started, you had men versus women. What was the ratio versus men versus women? In the data that we have? Yeah, so the data was we had more women than men in the patient cohort for the spatial repolarization data. And they were all healthy volunteers, too. This is kind of, we're moving this process as we want to collect data, which you're going to hear in the next talk, hopefully, of more different cases, like different age points as well. And we have looked at the data with respect to age, and there is a trend with the repolarization gradients change with age as well. And this is something we're looking at, and I think that's also a key to what you're alluding to as well. Yes. Thank you. Yeah, sure. Hi. So I think that this, you know, including these additional parameters, it's like great to be able to, you know, make these models more personalized and actually capture the variability within the population and whatnot. The thing that I want to know how, you know, you're thinking about addressing is every time we add a new parameter to these models, it's obviously adding another layer of complexity and sources of errors and uncertainties, especially because the data, like you say, is so sparse for how we're driving these changes. And so, you know, what do you think the way forward is between, like, we want to capture as much as we can, but we also don't want to introduce, you know, new errors because the data we're working with is so sparse? Like how do we balance that? That's a good question as well. Well, abundance of data is not a problem right now because it's not in the sex-specific world. Getting too much data is not an issue. So we are actually addressing our problems to the data we have right now. And it is important. At the very beginning, the question was arrhythmia initiation and maintenance, and it went to the literature, repolarization gradients, we know is one big important thing in terms of animal studies and this. So to get that, we only modify really at the tissue level, you know, where we do change it in a systematic manner, like potassium channel currents, things like this, but we don't really have in the model yet impacts, like we were talking about hormones and all that, from the cellular level to the organ level. That's a whole other problem. So to get just the idea of the arrhythmia initiation, I think this is enough to get an idea to ask more questions, but I think we have to get more detailed at the cellular level as well that might be more patient-specific, more age-specific, more sex-specific. There's so many questions that, yeah, the question should drive that data implement into the model. But I don't have a good answer to that yet about, you know, which data is needed yet because we're just using whatever we can. So it's not abundant, but yeah. Thank you. Thank you, Jason. Thank you for giving me the opportunity to give this presentation virtually. I do apologize for not being able to attend physically, and I hope that things will change and I can attend physically in the future again. I would like to talk about sex differences in activation and recovery, as we have imaged that with non-invasive electrocardiographic imaging. As a disclosure, next to my work as a scientist in Maastricht University, I'm also part-time employed by Philips Innovation and Strategy. I would like to highlight the difference of structural versus functional substrate when we're talking about certain cardiac arrests. For structural substrate, we understand the mechanisms fairly well. For example, in the border zone after myocardial infarction, provide a substrate for delayed conduction and re-entry, and we understand those mechanisms fairly well. For functional substrate, non-structural diseases, we have a much harder time understanding. Such abnormalities may come from conduction, reprolization, or may even be unknown, for example, in the case of idiopathic ventricular fibrillation, so we have a much harder time to understand those mechanisms. I want to illustrate with this case that we believe that repolarization in many of such functional substrates is actually a key mechanism. In this case in Maastricht, a R on T phenomena resulted in polymorphic VT. However, we could not find a real cause or a real substrate in this individual. We then applied non-invasive electrocardiographic imaging and discovered that repolarization in this individual was abnormal. You can see region 1 and region 2 on the right ventricle of this patient's heart. You can see that there is a region of early repolarization very next to a region of very late repolarization. That's what we call a repolarization gradient. Our hypothesis is that for unexplained string-guided death or for idiopathic ventricular fibrillation, repolarization and repolarization gradients will actually play a key role. We've also investigated this with explanted hearts, where we had a mapping of activation but also of repolarization. We had the ability to change repolarization in these hearts by infusing drugs in different regions of the heart, creating a very pronounced region of early repolarization and a pronounced region of late repolarization. When giving a stimulus in the early repolarizing region, we could easily induce reentry. We noticed that the steeper these repolarization time gradients by higher drug infusions, the easier it was to induce reentry. To really study electrical substrate, we should be studying repolarization. Currently we have a knowledge gap because electrical substrate is currently not part of risk assessment. We're used to look at ejection fraction or perhaps scar on imaging, but we do not have the ability to in detail study the electrical substrate. Non-invasive mapping of electrical characteristics would therefore be very useful. We want to use electrocardiographic imaging or ECGI for that purpose. Electrocardiographic imaging allows us to combine a multitude of body surface potential recordings with imaging to non-invasively reconstruct the potentials directly at the epicaleal surface. You can then visualize these epicaleal potentials as electrograms and from the activation and recovery times that you determine per electrogram, you can then display these 3D visuals of the patient's heart with activation and recovery isochrones. If you want to compare patients, however, it's often more useful not to study these 3D structures, but to actually map this down again to a bullseye plot where we map the activation and recovery times on a bullseye that shows both the left and the right franticle in the same picture. This technology allows us to revisit the electrical activity of the healthy heart. I'm sure you're familiar with the Dürer studies from the 1970s, where Professor Dürer explanted human hearts of healthy deceased individuals, and he studied activation in great detail. We can use ECGI to do something similar now and also study on top of that repolarization. I will be showing you the work of my PG student Job Stocks, who has done all of the work I'll be displaying here, and this to me is one of the most intriguing findings. These are three healthy individuals. You can see lead II of their ECG, and I think you'll appreciate that lead II of their ECG looks very similar between these three individuals. However, applying electrochromographic imaging shows that the activation times are actually vastly different. So the activation bullseye look very different with very different regions of earliest and latest activation. And you can see that here for all the individuals that we studied, so these are 22 healthy individuals, and you can see that their lead II electrocardiograms are often very similar, but that their bullseyes for both activation and recovery are very, very different. In other words, the ECG does not capture the complexity and the sequence of activation that we see for both activation and recovery. If we stick to two individuals and we follow them over time, in the time span of some beats or even a few minutes between beats, we do see that activation and recovery are very stable within each individual. So whereas the differences between individuals are large, the similarity between an individual is actually very similar and very stable. Applying this in our toolbox now allows us to study sex differences in cardiac electrophysiology. Here you can see that we studied activation, and perhaps not to our surprise, activation is not different between males and females. Not if we study the activation as an average activation time for each of these hearts. Also not when we split it between activation time on the left ventricle versus right ventricle. Activation does not differ between sexes. The story is very different for repolarization. Here you see the average repolarization for each of the males and females in our cohort. And obviously, as we would have expected, repolarization is delayed in females. Interestingly, this is the case both for the repolarization of the left ventricle and the repolarization of the right ventricle. We can zoom in even further, and then we start to really discover interesting differences. So what you see here is not the average repolarization time, but the average dispersion of repolarization, or in other words, how long between the earliest repolarization time and the latest repolarization time. You can see that on the whole heart, this is not different between males and females. But if we split it up between the left and right ventricle, we see that females have shorter durations of repolarization. We can also study when repolarization starts, and this is earlier in females. Combining this, and trying to use my drawing skills for the best purposes, we can then conclude that repolarization is vastly different between males and females, and that this has several components. Firstly, repolarization starts earlier in males, it ends earlier in males, and it takes longer in males still. Whereas in females, repolarization starts later, it also ends later, but still it has a shorter dispersion. Knowing things like this then allows us to better understand disease. Here you can see three examples. The first is a healthy individual, where we show you the ECG, the activation and repolarization isochrones, as well as the histograms of activation and repolarization times. And then for long QT patients, patient A and patient B, you can see that obviously repolarization is very, very abnormal. Understanding this in the context of sex differences, of course, is hugely important. We can go one step further and now start to understand this with computer models. So what we can do is implement abnormalities that we see in patients. We create a computer model that mimics such abnormalities and allows us to induce virtual arrhythmias. And then study such virtual arrhythmias with various characteristics, trying to match males versus female sex characteristics, for example. This allows us to study the vulnerable windows in each of these conditions, trying to understand when such repolarization differences are actually arrhythmiogenic and when perhaps they're benign. All this work has led us to believe that we can extend on the triangle of Kummel and what we have called the circle of reentry, that more explicitly takes into account the dispersion of excitability, both in time and in space, as well the characteristics of the trigger that initiates reentry, also in spatiotemporal characteristics. So what I think the main messages are is that in females, the recovery time is generally later. This is both due to a delayed onset of discovery and despite a shorter dispersion of recovery in females. Activation is very similar between males and females. And each individual, while being very different from other individuals, each individual has a very stable activation recovery pattern, at least in the time span of minutes. We believe that such female-specific recovery characteristics may help to explain women's different susceptibility to certain arrhythmias and their response to certain medications. And I think it's important to realize that such important differences between sexes are actually very easily missed on standard electrocardiograms, but may be uncovered by electrocardiographic imaging. And with that, I would like to thank everybody who contributed to this work, and I would like to thank you for your attention. And I hope that everything works out, I can actually answer some of the questions from the audience. Thank you very much. And Matthijs actually can hear us right now, and he's ready to take any questions from the audience. Yeah, hi, Susan Hallett from Canada. Just wondering whether, and I'm not sure if you said this, I might have missed it, whether you looked at age, thinking a little bit about sex steroid hormones and how they might change with age. And I don't know if you got there yet, just interested in your thoughts. Could you hear the question? Okay, yeah, the question, if you thought about age, and specifically, you know, the effect of hormones, if you've got to that point yet. Just wait a sec, wait a sec, wait a sec. Okay, try again. Let me know when I can talk. Yes, now we can hear you. Yes, great, thanks. Thanks for the opportunity to answer questions live. Much appreciated for the support by my chairs. And thanks for the question, that's a very interesting question. As you may have seen, we only had 22 healthy individuals. We did do an age investigation that I think I removed ultimately from the slide deck, so sorry for not showing you that. But we did see changes between patients of different ages. We didn't have the power to differentiate between sexes. I think age in several way impacts electrophysiology, but definitely sex hormones will play a role there as well. And I think we need to have larger studies to really investigate that with electrochirographic imaging. But I also really appreciate the power of computational models, because we do know quite a lot about sex hormones. And we do have, of course, the ability to at least do some studies already through computational modeling before we try to see that in larger patient studies. That will be fascinating, thank you. We have another question. Hey, Mattia, it's lovely to hear your voice. I'm sorry you couldn't make it here. I was wondering this modal fusion between the ECGI and the computational modeling. It's fascinating to see these two brought together. I wonder if you could speak a little bit of, do you see this as kind of the next step in applying this clinically? Because for a while now, we've had people attempting to apply computational models in the clinic and applying ECGI separately in the clinic. Is this the way forward is to try and use them together? And if so, what's missing? What do we need next to kind of bring that fusion together? Can you hear? Great, thank you. I could actually hear Pat's question very well. So thank you very much for that question. So you're asking me whether I believe in digital twins, right, for clinical purposes? Yeah. And so I have two hats, right? I have the industry hat, where I actually did work on digital twins for a while. And I have the academic hat. I think you also need two different approaches if you're working with digital twins. So for scientific insights, for example, studying the effect of sex hormones, I think such approaches are hugely valuable to get new mechanistic insights. I think if you want to bring such tools to clinics, we need to simplify things considerably because we usually do not have sufficient data to really create digital twins, either in the time span allowed by us or to us by the physicians, or simply because we don't have the data and we have way too complex models. So for that purpose, I think we need to simplify things considerably. And we really need to pick our battles. I do believe there are certain applications in certain cargo risk stratification where simplified models would be useful. But whether that's the highly detailed 3D structures built on high quality imaging with invasive mapping and non-invasive mapping fused, I think that's still a question to answer. I'm not sure whether we're going to answer that question positively. Thank you. Thank you. We thank Matthijs once again. And it is my pleasure to invite to the podium our last speaker to wrap up the session talking about the clinical implications of sex differences in cardiac arrhythmias. Anne Curtis from the University of Buffalo. Thank you, I found it fascinating to hear some of the latest results from basic research because they do influence what happens in the clinic and that's my focus. So I want to talk about, there's a lot to cover in a very short period of time but just really an overview of some of the sex differences in cardiac arrhythmias and you can see the range here from supraventricular tachycardia and AFib through catheter ablation and ventricular arrhythmia as an implantable device therapy. So first of all, we saw some action potentials and also some electrocardiographic signals and there are differences between men and women that are well known but given this table I just really want to highlight two of them. The first is that QRS duration is shorter in women than in men and seven milliseconds may not look that much but it does have an impact on some of the treatments that we do particularly for heart failure. And then the second one I want to highlight is the QT interval or the corrected QT interval which is longer in women than in men and that goes to some of the findings that we saw about repolarization in the heart. So first there are several different kinds of arrhythmias that we take care of in the clinic. There's a condition called inappropriate sinus tachycardia. It is much more common in women. I have almost never seen a man with it and particularly in younger women and there's also a condition called postural orthostatic tachycardia syndrome and basically what these are are heart rates, sinus heart rates that are much faster than would be expected for the level of activity often at rest that the patient has. And then there's some other reentrant supraventricular tachycardias, AV nodal reentrant tachycardia and AV reentrant tachycardia, the latter associated with the Wolff-Parkinson-White syndrome that have radically different frequencies in men and women. This is an old slide but it does show what we see as the general distribution. So if I see a woman with a narrow complex tachycardia and I look at the electrocardiogram and I can't see a P-wave, I'm almost certain I'm going to be dealing with AV nodal reentrant tachycardia which informs how we do the ablation procedure to cure it. And you can see the distributions, AT is atrial tachycardia and that has about the same frequency in men and women. So atrial fibrillation, the incidence is lower in women. So the female to male incidence is 1 to 1.5. But the prevalence remains unchanged with aging and because women have greater longevity, there are more women with AFib in the elderly population and so therefore the total number of patients is a little bit higher in women. So women with atrial fibrillation tend to be older, they have a lower quality of life, more significant comorbidities and more symptoms. The CHADS-VASc score, so for those who are clinicians in the audience, know this as how we determine whether patients with atrial fibrillation need anticoagulation. The last two letters SC stand for sex category or in other words female sex which is a modifier of stroke risk and has been viewed as an increased risk of stroke in women. Antiorrhythmic drugs are used to treat arrhythmias. The class one antiorrhythmic drugs are sodium channel blockers. Class threes are potassium channel blockers which would impact action potential duration. And these drugs more often cause torsades de pointes in women. So the polymorphic ventricular arrhythmias we see with QT prolongation. Efficacy of antiorrhythmic drugs is about the same in men and women. But in general women are prescribed antiorrhythmic drugs less often than men. And so treatment in general whether it's ablation, actually more so than antiorrhythmic drugs, is just less often used in women. So catheter ablation is a treatment for atrial fibrillation that has become more and more popular. You see a lot of that at this meeting for the Heart Rhythm Society. And when you look at women having worse quality of life in atrial fibrillation and more adverse effects from antiorrhythmic drugs, you would think that they would be referred more often for catheter ablation. And the opposite actually is true. They are referred less often. And what are the reasons for that? Some of it may be that they develop atrial fibrillation later. As I showed in younger age groups, men are more likely to get it. And then there's also a possibility of later referral for the procedure. If they get referred for catheter ablation, the success rates are similar between the sexes, although the rate of complications in women from the procedure itself has tended to be higher in some registries. So moving on to sex differences and ventricular arrhythmias and sudden cardiac death, which actually I think was more spoken to in some of the data that we saw about the ventricles and repolarization. There are a number of different ventricular arrhythmias that we do not have time to go through in detail. I just want to highlight two right here. And so one is sudden cardiac death, which is more common in men than in women. And the acquired long QT syndrome, by which we mean when it occurs because of the use of drugs that can prolong the QT interval or electrolyte abnormalities, that is more common in women than in men. So in data looking at the incidence of sudden unexpected death, you can go back into the 1990s with some very nice data about this, but this is a more contemporary look at it. And things haven't changed. So if you look at how often men versus women have sudden cardiac death, much more common in men. And you can see the red line there, and you can see the incidence per 100,000. And you can also see that the mean age is about 70 in women and 65 in men. If you look at underlying heart disease in people who have survived a sudden cardiac arrest, you can see that overwhelmingly coronary artery disease is the underlying substrate in men, with the next most common diagnosis being dilated cardiomyopathy. In women, fewer than half of those who are resuscitated from sudden cardiac death have coronary disease. And dilated cardiomyopathy is about twice as common as it would be in men. When one survives an out-of-hospital cardiac arrest, it's a big save. But when we look at the types of arrhythmias that are presenting at the time of cardiac arrest, there are sex differences that are seen there as well. So in this study from Dallas, Texas, they showed that if you looked at out-of-hospital cardiac arrest, women were older at the time. This is a consistent finding. They interestingly had higher resuscitation rates. And the reason why I say that's interesting is that the easiest arrhythmia to save somebody from as a cardiac arrest is ventricular tachycardia or fibrillation, which is more common in men than in women. You can see 41 percent versus 30 percent, whereas asystole and pulseless electrical activity, which tends to have the lowest survival rates, are more common in women than in men. In the long QT syndrome, there's congenital long QT syndrome, and that's what I'm talking about here, LQT1, 2, and 3. The risk of cardiac events in adult women with long QT syndrome is three times higher than in men, and the highest rate of events is with LQTS2, which is a potassium channel problem, and men at the earliest ages are going to have more often have sudden cardiac arrest. And you can see differences in the event rates there. So there's some clear-cut sex differences in congenital long QT syndrome. There are also perimenopausal changes in the risk of cardiac events in patients with long QT syndrome. So you can see with long QT1, the incidence goes down. The later one gets in the menstrual age, whereas it goes up in LQTS2. Moving on to implantable device therapy, the main ones I want to talk about are defibrillators and cardiac resynchronization devices. Implantable defibrillators are meant to save patients from ventricular arrhythmias. I've got the same laryngitis as one of the previous speakers. But anyway, I looked at this some years back with one of my fellows at some of the early defibrillator trials, and AVID was one of them. The rest of the trials that are listed here were primary prevention trials. When we say primary prevention, we mean that a patient is at risk for having a ventricular arrhythmia but hasn't had one yet. And in looking at the studies, what was interesting to me in looking at this, most of the time, no one even stratified them by men versus women. And if they did, frequently, there really wasn't much of a difference. And so we need to know more about this in men and women. A meta-analysis of ICD trials that looked at almost 1,000 women looked at the effect of ICD therapy for primary prevention of sudden cardiac death in patients with heart failure and actually didn't find any significant decrease in all-cause mortality in women with heart failure who received ICDs. It's a very concerning finding because our guidelines actually dictate that we don't treat men and women differently. The problem is that with the studies that we've done so far, the fact that they were so heavily male-dominated, the number of women in the studies was so small, it really made it difficult to tell the difference. And yet, how could one do the study today to try to tease that out a little bit better? So we're left with a little bit of uncertainty there, although the guidelines still dictate similar treatment. In terms of sex differences with CRT, cardiac resynchronization therapy is a treatment for heart failure in which patients who have dyssynchrony in the ventricles that is manifested by a prolonged QRS duration, usually left bundle branch block, by pacing the left and right ventricles simultaneously, we resynchronize the heart. It's a very effective treatment for heart failure. This is another study I did with one of my colleagues, and we looked at women versus men in one of the early ICD trials, CRT trials, and we found that women had a much better result with resynchronization therapy than men. And I think this speaks back to what I said originally about QRS duration. Women have shorter QRS durations to start with, so when you get a prolonged QRS duration in men and women, it's a relatively larger difference in women, and therefore, I think they're more likely to respond to CRT. And in other studies looking at this, we've seen similar findings whereby women respond better to cardiac resynchronization than men. I don't have time to talk about arrhythmias in pregnancy, but there are clear-cut differences that we keep in mind when we take care of these patients in the clinic. So in conclusion, there are significant sex-based differences in the incidence, prevalence, and clinical characteristics of arrhythmias. The outcome of pharmacologic and non-pharmacologic treatment of arrhythmias is similar in women and men, but there are notable sex differences in the incidence of sudden cardiac death and the response to cardiac resynchronization therapy for heart failure. Thank you. Hello. Thank you for that really great overview, and I'm going to your topic with CRT. It seems interesting. So females, from our side, from the modeling, the APD is always very long, right? So whether you apply pacing or not, you're always going to see a change, probably, in the QRS, where the male heart, you may or may not, right? So is this discrepancy maybe explain possibly why the females respond better? Yeah, well, the way the studies were done is there was a minimum QRS cutoff to be able to get into the studies. And so one of the ones I was heavily involved in, we set a cutoff of 130 milliseconds. And so if you have to have a QRS duration of at least 130 milliseconds, and the average to start with is 91 versus 98, that, you know, there was that relative bigger difference in women versus men. And so when you do the resynchronization, the QRS duration that you get to with resynchronization varies quite a bit. That hasn't been as predictive, and it has to do, to some extent, with where you place the leads. So it's really hard to get into that right now. But if you have a lead in the right ventricle and one in the left ventricle, depending on how posterior or inferior it is, you can get more resynchronization versus less. And so that also dictates some of the results that we see. OK. Cool. Thank you. Hi. Thank you. I'm very impressed by this. Thank you very much. Question for you. With, in regards to medications and using antiarrhythmic therapy, I first heard that, maybe correct me if I'm wrong, that females did not get the same outcome as males did with using antiarrhythmic therapy, correct? The efficacy is the same, but they have a greater incidence of adverse effects. OK. That's what I was wondering, if it was metabolism versus, you know. It has more to do with the QT interval, because so much of the adverse effects of antiarrhythmic drugs are because of QT prolongation. And since women have longer QTs to start with, they tend to be more susceptible to that. Thank you. Yeah. Thanks so much for your talk. So I'm wondering, especially in relation to the AF data that you talked about with women having worse comorbidities and outcomes, and I'm wondering what data exists before that in terms of, are there sex differences in terms of male versus female experience to diagnosis, for example, from when the patient first experiences events versus when it actually is diagnosed? Is there any difference in the time frame of males versus females, or when they are diagnosed, whether the severity is different? If I'm hearing you right, yeah, a lot of, I mean, there have actually been studies that show that a lot of times women with SVT are initially diagnosed as having panic disorders and anxiety, right? And then it's eventually figured out that there's actually an underlying substrate there, whereas with men, if they complain of palpitations, there's more of a tendency to believe there's got to be something real there and go look for it. So some delay in diagnosis, for sure, underlies some of the delays in treatment. Thanks. Hi. Thank you. It seems like, you know, the clinical definition of right or left bladder branch block complete is 120 milliseconds. It seems like it should be reduced for women. Yeah. Well, I mean, you know, I guess if you could actually look at a, I think that's true. And I'm actually very glad when we did the miracle trial, that was an early pivotal trial, I didn't realize it at the time, but I argued to get the QRS duration reduced from, you know, 150 down to 130 milliseconds. And I'm glad we did, because that's how we picked up that difference in women. If you have to go out that far, it just is fewer people are going to benefit from it. But absolutely. And, you know, some of the guidelines now are actually paying a little more attention to say, you know what, if your QRS duration is at least 120 milliseconds, and you've got bundle branch block, and you're a woman, that's more of an argument to go ahead with cardiac resynchronization therapy than men. So there is that distinction that's actually starting to show up. Thank you. We thank our speaker once again. Thank you, Dr. Curtis. Thank you. And this concludes our session. I want to thank all of the speakers and everyone in attendance for remaining engaged, and enjoy the rest of the conference.

sex-specific cardiac electrophysiology

cardiac differences

clinical settings

arrhythmias

sex hormones

calcium handling

repolarization gradients

electrocardiographic imaging

QT intervals

cardiac resynchronization therapy