It's a great pleasure to introduce Mario Delmo, who's going to tell us about the many faces of ARVC. So we don't have pointers because I can't do this. Just use the, this one here. Yeah, but I, do you see my arrow there? You do? Yeah, that's the problem. I had none there. All right. Okay. Well, we, I think we passed the disclosure slide, but you saw that I have no disclosures. And so if you want to give me money, your name could be in that, in that slide. I was asked to talk about the multiple faces of ARVC. And so I will start first by giving you a quick reminder, though this audience may not need it, of the main features of ARVC. This is the slide that has been shown, I think many times, is the heart of a 16 year old boy who died suddenly during a soccer game. This is the postmortem MRI and you can see the loss of ventricular mass in the free wall of the right ventricle that has been substituted, as per this histology, by adipose and collagen tissue. There is very little muscle remaining on the right side. On the other hand, on the left side, there is quite a bit of preservation of the muscle, although you can already see collagen infiltration. So it's not unique and exclusive of the right ventricle, but it has a right ventricular predominance. And you can see that the septum, as in many cases, is spared. This disease has been associated with mutations in genes that have been first identified in the desmosome. And so I think that the paradigm has been classically this one. There is one gene of the desmosome that causes one phenotype, arrhythmogenic right ventricular cardiomyopathy. I would like to say that this sort of a box, this monogenic model of one gene, one phenotype, is really not consistent with what we know. In fact, I take this slide from Connie Bezina. I like it because I think it speaks about multiple genetically determined conditions in which you rarely have this. You rarely have the yellow gene being with such a high penetrance that it goes from disease susceptibility all the way to your disease threshold and crosses it and gives you a disease phenotype. I think it is more common, and we are learning to be more common, that you have either a near monogenic situation or perhaps the most common one, where you have the summation of multiple genes, some with a small effect, some with a large effect, but that in a combination give way to a phenotype. And this is really moving the paradigm from classic monogenic diseases, from Mendelian distribution, to complex genetics. We do not know in ARVC what these genes are. We do not know, actually, in many of the inheritable arrhythmia diseases, but I think that a major frontier in the knowledge of ARVC is trying to figure out what are these genes. So we moved from the model of one gene, one disease, to the complex genetics model, where multiple genes in ways that add up, in ways that we don't quite understand so far, give us one phenotype. Now, this is certainly very important, but what I would like to emphasize in this conversation is the additional reverse problem, and that is, this is multiple genes to one phenotype. I would like to speak about one gene and multiple phenotypes, and this is called pleiotropism. So we have about 20,000 genes being expressed in the heart, and if you think of each one of these genes as being capable of doing only one thing, you really don't have enough genes to do everything that a cardiac cell would do. It is more logical to think that the gene codes for a protein that in itself has multiple functions. And I often, and you may have, some of you that have been in presentations that I've been in before may have heard me of this example, that in molecular cardiology we made the same mistake that endocrinologists made in the 19th and 20th century, and that is to name hormones after the very first thing they found them to do. So growth hormone is very important for growth, but I today now need growth hormone, and it's not because I have any hopes of getting any taller. It's because there are other functions to the same molecule. In the same way, connexin 43 is a gap junction molecule. There is no doubt that it makes gap junctions, but it is capable of doing things other than gap junctions, and it is in that pleiotropism where we may find answers to important questions that remain unsolved in the mystery of inheritable arrhythmias or cardiomyopathies. In the particular case that I am describing today, you can actually have one gene giving you multiple phenotypes, but that phenotype may go from phase one, from phenotype one, to phenotype two, to phenotype three, and so on. So you may have a pleiotropic disease where the manifestations don't necessarily have to present at the same time. So if you want to diagnose a disease only by the presence of all the elements that you classically have defined as the phenotype of the disease, you may be missing pieces. We have taken for years that approach to the study of placofilin-2-related disease because, frankly, I think that placofilin-2 is a pleiotropic gene, and arrhythmogenic is a trait, and cardiomyopathy is an independent trait, and right ventricular predominance is an independent trait. There is no doubt that you can have arrhythmogenic right ventricular cardiomyopathy, but you don't necessarily have to have all of them at the same time. You may have elements of PKP-2 that lead you to arrhythmogenicity or to the right ventricular predominance or to the cardiomyopathy. The important thing is to discover what are what we call the endophenotypes of the gene, and that is the phenotypes that are directly related to the expression or not of the gene. And that is where knockout models in mice or in large animals, when it is possible, are so helpful. It's not that when you make a knockout model of a gene, you are reproducing the disease. This is the way. This is one knockout that we made and that I will show some data about. This is a knockout of an adult mouse that grows happily until we inject it with tamoxifen, and we inject it with tamoxifen, only the placofilin-2 gene, only in the myocytes, and in both alleles, it's going to get knocked out. Does this mouse have ARVC now? Right? And frankly, and with all due respect to my friends and colleagues, there is no mouse or cell model of ARVC that, by that definition, reproduces ARVC. But what we're trying to do is to find out what are the endophenotypes of PKP-2, and from there extrapolate the mechanisms by which it causes the disease. When we inject this mouse with tamoxifen, the phenotype progresses, and I'm not going to go into details, by 21 days after we injected the mice into this. This is the heart of the mouse. If we have exaggerated the contrast for collagen, just to show you that I have a cardiomyopathy of right ventricular predominance, similar. Of course, this is, I'm not saying it's Photoshop, but it is just for displaying purposes, similar to what you find in the patient. And this is a very arrhythmogenic phenotype, so by knocking out a gene, we show that there are three endophenotypes of PKP-2, arrhythmias, right ventricular predominance, and cardiomyopathy. Now, the beauty of these models is that then you can take a step back. I can see now what would be the clinical phenotype of these mice, but I can go to subclinical phenotypes all the way to cells and molecules because I can interrupt the life of this mouse at whatever point after I have induced the knockout I want it to be. And what we have found, because we have been very interested in the mechanisms of arrhythmias in these mice, is that we go back to day 14, when these mice structurally are normal, when they are perfectly capable of running and, you know, having a normal life. We take their cells, and I just want you to focus on the difference between this red bar, which is the eagerness of the junctional sarcoplasmic reticulum for releasing calcium, which, of course, you know, would be leaved in to triggered activity, versus the left ventricular cells of the knockout or the right or left ventricular cells of the knockout. These mice, at this stage, have, in the right ventricular myocytes only, not in the left, a very high propensity to release calcium. And this translates in that the right ventricular myocytes have a very high propensity to initiate triggered activity. The loss of placofilin II causes a major dysfunction of the ability of the heart myocyte to control intracellular calcium. So this leaves us with the idea that there is, in ARVC, a structural substrate, no doubt about it. But as Igor just showed, actually that structural substrate is more dangerous when you still have a lot of ventricular mass and you have triggered activity that can be the trigger for the arrhythmias. And indeed, with Greg Morley, we have shown that you can have, at this stage, in which you have a structurally normal heart, base-induced ventricular fibrillation. And this is a camera focused on the right side, just to show you that you actually don't see any interruption of conduction. You just see arrhythmia. So what I would like, in the spirit of following the theme that I was asked to speak about, to tell you that placofilin II and leave you with a message that placofilin II is a pleiotropic gene. And this may not be the only pleiotropic gene that causes disease in an inheritable manner in the cardiac sphere. There is work coming up showing that structural genes that have been associated with cardiomyopathies can have, as an endophenotype, arrhythmias, independent of the changes in the sarcomere. So in the case of PKP2, we have known for a while that can affect the adjunctions, that can affect sodium channel complex, and that can affect cell adhesion, as well as, through transcription, changes in intracellular calcium. Now, this can give us impaired conduction and excitability. This can give us impaired contractility and increased cell death. This here can give us triggered activity. Altogether, these ingredients would lead us into arrhythmia and sudden death. These ones to cardiomyopathies that, of course, through fibrosis would feed into the arrhythmia mechanism. All of this would be arrhythmogenic right ventricular cardiomyopathy. But the important thing, and the point I want to leave you with, is that these are endophenotype manifestations of a pleiotropic gene in such a way that I don't have to come down all four arms at the same time. If I come down this arm at some point, I may think that this patient has Brugada syndrome. It doesn't have classic sodium dysfunction Brugada syndrome. It is a phase in the pleiotropism of a PKP2-related disease. If I come down this arm, I may think that my patient has CPVT. It is not classic RYR2-dependent CPVT. Now, if I come down this way, I may end up with a patient that has a dilated cardiomyopathy. But it is part of the whole spectrum that, when it is presented together, can be diagnosed as arrhythmogenic right ventricular cardiomyopathy. Thank you very much for the invitation and for your attention. Fabulous. One of the best talks I've ever heard. Do you think this pleiotropy exists in the hypertrophic cardiomyopathy genes, the sarcomeric genes, even something as classically monogenic as Long QT syndrome? I think that in Long QT syndrome, someone presented this morning a case of Long QT syndrome that some members of the family had also a cardiomyopathy. The only thing that they can find is a mutation that relates to the Long QT. There is also work, I know, that has been done by Bjorn Ullman, showing that in one of the sarcomeric, using IPS-derived cardiomyocytes, that some of the mutations, I want to say in myosin-binding protein C, change the affinity of the sarcomere for calcium. And in doing that, can lead to arrhythmias, regardless of the contractile state. And so, I think that we, of course, know that cardiomyopathies can go hand-in-hand on arrhythmias. The mechanisms of arrhythmias in cardiomyopathies, and I think this is an extreme case of that, does not have to do re-entry. It has to probably include re-entry, but the trigger for the re-entry can come from a mutation in the sarcomere. The trigger for the re-entry can come from endophenotypes of the cell that facilitate triggered activity or automaticity. Okay, we'll go on with our next talk. Thank you.

arrhythmogenic right ventricular cardiomyopathy

monogenic disease

mutations

desmosome

complex genetic condition