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Orthobiologics & Regenerative Medicine Series: Mus ...
Muscle Regeneration
Muscle Regeneration
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Good evening, everybody. I first want to thank all those who have attended tonight. My name is Shannon Strader, and I am this year's RFC research representative. I'm also the moderator and creator for the AAP and ARTS Orthobiologics and Regen Med series. The goal for this webinar is to provide comprehensive education for physicians in training and physiatrists interested in regenerative rehabilitation while reducing stigma, misinformation, and encouraging responsible advancements for the regenerative field. ARTS is offering certificates for those who attend eight webinars this year, and if during this session you have a question, please write in the chat box, and we will answer as many as time permits. It gives me great pleasure in introducing our brilliant speaker tonight and my personal favorite PI, Dr. Dhammageddy. Dr. Dhammageddy started his scientific career at the University of Geneva in Switzerland, where he obtained his diploma in biology. He then joined the University of Melbourne in Australia, where he obtained his PhD in physiology. From 2007 to 2015, he worked as a postdoctoral fellow and project scientist at the University of California in San Diego. Since 2015, he serves as a research scientist and principal investigator at Shirley Ryan Ability Lab and Northwestern University PM&R Department in Chicago. Dr. Dhammageddy works to identify new preclinical and translational mechanisms relating to skeletal muscle impairment and neurological and neuromuscular conditions, as well as generic and epigenetic biomarkers affecting patients, therapy, and disease outcomes. Some of his clinical and translational interests include drug repurposing and identification of macular and cellular biomarkers for the development of new therapeutics and muscle and nerve rehabilitation. Thank you so much, Dr. Dhammageddy. Well, thank you, Shannon. I just want to make sure that you're hearing me correctly. We spoke, but yeah, I assume that it's good. I would like to thank you so for the kind introduction first, and we'd like to thank also the AAPS residents and fellow council and ART3T to give me this opportunity to speak tonight. And as you can see today, I'm going to speak about treatment of muscle and motor dysfunction in children with cerebral palsy. Is there the possibility eventually to unlock the potential of the resident muscles themselves to treat muscle impairment in these patient population? So I'm going to go through the three points that I already indicated before, but before to do so, I'm just going to be starting my presentation by telling you where I'm located. I'm actually working in my lab. It's located within the Shealy Ryan Ability Lab, which is a rehabilitation PMNR focused hospital affiliated with Northwestern University School of Medicine in Chicago. It used to be known as the Rehabilitation Institute of Chicago, but it's been moved into this fantastic new building since 2017. And thanks also to the vision of our late CEO Johan Smith, the new Shealy Ryan Ability Lab built a fantastic biomedical research facility within the 26th floor of the building, which is a fully equipped and fully functional locations where you can perform a lot of molecular tissue and biological research and integrate that with translational research and clinical research within the hospital. So it's a fantastic place where to work and develop new discoveries for the sake of and for the good of the patients. So within the biologics lab, which is this biomedical facility, we are several different participants, principal investigators that with our own expertise cover pretty much these different components of different system of the body going from the central nervous system to the peripheral nerve system, to the musculoskeletal component. I believe that Dr. Jaya Balan would also give a talk on this series in September or October. So you will be hearing more about the type of research that we conduct in terms of osteoarthritis and the skeletal system. But my specialty as one of the PIs in the biologics lab is a striated muscle. So I, my education going back to many years in Switzerland and Australia was the cardiac tissue. So another type of striated muscle. So I worked in the cardiac tissue for many years, but eventually six, seven years ago, I saw the light and I moved to a better striated system, which is the skeletal muscle. And there are several reasons why, first of all, because of the clinical implication that came to light in my career, but also probably that's the most important things compared to the cardiac tissues is an extremely remarkable regenerative tissue. So, and the process, obviously it's dependent on the presence of resident stem cells within the muscle. So as you know, the skeletal muscle tissue is a very highly hierarchically structured tissue and organs. I'm not going to go into the details of that, but what is important is among the structure and the complex structure, you have the presence of multiple different types of stem cells. The one, one of which, which is called the satellite cell is absolutely essential and necessary for the proper growth, postnatal growth and regeneration of the muscle tissue. So satellite cells were discovered in the early sixties as small cells that reside in very close proximity to the muscle fibers and they reside under a small layer of connective tissue that wraps the muscle fibers, which is called the basal lamina. It is mostly made of laminin and under these basal lamina, you can detect these niches of muscle stem cells that stand positively for this transcription factor called PAC7. So we generally in the field, we call these cells, this stem cell, PAC7 positive satellite cells. And as I will show you in a second, they are extremely important for the proper maintenance and homeostasis of the skeletal muscle. So as I said, satellite cells are stem cells. The property, it's a little bit peculiar in the field of stem cells. You probably are aware of a different type of stem cell that you can find in a body. Obviously you have the embryonic stem cells, the iPS cells. Satellite cells or resident muscle stem cells are what we call adult stem cells. They maintain this capacity like whole stem cells to replicate and self-renew themselves by different processes of division that could be symmetric or asymmetric, but contrarily to embryonic stem cell or iPS cell, they lost most of their potency in the sense that they can only make muscle tissue. So because of their location, because of the expression of specific factors, like the PAC7 transcription factors, if you take these cells out of the body and you grow them in culture, they will make only muscle. And indeed, this is something that has been investigated thoroughly and published over the last 60 years. If you take a muscle biopsies, a chunk of muscle tissue, and you digested it and you isolate through sorting procedures, the satellite cells, and you put them into cultures and you give them a medium, very, very rich in growth factors like high serum, these cells will expand and grow into a population of stem cell, a progenitor cell called myoblast. And eventually if you withdraw these rich medium and give them a medium, which is more of a salvation medium, they will change their programs and it will start to fuse and put all their cytoplasm and nuclei together to form these multi-nucleated myotubes in vitro that recapitulate the muscle fibers or myofibers that you find in the tissue. So from these satellite cells, you can leave formations of myotubes and muscle fiber in a dish through this process that is called myogenesis. And as evidence has shown and has been published, this process, like most of all the processes in stem cell differentiation, is associated with a timely expression of genes that drive the myogenic program. It starts, obviously, with the presence of these pac-7 transcription factors that lead the activation of other myogenic factors, including myoD, which is the master of myogenic regulators, which will dictate in a timely manner the expansion of these cells and then the fusion and the formation of these myotubes in vitro. So the role of satellite cells in postnatal muscle development is critical. What data have shown, mostly it's data that was produced in animal models, shows that satellite cell plays a critical role both in postnatal tissue growth and then establishment of a permanent population of adult quiescent resident stem cell in a muscle. So obviously a critical component of postnatal growth is to grow your muscle from the size of a toddler to the size of an adult, and at the same time to have this muscle to gain in strength and function. There is evidence that's showing that these satellite cells plays a critical role, at least up to the puberty, in the process of growing muscle fibers in size. And ultimately, with the establishment of an adult population of resident satellite cells, these cells plays a critical role, a fundamental role, in adult muscle repair and regenerations. If a little felt sore after running a kilometer or so, and this is the process of muscle repair and regenerations, that's when satellite cells activate the process of myogenesis, they expand, they migrate in the areas of muscle injury, and then they recreate or reform new muscle fibers by processes of fusion, and this is critical. Another important component of these satellite cells is the fact that, as all stem cells, they wither in number and in potential to the process of aging, and they are also associated to disease conditions. Some of these are, for example, the process of sarcopenia, but also they are associated with other different types of muscle impairment and motor dysfunction. One of these processes of muscle impairment and muscle dysfunction is manifested in what we call cerebral palsy. So cerebral palsy, I will explain to you in a second about what cerebral palsy is, is obviously a condition that is very dear and close to me because I've been working on with patients with cerebral palsy for the last six, seven years to try to find out new ways to improve their motor function and muscle capacity in my lab. So cerebral palsy, it's a group of lifelong impairments of movements and posture, which is caused by a non-progressive injury to the developing brain. So the primary injury is within the brain, affecting the upper motor control centers, but most of the clinical manifestations that are observed in cerebral palsy are at the musculoskeletal level. Often cerebral palsy is caused by a stroke around birth up to the age of two years, and this is known to be one of the most common cause of physical disability in children. Cerebral palsy can be classified accordingly to the involvements, what part of the central nervous system that is basically injured, and it can be divided into spastic form. This is about 70% of all cases of cerebral palsy, which are associated with an insult to the part of the cortex associated with the pyramidal tract, or it could be also dyskinetic or toxic, affecting the extrapidaminal components of the central nervous system. Most importantly, cerebral palsy that causes obvious motor and muscle dysfunction, it can be classified using this gross motor function classification systems, or GNFCS scales, that goes from level one to level five, level one being children having almost no restrictions in their motor capacity and working capacity, two being fully assisted and wheelchair bound at the level five. One of the most debilitating causes that develops postnatally in children with cerebral palsy is the emergence of what we call limb of muscle contractures. These contractures that can occur in the upper or the lower limbs can be defined as a biological structure or mechanical change to the muscle tendon complex that leads, and that's very important, to a reduced range of motion around the joints. So development of these muscle contractions will occur in about 30 to 40% of children with cerebral palsy by the age of 18 is extremely debilitating. It reduces, obviously, quality of life in these patient population. It can be very painful, and when children become adults, these muscle contractures, which are associated with a more sedentary life, can cause also many cardiometabolic complications associated to that. Obviously, these are also associated with costly physical therapies, and unfortunately, there are no cure for contractures at this stage. But obviously, there are a lot of therapies in the field. So most of the therapies that try to tackle impaired motor and muscle dysfunction in children with cerebral palsy and contracture is really to try to boost muscle mass, muscle strength, and function, and try to manage all the complications associated with the brain injury, including spasticity that could develop as well, and development of muscle joint contractures. So as you know better than me, as clinicians, there are several different type of approaches and therapies. Obviously, there are what we call traditional physical therapy and occupational therapy approaches. There are also some pharmacological components like muscle nerve decouplers, like Botox, that can manage spasticity. But ultimately, many of these children will have to undergo surgical procedure to release the contractures that form. Now, as I said before, there are no cure for development of muscle contractures, and the effectiveness in the long term to prevent or reduce these contractures is relatively marginal and limited if you follow these therapeutical approaches. And the recurrence of contractures following surgical procedures are often an additional complication for these children. So in my lab, like in the lab, the biologics lab, we try to investigate all the biological changes that are associated with contracture developing cerebral palsy. So compared to typically developing children, so typical development that I will from now on mention as a TD, so compared to typically developing conditions, cerebral palsy, as I said, is associated with a progressive thinning, shortening of the muscle and stiffening of the muscle nerve complex, which is associated with impairments of the growth of the muscle while the skeletal component grows. This leads obviously to a reduced range of motion around the joints. So if you cut across a muscle, you will notice that the muscle fibers, dimensions and diameters and lengths are much smaller and shorter and thinner compared to typically developing conditions. And there is very often a penetration of fibrofatic infiltrations. So these muscles are not only shorter and thinner, but also more fibrotic. And this is work that was performed over multiple years in Dr. Lieber's lab. This condition leads to an increase in sarcomere length in muscle fibers in cerebral palsy that obviously reduces the forces that these muscle fiber can generate leading to a muscle weakness as well. Another important discovery that was done by Dr. Smith and Dr. Diomedes over the past few years is that the number of satellite cells, of resident satellite cells in contracture muscle in CP is significantly reduced. So this overall it's bad news because a lower number of satellite cells in the muscle of stem cell in the muscle means lower capacity to regenerate and heal the muscle or to grow the muscle during postnatal development. In my lab, we have moved even further and we proceeded with isolating the satellite cells from hamstring muscle that were kindly donated by children with cerebral palsy and typically developing children during surgical procedures. And by looking at the process of myogenesis that I explained before, we discovered that when compared to typically developing conditions, these isolated satellite cells are impaired in their capacity to generate new muscle fibers in vitro. This is an important discovery because it shows that these satellite cells isolated from their environment still maintain an intrinsic impairments or fail to grow muscle fibers in a dish meaning that there is something intrinsic to the satellite cell that is incapable to grow and differentiate them into muscle fibers. This can be quantified by several measure. One of these measure, it's looking at the fusion index which is the percentages of number of myoblast that they grow out of satellite cell that can fuse and put their nuclei within these muscle fibers in vitro. And the number of the percentage of these nuclei within these muscle fiber is significantly reduced in CP compared to typically developing conditions. And another experiment that we're performing we published three years ago is that this process of fusion, it's also associated with repression of the myogenic program of those genes that are critical to drive this myoblast, mononucleated myoblast to fuse and form these polynucleated myotubes. You also move into another path and then we took these cells, this myoblast and we checked how capable they were now to expand to proliferate in a dish and even to migrate through a membrane. And we noticed that while these cells were really severely impaired in their differentiation problem, in their fusion capacity they proliferate very well. And actually we noticed that they had an increased capacity to proliferate and expand. This was measured by looking at their doubling time which is the time that a cell culture takes to double inside. So a shorter doubling times mean higher proliferation rate and they also have a higher tendency or trends to move through a membrane and penetrate through a membrane and pass through the other side. So reduced, in summary, reduced number of residents' satellite cells in developing children with cerebral palsy increased capacity of these cells to proliferate, expand very rapidly. And however, an impaired capacity for these cells to differentiate and create muscle fibers, myofibers, at least in a dish. This would suggest to us that at least in cerebral palsy, the mechanism of early myofiber growth and establishment of a population of healthy adult muscles, satellite cells, stem cells could be severely compromising children with cerebral palsy. And therefore this process of growing muscle postnatally from a toddler to an adult could be severely compromised. Now, is this mechanism that cause development of a contractual or formation of a contractual cerebral palsy? At this stage, we don't know. We don't have these questions. It's a very difficult questions to answer. However, we know that by using an animal model, a mouse, in which we knocked down the number of satellite cells to the level that you could find in cerebral palsy, we know that when we cast these knocked down mice and we create a muscle contracture artificially in these mice, they have a hard time to recover from these contractures with such a slow number of satellite cells. So overall, we know that a reduced number of satellite cells is definitely a bad news in terms of muscle growth, muscle regeneration, muscle recovery. We also try to understand whether the depletion or a premature depletion of the satellite cell pool in postnatal development could lead to a premature tissue aging and senescence. And these are currently experiment that we are performing in the lab using both in vitro and in vivo model to test these hypotheses. Finally, and this is what I'm gonna spend the rest of my talk is to understand how we could potentially unlock or wash off these pathological phenotype in satellite cell from CP to try to improve muscle growth in these children over the period of post development. So I'm just gonna stop briefly here and ask if you have any questions or not. If not, I'm just gonna move forward. Just let me know in the next few seconds, Shannon, if you see that there are any question in the chat. If anybody has any questions, you can place them in the chat now, but I don't see any right now. Okay, so I'm just gonna move forward. I'm just gonna spend the next, the last part of my talk to discuss about a potential approach to try to reactivate the satellite cells and provides a potential relief in the terms of treating this patient with new therapeutic approach. So how can we potentially prevent development of Q or a muscle contracture in CP? We know that traditional therapies do not work, but we know that by looking at the resident population of satellite cells, these cells during postnatal development in CP have increased proliferation expansion rates. So they have a tendency to deplete faster because the number of times that a cell can divide is not forever. And then they also have impaired capacity to differentiate. This is a typical phenotype of hyperproliferative condition like you could find in cancer. Another, obviously we're not talking about cancer cell, but we're talking about cell that are highly proliferative and then they are not capable to differentiate. Another interesting discovery that we made in our lab is that these cells share another common mechanism that is associated with cancer cells, which is they have an increased level of DNA methylation. So satellite cells or myoblasts that are from the satellite cell in proliferation seems to have a higher level of methylated DNA compared to typically developing conditions. So what is DNA methylation? Well, DNA methylation is associated with what we call CPG site or CG site, which are regions of the DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of base along its five prime to three prime directions. So the cytosine of these denucleotides can be methylated. So it's a biochemical modification by a class of protein called DNMNT, so DNA methyltransferases. The addition of these methyl group to the C in the CPG denucleotide can occur in multiple regions in the genome. Actually, it is thought that about six to 8% of all the CPGs denucleotide in the genome could be methylated at a specific time. But they can occur actually in specific regions relative to the genes, which are very important in terms of how this gene will be able to be activated or repressed. So when these CGs or CPGs are residing high frequency in high series in specific region, they are called, they form what we call a CPG island. So our regions where there can be highly differentially methylated. So the CPG islands or series of CGs can occur, for example, in the promoter region of genes, or they can also occur in the body of the genes. When they occur in the promoter regions and the CPG islands highly methylated, this normally leads to repression of gene expression because of changes in conformation of the chromatin that leads to, that prevents transcription factor or RNA polymerase to bind the DNA and proceeds with gene expression. When DNA methylation occur in the body of the genes a little bit more complicated because it could be associated with both activation of repression of the gene. So in cancer field, this is a very important mechanism that is highly studied and investigated because cancer is often associated with an increased methylation in many promoter of genes that are associated with differentiation, or it could also be associated with hypermethylation or an increase in methylation in the body of the gene that activates a lot of genome which are associated with proliferation. So this increase in methylation in cancer cell can repress differentiation and activate proliferation. Now, what is very interesting is that in the cancer field, there's been several drugs that have been developed that can potentially restore these patterns of DNA methylation. And one of these class of drug are called cytidine analogs, like 5-azacytidine, which actually inhibits the activity of this DNA methyltransferases to methylate the CpG denucleotides. So cytidine analogs pretty much work as DNA-methyl inhibitors and hypomethylate the DNA, reduce the level of methylation in proliferating cells. So these class of drug is used in cancer in order to stop these cells to proliferate and promote potentially their differentiation. So 1-azacytidine, for example, has already been approved for many years by the FDA and is used to treat patients' neurodysplastic syndromes, which are pre-leukemic cancers where this class of drug is used to stop hematopoietic cells to be released prematurely into the blood and help them to differentiate in the hematopoietic system, the bone marrow, and prevent and afford to develop leukemic cancers. So what we thought was, can we potentially use and repurpose this drug and apply it to our hyperproliferative stem cells in cerebral palsy and restore their capacity to differentiate? So that's what we tested. As a reminder, differentiation of satellite cell into myofibers in Cp is compromised compared to typical developing condition. Now, we tested different concentration of these 5-azacytidine or azacytidine, and we came to a perfect optimal concentration and exposure condition for the satellite cells in a dish of 5-macromolar of azacytidine treated for 24 hours. They lead pretty much to a restoration of the differentiation programs in these cells. So when we treat this proliferating myoblast, 5-macromolar or 5-azacytidine for 24 hours, we are capable to restore the myogenic potential of these satellite cells in a dish. And this corresponds with pretty much restoring or normalizing the level of DNA methylation in Cp to the level that is normally observed in typically developing stem cells. So this high level of DNA methylation is lower to a level that is comparable in typically developing condition. And this leads to restoration of the differentiation program in Cp. When we look at proliferation and migration, we noticed that treating these cells, these proliferating myoblasts with 5-azacytidine, leads to a reduction of the proliferating capacity. So it slows down proliferation and also slow down their migration or invasion capacity. So in summary, satellite cells derive myoblast progenitors that we isolated from contractual muscle in Cp have a DNA methylation patterns that favor the proliferation over quiescence and differentiation. And what is important for us is that treating these cells with azacytidine rescue the phenotype So it acts a little bit like what we could observe with hyperproliferating cells in the cancer field. And it seems to be a promising potential candidate to potentially treat these muscle stem cells in Cp to restore the capacity to create muscle. Now, what we wanted to check and to test even further is what genes or what regions of the genomes are affected following in Cp compared to Td and following treatment with 5-azacytidine. So what we did was to take these satellite cell derived myoblast progenitor cells. We took the DNA and the RNA out of it and we look at the DNA methylation patterns throughout the genome. And we did some RNA sequencing to look at what genes were differentially regulated in all these conditions. So these are some of the data that came out from this study. What we did in terms of differentially methylated region in the satellite cell from Cp, we observe about 18,000 differentially hypermethylated region in the genome of satellite cell from children with cerebral palsy. So these 18,000 plus regions in the genome were hypermethylated compared to typical developing condition. Then when we treated with azacytidine, we noticed that at least 15,000 of these regions reduced the level of methylation. So those were 15,000 differentially methylated region that we can tweak around by treating the cell with 5-azacytidine. But where are they located? So we noticed that these 15,000 differentially methylated regions were located in about 9,000 different genes or transcribed sequences. Three quarter of them are protein coding sequences and a quarter of them are what we call long non-coding RNA which there is another interesting elements to the fact that long non-coding RNAs and non-coding RNAs could play an important role in satellite cell homeostasis in general. So relative to the genes, we also interestingly noticed that about three quarter of these sequences were actually located in the gene, in the body of the genes and only about a quarter of them were located in the CpG island. So usually when there is hypermethylation in CpG island, you have gene repression, but when three quarter of them are in the body of the gene, this could be associated with actually gene activation. So we ended up doing also a thorough RNA sequencing analysis of all the genes that were, that could be differentially regulated. And what we ended up with was to pick up about 530 genes that we found differentially upregulated in these proliferative myoblasts in culture in Cp. I don't wanna go into too many details here, but when we did a cluster analysis of all these genes, we noticed most of these genes were pertaining to specific paths that are associated with the cell cycle. So cell cycle regulation, mitosis, chromosome condensation and segregation, as well maintenance, chromosome maintenance, and then DNA repair. We call this mechanism to be upregulated in CP compared to TD in this myoblast, which are obviously hyperproliferative. So that was not something that we found surprisingly. However, when we treated with fibrosacitidin, we noticed that these genes overall downregulated in expression. So we were able to suppress the expression of these genes using fibrosacitidin, and also we were able to reactivate and upregulate other genes, which are mostly associated with protein maturation, secretion of membrane bound, formation of extracellular matrix, as well activating specific genes, which are more prone to leads to a satellite cell stemness, quiescence over division and proliferations. Meaning that this process of treating these satellite cells with azacitidin leads to a mechanism that's in the field of stem cell research, who we call mesenchymal to epithelial transition, which is an important mechanism that lead to increase in pluripotency and potency in these cells. In this very complicated slides, we enter a little bit more into the signaling pathways that we were able to identify to be upregulated in CP, but then normalizing expression by treating the cell with azacitidin and by using so demethylating or hypomethylating these differentially methylated regional CPG islands into these specific genes. So among the different pathways that we discover was obviously the JAK-STAT pathways, the PI3 kinase signaling pathways, as well other pathways associated with the anchoring of the cells to the extracellular matrix and movement of these cells in their environment. But overall, even if we don't wanna look into too many of the details of these signaling pathways, for us, it gives us a lot of targets that we can potentially look into in order to reactivate or wash off these pathologic epigenetic imprint into the satellite cells and reactivate the potential of these cells to grow muscle in children with cerebral palsy during their postnatal development. In terms of the myogenic programs, obviously we looked, we performed this experiment to proliferating myoblast, so we couldn't find a lot of late myogenic genes to be activated, but we found a lot of CPG islands and differentially methylated regions in many genes that are associated to these programs of differentiation. So we discovered many genes involved in two differentiation that could be a future target for our research, looking into different opportunities and possibilities to reactivate stem cell myogenesis into contracture muscle. So in summary, muscle contracture and CP are associated with a loss of satellite cell number, but also a loss of capacity of the cell to self-renew and differentiate and grow muscle fibers during their postnatal development. And this is associated with a differential DNA methylation profile. This is interestingly for us, can be reversed by lowering the DNA methylation patterns in the cells and this regulation of DNA methylation patterns, one way or another, we're still looking into the way to target this, could potentially rescue the satellite cell and myoblast homeostasis and normalize muscle growth potential in children with CP or at least provide, by providing this drug in one way or another to children's undergoing physical therapy, at least promote the stemness of their satellite cell and provide a better growth of muscle following physical therapy. So for us, we enter into these interesting patterns where we could potentially look into repurposing an anti-cancer drug, azacitidine, so their derivative and provide a new therapeutical approach to this patient population. At the moment, we're currently looking in vivo into study the safety of azacitidine, looking at different escalation, escalating doses and route of administration where we could potentially provide an injection of azacitidine topically for localized contracture of muscle and potentially reactivates the satellite cell that are there and to provide potentially another way to grow muscle in this patient population. So I'm gonna stop here by thanking, obviously the people that did most of the work that I presented today, starting with Lydia Sibley, who's now working as a PhD student at UVA and then all the sponsor for this research and then I'm open to questions. Thank you. Thank you so much, Dr. Domoghetti. If you have questions now, please put them in the chat and I'll read off. Okay. Are there cells to satellite cells that exist in tendons and ligaments or similar cells like satellite cells that exist in tendons and ligaments? Would this present a problem and fully treated or fully treating slash preventing muscle contractures when considering areas that are gradients and muscle tendons areas? This is actually a very interesting questions because obviously if you think overall when you postnatal grow of a muscle, it's obviously growth in size. You go from a very small muscle that is already formed during embryonic development, but then it has to grow from a small size to a much bigger size. And most of the growths that occur and very often occurs at the muscle tendinous junctions when you think about longitudinal growth. So the muscle tendon junction is actually an area that is known to be highly rich in satellite cells. So if you just sections your muscle, the whole muscle from tendon to tendon, and then you look and you stand and you look for the number of satellite cell residents in each sections, you will notice that many more of the satellite cell niche are located near the muscle tendinous junction. So the satellite cells, which are obviously myogenic cells are really highly proliferative and highly concentration exactly in the regions where it's needed for a lengthening of the muscle and the muscle fibers. So we don't have to forget that myofibers are among the largest or the biggest cells that you could have in the body. They are obviously not very thick in diameters. There are maybe a hundred, a hundred microns in diameters where they can reach maybe a foot of length. So up to 30 centimeters of length and they contains a lot of nuclei. So they are multi-nucleated. They need to maintain what we call nuclear domains along the length. Because if you think about these very, very long cells, it will need to grow both in thickness and in length and it needs additional nuclei to reactivate and to activate and produce proteins ectopically in the regions where the nucleus is located. So satellite cells fuse with these growing fibers along the longitudinal axis to provide obviously a bit of cytoplasm, but most importantly, a new nucleus to create and produce and synthesize proteins in that area of the muscle fiber. So when the muscle wants to grow longitudinally, satellite cells need to be highly concentrated along the muscle tendon function. So there are no satellite cell in the tendon itself but they are at the junctions and they're very concentrated and then they definitely provide a critical role into the process of lengthening the muscle during postnatal development. Thank you. Second question, what are the known neurologic and mechanical influences on satellite cell population? Is this strictly a neurological disease or does CP fundamentally affect muscle too? So if I understand the questions is, the question is whether or not, I don't have the chats, I can't see the chat right now, but if I understand well, is whether the neurological condition is the primary factor that drives this impairment with satellite cells, is it correct? Yeah, that's right. Yeah, so this is something that is not really known what drives this impairment of stem cell homeostasis in CP. And so the neurological conditions obviously leads to an impairments of the communication between the peripheral nerve system, the motor nerve system and the muscle fibers. So obviously muscle fiber are connected to the neuromuscular junctions to the motor nerve system. And obviously satellite cells are not directly connected. However, the formation of the neuromuscular system, the release of neurotrophic factors are topically locally where resident satellite cells could be, could create definitely an impairment of these satellites. So the nerve system could potentially have a drive on this impairment. And it is known already that the brain injury that causes cerebral palsy affect the DNA methylation of multiple tissues and organs in the body. For example, there are changes in DNA methylation in the brain following the injury. These patterns can also be detected in the blood. So if you isolate leukocyte and other mononucleated cells from the blood of a child with cerebral palsy, you can detect epigenetic changes in DNA methylation in the cell. Potentially this brain injury can affect systemically the satellite cells in a muscle and therefore cause the problem. An alternative could be that the nerve system impair. So cerebral palsy cause motor nerve hyperactivity that causes a hyper contractile state or hyper contractive states on the muscle. And this hyper contractive state is also one of the reasons why muscle don't grow postnatally. And this could indirectly impact the satellite cells which find themselves in an environment of constant repair. So the satellite cells might be constantly promoted in their expansion and activity because they have constantly to repair muscle. And therefore the prematurely senescent age because of these hyperactivity states. So those are some of the hypotheses that we're looking for. I guess I have a follow-up question after that. That's interesting. I didn't realize the blood, the epigenetics can be seen in the blood. Then I assume, could it be seen like in the cardiac heart as well with their cardiac, I guess having a predisposition to have cardiac problems and respiratory issues if possible, that can be the root cause as well. Yes. Well, the answer is that nobody knows. I don't think that a lot of people have looked into the cardiac tissue. It's very difficult to obtain a cardiac biopsy without injuring the heart, but maybe a cardiac puncture could be done. But definitely the patterns of DNA methylation is a pattern that is extensively and actually all these epigenetic mechanism are definitely investigated totally, not only in thermal policy, but in many other order dysfunction associated with neurological conditions like stroke, traumatic brain injury, and even spinal cord injury that seems to be changing a lot of the epigenetic mechanism that regulates gene expression and could potentially play a significant role why, for example, the nerve tissue cannot repair following these injuries, or in case of the muscle, which is what I'm interested in right now, impacts the stem cell population locally and prevents their capacity to regenerate or grow the tissue. So it is a new interesting field of interest. The result is a very complicated because DNA changes in DNA methylation not only impact, so DNA methylation not only impacts directly gene expression, but also impact the chromatin structure going a level up to the level of the histone modification and so forth. So there are all these cascade of different epigenetic changes that could dramatically affect the structure of the chromatin and impact how gene are expressed. The good news though, is that contrarily to mutations like Duchenne muscle dystrophy, you have a little mutation in dystrophin that causes the phenotype. The good news in epigenetic mechanism is that often they can be reversed. So in this case, for example, DNA methylation can be restored to a level by hypomethylating and using drugs against that. Other post-translational modification to histone can also potentially be reversed. So the good news about epigenetic mechanism, they can be restored somehow and could potentially provide new venues for therapeutic approaches. All right. I guess we'll take one more last question if anybody has one. If not, I have another question that I'm asked very often by clinician is that, but are you crazy? Are you guys gonna try to use a cancer drug to treat the muscle contraction in CP? Are you crazy? What about all these side effects? What about all these, and it is a really relevant questions and it's a relevant question that you observe very often in many parts of translational research where you try to repurpose old drugs for new therapies. And the question is very pertinent and obviously we're not there, but we'll definitely look into the possibility, not only to inject potentially ectopically or localize as a cytidine or other cytidine analogs into a contraction muscle to see if it has an effect, but we're gonna also look at potential stem cell therapies. It could be possible potentially to collect some tissue from these children's and then treat their satellite cells in vitro and re-inject them, for example, during a surgical procedure to promote growth post-surgical. So there are different ways that we can tackle these and not only as a cytidine, but there are also other mechanisms that lead to hypomethylation of the tissue. For example, one interesting and very practical way to lead to DNA hypomethylation is exercises training. This is known that different type of endurance training leads to DNA hypomethylation and actually can fight against senescence of satellite cells in the muscle of tissues over the course of a lifespan. So there are some interesting research perspective out there that can be used in order to tackle the problems of restoring the epigenetic footprints of these stem cells in disease conditions. Thank you so much again, Dr. Domaghetti. And this will be probably in a week posted for recording on AAP. So if anybody has peers or co-residents that missed this, you can check it out on the AAP website. Thank you again. Have a great night. No, thank you for listening. And I hope it was not too much information too fast.
Video Summary
In this video, Dr. Dhammageddy discusses the treatment of muscle and motor dysfunction in children with cerebral palsy. He focuses on the potential of activating the resident muscles themselves to treat muscle impairment in these patients. Dr. Dhammageddy explains that satellite cells, which are muscle stem cells, play a critical role in postnatal muscle development and repair. He discusses the impairments in satellite cell function seen in children with cerebral palsy, including reduced satellite cell numbers and impaired capacity for differentiation and muscle growth. Dr. Dhammageddy then explores the potential use of cytidine analogs, specifically azacytidine, to restore satellite cell function. He explains that these drugs can reduce DNA methylation and reactivate the myogenic potential of satellite cells. The potential pathways and genes involved in this process are also discussed. Dr. Dhammageddy concludes by suggesting that azacytidine could be used as a new therapeutic approach for children with cerebral palsy to improve muscle growth and function.
Keywords
muscle and motor dysfunction
children with cerebral palsy
activating resident muscles
muscle impairment
satellite cells
postnatal muscle development
muscle repair
cytidine analogs
azacytidine
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