I want you to be there. Good morning and welcome to our combined grand round. It's my great pleasure to introduce our speaker this morning, Dr. Richard Levy. Dr. Levy is visiting us from Columbia University where he's currently professor of anesthesiology and pediatrics and also vice chair for pediatric laboratory research in their department of anesthesiology. He obtained his undergraduate degree from SUNY Binghamton and medical degree from New York Medical College and then he completed a residency in pediatrics at CHOP followed by a residency in anesthesiology at the University of Pennsylvania. He then returned to CHOP and completed a fellowship in pediatric cardiac anesthesia and another fellowship in pediatric critical care medicine following this who's on staff at CHOP for a few years as a pediatric cardiothoracic anesthesiologist and assistant professor of anesthesiology at Penn. And then he became chief of pediatric pediatric cardiothoracic anesthesiology at Maria Ferrari Children's Hospital and Westchester Medical Center. And then following this he was vice chief of anesthesiology and pain medicine and director of cardiac anesthesia at Children's National Medical Center and George Washington University in Washington, DC. And then in 2014 he moved to Columbia University where he is where he holds his current position. His initial research focused on acquired mitochondrial dysfunction in the muring septic heart and his lab identified several novel therapeutic interventions, tardic targeting sepsis induced myocardial depression, but recently is focused his investigation on the effect of different environmental exposures on the developing brain as well as mitochondrial function and the developing brain of patients with fragile X syndrome. He has published over 75 original manuscripts and he's an associate editor for survey of anesthesiology and a regular ad hoc reviewer for many other journal journals. Today he's going to be speaking to us about purple ball toxicity in the cardio myocytes my mitochondria of developing mice. Thank you, Dr. Levy. That was the best introduction I've ever heard. Thank you. I want to thank Dr. Able, Dr. Hickie for the invitation to speak with you guys this morning. And we're going to present some of our work on purple ball infusion syndrome. So I have no conflicts of interest, no disclosures. Purple ball was first introduced in 1977 and then first introduced for commercial use and clinical use in the US in 1989. It is the most commonly used intravenous sedative hypnotic worldwide for general anesthesia and because of its desirable pharmacokinetics, it's also commonly used in the ambulatory setting as an anesthetic as well as in the ICU's for non-procedural sedation. Purple ball infusion syndrome or purple ball related infusion syndrome is a potentially life threatening consequence of an adverse consequence of drug administration. It was initially reported in a cohort of children in the early 1990s and is thought to be related to cumulative dose and duration of infusion. It is a diagnosis of exclusion and it's characterized by one or more of the following features in the context of a purple ball infusion. Usually it doses that are at or exceed 45 milligrams per kilo per hour and usually at about 48 hours in duration or more. The features here in yellow are the primary features and you can see metabolic acidosis, lactic acidosis with a probably the most common. Many folks develop raptomyalysis and then EKG changes are very common too. The secondary features are listed there in white and I just want to draw your attention to cardiac failure and then fever. And fever is going to be an important thing that will focus on a little bit. This is the first notable publication of a purple ball infusion syndrome in the literature appeared in British Medical Journal in 1992 and it described the death of five children in the pick you who were being sedated with purple ball. And they each developed severe metabolic acidosis and myocardial failure and then obviously unfortunately died. Here are the children you can see that range in age from about four weeks in age to six years of age. The duration of infusion time was somewhere between 66 hours to 115 hours so a little more than two days up to almost five days in duration. And the maximum average rate of purple ball administration was 10 milligrams per kilo per hour which equates roughly to 166 mics per kilo per minute. Bray was the first one to coin the term purple ball infusion syndrome and he did so in the mid to late 90s and he tried to identify the features of the syndrome and he did so at first by looking at the first 18 cases of purple infusion syndrome. And you can see the majority of kids were relatively young. Most of them were in the pick you with respiratory failure from some viral infection viral etiology. Many of them had enlarged livers and most of them had a metabolic acidosis. Some of them were lipemic. The majority of them had cardiac involvement with bradycardia progressing to asistally as the most common effect. Many of them were febrile, a number of them developed rhabdomiosis and the majority of them died. Up to about 2019 there were 44 reported cases in the literature of pediatric purple infusion syndrome. There's probably more that cases that either went under reported or were misdiagnoses. But you can see it's highly lethal with a mortality rate of 52 percent once someone developed the syndrome. This is an interesting and concerning report that was in the literature lab in 2018. This is a baby out of in Mexico. It was a healthy full-term newborn who was undergoing a C camera section and developed the phoblo and syndrome with a single induction dose of purple fall 3 milligrams per kilo. Baby developed severe metabolic acidosis, bradycardia hypotension, rhabdomiosis. This baby ended up surviving. So there's at least 45 cases now and again maybe more. In 2001 there was an unpublished randomized controlled trial because there was concern at the time. This was 327 children who were in the PICU. They were randomized to either the propyl sedation or conventional agents. Those who received propyl forward it was initiated at 5.5 milligrams per kilo per hour, about 90 mikes per kilo per minute, and then titrated to effect. And the investigators followed 28 mortality and what you can see is that compared to the 105 children that received conventional agents, the mortality rate in the mortality in the children who received propyl was 2 to 3 times higher. In response, the FDA actually warned against the use of propyl fall for non-procedural sedation of children in the PICU. And AstraZeneca at the time relabeled their product, Dipper van, such that it was no longer indicated for sedation of children in the PICU and only indicated for adults. And that's where it stands currently. But because propyl fall is such a superior sedative and hypnotic many PICUs around the world and certainly in North America continue to use propyl fall for sedation. And although the number of respondents in this survey that then this came out late 2019, the number of respondents was not that high. It tells the story that many PICUs continue to use propyl fall for sedation even up to 72 hours. This is the best review article to date on the issue. This appeared in the British Journal of anesthesia in 2019. They looked at both the adult and pediatric cases to date. The number of patients who have developed and succumbed to PRIS is probably too small to do a head to head comparison of children in adult. But even in the small numbers, the story is starting to manifest that there may be differences in the clinical features and the biochemical features between children and adults. And mechanisms in the manifestations may actually differ. I want to draw your attention to EKG changes which in children 75% of cases developed EKG changes. So very common in children even 62, 63% in adults. But fever is very interesting here. So the number of patients who developed pediatric propyl infusion syndrome had fever. But importantly, if you developed fever, it was significantly associated with mortality with a 7.8 odds ratio. So there's something about fever that's very important. So what's the path of physiology? What's the cause? And the short answer is no one knows. And it's really been fairly understudied. I just believe that there's a mitochondrial origin and etiology for the propyl toxicity. There is literature to suggest a number of studies have shown that propyl inhibits the electron transport chain at complex one, complex four, and may interfere with co-ins on Q-READOX activity. There's also evidence that propyl may inhibit fatty acid oxidation and impair carnitine palmitolial trans-race one. So here's the electron transport chain. We'll talk a little bit more about this in a second. But probable fault has been shown to inhibit complex one, complex four, and then co-ins on Q-READOX activity. And then with regard to carnitine palmitolial trans-race, the inhibition here prevents the transfer of fatty acids from the cytosol into mitochondria to contribute to beta oxidation and oxidative phosphorylation and ATP production. The problem with all the studies that have been done to date is they've all been done in adult or mature animals or hosts. So no studies have been done in developing or immature organisms. So we don't know if what the mechanism is in the context of children, infants, and the developing human. And the other thing is that the majority of studies have been looked at tissues that are probably less of interest with regard to morbidity mortality. So a lot of studies have looked at liver, brain, skeletal muscle, but very few studies have looked at cardiac and cardiac mitochondria. And that seems to be the one certainly in children that is most affected is the heart. And if the heart becomes infected, the risk of mortality is quite high. So we wanted to ask the question is, could we define a mechanism in immaturity and developing organisms? And we wanted to basically determine the mechanism of propyl toxicity in cardiomyosite mitochondria specifically and in immature mice. And again, we focused on heart because the heart seemed to be often impaired and affected in the pediatric form of the disease and had a very high association with mortality. We hypothesized that propyl would induce discrete defects within mitochondria from immature animals. So the way we went about to study it was to study 10-day-old C57-Black-6 mice. We isolated mitochondria from cardiomyosites from the ventricles. And we chose to first look at an in vitro propyl folx fogear and we chose different concentrations of propyl folx. So the reason why we chose 10-day-old is a very important time point for us. It's analogous to a postnatal time point in human infancy somewhere around 68 weeks of life. So very vulnerable time point in infancy. And we obviously wanted to focus on the heart because that seems to be a very important organ system in this disease state. And with these exposure concentrations, we wanted to choose concentrations that were well above the therapeutic levels. So these are super therapeutic solidly in the toxicological range of propyl folx. Okay. So quick review of oxidative phosphorylation and electron transport chain. So the majority of ATP in mammalian cells is generated by oxidative phosphorylation within mitochondria on the intermitocondrial membrane by a series of enzyme complexes called the electron transport chain. In the process, you have NADH and FADH2, which are generated by the crev cycle. And they enter the chain either a complex one, which is NADH or a complex two, which is FADH2. And what happens is electrons are transferred from complex one or complex two to coenzyme Q. Coenzyme Q then transfers its electron to complex three. Cytocrone C is the mobile electron carrier between complex three and complex four. The reduced cytocrone C will then reduce oxygen to water. And the purpose of all this is the electron flux is coupled to the hydrogen ion pumping across the intermembrane by complex one, three and four. And these enzyme complexes pump hydrogens to generate something called a proton motive force or hydrogen ion gradient or the mitochondrial membrane potential. They're all fairly synonymous terms. The ATP synthase, which is complex five, then uses this hydrogen ion gradient to then move electron to move hydrogen ions back into the matrix. And movement of hydrogen ions through the ATP synthase is coupled to the synthesis of ATP from ATP in an organic phosphate. So that's oxidative phosphorylation in a nutshell. Now, hydrogen ions can move independent of the ATP synthase such that ATP is not generated. And that's called leak. And we'll talk more about that in a second. Okay, so how do we measure mitochondrial function in vitro? One of the best ways is to measure oxygen consumption using a platinum coated electrode or something called a Clark type electrode. We can measure oxygen content in buffer with mitochondria. And we can actually measure the rate of oxygen consumption by the electron transport chain. And so the readout here is really complex four, but it's the electron transport chain that's linked in situ that the O2 consumption is basically indicative of electron flux and functioning of the electron transport chain as a unit. So what you do is you basically put mitochondria in solution. You give it substrate in this in this scenario. We've given peruvate and mallate, which are NADH donors. And the right mitochondria start to respire. So as they are respiring, the oxygen content will decline. And the rate of decline tells you about how fast or how slow the the mitochondria functioning and consuming oxygen. So this is called state two respiration when you just add substrate. We can then give ADP, which will stimulate the mitochondria to rapidly make ATP and they will burst in their oxygen consumption. And that's what you see here as you see a rapid increase in O2 consumption, a rapid decline in oxygen content here. And that's called state three convention, a state three respiration by convention. Once all the ADP is consumed, the mitochondrial then will convert to a slower rate of respiration, which is called state four. And that's more more reflective of this leak type of hydrogen ion movement. We can then do it again, give ADP, get nice state three, state four. And then we can give an antibiotic called oligamycin. And that inhibits complex five here so that you no longer can make ATP. So it markedly reduces the oxygen consumption of the electron transport chain. And it gives a rate that's called oligamycin state four respiration. And again, that's more indicative of leak of hydrogen ions. And then we can give agents which are called on couplers or ionophores, FCCP or DNP. They basically poke holes in the inner membrane to allow hydrogen ions to escape. And you get a loss of the membrane potential. And in response, the mitochondria really ramp up their respiration to try to defend the membrane potential. And that's what you see here. So these are the different states. So what did we see? So this is our healthy control mitochondria from the 10-day old heart. You get a nice state two respiration here. We give them ADP. You see very nice state three, state four respiration. Then we give them oligamycin. You get an oligamycin rate. And then we give them the uncoupler DNP and you see what the rate is here. So the numbers are the rate of respiration. And here now we did a dose response. We have 100 micromolar of propyl fall and 400 micromolar propyl fall. And probably the most glaring thing that you see is a dose dependent increase here in state four and in oligamycin state four respiration, which is highly suggestive of proton leak. And not really glaring changes in the other rate states of respiration. When we quantify this, so this is complex one dependent respiration. This is complex two. We see dose dependent increases here and now here are the concentrations of propyl fall compared to control. You see dose dependent increases in state four respiration in both substrates. And then dose dependent increase in state four oligamycin in complex two dependent respiration. And again, these are consistent with leak. We actually don't see much in the way of a change in state three respiration with either substrate types. And we only see a decrease in state three DNP at the highest level of propyl fall in both. And so if you were to see electron transport chain inhibition like the adult data has shown, we would see it mostly in state three because this is where really the electron transport chain is making the membrane potential. And we don't see that. But we're here. We're seeing leak and the respiratory control ratio, which is state three divided by state four is dose dependent lead decreased as the concentration of propyl fall increases, which is largely on the basis of the state four changes, which indicates leak. Okay, so next we moved on to measuring the individual complexes, steady state activities using spectral photometry and isolation with within without propyl fall. We then we use the highest concentration of propyl here. And what we found was a profound inhibition of complex one with the highest concentration of propyl fall. And that is consistent with what the adult and a mature animal shows. We actually found complex five to be looks like to be activated. And we didn't find any effect on complex two, three or four. We then you can then measure linked activity of complex one plus three and two plus three. And what that is is an indirect way to measure the read acts activity of coins on cue and the coins on cue pool. We found with complex one three activity that was significantly decreased and that was probably on the basis of complex one inhibition because it's a linked assay. But interestingly, we found complex two and three activity was increased. And that may be due to direct increase in in coqueure docs activity. All right, so we focused in on complex five here. Why was this activated? So this is now oxygen consumption on the above panel simultaneous membrane potential that we're measuring on the below panel here, control and red and propyl fall here in black. Now, so normally the ATP synthase complex five when it works in the forward direction will move hydrogen ions into the matrix. And so when you give them ADP and they stimulate respiration, what you see here in the membrane potential, it will drop because hydrogen ions are moving rapidly into the matrix to make ATP. When you give oligomice in which will poison this and stop the flow of hydrogen ions, you then get a restoration here of the membrane potential and it stabilizes what you see here in red with the propyl fall, you see that there's a little bit of a bump down here with with ADP. So the end times working a little bit in the integrated direction. So I give oligomice and you see here that it starts to rise, but then it just falls. And what that tells us because it didn't stabilize increase and stabilize is that the ATP synthase is actually also using the reverse mode. And the reverse mode, it will hydrolyze ATP and it's purposeful so that ATP synthase can then act as a fourth pump to help try to defend the membrane potential. So this probably explains why complex five is increasing its activity. All right, so this is all very confusing and we don't know, we don't have a mechanism. We have a lot of interesting abnormalities, but it doesn't really explain a mechanism. And the reason why that is is because when we do oxygen consumption, it's important to recognize that in vitro as well as in vivo, all a lot of these processes are happening at the same time. And individual processes can confound each other, can be additive and also can cancel each other out. So if you've got substrate oxidation and ATP turnover and they're in different directions, they basically make state three look normal and it can be very abnormal. One of these processes are both. So we have to do more sensitive assays and more complicated assays. And so this is something called modular kinetics. So everything in mitochondria revolves around the membrane potential or the proton mode of force or the hydrogen ion gradient. It is the brain stem of mitochondria. Everything they do is based on this membrane potential. There is only one process or module that generates or produces the membrane potential. And that's substrate oxidation. So that's the process of the electron transport chain oxidizing substrates and a DH and FADH2 to basically pump hydrogen ions to generate this hydrogen ion gradient. That is the only process that does that. There are two processes that consume or degrade the membrane potential. One is ATP turnover, which is synthesis and turn turnover of ATP. And the other is proton leak. And proton leak is a necessary process that moves hydrogen ions independent of the ATP synthase back into the matrix. And we can measure each of these processes independently. Now leak is very important. We're going to talk about it in a second. So the analogy that I like to think about is your bathroom sink. So if you think about the water level in your bathroom sink as the membrane potential or the hydrogen ion gradient, the mitochondria want to fill up that sink. So they want to maintain a membrane potential. And the flow of water here is the electron transport flow. It's the flow of electrons. So you want to maintain a nice healthy membrane potential. Too high of a membrane potential is actually really bad for mitochondria. They can generate oxidative stress, reactivative oxygen species and can be very damaging. So they want to limit that and have a pop off. And the pop off are these leak channels and proteins that allow a pop off of hydrogen ions to move back into the matrix to prevent the membrane potential getting too high. And it's analogous to that hole that's at the top of your sink to prevent it from overflowing. So that's called physiological leak. It's necessary and it's important. But there can also be pathological leaks. If you have a hole in your drain and you have a massive leak, you still need to make ATP and you still need to generate a membrane potential. So what mitochondria need to do in the setting of pathological leak is they need to flow a lot higher, the electron transport changes to work a lot harder, a lot higher O2 consumption rates to maintain a membrane potential in order to maintain ATP. So path leak can be pathological and mitochondria can accommodate. All right, so this is how we basically do this. This is all three modules on one graph just as a show you schematically what it looks like. We do these separately independently. So we can measure proton leak and sub-shred oxidation. We start in state four with oligomison and then we titrate the membrane potential using inhibitors or on couplers. And for ATP turnover, we start in state three and titrate the respiration with inhibitors. And the purpose we're doing this is you want to walk the mitochondria down a membrane potential to see how they respond with regard to respiration and you can compare them in a dynamic setting. It's this to me is analogous to doing pressure volume loops in the heart to measure cardiac function. You get an echo at one point in time. It's normal. It really doesn't tell you much about underlying dysfunction. You have to stress the heart by either volume loading or after loading the heart. To understand how the heart responds and this is the same thing. This is basically the stress test for mitochondria is if you challenge them by changing the membrane potential, you can see how they change their respiration in response and it can really unmask and uncover real pathology. All right, so here's sub-shred oxidation. This is control and red and this is a propyl fall exposed in black. So this is a normal sub-shred oxidation curve as the membrane potential falls. The mitochondria will try to defend that by increasing respiration. You can see the propyl fall exposed mitochondria. The curve is absolutely flatter and it's shifted down here with regard to membrane potential. So the membrane potentials are not totally normal. But the way you compare these curves is you take the highest common membrane potential and you compare the rates of respiration. And you can see the propyl fall exposed mitochondria have significantly higher respiration at the highest common membrane potential. This tells us that sub-shred oxidation is actually increased during propyl fall exposure which goes against electron transport chain inhibition. If one was to have inhibition respiration would be lower and we don't see that. So the mitochondria actually appear to be stimulated but they can't generate a normal membrane potential. So that's interesting and important. When we compare the curves here you see a left shifted propyl fall curve, highest common membrane potential just like sub-shred oxidation is significantly higher than the respiration rate than the control indicating that ATP turnover is increased. And this is likely because of that increase in complex five activity and the reverse activity of complex five to try to generate the membrane potential because you see the membrane potentials here are not at control levels. So there's something that is not allowing the mitochondria to generate a normal membrane potential. And this is the reason why proton leak is massively increased. So if you compare the highest common membrane potential respiration rate here is markedly and significantly higher than the propyl fall exposed mitochondria compared to control. That tells us that leak is induced by propyl fall. It is massive and this is likely the mechanism that prevents the mitochondria from generating the membrane potential in the setting of increased sub-shred oxidation and increased ATP turnover. So this appears to be pathological. So we think that propyl fall is inducing a leak channel to open to basically cause a pathological dissipation of the membrane potential. So what are the sources of leak in mitochondria? So there are three major sources that we know of. The A and T which is a translocase that normally takes ADP and translocates it with ATP in and out of mitochondria is also the major source of physiological leak. So this is the important one that pops off normally. It causes about 80% of leak in normal healthy mitochondria. We then have un coupling proteins that can also mediate leak. The classic one is un coupling protein one which is in brown fat mitochondria. And this is what brown fat and infants use to generate heat without having to shiver. So this is purposeful and necessary as an un coupling protein in those types of tissue in mitochondria. And then the third one is the mitochondrial permeability transition for. And this is a very important channel which also can mediate leak. And this channel can open pathologically. It is thought to mediate traumatic brain injury. It is thought to be a mediator of the Schemery perfusion injury. And as well as neuro degenerative disorders and with aging. But it is also thought to be very important with regard to normal healthy development in immature cells and mitochondria. And we can inhibit specifically each of these channels so we can know which is a source. So the transition porous inhibited by the classical inhibitor cyclosporin A. The A and T is inhibited by carbaccia track psilocyte and then we can inhibit un coupling proteins with GDP. Okay, so here we have oxygen consumption on the top panel membrane potential simultaneously measured on the below panel. Propefol is in black and control is in red. Here we start in state four respiration in the presence of oligomycin. And you can see in the healthy control cyclosporin here are the rates of respiration. Cyclosporin doesn't have much of an effect. If you're looking for an effective inhibitor to identify the source, you're looking for a slowing in respiration and a bump up in the membrane potential or stabilization in the membrane potential. And we actually see the cyclosporin stimulates respiration here but has no effect on membrane potential. It continues to fall here. The A and T inhibitor and the un coupling protein inhibitor both slow respiration and stabilize the membrane potential. That indicates that the source of physiological leak in these mitochondria are the A and T and un coupling proteins and this is known. So these are this is physiological sources of leak and here's our propefol. So a couple of things to notice in the propefol exposed myto. First, the rate of respiration is markedly increased. So state four respiration, the leak respiration is really increased. Secondly, they don't generate the normal membrane potential. They cannot generate a normal membrane potential which is consistent what we saw before. Look at cyclosporin. We have a nice drop in respiration rate here and we have simultaneously a bump up in membrane potential. Same thing with A and T inhibitor. And then GDP, we have a further slowing stabilization. This change in the membrane potential and respiration suggests that the transition poor and the A and T are potential sources of the pathological leak with propefol compared to controls. Now the problem with the transition poor is although it's a known entity and you can inhibit it and you know it exists and you know it can be pathologically can open. To this date, nobody knows the exact prodenacious identity. And so this is a problem but this is the working model of what the where the poor is and what it may be consisting of. It's thought to reside within the ATP synthase complex five and it's thought that cyclophyll and the A and T and the phosphate carrier are co factors for the poor. Well, since the A and T is a co factor for the poor, we focused on the poor itself as the pathological cause of propefol induced proton leak and we tried to rescue it to see if it was indeed the cause of this pathological leak and that's exactly what we found. So as a control here, we use FK 506 because FK 506 is also a cow's and urn inhibitor like cyclosporin but it has zero activity as no biological activity on the mitochondrial transition poor. So we're comparing a control here to our cyclosporin which is known to inhibit the poor control exposure versus propefol. So here in red is our control FK 506 is proton leak the module we're looking at and then we were able to see that FK 506 of propefol were able to induce the leak like we did before significantly higher respiration and the highest common memory potential. When we expose my normal mitochondria just to cyclosporin, you get a right shift here slight right shift but it's not significantly different from the FK 506 control. In the mitochondria that are exposed to cyclosporin with propefol we see a pretty profound right shift here in the curve and the way to interpret this again look at the highest common memory potential. So if you just look at the compared to FK 506 control the respiration rate at this highest common memory potential is markedly and significantly lower and that tells you that these are much cyclosporin rendered these might are much more efficient than just control mitochondria. So we can compare them to propefol FK 506 it's markedly efficient to be able to maintain that membrane potential a much lower respiration rate. So we've blocked the leak with cyclosporin and so we think this is the pathological cause is the transition for interestingly you see an increase in memory an increase in respiration here at the highest common memory potential compared to cyclosporin control. So this indicates that either there's another source of leak that we didn't block or this could be due to that coins I'm Q stimulated respiration but the important thing to note here is with cyclosporin and propefol these mitochondria are able to generate normal membrane potentials and are markedly more efficient. So it indicates that the permeability transition for is probably the pathological source of leak in propefol exposure in the 10 day old cardiac mitochondria. Alright so why would the immature heart immature cells be at my immature mitochondria be at risk for the poor opening. So George Porter who is a pediatric cardiologist at Rochester is very nicely shown that during embryological development cells are relying mostly on anaerobic like colicis to generate ATP. The oxidative phosphorylation is very mature the electron transport chain is very mature and disconnected and what he has shown is that you've got complex one that's not active yet. And the permeability transition for is actually open but as the cells in mitochondria develop there is a trigger and the trigger is probably the transition for closing and as it closes that is a stimulus for the cells to then switch from glycolysis to oxidative phosphorylation. It also triggers cells to go from proliferation to differentiation it's very important. But what's interesting is that even after the poor has closed and oxidative phosphorylation is maturing this poor is still prone and vulnerable to opening in various states is Schemier repercusion or other non-customuli. This may be why neonatal hearts for example are at risk in a period of period and he showed that in a five-veiled mouse heart he could detect that the poor was actually still open and we found that the poor was actually still open in the 10-day old mouse heart. So this is mitochondria from 10-day old in red and an eight-week old mouse heart in gray. So this is a state-for-respiration here and simultaneous membrane potential. If you look at the membrane potential and the respiration in the eight-week old cyclosporin again has very little effect it doesn't drop respiration and it doesn't maintain or increase membrane potential. So the poor it's a these mitochondria relatively insensitive to cyclosporin so the poor is closed. So he does have an effect here by dropping the rate and bumping up the membrane potential in gray and that's physiological source of leak. But here in the in the 10-day old we see that cyclosporin has a very nice effect here on respiration rate it drops it and you see a really clear increase in membrane potential and stabilization indicating that the poor was actually is actually open in the 10-day old mouse heart. So we think that propylphal is actually binding to the poor and opening it in the immature mouse heart mitochondria causing the pathological leak and potentially causing the the toxicity. What evidence that we have of that this is work that we've done with Rod Eckenhoff and Penn. We did photo affinity labeling with propylphal and found that propylphal does indeed bind to the ATP synthase, beta subunit and binds to the phosphate carrier which is a known cofactor of the poor. So we think that it's binding directly to the poor and we need to flesh that out a little bit more. So here's our working model of how propylphal toxicity may actually be happening and how this may actually lead to the disease in infants and children. We think that propylphal is binding to the poor, opening it causing profound and massive leak. And as I should mention that this type of leak generates heat, it's thermogenic and this may be the source of fever in these mitochondria and in these in these children. And it causes a massive inefficiency in the ability of the mitochondria to maintain the membrane potential in generate ATP, relying on glycolysis for for ATP production, you get lactate production, which would account for the acidosis. And then if you drop your membrane potential enough, that will lead to apoptosis and the creosis and that could lead to the rabidomyiosis and cell death. Okay, so this is all in vitro. So the real thing is you got to go to the next step, right? We need to see what it's doing in the organ systems or preferably in the whole animal. But it's impossible and probably infeasible to put a 10-day old mouse on a 72 hour infusion of propylphal. You're going to raise so many confounders that it's going to make the interpretation of any data almost impossible. You'd have to intubate the animals. You'd have to basically put them in an ICU essentially for 48 to 72 hours. Control for temperature, control for blood pressure, control for nutrition. It's just too many confounders. So the next best thing that we decided was to basically develop a model of propylphal infusion syndrome in the isolated heart. So this is a this is a lanyl door of preparation and Oscar lanyl door developed this approach in the 1800s. It's been around for over 100 years and it's a really robust way to assess pharmacology and toxicology in an isolated heart, removing all confounders. You can relate the aorta and you profuse the coronaries in a retrograde fashion with an oxygenated oxygenated solution. You can then put a balloon through the mitral valve into the left ventricle and measure ventricular function. And you get some really robust information from it. Most of this has been done in rats and in rat hearts because the size. And in the last 10 to 20 years, people have moved to my ice. They've gotten smaller techniques, smaller instruments. People have gotten more fast out with it. We wanted to take it one step further and no one has ever done it in an infant mouse heart. And after this is like hot off we're it's not even in the press. This is really hot out of our lab. We have developed the technique to be able to successfully cannulate the 10 day old mouse heart and establish a lanyl door prep, which is. It's probably the coolest thing I've ever done in my career. This is a 26 gauge needle that we've adapted as our aorta canula. This is a 10 day old mouse heart. And for those who have been in the cardiac ORs, these are temporary pacing wires that we use in cardiac surgery that we've placed touching the heart to be able to measure surface EKG. And so you you profuse the heart with we use a crevice, crystalloid perfusion, profusate, which is oxygenated. It's buffered and it goes into the coronaries retrograde fashion. And then it comes out of the coronary sinus. Out of the right atrium and drips out of the heart we can measure a whole bunch of things that's coming out of the heart as well. And so this is this works. This is a video of a mouse heart actually beating. So this is the 10 day old mouse heart. If you look closely you can see the aorta cannula here in the aorta that is sutured here. It is a tiny structure that the this is about a 30 milligram structure. The the drip here is basically the size of the heart. So you can imagine the size is really blown up. And then for this it's to the hardest to small fragile to put a balloon into measure pressure. It's just way too fragile. We tear the heart. We rip it off. It just is unfit. It don't make balloons this small. So you have to do it with saran wrap and it just it doesn't work. So we went back to the original method that Langdorf described. We put a suture in the apex and were able to attach it to a force transducer and measure longitudinal force generated by the heart during systole. And this is basically what we get. So we're able to measure EKG very nicely with a pretty high fidelity and simultaneous LV force tension generated during systole. And you can see here this is the sinus rhythm. The intervals are quite tight because it's a mouse heart. The rate is actually a little slower than the adult. The adult mouse heart is usually about five to six hundred beats per minute. Here the infant is somewhere between two and four hundred. The sinus rhythm, nice p wave, very tight QRS and you see the T wave here and then the LV tension. So we expose the mouse heart to lipid. We got a nice baseline measurement here. And then we expose it to interlipid as a control for 30 minutes. And you see you get a slight slowing of the heart rate but no real change in the LV tension. We then expose the heart to propyl fall and we use the 400 micro molar because that was our real toxic dose. And what's this is a slight starting off a little slower baseline heart rate sinus, nice generated pressure. And you can see within two minutes you start to get bradycardia here and a drop in the LV tension. Here's a nice PVC. Then 10 seconds later progressive bradycardia you see PACs here, LV tension really starts to diminish. We readjusted here to try to increase the end isolic attention. Bradycardia persists rate is quite slow here still sinus very diminished. Another 15 seconds really diminished function and then by three minutes a systole. This is exactly what has been reported clinically. Bradycardia progressing to a systole. So we think we have developed the model of propyl infusion syndrome in the isolated heart. So just to wrap up so next thing is really we want to test our hypothesis to confirm that the mechanism is what we're seeing now in situ in the intact heart with regard to induced pathologically induced leak by opening the transition for. We think we now have a way to test this also with regard to therapeutic intervention and confirm that the poor is opening. So we think that proper fall on couples immature cardiac mitochondria by opening the poor. We think the open poor in the immature heart and the open probability renders the infant and young child vulnerable to proper fall induced opening in the poor. And we think that this is potentially targetable so that we could develop a proper fall formulation that actually may be safe for infants and children for long term sedation to reinstate proper fall as a safe sedative without the risk of proper long fusion syndrome. And so that we're working on the Lang and Dorf and that's where we plan to go. So I want to thank the folks who did a lot of work and contributed to it as well as my funding source. So thank you very much. Any questions. Chuck. Oh, there we go. Thank you, Dr. Levy for a very fascinating talk your work here is very, very interesting to see. Does anyone have any questions for Dr. Levy? Rick, that was spectacular. You showed at the end in the in vivo or the Lang and Dorf model, the the intro libid control. There's been all the hypothesis about whether the type of lipid or the carrier or free fatty acid generation are confounders of the effect. If you suspend proper fall in or with a protein that finds hydrophilic or hydrophobic substances or whatever, can you can you show in in all those previous experiments that lipids have no component of the toxicity. Yeah, that's a great question. So we've done some work in the in vitro work with with lipid as a control as well and lipid is not a great control. If you look at what's in propyl the formulation, the thing that we use commonly in the operating rooms, you know, soybean oil is is is the major. And then we can put it 10% in the lipid is almost identical to propyl. But it has biological activity on a lot of things as you said, it's an uncoupler, it does a lot of things. It's not a great control and we do see effects. We don't see a pathological effects that we see with propyl. And from a toxicological standpoint, because other folks have said, well, have you you know, have you tested the pro because you can buy a crop of fall from sigma and just as a compound and just put it in solution with DMSO and you avoid the lipid question. And we can do that. The for me, our patients are getting the formulation. So from it, it's like pollution, right? If you if you wanted to study the effect of a car exhaust on brain development, you need to study car exhaust as a as a conglomerate. You can't just cherry pick as I was told by many reviewers, you can't just cherry pick carbon monoxide. So here we're interested in the solution as a conglomerate. So all the things that are in it, lipid is a reasonable control, but I think to be complete, we would want to do propyl fall by itself, you know, in DMSO, DMSO by itself and then to get a story. I think the LANGADOR of effect there is very telling probable apps lipid absolutely has an effect, but it's not causing the heart to stop. And there is something called lipatoxicity syndrome, which you guys probably remember, you know, for the with the primus who were overdosed with intro lipid and it can cause death as well. So there's definitely something about the lipid. And the part of the molecule that's doing this is independent of GABA receptor binding. And people are looking at structure activity relations in in the molecule to try to separate those. Everything that we were testing for has nothing to do with GABA. So and the poor, you know, this is within mitochondria that protein that we can certainly target. And the caveat is we don't know that we can't identify the, because it's not known, the actual proteins, you can't do a knockout, you know, you can't target it with other types of agents, but, but we do have drugs that are known to be specific inhibitors that are helpful. There are knockouts that can help get us to with it from a cofactor like the CIPD knockout. But we think this is as truly within the mitochondria. We do think it's a mitochondrial effect. And we do think that it really explains all of the features that are seen in the in the true syndrome. Paul Hickey, I love your talk because it gives me a scientific basis for my fundamental skepticism and bias against use of propyl and very young children, certainly infants and certainly nianics. Do you have any indication of what the relative maturation curve is in the human. So when there's a vulnerability of this particular protein complex to propyl on doing young coupling, when does that fade away or is it variable amongst various populations? It's a great question and I, the short answer is nobody knows. And the poor, the poor, the poor will open in anybody. It'll open in adults too. And, you know, it's thought to be do, you know, open within the context of TBI in the context of the scheme of reperfusion injury. It's thought to underlie some Alzheimer's pathology, Parkinsonism. So we know that it can open. And probably I'm using syndrome happens in adults and actually if you look at the literature, each month there's another case of adult press because the adult I use are still using it routinely for long term sedation, especially in the neuro units. And I think that the new born case report, I was waiting for that to come out quite frankly because if the pathology, if the biology of the toxicology is a real thing, there's going to be somebody out there who is going to be most vulnerable in that child who got, you know, a therapeutic bowl as a single bowl is went right into the foam and syndrome. And now whether they had an underlying, you know, in indolent mitochondrial disorder, I don't, you know, I don't know if that baby was tested, but that, you know, if you look at the durations, we know that if you give it long enough, you give it to people for three weeks, probably everyone's going to develop it if you give it to people for 48 hours, maybe a small. So there's probably it's probably going to open at some degree in everybody, but I think the infants are going to be more vulnerable. The second question, is there any evidence that the mitochondria from different tissues have different vulnerability? Is it heart muscle different than skeletal muscle? Is that different than liver mitochondria? That's another, another great question. And we started, we started looking at skeletal muscle as well. It's a ton of work that went into this and we tried to describe even just developmental changes. There are developmental changes just in mitochondrial expression of units, function of units, the different enzymes. So the short answer is I think that probably is some differential vulnerability. To me, you know, liver toxicity, you get rid of the drug, you do hemofiltration or dialysis, the liver will get better, right? People don't, and for something that's acquired like that, people are probably not going to die if it's recognized from liver failure. Cardiac failure is another story, right? That's really what takes these patients out. The raddenum myelosis can obviously, if it's skeletal muscle can lead to secondary injury and potentially be life threatening, but a lot of those things I think are supportive with regard to those organ systems that fail secondary. The heart, I think that that's the one that is the most important from a prevention and from a mediator or something that leads to death. The last question. One of the things the cardiac group has been doing here has been harvesting undamaged mitochondria from skeletal muscle and infusing them into the heart to try to deal with the hearts that are simply no longer functioning because of the scheme of repercussions injury and other things. Is there any possibility that you could do such an auto transplant into the heart in a small child with severe risk? Yeah, so I think, you know, I'm not, it would be a timing issue and then, you know, the heart would have to uptake the mitochondria in terms of timing to take it up. And then if how quickly that would restore function. A lot of these kids, number of these kids were supported on ECMO and because they, you can't resuscitate them. Especially when they go asistologist, they just are non-resuscitatable. And then they get better. If you can get to them, once the problem falls out of the system, they get better. What I think is happening in the heart is its hibernation and we actually have some quick data of this. I'll show you. This is a Schemier reper fusion. We, we, we, at the end of in the 30 minutes of a Schemian an hour of reper fusion, we injected propitium iodide, which doesn't get into cells except if they're dead and dying and then plasma memory is breached and then it gets into the nucleus. And you can see, and this is a Schemier, Schemier reper fusion. You see all these, basically all these red stars, right? That's, this is the left ventricle and you just see all of these red nuclei that are basically dead dying cells. This is the lipid. You see some, here's the propifull. You see almost nothing. And this heart is stopped. This heart is alive. It's just not beating and it has no electrical activity. So I think what's happening is that I think it's just a, it's a metabolic shutdown. It's a hibernation response. The problem is that's lethal. But the solution would be to support the patient and get propifull out of their system. And in some of these probifull infused hearts, we've washed them out and they start beating again. It's really fascinating. So I don't, so I think the, you know, although the mitochondrial transplantation and infusion is really novice novel and really innovative, it's probably overkill for what would need to be the solution. Just get the propifull out of the system. If you've got a patient that you're supporting. Just one more question. Just a surgical question. So you have to lower your the IQ level. Yeah. Okay. So I was just wondering if in the whole animal, there's an increase in oxygen consumption. When one administers propifull. And if so, can one measure oxygen consumption as an early warning sign of propifull toxicity? That's a great question. So we've tried to do it in the isolated heart. We were, we were like, oh, we're, you know, we're going to be able to detect this because we can measure. We do ABGs on the, the inflow, the perfusate and on the effluent. So we can measure myocardial O2P extraction consumption glucose extraction lactate production. And what's the heart is really smart. The heart is shutting down for a reason. And actually it's just matching. It's matching the demand to the ability to make ATP. And so we see it, even though it's profound, the heart is stopped. The electrical activity is silenced. The O2 extraction doesn't budge. It's still extracting O2. There's no lactate production. The potassium is normal. The glucose extraction is normal. So we were expecting that too. So I don't know what would happen in the human to tell you the truth. I know what happens in the human when the poor opens. When you have leak, you do get fever. And we've looked at with our fragile X model. Those have in the brain. They have pathologic poor opening. And they're one degree hotter than their litter mates controls. So it's a great question. I don't know. The fever was seen in about 36% of the cases in children. But those kids also had group and viral ideologies. So it's unclear if it was due to the poor opening or not. I mean, those who do, you know, take care of them. We use probe folic crazy in the MRI scanner. A lot of these kids, I remember back in the day when we used other agents, they would all get cold. Now I see a lot of our kids are getting hot. Is that the probe fall? Is it the magnet? Is it new technology? Is it? I don't know. So these are great questions. These and but these are also great potential biomarkers and room to study further in kids. Thank you again, Dr. Levy. Unfortunately, that's all the time we have for questions. Thank you.
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