Welcome to the Empirical Cycling Podcast. I'm your host, Kolie Moore, joined as always by our Empirical Cycling co-host, Rory Porteous. Well, not as always, Kyle's still in the desert on balloon duties, but he will be back hopefully soon with lots of balloon tails. But anyway, thank you everybody for listening. And if you are new here and you like what you're hearing, please consider subscribing to the podcast. And if you are returning, which I assume is most of you, thank you so much for coming back. Really appreciate having you back. And if you would like to support the podcast, you can always give us a nice rating. You can share the podcast, especially on forums. or by word of mouth. That helps a ton. Thanks for all of that. And because we are also ad-free and we are totally free content, you can always donate to the show at empiricalcycling.com slash donate or if you would like to become a client, it is about to be September. And so if you are thinking about coaching for next year, we want to consult on your training so far or training for the next year. please reach out to empiricalcycling at gmail.com and if you want to follow me on Instagram I do weekend AMAs and then that's up in the stories if you want to ask questions for the podcast you can also do that up in my stories so give me a follow over there at empiricalcycling and go ahead and ask away or just follow along if you would like And for this episode, we are actually not doing listener questions because we have something on the podcast today that we've never really talked about, which is another adaptive pathway that is fairly unique and it's really cool. Well, I think so. And we've been going a bit more in depth on endurance stuff. Mitochondria, Energy State, that kind of stuff. And we're going to kind of start thinking more about more high intensity stuff at this point. We're going to do more oxidative things, oxidative stress. We're going to talk a little bit more about VO2 max, and we're going to start to broaden our scope out of the muscles into the larger parts of the body. So it's been a little while since we've talked about oxygen, really. They have, well actually, let me just jump to this. Rory, have you ever heard anybody talking about hypoxia inducible factor in like media or on forums or just whenever? Because I don't really hear about it that much. I don't think I hear anyone talking about any of the shit until you talk about it. It's a very, very low chance that anyone listening to this knows what's going on. I don't know what's going on, and we already recorded this podcast once. Yeah, and it was a bit of a mess. It was extremely ADHD. Yeah, I was very late in a diet. I've now kind of brought my macros back up to normal and I'm actually awake. Rory is actually, it's no longer 11 o'clock for him after a long day. So we are actually going to be awake and energetic and that is going to be awesome. And we're going to keep the ADHD-ing with each other to a minimum. Well, when applicable, we will try to keep it to a minimum. So this is one of the pathways that kind of runs, would you say, parallel to a lot of other adaptive pathways like AMPK? So if we do like a VO2 max interval, like five minutes max, something like that, we're going to have AMPK activation, like quite a lot of it. We're going to have, if we're kind of untrained, we're going to have leaky reanodyne receptors. So this pathway runs a bit parallel to all of that. And I don't think we've ever discussed it before, have we? Right. So we haven't. Can you do some refresher on like high intensity training for the folks out there? Or hit training as you like to call it? Hit training as you hate it. In fact, this is where we made the joke in the last recording. The ADHD has already started, folks. Hit training or there's grappling training. Yeah, so any sort of high intensity training is typically thought of as something that's going to work in the intensity domains above FTP. When we're talking about high intensity, we're specifically thinking about something that requires a conscious, hard effort, usually quite short. Often a repetitive sort of thing, like a 30-30s, which can be of various lengths. You could also do a 15-15s, 40-20s, but it's usually, there's people that like to do them as a VO2max sort of workout, and you probably get a bit more luck doing them as something where you're working on anaerobic power, more so than that. They're simple workouts. Short, hard, horrible. Everyone says they're excited to do them when you put them in their training plan and then they very quickly change their mind. Yeah, that's pretty normal. A lot of, oh my God, that sucked and some people actually say, I can't wait to do this again. I've had people volunteer for workouts like that. I screenshot people's comments when they say they're looking forward to it and then I send it back to them. You are exactly that kind of coach, yeah. So one of the things that happens during these high-intensity efforts is, like I said, AMPK activation is probably the most well-known one because AMPK is very, very well studied at this point. HIF in the muscle, like we're looking at today, is actually very infrequently studied. And I've pretty much read, I think, just about every paper that's been published on hypoxia-inducible factor in the muscle with exercise. That's not the usual thing. Usually I have to glance over a couple papers, see, okay, because I've got to triage my time. Like I can't read every paper when I'm preparing for an episode. But this one, there's so little research, but there's some good research that I was fortunate enough to kind of have a very good scope of. the actual adaptations. So when it comes to AMPK, that's energy state. We're looking at ATP becoming ADP and the AMP levels in the muscle rising and AMPK gets activated and that yields a whole bunch of other adaptations we've talked about before. But this one senses oxygen. So HIF, hypoxia inducible factor, is a low oxygen sensor. And it's actually in every tissue in the body, but we're really thinking about muscles. So we'll think about things like kidneys later when it comes to other pathways. But basically what happens is as you sustain high intensity exercise, the oxygen tension drops in the muscles. So basically what happens is hypoxia inducible factor activates, kind of like PGC1 alpha in a way, where they're both transcription factors. So HIF will activate and move into the nucleus to help genes transcribe in response to low muscular oxygen levels. And I think that that's really cool. And the discovery of HIF is actually... worth noting too, because it led to a Nobel Prize for, quote, the discovery of how bodies sense and adapt to oxygen availability. So basically what investigators were looking at, they were looking at the origins of erythropoietin, EPO creation in the kidneys and the liver, and that's where they discovered HIF. They basically took snippets of a DNA sequence from an enhancer region of the EPO gene, and they basically went fishing. They said, okay, we've got this thing, we're going to dangle this little DNA strand and see if anything binds to it. And that's how they pulled out HIF from the pool, so to speak. And that's like an old school, I mean, well, this happened, I think, in the 80s and 90s, if I'm not mistaken. So that's like a really old school technique. I think actually, Rory, last podcast we recorded on this, you said that people were still doing a lot of this kind of stuff. I think so. But like just generally like one of the interesting things about any sort of genetic study is often when you're using like a model animal or plant like Arabidopsis. and the way those studies often try to work out how a gene works is to remove it and just see what changes which is a really like when you describe it like that it's quite a funny way to do science it's just it's definition fuck about it and find out but like that is the best way to do it because if you can't if you don't know what the gene is doing before You actually conduct your experiment, then the only way you really have to find out is to breed up an organism that is missing the gene that you are interested in looking at and just seeing what's different. The downside is... Sometimes they don't survive. Yeah, like catastrophic. Is that what a karyotype malfunction is in humans? That's the sort of thing that causes major developmental disorders. Yeah, something like, I believe, well, karyotype is when you make all of your chromatin or chromosomes shrivel up into the compact version that happens during cell division, and you look at them under a slide, basically. Yeah. So if you Google, like, what are, like, you know, all, you know, genes, blah, blah, blah, in the body, like, you're probably going to see. All of the human genes. And it's varied throughout the animal kingdom. But one of the things that we're going to do in the future, because I have an idea to do four episodes on this. And the next two episodes, we've got a knockout. So that in the gene studies is what it's called when you get rid of a gene. We've got a knockout study. And we also have a, we're going to make this obligately active study for HIF. And both of those are really cool. And then we've also got one on well-trained athletes that we're going to look at last, I think. So I think it's going to be a really cool arc to look at this stuff. And we're not going to have a ton of super applicable takeaways, but we'll have some for sure. But I think it's just worth really considering on how all this stuff happened. Actually, I can link to a good... study on, or a paper on the discovery of HIF and EPO and stuff. And actually the really excellent health outcomes that have happened because of this research, which is one of the reasons it won the Nobel Prize in Medicine. So, because I think also for our audience at the moment, they're probably, if you've heard of it, you probably know about it in the kidneys. because kind of like we mentioned, it has a big role in activity in the kidneys for regulating EPO production, which in turn will increase red cells. On the other hand, in the muscle, it's, yeah, I don't really hear a lot of people talking about it and we'll kind of see why that might happen maybe by the end of this episode or maybe a little bit into the next couple. But I think first, Let's revisit the dynamics of blood flow into muscle and oxygen distribution. So oxygen gets into the muscles by passive diffusion. So what's, and this is like, how many science classes have you taken, Rory? We've both taken a bunch at this point, where day one, you've also taught them, where day one is transport. So, embarrassingly, diffusion and osmosis were two things that I always forgot to talk about when I was lecturing. But yeah, it's one of those things where it's such a basic component of understanding biology that you assume everyone knows it, which as a lecturer is very irresponsible. Yes, that's absolutely true, which is why we're talking about it right now. Passive diffusion means there's no energy input. I'll give you an example of passive diffusion way back in the day. We had an experiment looking at passive diffusion across erythrocyte membranes, so red blood cells. And what we did was we basically put different alcohols with different lengths of carbon chains into a solution with red blood cells. and the faster it diffused into the red blood cell the faster those cells would explode and become transparent and after a little bit you could actually see like a black line on the other side of the test tube and you basically timed how long it would take and the entire process like you didn't have to heat it there was there's like no whatever like it just it just shoots into the into the red blood cells and explodes them. And that's passive diffusion. There's no input. It just happens based on very basic principles in physics of just basically entropy trying to equilibrate everything on each side of a membrane where they're capable of diffusing through. So is that reasonable? Yeah. Things move from a region of high concentration to a region of low concentration until that equilibrium's been reached. And then there's no concentration gradient for it to flow down. Yeah. Here's an example is if you fart in the corner of a room, it's not going to stay in the corner of that room. The fart's going to distribute through the room going from the area of high concentration next to your butt to the areas of low concentration, which is everywhere else and potentially into the nostrils of very unhappy friends. So the fart model of entropy is what I learned in Chem 102. My professor was very proud of that one. The fart model of entropy. So the partial pressure of oxygen in the atmosphere is about 160 torr. One torr is approximately one millimeter of mercury for the Americans out there who are familiar with millimeters of mercury and not real units. So what is a partial pressure, Rory? Why isn't the oxygen pressure in the air just one atmosphere? Because The air at sea level is one atmosphere. Pressure. So if you think of air pressure as a single homogenous whole, then yeah, you have one whatever pressure unit you're deciding to use. In this case, most people are just talking atmospheres, I think, nowadays. Partial pressure is how is that pressure made up? So how is the entire pressure of the gas you're talking about? Basically, what's the division on it? So I can't remember the specific divisions of atmospheric air. It's like nitrogen, oxygen. Nitrogen, oxygen, carbon dioxide are the main ones. Yeah. And so if oxygen made up 50% of the air, Then we could say the partial pressure of air or of oxygen in the air at 160 torr is just straight up 80, 80 torr. Yeah. So 50%. It's actually 21%. Yeah. Yeah. 21%. So it's 21% of the yada yada. Anyway, you guys get it. So what happens is that pressure continues all the way through your body into your muscles and other tissues. Oh, quick note, partial pressure, I'm sorry for this, is also called tension. So when we say oxygen tension, we are also talking about oxygen partial pressure, and we're going to use those interchangeably. And for that, we apologize for whatever confusion that causes. But anyway, so by the time the oxygen gets to your lungs, diffuses into your capillaries and pumped into your muscles, well, not really pumped, it just diffuses. So that's, your heart's pumping the blood there. Passive diffusion. Passive diffusion. No energy input. There's no pumping except getting it in the area. And then it diffuses into the muscles. At rest, the oxygen tension in your muscles is about 40 tor at sea level in healthy people. And so working mitochondria, where we turn oxygen into water at complex four, they basically have a spot of zero partial pressure. where the oxygen gets reduced to water. And this is probably a bad analogy, but I think I've used it before in the old Wattstock series on VO2 Max where it's kind of like a little black hole or a little vacuum. And so as the oxygen goes away from that, you've got a spot for the oxygen to diffuse into. And you can think about this throughout your entire body. That's pretty much how I think about it. And so otherwise, if you didn't have the oxygen disappearing from there, if it just sat there, You'd be saturated and there would be no need to actually pump blood around your body at all, which would be very strange. But luckily that's not what happens. So we can think about it. It's almost like falling down a concentration gradient because there's less oxygen in the next compartment. There's less oxygen in the next spot and less oxygen in the next spot. And so the high concentration goes to the low concentration. So that's basically what's happening. And you can probably extrapolate that out now, listeners, too. If you think about high altitude where the pressure of air is lower, thus the partial pressure of the oxygen in that air is lower, which is why you struggle to get enough oxygen in altitude slash Y altitude is beneficial when it comes to something like aerobic training. Well, you are getting ahead of us. But also, don't forget in the AMPK episode, because the power outputs were lower at altitude, There was less AMPK activation. So there is a trade-off, but it certainly does, and we're going to see this in a future episode, it certainly does activate hypoxia-inducible factor in many, many tissues, and it has a lot of interesting implications for performance. But anyway, we're not talking about that today. We're talking about how in the muscles during high-intensity exercise, so I said at rest, oxygen tension is 40 tor. During absolute max exercise, This is very difficult to measure, but the approximate measurements we have are about one to four tor of oxygen tension in the muscles at like VO2 max. Like when you are doing a five minute max effort, right before your legs just give out, there's, you're damn close to zero oxygen in your legs. It's not zero, but it's low, low partial pressure, very low. The oxygen is flying into the legs and immediately getting consumed essentially. Yes, that is exactly what's happening. And so, but what happens if we go to like below sea level or if we go into a hyperoxic environment? Now partial pressure goes up and we can put out a ton more power and that has its own kind of ups and downs when it comes to training and racing, which we will discuss when we talk about altitude one of these days. So we should also be careful of our terms because I don't want anybody to think of this as anaerobic. Anaerobic has a very specific definition because this is not anaerobic, this is hypoxic. And we'll now over-explain the difference as usual. So when low oxygen tension happens during exercise, it's approaching anaerobic oxygen tension because we're getting close to zero, but it's definitely not zero because it's also highly aerobic. We're at VO2 max. There's a lot of oxygen flux happening. Metabolism is happening at as high a rate as it can, but being basically oxygen supply limited, we are having a very large amount of oxygen flux. So the flux meaning there's a lot of oxygen passing through and being utilized at a rapid rate. So even though there's low O2 tension, there's still a large amount of oxygen being used, which is highly aerobic. And so here's my analogy for this. I get cereal every time I go out and get food. Rory knows what's coming. And so if I'm eating cereal at a rapid rate, at most points of the day, you could go look at any one of my cereal boxes and go, there's nothing in here. And so the question is, I'm consuming a large amount of cereal, but am I in a high cereal environment or a low cereal environment? In a sore tummy environment. Potentially that too. So the buy and eat cycle for me in cereal is what happens in the muscles when you're doing high intensity intervals. You've got periods of high flux during exercise and momentary low levels, just like cereal in my kitchen. And I think the last thing to think about hypoxic conditions with is that Hypoxic comes from the words hypoxic. And so if you are thinking about, you know the word peroxide? So peroxide comes from hyperoxide. You just take out the HY off of it. And so hypoxic, hypoxic, because if you put two O's, it would be hypoxic, and that would look strange, I think. as opposed to normoxic conditions that we normally have at sea level. And then we have hyperoxic conditions if we have supplemental oxygen or we go below sea level or something like that. So I hope all that makes sense. It makes a lot of sense, listeners, if you look at a diagram of peroxide and realize that there's two oxygen molecules. And also two hydrogens. And two hydrogens. And that's, yeah, anyway. I would get into a thing about catalase right now, but we don't want to ADHD any more than we've already done. Okay. Yeah. So hypoxia inducible factor, that's our low serial sensor. And basically what it is in all of your cells in your body, or just about all of your cells, it's a constitutively active protein. And in skeletal muscle, it has a role of, as usual, sensing low oxygen levels. and turning that into a change in phenotype. There's two subunits to hypoxia inducible factor, HAF1 alpha and HAF1 beta. And so they're not usually attached to each other, but they can be. So if we are not talking about a specific subunit, we're going to talk, we're going to just say HAF1. HAF-1 alpha has what's called a regulatory domain on the protein. And so when oxygen is present in sufficient quantity, HAF-1 alpha gets modified with certain hydroxyl groups on some proline residues. And basically what this happens is it makes it a target. for VHL E3 ligase protein complex. So VHL is von Hippel-Lindau. So if you are like a cancer researcher, you're probably extraordinarily familiar with this. If you've researched the ubiquitin pathway, you're probably very familiar with this too. So basically what happens is it marks it for degradation. It's like getting marked for death. You get this complex on your HIF-1 alpha and your HIF-1 alpha is not long for this world. That means it cannot be active. And the ubiquitin pathway degrades it. And by the way, it's called ubiquitin because it's absolutely everywhere. And it seems to be mostly well known for tagging proteins for degradation, but it seems to have some non-degradation functions that I'm not too familiar with because I'm not that deep into that area of research. But that's something cool to note. Anyway, when our oxygen pressure in the muscle starts to drop with high-intensity exercise, altitude, or Injury or Ischemia. So Ischemia means like just cutting off blood flow. When all that stuff happens and you've got low oxygen levels, the things that would bind the alpha subunit and tag it for degradation are now going to bounce off of it. They are no longer going to bind to degrade HAF-1 alpha. By the nature of it not being degraded, it's now what's known as stable. And so when it's stable, it means it's just hanging out going, do we have HAF-1 beta around anybody? and occasionally it's going to run into an HIF-1 beta and that binds them together. And now that they are together and active, they can go into the nucleus and target gene expression. So how fucking cool is that as a mechanism? Like you've got this thing, you're going to make it constantly. Let's say you've got a day where you're doing four by five minute VO2s out of 1,440 minutes per day. You spend 1,420 minutes per day getting rid of HAF-1 alpha pretty much. Not exactly, but approximately. I think this is something we'll talk about a little bit later, but this is very much, it kind of goes against the grain for what you'd expect cells to want to do, because this is your cells constantly creating this protein purely for the purpose most of the time. Especially in sedentary people, for it just to get destroyed again. And its actual use case is when it doesn't get destroyed, but all that energy goes into creating it. And I think we're going to get into a wee bit later why that's actually quite an interesting twist in what you'd normally expect a cell to want to do from an energy preservation purpose. Yeah, I mean, because it's spending energy, basically. I think my analogy for it is like if you're if you have a fire in your kitchen and that's when you order a fire extinguisher on Amazon. Yeah. Like it's going to take you, you know, if you got Prime, it'll take you a day or two for it to show up depending on where you live. And by then you've hopefully put the fire out or it's burned your house down. So so it's good to have around, but it's like imagine if you had to. If every like 20, 30 minutes your fire extinguisher expired and you had to get a new one, but by the nature of being alive, you know that there's going to be a fire in your kitchen, metaphorically speaking, potentially a couple times a week. And so not having one becomes very, very, very dangerous if you get that fire in your kitchen or you run into low oxygen levels. Because if you had low O2 tension and the partial pressure just drops because you're doing super high intensity exercise. And then you start to transcribe HIF-1 and then you can adapt. It's like the stimulus is gone, you know, a while before that protein actually shows up. It's like, you know, it's like a firefighter showing up at your burned down house and going, well, I guess I'll go home. Nothing for me to do here. Call me earlier, guys. So that's kind of what it's like. Google futile cycles in biology. There are tons of them. And there's some in glycolysis. There's a bunch like this in terms of proteins that are made and degraded except for in emergency situations. And in that respect, it makes a lot of sense to why you would want to spend the energy to make these proteins despite the fact that most of the time they're just... Not Being Used. Rory's giving me the nod like I've beat this horse to death. So, okay. I'm trying not to ADHD again. This is how it happened. All right. So, the sensing mechanism of HAF also means that activation or dimerization is not all or nothing because we've got more than one protein per muscle cell. So we've got a lot of proteins and the number of proteins that are activated would be in response to the magnitude and duration of the hypoxic stimulus. So you can imagine if you're at altitude and the O2 tension levels in your muscles drop a little bit, you're going to have, I don't know, maybe like 10% activation or something like that if you go from like 40 to 30 tor approximately. It's difficult to measure and everybody's going to be a little different depending on their training status and yada yada. Don't take these numbers to heart, but let's say you go up to altitude and you're like 10% activated because you now have like 30 tor in your muscles. So you're going to have a low level of stimulus for a long time as long as you're at altitude. And so that's very different than being at sea level and doing five minutes of VO2 max and going, wow, I'm dead. And your muscles are also going, wow, there's a... Big Stimulus. So those are the two ways that we can think about this. So it's like a proportion of activation of the total proteins in the cell. But as O2 tension drops, the increase, the function is actually exponential. We get exponentially more HAF1 activation. And so one of the problems, though, with thinking about exact measurements, because I had a couple in here in the notes, and I was like, you know what? It's difficult to measure in practice because like the closest we've got is like a MOXIE or any kind of NIRS monitor. And that's only looking at like, you know, what, a centimeter below the surface, depending on how much adipose tissue you've got in the way, depending on blood flow and all that kind of stuff. So it's not amazing. And it also doesn't give you absolute units. It's just looking at the proportion of hemoglobin saturation and desaturation. So we've got some... We're not doing any mid-VO2 max interval muscle biopsies. No, that sounds fun, but unnecessary. So, I mean, so there's not a lot of practical knowledge that we can take away from thinking about this. But also, the other thing is that as a basic principle of adaptation, we are going to have a phenotypic change. to alleviate a stressful condition. And the stress means disturbing your equilibrium of your cells. And so when we think about this happening, we could think about it in the same way as like AMPK activation or anything else where we get a certain level of activation, but as we get better and better trained, we expect that activation to drop off at the same relative or absolute exercise intensity. Essentially, we've got A mechanism within the muscle that's able to detect and respond and initiate specific responses to a state of low oxygen, not no oxygen, but low. And when we come into some of the training stuff that you'll probably talk about in a future podcast, this is one of those things that the training is meant to be targeting a little. And it's also one of those things that... Maybe if you're a bit newer to the sport, you are going to have a much easier time getting an effect from rather than if you are a 10-year racer. Yeah. I mean, and this is kind of what we see too. It's like, I've got some... You know, we've got a bunch of podcast ideas. And one of them I've got is training mistakes for like intermediate and advanced athletes. Well, we'll probably do this two different episodes. But one of the mistakes is thinking that the plan that you did when you started training, like started structured training, and you saw all those big improvements, thinking that that plan is going to be what's going to get you your next big bunch of improvements is probably... Not going to be the case. And this is one of the reasons why. And actually, we're going to see some hard data on individual variation in this stuff in the paper that we're going to look at today. So yeah, so that's, yeah, all really, really good thoughts. So what adaptations do we get from HIF1 in the muscle? So the genes that it targets... There are several hundred at least. That's the last count. That's the latest number I saw like as of a handful of years ago. And a bunch of those are like tumor suppressors, cytokines, epigenetics, apoptotic genes. But for our purposes, we want to think about things that help us with exercise and performance. So the big one is oxygen delivery. How do we get more oxygen to our muscles? Well, in the kidneys, they're going to signal for more red blood cells. But for muscle, the really big ones are going to be, well, let's think about what's happening. High intensity exercise, you have a reduction in oxidative availability. And so what's our energy pathway that's going to help us in the meantime? Yeah, you're rate limited in terms of how much oxygen you're actually capable of using. So if you have to continue working, how do you shoulder that load? Yeah. So what we're going to do is we're going to start making a bunch of genes. that will improve our glucose uptake and metabolism. So with low aerobic glycolysis, we're going to have a lot of anaerobic glycolysis. So you're going to need to pull glucose in from the bloodstream, so you get a lot more of those transporters. And we're also going to, especially early on in training, we're going to improve a bunch of enzymes that would help us with glycolysis, just straight up anaerobic glycolysis. And so like we'll see actually, I think we'll see a transcription of like hexokinase in our study today. But we're also going to upregulate genes involved in pH, like buffering and lactate transport. And so especially during hypoxic conditions, we've got a lot of acidity happening and unrelated, by the way, we're going to have a lot of lactate made. And one of the big lactate transporters will export. a lactate plus a proton, like as a package deal. And they're going to just shove those out into the bloodstream and be like, all right, get out of here. It's too busy in the cell. Go away. So we're going to get a lot of those. And we'll even activate some genes that are related to iron handling by the muscles. Because of course, we have a lot of proteins that have iron in them, an electron transport chain and myoglobin and all that kind of stuff. And so the muscular response to HIF seems actually to be Down-regulating the muscles iron uptake proteins. Down-regulating. So we're going to make less of these things in order to keep, seems to be in order to keep, this is my interpretation by the way, iron in the bloodstream available for red cell formation. So maybe a little counterintuitive that one. Yeah, remember that one of the things that's coming down the line here as a result of this sort of pathway is, You're hopefully going to get some more red blood cells out of it. And if the body wants to be able to do that, it needs to make sure that the resources are available to do that. And one way to affect that is to block the uptake of iron into places that at that moment in time don't need it. But the body is determining, basically as a safety mechanism, I need more red blood cells. I need iron. Stop using the iron. We have an iron shortage. Yeah. Speaking of which, get your ferritin checked, kids. And so one of the other, one of the really, really big ones, and I kind of wanted to build up to the main one that people think about with HIF and the muscles, is the vascular system. We're going to start creating more capillaries around our muscle cells. And the signal pathway... for HIF also targets the VEGF gene, the vascular endothelial growth factor gene that signals for more capillary growth. And so we're actually going to see a knockout paper, like I said, that will look at the fewer number of capillaries around a muscle fiber. In relation to that, we're also going to get an upregulation of genes involved in nitric oxide signaling that are used to dilate blood vessels. They're not just for boner pills. This is something that happens in well-trained people. And usually, this is my interpretation anyway, it's one of the reasons you don't necessarily need beetroot juice if you're well-trained because you are training your body to do this already. So... Yeah, this is the other side of the make more red blood cells. Component here is because the general problem is oxygen availability, one of the ways in which the body will respond is how can I get more oxygen to this area, which is essentially a means of increasing the surface area of blood vessels available within that area, which is just, you know, in these tissues, more capillaries, you're not growing an entire new artery. It's trying to just... Give as many opportunities for blood to pass by this area if it is in demand of oxygen. Obviously, not within the workout you're doing, but for hopefully the next time you do the workout. Yeah. And I've already started digging into the literature on blood vessel formation, and there's a lot out there. It's really, really, really cool stuff, and I'm super excited to get into that in the future. But for now, I'd like to look at our paper for today. And so you can probably see the timeline of the episode and know that there's going to be a short episode or short for us. And so we're not far from the end, but I think this paper is going to be really cool because I want to talk about the kind of training when HIF is highly activated in the muscle. But I also want to use this to kind of think about our kind of more basic principle of the Phenotypic changes that are going to alleviate the stressful condition. And we're going to see a little bit of data on how that actually starts happening. So we've got a link to this paper at empiricalcycling.com under the podcast episode for the notes. And I also have to say that a lot of the papers that we're going to be talking about for HAF are not my favorite. But it's the field of exercise physiology. So it's slim pickings for HIF studies, especially in muscle. But like I said, I actually managed to read through almost all of them. And this is one of the really, really good ones. And it's in part because of some of the techniques that they use that we're going to talk about. So the paper is a HIF-1 signature dominates the attenuation in the human skeletal muscle transcriptional response to high intensity interval. Training. All right. In this study, they had 11 healthy, subtext untrained, men do nine sessions of HIT over three weeks. So they hit these guys nine times in three weeks. That's probably the not right way to say hit training, but there we go. The sessions were 10 by four minutes at 91% of max heart rate with two minutes rests. Again, not my favorite, but there's not a lot of good comps for this paper. And by that, I mean none. So they didn't focus much on our old friend PGC1-alpha in this study. So fortunately, the main focus was hypoxia reducible factor. So the training session that they did, 10 by 4 minutes at 91% max heart rate with 2 minutes of rests, these were progressive in nature because They were controlling power output. And as the heart rate would drop, as people got better trained, the power output that they could do was going up. They had to improve the power outputs. So they raised the power, keep the heart rate within the target range. And over the course of the study, so this is three weeks, the VO2 max increased an average of 8.7%, which is a really good response for three weeks of fairly hard training. So what they did was before and three hours after the first and last bouts of exercise, they biopsied the participants. The biopsy checked muscular transcriptional response to exercise. And so what we're after here in the paper is going to be in figures 2A and 2B for most of what we want. So what these are, are ridge plots. and the x-axis is log twofold change. So we discuss a little bit of standard genomics work in Wostok 41. Basically, more log twofold change, more transcriptional activity. I was joking before about trying to make papers legible for people. This is one of those figures that doesn't immediately jump off the page as easy to understand. Essentially, because you're... X-axis here is representing the log-fold change. As you go from left to right on these charts, the magnitude of improvement is going up quite significantly. So essentially expression is very high if you're on the right-hand side of the chart and it is lower if you're on the left-hand side of the chart. And what you're hopefully able to see if you're looking at these is between the first bout and the ninth bout. You're seeing that there is usually some sort of shift towards the left as they've gone through this exercise program over nine bouts. Yeah, and I think that probably the most important ones to look at, well, we're going to, let's look at our basic, the top left. You know, we'll go to like the top one, top ridge plot for 2a. And this is looking at the VEGF signaling. So this is a big target of the HIF pathway. And we see from the first of the ninth bout, there is a basically non-significant difference between the two. And what we see is probably a little bit of leftward shift for the ninth bout. It's not significant, but it's starting to trend that way. And basically what this means, and there's also like a downward shift a little bit. So the peak. kind of moves up a little bit over the, what is that about 0.25, 0.3 log two fold change. And all the genes kind of over on the right are starting to drop. The magnitude of that intensity is dropping for the ninth bout. And that's kind of saying that all the genes that were over here that were more upregulated after the first bout have kind of been shifted over to the bin of like, yeah, these are a little less upregulated than before. You've gone from a high response to a lower response. Yeah, it's not low by any stretch of the imagination, but it's lower, not significant, but trending down. And if we just look at the VEGF specifically, they've got like a subplot for this one. It's log CPM, and we're looking at before the first bout, the box plot is somewhere around like the high sixes. And after the first bout, it's all over eight, basically. And the error bars are actually fairly small for this. And for the ninth bout, beforehand, it's also kind of in the high sixes, below seven. And then after the ninth bout, we're looking at the VEGF being in the seven and a half to eight for the box plot. So we're showing a decrease, but it's not... You know, it's not massive, but we're seeing a shift towards a smaller signal as you do the training. Yeah. And this becomes more obvious in some of these other ridge plots, especially as you get down towards the ones that show a statistically significant difference when they've actually assessed it, which is platelet-mediated interactions with vascular and circulating cells and glycolysis and gluconeogenesis, Possibly the most significant one. Let's dig into that one because that is a really, really big one. So after the first bout, we basically have a bimodal plot. We have a giant bump around like 0.8 log twofold change. We've got another giant bump around like 2.2, two and a quarter-ish. And for the post ninth bout, that bump... at two and a quarter is gone, completely gone, it's just flat. And all of those genes have basically shifted into the like 0.75 log twofold change ridge, which is more or less, to my eyeball, looks like it's about doubled in size in terms of the area under the curve. Yeah, like you've gone from this pathway exhibiting some sort of massive training response and a non-training response to basically just the non-training response. Yeah. So, well, and that's actually very common that we see in literature is, especially with glycolysis and gluconeogenesis, the response for basically all types of training is fairly rapid. And even with like FTP training, if my memory serves correctly, like, or as they would have called it in the paper, like lactate threshold training, probably done in the like 70s to 80s, where the Glycolysis enzymes showed a big response initially, and then they kind of tapered off. And that's pretty common, especially when you think that, well, glycolysis, if you just isolate those enzymes, they've got way more capacity to actually break down glucose and provide ATP than we can have in the cell. But kind of like we talked about in, what was it, Wastlock 40, where we're talking about the larger mitochondrial reticulum and that surface area and serving... the cell better in terms of energy state maintenance. We're looking at the same thing when we make more of these enzymes. So despite the fact that we're probably not really tapping out the true capacity for the system, we are actually going to be serving a wider area better. I think one of the other cool things is think about how this might change in trained people. We don't have the data, but we can make a reasonable estimation that you would probably see a fairly strong response in the VEGF pathway, but probably not to the same degree. And we would probably see a similar response in the glycolysis and gluconeogenesis, et cetera, et cetera. And it's kind of difficult to say exactly what otherwise until we actually see some data on this. Yeah, if you're just making the educated guess, you'd assume that someone who's very well trained is just going to shift to the left, the better trained they get. Yeah. All right. So let's look at figure two B now. So over on the other side. And what we're looking at here is we're looking at the pre and post first bout. And then we are comparing it to what they call a training steady state. And the training steady state means before the ninth bout. So what's basically happening at the muscular level in terms of what pathways are active. from before starting to train, and then what's more or less constitutively active to a low level that's changed when you've been training hard for three weeks minus one session? Well, actually, our glycolysis and gluconeogenesis is basically the same, even though they misspelled gluconeogenesis. That's gluconeogenesis. Oh, you're probably right. Editors, every time, right? But what I think is most interesting here is the log twofold change in the TCA enzymes. So we've got actually a fairly large bump up. It's not massive. So we go from the log twofold change from before the first bout being about About zero, actually. That is a, that's a ridge plot that's centered around, it's just a big, tall, normal distribution centered basically around zero, maybe a little below. And then our training steady state, quote unquote, is just a little bit above zero. It's at like 0.5 for log twofold change. About the same magnitude too, but it's significantly different. And especially When we look at the single gene that they've pulled out to represent the whole thing, they pulled out citrate synthase, which we've talked about a lot in the podcast, the entry point to the Krebs cycle. The citrate synthase before and after the first bout and after the ninth bout, we actually see a drop in the expression of citrate synthase, which I think is very, very telling of one of the, it's, how do I put this? Let's say it's foreshadowing for some of the trade-offs that we make when we activate this pathway. And there are trade-offs. So your thoughts on these bumps in citrate synthase, Rory, so far? It's interesting that it shifts right before the work period has begun. But yeah, I'd have liked to see the ridge plot of one after it as well. See if that pattern maintains. Yeah. So I think kind of what we can take away from this so far, and this is just my interpretation of this data, is that in untrained people, a sufficiently high training intensity should be enough to keep hypoxia inducible factor one activated, along with many other adaptive pathways. Like I'm sure that if we looked at AMPK for this, we would be seeing something somewhat similar. And it also corroborates a lot of the other literature that sees a large and fast increase in things like muscle capillarization, mitochondrial density, glycolytic enzymes, and et cetera, et cetera, et cetera, in terms of normal fast training response. But the response tapers off if you do keep people training for long enough. And I'm sure part of it's desensitization. And part of it's fatigue. Part of it may be like nutrition or off, you know, off-bike related. But I think that it's really interesting to see this in untrained people because this is probably the first time we've actually looked at hard data that shows the... the decrease in training response, even in untrained people who you'd expect, oh, three weeks, okay, yeah, they're going to be flying, there's going to be no difference in response, but like, actually, we're starting to see a difference in response in some pathways and a giant difference in response in other pathways. Yeah, if you have gotten into cycling in any sort of serious way in the last year and you think about, you maybe don't feel it at the time, but if you think about how fast you got in the first month, two months, three months, You probably notice the biggest difference in the first month if you're really honest with yourself. And part of what you're seeing here in this paper is here are the potential reasons why, not that you didn't get faster from there, but why it isn't unfortunately a linear increase in fitness when you start. Yeah. Yeah. So like I said, we're also... We can only speculate based on our coaching experience on the difference that would be in trained people. And this is definitely a study I would really, really like to see. But in a couple episodes, we are going to look at a study on trained people. We're not going to see a difference from the untrained state, but we will definitely see how the signature changes in trained people. But anyway, in this study, I think one of the coolest things that they did was they actually looked at individual variation in a couple genes. in terms of the transcriptional activity. So they looked at individual variation of PGC1-alpha. So they're looking at the transcription of that of the aerobic pathway, a MAPK target gene. And they are also looking at, well, NR4A1, don't even worry about it. Between the first and the ninth session, they found a massive amount of variability in all genes. So now this is looking at individual training response, remember. So for PGC1 Alpha, variance alone was 25% in the first session and 14% in the last session. And for our MAPK representative gene, 57% after the first session and 36% first to the last workout. So we're seeing a massive difference in individual variation. of these genes and how people respond to training. Rory, you've got the furrowed brow. I'm trying to remember how this chart works, but I now understand it. And yeah, like one of the things that stands out about it is there's an enormous degree of variation between all the people that took part in this study, which you can potentially extrapolate out to whatever population they came from. But yeah, like... Some of these subjects are getting a close to 20-fold change in PGC-1 alpha expression, whereas others, I think the lowest is under 10. But interestingly, the average compared, so they give you the initial response after the first bout, they give you the last response after the ninth bout, and then they work out an average normalized for basically what the original was to what the new one is. And the average change is actually much more clustered than the peak expression, which is interesting. So there is some... The actual interesting part of this is where the average is set is around between five to tenfold, unfortunately not 15 to 20, as some of these people get on their final one. Yeah. Yeah. And actually, and looking at HAF1 Alpha itself, that was upregulated 2.8 fold after the first workout down to two fold after the second. And this is not from this figure. I think this is from the body of the paper, but... The first workout made our old friend PGC-1 Alpha go 13-fold upregulation down to 7.5-fold after the ninth. So we're seeing that drop in training response, but the huge amount of individual variation. And I would have loved to have been in the study and found out that my PGC-1 Alpha response was like, oh yeah, you're at the bottom of the barrel. One of the things I need to note on this, because actually just talking about the difference in PGC-1 alpha, NHF-1 transcription kind of bugs me though, but like I said, we don't have a lot of other comps for this paper, is that they're transcription factors. They're a step on the way to the adaptation, which is a step on the way to improve performance. And we can take the transcription amounts here to be roughly representative of the activity of HIF-1 and PGC-1 alpha via their need for replacement, but it doesn't really say anything about the subsequent steps. And this is one of the big reasons I have actually excluded a ton of studies on the podcast in the past because They look at two different training interventions and they look at PGC-1 alpha transcription only. I want to see activation. I want to see genes downstream of that. Then I want to see performance. And just as it is now, I'm not super stoked about this one, but it's, you know, I think in the context that I mentioned that we might... be able to look at it as representative of their relative activities by their need for replacement. I think that's probably the best way to think about this little piece of data here. It's interesting enough to stand out. Yeah, it is. The last thing they did was look at what could have been causing the blunting of the pathways. And what they found was, and we don't need to get into all the yada yadas on it, but it's basically negative regulation of HAF-1 alpha. So what's negative regulation? They're making the pathway less active. And so basically increasing the proteins that would degrade HAF1-alpha to make that pathway less active. So if we got less protein for the same amount of hypoxia in the muscle, we are going to get a smaller response. And to put it another way, what is in short supply at high intensity exercise? Oxygen. What adaptive response would make the most sense in the short term versus the long term? So in the short term, it would make the most sense, and we've kind of touched on this already, to decrease the cell's reliance on oxygen because you don't make sufficient adaptations after one workout to fully alleviate the stressful condition. It takes repeated exposure to the same stress in order to... have some kind of response. But in the long term, what we're going to do is we're going to increase the cell's oxygen supply and alleviate the stressful condition. And when we start alleviating the stressful condition, we're going to, by just definition, decrease the amount of the stimulus that we give the muscle cell. Yeah, this is relating back to what we were saying earlier on of long term. Want more red blood cells, want to produce more capillaries to try and maximise how much oxygen we can get to tissue. But there and then in the moment, you've got a demand for energy to be created and burdened and created and burned. And the only way that you're going to actually be able to do that is not by increasing the supply of oxygen at that moment, because you don't have the ability to do that beyond breathe a bit harder and faster, which you're probably already doing. You have to start. Burning an Alternative Source of Energy, which is why you're seeing that up-regulation of glycolysis. Yeah, but we're also seeing a down-regulation of citrate synthase expression. Remember that one. Because, I mean, this is the real two-headed beast that HIF1 is. And we're going to see a ton more on this in the next couple of podcasts, which I am so looking forward to because they're absolutely fascinating when we start knocking out and what happens. And a lot of it's counterintuitive until you kind of go, oh, right, this is in the short term, this makes sense. In the long term, this makes sense. So the authors conclude their paper by saying that despite the workout stimulus remaining constant, their deeper analysis suggests that the blunted response was across all individuals in the study. There wasn't one person who was like, hey, I still got the same as before. It's cool. Like everybody. And this means that the inhibition of HF1's activity is active and not passive. And so we're going to investigate more of that in the next couple episodes. So yeah. So maybe I think for practical takeaways, I think we've really seen a lot on the basics of adaptation today. And this is a great example of it because I think when it comes to endurance adaptations in the muscles in order to maintain energy state, it's more obtuse until you spend a lot of time around these concepts in biochem. But I feel like this one's a lot more intuitive. You can imagine a lot of oxygen flux in the muscles. You can imagine low levels. You can easily imagine low O2 tension and making more capillaries to increase the O2 pressure around the muscle cell. Higher Detention by, you know, more red blood cells. Like, it all makes very intuitive sense, right? Yeah. I mean, it's, think, again, back to your early days of starting out cycling or running or any other kind of exercise. Like, one of the things you probably remember doing is being out of breath a lot. And during that period, bet HIF was having a great time. Yeah. Yeah, I mean, and, you know, think about the short term and the long term, too. Like, we've got a pretty stark condition, which is hypoxia. And the adaptations that deliver more oxygen, you know, we're going to also tweak parallel pathways. So we're going to increase glycolysis and glucose uptake. But something that would worsen the condition, like having more oxidative metabolism, we're going to tamp that down a little bit. Doesn't mean the pathway's not active. We clearly saw it very, very, very active. But it's going to go, hold on, buddy. Not yet. We're going to do a little bit of a trade-off. And in some of the future papers that we're going to see, we're going to see potentially more trade-offs. So hashtag foreshadowing. So when we come to training adaptations, when would we expect HAF to be the most activated? As in, when do we expect low O2 tension? So we've got times of maximal oxygen demand, very intense exercise, VO2 max stuff. But a note, a proxy for this is like elevated breathing, high heart rate, et cetera, et cetera. But these are proxies for the conditions. So just because your heart rate's elevated, just because you're breathing hard does not actually mean you are creating the stimulus. You know, it's like everybody kind of intuitively knows this. Like if you're watching a movie, like if you're watching Avengers or something like that, and you're on the edge of your seat and you're breathing hard, You're not necessarily making HAF1 active. You're excited, sure, but it doesn't necessarily meet all of the conditions. And so one of the things that probably illustrates this really well is that there seems to be a minimum duration. And this is just me interpreting the literature as far as I could see it. There seems to be a minimum duration for this, which I saw a paper on sprint interval training. I think it was in untrained people, but it might have been in trained people. I'll see if I can dig it up and put it in the show notes. But what they did was they looked at VEGF expression, so vascular endothelial growth factor expression after an endurance ride with and without 30-second sprints. And they saw no difference whatsoever. Think about it. It sounds like trained people. But 30 seconds on a long ride, insufficient for... Improving, you know, our, let's say, our example gene target of HIF. So you'd probably want to do like a full-on VO2 max workout for this kind of thing. But it's also difficult to progressively overload this pathway because the cost is fatigue for doing these kinds of efforts. And you can do a lot of them, but you're going to... Pay the cost on the other side. It's like do anything with super high intensity. It's like if you look at just general like adhesion, adhesion, strong word, adherence to workout programs for high intensity training programs, adherence over a couple of weeks, it drops for a lot of people. Some people love it and they can do it forever, sure. But most people on average, like they're not going to be able to adhere to a super intense, you know, day in, day out, super high intensity workout regime. There's also the problem of as fatigue begins to mount your ability to actually put into action the adaptations you want to make to get the benefit is going to fall off as well because at a certain point you're in too much of a deficit to benefit from much which is why you've got to balance rest and fatigue. I knew you were going to work rest into this at some point. All right. Rest of overload. Well, and here's the thing. It's like, it's hard to overload this because the intensity itself is the stimulus. You know, I mean, if you are a good responder to this stuff and individual response, as we've seen, is huge. And as we know from practical experience in coaching, the range of response is very, very, very wide. And so the intensity. is the stimulus. You cannot go more max than max with this stuff. It's like, okay, go 100%. Now go 110%. Well, if your 110% is actually your max, that's 100%, not 110. Your previous was like 90%. You can't move the needle up on the ceiling. Yeah, sometimes all out just has to be all out. But here's the other thing is that you can certainly miss different kinds of stimuli that would aid in types of racing by Over-focusing on certain types of training and ignoring other types of training. So I think from a larger picture perspective, a very experienced coach once told me very early on, it was a very good lesson to learn, was that no one type of training will benefit you the most. Basically, there's no silver bullet. And it's not endurance training, it's not threshold training, it's not VO2 max training, it's not sprint training. But if you kind of do a bit of everything in about the right proportions at the right times, you'll probably get there. I think the even bigger picture from here is actually what world tour people do in terms of altitude. Because if you really want to activate these pathways and you... You really want to improve red cell mass. You want to improve capillarization, things like that. You go to altitude and you go there for a while. And this is one way to reliably overload this thing. But as they've seen, and as I've seen in coaching, and I'm sure you have too, Rory, is that people have different responses to altitude. Some people can go to like, you know, 1,500 meters and have a good response. And some people need to go to like 2,000, 3,000 meters and have a really good response and don't until then. People go up to altitude and just have a really fucking bad time without even riding a bike. Also true. If that's you, hydrate. Hydrate more. And also, eat more carbs. Because one of the things that we're going to look at in the future episodes is we are going to look at more on the reduction of – and it's not cessation, by the way. This is a slight reduction of reliance on the aerobic pathways. and an increase in reliance on the glycolytic pathways. And so even for, if I recall correctly, even for these same relative and absolute exercise intensities, well, especially relative, when you go to altitude, you are going to see more carbohydrate usage. And also at altitude, the air is very dry. You are going to breathe out a lot of water very fast. Eat more carbs, drink more water, and you're going to have a good time at altitude. Anyway, more on that in a future episode. But yeah, I really want to take a look at on the other side of the HAF pathway and muscles because we do kind of see it in practice and we'll talk about that a little bit. Everybody, after I tell you a couple things, you are free to go. So as usual, if you would like to reach out for coaching or consultations, empiricalcycling at gmail.com if you'd like to ask questions. for our future podcast episodes and check out the weekend AMAs. 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