James Spudich (Stanford) 1: A brief history of muscle biology 1864-1969

Hello. My name is James Spudich and I'm from Stanford University. And I'm very excited to have the opportunity to give you a series of talks on a brief history of muscle biology. And in this, Part 1, we're going to be dealing with a long period from 1864 to 1969. And it's going to be on the origins of muscle research through what is called the sliding filament theory. So, we all know that all animals move by coordinated contraction of skeletal muscle. And ATP is the energy source. This is our body's fuel, the same way gasoline is the fuel for an automobile, and in fact, as you'll see, there are a lot of connections between man-made machines and those we find in biology. What you may not know is that, in the early part of the 20th century, it was unclear what the energy source was. In fact, ATP was not discovered until 1929, and direct proof for ATP hydrolysis during contraction of muscle came only in 1962. So, here's the comparison between an automobile and human muscle. There are a lot of similarities, for example, if you just look at the power to engine weight ratio, they're pretty close. An automobile moves at about 200 to 250 horsepower per 400 pounds, whereas we humans have about 50 horsepower per 400 pounds of weight. Gasoline is the fuel for the automobile; ATP is the fuel for us. The automobile burns, if you're an average driver, about 9 pounds per day, and it's about 10% efficient. Most comes out as heat. Whereas, in the muscle, if you count an ATP molecule being burned every time it's hydrolyzed into ADP and phosphate, you actually burn about 150 pounds of ATP a day doing just your normal routines. So, almost your body weight of ATP is used. And the machine is much more efficient than that of an automobile. Let's look at a quick overview of skeletal muscle architecture. So, your muscle is attached, by way of tendon, to bone and, if you look at this part of muscle coming out, what you have is a bundle of muscle fibers. And here's one muscle bundle coming out that's surrounded by connective tissue, and that bundle is called a fascicle. And then out of that is one muscle fiber. Now, that muscle fiber is the muscle cell. So, that fiber is surrounded by a normal plasma membrane and you can see it's very, very long. So, even though it's a single cell, it can be several centimeters, or even much longer than that, in length. And it's very, very thin, 10 to 100 microns. And it's filled with cytoplasmic constituents which are called myofibrils, which we'll be looking at shortly. So, here's an image, a light microscope picture of skeletal muscle structure showing three muscle fibers. Again, each of these fibers is a single muscle cell. It's a syncytium, meaning that it arrived by fusion of many skeletal myoblasts and the syncytium, as I said, can be many, many centimeters long and about 10 to 100 microns in diameter. Cardiac muscle is quite different, or somewhat different, I should say. I mean, the heart contracts, again, because the cell contracts, the same way that the skeletal muscle works. But in the case of the heart, you don't have a syncytium of fused cells. The cardiac muscle cell is an individual cell. A lot of them have two nuclei, and many have just a single nucleus. But the cells are very firmly attached to one another. They're about 20 microns in diameter and about 100 microns long. But, in both cases, you can see that the cardiac cell and the skeletal muscle cell have striations. And both of these muscles are therefore called striated muscle. So, what are those striations all about? Well, here's looking at just one segment of the myofibril within the cell, blown up here, you can see that you have something which is called a sarcomere. And this sarcomere is a set of overlapping thin and thick filaments. So, now we're looking at something that's just a couple microns from one end of the sarcomere to the other, which is about the size of a bacterium. So, it's still not tiny. But the important thing is that you have this overlapping set of thin filaments, which are made up of a protein called actin, and thick filaments that interdigitate between the thin filaments, that are made up of a protein called myosin. Okay. And muscles contract because their cells contract, and the cells contract because their myofibrils within the cell contract, and the myofibrils contract because the myofibrils are made up of these sarcomeres that are hooked together in series throughout the myofibril. And, when the sarcomere contracts, the whole myofibril contracts. So, every sarcomere contracts within the myofibril. And the contraction, and I'm gonna give you the answer, but then we'll go back in history and see how this was determined, the answer is they contract by the relative sliding of the thin filaments past the thick filaments, to go from a relaxed state, shown above, to a contracted state, shown below. Okay. This is a cardiac cell contracting and, when I first made these diagrams a couple of years ago for a review that I wrote, I was determined to draw everything to scale. And I was very surprised to see that for cardiac muscle the contraction is really a very short, 10% shortening. We're kind of used to thinking about skeletal muscle, where the upper figure almost looks like a fully contracted sarcomere. In the skeletal muscle, the contraction is much more extensive, more like 30%. But the cardiac is really interesting because the contractions are very short, just about 10% shortening, as shown here. Okay. Now, how do we know that this is in fact the way the muscle works? And so I want to go back in history, but I want to make a really major point here and this is really a very key point of the story I want to tell you and what I want you to think about, especially those young people in the crowd who may not realize how easy it is to get caught in a trap of the dogma of the day. Okay? So here's a warning for you. And the history of muscle research is filled with examples of how the field didn't move forward because of dogma that the muscle community couldn't let go of. And so that's going to be a major part of what I'm talking about today. So, let's go back to the origins of muscle research. Well, we could start in the 17th century, when Leeuwenhoek designed the light microscope and first observed these striated patterns. But I'm gonna really start in the 19th century, when, in 1864, Wilhem Kuehne in Leipzig, Germany, ground up muscle, got out some gooey material, which was the main proteinaceous component of the muscle, and since muscle is 'myo' he named this material myosin. Okay. The same year, Leon Fredericq, at the University of Liege in Belgium, looked at these striations in more detail. He named the dark bands A bands, meaning they're anisotropic, which means they show birefringence, and the light bands he named I bands because they are isotropic and don't show birefringence. And, in an amazing experiment that even most of my muscle colleagues, I think, don't really know about or remember, is that he showed, way back in 1864, that when the muscle shortens and what we're looking at here is the A-I, I-A-I distribution of bands across, as a function of time of shortening going down here. And you can see that, as the muscle shortens, the A bands do not change in size but the I bands get smaller and smaller and disappear. Now, why don't we know about this from this period? This was completely forgotten for the first half of the 20th century, because it didn't fit the prevailing theory for muscle contraction, which was, clearly, muscle had to contract by a folding or coiling of continuous filaments of some nature. And that was the prevailing model for a long, long time. And a model that was very difficult for muscle biologists to get out of their minds. So, before 1954, contraction was believed to be due to this folding or coiling of this rod-shaped 'myosin', quote. And these were thought to be structural elements that are being acted upon by some soluble ATPase enzyme. Why soluble? Because all enzymes were known to be soluble. And so there had to be a soluble ATPase working on this structural myosin. And, somehow, that soluble ATPase induced this coiling or changing in shape, folding, that gave you contraction. That was the dogma. There was a major breakthrough, but not until 1939, when Englehardt and his wife Liubimova showed, in a one-page Nature paper, so, you know, all of the best papers out there are really short and you don't even need to put things even online in supplementary materials if you really do the right experiment. And the right experiment, here, was the definitive proof, against all odds, that in fact this structural myosin was the ATPase, itself. Okay? A structural protein? An ATPase? Who would have thought? Okay, so this was a major breakthrough that changed the way people thought about this. And then, just a few years later, in 1942, another major breakthrough. And that was the discovery, by Albert Szent-Gyorgyi and Bruno Straub, in his lab in Hungary, of an activator of the true myosin, because they realized that this myosin that Wilhem Kuehne purified in 1864 was actually a heterogeneous mixture of two major proteins, one of which they continued to call myosin and, for a long time, the first myosin was called myosin A and the second one myosin B, but anyway, everyone now refers to the real myosin that Szent-Gyorgyi described as myosin. And the other component that was in the original myosin prep was an activator. And, since it was activating this myosin, they named it actin. Okay? And then Szent-Gyorgyi did a really interesting experiment. He took this actin-myosin mixture and put it in high salt, in which case it was clear that it was very soluble. But if you squirted it out of a syringe into a low salt buffer it became insoluble and turned into a thread, such as shown here. And then, when he added ATP to this, in fact it shortened, as shown just below. And this was amazing because it was showing with two purified proteins that you could reconstitute muscle contraction in what was clearly a continuous mixture of actin and myosin. No bands, no I bands, no A bands. And yet you can get shortening. And so, the idea was, well, whatever kind of folding is going on, that it's being reproduced here. And, in fact, it turns out that Szent-Gyorgyi remained bitterly opposed to the sliding filament theory, which was proposed some years later. Okay. Then, in the 1950s, many of you know that electron microscopy became a new tool. And, of course, one of the first things that people wanted to look at was, what did muscle really look like if you looked at it at higher resolution? And this was going on in the laboratory of Francis Schmitt at MIT, and one of his colleagues, Hall, was getting pictures such as shown here, which, guess what, shows you these A bands and I bands. But they seem to not pay much attention to the fact that you have these cross striations of A and I bands. They still believed that this somehow was a continuous network of actin and myosin that was coiling or folding. And it was Hugh Huxley and Jean Hanson who arrived at Schmitt's lab a few years later, because they wanted to learn this new technique, and they were both very interested in muscle. Hugh was actually doing low angle X-ray scattering, and I'll get to that in a moment or in a later lecture, having to do with looking at muscle contraction by X-ray scattering. But now he wanted to learn this new technique, and so he and Jean Hanson went to MIT and worked in Schmitt's lab and in their experiments, they began to come up with the idea of these overlapping sets of filaments that might be sliding. Okay? And, in fact, Schmitt's lab really, Schmitt himself didn't believe this, because the idea of folding and/or coiling of filaments was so attractive and so obvious to be couldn't possibly be wrong. Okay? Ignoring the A and I bands. Alright. Forward to 1954, the sliding filament theory. Two Huxleys, totally unrelated, Andrew, shown here, with his colleague, Ralph Niedergerke, were using live muscle to look at the behavior of these A and I bands as the muscle shortens. And, as you can see, the A band, the dark band, doesn't get any smaller, but the I band, the light band, which it turns out is where the actin is, gets smaller and smaller. Guess what? We knew this from a paper in 1864 that everybody has forgotten. Meanwhile, Hugh Huxley, totally unrelated to Andrew, and his colleague, Jean Hanson, were doing the same kind of experiment but in isolated myofibrils, taking the myofibrils out of the muscle, and they saw the same thing. So, A bands do not change as contraction occurs, but the I bands get shorter and shorter and shorter. Okay? These two papers are very famous in muscle biology. They were back to back in Nature, and they were the papers that caused both of these workers to propose the 1954 sliding filament theory, which didn't catch on right away, even though this evidence was there. In fact, as I said, there was already evidence for something like this way back in 1864. But it didn't catch on, because the dogma was these muscle fibers had to be contracting by some folding. And the idea of a ratcheting of overlapping filaments just didn't sit well in peoples' minds. So, in summary, here's the 1954 sliding filament theory where the I band, made up of actin, slides past these myosin thick filaments, that make up the A band, to give you a shorter sarcomere, and therefore a shorter myofibril, and therefore a shorter muscle. So, further experiments, myosin is in the A band and actin is in the I band. I've already been telling you that, but how do we know that? Well, we know that because Hanson and Huxley, in 1956, in this paper, I think it's 1955, actually, in this paper, showed myofibrils before extraction with anything, so the myofibrils have been extracted from the muscle cell but not treated with anything, and you see the A and the I bands quite nicely. And now, when you extract those isolated myofibrils with pyrophosphate, the A band disappears. And when they look in solution, they find that the myosin has been solubilized. So, the A band is myosin, but you can still see the gray background of the I bands. And if you treat, now, what's remaining with potassium iodide, you extract what the I band is made up of, and you look in solution and in fact you find the actin. So, it was clear where which protein was and, with all of this, the sliding filament theory was still not immediately embraced, mainly because it flew in the face of this dogma. But also because the EM data to date were not fully convincing that the filaments were not contiguous or continuous. So, that required a lot finer resolution electron microscopy, which Hugh Huxley decided to pursue. And he obtained very thin sections of well-preserved muscle with spectacular electron microscopy results, just another indication that technology development is always essential to make the next step in understanding some biological problem. And, of course, most of you have seen such images, even in your high school textbooks, because they're so beautiful and so remarkable. And he was able to show, in very, very thin sections that in the overlapped region between the thick and the thin filaments there are little cross bridges, which turn out to be the heads of the myosin molecule that we're going to talk about later. And those myosin molecule heads are seen, here, reaching across from the myosin-containing thick filament, binding to actin in the thin filament. Okay? In cross-section, there's this beautiful hexagonal array that you can see in such fibers and sarcomeres, where, in skeletal muscle, the actin filament, shown as the little dot as you're looking at the filament on-end in the cross-section, is in a trigonal position between three different thick filaments. And so the muscle has this wonderful, beautiful, almost crystalline organization set up to maximally utilize these protein components and ATP to give you a very efficient muscle contraction. Then, another kind of experiment was done. And this, now, takes us to about 1966, okay, and that era. This particular paper that shows this image is from Gordon and AF Huxley, published in The Journal of Physiology. And what they did was they studied the effect of the overlap of the thin and the thick filament in terms of how sarcomere tension or force production changes with overlap. Okay? And the important part of this is just shown, here, where we look at the top is no overlap at all between the thin and thick filaments, so they've taken the muscle and they've stretched it such that they removed any overlap that was there. So, the muscle doesn't usually start in that position. And then they allowed it to shorten and shorten and, at each point between this top figure and this second, bottom figure that shows complete overlap, they asked, how does the tension change? And the answer is shown by this red, dotted line, which starts at no overlap, down here, where you have no tension, and there's no myosin heads interacting with actin, and then, as you go to complete overlap, you have a linear progression of tension development. So, this suggested that, in fact, these heads that are projecting out and reaching across to the actin filaments, may be acting as independent force generators and, depending on how many heads were interacting with the actin filaments, you had a linear relationship between the force and those number of heads. So, a really elegant experiment from AF Huxley's lab. Meanwhile, as I already mentioned, HE Huxley, Hugh Huxley, even in his PhD thesis, had used low angle X-ray diffraction of live muscle to complement the work that he was doing with electron microscopy. And I don't have a lot of time here to go into exactly the theory of this, but you may remember from high school physics that when you have planes of density, or, in high school, you've studied the effect of shining light through slits that were spaced by certain distances, and you got a diffraction pattern behind it. Well, in this case, you can see that you have two different lattice planes. One's called the (1,0) lattice and that's the thicker black lines that are going through the thick filaments in cross-section, here shown in cross-section. And then you have these thinner lines that are going, called the (1,1) lattice, that's going between thick and thin filaments. And, you know, the (1,0) lattice is a little stronger in density than the (1,1) lattice. Those give reflections in a diffraction pattern that are shown here. Here's the (1,0) reflection and here's the (1,1) reflection and they're sort of a similar strength, okay? (1,0) reflection, as I said, may be a bit stronger. But look what happens during contraction. During contraction, the (1,1) reflection divided by the (1,0) reflection, that ratio, increases about fivefold. And what this means is mass is moving from the thick filament toward the thin filament, which gives rise, then, to this change. And we're going to come back to this (1,0) / (1,1) reflection in a later lecture. But this is just to give you a little introduction to this and I'd encourage you to go and read about diffraction theory a little bit to understand this. Okay. So, given all of this, Hugh Huxley, in 1969, wrote a famous Science paper which summarized his views on how the muscle really worked. And his view was that the head of the myosin molecule, and we're going to look at myosin in more detail in a subsequent lecture, but the myosin has a head and a long coiled-coil tail, and the far end of the coiled-coil tail is assembling into these thick filaments, but that head, which is only about 15 nanometers long, by his swinging crossbridge hypothesis, binds to an actin filament and then undergoes a rotation. And that rotation would move the actin filament relative to the thick filament, about 5 to 10 nanometers, no more than that, because the head is only 15 nanometers long. And it would then come off and somehow recock and bind again, and stroke again. And that was his swinging crossbridge hypothesis. Okay. So, just to summarize this Part 1, muscle biology has its origins prior to the 19th century. A really important point that I want you to get out of this lecture is that constant new technology advances are really necessary to elucidate the mechanism of whatever biology you're trying to study. The dogma here was so difficult to let go of, it really pervades the history of muscle and it's something you should think about and avoid in your own work. Sliding filament theory was finally accepted by the 1960s, but it took a long time. And then the swinging crossbridge model that was proposed by HE Huxley in 1969 really became the focus of attention going forward. So, I very much appreciate your listening to this Part 1 of the brief history of muscle, that's covered a lot of time and of course not nearly a significant part of a tremendous amount of research by many investigators has been left out, but I've tried to hit high points to show you how we got from 1864 to 1969. And I hope you've enjoyed this lecture and I hope you'll return for Part 2, where I continue with a brief history of muscle biology. If you do, I look forward to seeing you soon. Thank you.