hello my name is James footage 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 one 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 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 you're pretty close the nominal bill moves at about 250 horsepower through 400 pounds whereas we humans have about 50 horse powers 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 P molecule being burned every time it's hydrolyzed into adp and phosphate you actually burn about a hundred and fifty 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 ten 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 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 ten to a hundred microns in diameter cardiac muscle is quite different 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 a few 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 a hundred microns long but in both cases you can see that the cardiac cell and the skeletal muscle cell has 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 we've 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 entered 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 they're 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 contract within the myofibril and the contraction I'm gonna give you the answer but then we'll go back in history and see how this was determined the 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 this 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 in 17th century when Leland hooked designed the light microscope and first observed these striated patterns but I'm gonna really start in the 19th century when in 1864 we'll have tuna in Leipzig Germany ground up muscle got out some gooey material which was the main proteinaceous component of the muscle and since muscle is Myall he named this material myosin the same year Leon Frederick at the University of liège in Belgium Belgium looked at these striations in more detail he named the dark bands a bands meaning they're anisotropic which 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 and what we're looking at here is the ai-ai-ai-aight 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 eye bands get smaller and smaller and disappear now why don't we know about this from this period this was completely forgotten through 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 angle Hart and his wife Luba mova 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 you don't need to put things even on line in in its 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 it 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 Elbert st. George II and Bruno Straub in his lab in Hungary of an activator of the true myosin because they real that this myosin that Willie tuna 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 st. George II 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 and then st. George II did a really interesting experiment he took these 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 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 turns out that st. George II remained bitterly opposed to the sliding filament theory which was proposed some years later okay then in the 90 50s 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 Schmidt at MIT and one of his colleagues Hall was getting pictures such as shown here which guess what shows you these a bands and eye bands but they seem to not pay much attention to the fact that you have these cross striations of Ani 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 gene Hanson who arrived at Smith's MIT's lab a few years later because they wanted to learn this new technique and and they were both very interested in muscle hew 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 with looking at at muscle contraction by x-ray scattering but now he wanted to learn this new technique and so he and gene Hanson went to MIT and worked in Schmidt's lab and and in their experiments they began to come up with the idea of these overlapping sets of filaments that might be sliding okay in fact Schmidt's lab really Schmidt 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 they ignoring the A&I bands all right forward to 1954 the sliding filament theory to Huxley's totally unrelated Andrew shown here with his colleague Ralph Nader gurkey we're using live muscle to look at the behavior of these Ani bands as the muscle shortens and as you can see the a band the dark band doesn't get any smaller but the eye band the light band which 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 did not change as contraction occurs but the I bands gets 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 that 1954 sliding filament theory which didn't catch on right away even though this evidence was 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 of overlapping filaments just didn't sit well in people's minds so in summary here's the 1954 sliding filament theory where the Iban 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 ban 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 a myofibrils before extraction with anything so the myofibrils have been extracted from the muscle cell but not treated with anything and you see the EA and the I bands quite nicely and now when you extract those m''e 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 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 e/m 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 your hi-c textbooks because they're so beautiful and so remarkable and he was able to show in very very thin sections that in the overlap 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 in cross-section there's this beautiful hexagonal array that you can see in such fibers and sarcomeres where in skeletal muscle of the actin filaments shown as the little bot 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 and this now takes us to about nineteen sixty-six okay and that era this particular paper that shows this image is from Gordon and af Huxley published in journal 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 has just shown here where we look at at the top is no overlap at all between the thin and thick filaments so they've taken the MA 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 through 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 H e Huxley hugh huxley even in his PhD thesis had used low angle x-ray diffraction of a 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 one zero 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 one one lattice that's going between thick and thin filaments and you know the one zero lattice is a little stronger in density than the one one lattice those give reflections in a diffraction pattern that are shown here here's the one zero reflection and here's the one one reflection and they're sort of a similar strengths okay one zero reflection as I said maybe a bit stronger but look what happens during contraction during contraction the one one reflection divided by the one zero 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 one zero one one 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 ok 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 coil coiled tail and the far end of the coil coil tail is assembling into these thick filaments but that head which is only about 15 nanometers long by his swinging cross bridge 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 510 animators no more than that because they had is only 50 nanometers long and they would then come off and somehow rikako and bind again and stroke again and that was his swinging cross bridge hypothesis okay so just to summarize this part one 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 really pervades the history of muscle and 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 cross bridge model that was proposed by H E Huxley in 1969 really became the focus of attention going forward so I very much appreciate your listening to this part one of the history of our 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 the 1969 and I hope you've enjoyed this lecture and I'm hope you'll return for part two where I continue with a brief history of muscle biology if you do I look forward to seeing you soon thank you you