Muscular Contraction: Progress and Uncertainties
Huxley, Andrew
Research on muscular contraction in this century has exemplified Kuhn's proposition that progress in science is of two kinds: occasional "revolutions" when a current set of ideas is overthrown, and intervening periods when new discoveries fit into the current scheme. The first use of the word "revolution" in connection with muscle was in the title of A.V. Hills review of 1932. The "revolution" in question was the demise of the lactic acid theory: for thirty years it had been firmly believed by all influential muscle people that producton of lactic acid brought about shortening of long protein chains by neutralising negative charges whose repulsion was keeping the chains extended. As regards the involvement of lactic acid, it was indeed a revolution: lactic acid production was relegated to the role of a back-up process. On the other hand, the idea of long protein chains that folded or coiled as a result of chemical events survived for more than twenty years. A substitute for lactic acid as the chemical involved was already at hand: the liberation of inorganic phosphate from phosphocreatine during contraction had been discovered three years earlier. The rise of classical biochemistry at the turn of the century led to recognition that "contraction is a molecular event". This was a revolution in the sense that attention ceased to be paid to structural changes that had been well established with the light microscope in the 19th century. This change of attitude held up progress on the structural aspects of the contraction mechanism for half a century. There were many important discoveries during the 1930's and 1940's: displacement of the dephosphorylation of phosphocreatine by that of ATP as the primary chemical event; recognition that "myosin" is an ATPase; and the separation of what had been called "myosin" into two components, actin and what is now called "myosin". I do not regard these as "revolutions" because they fitted into the prevailing idea that shortening occurs by folding of long protein chains under the influence of a chemical reaction. The sliding filament theory of 1953-4 was genuinely a revolution, however, because it made this idea obsolete. As regards the question how a relative force between the two sets of filaments is generated, two aspects now agreed were suggested by observations made more than ten years earlier, namely the decrease of active tension when the length of a muscle fibre is increased beyond an optimum, and the relation between rate of energy liberation (heat plus work) and speed of shortening. Neither of these could be accommodated in the idea of contractile elements changing progressively from a long to a short state. They led respectively to the ideas (1) that contributions to force are generated by "active sites" uniformly distributed along each zone of overlap between thick and thin filaments and (2) that each of these acts cyclically. Observations with the electron microscope showed that these active sites were "cross-bridges" composed of the heads of myosin molecules extending from a thick filament and attached to the thin filament; further, the orientation of these cross-bridges changed when force was generated, displacing the filaments relative to one another. Several questions remained, none of which has yet been completely solved: 1.Does the whole cross-bridge tilt or is the attachment to the thin filament rigid and a "conformational change" happens within the myosin head? Very recent evidence suggests that both may occur. 2.Is the change of attitude a single event or does it consist of two or more steps? 3.How is the hydrolysis of ATP coupled to these step(s)? It has long been clear that one action of ATP is to bind to the myosin head, leading to dissociation of the myosin from actin; are steps in the hydrolysis of ATP (e.g. release of inorganic phosphate and of ADP) linked to steps in the shape change of the cross-bridge? 4.Despite many impressive experiments measuring the effects of single myosin-actin interactions, these are still wide disagreements about (1) the numer of myosin heads that are attached at any moment during contraction, (2) the range of movement associated with the utilisation of one molecule of ATP, and (3) the amount of force generated by a single interaction between a myosin head and an actin filament.
 Thank you very much, Professor Westling, for your kind introduction. And I would like also to express the thanks of all of my wife and myself to the Coratorium and to Countess Sonia for inviting us once again to this extremely interesting and enjoyable event. Well, as Professor Westling mentioned, it was in 1952 that Alan Hodgkin, who sadly died last December, and I completed the work for which we shared a Nobel Prize with the late Sir John Eccles. And that was about the ionic mechanism of conduction in nerve. And at that time, when we'd finished that work, we could not see what to do next to elucidate the mechanism of nerve conduction and excitation. We realized that channels must exist for the ions, but the enormous advances that have taken place since were quite unforeseeable at that time. We just could not imagine recording the currents through single channels, as of course was done by a Winn-Nair and that Satman, when was it, yes, 30 years later. So we moved into other fields. For a time, Hodgkin worked on active transport of ions, and then he moved to the electrical responses of rods and cones in the retina. And I moved on to the study of muscle contraction. Well, I'm long retired. I gave up active laboratory work a year or two ago, so I have no new experiments to report to you. But with the approach of the end of this century, it seemed appropriate to review the changes in what people have thought about muscle contraction since 1900. And to a considerable extent, these changes have followed the, well, the path that was suggested by the philosopher of science, Kuhn, in his book, The Nature of Scientific Revolutions. He proposed that scientific advance in any field takes place partly through occasional revolutions when a new observation or discovery makes previous ideas obsolete and one has to change one's, all one's thinking. And in between these revolutions, there's, well, normal science in which much progress is made but without a change of the framework of ideas within which one works. Well, starting at 1900, that was just about the time when classical biochemistry really started. And this stimulated an interest in the chemical changes in muscle. And at that time, the only chemical change known during contraction was the production of lactic acid, which had been observed, an extraordinary achievement by the Swedish chemist Batcellius in 1807. So that was the only thing known in 1900. First slide, please. And all the, everybody believed that this production of lactic acid was directly the cause of contraction. It was supposed that there were long chains of protein molecules which were kept extended by the repulsion of their negative charges. These charges were neutralized by the hydrogen ions from lactic acid that allowed them to fold up. And this theory met difficulties in the 1920s, but the biochemists stood firm on the theory, notably Meyerhoff, until 1930 when the Danish biochemist Lundsgaard experimented with muscle poisoned with iodoresetic acid and found that the muscle could give many normal contractions without producing any lactic acid. Now that was the occasion for the first use of the word revolution in connection with muscle. Next slide, please. That was by A.V. Hill, famous particularly for his work on the heat production of muscle. He shared a Nobel Prize with Meyerhoff in 1923. And yes, he described this demise of the lactic acid theory as a revolution. Next slide, please. And here is an excerpt from the review which he entitled, entitled, The Revolution in Muscle Physiology. Well, a substitute for the production of lactic acid was already in the literature. Two groups, Fisk and Soborow in the United States and the two Egltons, Philip and Grace, in Scotland. One of the many husband and wife teams in muscle physiology had discovered that phosphate is released during contraction and this came from a substance known by the general name Phosphogen and Fisk and Soborow identified this substance as phosphorylcreatin. So textbooks came to say, well, this sort of statement in which hydrolysis of phosphorylcreatin or phosphogen took the place of lactic acid production. Well, although this was the first use of the word revolution connected with muscle, one could say that the switch of interest to chemical events in 1900 was a revolution. People realized and the statement is made in influential papers that contraction is a molecular process. And of course, you can't see molecules with a light microscope and it was concluded that you won't learn anything from what you can see with the light microscope. And the result was that interest switched away from what you can see with the microscope. The striations of muscle had been much studied in the 19th century. Next slide, please. Now this is from one of the early electron micrographs of muscle published in 1949 in Australia by Draper and Hodge just to illustrate the nomenclature of the parts of the pattern. This is a shadowed picture, you can see a shadow here indicating that there's more material here in the band which is called A standing for anisotropic because as well as having much material and therefore a high refractive index in the intact muscle, it is also optically anisotropic. It is birefringent or doubly refracting and shows up in the polarizing microscope. And then there's a less dense band, isotropic because it is not birefringent and that's intersected by the z-line, the Tsvyschensheibe, which again has much material. But that's just to illustrate the nomenclature. This structure is a myofibril about one micrometer across and a single muscle fiber consists of many thousands of these which come apart when the muscle is fragmented. Well as I said much was learned about this astriation pattern in the 19th century but as a result of the switch of interest to chemical events, this knowledge gained between 1850 and 1900 was almost completely lost. So it was a revolution and the previous ideas were overthrown but like many political revolutions its results were really a disaster for muscle. And the loss of interest in this striations was reinforced by for example the fact that our involuntary muscles which don't have striations, not clear visible ones, they're able to contract and it was argued therefore the striations are not of any fundamental significance. And at about the same time confidence in microscopy was shaken by several papers, one notable one in 1899 by W. B. Hardy, one of my heroes for his work, pioneer work on polymer chemistry and colloids but he showed that many of the sort of appearances that you see with the light microscope in fixed preparations could be produced in simple protein like egg albumin by treating with chemical fixatives. And as a result of people no longer using the microscope as the principle tool, the skills required in studying unstained preparations, living muscle with the microscope were lost. And there was some bad microscopy in the first half of this century and some of what had been known in the 19th century was back to front in the textbooks of 1950. Well there was much progress in the 1930s and 1940s, next slide please. The splitting of phospho-creatin was replaced by this hydrolysis of adenosine triphosphate producing ADP and the role of phosphogen or just to say phosphoryl-creatin was relegated to being a backup process. Then it was found by Engelhardt and Glebimova in Moscow, yet another husband and wife team in 1939. They showed that the contractile protein myosin is in fact an ATPase, that's to say it is an enzyme capable of bringing about this hydrolysis of ATP. And then it was found in the laboratory of Alberts and Gerger that what had been called myosin originally described by Cuner in 1864 is a complex of the protein that we now call myosin and another protein to which they gave the name Actin and that was the work of F.B. Straub who later for a time was president of Hungary. But these developments all took place with no change in the general framework of ideas. Everybody still thought that the contractile elements were long chains of protein that were caused to fold up in some way and of course the protein became Acto myosin but it was still thought of as long filaments. And indeed the existence of continuous filaments in muscle was claimed by the authors. Now this is from the first useful published electron micrographs of muscle by Hall, Jacobs and Francis Schmidt published 1946, they worked at MIT in Boston. And here again are myofibrils, this time stained with a heavy metal. Again you see the familiar pattern of A bands but the authors claimed that they could see continuous filaments running through both bands supporting the current idea. Still with hindsight I think one has to say there was an element of wishful thinking in seeing what they thought must be there. But on the good side of this work it convinced people that the details of the striations which had been described in the 19th century not merely the A and I bands and the Z line which are conspicuous but further details a little middle-shiber, the M line in the middle of the A band, a less dense zone in the middle of A called H after Henson and even some dark things, a flanking Z line, Neybenscheiben or N bands. And these were all things that had been described in the second half of the 19th century and had been dismissed as, well, figments of the microscopist's imaginations. So this restored some confidence in structure and microscopy. Well I became interested in muscle through being asked to give lectures on it to the final year students at Cambridge and I was given the lecture notes of my predecessor and from then I learned of something that I had not learned as a student. Next slide please. Now this is from a paper of Engelmann in, well he worked in Utrecht but originally and finally in Germany. A schematic picture of a muscle showing this striation pattern going from rest up here to a shortened state. This is a fiber from probably the leg of an insect and these fibers when teased out undergo spontaneous contraction waves. And well dark in this image corresponds to high refractive index, simply high concentration of protein. Here's the appearance in polarized light, the A bands being birefringent show up bright against the dark background. But you see that when the muscle is heavily shortened the most dense region here is at the position that corresponds to the middle of the eye band, the low refractive index band. So this phenomenon was called the reversal of striations. The part that had been the least dense, the eye band, became the most dense. And no one had paid attention to this phenomenon for some 50 years. I'm speaking of about 1950. So I thought it was an interesting thing to reinvestigate. And to study it in intact fibers from a vertebrate, a frog notably, which are these are rather thick fibers and with narrow striations, very difficult object for the ordinary microscope. But it needed an interference microscope that would show up phase differences due to differences of refractive index, while the ordinary microscope is designed primarily to show up differences of absorption of light. And muscle doesn't absorb light appreciably. Things appear dark or light according to refractive index. But the appearance that you get depends on how you focus the microscope, how thick the specimen is. Of course, phase contrast, invented by Zernicker, does show up phase differences due to refractive index differences, but it does not work satisfactorily on a thick specimen. And I had a boyhood interest in microscopes. I had an idea of how to build an interference microscope. So here was an opportunity to exercise my own hobby at the same time as investigating what might be an interesting phenomenon. So we built this microscope, got the optical parts made by the firm of Beck in London, and looked at isolated living fibers from frog muscle, jointly with a German post-doctoral colleague, Grolf Niedergärke. And the first thing we saw, next slide please, these are images of a frog muscle fiber with the instrument adjusted so that high refractive index regions appear dark. So these dark bands are the A bands. And we're here, we're simply stretching a fiber, not stimulated, and you see that as we, this is the repeat distance of the striation pattern, and as we stretch it from rest a little over two micrometers up to more than double, the width of the A bands hardly changes. Well now that was the reverse of what was in all the textbooks at that time. Well, as I said, we were wanting to investigate the reversal of striations, so we stimulated one of these fibers under the microscope. And next slide please. I hope you can see here, well here it is at rest, again A bands dark, and I hope you can see here that there are some very narrow dense lines in this region that is contracted actively near the edge of the fiber. But these dense lines were not where we expected to find them, so watching the fiber under the microscope or watching the film from which these still photographs are taken, you can see that these narrow dense lines are at the middle of the A band. And it was only on further shortening that we did see a further lot of dense lines at the middle of where the A bands had been. Well seeing the constant width of the A bands, of course immediately suggested that the high refractive index of the A bands was due to material in little rodlets that went from one side of the A band to the other and were not individually stretched when the fiber was stretched. And then the contraction bands at the middle of the A band would be due to these rods colliding and either folding up or overlapping. So these little bands at the middle of the A band might be due to a second lot of filaments that slid in and then when they overlapped or folded up where they collided would cause these bands. So that was how we got onto sliding filaments. And at exactly the same time, this is early 1953, Hugh Huxley, together with Gene Hansen who sadly died quite young afterwards, well they approached not through living muscle but through electron micrographs, transfer sections of muscle and also phase microscopy of separated myofibrils and they reached, well we both reached just the same conclusions. And next slide please, you're probably all familiar with diagrams like this based on Hugh Huxley's work. It's pure coincidence that we have the same surname. We haven't yet been able to trace any family connection. So there were these thick filaments and now they also and Hasselbach in the laboratory of HH Weber showed that the protein myosin was in the A bands and when it was dissolved away the remaining material was distributed like the thin filaments and that was actin. Well my colleague, Rolf Niedergärke had been at Göttingen and when he was there he had been at a seminar where the old work on the striations had been discussed and he had a memory that Krause, one of the 19th century microscopists had done something interesting. And I followed this up in the library of our department and found many very interesting and surprising things. First a paper by Ernst von Brücke in Austria, a beautiful paper of 1858 in which he measured the strength of birefringence of muscle fibers and found that when he stretched one of these fibers the strength of birefringence did not increase. If you take most biological fibrous materials and stretch them they become much more strongly birefringent. And von Brücke concluded, next slide, that the birefringence was due to little rodlets distributed well through the A-band and that when the muscle was stretched or shortened these rodlets changed their positions but were not individually stretched. And that was a perfectly valid conclusion and it's still valid now. And he gave the name distioclasts which if you translate from the, by parts from Greek into Latin it comes out as English in English as birefringent elements. And then a decade later Krause finding that the A-bands stayed at constant width on stretch concluded that these distioclasts were rodlets going from one side of the A-band to the other and he treated muscle with solutions known to dissolve myosin. The A-bands disappeared so he concluded that these distioclasts were rodlets of myosin. So the thick filaments made of myosin were there in place by 1869. And then next slide a little later I came across a paper by Leon Friedrich in Belgium. This is a composite diagram showing the appearance of the striation pattern in ordinary light. Dark is higher refractive index. Here at rest, here moderate shortening, here extreme shortening. And at extreme shortening the dense lines are at the position of the middle of the I-band. So there the contraction bands that Engelmann had seen in the 1870s and 80s. But at a degree of shortening before those bands appear you can see a dense line in the middle of the A-band appearing. And that was what put Rolf Niedergerker and myself on to thinking of a second lot of filaments. So all the evidence necessary for thinking of sliding filaments was there by 1876. But nobody thought of it, these observations did not get built into any theory and largely as a result of that they were forgotten. Well, the theory of sliding filaments and its acceptance fairly rapid was indeed a revolution. All previous ideas about shortening due to long filaments folding up were obsolete. And well you might regard it also as a counter revolution reversing that of 1900. It emphasized the importance of structure in muscle and it opened up new ways of thinking and of interpreting even existing observations. And there were two very significant observations already in the literature. One, this is another husband and wife team in the United States, Robert Ramsey and Sybil Street measuring the rise of tension when an isolated fraud muscle fiber is stimulated starting at different lengths. Maximum tension at 100%, that's the slack length about two micrometers per repeat of the pattern. And this linear decline coming down to zero, no tension produced on stimulation at about double the slack length. Well that suggested that perhaps tension is proportional to the amount of overlap between the two lots of filaments, full overlap up here and this might be coming down to zero. Well qualitatively that was okay but this length is actually more than the sum of the lengths of the thick and thin filaments so it wasn't quite right. But we found that that was due to a simple artifact. When muscle fiber is stretched the end parts don't stretch as much as the middle so there was still overlap at the ends of the fiber even though in most of the length of the fiber was no overlap. So we had to use a trick to keep constant the length of a middle segment of the fiber where the striation spacing was nice and uniform and when we did that, next slide, yes we found the relationship between tension produced and length of the fiber in here expressed in micrometers of the striation repeat. It followed very closely a straight line over this region coming down to a length just about equal to the sum of the lengths of the thick and thin filaments reaching a maximum here where all these little cross bridges on the thick filaments are overlapped by the thin filament and then a plateau and then a drop when collision has begun. So this implied that the tension was produced by contributions from a number of sites uniformly distributed within each overlap zone. And then another observation already in the literature, this is from the work of A.V. Hill again whose photograph I showed, paper of 1938. This is showing the increase of total rate of energy liberation which is partly coming out as work and then an additional amount of heat. Well this would represent the increase of total rate of chemical change and Dorothy Needham, wife of yet another husband wife team in Mussel who is the wife of Joseph Needham whose name you may know as a historian of the science in China. And she suggested that it was pointed to the analogy between this hyperbolic relation and the Michaelis-Minton relationship between the rate of an enzymic reaction and the concentration of the substrate molecule. And this of course enzymes work in a cyclical manner repeatedly, well hydrolyzing or whatever it is, the substrate. So she suggested this implied that the contractile elements in Mussel operated in a cyclic manner during a contraction, each one going through many cycles. Well both of these features, force proportional to overlap and cyclic operation were really inconsistent with the idea of continuous filaments which required a progressive change from a long state to a short state. And I think all current theories do incorporate these two features, though many theories were put forward after sliding filaments were well accepted that did not incorporate these features but were progressive as opposed to cyclic theories and requiring a progressive change from an elongated state to a shortened state. So in 1954 I worked out a very speculative theory incorporating these features and next slide I won't spend time on this, assuming that a little cross piece on the thick filament had properties, it was elastic, represented by drawing a spring and it could attach to sites on the actin filament and if it was in this sort of position, if it was to this side it would produce a contribution to positive tension and if some sliding takes place pulling this to the left, it's compressing this spring, it will produce a negative contribution to tension. And by assuming that the rate constant for making an attachment between this Myosin site and an actin site had positive values over a certain range on the positive side where the force would be generated, the rate constant for detachment was very low until sliding had brought the attachment to a position where it was going to produce negative force and then there's a very high rate of detachment and that gave a sufficient fit to the steady state relations found by AV Hill. So those features I think need to be incorporated in any current theory but they're very far from complete, particularly they don't begin to explain the transient responses when the length of a muscle is suddenly changed, if we shorten it by over half a percent of its length, here we've stimulated, tension rises, this is 100 milliseconds, a slow record, tension drops simultaneously with the shortening, recovers part of the way very fast and then the rest of the way comparatively slowly. And here on a 100 times faster time scale, here's the initial drop of tension and this quick recovery in one or two milliseconds. Still this is clearly in some sense the working stroke of the muscle fiber. And in 1957 Hugh Huxley published some of the most beautiful electron microscopes yet seen in which in the same section you see a thick filament and then there are two thin filaments connected to the z-line and there are little bridges going between them and the little bridges were clearly projections from the thick filaments, the mass in filaments. So it was natural to identify these cross bridges with the sites at which contributions to force are produced. Well, that leaves open the question how they produce force and a paper a few years later by Reedy Holmes and Triguer showed that a change of their angle took place so that it was like a lever tilting about the attachment to the thin filament that pulled the thick filament along. And then in the early 90s the atomic structures of actin and of the head part of myosin, the part that makes these cross bridges were solved by X-ray crystallography and these structures suggested that it wasn't the whole myosin head that tilted but that there was a hinge in the middle of the myosin head where the angle would change and part of the myosin head would act as a lever. And this is a diagram now familiar, the cycle goes round clockwise and the working stroke is between from here to here. This part of the myosin head is assumed to be rigidly attached to the actin filament and the working stroke is that this part of the myosin head changes its angle pulling on the thick filament and moving it along. And then, well, rest of the cycle I think we haven't got time to look into. And this is the generally accepted idea at the present time. But there are still many uncertainties, great disagreements about how many of the myosin heads are attached at any one time. It's known that the actin filament is a helical structure and you would expect that when myosins attach to this they would follow the helical pattern and the features of the x-ray diffraction pattern coming from this helix would be strengthened. But only very slight strengthening was found until, well, in the last year or two, two Russians, Bershitsky and Saturyan, made rapid rise of temperature in active muscle, tension rises, or up to perhaps double what it had been at the low temperature, not very fast, not as fast as the tension recovery after a little release. And they found also that during this tension rise after a temperature jump the strength of these reflections from the actin in the x-ray pattern became stronger. And this immediately suggested that there are two mechanisms at work. The one what was generally believed, this represents the little lever which tilts by a conformational change at the attachment here. And that would be responsible for the rapid recovery of tension after a short release. But the response to temperature rise might be the whole head tilting because in this state it follows the actin helix, I haven't drawn this as helical but it is, but in this state one would have to assume that, well, it's just a two-point attachment so it's free to rotate about the long axis of the filament so it wouldn't really follow even if the attachments were at points following the helix, the mass of the myosin would not follow the helix. So this is a suggestion that perhaps movement is generated at two places, one this conformational change making the lever or move, the other the whole head tilting. And suggestions of this have been seen in electron micrographs but this is still very speculative. Well, in the last ten years or so there have been astonishing developments in another direction that's recording the tension produced by single myosin molecules. Next slide please. High-tech method, an actin filament is held between two beads each of which is held in a light trap, a strongly focused beam of laser light and then you can move these beads by displacing this focus. And then there's myosin on another bead here, you bring the filament down onto it, there may be a connection between actin and myosin and when there is the beads are displaced and that's shown by these upward jerks. Here the ATP concentration, the more rapidly they detach again, here with a very low ATP you get these quite long lasting displacements and yes here there are displacements of a few nanometers. Still and then even more surprising just published a couple of months ago from the University of York using the same technique but with a myosin not from a muscle but it's a much slower muscle from brush border, here are these displacements due to single interactions between the myosin molecule and you see that in each case it goes in two steps, the second arrow shows the second displacement. Well now whether that happens also with muscle myosin we don't know, you don't see a second step but it's still possible that it's so rapid if it happens within one millisecond you wouldn't see it with the time resolution that's now available. So that's yet another uncertainty. Well we may be undergoing yet another revolution in which interest concentrates on these single molecule events and other high-tech methods, so not a revolution like the end of the lactic acid story or the sliding filaments, these ideas are still within the general framework of cross bridges forming and pulling the filaments along but it may be like the revolution of 1900 in switching interest away from what had been a valuable source of knowledge and a recent review article in Nature is based entirely on well results of this kind disregarding much that's known from whole muscle work and misinterpreting other observations and in a couple of recent meetings about muscle there's been a similar emphasis on this kind of work and almost nothing said about what you can learn from living muscle. So I have a nasty suspicion that perhaps history is repeating itself after 100 years and that much valuable knowledge is being lost through loss of interest. May seem inconceivable to you that this could happen now but it's no more inconceivable than the loss of the 19th century microscopy would have seemed in 1900 and there are many pressures now that make it more likely the bulk of literature being published increasing specialization, computer databases don't go back far enough and it's reinforced by the same story being told repeatedly at symposia. And well there was early this century I mentioned there was bad microscopy that got some of these things back to front it would have been criticized by people like Engelmann and Kölnick and to stay alive as long as I can and remind people of things that may be in danger of being lost. Well thank you for listening. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you.
 Thank you very much, Professor Westling, for your kind introduction.  And I would like also to express the thanks of all of my wife and myself to the Coratorium  and to Countess Sonia for inviting us once again to this extremely interesting and enjoyable  event.  Well, as Professor Westling mentioned, it was in 1952 that Alan Hodgkin, who sadly  died last December, and I completed the work for which we shared a Nobel Prize with the  late Sir John Eccles.  And that was about the ionic mechanism of conduction in nerve.  And at that time, when we'd finished that work, we could not see what to do next to  elucidate the mechanism of nerve conduction and excitation.  We realized that channels must exist for the ions, but the enormous advances that have  taken place since were quite unforeseeable at that time.  We just could not imagine recording the currents through single channels, as of course was  done by a Winn-Nair and that Satman, when was it, yes, 30 years later.  So we moved into other fields.  For a time, Hodgkin worked on active transport of ions, and then he moved to the electrical  responses of rods and cones in the retina.  And I moved on to the study of muscle contraction.  Well, I'm long retired.  I gave up active laboratory work a year or two ago, so I have no new experiments to report  to you.  But with the approach of the end of this century, it seemed appropriate to review the changes  in what people have thought about muscle contraction since 1900.  And to a considerable extent, these changes have followed the, well, the path that was  suggested by the philosopher of science, Kuhn, in his book, The Nature of Scientific Revolutions.  He proposed that scientific advance in any field takes place partly through occasional  revolutions when a new observation or discovery makes previous ideas obsolete and one has  to change one's, all one's thinking.  And in between these revolutions, there's, well, normal science in which much progress  is made but without a change of the framework of ideas within which one works.  Well, starting at 1900, that was just about the time when classical biochemistry really  started.  And this stimulated an interest in the chemical changes in muscle.  And at that time, the only chemical change known during contraction was the production  of lactic acid, which had been observed, an extraordinary achievement by the Swedish chemist  Batcellius in 1807.  So that was the only thing known in 1900.  First slide, please.  And all the, everybody believed that this production of lactic acid was directly the  cause of contraction.  It was supposed that there were long chains of protein molecules which were kept extended  by the repulsion of their negative charges.  These charges were neutralized by the hydrogen ions from lactic acid that allowed them to  fold up.  And this theory met difficulties in the 1920s, but the biochemists stood firm on the theory,  notably Meyerhoff, until 1930 when the Danish biochemist Lundsgaard experimented with muscle  poisoned with iodoresetic acid and found that the muscle could give many normal contractions  without producing any lactic acid.  Now that was the occasion for the first use of the word revolution in connection with  muscle.  Next slide, please.  That was by A.V.  Hill, famous particularly for his work on the heat production of muscle.  He shared a Nobel Prize with Meyerhoff in 1923.  And yes, he described this demise of the lactic acid theory as a revolution.  Next slide, please.  And here is an excerpt from the review which he entitled, entitled, The Revolution in Muscle  Physiology.  Well, a substitute for the production of lactic acid was already in the literature.  Two groups, Fisk and Soborow in the United States and the two Egltons, Philip and Grace,  in Scotland.  One of the many husband and wife teams in muscle physiology had discovered that phosphate  is released during contraction and this came from a substance known by the general name  Phosphogen and Fisk and Soborow identified this substance as phosphorylcreatin.  So textbooks came to say, well, this sort of statement in which hydrolysis of phosphorylcreatin  or phosphogen took the place of lactic acid production.  Well, although this was the first use of the word revolution connected with muscle, one  could say that the switch of interest to chemical events in 1900 was a revolution.  People realized and the statement is made in influential papers that contraction is  a molecular process.  And of course, you can't see molecules with a light microscope and it was concluded that  you won't learn anything from what you can see with the light microscope.  And the result was that interest switched away from what you can see with the microscope.  The striations of muscle had been much studied in the 19th century.  Next slide, please.  Now this is from one of the early electron micrographs of muscle published in 1949 in  Australia by Draper and Hodge just to illustrate the nomenclature of the parts of the pattern.  This is a shadowed picture, you can see a shadow here indicating that there's more material  here in the band which is called A standing for anisotropic because as well as having  much material and therefore a high refractive index in the intact muscle, it is also optically  anisotropic.  It is birefringent or doubly refracting and shows up in the polarizing microscope.  And then there's a less dense band, isotropic because it is not birefringent and that's  intersected by the z-line, the Tsvyschensheibe, which again has much material.  But that's just to illustrate the nomenclature.  This structure is a myofibril about one micrometer across and a single muscle fiber consists  of many thousands of these which come apart when the muscle is fragmented.  Well as I said much was learned about this astriation pattern in the 19th century but  as a result of the switch of interest to chemical events, this knowledge gained between 1850  and 1900 was almost completely lost.  So it was a revolution and the previous ideas were overthrown but like many political revolutions  its results were really a disaster for muscle.  And the loss of interest in this striations was reinforced by for example the fact that  our involuntary muscles which don't have striations, not clear visible ones, they're able to  contract and it was argued therefore the striations are not of any fundamental significance.  And at about the same time confidence in microscopy was shaken by several papers, one notable one  in 1899 by W. B. Hardy, one of my heroes for his work, pioneer work on polymer chemistry  and colloids but he showed that many of the sort of appearances that you see with the  light microscope in fixed preparations could be produced in simple protein like egg albumin  by treating with chemical fixatives.  And as a result of people no longer using the microscope as the principle tool, the  skills required in studying unstained preparations, living muscle with the microscope were lost.  And there was some bad microscopy in the first half of this century and some of what had  been known in the 19th century was back to front in the textbooks of 1950.  Well there was much progress in the 1930s and 1940s, next slide please.  The splitting of phospho-creatin was replaced by this hydrolysis of adenosine triphosphate  producing ADP and the role of phosphogen or just to say phosphoryl-creatin was relegated  to being a backup process.  Then it was found by Engelhardt and Glebimova in Moscow, yet another husband and wife team  in 1939.  They showed that the contractile protein myosin is in fact an ATPase, that's to say it is  an enzyme capable of bringing about this hydrolysis of ATP.  And then it was found in the laboratory of Alberts and Gerger that what had been called  myosin originally described by Cuner in 1864 is a complex of the protein that we now call  myosin and another protein to which they gave the name Actin and that was the work of F.B.  Straub who later for a time was president of Hungary.  But these developments all took place with no change in the general framework of ideas.  Everybody still thought that the contractile elements were long chains of protein that  were caused to fold up in some way and of course the protein became Acto myosin but  it was still thought of as long filaments.  And indeed the existence of continuous filaments in muscle was claimed by the authors.  Now this is from the first useful published electron micrographs of muscle by Hall, Jacobs  and Francis Schmidt published 1946, they worked at MIT in Boston.  And here again are myofibrils, this time stained with a heavy metal.  Again you see the familiar pattern of A bands but the authors claimed that they could see  continuous filaments running through both bands supporting the current idea.  Still with hindsight I think one has to say there was an element of wishful thinking in  seeing what they thought must be there.  But on the good side of this work it convinced people that the details of the striations which  had been described in the 19th century not merely the A and I bands and the Z line which  are conspicuous but further details a little middle-shiber, the M line in the middle of  the A band, a less dense zone in the middle of A called H after Henson and even some dark  things, a flanking Z line, Neybenscheiben or N bands.  And these were all things that had been described in the second half of the 19th century and  had been dismissed as, well, figments of the microscopist's imaginations.  So this restored some confidence in structure and microscopy.  Well I became interested in muscle through being asked to give lectures on it to the  final year students at Cambridge and I was given the lecture notes of my predecessor  and from then I learned of something that I had not learned as a student.  Next slide please.  Now this is from a paper of Engelmann in, well he worked in Utrecht but originally and finally  in Germany.  A schematic picture of a muscle showing this striation pattern going from rest up here  to a shortened state.  This is a fiber from probably the leg of an insect and these fibers when teased out undergo  spontaneous contraction waves.  And well dark in this image corresponds to high refractive index, simply high concentration  of protein.  Here's the appearance in polarized light, the A bands being birefringent show up bright  against the dark background.  But you see that when the muscle is heavily shortened the most dense region here is at  the position that corresponds to the middle of the eye band, the low refractive index  band.  So this phenomenon was called the reversal of striations.  The part that had been the least dense, the eye band, became the most dense.  And no one had paid attention to this phenomenon for some 50 years.  I'm speaking of about 1950.  So I thought it was an interesting thing to reinvestigate.  And to study it in intact fibers from a vertebrate, a frog notably, which are these are rather  thick fibers and with narrow striations, very difficult object for the ordinary microscope.  But it needed an interference microscope that would show up phase differences due to differences  of refractive index, while the ordinary microscope is designed primarily to show up differences  of absorption of light.  And muscle doesn't absorb light appreciably.  Things appear dark or light according to refractive index.  But the appearance that you get depends on how you focus the microscope, how thick the  specimen is.  Of course, phase contrast, invented by Zernicker, does show up phase differences due to refractive  index differences, but it does not work satisfactorily on a thick specimen.  And I had a boyhood interest in microscopes.  I had an idea of how to build an interference microscope.  So here was an opportunity to exercise my own hobby at the same time as investigating  what might be an interesting phenomenon.  So we built this microscope, got the optical parts made by the firm of Beck in London, and  looked at isolated living fibers from frog muscle, jointly with a German post-doctoral  colleague, Grolf Niedergärke.  And the first thing we saw, next slide please, these are images of a frog muscle fiber with  the instrument adjusted so that high refractive index regions appear dark.  So these dark bands are the A bands.  And we're here, we're simply stretching a fiber, not stimulated, and you see that as  we, this is the repeat distance of the striation pattern, and as we stretch it from rest a  little over two micrometers up to more than double, the width of the A bands hardly changes.  Well now that was the reverse of what was in all the textbooks at that time.  Well, as I said, we were wanting to investigate the reversal of striations, so we stimulated  one of these fibers under the microscope.  And next slide please.  I hope you can see here, well here it is at rest, again A bands dark, and I hope you can  see here that there are some very narrow dense lines in this region that is contracted actively  near the edge of the fiber.  But these dense lines were not where we expected to find them, so watching the fiber under  the microscope or watching the film from which these still photographs are taken, you can  see that these narrow dense lines are at the middle of the A band.  And it was only on further shortening that we did see a further lot of dense lines at  the middle of where the A bands had been.  Well seeing the constant width of the A bands, of course immediately suggested that the high  refractive index of the A bands was due to material in little rodlets that went from  one side of the A band to the other and were not individually stretched when the fiber  was stretched.  And then the contraction bands at the middle of the A band would be due to these rods colliding  and either folding up or overlapping.  So these little bands at the middle of the A band might be due to a second lot of filaments  that slid in and then when they overlapped or folded up where they collided would cause  these bands.  So that was how we got onto sliding filaments.  And at exactly the same time, this is early 1953, Hugh Huxley, together with Gene Hansen  who sadly died quite young afterwards, well they approached not through living muscle  but through electron micrographs, transfer sections of muscle and also phase microscopy  of separated myofibrils and they reached, well we both reached just the same conclusions.  And next slide please, you're probably all familiar with diagrams like this based on  Hugh Huxley's work.  It's pure coincidence that we have the same surname.  We haven't yet been able to trace any family connection.  So there were these thick filaments and now they also and Hasselbach in the laboratory  of HH Weber showed that the protein myosin was in the A bands and when it was dissolved  away the remaining material was distributed like the thin filaments and that was actin.  Well my colleague, Rolf Niedergärke had been at Göttingen and when he was there he had  been at a seminar where the old work on the striations had been discussed and he had a  memory that Krause, one of the 19th century microscopists had done something interesting.  And I followed this up in the library of our department and found many very interesting  and surprising things.  First a paper by Ernst von Brücke in Austria, a beautiful paper of 1858 in which he measured  the strength of birefringence of muscle fibers and found that when he stretched one of these  fibers the strength of birefringence did not increase.  If you take most biological fibrous materials and stretch them they become much more strongly  birefringent.  And von Brücke concluded, next slide, that the birefringence was due to little rodlets  distributed well through the A-band and that when the muscle was stretched or shortened  these rodlets changed their positions but were not individually stretched.  And that was a perfectly valid conclusion and it's still valid now.  And he gave the name distioclasts which if you translate from the, by parts from Greek  into Latin it comes out as English in English as birefringent elements.  And then a decade later Krause finding that the A-bands stayed at constant width on stretch  concluded that these distioclasts were rodlets going from one side of the A-band to the other  and he treated muscle with solutions known to dissolve myosin.  The A-bands disappeared so he concluded that these distioclasts were rodlets of myosin.  So the thick filaments made of myosin were there in place by 1869.  And then next slide a little later I came across a paper by Leon Friedrich in Belgium.  This is a composite diagram showing the appearance of the striation pattern in ordinary light.  Dark is higher refractive index.  Here at rest, here moderate shortening, here extreme shortening.  And at extreme shortening the dense lines are at the position of the middle of the I-band.  So there the contraction bands that Engelmann had seen in the 1870s and 80s.  But at a degree of shortening before those bands appear you can see a dense line in the  middle of the A-band appearing.  And that was what put Rolf Niedergerker and myself on to thinking of a second lot of filaments.  So all the evidence necessary for thinking of sliding filaments was there by 1876.  But nobody thought of it, these observations did not get built into any theory and largely  as a result of that they were forgotten.  Well, the theory of sliding filaments and its acceptance fairly rapid was indeed a revolution.  All previous ideas about shortening due to long filaments folding up were obsolete.  And well you might regard it also as a counter revolution reversing that of 1900.  It emphasized the importance of structure in muscle and it opened up new ways of thinking  and of interpreting even existing observations.  And there were two very significant observations already in the literature.  One, this is another husband and wife team in the United States, Robert Ramsey and Sybil  Street measuring the rise of tension when an isolated fraud muscle fiber is stimulated  starting at different lengths.  Maximum tension at 100%, that's the slack length about two micrometers per repeat of  the pattern.  And this linear decline coming down to zero, no tension produced on stimulation at about  double the slack length.  Well that suggested that perhaps tension is proportional to the amount of overlap between  the two lots of filaments, full overlap up here and this might be coming down to zero.  Well qualitatively that was okay but this length is actually more than the sum of the  lengths of the thick and thin filaments so it wasn't quite right.  But we found that that was due to a simple artifact.  When muscle fiber is stretched the end parts don't stretch as much as the middle so there  was still overlap at the ends of the fiber even though in most of the length of the fiber  was no overlap.  So we had to use a trick to keep constant the length of a middle segment of the fiber  where the striation spacing was nice and uniform and when we did that, next slide, yes we  found the relationship between tension produced and length of the fiber in here expressed  in micrometers of the striation repeat.  It followed very closely a straight line over this region coming down to a length just about  equal to the sum of the lengths of the thick and thin filaments reaching a maximum here  where all these little cross bridges on the thick filaments are overlapped by the thin  filament and then a plateau and then a drop when collision has begun.  So this implied that the tension was produced by contributions from a number of sites uniformly  distributed within each overlap zone.  And then another observation already in the literature, this is from the work of A.V.  Hill again whose photograph I showed, paper of 1938.  This is showing the increase of total rate of energy liberation which is partly coming  out as work and then an additional amount of heat.  Well this would represent the increase of total rate of chemical change and Dorothy  Needham, wife of yet another husband wife team in Mussel who is the wife of Joseph Needham  whose name you may know as a historian of the science in China.  And she suggested that it was pointed to the analogy between this hyperbolic relation and  the Michaelis-Minton relationship between the rate of an enzymic reaction and the concentration  of the substrate molecule.  And this of course enzymes work in a cyclical manner repeatedly, well hydrolyzing or whatever  it is, the substrate.  So she suggested this implied that the contractile elements in Mussel operated in a cyclic manner  during a contraction, each one going through many cycles.  Well both of these features, force proportional to overlap and cyclic operation were really  inconsistent with the idea of continuous filaments which required a progressive change from a  long state to a short state.  And I think all current theories do incorporate these two features, though many theories were  put forward after sliding filaments were well accepted that did not incorporate these features  but were progressive as opposed to cyclic theories and requiring a progressive change  from an elongated state to a shortened state.  So in 1954 I worked out a very speculative theory incorporating these features and next  slide I won't spend time on this, assuming that a little cross piece on the thick filament  had properties, it was elastic, represented by drawing a spring and it could attach to  sites on the actin filament and if it was in this sort of position, if it was to this  side it would produce a contribution to positive tension and if some sliding takes place pulling  this to the left, it's compressing this spring, it will produce a negative contribution to  tension.  And by assuming that the rate constant for making an attachment between this Myosin site  and an actin site had positive values over a certain range on the positive side where  the force would be generated, the rate constant for detachment was very low until sliding  had brought the attachment to a position where it was going to produce negative force and  then there's a very high rate of detachment and that gave a sufficient fit to the steady  state relations found by AV Hill.  So those features I think need to be incorporated in any current theory but they're very far  from complete, particularly they don't begin to explain the transient responses when the  length of a muscle is suddenly changed, if we shorten it by over half a percent of its  length, here we've stimulated, tension rises, this is 100 milliseconds, a slow record, tension  drops simultaneously with the shortening, recovers part of the way very fast and then  the rest of the way comparatively slowly.  And here on a 100 times faster time scale, here's the initial drop of tension and this  quick recovery in one or two milliseconds.  Still this is clearly in some sense the working stroke of the muscle fiber.  And in 1957 Hugh Huxley published some of the most beautiful electron microscopes yet  seen in which in the same section you see a thick filament and then there are two thin  filaments connected to the z-line and there are little bridges going between them and  the little bridges were clearly projections from the thick filaments, the mass in filaments.  So it was natural to identify these cross bridges with the sites at which contributions  to force are produced.  Well, that leaves open the question how they produce force and a paper a few years later  by Reedy Holmes and Triguer showed that a change of their angle took place so that it  was like a lever tilting about the attachment to the thin filament that pulled the thick  filament along.  And then in the early 90s the atomic structures of actin and of the head part of myosin, the  part that makes these cross bridges were solved by X-ray crystallography and these structures  suggested that it wasn't the whole myosin head that tilted but that there was a hinge  in the middle of the myosin head where the angle would change and part of the myosin  head would act as a lever.  And this is a diagram now familiar, the cycle goes round clockwise and the working stroke  is between from here to here.  This part of the myosin head is assumed to be rigidly attached to the actin filament  and the working stroke is that this part of the myosin head changes its angle pulling  on the thick filament and moving it along.  And then, well, rest of the cycle I think we haven't got time to look into.  And this is the generally accepted idea at the present time.  But there are still many uncertainties, great disagreements about how many of the myosin  heads are attached at any one time.  It's known that the actin filament is a helical structure and you would expect that when myosins  attach to this they would follow the helical pattern and the features of the x-ray diffraction  pattern coming from this helix would be strengthened.  But only very slight strengthening was found until, well, in the last year or two, two  Russians, Bershitsky and Saturyan, made rapid rise of temperature in active muscle, tension  rises, or up to perhaps double what it had been at the low temperature, not very fast,  not as fast as the tension recovery after a little release.  And they found also that during this tension rise after a temperature jump the strength  of these reflections from the actin in the x-ray pattern became stronger.  And this immediately suggested that there are two mechanisms at work.  The one what was generally believed, this represents the little lever which tilts by  a conformational change at the attachment here.  And that would be responsible for the rapid recovery of tension after a short release.  But the response to temperature rise might be the whole head tilting because in this  state it follows the actin helix, I haven't drawn this as helical but it is, but in this  state one would have to assume that, well, it's just a two-point attachment so it's  free to rotate about the long axis of the filament so it wouldn't really follow even  if the attachments were at points following the helix, the mass of the myosin would not  follow the helix.  So this is a suggestion that perhaps movement is generated at two places, one this conformational  change making the lever or move, the other the whole head tilting.  And suggestions of this have been seen in electron micrographs but this is still very  speculative.  Well, in the last ten years or so there have been astonishing developments in another direction  that's recording the tension produced by single myosin molecules.  Next slide please.  High-tech method, an actin filament is held between two beads each of which is held in  a light trap, a strongly focused beam of laser light and then you can move these beads by  displacing this focus.  And then there's myosin on another bead here, you bring the filament down onto it, there  may be a connection between actin and myosin and when there is the beads are displaced  and that's shown by these upward jerks.  Here the ATP concentration, the more rapidly they detach again, here with a very low ATP  you get these quite long lasting displacements and yes here there are displacements of a  few nanometers.  Still and then even more surprising just published a couple of months ago from the University  of York using the same technique but with a myosin not from a muscle but it's a much  slower muscle from brush border, here are these displacements due to single interactions  between the myosin molecule and you see that in each case it goes in two steps, the second  arrow shows the second displacement.  Well now whether that happens also with muscle myosin we don't know, you don't see a second  step but it's still possible that it's so rapid if it happens within one millisecond  you wouldn't see it with the time resolution that's now available.  So that's yet another uncertainty.  Well we may be undergoing yet another revolution in which interest concentrates on these single  molecule events and other high-tech methods, so not a revolution like the end of the lactic  acid story or the sliding filaments, these ideas are still within the general framework  of cross bridges forming and pulling the filaments along but it may be like the revolution of  1900 in switching interest away from what had been a valuable source of knowledge and a  recent review article in Nature is based entirely on well results of this kind disregarding  much that's known from whole muscle work and misinterpreting other observations and in  a couple of recent meetings about muscle there's been a similar emphasis on this kind of work  and almost nothing said about what you can learn from living muscle.  So I have a nasty suspicion that perhaps history is repeating itself after 100 years and that  much valuable knowledge is being lost through loss of interest.  May seem inconceivable to you that this could happen now but it's no more inconceivable than  the loss of the 19th century microscopy would have seemed in 1900 and there are many pressures  now that make it more likely the bulk of literature being published increasing specialization,  computer databases don't go back far enough and it's reinforced by the same story being  told repeatedly at symposia.  And well there was early this century I mentioned there was bad microscopy that got some of  these things back to front it would have been criticized by people like Engelmann and Kölnick  and to stay alive as long as I can and remind people of things that may be in danger of  being lost.  Well thank you for listening.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.  Thank you.
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Thank you very much, Professor Westling, for your kind introduction. And I would like also to express the thanks of all of my wife and myself to the Coratorium and to Countess Sonia for inviting us once again to this extremely interesting and enjoyable event. Well, as Professor Westling mentioned, it was in 1952 that Alan Hodgkin, who sadly died last December, and I completed the work for which we shared a Nobel Prize with the late Sir John Eccles. And that was about the ionic mechanism of conduction in nerve. And at that time, when we'd finished that work, we could not see what to do next to elucidate the mechanism of nerve conduction and excitation. We realized that channels must exist for the ions, but the enormous advances that have taken place since were quite unforeseeable at that time. We just could not imagine recording the currents through single channels, as of course was done by a Winn-Nair and that Satman, when was it, yes, 30 years later. So we moved into other fields. For a time, Hodgkin worked on active transport of ions, and then he moved to the electrical responses of rods and cones in the retina. And I moved on to the study of muscle contraction. Well, I'm long retired. I gave up active laboratory work a year or two ago, so I have no new experiments to report to you. But with the approach of the end of this century, it seemed appropriate to review the changes in what people have thought about muscle contraction since 1900. And to a considerable extent, these changes have followed the, well, the path that was suggested by the philosopher of science, Kuhn, in his book, The Nature of Scientific Revolutions. He proposed that scientific advance in any field takes place partly through occasional revolutions when a new observation or discovery makes previous ideas obsolete and one has to change one's, all one's thinking. And in between these revolutions, there's, well, normal science in which much progress is made but without a change of the framework of ideas within which one works. Well, starting at 1900, that was just about the time when classical biochemistry really started. And this stimulated an interest in the chemical changes in muscle. And at that time, the only chemical change known during contraction was the production of lactic acid, which had been observed, an extraordinary achievement by the Swedish chemist Batcellius in 1807. So that was the only thing known in 1900. First slide, please. And all the, everybody believed that this production of lactic acid was directly the cause of contraction. It was supposed that there were long chains of protein molecules which were kept extended by the repulsion of their negative charges. These charges were neutralized by the hydrogen ions from lactic acid that allowed them to fold up. And this theory met difficulties in the 1920s, but the biochemists stood firm on the theory, notably Meyerhoff, until 1930 when the Danish biochemist Lundsgaard experimented with muscle poisoned with iodoresetic acid and found that the muscle could give many normal contractions without producing any lactic acid. Now that was the occasion for the first use of the word revolution in connection with muscle. Next slide, please. That was by A.V. Hill, famous particularly for his work on the heat production of muscle. He shared a Nobel Prize with Meyerhoff in 1923. And yes, he described this demise of the lactic acid theory as a revolution. Next slide, please. And here is an excerpt from the review which he entitled, entitled, The Revolution in Muscle Physiology. Well, a substitute for the production of lactic acid was already in the literature. Two groups, Fisk and Soborow in the United States and the two Egltons, Philip and Grace, in Scotland. One of the many husband and wife teams in muscle physiology had discovered that phosphate is released during contraction and this came from a substance known by the general name Phosphogen and Fisk and Soborow identified this substance as phosphorylcreatin. So textbooks came to say, well, this sort of statement in which hydrolysis of phosphorylcreatin or phosphogen took the place of lactic acid production. Well, although this was the first use of the word revolution connected with muscle, one could say that the switch of interest to chemical events in 1900 was a revolution. People realized and the statement is made in influential papers that contraction is a molecular process. And of course, you can't see molecules with a light microscope and it was concluded that you won't learn anything from what you can see with the light microscope. And the result was that interest switched away from what you can see with the microscope. The striations of muscle had been much studied in the 19th century. Next slide, please. Now this is from one of the early electron micrographs of muscle published in 1949 in Australia by Draper and Hodge just to illustrate the nomenclature of the parts of the pattern. This is a shadowed picture, you can see a shadow here indicating that there's more material here in the band which is called A standing for anisotropic because as well as having much material and therefore a high refractive index in the intact muscle, it is also optically anisotropic. It is birefringent or doubly refracting and shows up in the polarizing microscope. And then there's a less dense band, isotropic because it is not birefringent and that's intersected by the z-line, the Tsvyschensheibe, which again has much material. But that's just to illustrate the nomenclature. This structure is a myofibril about one micrometer across and a single muscle fiber consists of many thousands of these which come apart when the muscle is fragmented. Well as I said much was learned about this astriation pattern in the 19th century but as a result of the switch of interest to chemical events, this knowledge gained between 1850 and 1900 was almost completely lost. So it was a revolution and the previous ideas were overthrown but like many political revolutions its results were really a disaster for muscle. And the loss of interest in this striations was reinforced by for example the fact that our involuntary muscles which don't have striations, not clear visible ones, they're able to contract and it was argued therefore the striations are not of any fundamental significance. And at about the same time confidence in microscopy was shaken by several papers, one notable one in 1899 by W. B. Hardy, one of my heroes for his work, pioneer work on polymer chemistry and colloids but he showed that many of the sort of appearances that you see with the light microscope in fixed preparations could be produced in simple protein like egg albumin by treating with chemical fixatives. And as a result of people no longer using the microscope as the principle tool, the skills required in studying unstained preparations, living muscle with the microscope were lost. And there was some bad microscopy in the first half of this century and some of what had been known in the 19th century was back to front in the textbooks of 1950. Well there was much progress in the 1930s and 1940s, next slide please. The splitting of phospho-creatin was replaced by this hydrolysis of adenosine triphosphate producing ADP and the role of phosphogen or just to say phosphoryl-creatin was relegated to being a backup process. Then it was found by Engelhardt and Glebimova in Moscow, yet another husband and wife team in 1939. They showed that the contractile protein myosin is in fact an ATPase, that's to say it is an enzyme capable of bringing about this hydrolysis of ATP. And then it was found in the laboratory of Alberts and Gerger that what had been called myosin originally described by Cuner in 1864 is a complex of the protein that we now call myosin and another protein to which they gave the name Actin and that was the work of F.B. Straub who later for a time was president of Hungary. But these developments all took place with no change in the general framework of ideas. Everybody still thought that the contractile elements were long chains of protein that were caused to fold up in some way and of course the protein became Acto myosin but it was still thought of as long filaments. And indeed the existence of continuous filaments in muscle was claimed by the authors. Now this is from the first useful published electron micrographs of muscle by Hall, Jacobs and Francis Schmidt published 1946, they worked at MIT in Boston. And here again are myofibrils, this time stained with a heavy metal. Again you see the familiar pattern of A bands but the authors claimed that they could see continuous filaments running through both bands supporting the current idea. Still with hindsight I think one has to say there was an element of wishful thinking in seeing what they thought must be there. But on the good side of this work it convinced people that the details of the striations which had been described in the 19th century not merely the A and I bands and the Z line which are conspicuous but further details a little middle-shiber, the M line in the middle of the A band, a less dense zone in the middle of A called H after Henson and even some dark things, a flanking Z line, Neybenscheiben or N bands. And these were all things that had been described in the second half of the 19th century and had been dismissed as, well, figments of the microscopist's imaginations. So this restored some confidence in structure and microscopy. Well I became interested in muscle through being asked to give lectures on it to the final year students at Cambridge and I was given the lecture notes of my predecessor and from then I learned of something that I had not learned as a student. Next slide please. Now this is from a paper of Engelmann in, well he worked in Utrecht but originally and finally in Germany. A schematic picture of a muscle showing this striation pattern going from rest up here to a shortened state. This is a fiber from probably the leg of an insect and these fibers when teased out undergo spontaneous contraction waves. And well dark in this image corresponds to high refractive index, simply high concentration of protein. Here's the appearance in polarized light, the A bands being birefringent show up bright against the dark background. But you see that when the muscle is heavily shortened the most dense region here is at the position that corresponds to the middle of the eye band, the low refractive index band. So this phenomenon was called the reversal of striations. The part that had been the least dense, the eye band, became the most dense. And no one had paid attention to this phenomenon for some 50 years. I'm speaking of about 1950. So I thought it was an interesting thing to reinvestigate. And to study it in intact fibers from a vertebrate, a frog notably, which are these are rather thick fibers and with narrow striations, very difficult object for the ordinary microscope. But it needed an interference microscope that would show up phase differences due to differences of refractive index, while the ordinary microscope is designed primarily to show up differences of absorption of light. And muscle doesn't absorb light appreciably. Things appear dark or light according to refractive index. But the appearance that you get depends on how you focus the microscope, how thick the specimen is. Of course, phase contrast, invented by Zernicker, does show up phase differences due to refractive index differences, but it does not work satisfactorily on a thick specimen. And I had a boyhood interest in microscopes. I had an idea of how to build an interference microscope. So here was an opportunity to exercise my own hobby at the same time as investigating what might be an interesting phenomenon. So we built this microscope, got the optical parts made by the firm of Beck in London, and looked at isolated living fibers from frog muscle, jointly with a German post-doctoral colleague, Grolf Niedergärke. And the first thing we saw, next slide please, these are images of a frog muscle fiber with the instrument adjusted so that high refractive index regions appear dark. So these dark bands are the A bands. And we're here, we're simply stretching a fiber, not stimulated, and you see that as we, this is the repeat distance of the striation pattern, and as we stretch it from rest a little over two micrometers up to more than double, the width of the A bands hardly changes. Well now that was the reverse of what was in all the textbooks at that time. Well, as I said, we were wanting to investigate the reversal of striations, so we stimulated one of these fibers under the microscope. And next slide please. I hope you can see here, well here it is at rest, again A bands dark, and I hope you can see here that there are some very narrow dense lines in this region that is contracted actively near the edge of the fiber. But these dense lines were not where we expected to find them, so watching the fiber under the microscope or watching the film from which these still photographs are taken, you can see that these narrow dense lines are at the middle of the A band. And it was only on further shortening that we did see a further lot of dense lines at the middle of where the A bands had been. Well seeing the constant width of the A bands, of course immediately suggested that the high refractive index of the A bands was due to material in little rodlets that went from one side of the A band to the other and were not individually stretched when the fiber was stretched. And then the contraction bands at the middle of the A band would be due to these rods colliding and either folding up or overlapping. So these little bands at the middle of the A band might be due to a second lot of filaments that slid in and then when they overlapped or folded up where they collided would cause these bands. So that was how we got onto sliding filaments. And at exactly the same time, this is early 1953, Hugh Huxley, together with Gene Hansen who sadly died quite young afterwards, well they approached not through living muscle but through electron micrographs, transfer sections of muscle and also phase microscopy of separated myofibrils and they reached, well we both reached just the same conclusions. And next slide please, you're probably all familiar with diagrams like this based on Hugh Huxley's work. It's pure coincidence that we have the same surname. We haven't yet been able to trace any family connection. So there were these thick filaments and now they also and Hasselbach in the laboratory of HH Weber showed that the protein myosin was in the A bands and when it was dissolved away the remaining material was distributed like the thin filaments and that was actin. Well my colleague, Rolf Niedergärke had been at Göttingen and when he was there he had been at a seminar where the old work on the striations had been discussed and he had a memory that Krause, one of the 19th century microscopists had done something interesting. And I followed this up in the library of our department and found many very interesting and surprising things. First a paper by Ernst von Brücke in Austria, a beautiful paper of 1858 in which he measured the strength of birefringence of muscle fibers and found that when he stretched one of these fibers the strength of birefringence did not increase. If you take most biological fibrous materials and stretch them they become much more strongly birefringent. And von Brücke concluded, next slide, that the birefringence was due to little rodlets distributed well through the A-band and that when the muscle was stretched or shortened these rodlets changed their positions but were not individually stretched. And that was a perfectly valid conclusion and it's still valid now. And he gave the name distioclasts which if you translate from the, by parts from Greek into Latin it comes out as English in English as birefringent elements. And then a decade later Krause finding that the A-bands stayed at constant width on stretch concluded that these distioclasts were rodlets going from one side of the A-band to the other and he treated muscle with solutions known to dissolve myosin. The A-bands disappeared so he concluded that these distioclasts were rodlets of myosin. So the thick filaments made of myosin were there in place by 1869. And then next slide a little later I came across a paper by Leon Friedrich in Belgium. This is a composite diagram showing the appearance of the striation pattern in ordinary light. Dark is higher refractive index. Here at rest, here moderate shortening, here extreme shortening. And at extreme shortening the dense lines are at the position of the middle of the I-band. So there the contraction bands that Engelmann had seen in the 1870s and 80s. But at a degree of shortening before those bands appear you can see a dense line in the middle of the A-band appearing. And that was what put Rolf Niedergerker and myself on to thinking of a second lot of filaments. So all the evidence necessary for thinking of sliding filaments was there by 1876. But nobody thought of it, these observations did not get built into any theory and largely as a result of that they were forgotten. Well, the theory of sliding filaments and its acceptance fairly rapid was indeed a revolution. All previous ideas about shortening due to long filaments folding up were obsolete. And well you might regard it also as a counter revolution reversing that of 1900. It emphasized the importance of structure in muscle and it opened up new ways of thinking and of interpreting even existing observations. And there were two very significant observations already in the literature. One, this is another husband and wife team in the United States, Robert Ramsey and Sybil Street measuring the rise of tension when an isolated fraud muscle fiber is stimulated starting at different lengths. Maximum tension at 100%, that's the slack length about two micrometers per repeat of the pattern. And this linear decline coming down to zero, no tension produced on stimulation at about double the slack length. Well that suggested that perhaps tension is proportional to the amount of overlap between the two lots of filaments, full overlap up here and this might be coming down to zero. Well qualitatively that was okay but this length is actually more than the sum of the lengths of the thick and thin filaments so it wasn't quite right. But we found that that was due to a simple artifact. When muscle fiber is stretched the end parts don't stretch as much as the middle so there was still overlap at the ends of the fiber even though in most of the length of the fiber was no overlap. So we had to use a trick to keep constant the length of a middle segment of the fiber where the striation spacing was nice and uniform and when we did that, next slide, yes we found the relationship between tension produced and length of the fiber in here expressed in micrometers of the striation repeat. It followed very closely a straight line over this region coming down to a length just about equal to the sum of the lengths of the thick and thin filaments reaching a maximum here where all these little cross bridges on the thick filaments are overlapped by the thin filament and then a plateau and then a drop when collision has begun. So this implied that the tension was produced by contributions from a number of sites uniformly distributed within each overlap zone. And then another observation already in the literature, this is from the work of A.V. Hill again whose photograph I showed, paper of 1938. This is showing the increase of total rate of energy liberation which is partly coming out as work and then an additional amount of heat. Well this would represent the increase of total rate of chemical change and Dorothy Needham, wife of yet another husband wife team in Mussel who is the wife of Joseph Needham whose name you may know as a historian of the science in China. And she suggested that it was pointed to the analogy between this hyperbolic relation and the Michaelis-Minton relationship between the rate of an enzymic reaction and the concentration of the substrate molecule. And this of course enzymes work in a cyclical manner repeatedly, well hydrolyzing or whatever it is, the substrate. So she suggested this implied that the contractile elements in Mussel operated in a cyclic manner during a contraction, each one going through many cycles. Well both of these features, force proportional to overlap and cyclic operation were really inconsistent with the idea of continuous filaments which required a progressive change from a long state to a short state. And I think all current theories do incorporate these two features, though many theories were put forward after sliding filaments were well accepted that did not incorporate these features but were progressive as opposed to cyclic theories and requiring a progressive change from an elongated state to a shortened state. So in 1954 I worked out a very speculative theory incorporating these features and next slide I won't spend time on this, assuming that a little cross piece on the thick filament had properties, it was elastic, represented by drawing a spring and it could attach to sites on the actin filament and if it was in this sort of position, if it was to this side it would produce a contribution to positive tension and if some sliding takes place pulling this to the left, it's compressing this spring, it will produce a negative contribution to tension. And by assuming that the rate constant for making an attachment between this Myosin site and an actin site had positive values over a certain range on the positive side where the force would be generated, the rate constant for detachment was very low until sliding had brought the attachment to a position where it was going to produce negative force and then there's a very high rate of detachment and that gave a sufficient fit to the steady state relations found by AV Hill. So those features I think need to be incorporated in any current theory but they're very far from complete, particularly they don't begin to explain the transient responses when the length of a muscle is suddenly changed, if we shorten it by over half a percent of its length, here we've stimulated, tension rises, this is 100 milliseconds, a slow record, tension drops simultaneously with the shortening, recovers part of the way very fast and then the rest of the way comparatively slowly. And here on a 100 times faster time scale, here's the initial drop of tension and this quick recovery in one or two milliseconds. Still this is clearly in some sense the working stroke of the muscle fiber. And in 1957 Hugh Huxley published some of the most beautiful electron microscopes yet seen in which in the same section you see a thick filament and then there are two thin filaments connected to the z-line and there are little bridges going between them and the little bridges were clearly projections from the thick filaments, the mass in filaments. So it was natural to identify these cross bridges with the sites at which contributions to force are produced. Well, that leaves open the question how they produce force and a paper a few years later by Reedy Holmes and Triguer showed that a change of their angle took place so that it was like a lever tilting about the attachment to the thin filament that pulled the thick filament along. And then in the early 90s the atomic structures of actin and of the head part of myosin, the part that makes these cross bridges were solved by X-ray crystallography and these structures suggested that it wasn't the whole myosin head that tilted but that there was a hinge in the middle of the myosin head where the angle would change and part of the myosin head would act as a lever. And this is a diagram now familiar, the cycle goes round clockwise and the working stroke is between from here to here. This part of the myosin head is assumed to be rigidly attached to the actin filament and the working stroke is that this part of the myosin head changes its angle pulling on the thick filament and moving it along. And then, well, rest of the cycle I think we haven't got time to look into. And this is the generally accepted idea at the present time. But there are still many uncertainties, great disagreements about how many of the myosin heads are attached at any one time. It's known that the actin filament is a helical structure and you would expect that when myosins attach to this they would follow the helical pattern and the features of the x-ray diffraction pattern coming from this helix would be strengthened. But only very slight strengthening was found until, well, in the last year or two, two Russians, Bershitsky and Saturyan, made rapid rise of temperature in active muscle, tension rises, or up to perhaps double what it had been at the low temperature, not very fast, not as fast as the tension recovery after a little release. And they found also that during this tension rise after a temperature jump the strength of these reflections from the actin in the x-ray pattern became stronger. And this immediately suggested that there are two mechanisms at work. The one what was generally believed, this represents the little lever which tilts by a conformational change at the attachment here. And that would be responsible for the rapid recovery of tension after a short release. But the response to temperature rise might be the whole head tilting because in this state it follows the actin helix, I haven't drawn this as helical but it is, but in this state one would have to assume that, well, it's just a two-point attachment so it's free to rotate about the long axis of the filament so it wouldn't really follow even if the attachments were at points following the helix, the mass of the myosin would not follow the helix. So this is a suggestion that perhaps movement is generated at two places, one this conformational change making the lever or move, the other the whole head tilting. And suggestions of this have been seen in electron micrographs but this is still very speculative. Well, in the last ten years or so there have been astonishing developments in another direction that's recording the tension produced by single myosin molecules. Next slide please. High-tech method, an actin filament is held between two beads each of which is held in a light trap, a strongly focused beam of laser light and then you can move these beads by displacing this focus. And then there's myosin on another bead here, you bring the filament down onto it, there may be a connection between actin and myosin and when there is the beads are displaced and that's shown by these upward jerks. Here the ATP concentration, the more rapidly they detach again, here with a very low ATP you get these quite long lasting displacements and yes here there are displacements of a few nanometers. Still and then even more surprising just published a couple of months ago from the University of York using the same technique but with a myosin not from a muscle but it's a much slower muscle from brush border, here are these displacements due to single interactions between the myosin molecule and you see that in each case it goes in two steps, the second arrow shows the second displacement. Well now whether that happens also with muscle myosin we don't know, you don't see a second step but it's still possible that it's so rapid if it happens within one millisecond you wouldn't see it with the time resolution that's now available. So that's yet another uncertainty. Well we may be undergoing yet another revolution in which interest concentrates on these single molecule events and other high-tech methods, so not a revolution like the end of the lactic acid story or the sliding filaments, these ideas are still within the general framework of cross bridges forming and pulling the filaments along but it may be like the revolution of 1900 in switching interest away from what had been a valuable source of knowledge and a recent review article in Nature is based entirely on well results of this kind disregarding much that's known from whole muscle work and misinterpreting other observations and in a couple of recent meetings about muscle there's been a similar emphasis on this kind of work and almost nothing said about what you can learn from living muscle. So I have a nasty suspicion that perhaps history is repeating itself after 100 years and that much valuable knowledge is being lost through loss of interest. May seem inconceivable to you that this could happen now but it's no more inconceivable than the loss of the 19th century microscopy would have seemed in 1900 and there are many pressures now that make it more likely the bulk of literature being published increasing specialization, computer databases don't go back far enough and it's reinforced by the same story being told repeatedly at symposia. And well there was early this century I mentioned there was bad microscopy that got some of these things back to front it would have been criticized by people like Engelmann and Kölnick and to stay alive as long as I can and remind people of things that may be in danger of being lost. Well thank you for listening. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you.
the, well, the path that was suggested by the partly through occasional revolutions when a new obsolete and one has to change one's, all one's when classical biochemistry really started. And chemical change known during contraction was the long chains of protein molecules which were kept extended by the repulsion of their negative Now that was the occasion for the first use of the word revolution in connection with muscle. Muscle Physiology. Well, a substitute for the production of lactic acid was already in the that phosphate is released during contraction and that contraction is a molecular process. And of isotropic because it is not birefringent and completely lost. So it was a revolution and the previous ideas were overthrown but like many the light microscope in fixed preparations could microscopy in the first half of this century and some of what had been known in the 19th century there was much progress in the 1930s and 1940s, role of phosphogen or just to say phosphoryl-creatin was relegated to being a backup process. Then it was found by Engelhardt wife team in 1939. They showed that the contractile protein myosin is in fact an ATPase, about this hydrolysis of ATP. And then it was that what had been called myosin originally thought that the contractile elements were long convinced people that the details of the of striations. The part that had been the least no one had paid attention to this phenomenon for more than double, the width of the A bands hardly changes. Well now that was the reverse of what watching the fiber under the microscope or through living muscle but through electron myosin was in the A bands and when it was distributed like the thin filaments and that was myosin. So the thick filaments made of myosin any theory and largely as a result of that they were forgotten. Well, the theory of sliding indeed a revolution. All previous ideas about shortening due to long filaments folding up were counter revolution reversing that of 1900. It perhaps tension is proportional to the amount of overlap between the two lots of filaments, full lengths of the thick and thin filaments so it contributions from a number of sites uniformly distributed within each overlap zone. And then this is from the work of A.V. Hill again whose showing the increase of total rate of energy then an additional amount of heat. Well this between this hyperbolic relation and the cycles. Well both of these features, force really inconsistent with the idea of continuous from a long state to a short state. And I think attach to sites on the actin filament and if it to be incorporated in any current theory but when the length of a muscle is suddenly changed, contributions to force are produced. Well, that leaves open the question how they produce force the attachment to the thin filament that pulled here to here. This part of the myosin head is about how many of the myosin heads are attached double what it had been at the low temperature, temperature rise might be the whole head tilting a suggestion that perhaps movement is generated at two places, one this conformational change very speculative. Well, in the last ten years or held between two beads each of which is held in a and myosin and when there is the beads are displacements due to single interactions between yet another revolution in which interest concentrates on these single molecule events and filaments, these ideas are still within the pulling the filaments along but it may be like and a recent review article in Nature is based work and misinterpreting other observations and