ANTIQUITY AND THE HELLENISTIC AGE

In the development of thought about the bodies of men and animals there came a time when the age-old acceptance of undifferentiated body-substance, the biblical ‘flesh of rams’ or the meat on which Homeric heroes feasted, gave place to a realisation that it consisted of individual muscles. How early did this happen and when was the function of these muscles as instruments of movement realised? With these questions our story naturally begins.

The Hippocratic collection of writings on medicine and its philosophy, by a number of writers of his school as well as perhaps by Hippocrates of Cos himself, was put together before the end of the third century B.C. and includes works of the two previous centuries, some indeed containing ideas from still earlier times. There is thus no such thing as a single system of thought to be found in them; the different treatises of the Corpus, some sixty in number all told, represent several different, and even opposing, schools. Three of them have been attributed by some distinguished scholars to the great physician of Cos himself, and eight more are considered to date from his time (460 to 380 B.C.). The only certainly pre-Hippocratic one is the ‘Sevens’, a prognostic text which implies the humoral theory of disease and the doctrine of critical days.

In these Greek writings the tendons (which were confused with nerves) were endowed with the power of causing movement. In fact the same word neuron was used indiscriminately for both, just as phlebes was used indifferently for the veins and the arteries.

THE MACHINE AS ENZYME AND THE FUEL AS SUBSTRATE

The discovery by Engelhardt & Lyubimova in 1939 of the ATPase activity of myosin opened a new era in muscle biochemistry. Lundsgaard (8) had suggested that breakdown of ATP might be associated with restoration of the contractile substance, and D. M. Needham (1) that possibly ATP had some special spatial relationship to the myosin micellae. But the idea of the enzymic activity of the muscle machinery itself was an entirely new one, and the Russian workers fully realised its implications. They endeavoured to free the myosin from enzymic activity by repeated washing and reprecipitation but instead the activity rose to a fairly constant level. The purified myosin split off only one phosphate group, yielding ADP. They remarked on the great heat-lability of the ATPase, its activity being lost in 10 min at 37° and compared this with the low coagulation temperature of myosin known since the time of Kühne; they also noticed the similar sensitivity to acids of myosin as protein and as ATPase. These similarities served to increase the probability of the identity of the ATPase and myosin, but Engelhardt & Lyubimova considered that no final decision could be taken.

In 1941 Engelhardt, Lyubimova & Meitina (1) were the first to test the effect of ATP on myosin threads. These, though containing only about 2 % of protein, showed a certain amount of tensile strength; they were immersed in fluid and connected with the lever of a torsion balance so that when tension (about 200 mg) was applied the extensibility could be measured. They found that addition of 5 × 10−3M ATP caused considerable increase, 50–100%, in the extensibility.

The greater part of the eighteenth century brought no fundamental contributions to the elucidation of contractility in living organisms. A deeper understanding of the chemistry of inorganic and organic matter was really pre-requisite for this, and great strides now began to be made in these directions.

THE CHEMICAL BACKGROUND

The quantitative study of gases (which had begun with Robert Boyle in 1660) was continued vigorously during this next century, and interpreted in terms of the phlogiston theory of Stahl, enunciated in 1697. This theory explained combustion as due to the presence in combustible material of a principle of inflammability (sometimes credited with negative weight) termed phlogiston; material supporting combustion did so in virtue of its power to absorb phlogiston, and this principle was lost during combustion. The work of Black on fixed air (carbon dioxide) in 1755; of Cavendish between 1766 and 1784 on fixed air, inflammable air (hydrogen) phlogisticated air (nitrogen) and dephlogisticated air (oxygen); and of Priestley on dephlogisticated air may be specially mentioned. In 1774 Priestley prepared purified dephlogisticated air (to which Lavoisier a little later gave the name oxygen) by heating red oxide of mercury; he showed that this gas was better than common air for supporting combustion and life.

Lavoisier had also intensively studied calcination, and had shown that in this process tin for example gained in weight, while the air in which it was contained lost equally in weight and also diminished in volume.

Here we shall consider on the one hand the nature and arrangement of the proteins making up the muscle machine, and on the other hand the chemical substances and enzymic reactions providing the energy. A vast number of observations has been made on examples of the different phyla, but knowledge is still very incomplete and it is often difficult to generalise, particularly because important differences may turn up between closely related species. It will not be possible to deal in a comparative way with more than a part of the data. In the case of two important types, however, the adductor muscle in molluscs (responsible for the proverbial closing mechanism e.g. in the clam) and the fibrillar muscle of certain flying insects (the fastest and most active muscle known) very thorough investigations have been made. Formulation of detailed suggestions for the modus operandi of these two types has been possible.

It is necessary to say a few words about structure, which is very varied. Setting out from histological and electron-microscope observations, Hanson & Lowy (3) have distinguished three kinds of invertebrate muscle; (a) Striated, showing the A and I bands familiar in vertebrate striated muscle; in invertebrates these are found e.g. in insect muscle and in the phasic adductor of Pecten. (b) Muscles such as those in certain cephalopods and annelids showing ‘double-oblique striation’, which depends on the helical arrangement of thick and thin filaments.

Let us turn now for a while, in closing this story of the development of ideas concerning the nature of muscle contraction and the pathways of energy provision for it, to survey certain wider horizons which have come into view. In 1933 Hopkins (4), in his presidential address to the British Association, after briefly referring to the sequence of chemical events (as understood at that time) which led up to the mechanical response, continued:

And in conversation he was wont to emphasise the outstanding suitability of muscle as the material for studies on energy relationships, just because in this tissue it was easier to make quantitative measurements of the energy changes involved; he was confident that the relevance of such results to the behaviour of other tissues would in time emerge.

INTRODUCTION

In the last chapter attention was concentrated mainly on the various types of evidence which contributed to the knowledge of the fine structure of the myofibril at rest and contracted. The generally preferred conception emerged of the interaction of two types of filament, one consisting mainly of myosin, the other mainly of actin, intermittently linked by bridges from the myosin; and of contraction as depending not on shortening of the filaments, but on the degree of overlap (thus on the degree of possibility of interaction) of the two sets of filament. The manner in which such a conception could fit with such well-known facts as the effects of ATP on actin/myosin association, or energy provision by actomyosin-catalysed ATP hydrolysis, was explored in a general way. Now we have two tasks – first that of considering more specific theories attempting to explain how the sliding could take place and derive its energy; secondly and mainly that of describing experimentation of recent years which, in various ways, has set out to throw light on possible conformational changes in the proteins during contraction, or under conditions which might obtain during contraction, or which might resemble such conditions. In some cases the possibility that these changes might be relevant is simply stated; in other cases, the results are assembled to support certain detailed ideas concerning the mechanism of sliding and the provision of energy for it.

BACKGROUND TO THE THEORIES

MUSCLE STRUCTURE. Before introducing some of these conceptions entertained during the ten or fifteen years after the discovery of the interaction of myosin, actin and ATP, we may consider the re-orientation of ideas concerning interpretation of visible muscle structure. This closer look was necessitated by the discovery of actin, the more exact knowledge of the relative quantities of the muscle proteins and the early observations by means of the electron microscope. As we have seen, ‘myosin’ had been allotted by Noll & Weber (1) in 1935 to the A band, and the double refraction of the fibre had been explained as due to the rod and intrinsic double refraction of this protein. Weber (6) in 1956 remarked that this would mean that the I band must consist of other proteins – perhaps including globulin X and stroma. The assumption however was frequently made that it consisted of disordered myosin. Some observers recorded that the I band rather than the A band shortened on contraction, but the general opinion seems to have been that the material of the A band was that primarily concerned in the mechanism of movement, the changes in the I band being passive.

It is striking to see how many of the observations of classical histology were confirmed by the electron microscope – for example, the A and I bands, the H zone, and the Z and M lines could all be distinguished.

EARLY ADVANCES IN THE MICROSCOPY OF MUSCLE

In the immediately preceding chapters attention has mainly been concentrated on the processes of energy provision for contraction; speculations concerned with the nature of the muscle machine itself were vigorously canvassed in the seventeenth century as we have seen, but it is necessary now to consider in detail the intensive work on this question which began early in the nineteenth century.

This work followed two main lines: first, the microscopic examination of muscle sections and fibres by ordinary and by polarised light; secondly, since the muscle structure must consist mainly of protein, the biochemical examination of the extracted proteins. Early in the twentieth century, the methods of X-ray diffraction were called upon, and about the middle of the century the phase contrast, interference and electron microscopes began to play their part. This chapter then, leading up to 1939 when Engelhardt & Lyubimova (1) made their pregnant discovery of the adenosinetriphosphatase activity of the structural protein myosin, will be concerned with the microscopic structure of the muscle machine and the nature of its protein composition.

After the first microsopic examination of muscle by Leeuwenhoek in 1674 and his discovery of the cross-striations in 1682, there was little progress for more than 100 years. This is to be correlated with the fact that during the eighteenth century, though much experimentation went on, no optical but only mechanical improvements were made in the microscopes generally available.

FROM THE LIQUIDATION OF INOGEN TO THE FIRST BALANCING OF THE THERMOCHEMICAL BOOKS

In 1898 Fletcher, coming with an open mind to a subject in danger of being stifled with theorisation, published his first paper on survival respiration of excised muscle. This work may be regarded as the real beginning of quantitative muscle biochemistry. The new, rapid and comparatively micromethod of carbon dioxide estimation that he used made it possible to study the gas evolution over far shorter periods than formerly, and so to avoid the complications of putrefactive changes.

Fletcher first observed the behaviour of frog muscle kept in air or nitrogen. There was an initial fall in carbon dioxide output, followed by a small steady evolution during some hours; then an important acceleration accompanied by shortening – the onset of rigor. From the shape of the time curve, Fletcher explained the early fall as due to outward diffusion of carbon dioxide already present in the muscle; the plateau as due to evolution of the gas displaced from carbonates by slow production of acid. The survival carbon dioxide production of resting muscle in oxygen was some four times that in nitrogen. Stimulation to contraction in nitrogen had little effect unless the muscle was pushed to fatigue; but in oxygen there was always increased output roughly proportional to the number and degree of contractions.

RED AND WHITE MUSCLE; EARLY WORK

PIGMENTATION. The variations in colour of skeletal muscle, from the deep crimson of the pigeon breast to the whiteness of fish muscle, have long been a matter of discussion; in 1678 Lorenzini commented on the striking differences of colour in certain muscles of the rabbit. It was at first supposed that the red colour was due to a greater supply of blood, but Kolliker (1) in 1850 considered from his histological studies that the pigment was contained actually within the contractile substance of the fibres. Kuhne (4) in 1865 showed that this was indeed the case, by perfusing muscles to wash out all blood, and finding that a haemoglobin-like substance remained in the muscle plasma. In spectral analyses of the reduced and oxygenated forms of the substance, as well as of the carbon monoxide compound and the haematin formed from it, he found no significant difference from blood haemoglobin; but the work of Morner (1) in 1896 made it clear that in all these cases the absorption bands lie nearer the red end of the spectrum with muscle haemoglobin than with blood haemoglobin. He suggested the name myochrome, and the designation myoglobin now in general use was introduced by Günther (1), who confirmed the results of Mörner, only in 1921. Keilin has described the confusion in the literature during the first quarter of this century between myochrome and MacMunn's myohaematin, confusion only cleared up by Keilin's own work.

SOLUBILITY AND EXTRACTABILITY OF THE STRUCTURAL PROTEINS

In chapter 8 we were concerned with the growth of knowledge of actomyosin- ATP interactions from 1939 to about 1953. In chapter 9 we considered the theories of contraction (with their interesting variations) which resulted from the realisation of the importance of the actomyosin-ATP relationship. Here again the period chosen terminated about 1953, because at this time the idea of the sliding-filament mechanism began to emerge; as evidence has accumulated, this has gradually replaced almost all other postulated mechanisms. The story of this will occupy chapter 11. In the present chapter I want to discuss the properties of the individual structural proteins, to which another was added in 1946 by the discovery of tropomyosin by Bailey (2).

We have already discussed the results of earlier workers who estimated ‘myosin’, myogen, globulin X and stroma protein in muscle. After the discovery of actin and actomyosin, Balenović & Straub (1) were the first to try to estimate, albeit in an indirect way, the amount of actin present.

As the basis of this method they used the formation of actomyosin when actin was added to excess of myosin, the actomyosin being assessed by the fall in viscosity on addition of ATP. This decrease in specific viscosity as a function of the specific viscosity in presence of ATP, Straub (1) termed the ‘activity’ of unknown ‘myosin’ solutions, and he took the activity of myosin B solutions prepared from muscle in a standard way as 100%.

THE DISCOVERY OF PHOSPHAGEN AND EARLY IDEAS OF ITS FUNCTION

In 1927 P. Eggleton & G. P. Eggleton (1), and independently Fiske & Subbarow (2), reported the existence in muscle extracts of a phosphorus compound, very labile especially in acid solution; the figures for inorganic P content of muscle found by earlier workers, using methods involving acid treatment of the extracts, were therefore open to grave doubt.

Eggleton & Eggleton, using the Briggs method (1), in which the colour due to reduced phosphomolybdate is allowed to develop during 30 min in acid solution, found that the increase in colour during this time with inorganic phosphate solutions was only some 5 %; but with extracts from resting frog's muscle the increase was several 100%. They proposed the name ‘phosphagen’ for the labile substance. The value for the true inorganic P of resting muscle, found by extrapolation back to zero time when the rate of colour development was followed, amounted to about 25 mg/100 g muscle; the phosphagen P content to about 60 mg/100 g. Estimations made in neutral or slightly alkaline solution (as in the Bell-Doisy (1) method or by precipitation with magnesia mixture) gave results approximating to the extrapolated values of the Briggs method. In rapidly induced fatigue the true inorganic phosphate increased at the expense of phosphagen P, though not all the phosphate of the disappearing phosphagen was found as inorganic P. In aerobic recovery, phosphagen quickly reappeared at the expense of inorganic P, during a time when little lactic acid removal had yet taken place.

We have already discussed Lundsgaard's realisation in 1934 that phosphocreatine breakdown must be considered a recovery process, leading to the restitution of some unknown energy-rich substance. The evidence from his experiments on whole muscle seemed to rule out ATP as this substance, since ATP breakdown was observed only after stimulation to exhaustion, just before rigor supervened. Lohmann's experiments a little later on muscle extracts, however, bore the clear implication that ATP breakdown must precede phosphocreatine breakdown. The idea of ATP hydrolysis as the energy-yielding reaction closest to the muscle machine was greatly strengthened by Engelhardt & Lyubimova's discovery of the ATPase activity of myosin and the subsequent work on interactions in vitro of ATP and actomyosin gels. Since no decrease in ATP content of normally contracting, unexhausted muscle was observed, the confident assumption was generally made that resynthesis of the ATP used was too rapid to permit of measurement of its hydrolysis. This attitude was too complacent for although the assumption turned out to be right in the end, its rigorous proof was attended by extreme difficulties.

We shall consider first in this chapter the various ways and means which experimenters tried hoping to get light on the chemical reactions accompanying contraction, and the evidence which came out of such work for ATP dephosphorylation during a short series of twitches or a short tetanus.

Looking back on the years spent in writing this book, I feel sometimes that it was done primarily for my own enjoyment. I wanted to visualise in a single perspective the path of man's knowledge about the function of muscles, progressing so slowly for so many centuries but then during the last seventy years reaching speedily towards the goal in a rush of great discoveries. Professor A. V. Hill, whose fundamental work constantly appears in these pages, tells me that he never took theories of contraction seriously; but perhaps he would not disagree with Dr William Croone who in the seventeenth century reckoned ‘such speculations amongst the best entertainments of our mind’. Be this as it may, I hope that, in spite of its defects and omissions, this book will be useful to some of those for whom the fearful and wonderful phenomena of muscular movement retain all their fascination. We can respond to the words of Sir Thomas Browne (Religio Medici 1. 13):

The subject of muscle diseases is a very wide and intricate one, and here we shall deal only with biochemical research on two forms – progressive muscular dystrophy and glycogen storage disease. This chapter came to be written because the muscle biochemist is so often confronted with the question ‘Does all this research on muscle help in the cure of muscle diseases?’ The short answer at present is ‘No’ as far as these two types are concerned. Both are hereditary, and in the case of glycogen storage disease the defect has been traced in different types to lack of a particular enzyme. In the case of progressive muscular dystrophy it has been considered clear that the primary cause is in the muscle itself and not in any deterioration of its nerve supply, but in spite of much experimentation this cause has not been found. Nevertheless much ground has been cleared, and we can say a little about the various views held as to possible causes. It must, however, always be remembered in what follows that the work of the last few years on the effects of cross-innervation on the enzyme patterns of red and white muscles suggests that the nerve supply exerts a more subtle influence on muscle metabolism than had previously been realised. The possibility thus remains that in muscular dystrophy some specific change occurs in the nerve without visible degeneration.

THE CO-ENZYME FUNCTION OF ATP

In 1931 Meyerhof, Lohmann & Meyer (1) published their first observations on the co-enzyme function of ATP in the glycolytic system of muscle. Muscle kochsaft was prepared from extract which had been allowed to autolyse for 1 h at 37° before being boiled; such a kochsaft was incapable of restoring activity to a muscle extract which had lost its glycolytic power after many hours dialysis. Replacement of only a small part of this autolysed kochsaft with kochsaft prepared from fresh extract led to considerable lactic acid formation, though this small quantity had little effect alone. This suggested that the co-ferment system was made up of an autolysable part and a non-autolysable part. Earlier experiments of K. Meyer (1) had suggested that easily hydrolysable phosphate esters with insoluble barium salts were concerned in the co-enzyme activity, and the autolysable, easily hydrolysed component was now identified as ATP. Lohmann (8) showed a little later than the stable component was Mg. With purified ATP he confirmed Meyerhof's earlier finding with kochsaft that more was needed by the system the less the substrate was phosphorylated. The same was true for Mg.

The important implications of these observations became clearer with the experiments of Meyerhof & Lohmann in 1932 (9). It was already known that phosphocreatine synthesis could take place in muscle extracts in circumstances such that lactic acid formation could not supply more than a fraction of the necessary energy.