Muscular Activity

1 This account of the theories of Hermann and Pfliiger has been taken from the Croonian Lecture by Fletcher and Hopkins. Proc. Roy. Soc., 89B., p. 444, 1917. The change of outlook started from the experiments of Fletcher, on the gaseous exchanges of the isolated muscle, and on the effects of oxygen in delaying loss of irritability. A muscle left in nitrogen gradually stiffens and contracts: left in oxygen it remains flaccid without shortening: forced by stimulation towards stiffening it may be recalled by oxygen to its previous flaccidity. In oxygen its carbon dioxide output is increased. All this seemed to point to the oxidative removal of some product of activity, which is the basis of stiffening and fatigue.

Now lactic acid itself is the most obvious cause of the slowing of fatigue and of the stiffening of rigor mortis. Its artificial application can mimic the signs of both. Fletcher’s results led irresistibly to the conclusion that lactic acid produced by activity is oxidisable, or in some other way removable by oxygen, with an accompanying production of carbon dioxide. The next step was to obtain direct evidence of the changes undergone by lactic acid in the muscle. That step we owe to the classical investigation of Fletcher and Hopkins published in 1907.

Up to the time of Fletcher and Hopkins’s investigation there was hardly any single statement made with regard to the conditions of lactic acid appearance in muscle which observers. The inherent difficulty besetting the chemical examination of muscle lay in the fact that the necessary _ processes of extraction cause, at the moment of their application, profound structural and chemical alteration. The realisation and the overcoming of these difficulties is one outstanding merit of Fletcher and Hopkins’s work. Since 1907 the methods of studying lactic acid in muscle have been considerably quickened and improved by others,

especially by Meyerhof, and the investigations of the latter have helped to complete the structure of which the foundations were laid by Fletcher and Hopkins in 1907. It was : their pioneer work, however, which gave the initial impulse _ to all the work on which our present knowledge of the intimate chemical behaviour of muscles is based. I will cease now to follow the order of discovery and will describe the facts in their logical and not in their historical sequence. We will consider first the processes which occur in the complete absence of oxygen. ;

When a muscle is excited and fatigued, or sent into rigor by heat, chloroform or other similar agencies, or allowed to undergo a prolonged process of anaerobic survival, definite changes are recognisable: (D) Heat is produced proportional to the lactic acid formed. (£Z) The hydrogen ion concentration rises. The same amount of heat is liberated, per gram of lactic acid produced, whether as the result of stimulation, of survival, or of rigor. Clearly the same chemical process occurs in every case. Some 370 calories accompany each — 1 gram of lactic acid, by whatever process the latter is formed, provided that no oxidative reactions are allowed.

These are the only chemical reactions known definitely to occur. According, however, to Embden and his coworkers another process accompanies activity, the breakdown of a somewhat mysterious hexose diphosphate (“lactacidogen”) which they believe to be the immediate precursor of lactic acid. They have succeeded in isolating an osazone from muscle similar to that of hexose diphosphate, and have found that all the processes which liberate lactic acid result in the disappearance of their lactacidogen. Unfortunately the amount of hexose diphosphate present in muscle at any time is not capable of immediate and direct estimation: it is necessary to assume that this substance is unstable and breaks down gradually when the muscle is injured: its amount may then be deduced from the increase in inorganic phosphate between two moments, one immediately after and the other an hour or two after, mincing the muscle. The evidence is somewhat indirect and the supposed réle of hexose diphosphate as the immediate precursor of lactic acid requires wider and more direct verification. There is no doubt of the importance of phosphate in the lactic acid cycle: apart altogether from Embden’s experiments Meyerhof has shown that considerably higher lactic acid yields can be obtained from minced muscles, in the presence of phosphate buffers, than in any other way. In view, however, of the definite proof by Meyerhof (confirmed in Hopkins’s laboratory) that lactic acid is formed at the expense of glycogen, it is impossible to suppose that hexose diphosphate is other than an intermediary, in which case it is not easy to see why the formation of the end product should diminish the amount of the intermediary so long as theultimate precursor is still present. Clearly much more work must be done before we understand the role of phosphate in the carbohydrate changes of muscle.

The increase of hydrogen ion concentration in muscle, even in rigor or extreme fatigue, is comparatively small. At no moment during a short tetanus can an electrode stuck deep into the muscle substance detect any appreciable rise: only after prolonged stimulation does a measurable change occur. It is clear that the lactic acid which is - formed is rapidly and completely neutralised, and that if - it acts in producing contraction by virtue of its hydrogen

structure, and to compare the effect of acid bathing a complete muscle (as some have done) with its action in the living organised mechanism, is about as logical as to compare the effect of petrol poured over a motor car with its action in the engine. The lactic acid, locally produced during activity, may cause a local effect resulting in the mechanical response: that effect, however, is only fleeting, and is rapidly succeeded by the neutralisation of the acid and the relaxation of the muscle. ;

amount of alkaline salts (bicarbonate and phosphate) present in a frog’s muscle is inadequate to account for the large amount of acid known to be neutralised in severe fatigue. Moreover the rise of hydrogen ion concentration and the evolution of COs, if neutralisation by bicarbonate were the case, would be greater than are actually observed. It is necessary to suppose that some more effective buffer, something analogous to the haemoglobin of blood, is present in muscle, and strong evidence exists for the hypothesis of a sodium (or potassium) compound of protein, capable of neutralising acid according to the scheme:—

Nat+P- + Ht+L- — Nat+L- + #£424P (sodium protein) (lactic acid) (sodium lactate) (protein) This protein is a weaker acid than H.CO; or NaH.PO, and it acts as the first line of defence against a rise of hydrogen ion concentration. The substance in question has not yet been isolated from muscle: the evidence for its existence is indirect: it is impossible, however, to explain the facts if we deny its presence. The formation of lactic acid does not proceed until all the glycogen has disappeared: it stops short at a much

earlier stage. If the muscle be suspended in a solution maintained alkaline with phosphate buffers the breakdown can be pushed much further, and it is necessary to suppose that it is limited by the rise of hydrogen ion concentration and by the lack of available phosphates. The process is not yet clear, the presence of an excess both of alkali and of ‘phosphate seems necessary in order to continue the breakdown. It is natural to connect the necessity of phosphate with Embden’s hexose phosphate, but in what precise way remains to be determined: the necessity of alkali suggests other reactions which are “self-inhibited”’ by their resulting rise in hydrogen ion concentration. It is clear, in any case, that the lactic acid maximum and the extent of the breakdown are not determined simply by the amount of the lactic acid precursor, but rather by factors connected with the accumulation of the products of activity.

It seemed possible, although no chemical changes have ever been detected during muscular activity other than those described, that this was due rather to the failure of our means of detection than to their non-occurrence. Considerations of energy, however, negative this possibility. The total energy available in the formation of sodium lactate from dissolved glycogen may be calculated from the heats of combustion of glycogen and lactic acid and the heat of neutralisation of the latter. The heat of combustion of dissolved lactic acid is 3601 calories (Meyerhof). The older determinations of the heat of combustion of glycogen are seriously in error. A recent estimate by Slater places it at 3836 calories, for the dissolved hydrate (C,0;.0.)n. Slater used the glycogen of mytilus, giving an ultimate analysis corresponding accurately to the above formula, and complete freedom from ash. This value is disputed by Meyerhof, whose determinations give a mean

value 46 calories less. Meyerhof’s substance, however, contained 1.3 per cent of ash, irremovable by prolonged dialysis, whose combination (in some unknown form) with the glycogen may have led to some unknown heat change; it seems better to accept Slater’s value. The total energy therefore, in the formation of 1 gram of lactic acid from glycogen is 3836 — 3601 = 235 calories. To this must be added the heat of its neutralisation by alkaline protein salts, which according to Meyerhof amounts to 135 calories. The total, therefore, in the formation of sodium lactate, is 370 calories per gram of hydrated glycogen broken down. This is precisely the amount found in the formation of 1 gram of lactic acid in muscle, so that, if any other chemical reactions do actually occur, their total heat-production must be nil. It is unlikely, therefore, that reactions of any importance arise, in the complete anaerobic cycle, other than those already known.

If the formation of lactic acid from glycogen, and its subsequent neutralisation, be the only processes involved in the complete cycle of anaerobic activity, then we must inquire how these processes can evoke the mechanical response. We have spoken of the complete cycle: it is obvious that in the partial cycle of contraction (i.e., in the development of the mechanical response) other reactions must occur, which are reversed in relaxation: they cannot be examined by ordinary chemical means, since they are reversed so rapidly. Presumably the liberation of acid is highly localised, and produces a large local effect on certain elements or structures (Meyerhof’s “Verkiirzung- _ sorten’’), evoking the mechanical response. The resulting condition, however, is unstable for several reasons, and rapidly passes off:

(A) The acid is free to diffuse away into the spaces of the muscle fibre. (B) There, waiting for it, is an abundant supply of alkali ready to complete its neutralisation. (C) The system possesses a large amount of free potential energy, which acts as a ‘driving force’ tending to cause the reaction to be reversed. Of these (C) requires further explanation. Usually in chemical reactions we do not consider mechanical energy as affecting the conditions of equilibrium: in the laboratory the case is comparatively rare. If, however, a chemical reaction goes on with the liberation of mechanical potential

_ energy, if, for example, the pressure in a system rises as the result of a reaction, then the rise of pressure, the accumulation of potential energy, like that of any of the products of the reaction, must necessarily hinder its continuance, tend to cause its reversal. Given a suitable other path of reaction for the bodies concerned, involving a smaller accumulation of potential energy, that other path will tend to be taken. In the muscle, that other path is the neutralisation of the acid.

Unfortunately we have, at present, only the vaguest ideas as to the nature of the reactions which occur in contraction and are reversed in relaxation. There is a liberation of heat in contraction, amounting to about 60 per cent of the total initial heat, i.e., to 176 calories: in addition to this there is the appearance of mechanical potential energy. There is a liberation of heat in relaxation amounting to about 40 per cent of the total initial heat, i.e., to about 118 calories: in addition the mechanical energy disappears. It is natural to suppose that this mechanical potential energy, if unused to do external work, is degraded into heat in relaxation: the relaxation heat might represent

necessary. It would be much easier to explain some of Fenn’s results if we could believe that the muscle is able to reabsorb such of its mechanical potential energy as potential energy transformed into work cannot be reab- — sorbed while potential energy not so transformed can, we should have an explanation of the most important of Fenn’s results. Unfortunately, such a simple theory will not — explain why shortening during relaxation causes a dimin- — ished, and not an increased, production of heat. Fenn has provided us with some very difficult problems. Finally, for a long period after the contraction is over, there is a delayed anaerobic production of heat, amounting to about 74 calories (fig. 1). That also is difficult to

Fia. 1. Coursz or THE DELAYED ANAEROBIC HEAT-PRODUCTION IN MUSCLE, THE Timp oF Strmutus 1s Not Very SHortT The dotted curves represent two hypothetical simple curves making up the more complex actual one. In many cases the curve is similar to the second dotted one. The second curve provides nearly the whole of the delayed anaerobic heat. (Hartree and Hill, 1923). explain, unless we suppose with Slater that it represents the continued neutralisation of the acid, or rather the slow replacement of an initial local neutralisation with alkaline salt by a final general and more permanent neutral- - isation with sodium protein. The latter yields more heat and probably more free energy than the former, so that such a replacement might well occur. Relaxation may result from a rapid neutralisation of the acid, by any alkalies available in the locality of its appearance:

the anaerobic delayed heat may represent the change over from the temporary and local to the permanent and general condition of neutralisation. Unfortunately the subdivision of the complete anaerobic cycle into its several stages is little but speculation, aided by a very imperfect thermodynamic outline. Chemists must invent far finer and more rapidly reacting tools before they can hope to get inside the mechanism, and to examine anything but the initial and final products of activity. Perhaps light may come from a chemical examination of some much slower form of movement, occurring in some other cell or organism.

In a resting isolated muscle left in oxygen lactic acid does not appear: any originally there tends to disappear. In an isolated muscle stimulated in oxygen lactic acid is set free, but vanishes again during a subsequent resting period. During this “recovery process” so-called, CO; is produced equivalent in amount to the oxygen used, heat is liberated, and glycogen is restored. The amount of glycogen gained is not equivalent to the lactic acid lost: a certain amount of one or the other is oxidised, presumably to provide the energy to drive the endothermic reaction:

According to Meyerhof, about 1 molecule in 4 of the lactic acid removed in recovery is oxidised, the remaining three molecules reappearing as glycogen. In any attempt to follow, by direct chemical estimations in the isolated muscle, the reactions of recovery, the fundamental difficulty occurs that, owing to the relatively must be produced and removed, large quantities of glycogen must be restored. Now severe fatigue in the intact muscle may be rapidly recovered from, as is seen in the case of man after violent exercise: the rapidity is due to the plentiful supply of oxygen through the circulation. The study of the fundamental changes, however, must be made in the isolated muscle, since we cannot say how much lactic acid may be removed, or how much glycogen brought or carried away, by the circulating blood. In the isolated muscle, unfortunately, the oxygen supply has to depend on diffusion and is very poor, so that recovery from severe fatigue is extremely slow, the condition of the muscle is abnormal and the usual relations of recovery may be distorted. For this reason the myothermic method, by reason of its great sensitivity, yields more consistent and reliable results.

In an isolated muscle in oxygen, there is ample oxygen dissolved in the muscle itself to complete the recovery — from a short stimulus. A tetanus of 0.1 second produces, at 20°C., an immediate rise of temperature of about 0.02°C.: the complete cycle, therefore, of breakdown and recovery causes the evolution of about 0.05 calories per gram of muscle, which requires about 0.01 cc. of oxygen. The solubility in water of damp oxygen (at 1 atmosphere pressure) at 20°C. is 0.030, in muscle therefore about 0.025, so that about 2} times as much oxygen is present, ready dissolved and immediately available, as is required in recovery from 0.1 second tetanus at 20°C. Thus recovery after such a short interval of activity can go on at a rate entirely independent of the oxygen supply, presumably therefore at least as well as in the normal muscle with its — circulation intact. In such a muscle the heat-production may be studied, its total amount determined, its timecourse plotted out, with the following results:

(a) The total heat liberated in the complete cycle in oxygen is almost exactly twice the total heat of the partial cycle without oxygen: (b) The delayed oxidative heat production follows a complicated curve, rising rapidly to a maximum and falling slowly to zero again: ; (c) The speed of the recovery process increases rapidly as the temperature rises, decreases as the temperature falls: (d) The speed of the recovery process is affected by the hydrogen ion concentration, being greater in an alkaline, less in an acid medium.

The total heat in the absence of oxygen is 370 calories per gram of lactic acid set free. In the reversal, therefore, of the reactions of anaerobic activity 370 calories of energy must be absorbed. In oxidative recovery, however, about 370 calories of heat appear, so that a total of 740 calories of energy must have been liberated, 370 as heat, 370 as the chemical energy of the glycogen + sodium-protein restored. The net result of the whole process, breakdown followed by complete recovery, is that a certain amount (z grams) of glycogen has disappeared by oxidation, and 740 calories of heat have been evolved. Everything else has returned, so far as we know, to its initial condition, so that x X 3836 (the heat of combustion of glycogen) must equal 740; hence x = 1/5.2; 5.2 molecules of lactic acid have disappeared for every one oxidised. The precision with which this estimate can be made, the good condition of the experimental muscle, and the normality of its recovery conditions, make it clear that this estimate is not far from the truth. As a matter of fact, practically the same value is reached by experiments made on man, as will be described in my next lecture.

The speed of the recovery process, in the presence of adequate oxygen, is clearly determined by chemical reactions: so much is shown by its high temperature coefficient. In the isolated muscle the substance oxidised is carbohydrate or lactic acid of the same empirical formula: so much is shown by the respiratory quotient, In man, practically 1.00 may be observed during continued muscular exercise: the complete cycle, however, of exercise and recovery, in

have a respiratory quotient of unity, and involves therefore only the oxidation of carbohydrate. If the exercise be prolonged, fat is apparently transformed to reinstate the carbohydrate used: the primary fuel, however, of the muscle is carbohydrate. The matter will be discussed more fully in my next lecture, but experiments on man have fully confirmed Meyerhof’s observation on the isolated muscle, that carbohydrate only is oxidised in recovery.

mechanism as entirely distinct from the oxidative process in which energy is liberated to drive the reaction to the left? May we regard the breakdown and restoration as occurring in a separate system, analogous to a lead accumulator, having no connection with the oxidative reaction which provides energy to drive the dynamo—so to speak—by which the restoration is effected? Or should we consider some type of complex ‘“‘coupled’”’ reaction, represented empirically by the formula:

It is impossible at present to say. The decision is one of the major problems of the science of carbohydrate metabolism: we can only indicate the problem now. altogether unexpected. Other oxidations analogous to those occurring in living cells are similarly affected, e.g., the autoxidation of cystein or glutathione. It is striking that the effect of CO, in this respect is considerably greater _ than that of a corresponding change in the hydrogen ion concentration of a fluid bathing the muscle: the explanation of this may well be that CO, produces its effect by its greater capacity for penetrating the walls of the muscle cell, a property established by Jacobs on other cells. The effect of cyanide on the recovery process is to inhibit it completely, as in other oxidative systems.

When a muscle is stimulated with a tetanising current the ratio of the heat-production to the force developed and maintained is practically a linear function of the duration of the stimulus (p. 43). where H is heat-production, T is force, J is length of muscle, t is duration of stimulus. A and B are constants. A is independent of the temperature, having a value always about 1/5.5, B increases rapidly as the temperature is raised. ‘This relation was expected. A rise of temperature quickens the twitch, diminishes its scale of time so to speak: and a tetanus, which is a fusion of twitches, should have the same heat-production in a shorter time, or a greater heat production in unit time. This relation, therefore, is intelligible on general principles.