Principles of General Physiology

It is unnecessary to describe any particular experiment to show that work can be done by a muscle allowed to shorten. Everyday experience in the raising of weights is sufficient to prove this point. That this work is done at the expense of potential energy stored in the muscle, and not by an exothermic chemical reaction involving the burning up of some food-stuft' at the moment, is shown by the fact that an excised frog's muscle in nitrogen is capable of giving a maximal contraction every five minutes for two

hours and a half, before signs of fatigue appear (Fletcher, 1902, p. 491). The contractile power continues longer in oxygen, as we shall see presently We may note here that the onset of fatigue shows that something has been used up, and that it has not been replaced. Supply of oxygen to a muscle, fatigued in nitrogen, even in the case of an excised muscle, to whose interior the access of oxygen is difficult, brings about recovery to a very considerable extent (see Fig. 135, which shows also that spontaneous rigor is hindered by the presence of oxygen).

In order to discover what chemical change occurs in the act of contraction itself, we must exclude the influence of oxygen. We know that contraction can take place in its absence, so that the contractile process itself does not make use of it. An important part of the problem to be solved is, in fact, the part played by oxygen. That muscles stimulated in the absence of blood supply become acid, and that lactic acid is produced by muscle as it dies and enters into rigor mortis, are old observations, but the production of lactic acid in the normal contraction was first elucidated by the work of Fletcher and Hopkins (1907). Surviving excised

muscle forms lactic acid, slowly, as it dies, but the fact that chiefly concerns us here is that this production is greatly accelerated by stimulating the muscle to activity. These investigators have shown that muscle freshly removed, immersed in ice-cold alcohol and disintegrated therein, contains only the trace of lactic acid which might be expected to be formed during the slight unavoidable delay in the experimental procedures. An instructive experiment, showing its production on stimulation, is described on pp. 308 to 309 of the paper referred to. Hopkins' delicate thiophene test for lactic acid is used. ,

This, then, is the only chemical change that can be demonstrated to occur in the act of contraction itself. It is true that carbon dioxide is slowly given off by excised muscle in an atmosphere of nitrogen and, as we saw above (page 272), Hermann represents the products of the breakdown of his inogen substance as " myosin " (that is, the nitrogen-containing residue, after separation of lactic acid), lactic acid itself and carbon dioxide. But Fletcher (1902 and 1913, p. 374) has conclusively shown that the slow evolution of carbon dioxide in an atmosphere of nitrogen is to be accounted for by the action of the lactic acid on bicarbonates already present in the muscle; the carbon dioxide so formed gradually escapes. And, what is more to the point for our purpose is that stimulation does not increase this output. Consumption of oxygen in the contractile process is excluded by the continuous and prolonged activity of muscle in its absence.

As to the form of energy present in muscle itself, the chief experimental evidence is contained in the work of A. V. Hill (1911, 1, 1912, 2, 1913, 1 and 4, 1914, 1 and 2) on the formation of heat in muscular contraction. There is found to be a definite proportion between the tension developed and the heat given off. If a muscle is allowed to contract isometrically at one time and allowed The muscle is inserted in the conical cavity, with its tendon upwards, until it fits exactly and is in contact with the junctions, b, b, b. When contracting, it cannot slip away from the junctions.

a, a, a, External junctions, imbedded in the ebonite. Co., CM, Copper leads to the galvanometer. The muscle is tied to supports at both ends, or to a lever at the lower end. to shorten at another time, the heat formed is greater in the first case. It should be remembered that the heat measured in these cases represents the total energy change in the contractile process, the tension being allowed to disappear in the form of heat, and the weight raised, if such is done, is allowed to fall again. If we allow the muscle to shorten, by releasing it at the time that the maximal tension has developed, but not earlier, the heat is unaffected. The heat produced, or energy developed, is, in fact, directly proportional to the length of the fibres during the time that the contraction takes place and not to their volume. In other words, it is a surface phenomenon. This view was first clearly put forward by Blix (1902, p. 113). The tension developed thus depends upon the area of

certain surfaces running longitudinally in the muscle. After the maximal tension has been developed, its potential energy can be used for doing external work or may be converted into the equivalent quantity of heat. The initial process of contraction, with which we are at the moment concerned, consists in the development of this potential energy of tension, which can do work or be converted into heat. Hill has shown that, under optimal conditions, the total amount of heat developed in the actual contractile process is practically identical with that which would be derived from the energy of the tension, so that the "efficiency," in tinmechanical sense, of this first process is 100 per cent. (1913, 2, p. 463).

This high efficiency is clearly sufficient to exclude the possibility of the muscular machine being a heat engine; the chemical energy of the food taken by an organism is converted into work by a more efficient mechanism. As we shall setpresently, however, the efficiency of the total muscular process, although high, is only about half that of the first, contractile stage. This work on the production of heat obviously requires ^, very perfect experimental technique, details of which will be found in the papers by A. V. Hill referred to, especially that of 1913, No. 4. A sketch of the thermopile used, in its latest form, is given in Fig. 136.

The essential process in muscular contraction is, then, the development of a certain degree of tension. As this, if unused, is converted into the equivalent amount of heat, we can, by determining the total amount of tension developed in a series of contractions, deduce the amount of heat. The application of this fact will be seen presently. The stage in which we have now left the muscle is with a diminished store of potential energy in the complex physico-chemical system and with a certain amount of lactic acid, which has been separated from this system. In order to restore the muscle to its previous state, work must clearly be expended on it, otherwise we should be obtaining work from nothing. What do we know about the way this restoration is brought about 1

We have seen that, in order to detect the production of lactic acid, the muscle must not have oxygen at its disposal, and it was definitely shown experimentally by Fletcher and Hopkins (1907) that the lactic acid disappears under the action of oxygen. It is natural to suppose that it might be oxidised to carbon dioxide and water, since, although muscular activity, in the absence of oxygen, is not associated with evolution of carbon dioxide, this gas is given off when oxygen is present. It is a remarkable fact that experimental evidence shows that this is not the way in which lactic acid disappears. Fletcher and Hopkins have shown that, when muscle is allowed to enter into a state of spontaneous " rigor," which can be accelerated by raising the temperature to 45°, a certain definite amount of lactic acid comes from the decomposition of the " inogen " substance.

The experiment is done as follows : Muscle is stimulated in absence of oxygen, so that lactic acid is formed ; it is then exposed to an atmosphere of oxygen, and the lactic acid disappears ; the stimulation and exposure to oxygen are repeated nine times. Finally, the muscle is put into heat rigor and the lactic acid estimated. If it had been oxidised, it is clear that the final amount to be obtained would be diminished. In point of fact, it is found to be the same as if the muscle had not been stimulated and exposed to oxygen. It must, therefore, have been replaced, by some means, in the system from which it was split off on stimulation (see the paper by Fletcher and Hopkins, 1907, pp. 292-296, and Fig. 137 below).

In case any doubt may arise in the mind of the reader as to the origin of the lactic acid in contraction and in rigor being the same, the work of Peters (1913) may be consulted. Further evidence as to the fact of the non-oxidation of lactic acid is given in a calculation by A. V. Hill (1914, 2) ; but we must first consider the facts known with regard to the consumption of oxygen and the evolution of carbon dioxide connected with the removal of lactic acid.

It may be repeated that there is no evolution of carbon dioxide, and obviously no consumption of oxygen, in the absence of this gas, whereas both processes occur in its presence. Further, the difference between the two cases is that, in the first case, the lactic acid remains, while in the second, it disappears. This fact indicates that the oxidation process is a stage secondary to that of contraction and concerned with the restoration of the system to its initial state, with the replacement of the lactic acid. The oxidation process, then, has only an indirect relationship to the actual contractile process, although it is, of course, an essential one, since it provides energy for the subsequent work to be done by the muscle in contracting. Verzar (1912) showed that the consumption of oxygen by the gastrocnemius muscle of the cat was increased for some minutes after the end of a tetanus of about half a minute; Hill (1911, 1) showed that production of heat in the frog's muscle continued for a considerable time after the end of contraction and also (1913, 1, p. 43) that, dunng this after-period, the heat production associated with the recovery process is about equal to that obtained in the contractile process when the tension is allowed to become degraded to heat. Peters (1913, p. 264) showed that the heat evolved when a muscle is stimulated to fatigue in absence of oxygen is about 0'9 calorie per gram of muscle. Therefore, we may reckon, from Hill's result, that the heat

FIG. 137. REPLACEMENT OF LACTIC ACID IN MUSCLE AFTER CONTRACTION. — At the beginning there are two estimations of maximum lactic acid, produced by heat rigor, in control muscles. At the end there are two similar estimations which have the same value, although the muscles had gone through nine periods of severe stimulation, alternating with rest in oxygen. The continuous part of the line shows the course of acid loss in oxygen as actually determined by estimation. The dotted line shows the presumed course of acid loss and gain during the other periods of rest and stimulation respectively.

evolved in the oxidative process of recovery is another 0'9 calorie, being equal to that produced in the contractile process. We require further to know the amount of lactic acid which disappears in the recovery process, which we can obtain from the work of Fletcher and Hopkins (1907). The maximum yield in heat rigor is 0*003 to 0'004 g. per gram of muscle and, when stimulated to fatigue about half of this (p. 280). We have now the fact that the disappearance of 1 g. of lactic acid, in the recovery process, is associated with the production of 450 calories (see the paper by A. V. Hill, 1914, 2). But 1 g. of lactic acid on oxidation gives 3,700 calories, or eight times as much as that actually obtained in the muscle process, a difference far too large to be experimental error. We must conclude that if lactic acid is oxidised at all, only a small part of it disappears in this way, and the results of Fletcher and Hopkins show that no detectable part is oxidised in any case.

For example, in their experiment of p. 293, about 0'5. g. of lactic acid had been produced and had disappeared again, but, at the end, the total amount in rigor was rather higher than that of the companion set, 0'540 against 0-500. If only one-eighth had been oxidised, its disappearance would have been detected. As Hill puts it, " the lactic acid is part of the machine and not part of the fuel." If we may compare the muscle to a gas engine, the lactic acid corresponds to some essential moving part, say the piston, which merely undergoes change of position. Its change of position, however, leads to the liberation of energy.

The energy required to put back the lactic acid and restore the high potential energy of the resting, unfatigued muscle must, accordingly, come from some independent reaction, involving the oxidation of a non-nitrogenous carbon compound. But, if we accept Ostwald's position (see page 30), a "coupled reaction," in order to give chemical energy to another reaction, must have components in common with it, and it is difficult to see what these can be in the case of muscle, if we look upon the potential energy of the " inogen " as chemical energy. Moreover, this inogen is not analogous to a combustible substance containing an excess of oxygen, such as nitro-cellulose, for example. There is direct evidence, as we shall see later, that oxygen is not taken up in this " intra-molecular " form, and, even if it were, the products of contraction in anaerobic conditions would contain considerable amounts of carbon dioxide, which is not the case. The formation of lactic acid from glucose or similar substance is only associated with the giving off of minimal amounts of energy. Again, as Hill points out (1913, 1, p. 77), if the lactic acid precursor could be restored in the presence of oxygen without the evolution of heat, we should be justified in concluding that the oxygen is built up into the precursor, and that the breakdown of the precursor is, in fact, an oxidation with the liberation of heat. But this is not so, "at least as much heat is used in the restoration of the contractile tissues to their previous condition as in their breakdown, so that the oxygen cannot be merely built up as intra-molecular oxygen, but must be utilised in some way in oxidation processes." In fact, one cannot imagine a chemical compound of high potential energy which would satisfy the conditions required.

It would be rash to deny its existence, nevertheless, since there are such substances as nitrogen iodide. On the whole, we are driven back to the assumption of a system whose energy is more of the nature of surface energy, a view confirmed by the relation of heat and lactic acid to length of fibres. And, if this be so, the difficulty with regard to the coupled reaction vanishes. With the data at our disposal, we can obtain some further idea of the nature of this secondary oxidation process. A. V. Hill (1914, 2) has determined the total energy that can be afforded by isolated muscles stimulated in oxygen. He had previously shown (1913, 2, p. 462) how the heat production in relation to the tension developed can be estimated, and found it to be 5 x 10""8 calories (5 microcalories) per gram-weight of tension developed per centimetre of muscle length. This is in the absence of oxygen, and refers, therefore, to the heat set free from the actual contractile process only. If the heat of the recovery process in oxygen is added, the value becomes 10 micro-calories. If we obtain a record of the tension produced in a muscle in a series of isometric twitches, we can estimate, therefore, the heat produced. Hill has compared the total heat produced when sartorius muscles were stimulated to exhaustion in air, on the one hand (that is, in insufficient oxygen), and in oxygenated Ringer solution, on the other hand. In this latter, they can be stimulated five times a minute for nearly two days, giving, on an average, 30 calories of heat per gram, whereas, as we saw, Peters found only 0'9 calorie in absence of oxygen. In Hill's experiments in air, or nearly complete absence of oxygen, the value l-4 was obtained. Now, to. afford these 30 calories, 0-008 g. of lactic acid would have to be burned. The amount that actually disappears is 0-002 g., at the most. About the same amount of carbohydrate would be required, and muscle is stated to contain about 0'004 to O'Ol g. per gram of its weight. The higher figure is more than sufficient to account for the energy production.

We shall find presently more evidence as to the nature of the substance burned, but it would be interesting to have actual values of the carbon dioxide produced and oxygen consumed under the conditions of Hill's experiments. Comparing again the muscle system to a gas engine, it is as if the energy of the combustion of the fuel were not used at once to drive machinery, such as drills, hammers, and so on, by means of shafting and belts, but as if an air compressor

were driven and a store of air at high pressure obtained. The engine might then be stopped and the compressed air used to drive pneumatic tools. This would be similar to the anaerobic muscular contraction. Or the compressor might be kept continuously at work, as is usual, in order to replace the potential energy of the air consumed by the tools ; this corresponds to the work of a muscle under normal oxygen supply. If it be preferred to regard the potential energy of the muscle as chemical, then one might take the case of an engine driving a dynamo, which is itself charging accumulators ; the current from the accumulators is then used for electric motors. In either case, the energy of the fuel is not used directly, just as that of the oxidation of carbohydrate in the recovery process of muscle is not so used. The latter process is more efficient than the ordinary heat engine, since the transformation of the chemical energy does not pass through the stage of heat, although part of it appears to be lost in this way, even in muscle.

It may be mentioned here that von Frey (1909, p. 497) clearly expresses the view that only a part of the energy set free in active muscle is connected with the contractile process itself, the other part being due to a subsidiary reaction, for which oxygen is necessary. I venture to think that the phrase "oxidative removal" of lactic acid, although correct, may be apt to give the mistaken impression that the lactic acid itself is oxidised. Perhaps "restitution by another reaction" might be preferable, leaving the nature of the reaction to be stated separately.

Lactic acid, then, clearly undergoes no chemical change, and is merely put backwards and forwards in some kind of physico-chemical system. It seems remarkable that lactic acid, which is a common derivative of glucose, should apparently have no connection with carbohydrate in the muscle. If one may say so, it seems just calculated to put the physiologist on the wrong scent. It is no more obvious why lactic acid particularly should have the properties necessary for the act of contraction ; as a stage in the oxidation of glucose, it is at hand. In the present state of knowledge as to the intimate nature of the process, any suggestions must be purely speculative. But it seems probable that hydrogen ions, arising from dissociation of the acid, play an important part in the polarisation of the cell membranes, and also in the separation of inorganic salts from adsorption by colloids in the sarcomeres, as in Macdonald's theory (1908), which is similar to that already referred to in the case of nerve. In muscle, however, these electrolytes which are set free owing to aggregation of colloids, are represented as increasing the osmotic pressure of the contents, and causing shortening by attracting water from one part of the fibre to another.

It is an experimental fact that fatigued muscle has a higher osmotic pressure than resting muscle, since it swells in a solution which is isotonic for the latter, and models have been made which shorten when distended by forcing in water. Roaf (1914) has calculated that the rate of inflow of water may be sufficiently great to offer no difficulty in this theory of contraction. It must also be admitted that, although the energy of a contraction is a function of the area of certain surfaces in the fibre, the fact does not necessarily exclude the possibility of the intervention of volume energy due to the osmotic pressure of the electrolytes split off from these surfaces.

The method described by Roaf (1913), by which electrodes of various types are used to detect changes in the concentration of particular ions on the surface of muscle in contraction, will probably afford valuable information, when complete, as to the time relations of the muscle processes. This investigator has found an increase of hydrogen ions, a probable increase of chlorine ions, and a diminution of oxygen tension in this way. The hydrogen ions, no doubt, come from lactic acid and the chlorine ions from potassium chloride, which might either be set free from adsorption or escape owing to changes of permeability.

There is a further group of theories which attributes the development of tension in a muscle to changes of surface tension at the contact of fibrillae with sarcoplasm. That changes in surface tension are a controlling factor in the development of the energy of muscular contraction is made practically certain by the observation of Bernstein (1908), who found that the maximal tension developed by a particular muscle, for example, was 375 g. at 0° and 205 g. at 18°. This means that the energy in question has a negative temperature coefficient, and of all the possible forms of energy involved in muscular processes, surface energy is the only one that has a negative coefficient. This follows from the fact that the surface tension at the interface between a liquid and its vapour becomes zero

at tincritical point, and a negative coefficient is also found experimentally (Freundlich, 1909, p. 32). In the paper by Bernstein s<>mc detttmmattODB of the temperature coefficients of the surface tensions of colloidal solutions are given, and shown to he negative (see also page 61 above). Mines (1913, 1, pp. 14-16) brings forward good reason for regarding the production of lactic acid as responsible for the changes of surface energy, and shows that, owing to there being an optimal hydrogen ion concentration for the contract ile response, the first effect of the production may be an increase of this factor to the optimal value; hence the phenomenon of the "staircase.'' Although the lactic acid is rapidly removed, its disappearance cannot be instantaneous, and it will probably attain a finite concentration in tetanus; at this concentration it will be produced and removed at an equal rate. This concent rat ion is, no doubt, above the optimal one, and hence the decrease in height of each succeeding twitch in the summation of tetanus. These effects on excitability and tone are supposed by Mines to be due to the diffusion of the lactic acid, first formed at the active surfaces responsible for the production of the tension of the twitch. It will !»• noted, however, that we have, as yet, no explanation of the manner in which the lactic acid is liberated by the stimulus, and why the process appears to be a surface phenomenon.

A further account of the question will be found in Macallurn's article (1911). It seems clear that a sufficient change in tension might be obtained from surface energy, but a decision on the point is not yet possible. In all probability, the change of surface tension is the primary factor, but osmotic pressure may play a part subsequently, although it seems somewhat doubtful whether sufficient tension could be produced by this means alone, which acts rather at a disadvantage. The movement of water, on the other hand, is most readily accounted for by changes of osmotic pressure, but it may be merely incidental.

Haber and Klemensicwicz (1909, p. 390), in their work on the force^ pie-cut at the boundaries of phases, express the view of the intervention of surface tension as follows : " The relation between the chemical process and the mechanical effect of muscle is to be regarded in this way : production of acid alters the electrical forces at the phase boundary ; this electrical change involves one of the surface tension also, and it is this change of surface tension that brings about the mechanical deformation of the muscle."

With regard to several of the points discussed in the preceding pages, the recent work of Weizsiicker (1914) gives important information. By means of a method devised by A. V. Hill and himself (1914), experiments could be made on the heat evolved by muscle immersed in various solutions. The "initial heat production " is exactly the same with or without the presence of oxygen. It is also unaffected when oxidation is prevented by potassium cyanide. With respect to the action of this substance, the facts given in Chapter XX. may be referred to. This part of the muscle process is, then, not an oxidation. It has been mentioned above that the tension developed has a negative temperature coefficient, and the same fact is shown by Weizsiicker to hold for the initial heat production. Alcohol prevents the development of the tensile stress, while one-third or more of the initial heat production remains. There are thus two parts or stages in the contractile mechanism; namely, one part providing free energy, and another which transforms this energy into mechanical potential energy or work. Both of these are abolished, reversibly, by the use of hypotonic Ringer's solution, and at the same time. The three different components of the act of contraction can tlms be acted on. (1) Cyanide acts on the oxidations. (2) Temperature or hypotonic saline on the initial liberation of energy. (3) Temperature or alcohol on the transformation of this energy into the mechanical response. The oxidative recovery process is affected by temperature in the same way as a chemical reaction. The oxygen used increases, while the heat production falls, with a rise of temperature.