J. Physiol. (I953) 12I,

Transcription

1 275 J. Physiol. (I953) 12I, THE ONSET OF RIGOR MORTIS IN VARIOUS MUSCLES OF THE DRAUGHT HORSE BY R. A. LAWRIE From the Low Temperature Stationfor Research in Biochemistry and Biophysics, University of Cambridge, and Department ofscientific and Industrial Research (Received 29 December 1952) It has been shown by Bate-Smith & Bendall (1947) that loss of extensibility during the course of rigor mortis in the psoas muscle of the rabbit is directly related to a rapid fall in the level of adenosinetriphosphate (ATP) from its value in the muscle at rest. For psoas muscles having the same initial ph and thus, by inference, being in a comparable physiological condition, the time elapsing before the onset of the rapid phase of rigor mortis, at any given temperature, depends on the magnitude of the glycogen reserve (Bate-Smith & Bendall, 1949). As long as the glycogen supply lasts, the process of anaerobic glycolysis can normally continue, 1-5 moles of ATP being produced from adenosinediphosphate (ADP) for every mole of lactic acid arising from glycogen (Lipmann, 1941). The maintenance of a high ATP level, and hence of pre-rigor extensibility, depends not only on the resynthesis of ATP by glycolysis: the level of creatine phosphate (CP), which serves as a reservoir for the formation of ATP from ADP, is also important. This is apparent from the fact that, irrespective of the glycogen reserves, the ATP level apparently diminishes rapidly as soon as 80% of the CP initially present has been broken down (Bendall, 1951). In the present study, comparative observations have been made on the onset of rigor mortis in the longissimus dorsi, psoas, diaphragm and heart muscles of the horse. It has been confirmed that the loss of extensibility, characteristic of rigor mortis, is associated with a decrease of ATP in all four muscles. In the psoas of the rabbit, the onset of the rapid loss of extensibility occurs when the ATP level has fallen to about 60-65% of its initial value. On the other hand, the four horse muscles studied do not lose their pre-rigor extensibility appreciably until the ATP level has decreased to 30% of its initial value. The maintenance of ATP levels in these muscles appears to depend on a CP reservoir to a significantly different extent in each and in 18-2

2 276 R. A. LAWRIE various other respects the horse muscles differ both from rabbit psoas and from each other. These differences appear to bear a systematic relationship to the activity of the respective muscles, as will be shown in the discussion. METHODS Conditions of slaughter Bate-Smith & Bendall (1947, 1949) ensured a uniformity of material in their experiments by injecting the muscle relaxant, myanesin, into the rabbit before decapitation. This could not be done with the horses in the present work. Samples had to be transported, moreover, from the knackery to the laboratory, and in these circumstances a wide scatter in the biochemical data, and in the behaviour of the muscles, was anticipated: the only standardization possible was to make certain that exactly 1 hr elapsed between the death of the animal and the start of the experimental work. Despite this difficulty, the initial condition of the skeletal muscles studied was found to be remarkably similar, as the data reported below will demonstrate. The terms 'initial' and 'final', as used in this paper, will refer respectively to the conditions obtaining exactly 1 hr after the death of the animal and after the completion of the rigor changes, as determined by the point at which no further loss of extensibility took place. The demonstration of rigor mortis in heart muscle necessitated certain alterations in procedure which will be considered later. Sampling Samples of the four muscles studied were taken from the following regions of the carcasses: (1) heart, left ventricular wall; (2) diaphragm, junction of pars sternalis with pars costalis on righthand side of carcass; (3) psoas, portion of psoas major at level of 1st sacral vertebra on right-hand side of carcass; (4) longissimus dorsi (1. dorsi), portion posterior to 14th dorsal vertebra on righthand side of carcass. Thin strips, about 0-8 cm wide and about 3 cm long, were dissected from the muscle samples. With psoas and diaphragm, it was relatively easy to secure strips in which the fibres ran parallel to the long axis. This was found to be considerably more difficult with the 1. dorsi (in which the fibres are much convoluted) and with the heart (in which the fibres form a syncytium). Nevertheless, even from these muscles it was possible to dissect portions sufficiently elastic to permit the extensibility changes of rigor to be readily detected. The strips thus obtained were wrapped at each end with adhesive tape and the ends were fixed by clips in the apparatus for the measurement of extensibility described by Bate-Smith & Bendall (1949). Measurement of extensibility Throughout these experiments the apparatus was set up in a room held at 370 C. This was done because the duration of the delay period before the onset of the first phase of rigor in the rabbit is decreased by raising the temperature, the Q1o being 1-6 (Bate-Smith & Bendall, 1949). In the present comparative study, absolute rates of change during the rigor processes were unimportant. A stream of moist nitrogen was passed over the muscle to maintain anaerobiosis during the period of observation, which lasted from exactly1 hr after the death of the animal until the rigor changes were completed. A load of 25 g was automatically applied to the muscle every alternate 8 min. Extensibility at any period after the start of the experiment was expressed as a percentage of the initial value. Chemical estimations Portions of the muscles adjacent to those providing the strips used for measuring extensibility changes were also placed under nitrogen in the 37 C room and were used for the following determinations: ph. Roughly1 g of muscle was macerated in 10 ml. 0-01M-sodium iodoacetate in a Marsh-Snow homogenizer (Marsh & Snow, 1950) and the ph measured by glass electrode.

3 RIGOR MORTIS IN HORSE MUSCLES 277 Buffering capacity. 2 g of muscle were homogenized in 10 ml M-sodium iodoacetate and 0-05 N-sodium hydroxide or hydrochloric acid were added in 0 5 ml. quantities over the ph range 5-8, the ph being measured after each addition (cf. Marsh, 1952). Buffering capacity could then be expressed as 10-5 equivalents of base required to change the ph of 1 g of muscle by one unit. Determination of phosphate About 1 g of muscle was macerated in 20 ml. ice-cold 5% trichloroacetic acid (TCA) using the Marsh-Snow homogenizer. The extracts were immediately neutralized to phenolphthalein with N-sodium hydroxide (to minimize the breakdown of CP: Bailey & Marsh, 1952) and kept at 00a to await analysis. Phosphate esters in the muscle extract were determined as inorganic phosphate by Allen's method (Allen, 1940) in a photoelectric absorptiometer. Inorganic phosphate was split from these esters as follows: Creatine phosphate (CP). In 36 min at 170 C creatine phosphate is hydrolysed to creatine and inorganic phosphate by the amidol perchloric acid reagent (Bendall, 1951). Adenosinetriphosphate (ATP). In 7 min at 1000 C, N-HCI splits off the two easily hydrolysable phosphate groups from ATP as well as hydrolysing CP. Total soluble phosphate (TSP). Digestion in 60% (w/v) perchloric acid for 3 hr converts all the phosphorus in the TCA extract to inorganic phosphate (Allen, 1940). As indicated above, the symbols ATP and CP will be taken to refer to the adenosinetriphosphate and creatine phosphate molecules. It will be understood, however, that where quantitative data for ATP and CP are quoted, the values refer to the phosphorus liberated from ATP and CP by the procedure outlined above. Such values are, of course, strictly proportional to the quantities of the corresponding esters present and give a true measure of the latter. The ATP values were corrected for the presence of hexose diphosphate and of triose phosphates in the extract by the procedure of Bailey & Marsh (1952). The data for ATP and CP were expressed as a percentage of the TSP in aliquots of the extract. To relate them to the wet weight of muscle, a series of independent determinations of TSP on weighed samples had to be carried out. About six ph determinations and six TCA extracts were made from the muscle samples at appropriate intervals during the measurement of the extensibility changes in the corresponding muscle strips. RESULTS AND DISCUSSION Time course of rigor mortis in longissimus dorsi, psoas and diaphragm muscles of the horse In Figs. 1-3 the mean changes (six animals) in ATP, CP, ph and extensibility, for 1. dorsi, psoas and diaphragm respectively, have been recorded as a function of time in nitrogen at 370 C. (Zero time: 1 hr post-mortem.) In practice there is often a slow loss of extensibility before the major changes of rigor mortis occur. This probably represents the onset of rigor in a small number of damaged fibres of the muscle strips and has been ignored in the present work. The time of onset of the fast phase of rigor, when the muscle as a whole loses its extensibility, has been found by extrapolation. (For this purpose the rate of change of extensibility during the fast phase of rigor was assumed to hold, from the moment when the muscle was last fully extensible, until the rigor changes were complete.) In that they demonstrate a general correspondence between the onset of the fast phase of rigor and a decrease in ATP, CP and ph, the present data confirm the general findings of Bate-Smith & Bendall (1947, 1949) and of

4 278 R. A. LAWRIE Time (min in nitrogen at 370C) Fig. 1. The onset of rigor mortis in 1. dorsi muscle of the horse: changes in ATP, CP, ph and extensibility as a function of time in nitrogen at 370 C. Zero time: 1 hr post-mortem. Curves represent mean observations from six animals in each case. Adenosinetriphosphate (ATP) and creatine phosphate (CP) determined in trichloroacetic acid extracts made from the muscles at various time intervals. ATP-P and CP-P expressed as mg P/g wet weight of muscle. Extensibility expressed as % of initial value. O-EO, ATP; A-A, CP; 0-0, ph; 0-0, extensibility. ph E u ~ Fig. 2. Time (min in nitrogen at 37 C) The onset of rigor mortis in psoas muscle of the horse: changes in ATP, CP, ph and extensibility as a function of time in nitrogen at 370 C (details as in Fig. 1).

5 RIGOR MORTIS IN HORSE MUSCLES 279 Bendall (1951) for the rabbit psoas. The other salient features of Figs. 1-3 are summarized in Table 1, together with the standard error of the observations from which the graphs have been derived. The table shows that the initial ph values for all three muscles are high, being not greatly different from the Fig. 3. Time (min in nitrogen at 370C) The onset of rigor mortis in diaphragm muscle of the horse: changes in ATP, CP, ph and extensibility as a function of time in nitrogen at 370 C (details as in Fig. 1). TABLE 1. Comparative data on the course of rigor mortis in various horse muscles Time before Duration ph onset of of rapid A rapid phase At onset phase of of rigor of rapid Muscle rigor (min) (min) Initial phase Final L. dorsi 214±57 72± ±0-12 ±0-24 ±0-13 Psoas 173±37 63± ±004 ±0-23 ±0-10 Diaphragm 148±20 75± ±0-06 ±0-20 ±0 04 ATP (mg P/g) At onset of rapid Initial phase Final ±005 ±007 ± ±0-06 ±0t05 ±0t ±0 03 t004 ±0-02 CP (mg P/g), A At onset of rapid Initial phase Final ± ±0u01 ±0t01 0Q ±003 probable in vivo resting values. It may also be noted that the scatter of the data is small. These facts emphasize the remarkable uniformity of the experimental material and indicate that horse muscles undergo little struggling at death. This, of course, is supported by observation: the horse is an exceedingly passive animal, showing little excitement even at the slaughter house. It can also be seen that the duration of the rapid phase of rigor and the levels of ATP in the three muscles, both at the onset of the rapid phase and finally, do not significantly differ from one another despite the fact that the initial value of ATP in the 1. dorsi is significantly higher than in psoas or

6 280 R. A. LAWRIE diaphragm. The present data suggest that the residual 'ATP' in psoas is greater than that in 1. dorsi or diaphragm. The nature of this residual 'ATP' is uncertain: it seems unlikely to be, in fact, true ATP. Bendall (1951) mentions a similar finding -in rabbit psoas. The phosphorus of hexosediphosphate and of triose phosphates cannot be implicated as correction for these esters has already been made. Its final identification must await further study. The time which elapses at 370 C in nitrogen before the onset of the rapid phase of rigor is apparently characteristic of each type of muscle studied. This will be discussed below. Particularly striking is the difference between the initial levels of CP in the three types of muscle. While quantitatively similar to the initial level of ATP in the 1. dorsi, the CP in the psoas is only about 54 %, and in the diaphragm only about 13%, of the corresponding ATP levels. This finding might be regarded as evidence that post-mortem glycolytic breakdown had progressed to a characteristically different extent in each type of muscle when initially sampled. If, however, the low initial CP level in diaphragm signified an advanced stage, and the high initial CP level 1. dorsi an early stage, in the same breakdown pattern, it would be difficult to understand why the corresponding initial ATP levels were maintained for a similar period in both these muscles. This is more clearly seen in Fig. 4a. There is a significant difference between the final ph values for 1. dorsi, psoas and diaphragm. Assuming that lactic acid production is the only important factor causing post-mortem fall of ph (Bate-Smith & Bendall, 1949), and knowing the buffering capacity of the muscles (6-07 x 10-5, 4 07 x 10-5 and 4-51 x 10-5 equivalents/ph/g for 1. dorsi, psoas and diaphragm respectively), it may be calculated that glycogen reserves at the moment of death are likely to be highest in 1. dorsi, intermediate in diaphragm and least in psoas. This assumes that the final ph values are derived from the conversion to lactic acid of all glycogen remaining in the muscle at the moment of death. That the psoas should be apparently so deficient in glycogen is difficult to reconcile with indications in the literature-and from the palate-that horse muscles have an abnormally high glycogen content. It is possible, of course, that in the psoas, for example, the breakdown of glycogen under post-mortem conditions is subject to some inhibition not obtaining in the 1. dorsi. Glycogen determinations were not undertaken in the present study, however. Apart from the final ph values, the only striking difference in the ph-time curves represented in Figs. 1-3 is that the rate of ph fall in the diaphragm, during the first 120 min, is considerably higher than in the psoas and 1. dorsi. The significance of this difference will be discussed below.

7 RIGOR MORTIS IN HORSE MUSCLES 281 The level of ATP and the onset of the rapid phase of rigor in longissimus dorsi, psoas and diaphragm Although, even on an absolute basis, the onset of the rapid phase of rigor mortis in 1. dorsi, psoas and diaphragm coincides, in each, with a similar level of ATP-P ( mg P/g), it is not immediately obvious from Figs. 1-3 why the ATP should have fallen to this value after such different intervals as 214, 173 and 148 min respectively. In the case of the diaphragm with its small initial CP content, the reason why the initial ATP can be maintained for over 50 min is obscure, especially as the subsequent onset of rigor mortis occurs considerably earlier in this muscle than in psoas or 1. dorsi. To appreciate this phenomenon more fully, and to establish a more valid basis for comparison, the data on ATP and CP have both been expressed as percentages of the initial ATP level for each muscle. They have been recorded in Fig. 4a. The vertical arrows indicate the ATP level and the time at the onset of the rapid phase of rigor mortis. It may be seen that the CP level at which maintenance of the initial ATP is no longer possible is about 35% of the latter in psoas and 1. dorsi, whereas it is only 5% in the diaphragm. There must thus be some mechanism for ATP production available to the diaphragm, other than synthesis by the interaction of CP and ADP, which can initially supply ATP more effectively than in the other two muscles. This mechanism appears to be anaerobic glycolysis. As mentioned above, the production of 1 mole of lactic acid from glycogen may be regarded as synthesizing 1-5 moles ATP from ADP. Knowing the buffering capacity of the muscles (above) it may readily be calculated that a ph fall of one unit represents a synthesis of 2*82, 1*89 and 1-80 mg ATP-P/g in 1. dorsi, psoas and diaphragm respectively. The ph-time curves of Figs. 1-3 can thus be redrawn as rates of synthesis of ATP at various times during the course of post-mortem glycolysis. To allow comparison with the data in Fig. 4a, these rates have been expressed as percentages of the corresponding initial ATP level/min/g, in Fig. 4b. An explanation can thus now be advanced for the maintenance of the initial ATP in the diaphragm, despite the relative absence of CP. From Fig. 4b it may be seen that the rate of ATP synthesis by glycolysis in the diaphragm (4 %/min/g) is considerably greater than the corresponding rates in psoas and 1. dorsi (2.5 and 2-2 %/min/g respectively) during the first 10 min in nitrogen at 370 C. This high rate soon flags, however, falling after a further 10 min below the rates for the other two muscles, and although it subsequently rises to a maximum 170 min after the start of experiments it remains lower than in the psoas and 1. dorsi. This probably accounts for the greater rate of ATP decrease when, eventually, it falls from its resting level in the diaphragmand consequently for the early onset of rigor in this muscle. After 170 min the ATP synthesis rate for psoas falls off more quickly than that in the 1. dorsi,

8 282 R. A. LAWRIE L 60.9 C- St o 6h0, E - tc _ Time (min In nitrogen at 370C) Fig. 4. Biochemical changes associated with rigor mortis in various horse muscles as a function of time in nitrogen at 370 C. Zero time: 1 hr post-mortem. Curves represent mean observations from six animals in each case. Adenosinetriphosphate (ATP) and creatine phosphate (CP) determined in trichloroacetic acid extracts made from the muscles at various time intervals. a, changes in ATP and CP, expressed as % initial ATP. O-0, ATP in 1. dorsi; A-A/ ATP in psoas; 0-0, ATP in diaphragm; O - ---, CP in 1. dorsi; A - ---A, CP in psoas; , CP in diaphragm. Vertical arrows represent times of onset of fast phase of loss of extensibility in diaphragm, psoas and 1. dorsi (reading from left to right). b, changes in the rates of ATP synthesis by glycolysis. 0-O, % initial ATP/min/g in 1. dorsi; AL-A, % initial ATP/min/g in psoas; 0-0, % initial ATP/min/g in diaphragm. Rates calculated from ph curves and buffering capacity as described in the text.

9 RIGOR MORTIS IN HORSE MUSCLES 283 and this factor, together with the higher initial CP in the latter, may explain why the onset of rigor in the 1. dorsi is delayed for so long. A satisfactory explanation for the time courses of rigor mortis characteristic of the three muscles considered above can thus be given in terms of the maintenance of ATP, above the level necessary for normal extensibility, by (a) the initial store of this ester held as CP, and (b) the rate of its synthesis by glycolysis. The onset of rigor mortis in longissimus dorsi, psoas and diaphragm of the horse in relation to that in rabbit psoas A point of marked dissimilarity from the rigor characteristics of the rabbit psoas may be mentioned. At 370 C, for a rabbit psoas the level of ATP at which rapid loss of extensibility occurred is about 60-65% of its initial value (Bendall, 1951). In horse 1. dorsi, psoas and diaphragm, on the other hand, it has been shown to be %. It may also be mentioned that in the rabbit the time before the initial level of ATP begins to fall is about 140 min at 370 C. In view of the high initial CP in the rabbit (160% of the initial ATP; cf. Bendall, 1951), the present findings (Fig. 4 a) that the corresponding delays in 1. dorsi and psoas of the horse are 80 and 40 min, with CP levels 90 and 54% of the initial ATP, respectively, are not unexpected. The correlation between the fall of the rate-ratio (i.e. ATP formed/atp split) and the onset of the fast phase of rigor mortis, which Bendall (1951) found to be even closer than that between the latter and the fall of ATP, could not be verified. In agreement with Bendall's work on the rabbit psoas at 370 C, marked shortening was observed to coincide with the loss of extensibility for all muscles in the present series of experiments. Time course of rigor mortis in horse heart muscle In parallel with studies on various skeletal muscles of the horse, a series of observations were made on the onset of rigor mortis in horse heart. These observations, again at 370 C and in nitrogen, were likewise commenced exactly 1 hr after the death of the animals. The first experiments failed to show any change of extensibility whatsoever during the period of observation. There was little initial ATP, no CP and the ph ( ) changed inappreciably. It thus appeared that rigor mortis either did not occur in horse heart or that the onset was so swift that its detection was impossible at 1 hr post-mortem. When facilities were provided to secure the heart muscles within 5 min of the death of the animal the second alternative proved to be correct. Samples were cooled immediately in ice and transported, as quickly as possible, to the laboratory, so that a strip could be dissected out and tested

10 284 R. A. LA WRIE for extensibility (at 370 C) on the rigor machine within 30 min of death. With these limitations, it was possible to examine heart muscles with an initial ph of A typical experiment is shown in Fig. 5a. Although, for the above reasons, heart muscle is not directly comparable with the skeletal muscles, it is obvious that (a) rigor mortis does occur in heart, but that the onset of the rapid phase takes place very considerably earlier than it does in 1. dorsi, psoas and diaphragm, (b) the onset of the fast phase corresponds to a point where the ATP has fallen to 50% of its initial value, and ph (a) tx * E (b) C -~5 O 5* Time (min in nitrogen at 370C) Fig. 5. Biochemical changes associated with rigor mortis in horse heart as a function of time in nitrogen at 370 C. Zero time: 30 min post-mortem. Muscle precooled in ice. Curves represent adenosinetriphosphate (ATP) and creatine phosphate (CP) determined in trichloroacetic acid extracts made from the muscle at various time intervals. a, changes in ATP and CP; expressed as % initial ATP, and of ph. x - x, ph; 7-V, ATP; -A-, CP. Vertical arrow represents time of onset of fast phase of stiffening. b, changes in the rate of ATP synthesis by glycolysis. O-O, % initial ATP/min/g. Rate calculated from ph curve and buffering capacity, as described in the text. (c) since there is virtually no CP, any delay in the fall of ATP must be due to synthesis of ATP by glycolysis. The data in Fig. 5 b show how high this rate is (the scale is one-quarter of that in Fig. 4b). It is particularly noteworthy that, despite this fact, the initial level of ATP cannot be maintained for more than about 10 min, which suggests that there is a spatial separation of the sites of ATP breakdown by myosin ATP-ase and of ATP synthesis by glycolysis. The same question has been considered by Bendall (1951) who found that, despite

11 RIGOR MORTIS IN HORSE MUSCLES 285 a vigorous glycolysis, the initial level of ATP in the psoas of the rabbit began to fall whenever the CP had decreased to about 50% of the initial ATP. (This has been found to occur in 1. dorsi and psoas muscles of the horse at a level of CP equivalent to 30-35% of the initial ATP.) Bendall suggests that diffusion of ATP from the sites of glycolysis to the myosin ATP-ase may become the limiting factor. This implies that the apparently more effective maintenance of the initial ATP by CP is due to the latter occupying a site fairly close to the active centres of the myosin. If such a hypothesis be accepted a further reason must be sought for the maintenance of the initial ATP in the diaphragm. The content of CP is low and the rate of glycolysis is, after all, only about twice as great as that in the psoas and 1. dorsi (Fig. 4 b). Possibly a twofold increase in the rate of glycolysis at the higher ph levels obtaining at the start of the experiments can supply ATP to the myofibrillar system more effectively than can even greater increases at the low ph values subsequently attained in the dying muscle. In the heart the ph falls from its initial value very swiftly to 5-8 so that low ph may early weigh against the higher rate of ATP production, which the heart's high rate of glycolysis would otherwise signify. Moreover, the nature of heart muscle fibres, forming a syncytium, may create an additional obstacle to the diffusion of ATP to the sites where it is required to maintain extensibility. It must, of course, be admitted that, in accordance with the Embden-Meyerhof scheme (Needham, 1942) the rate of glycolysis is largely determined by the availability of inorganic phosphate, the latter being liberated as ATP is broken down. Thus a high rate of diffusion of inorganic phosphate 'outwards' from the ATP-ase sites must occur, even if the diffusion of ATP 'inwards' becomes increasingly difficult as the ph falls. The considerably smaller molecular size of the phosphate radical would ensure freedom from much of the restriction of intracellular movement to which the ATP molecule would be subject. It may be argued that the initial CP store in the heart is low merely because it is depleted by several anaerobic heart beats after the death of the animal and before it is possible to carry out chemical determinations. This has been shown to be unlikely by an experiment in which an extract for analysis was prepared from a piece of goat heart, 10 sec after its removal from the intact organ. The heart was beating in the animal, with full oxygen supply, before and after removal of the sample. The CP was found to be only 2% of the initial ATP value. A low CP store in heart in vivo may thus be regarded as feasible. The high rate of anaerobic glycolysis, and thus of ATP synthesis, in heart muscle during changes post-mortem (Fig. 5b), must, according to the Embden- Meyerhof scheme, involve a very high rate of ATP-ase activity under these conditions. The ATP-ase rate must indeed be considerably greater than that

12 286 R. A. LA WRIE in horse diaphragm, psoas and 1. dorsi. Yet when ATP is added to homogenates of these four muscles, the rate of splitting ATP is relatively much lower in heart, being 1*8 mg ATP-P/g/hr, compared with 5 4, 4-8 and 9 0 mg ATP-P/g/hr in diaphragm, psoas and 1. dorsi respectively (Lawrie, 1953). This phenomenon might suggest that the degree of organization in the intact heart is particularly effective for splitting ATP, but the question merits further investigation. Characteristics of rigor mortis in horse muscles in relation to muscle function It has previously been shown (Lawrie, 1952, 1953) that the capacity for respiratory metabolism in enzyme preparations from various horse muscles, as measured by the maximum rate of oxygen uptake catalysed by cytochrome oxidase, has the following order of decreasing activity: heart, diaphragm, psoas and 1. dorsi, the values, calculated on a wet weight basis, being 32-4, 13-6, 11-2 and 2-7 ml. oxygen/g/hr, respectively. Moreover, a high rate of oxygen uptake is accompanied by a high capacity for the aerobic synthesis of ATP (Lawrie, 1953). These findings agree with physiological observations. The heart possesses an excellent blood supply and the adequate oxygen thus made available will ensure a highly efficient production of ATP, catalysed by the cytochrome system of enzymes. A constant supply of this ester would be particularly necessary in an organ which is continuously in action. Such a muscle could not be dependent on a potential store of ATP, such as CP, for use during activity and to be replenished during periods of relative inactivity. By its nature, the heart must always function aerobically, synthesizing the energy-yielding and extensibility-maintaining ATP as fast as it is used up. The character of the time course of rigor mortis in the heart tends to confirm this view. There is an almost negligible CP store and thus, under the anaerobic conditions employed in the present study, the ATP level falls quickly. The onset of the fast phase of rigor is early in consequence. On the other hand, the 1. dorsi has a relatively large store of CP and, judging by its low final ph (Table 1), of glycogen. It thus has a capacity for prolonged ATP synthesis from anaerobic glycolysis. The onset of rigor, as a result, is delayed for a very considerable time even at 370 C in nitrogen, as has been shown. In the horse the 1. dorsi in vivo is capable of short intense bursts of anaerobic activity, but these are followed by periods of comparative inactivity, during which aerobic synthesis of ATP, catalysed by the cytochrome system, can restore the resting equilibrium. The activity of the cytochrome system, necessary for such restitution, would not require to be as high as in a muscle such as the heart. It has been shown that the capacity for oxygen uptake of the 1. dorsi is indeed relatively very low (Lawrie, 1953). As measured by the time before the onset of rigor mortis, the capacity for

13 RIGOR MORTIS IN HORSE MUSCLES 287 anaerobic ATP formation in psoas and diaphragm is intermediate between that of 1. dorsi and heart. At the physiological level their action may be considered of intermediate severity, although of a different nature in each' muscle-and their capacity for oxygen uptake is also intermediate. The time course of rigor mortis characteristic of the four muscles may thus be seen to bear a systematic relationship to the corresponding muscle function. SUMMARY 1. Changes in ph, extensibility, creatine phosphate (CP) and adenosinetriphosphate (ATP) were followed during the course of rigor mortis in various skeletal muscles of the draught horse in nitrogen at 370 C. Observations commenced at exactly 1 hr post-mortem (zero time) and continued until the loss of extensibility ceased. 2. Under these conditions the time elapsing before the onset of the rapid phase of rigor mortis was , , and min in 1. dorsi, psoas and diaphragm respectively. 3. The initial values of ATP-P were , and mg P/g in 1. dorsi, psoas and diaphragm respectively: corresponding values of CP-P were , and mg P/g; and for ph, , and In psoas and 1. dorsi the initial ATP level began to fall when the CP had decreased to a value corresponding to 30% of the initial ATP. The latter was maintained in the diaphragm for a considerable time despite low initial CP. This was found to be due to the high initial rate of ATP synthesis from glycolysis (4% of the initial ATP/min/g) in the diaphragm: the corresponding values for psoas and 1. dorsi were 2-5 and 2-2 %. 5. In these three muscles the maintenance of pre-rigor extensibility was shown to depend on conservation of the ATP above 30-35% of its initial value by (a) the magnitude of the initial CP store, and (b) the rate of ATP synthesis by glycolysis. 6. By taking special precautions, it was possible to demonstrate the onset of rigor mortis in horse heart. Even in the most favourable conditions the rapid phase occurred after only about 50 min at 370 C in nitrogen. The CP store appeared to be practically nil, although the initial ATP level was similar to that in the three skeletal muscles. 7. The initial ATP level in heart muscle was maintained for about 10 min, apparently by virtue of a very high rate of ATP synthesis by glycolysis amounting to 16% of the initial value/min/g. 8. A high rate of synthesis of ATP by glycolysis could not apparently in itself long maintain ATP above the level at which the rapid phase of rigor occurred. This tended to support the contention of Bendall (1951) that the sites of glycolysis and of myosin ATP-ase were spatially distinct.

14 288 R. A. LAWRIE 9. The time-course of the biochemical and biophysical changes in the four horse muscles appeared to be characteristic of each, bearing a systematic relationship to muscle activity and an inverse correlation with the capacity for aerobic metabolism. I am grateful to Mr J. R. Bendall for helpful discussions and criticism, and to Dr J. K. Walley, who performed the partial left ventricular myocardectomy on the goat. Mr W. A. Deer gave technical assistance throughout this investigation. The work described in this communication was carried out as part of the programme of the Food Investigation Organization of the Department of Scientific and Industrial Research. REFERENCES ALLEN, R. J. L. (1940). The estimation of phosphorus. Biochem. J. 34, BAILEY, K. & MARSH, B. B. (1952). The effects of sulphydryl reagents on glycolysis in muscle homogenates. Biochim. biophy8. acta, 9, BATE-SMITH, E. C. & BENDALL, J. R. (1947). 106, Rigor mortis and adenosinetriphosphate. J. Physiol. BATE-SMITH, E. C. & BENDALL, J. R. (1949). J. Physiol. 110, Factors determining the time-course of rigor mortis. BENDALL, J. R. (1951). The shortening of rabbit muscles during rigor mortis: its relation to the breakdown of adenosinetriphosphate and creatine phosphate and to muscular contraction. J. Physiol. 114, LAWRIE, R. A. (1952). Biochemical differences between red and white muscle. Nature, Lond., 170, 122. LAWRIE, R. A. (1953). The relation of energy-rich phosphate in muscle to myoglobin and cytochrome-oxidase activity. Biochem. J. (in the Press). LIPMANN, F. (1941). The metabolic generation and utilization of phosphate bond-energy. Advanc. Enzymol. 1, MARSH, B. B. (1952). Observations on rigor-mortis in whale muscle. Biochim. biophy8. acta, 9, MARSH, B. B. & SNOW, A. (1950). A simple tissue homogenizer. J. Soc. Food Agric. 1, 190. NEEDHAM, D. M. (1942). 36, The adenosinetriphosphatase activity ofmyosin preparations. Biochem. J.