From the Karolinska Institutet, Department of Experimental Histology, Stockholm Director: Prof. Hj. Holmgren MICROCINEMATOGRAPHICAL OBSERVATION OF THE SHORTENING OF ACTOMYOSIN THREADS AND MUSCLE FIBRES ON THE EFFECT OF ADENOSINTRIPHOSPHATE by A. G. MATOLTSY and V. HANZON In the foregoing paper H j. Holmgren and V. Hanson described a microcinematographic apparatus constructed and used for vital microscopical investigations. The apparatus is useful for photogra phing phenomena of contraction of muscle fibres as well as the pheno mena of shortening of actomyosin threads too. The shortening of actomyosin threads on the effect of adenosintriphosphate, was investigated in the last years, in the laboratory of Prof. Szenl-Gyorgyi (1). We repeated these experiments, and photogra phed the phenomena, and made a complete microfilm1). In this paper we detail some of our observations and give a des cription of the phenomena which we have photographed. l) The first part of the film shows the method of Szent-Gydrgyi and Gerendds. used in the preparation of actomyosin threads. The second part shows the iso- and anisodimensional shortening of actomyosin threads as ATP was added. (The pic tures were taken in normal light, polarised light or in dark field illumination.) In the third part are shown fresh muscle fibres and fibres which have been washed in water for 21 hours and their contraction caused by ATP. Downloaded by: Vanderbilt University Library 188.8.131.52 - 10/28/2017 4:23:43 AM Introduction. The Szent-Gydrgyi school has shown that the contractile sub stance of muscle is built up not only of myosin, hut it contains another protein too called actin detected hv Straub (2). W hen these two pro teins were mixed, the form ation of a new substance was observed cal led actomyosin. Actomyosin was found to he a fibrous colloid, highly viscous and containing long micellar particles. If a concentrated solution of actomyosin was blown through a pipette in distilled water or in weak KC1 solution, threads could he M atoltsy an<l H anzon. Mirrorinematographical observation... 123 prepared. On the addition of adenosintriphosphate (ATP) the threads shortened. The shortening of the micels on the effect of ATP was re garded to take place in a similar way as is the case in muscle if it con tracts (3). (For details see literature.) On the basis of these experiments we photographed the short ening of aetomyosin threads and compared it with that of muscle fibres. Vie made the following preparations and investigations: The microcinematrograph apparatus and the photographic technique. The apparatus is described in the foregoing paper (5). Pictures were taken partly in norm al light partly in u. v.-rich light. (Philora S. P. 500 W.). To obtain well defined pictures the micro scope was used without a condensor or to it was applied a cardioid condensor. The film was Kodak super X X . Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/28/2017 4:23:43 AM The preparation o f the aetomyosin threads. Aetomyosin w as extracted from a young rat muscle in an essen tially similar m ethod as that described by Szent-Gydrgyi (4). The animals were killed, the abdominal and thigh muscle were cut out rapidly and immersed in a small quantity of ice cold Edsallsolution. and minced in a homogeniser. After mincing it was filled up w ith Edsall-solution. to contain 3 volumes of the solution for every g. of muscle. It was mixed for a few minutes and left for 24 hours at 0° C. After 24 hours it was diluted (the aetomyosin extracted is a highly viscous fluid) and the undissolved muscle particles were separated by centrifugation. The extract was further diluted to an end concentral ion of 1.8%. The prepared aetomyosin was sucked into a glass pipette. The pipette was dipped into a basin filled with a solution containing 0.1 M KC1 and 0.001 M MgCl2. Bv blowing into the pipette and at the same time moving it to right and left. (Fig. 1) uniform threads were prepared. The threads were cut with a celluloid spatula into pieces 2 mm long. For the microcinematographical observation the threads were [)ut into a small basin (diam. 3 cm.) filled with a solution containing 0.1 M KC1 an 0.001 M MgCl2. The K-salt solution of ATP was added from a small pipette under a strong pressure. In other cases the thre ads were suspended in a small drop of the KCl-solution on a glass objectholder. and ATP was added from a micro-pipette. The concen tration of the ATP solution used was 0.1%. M atoltsy and Han /.on, Microcinematographical observation Fig. I. Actomyosin thread blown into a basin filled with a solution containing 0.1 M KC1 and 0.001 M MgCl2. Fig. 2. The shortening of an actomyosin thread on the addition of ATP-solution. a. before ATP was added, b. after ATP was added. Fig. 3. ATP-solution diffused in the thread from the left, it curls up on this side. Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/28/2017 4:23:43 AM 124 of the shortening of actomyosin threads and muscle (ihres... 125 Fig. 4. „Crocodile’s skin figure“ ATP diffuses rapidly into the outside of the thread, and it breaks up there, (b. c.) Fig. «. On the outside of the thread appears the „crocodile’s skin figure“. />, c. Later the contraction will be so violent that the thread breaks up. Fig. 6. The shortened thread squeezes out a substance (myo globin). b. Fig. 7. The shortening of an orientated actomyosin thread, b. It will be shorter and thicker. Fig. !l. The contraction of a muscle strip (washed in distilled water for 24 hours). «. relaxed state, b. contracted state after ATP was added. Fig. 9. Fresh llydrophilus muscle fibre. «. The lower end of the fibre was cut. b. ATP diffuses to this end and the myofibrillarstructure will be disor ganised. Fig. 10. A piece of a fresh llydrophilus muscle fibre, a. If ATP is added it contracts rapidly and violently, b. The sarcolemma breaks up. and at this point the sarcoplasm is squeezed out. The shortening o f the aclomvosin threads. We observed the following morphological changes of the threads as ATP was dropped in the suspending solution. We found, as soon as ATP diffused into the threads, th a t shor tening took place. No latent period was detectable. The shortening was isodimensional. th at is. the threads were shorter and smaller, as described by Szent-Gyorgyi. (Fig. 2). If ATP diffuses sidewards into the thread an irregular shortening takes place, the thread curls up towards that side. The curling up of the thread indicates, th at the actomyosin micels shrink only in that part of the thread where they come in contact with the ATP molecules. As ATP diffuses more deeply a continous shortening of the whole thre ad takes place. (Fig. 3). If A TP diffuses rapidlv into the thread it happens that the outer part shortens earlier than the inner. In such a case, the outer part breaks up and fissures. (Fig. 4). Szent GvSrgyi called this morphologi cal appearance of the thread on account of its sim ilarity to a crocodile skin: "crocodile skin figure” . If the action is too violent we observed, th at the whole thread could break up too. (Fig. 5). We noticed also, th at during shortening of our threads a sub stance was diffusing outward from the thread. (Fig. (>). The quantity of this substance sometimes varied from more to lees. We believe this substance to be myoglobin because it was not removed from our solu Downloaded by: Vanderbilt University Library 18.104.22.168 - 10/28/2017 4:23:43 AM Before flu* addition of ATP to the threads 10 exposures were taken. As ATP was added and the shortening appeared, we generally took 1 exposure every 5 seconds. 126 M atoltsy and H anzon, Microcinematographical observation tion used for the preparation of the threads. Myoglobin is squeezed out of the threads as the actomyosin micels become shrunken. The size o f the shortened threads. We observed microcinematographically the shortening of 12 threads. These threads were about 2 mm long, and 0.4 mm in width. Copies were made from the film and the degree of shortening measured directli on them . The values are given in Table 1. Table 1. No. . Shortening in Length % \\ idth % 36.5 34.5 2. 40.5 37.0 42.5 3. 37.5 4. 31.2 42.5 42.5 26.7 5. 42.4 6. 48.5 43.0 7. 52.0 41.1 8. 52.5 38.1 52.5 9. 10. 54.5 56.8 11. 57.0 22.5 12. 45.5 58.3 47.4 % 38.9 % Average ± 6.7 ± 7-4 The results show th a t the threads will he shorter by 47 % and thinner by 39%. T hat is. the threads shorten in both dimensions. whereas muscles shortened in length and will he thicker. Our numerious values measured. differ from those of SzentGydrgyi and Gerendus. They used rahbit muscle, whereas we used rat muscle. The values obtained by Szenl-Gybrgyi and Gerendas on rabbit and our results on rat muscle are shown in Table 2. 1 Animal R abbit R at Shortening; in Length % 62 47 \\ idth % 58 39 Downloaded by: Vanderbilt University Library 22.214.171.124 - 10/28/2017 4:23:43 AM Table 2 if the shortening of aetoniyosin threads and muscle fibres. 127 The values show th at aetoniyosin of rat shortens on a lower value. The differences may indicate differences in nature of the aetoniyosin extracted from various animals. The shortening o f orientated aetoniyosin threads. The actoiny osin micels in muscle are orientated coaxially. those in the threads are inorientated. The question is how are the micels of the aetoniyosin thread orientated and how is it shortened. If we inves tigate a native aetoniyosin thread in polarised light, we find that it has only a very weak double refraction. It indicates that only a minute part of the micels are orientated coaxially . B etter orientation could be obtained by stretching the thread. But native threads are very soft, they cannot be stretched, because they break up. Gerendds ((>) immersed the threads in glycerin or in ZnS()4-solution and found Downloaded by: Vanderbilt University Library 126.96.36.199 - 10/28/2017 4:23:43 AM 77ie comparison o f the contraction o f Hashed muscle fibres and the shortening o f aetoniyosin threads. Szent-Gyorgyi found that aetoniyosin threads shortened in length at a similar rate as muscle fibres. In our observations we found the same phenomenon. To compare our values obtained on the aetoniyosin threads, we measured the contraction size of muscle fibres. For this reason we cut out 2 mm long strips parallel to the fibre axis from the abdominal muscle of the same rat used in the preparation of the aetoniyosin solution. The strips were put in distilled water for 24 hours at 4°C. The water was changed three times. Ii\ this treatem ant ATI’ present in the muscle was washed out and the muscle strips became comple tely relaxed. The strips were put in the same KCl-Mg(’.l., solution used for the threads. ATP was added and their contraction was registered by the microcinematographic apparatus. Measurement on these muscle strips showed that they were shor ter by 48% and thicker by 18%. Comparing the values, we can con clude th a t the threads shorten in their length at a similar rate as muscles. The shortening of the threads was 47.8% , that of muscle was 48.8'’,,. But there are differences in the width, the threads were thinner by 40% , and the muscle was thicker by 18%. These differen ces may be caused because the micels are not orientated with the threads, whereas those of muscle are orientated coaxially. 128 M atoltsy and Ilanzon. Microcinematographical observation The change o f the morphological structure o f washed and of fresh muscle fibres on the effect of A T P . The physiological effect of ATP on fresh muscle fibres w as de scribed by Buchthal. Deulscli and Knappeis (7) (1944). It was found th at ATP causes twitches or tetanus-like contractions and Rozsu found th a t only 0.5 y of ATP per ml is sufficient to scaue contraction of fresh fibres. We investigated the microscopical structure of the fibres when ATP was added. In the case of washed fibres no anomalous change of the structure could be detected. In the case of fresh fibres a violent and rapid contraction was observed. If sarcolemma of the fibres was cut or broken, the ATP diffused directly to the myofibrils. In that case an extrem ely violent contraction took place, so strong that the myofibrils broke up and curled up. This phenomenon was observed in isolated fibres of Hvdrophilus and it is shown in Fig. 9. The low'er end of the fibre was cut by Downloaded by: Vanderbilt University Library 188.8.131.52 - 10/28/2017 4:23:43 AM that the threads could he stretched without breaking and the micels were orientated. The shortening of a thread treated in such a way was anisodimensional like that of muscle. We found th at we could obtain an orientation of native threads too in the following way: We put the native thread on a glass objectholder and covered it with just enough KC1 solution to prevent it from drying. In this way the lower surface of the thread was strongly adhesive to the surface of the objectglass. If we added ATP from a micro-pipette the thread began to shorten, but its lower surface adherent to the glass held it in extension. In polarised ligth. while shortening, we observed the rise of double refraction, indicating the better orientation of the micels. These threads shortened in dimensions similar to muscle. We registered this kind of shortening also m icrocincm atographicalh. A photographed thread is shown in Fig. 7. It was shorter by 47% and thicker by 13.5%. A muscle strip investigated under the same con ditions was shorter by 50% and thicker by 12.4 % (Fig. 8). These val ues correspond to each other and indicate that actomyosin threads, when orientated, can shorten in dimensions similar to muscle fibres. But the anisodimensional shortening of the threads was not always found to be so favourable. For example another thread photographed, shortened only by 15.4% and became thicker by 15.8%. of the shortening of actoniyosin threads and muscle libres... 129 LITTERATURE. 1. Szent-Györgyi. A. a. co-uorkers : Studies from the Institute ofMed. Chemi stry Univ. Szeged. Vol. I. II. III. 1941-1943. - Studies on Muscle. P’rom the Institute of Medical Chemistry of the Univ. Szeged. 1944. 2.Szent-Györgyi. A. : .1. of colloid Sei. I. 1-19. 1946. - 3. Straub. F.D.: Studies from the Institute of Med. Chemistry. 1 niv. Szeged. Vol. II. 1. 1942. —4. Szenl-Györgyi. A.: Chemistry of Muscular Con traction. Acad. Press. New York 1947. p. 145. - 5. Holmgren. Hj. and V. Hanzen Acta Anat. 8. 113. 1949. - 6. Gerendâs. M.: Studies from the Institute of Med. Chemistry. Univ. Szeged. I, 47. 1941-1942. - 7. Iiuclilal. A. Deutsch and G. G. Knappeis: Acta Phys. Scand. 11. 524, 1946. Received Dezember 1948. A rta Anatómica, Vol. V III, Fase. 1/2 (1949) 9 Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/28/2017 4:23:43 AM a sharp knife. At this point ATP diffused directly to the myofibrils, and its structure was disrupted. We observe in some cases that if the contraction is too violent and rapid, the sarcolemma is not elastic enough to allow the increase in width : it breaks up and fissures, and the sarcoplasm is squeezed out (Fig. 10). The observation made on fresh muscle fibres indicate the great sensitivity of the myofibrils if they came in contact with ATP-solution. The contraction of the actoniyosin micels of the myofibrils is so strong th a t the microscopical structure of the myofibrils is disorganised. Summary. A microfilm was made on the phenomenon of shortening of actomvosin threads and muscle fibres on the effect of adenosintriphosphate. The method of Szenl-Györgyi and Gerendâs was photographed. We discrihed the technical methods we used, and the preparation and phenomenon of shortening. Résumé. L 'auteur a pris un microfilm pour dém ontrer les processus de réduction des filaments d'actomyosine et des fibres musculaires sous l'influence de triphosphate d adénosine. La méthode de Szenl-Györgyi et Gerendâs est présentée à l'aide de la photographie: l'auteur en donne les détails et les méthodes de préparation. '/. u sa mmenfassung. Es w urde ein Mikrofilm hergestellt, der die Verkürzungsvorgänge von Aktomyosinfäden und Muskelfasern nach Zusatz von Adeno sintriphosphat zeigt. Die Methode von Szenl-Györgyi und Gerendâs wurde photogra phisch dargestellt. Die gesamte Methodik, die Herstellung des Prä parates sowie das Verkürzungsphänomen werden beschrieben.