Fine Structure of Myocardial Mitochondria in Rats after Exercise for One-Half to Two Hours

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1 Fine Structure of Myocardial Mitochondria in Rats after Exercise for One-Half to Two Hours By Ruben P. Laguens, M.D., and Cesar L. A. Gomex-Dumm, M.D. ABSTRACT Acute exercise (swimming in water at 24 C) provoked marked changes in the fine structure of the mitochondria of heart muscle in rats. At 60 min after the start of exercise, a large number of imaginations appeared in the mitochondrial wall, suggesting the existence of replication phenomena. In the interior of the invaginations, electron-dense granules considered to be ribosomes were seen. After 90 and 120 min of swimming, mitochondria of unusual size, measuring several sarcomeres in length, were seen. The enlarged mitochondria showed no structural alterations. The planimetric measurement of the micrographs of myocardium of the control animals and of those that exercised showed a statistically significant increase in the whole mitochondrial mass in the latter. On trie basis of these observations it is suggested that acute exercise can produce an increase in the mitochondrial mass of the heart muscle cell in a short time. ADDITIONAL KEY WORDS mitochondrial enlargement acute exercise mitochondrial replication heart mitochondrial mass mitochondrial protein synthesis In a previous report (1) it was shown that acute and exhaustive exercise provokes a marked increase in the mitochondrial mass of the dog's myocardium. In that paper no attempt was made to express the quantitative increase in the mitochondrial mass and no studies were made on the morphologic sequence of events that led to the mitochondrial hypertrophy. The same experiment repeated in rats showed changes similar to those found in the dog. In the present experiments, samples of the myocardium of rats were removed for study with the aid of the electron microscope at 30, 60, 90, and 120 min after the start of exercise, and the mitochondrial modifications were evaluated by quantitative methods. Material and Methods Male adult rats of the Wistar strain, weighing 180 to 200 g exercised continuously by swimming in a tank filled with warm water (24 C). For each experiment 5 rats were used: 1 served as From the Department of Electron Microscopy, Comisi6n de Investigaci6n Cientifica de la Provincia de Buenos Aires, La Plata, Argentina. Accepted for publication July 10, control and the remaining 4 were killed 30, 60, 90 and 120 min after they started swimming. Ten experiments were performed. The animals were killed with a blow on the head, the hearts were exposed, and while still beating, small pieces of atrium and ventricle were removed and fixed in cold 1% osmium tetroxide in barbital acetate buffer or in phosphate buffer (2) and embedded in epoxy resin (3). Thin sections were cut with an ultramicrotome (LKB Ultratome). They were stained with potassium permanganate (4) or lead citrate (5) and examined with a Philips 200 electron microscope. Different magnifications were used in different parts of this study, for a quantitative analysis of the data; one series of micrographs was enlarged at 7,450 diam for each animal. In each of these micrographs a planimeter was used to determine the individual mitochondrial areas and the percentage of cytoplasm occupied by the mitochondria. The mean, the standard deviation, and the standard error of the mean were computed for tissues of each rat as well as for the control and the experimental groups. The i-test was used to determine the statistical difference between the means, and a P value < was chosen as the level of significance. Results In the control group, the fine structure of the myocardium was similar to that previously reported (6, 7) (Fig. 1). In the rats that were Circ*U)io* Ruwcb, Vol. XXI, Stptrmbtr

2 272 LAGUENS, G6MEZ-DUMM FIGURE 1 Electron micrograph of a section from the myocardium of a control animal. The mitochondria ate evenly distributed among the myofibrils and most of them measure 1 sarcomere in length. killed after 30 min of exercise, no modifications were found at the ultrastructural level. In the rats that exercised for 60 min, the electron micrographs showed marked modifications of the mitochondrial shape. Both in the control rats and in those that exercised for 30 min, most of the mitochondria were roundish or oval. The constancy in shape was independent of the orientation of the sections or of the localization of the mitochondria within the muscle cell. The mitochondrial wall was smooth. In the heart of the animals killed 60 min after the start of exercise, mitochondria of unusual shape represented a significant percentage of all mitochondria. The commonest modification consisted of invaginations in the mitochondrial wall, which involved both the outer and the inner membrane (Figs. 2 and 3). The invaginations were of various depths and often multiple (Fig. 3); as many as 4 could be observed in a single mitochondrion. At times the invaginations were double and symmetrically located opposite to each other, separating pieces of mitochondria that resembled lateral buddings (Fig. 4). The sarcoplasm located within the invaginations was of a very low electron density and generally presented a varying number of electron-dense granules 150 A in mean diameter, considered to be ribosomes emulation Rtstercb, Vol. XXI, Stpumitr 1967

3 HEART ULTRASTRUCTURE AFTER EXERCISE 273 FIGURE 2 Appearance of the mitochondria after 60 min of exercise. The arrows point to mitochondria with transversauy and longitudinally sectioned wall invaginations. In the interior of the invaginations electron-dense granules may be seen. (Fig. 5). The modified mitochondria showed no preferential localization, and were as common among the myofibrils as in the perinuclear or subsarcolemmal areas. In the rats that exercised for 90 min, the mitochondria with invaginations were less common. The most striking findings consisted of the appearance of mitochondria of unusual size. In the sections oriented parallel to the myofibrils, 97.5$ of the mitochondria in the control animals measured 1 sarcomere or less in length, 2.1% measured between 1 and 2 sarcomeres and only 0.4% exceeded that size. In the animals that exercised 90 min, mitochondria 2 or more sarcomeres long were often seen (Fig. 6 and Table 1). The increase in.-, mitochondrial size was apparent not only in the longitudinal sections; mitochondria with a diameter double that of normal ones could also be seen in the transverse sections. The TABLE 1 Length of Mitochondria Relatvoe to Length of Sarcomere Exercise cise None 30 min 60 min 90 min 120 min < 1 to 1 urcomere long Percent of mitochondria > 1 to 2 sarcomeres long >2 urcomeres long CtnmUtion Kumrcb, Vol. XXI, Sepumber 1967

4 274 LAGUENS, G6MEZ-DUMM I» FIGURE 3 Electron micrograph after 60 min of exercise. The mitochondria are irregular in shape and shew invaginations. The arrow points to a bizarre mitochondrion with four invaginations. appearance of enlarged mitochondria was not diffuse; they were often seen alongside others of normal size. In the rats that exercised 120 min, the increase in mitochondrial size was more marked than in those that exercised 90 min, and more mitochondria were enlarged (Table 1). The increase in size was not associated with structural abnormalities (Fig. 7). The appearance of the cristae, matrix, and outer membranes was normal according to currently accepted morphologic criteria. In this group almost no invaginations of the mitochondrial wall could be observed. The mitochondrial modifications were similar in the walls of all four cardiac chambers. However, they were more marked in the ventricles. In an attempt at a statistical approach, the areas of individual mitochondria and the percent of cytoplasm occupied by them were determined, with the aid of a planimeter, in the electron micrographs of samples taken at random from the right and left ventricles of all Circulation Restarcb, Vol. XXI, Sepumitr 1967

5 HEART ULTRASTRUCTURE AFTER EXERCISE 275 y FIGURE 4 After 60 min of exercise. The arrow indicates an imagination that has produced a budlike projection. the groups. In all, the area of 117 heart muscle cells and of 14,520 mitochondria were measured. The results of this study are shown in Tables 2 and 3. In the rats killed after 30 and Percent of ExerciBe time None 30 min 60 min 90 min 120 min TABLE 2 Cytoplasm Occupied Mean by Mitochondria ±SD ±SD * 1.97* 2.67* 1.43* 'P < The values were obtained by comparing the experimental with the control group. = right ventricle; = left ventricle. Circulation Rtsttcb, Vol. XXI, Sfpttmttr mib of exercise the mitochondrial area was not significantly increased over that of the controls. In those killed after 90 and 120 min of exercise, it was significantly increased Individual Exercise time None 30 min 60 min 90 min 120 min TABLE 3 Mitochondrial Area ( i Mean ±SD ±SE * 0.035* 0.018* 0.040* *P < The values were obtained by comparing the experimental with the control gtoup. = right ventricle; left ventricle.

6 276 LAGUENS, G6MEZ-DUMM i JJ FIGURE 5 After 60 min of exercise. High-power view of a mitochondrion with two invaginations. In the interior of the invaginations electron-dense granules interpreted as ribosomes can be observed. (Table 2). The individual mitochondrial area was also larger in the animals submitted to 90 and 120 min of swimming than in those of the other groups studied (Table 3). The increase in the percentage of cytoplasm of the heart muscle cells occupied by mitochondria could be ascribed to an increase in the size of the individual mitochondria. The number of mitochondria per 1,000 (A 2 of fiber area was similar in the control and the experimental groups (Table 4). With the exception of the mitochondria, the remaining components of the heart muscle cell showed no striking or constant modifications in any of the rats. Discussion The results reported in this paper indicate that, after 90 and 120 min of exercise, the mitochondrial mass of the cardiac muscle cell increases significantly. This is apparently due to hypertrophy of the individual mitochondria rather than to an increase in the number of mitochondria. Enlargement of individual mitochondria has been reported after various degrees of ischemia (8-12). However, the enlarged mitochondria described after ischemia appear to be altered, presenting a swelling and decrease in the electron density of the matrix TABLE 4 Number of Mitochondria per 1,000 Square Micra Exercise time None 30 min 60 min 90 min 120 min = right ventricle; = left ventricle CircuUtion Rtsurtb, Vol. XXI, Stptmbtr 1967

7 HEART ULTRASTRUCTURE AFTER EXERCISE FIGURE 6 After 90 min of exercise. The arrow points to an enlarged mitochondrion measuring more than 3 sarcomeres in length with two invaginations. and a decrease in the number of cristae. In our material, the hypertrophic mitochondria had no structural alterations. This fact induces us to suppose that the increase in mitochondrial size is a response to exercise rather than to a relative ischemia. In cardiac hypertrophy following aortic stenosis (13) and experimental renal hypertension (14), various degrees of mitochondrial enlargement have been described; in these cases the enlarged mito- 1JJ chondria presented structural alterations. On the other hand, the hypertrophic hearts were examined a long time after the aortic stenosis or the renal hypertension had been established. To our knowledge, no experiments have yet been described in which the mitochondrial mass of the myocardium increases as rapidly as in the present studies of the effect of exercise. Apparently the increase in mitochondrial CirctiUuan Rttetrcb, Vol. XXI, Stpitmbtr 1967

8 278 LAGUENS, G6MEZ-DUMM 1JLJ FIGURE 7 After 120 min of exercise. Electron micrograph shows many enlarged mitochondria measuring from 2 to 4 sarcomeres in length. mass observed 90 and 120 min after the start of exercise was preceded by marked modifications of the mitochondrial shape. Although no definitive evidence exists, it is tempting to speculate that the presence of these alterations is specifically related to mitochondrial hypertrophy. The appearance of multiple invaginations and lateral buddings has been considered as morphologic evidence of replication of mitochondrial components (15). This fact would be supported by the constant presence of ribosomes in the sarcoplasm located within the invaginations. However, in our studies, we did not find an increase in the number of mitochondria. It is known that besides their role in the production of energy, mitochondria can synthesize their own proteins (16, 17). The ribosomes present within the invaginations might play a role in the synthesis of the new mitochondrial components that would lead to the increase in size with preservation of the structural integrity. Apparently cardiac mitochondria possess the ability to enlarge in response to stimuli that place an increased load on the myocardium. Whether the increase in mitochondrial mass is related to an increase in their metabolic functions, remains to be determined. Acknowledgment The technical assistance of Miss Susam Rivas, Miss Patricia Cabeza-Meckert and Mr. Osvaldo Neira is much appreciated. References 1. LACUENS, R. P., LOZADA, B. B., G6MEZ-DUMM, C. L. A, AND RUIZ-BERAMENDI, A. L.: Effect of acute and exhaustive exercise upon the fine structure of heart mitochondria. Experientia 22: 244, MILLONIG, G.: Further observations on a phosphate buffer for osmium solutions in fixation. In Fifth International Congress for Electron Microscopy, edited by S. S. Breeze, Jr. New CkeuUtion Rtietrcb, Vol. XXI, Septtmbtr 1967

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