Tohoku J. exp. Med., 1967, 91, 239-248

Electron Microscopic Study on the Effects of Adrenalectomy on the Heart Muscle

Tsuneo Suzuki

Department of Forensic Medicine (Prof. S. Akaishi), Tohoku University School of Medicine, Sendai

To elucidate the relationship between adrenal insufficiency and the so-called 'lymphatic constitution', the electron and light microscopic changes of the heart muscle, the changes of electrocardiogram and the general condition of adrenal ectomized rats were studied. The adrenalectomized rats exhibited decrease in both the body and heart weights, cardiac arrhythmia, bradyeardia and disappearance of P wave. Light microscopically, the muscle fibers were atrophic, edematous and uneven in staining. Electron-microscopically, the mitochondria enlarged, their external membranes were destroyed and the electron density of mitochondrial matrix was diminished. The cristae decreased and became fragmented. Edema and fat droplets appeared around the mitochondria. Glycogen granules decreased noticeably in number. The myofibrils became slender and fragmented. The above results suggested that these ultrastructural changes might be induced by due to spasm of the coronary artery which was causable by parasympathetic excitation in adrenal insufficiency, and the changes seemed to contribute to reduction of the reserve power of the heart and to development of cardiac failure.

Based on the findings in cases of children who died suddenly from unknown causes, Paltaufl introduced the concept of 'lymphatic constitution' into the study of constitution. Later, Bartel2 reported that hypoplasia of the heart, aorta, adrenals and gonads was observed in a group of children with such a constitution. Jaffe,3 Marine et al.4 and Simpson et al.5 observed the enlargement of the thymus and lymphatic nodules of adrenalectomized animals, and a certain relationship was suggested between the adrenal insufficiency and the lymphatic constitution. Recently it has been known that sudden death in the case of adrenal insuf ficiency is caused by acute circulatory failure and that the findings are similar to those in cases of shock. The heart is often small in size with increased lipochrome pigments in muscle fibers. Patchy myocardial degeneration is sometimes present and may be quite severe.6 However, the relationship between sudden death by slight stimuli and adrenal insufficiency has not been clarified. The present report is concerned with the relationship between acute cardiac

Received for publication, November 15, 1966. 239 240 T. Suzuki

failure and adrenal insufficiency from the view-point of the myocardial ultrastruc ture of adrenalectomized rats.

MATERIALS AND METHODS Seventy-sevenWistar breed rats were divided into three groups of 25 to 27 rats each; Group 1 was 40 days, Group 2 was 70 days and Group 3 was 150 days after birth. Bilateral adrenalectomy was conducted by the usual dorsal approach. Three to five rats from each group were exempted from adrenalectomy and received only the same incision as in adrenalectomy. All the rats were weighed every day and the electrocardiogramwas taken immediately before sacrifice. To examine the cardiac muscle of rats surviving a long period after adrenalectomy, the hearts of the surviving one-third of one group were excised when the two thirds of the group had died. The rats in groups 1, 2 and 3 were sacrificedunder anesthesia with ether on the 5th, 10th and 13th days of the operation, respectively. The still beating hearts were excised immediately after thoracotomy and weighed with a torsion balance. Two slices were taken out of the left ventricular wall, cut in cubes of 1 to 2 mm on a side, fixed in 1% osmic acid solution adjusted to pH 7.4 with veronal-acetate buffer for about three hours in an icebox,7 washed in water, dehydrated in a series of ethanol solutions, and embedded in epoxy resin. The blocks were sectioned by a Hitachi type UM-3 ultramicrotome, and the sections showing interference colors of golden to silver hue were picked up on the collodion-coated mesh grids and doubly stained with uranyl acetate and lead citrate.8 Micrographs were taken at magnifications of 4,000 to 10,000 on an Hitachi type HS-7 electron microscope and photographically enlarged to a desired size. The hearts of all experimental animals were fixed in 10% formalin for light microscopy.

RESULTS Gross and light microscopic findings Most adrenalectomizedrats lost their weight rapidly for two to four days after operation and then continued to lose weight slowly, although some rats kept weight after its rapid decrease. The control rats lost weight as much as the ad renalectomized rats for two to four days followingthe operation but they gained weight slowly thereafter (Fig. 1). The heart weight of the adrenalectomized and control rats per 100g of body weight averaged 360.5 and 403.2 mg in Group 1, 295.0 and 337.5mg in Group 2, and 263.0 and 286.5 mg in Group 3. The mean heart weight of the adrenalectomizedrats in Groups 1, 2 and 3 was less by 10.6%, 12.6% and 8.2% than those of the control, respectively (Fig. 2). The heart rate of the adrenalectomized rat was about 150 to 300 per minute , while that of the control was about 400 to 500 per minute . In some rats, Effects of Adrenalectomy on Heart Muscle 2 41

Fig. 1. Changes in body weight after adrenalectomy ,

Fig. 2. Mean heart weight (per 100 g of body weight).

arrhythmia or disappearance of P wave was observed. Histologically, capillary congestion was partly remarkable in the myocardium . In the sections in which capillary congestion was pronounced, there was usually an unevenness in the staining of the myocardium. There were foci of edematous and atrophied myofibrils, although the nuclei of muscle fibers appeared normal in these regions. 242 T. Suzuki Electronmicroscopic findings The heart of the control rats showed essentially the same arrangement of the fine structures as that of the normal mammalian heart described by many investigators.9-14 No change attributable to anesthesia or surgical manipulation was noticed (Figs. 4 and 5).

1) Group1 (adrenalectomizedon the 40th day of life) The mitochondria showed marked swelling. The major axis of the mitochondria averaged 1.12,ƒÊ, while that of the control averaged O.87ƒÊ (Fig. 3).

The cristae were extremely fragmented and decreased in number. A few mito chondria lost a majority of cristae and the electron density of their matrices, and appeared as vacuoles. Most mitochondria were irregular in shape.

Discontinuity of the mitochondria) membranes was commonly seen. In many cases, groups of mitochondria were fused, and lucid spaces around the mitochondria were enlarged. This showed that liquid accumulated around the mitochondria and caused edema. The sarcoplasmic reticulum was slightly enlarged and the myofibrils were fragmented in many places. The heart muscle cells in rats of this group showed a marked reduction of glycogen granules (Figs.

6 and 7).

Fig. 3. Distribution of major axis of mitochondria.

2) Group 2 (adrenalectomized on the 70th day of life) The mitochondrial enlargement was not so distinct as in Group 1. The major axis of the mitochondria averaged 1.04,u, while that of the control averaged 0.85,u (Fig. 3). The mitochondria were pale because of a decrease in the electron density of the matrices. The cristae were moderately dense in some mitochondria, but they were sparse and fragmented in general. The external membranes of the Effects of Adrenalectomy on Heart Muscle 243 mitochondria were uneven and some of them were fused. Many fat droplets were seen close to the mitochondria or the sarcoplasmic reticulum. They were about 0.2 to 0.6,u in diameter and irregular in shape. Intracellular edema was as severe as in Group 1. Glycogen granules were hardly observed in the cytoplasm. The cisternae of the sarcoplasmic reticulum were markedly swollen in a few muscle fibers. Some of them appeared as vacuoles, or they were ruptured. The myofibrils were slender and a few of them were fragmented (Figs. 8-10).

3) Group 3 (adrenalectomizedon the 150th day of life) Mitochondrial swelling was slight in general. The major axis of the mitochondria averaged 0.95ƒÊ, while that of the control averaged 0.84,a (Fig. 3).

The electron density of the mitochondrial matrix was almost normal. A few mitochondria were irregular in shape and were fused. In some mitochondria, their cristae were sparse and fragmented. The sarcoplasmic reticulum was moderately enlarged. Fat droplets and edema were found around the mito chondria. Glycogen granules were far less than in the control rats. Fragmented myofibrils were hardly observed. The changes in this group were in general not so conspicuous as those in the younger groups (Fig. 11).

DISCUSSION

Many investigators reported that plasma volume decreased in the adrenalectomized animals. Some of them15,16 pointed out that the plasma deficiency was caused by rapid passage of a large volume of water from the extracellular phase into the cells as a result of a certain non-specific stress and it was one of the important factors in the susceptibility of the adrenalectomized animal to shock. Britton and his co-workers" demonstrated the depletion of glycogen and accumulation of water in the heart muscles of American monkeys in adrenal insufficiency and suggested that carbohydrate metabolism was disturbed in adrenal insufficiency. The adrenalectomized rats showed conspicuous bradycardia, arrhythmia, and disappearance of P wave in the electrocardiogram. Bradycardia in the adrenalectomized animals has been reported by several workers.18-21 Nilson20 believed that heart failure due to severe bradycardia was one of the causes of sudden death in adrenal insufficiency. Hall and Cleghorn21 observed bradycardia, arrhythmia and disappearance of P wave, and considered that these changes were caused at least in part by relative overactivity of the parasympathetic nervous system which resulted from adrenal cortical insufficiency. They also found rupture and edema of the inner coat of the coronary artery and coronary thrombo sis, and suggested that these changes resulted from ischemia due to spasm of the coronary artery caused by parasympathetic excitation. Hall and his co-workers22 observed coronary spasm, coronary thrombosis, and of the muscle fiber, when the functional equilibrium between the sympathetic and parasympathetic 244 T. Suzuki

nerves was disturbed in adrenal insufficiency. Atrophy of the muscle fibers which was found in the present study was considered to have resulted from ischemia due to coronary spasm and seemed to be a cause of reduction of heart weight. Electron-microscopically, the myocardial changes in adrenal insufficiency were similar in some respect to the changes of the cardiac or skeletal muscle in anoxia or ischemia, which included swelling of mitochondria, changes of sarcoplas mic reticulum, appearance of fat droplets and depletion of glycogen granules.23-26 From the fact that the adrenalectomized rats showed excitation of the parasym pathetic system in the electrocardiogram, these ultrastructural changes were considered to be caused by ischemia due to coronary spasm. The heart muscle has an enormous number of mitochondria which take part in carbohydrate oxidation. A marked loss of myocardial contractibility, which was observed by Webb and his co-workers27 in the adrenalectomized dogs seemed to result from such mitochondrial destruction as shown in the present study. Disturbance of carbohydrate metabolism may cause intracellular edema, appearance of fat droplets around mitochondria and depletion of glycogen granules. Fragmentation of the myofibrils may result from the compression by swollen mitochondria, enlarged sarcoplasmic reticulum and intracellular edema. The sarcoplasmic reticulum is concerned with intracellular conduction of excitation and relaxation of myofibrils.28 Accordingly, the enlargement or rupture of the sarcoplasmic reticulum suggested the disturbance of intracellular conduction of excitation and relaxation of the myofibril. All of these ultrastructural changes are considered to deprive the cardiac muscle of its reserve power and apt to cause cardiac failure on slight stimuli. The ultrastructural changes in the oldest group which had survived a longer period following the adrenalectomy were slighter than those of younger groups. However, cardiac failure developed within 12 days following the adrenalectomy. It seemed that the cardiac failure in this group was caused not only by these myocardial changes but also by other factors such as decrease in plasma volume due to adrenal insufficiency.

Acknowledgment

The author wishes to thank Dr. T. Murakami, former Professor of Forensic Medicine , Tohoku University, for his helpful suggestions.

References

1) Paltauf, A. Ober die Beziehungen des Thymus zum plotzlichen Tod . Wien. klin. Wschr., 1889, 2, 877-881. 2) Bartel, J. Uber die hypoplastische Konstitution and ihre Bedeutung . Wien. klin. Wschr., 1908, 21, 783-790. 3) Jaffe, H.L. The influence of the suprarenal gland on the thymus . I. Regeneration of the thymus following double suprarenalectomy in the rat. J. exp . Med., 1924, 40, 325-342. 4) Marine, D., Manley, O.T. & Baumann, E.J. The influence of thyroidectomy , gonad Effects of Adrenalectomy on Heart Muscle 245

ectomy, suprarenalectomy and splenectomy on the thymus gland of rabbit. J. exp. Med., 1924, 40, 429-443. 5) Simpson, S.L., Dennison, M. & Korenchevsky, V. Some effects of adrenalectomy in male rats. J. Path. Bact., 1934, 39, 569-590. 6) Anderson, W.A.D. , 4th ed., C.V. Mosby Co., St. Louis, 1961, pp. 1039 1047. 7) Caulfield, J.B. Effects of varying the vehicle for Os04 in tissue fixation. J. biophys. biochem. Cytol., 1957, 3, 827-830. 8) Reynolds, E.S. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol., 1963, 17, 208-212. 9) Beams, H.W., Evans, T.C., Janney, C.D. & Baker, W.W. Electron microscope studies on the structure of cardiac muscle. Anat. Rec., 1949, 105, 59-81.

10) Kisch, B. Studies in comparative electron microscopy of the heart. II. Guinea pig and rat. Exp. Med. Surg., 1955, 13, 404-428.

11) Linder, E., Die submikroscopische Morphologic des Herzmuskels. Z. Zellforsch., 1957, 45, 702-746. 12) Moore, D.H. & Ruska, H. Electron microscope study of mammalian cardiac muscle cells. J. biophys. bioehermx. Cytol., 1957, 3, 216-268.

13) Kisch, B. Electron microscopic investigation of the heart of cattle. Exp. Med. Surg., 1959, 17, 1-14. 14) Kisch, B. Electron microscopy of the cardiovascular system, Charles C. Thomas, Springfield, 1960, pp. 10-103. 15) Swingle, W.W., DaVanzo, J.P., Crossfield, H.C., Gleinister, D., Osborn, M., Rowen, R. & Wagle, G. Glucocorticoids and maintenance of blood pressure and plasma volume of adrenalectomized dogs subjected to stress. Proc. Soc. exp. Biol. Med., 1959, 100, 617-622. 16) Swingle, W.W., Remington, J.W., Drill, V.A. & Kleinberg, W. Differences among adrenal steroids with respect to their efficacy in protecting the adrenalectomized dog against circulatory failure. Amer. J. Physiol., 1942, 136, 567-576. 17) Britton, S.W., Silvette, H. & Kline, R.F. Adrenal insufficiency in American monkeys. Amer. J. Physiol., 1938, 123, 705-711. 18) Harrop, G.A., Soffer, L.J., Ellsworth, R. & Treseher, J.H. Studies on the suprarenal cortex. ‡V. Plasma electrolytes and electrolyte excretion during suprarenal insufficiency in the dog. J. exp. Med., 1933, 58, 17-38.

19) Rogoff, J.M. & Stewart, G.N. Studies on adrenal insufficiency in dogs. I. Control animals not subjected to any treatment. Amer. J. Physiol., 1926, 78, 683-710. 20) Nilson, H.W. Corticoadrenal insufficiency, metabolism studies on potassium, sodium and chloride. Amer. J. Physiol., 1937, 118, 620-631. 21) Hall, G.E. & Cleghorn, R.A. Cardiac lesions in adrenal insufficiency. Caned. med. Ass. J., 1938, 39, 126-133. 22) Hall, G.E., Ettinger, G.H. & Banting, F.G. An experimental production of coronary thrombosis and myocardial failure. Caned. med. Ass. J., 1936, 34, 9-15. 23) Molbert, E. Die Herzmuskelzelle nach akuter Oxidationshemmung im electronen mikroskopischen Bild. Beitr. path. Anat., 1957, 118, 421-345. 24) Bryant, R.E., Thomas, W.A. & O'Neal, R.M. An electron microscopic study of myocardial ischemia in the rat. Circulat. Res., 1958, 6, 699-709. 25) Caulfield, J. & Klionsky, B. Myocardial ischemia and early infarction, an electron microscopic study. Amer. J. Path., 1959, 35, 489-524. 26) Stenger, R.J., Spiro, D., Scully, R.E. & Shannon, J.M. Ultrastructural and

physiologic alterations in ischemic skeletal muscle. Amer. J. Path., 1962, 40, 1-20. 27) Webb, W.R., Degerli, Z.U., Hardy, J.D. & Unal, M. Cardiovascular responses in adrenal insufficiency. Surgery, 1965, 58, 273-282. 28) Porter, K.R. The sarcoplasmic reticulum. Its present history and present status. J. biophys. biochem. Cytol., 1961, 10, Suppl., 219-226. 246 T. Suzuki

Legends for Figs. 4-11

ss, mitochondrion er, crista er, sarcoplasmic reticulum sl, sarcolemma mf, myofibril pf, protofibril z, Z band m, M band id, intercalated disc n, nucleus f, fat droplet g, glycogen granule e, edema i, interstitial cell Scale for all electron micrographs: i micron

Fig. 4. Heart muscle cell of a control rat. Longitudinal section. Mitochondria have dense cristae and are arranged in rows between the myofibrils in the long axis of the muscle. Within the myofibrils, Z and M bands are distinctly visible. Sarcoplasmic reticulum is conspicuous at the level of Z band and extends to the indentations of the sarcolemma. Glycogen granules scatter in the sarcoplasm. •~11,000. Fig. 5. Heart muscle cell of a control rat. Cross section. Note the different packing of

protofibrils. Sarcoplasmic reticulum is cut transversely beneath the sarcolemma and between the myofibrils. •~ 13,000. Fig. 6. Heart muscle cell of a rat adrenalectomized on the 40th day of life and sacrificed on the fifth day of the operation. The mitochondria are enlarged noticeably and some of them are ruptured. The electron density of the mitochondrial matrix diminishes extremely. The cristae are sparse and fragmented. Some of the mito chondria appear as large vacuoles (•ª). Edema is observed around the mitochondria and the nucleus. •~ 15,000. Fig. 7. Heart muscle cell of a rat adrenalectomized on the 40 day of life and sacrificed on the fifth day of the operation. Groups of mitochondria appear fused (•ª). Enlarge ment of sarcoplasmic reticulum is conspicuous. The myofibrils are fragmented.

•~ 16,000. Fig. 8. Heart muscle cell of a rat adrenalectomized on the 70th day of life and sacrificed on the 10th day of the operation. Note marked edema around the mito chondria and myofibrils. The mitochondria are moderately swollen and the cristae are fragmented. The myofibrils are slender and fragmentary. Some fat droplets are seen near the mitochondria. Glycogen granules are hardly observed.

•~ 8,000. Fig. 9. Heart muscle cell of a rat adrenalectomized on the 70th day of life and sacrificed on the 10th day of the operation. Edema and fragmentation of the myofibrils are ermarkable. Enlargement of the sarcoplasmic reticulum is noticeable. Some fat droplets are seen, but glycogen granules cannot be found. •~ 15,000. Fig. 10. Heart muscle cell of a rat adrenalectomized on the 70th day of life and sacrificed

on the 10th day of the operation. Moderate enlargement of mitochondria and sarcoplasmic reticulum is noticed. A fat droplet appears, but glycogen granules are hardly observed. •~ 10,000. Fig. 11. Heart muscle cell of a rat adrenalectomized on the 150th day of life and

sacrificed on the 13th day of the operation. The sarcoplasmic reticulum enlarges slightly. A few fat droplets and glycogen granules are visible. In some mitochondria

the cristae are fragmented. but the matrices have almost normal electron density. •~ 15,000. Effects of Adrenalectomy on Heart Muscle 247 248 T. Suzuki