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 ischemia 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 autopsy 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.