Glycogen Aggregates in Cardiac Muscle Cell: A Cytopathological Study on Endomyocardial Biopsies

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1 Arch. histol. jap., Vol. 45, No. 4 (1982) p Glycogen Aggregates in Cardiac Muscle Cell: A Cytopathological Study on Endomyocardial Biopsies Kazumasa MIURA, Tohru IZUMI, Junichi FUKUDA, Masaru YAMAZOE and Kohji TAMURA Departments of First Internal Medicine (Prof. A.SHIBATA), Third Anatomy (Prof. T. FUJITA) and Second Surgery (Prof. S. EGUCHI), Niigata University School of Medicine, Niigata, Japan Received October 30, 1981 Summary. Glycogen aggregates in the cardiac muscle cell, which have been frequently demonstrated in endomyocardial biopsies under the transmission electron microscope, were studied using the optical microscope in order to clarify their histopathological significance in various heart diseases. The right ventricular muscle biopsies were obtained from the following 120 patients: 24 cases of congestive cardiomyopathy, 26 of hypertrophic cardiomyopathy, 28 of atrial septal defect, 6 of primary pulmonary hypertension, 31 of bradyarrhythmic hearts and 5 controls. The tissue specimens were fixed with glutaraldehyde and osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections from these specimens were dyed with tribasic staining originated by KUROTAKI (1972). Under the optical microscope, glycogen is clearly identified by its forming red stained areas which are seen not only in the subsarcolemmal layers and the perinuclear region, but also in the intermyofibrillar zones. The glycogen aggregates are more frequently observed in the specimens from the atrial septal defects and the bradyarrhythmic hearts than in the other cases. Thus, observation of glycogen aggregates reveals notable differences in appearance depending on the kinds of diseases. In the bradyarrhythmic hearts, the glycogen aggregates can be more readily observed in specimens from younger patients than in elderly ones. Furthermore, the glycogen aggregates appear regardless of both the grades of hypertrophy and degeneration in myocardial cells. These results do not agree with previous reports that the occurrence of glycogen is proportional with the grade of cardiac cell hypertrophy and/or damage. In the cardiac muscle cell, glycogen particles are visualized as granules nm in diameter with high electron density under the transmission electron microscope. These granules, called beta-particles, have been reported to occur in mitochondria and nuclei and to form rosettes, alpha-particles, in diseased muscles (FERRANS et al., 1973; JONES and FERRANS, 1973; MARON and FERRANS, 1974). However, there has been no agreement concerning the histopathological significance of the intracellular glycogen in diseased human myocardium. We previously have reported that aggregations of glycogen could be successfully observed under the optical microscope by application of the tribasic staining originated by KUROTAKI (1972) to the myocardial biopsy specimens (FUKUDA et al., 1980). Thus, the present study aims to clarify morphological 347

2 348 K. MIURA et al.: characteristics and the pathological significance of the glycogen aggregates in diseased human cardiac muscles. MATERIALS AND METHODS The right ventricular muscle specimens were obtained from the following patients (Table 1): 24 cases of congestive cardiomyopathy (COCM), 26 cases of hypertrophic cardiomyopathy (HCM), 28 cases of atrial septal defect (ASD) caused by right ventricular volume overload, 6 cases of primary pulmonary hypertension (PPH) by pressure overload, 19 cases of sick sinus syndrome (SSS) and 12 cases of high grade atrioventricular block (A-V block, including cases over the second degree block except the Wenchewach type) causing bradyarrhythmic hearts. These patients were diagnosed by clinical histories, the data having been obtained by the non-invasive and/or the invasive techniques, i.e., cardiac catheterization and angiography. The diagnosis of idiopathic cardiomyopathy was accorded to the criteria decided by the Idiopathic Cardiomyopathy Research Group (Chief director Prof. C. KAWAI, 1976). All patients with the bradyarrhythmic heart were implanted with an artificial pacemaker after biopsy. Examinations with echocardiogram, cardiac catheterization and angiogram confirmed that these patients of bradyarrhythmia did not have left ventricular hypofunction except for electrical failure. The diagnosis of five controls was rate dependent left bundle branch block, complete right bundle branch block, 2 of spontaneous occurrence of ventricular tachycardia and intramural cardiac tumor. The ages of the examined cases ranged from 5 to 72 years and averaged 45 years. Among these cases, primary pulmonary hypertension was found the youngest patients, averaging 36 years in age, while the cases of bradyarrhythmic heart were in the oldest, 53 years in average (Table 1). The muscle specimens were fixed with 2.5 o glutaraldehyde, post-fixed with 2% osmium tetraoxide, dehydrated with ethanol and embedded in epoxy resin according to the conventional method for transmission electron microscope samples. Semi-thin sections were cut from these blocks and observed under the optical microscope after Table 1. The kinds of diseases and the mean cardiac cell diameters

3 Glycogen Aggregates in Cardiac Muscle Cell 349 staining with 0.5% malachite green, toluidin blue and 0.5% basic fuchsin according to KUROTAKI'smethod (KUROTAKI,1972; FUKUDAet al., 1980). Calibers of 200 muscle fibers, only crossly cut profiles, were sequentially measured by sonographic pendigitizer computer system (Ohsawa, Oscon). The cardiac muscle cell diameters, the muscle fibers containing nuclei in the section, were determined from those measurements for each case of cardiac disorder. RESULTS AND DISCUSSION Using KUROTAKI'Stribasic staining, glycogen aggregates, i.e., accumulations of glycogen particles were clearly stained as dark-red or pinkish-red areas in the cardiac muscle cell and could be easily detected ranging over a wide visual field under the optical microscope. Figure 1 A represents typical microscopic images of glycogen aggregates, which represent saccular forms protruding in the subsarcolemmal layer. Figure 1 B is a transmission electron micrograph showing glycogen aggregates similar to those in Figure 1 A. In the center of the figure, a blood capillary can be seen. Glycogen aggregates in the subsarcolemmal layer frequently appeared close to a capillary. Figure 2 A and 2 B are pictures of glycogen aggregations among the myofibrils and Figure 3 A B A Fig. 1. Glycogen aggregates in the subsarcolemmal layer glycogen aggregates protruding in subsarcolemmal rows). Co collagen fibers. KuROTAKI's staining. subsarcolemmal layer under the electron microscope. (SSS, 32 year old female). layer under the optical B. Glycogen aggregates C blood capillary. A. Saccular microscope (arprotruding in

4 350 K. MIURA et al.: B A Fig. 2. Intramyofibrillar glycogen aggregates (SSS, 53 year old female). A. intermyofibrillar glycogen aggregations stained by KUROTAKI'S method. glycogen particles gathered close to the mitochondria and myofibrils. The arrows B. Electron show dense and 3 B are those in perinuclear areas. In Figure 3 B, the arrows in the upper part indicate an adjacent cardiac muscle cell, where glycogen particles are less densely accumulated. Figure 3 C and 3 D are high magnifications of the glycogen aggregate. The glycogen particles differed in amount between these two figures: they are much more densely gathered in C than in D. Under the optical microscope, the difference in amount is reflected in color changes from dark-red (dense assembly) to pinkish-red (loose aggregation). We can see the color difference in Figuer 3 A. The figures of the glycogen aggregations, however, must be differentiated from the following two fuchsinophilic structures. One is a non-granular pinkish zone in the sarcoplasma, where few glycogen particles could be seen under the transmission electron microscope. The other was a fine granular and well demarcated pinkish-red zone, which proved to be the well-known basophilic degeneration by transmission electron microscopy. These figures could be easily differentiated from glycogen aggregates under the optical microscope. The occurrence and nature of glycogen in the cardiac muscle were reported in detail by FERRANrset al. (1973) using both the transmission electron microscope and the cytochemical techniques. However, no investigation seems available which examines the glycogen aggregates in a wide optical visual field and demonstrates as to where glycogen aggregates are apt to appear in the cardiac muscle cell. As shown in Figures 1, 2 and 3, the occurrence of glycogen aggregates differed in both frequency and site from cell to cell. This variability may be related to different glucose metabolism in

5 Glycogen Aggregates in Cardiac Muscle Cell 351 the cardiac muscles. In order to clarify this question, further experimental pathological studies will be necessary. The next question to arise is: on what cardiac disorders do the glycogen aggregates reflect? In the present study, the kind of disease, age of the patient, grade of muscle cell hypertrophy and degree of cellular degenerative change were checked. The intensity of glycogen aggregate was classified into 4 grades, negative () in which the aggregations are not detectable at all in any muscle samples stained with KUROTAKI's tribasic staining; slightly positive (+) in which they are in a few muscle cells in some visual fields: moderately positive (-H-) in which they are detectable in several cells I B C D Fig. 3. Perinuclear glycogen aggregates (SSS, 63 year old male). A. The bold arrow shows a darkred stained glycogen aggregate and the slim arrow shows a pinkish one. B. Electron dense glycogen particles gathered around the nuclei. C and D. High magnifications of the glycogen glycogen aggregate. particles. C. Dense assembled glycogen particles. D. Loose aggregation of This difference reflect in color changes under the optical microscope.

6 352 K. MIURA Fig. Fig. 4. et al.: Percentage of the glycogen 5. Relationship between the age of the patient and the frequency of the glycogen aggregates in the bradyarrhythmic heart. aggregates among the heart diseases. within a visual field; and highly positive (Th-)in which they are numerously found within a visual field. The cardiac muscle cells at the subendocardial layer were excluded in the present evaluation because the cells might possibly belong to the conduction system. Figure 4 gives the relation of the degree of glycogen aggregate to the kind of heart disease. Moderately and highly positive (* and {f ) cases made up 12% in congestive cardiomyopathy, 1910 in hypertrophic cardiomyopathy, 47 o in atrial septal defects, 0% in primary pulmonary hypertension, 61% in bradyarrhythmic heart and 20% in the control. In brief, both cardiomyopathies, which are characterized by a primary disorder of the cardiac muscle, are not significantly different from the control with regard to the occurrence of glycogen aggregates. Interestingly, an opposite result is recognized in the overload heart: there were numerous glycogen aggregates in the volume overload heart, i.e., atrial septal defect and very few of them in the pressure overload heart, i.e., primary pulmonary hypertension. Bradyarrhythmic hearts in which no cardiac pump failure had been confirmed by the usual clinical studies, also revealed significantly higher percentage occurrences of glycogen aggregates than in the control. These results suggest that the glyco- gen aggregates differ in frequency according to the type of heart disease. Concerning the age of the patients, glycogen aggregates were studied in only the bradyarrhythmic cases because this disease seemed to most faithfully reflect the morphological changes based on aging among the present cases. As shown in Figure 5, the mean age was 60 years in the negative (-) group and 58 years in the slightly positive (+) group, 52 years in the moderately positive (-}{) group, and 43 years in the highly positive (t}}) group. These results hardly indicate a correlation between the

7 Glycogen Aggregates in Cardiac Muscle Cell 353 frequency of glycogen aggregates and aging in this disease. A tendency that glycogen aggregates are apt to appear in younger cases rather than in older ones was also recognized in the control cases, though the examined number was very small. The relation of glycogen aggregates to muscle cell hypertrophy was investigated Atrial septal defect Primary pulmonary hypertension Fig. 6. Relation between the cardiac muscle cell diameter and the frequency of the glycogen aggregates in the right ventricular overload'heart. Congestive cardiomyopathy Bradyarrhythmia Fig. 7. Relation between the grade of degeneration and the frequency of the glycogen aggregates in the congestive cardiomyopathy and bradyarrhythmic heart.

8 354 K. MIURA et al. in both the volume overload hearts and the pressure overload hearts. As shown in Figure 6, no definite tendency could be detected in either disease. In the same way as mentioned above, cytologically degenerative changes in myofibrils, mitochondria and sarcoplasma were assessed using three-grade classification in both congestive cardiomyopathy which is characterized by a primary muscle weakness, and the bradyarrhythmic heart without cardiac pump failure (Fig. 7). These degenerative changes did not reveal any definite tendency in relation to the occurrence of glycogen aggregates. Though MARON and FERRANS (1975) considered the glycogen accumulation to perhaps result from either muscle cell hypertrophy or degeneration, the present study does not support this view. To conclude, glycogen aggregates appear more frequently in the right ventricular volume overload (atrial septal defect) and the bradyarrhythmic heart than in the other disorders examined, and their occurrence correlates neither with the grade of muscle cell hypertrophy nor with the severity of cellular degeneration. REFERENCES Ferrans, V. J., L. M. Buja and M. Jones: Ultrastructure and cytochemistry of glycogen in cardiac diseases. In: (ed. by) N. S. Dhalla: Recent advances in studies on cardiac structure an metabolism. Vol. 3. Myocardial metabolism. University Park Press, Baltimore, 1973 (p ). Fukuda, J., S. Eguchi, K. Miura and T. Izumi: Kurotaki's staining on myocardial biopsies. J. Electron Microsc.13: (1980). Idiopathic Cardiomyopathy Research Group (Chief director Prof. C. KAWAI): Guidance for diagnosis of idiopathic cardiomyopathy. In: Annual report of idiopathic cardiomyopathy research in 1975, (in Japanese). The Japanese Welfare Ministry, 1976 (p. 246). Jones, M. and V. J. Ferrans: Intramitochondrial glycogen in hypertrophied infundibular muscle of patients with congenital heart diseases. Amer. J. Pathol. 70: (1973). Kurotaki, M.: A differential staining of semi-thin sections of epon-embedded tissues and a short review on the optical microscopy. Amer. J. Pathol. 71: (1972). Maron, B. J. and V. J. Ferrans : The occurrence of alpha-glycogen in human cardiac muscle cells. J. mol. cell. Cardiol. 6: (1974). - : Intramitochondrial glycogen deposits in hypertrophied human myocardium. J. mol. cell. Cardiol. 7: (1975). Dr. Kazumasa MIURA First Department of Internal Medicine Niigata University School of Medicine Asahimachi, Niigata 951 Japan