3ournal of Molecular
and Cellular
Comparative in Adult
Cardiology
7, 195-202
Electron Microscope Rats Fed on Normal HAUKUR
Detartment
(1975)
MELAX
of Anatomy, (Received
AND
University
27 August
Studies of the Myocardium and Cholesterol Diets*
THOMAS
S. LEESON
of Alberta,
Edmonton,
1973, and accepd
18 March
Alberta,
Canada
1974)
H. MELAX AND T. S. LEESON. Comparative Electron Microscope Studies of the Myocardium in Adult Rats Fed on Normal and Cholesterol Diets. Journal of Molecular and Cellular Cardiology (1975) 7, 195-202. In young adult rats which were fed on cholesterol diet for four weeks, moderate morphological changes occurred within the myocardium, as compared to the myocardium in rats of the same age which were fed on normal rat diet only. After a period of ten weeks on the cholesterol diet, further changes or degenerations of the heart wall were observed. In general, the primary changes were thickening of the basal laminae of the endothelial layer of the endocardium and of the capillaries. In the extracellular spaces, numerous membrane-bound bodies or vacuoles were present. Within the myocardial cells, swelling of mitochondria occurred as well as their conversion into myelin bodies. Large numbers of formed myelin structures were present within the sarcoplasm which lead to a disruption in the normal relations of the myofibrils, the sarcoplasmic reticulum, and the transverse tubular system. Increased complexity (jigsawpattern) of the intercalated discs, and formation of an electron dense zone inside the apposed plasma membranes occurred. Eventually the intercellular gap of the intercalated discs became enlarged and irregular and finally adjacent myocardial cells became completely separated. WORDS: Cholesterol administration; Intercalated discs; Vacuoles.
KEY
Myocardial
degeneration;
Myelin
bodies;
1. Introduction In recent years ultrastructural studies have been carried out on the various parts of the heart in the normal rat, e.g. by Jamieson and Palade [15], Bozner et al. [4], Hadek and Talso [II], Hibbs and Ferrans [13] and Melax and Leeson [23-261. More recently some attention has been focused on the ultrastructural changes or degeneration which have resulted from excessive exercise or following drug administration Sohal et al. [36] revealed ultrastructural changes in the intercalated disc of exercised rats, and their report indicated a considerable increase in the intercellular space of the intercalated discs, or local swellings of that region. Lin and Duffy [ZO] described cobalt-induced myocardial lesions in rats. With the increase in heart disease, any information on morphological changes in the heart tissue induced or otherwise is of value. The present morphological observations and comparisons reveal evidence of direct damage to the myocardium in rats fed on a high cholesterol diet. * This work
was supported
by the Medical
Research
Council
of Canada
Grant
MA-4603.
196
H.
MELAX
AND
2. Materials
T. S. LEESON
and
Methods
Twelve juvenile male albino rats were selected from three litters fathered by the samemale. Each rat weighed about 180g. One third of the rats were fed on normal rat diet only. The others were fed on a mixture of normal rat diet containing pure cholesterol-the cholesterol being approximately l--+ to 2% of the total diet by weight. After four weeks, half of the rats were sacrificed. The rats in each group weighed about 210 g although those on the cholesterol diet were slightly heavier. Six weeks thereafter the remainder of the rats were sacrificed. The rats which were fed on normal rat diet had only slightly increased their weight in that period of time, while the ones fed on cholesterol diet for a total of ten weeks had gained considerable weight, and on the average they were 25 to 30 g heavier than those on normal diet. After sacrifice, the hearts were excised, dissectedinto several pieces, and small specimensfrom known regions were collected separately. The small blocks were fixed in 1% 0~04 adjusted to pH 7.4 with phosphate buffer. In some casesthe specimenswere fixed in a combined formaldehyde-glutaraldehyde fixative [16J followed by postfixation in 1o/o buffered 0~04. Epon 812 was used as embedding medium. The sectionswere variously stained with (1) an aqueoussolution of lead citrate [33], (2) 2% potassium permanganate in aqueous solution for 15 to 20 min, or (3) 3% uranyl acetate for 3 to 5 h. They were examined in a Philips EM100 or Philips EM200 electron microscope.
3. Results In the rats which were kept on norma rat diet, the myocardium showed no abnormalities or degenerations. Its ultrastructure was identical to the well-established ultrastructural pattern for the myocardium in the normal rat. However, in the rats which had been kept on the cholesterol diet for a period of four weeks,abnormalities within the heart wall were evident. Further, these abnormalities or degenerations which occurred within the myocardium were more advanced in those rats which were kept on the cholesterol diet for the longer period of time, i.e. ten weeks. The degeneration of the myocardium was evenly distributed, no specific areas or locations of the heart wall showing more excessivedegeneration than others. After four weeks on the cholesterol diet, the organelles of the myocardial cells showed displacement within the sarcoplasmand degenerationswere evident. Large numbers of mitochondria appeared swollen or vacuolated, while others displayed an unusually electron dense matrix., Mitochondria in groups or clusters often appeared to be densely packed between the myofibrils (Plates 2,5 and 7). Throughout the myocardium, mitochondria were found containing a myelin structure or
MYOCARDIAL
DEGENERATION
IN RATS
FED
CHOLESTEROL
197
structures (Plate 7). It appeared that the inner membrane structures of the mitochondria, i.e. cristae mitochondriales, did break up, and then converted into myelin-like whorls. Within the sarcoplasm of the myocardial cells lipid droplets in close association with the mitochondria and glycogen particles were developed (Plates 5 and 6). As these changes occurred the myofibrils were dislocated within the sarcoplasm, as well as the sarcoplasmic reticulum which also became dilated, and the relatively poorly developed transverse tubular system disintegrated. The nuclei which are spherical to ovoid in normal myocardial cells become heavily indented or even lobulated (Plate 5). After ten weeks on the cholesterol diet, most if not all myocardial cells had developed relatively large myelin structures within their sarcoplasm (Plates 8, 9 and lo), and further dislocation of the organelles occurred. The myelin structures, pleomorphic in shape and size, often appeared to be composed of several smaller myelin structures. Generally a myelin structure was composed of several membranous concentric rings enclosing a central core. The matrix of the center resembled the sarcoplasm with some glycogen particles and small vesicles present within it. Also present, within a group of myelin structures, were small tubules with a diameter of about 550 A (Plates 8 and 9). They were commonly found in parallel rows, and serial sections indicated that these are straight tubules several microns long. The extracellular content of the myocardial tissue space and the subendocardium of normal rat is known to consist of a rather electron lucent, homogeneous matrix, containing some native collagen fibrils as the only formed extracellular elements. However, in all those rats which were kept on the cholesterol diet a considerable number of membrane-bound bodies or vacuoles were present in the extracellular matrix (Plates 2, 3, 11 and 12). Serial sections indicated that these vacuoles were spherical in shape and of various sizes. Most of them contained an electron lucid homogeneous matrix, although a few also contained membranebound bodies. In those rats which were kept on the cholesterol diet, no obvious changes or degeneration were observed in the cellular components of the myocardial tissue space or the endocardium. However, an increase in number of the so-called myocardial foam cells (Plate 4) was noted [Xl. The endothelial cells of the capillaries and of the endocardium displayed a similar pattern in all cases, except for the basal laminae. In the normal rat, the capillaries and the endothelial endocardium displayed uniform homogeneous basal laminae of approximately 500650 A. In the experimental rats a thickening of the basal laminae occurred, measuring up to 2000 A or more (Plates 2 and 11). I n many instances electron dense bodies or structures were found within, or in close association with, the thickened basal laminae of the endothelium. In normal rats, the basal laminae of myocardial cells is identical structurally to that of the endothelium. A thickening of the basal laminae of cardiac muscle cells occurred in those rats fed on the cholesterol diet for 10 weeks, but this increase in thickness was lessthan that found in endothelial basal laminae.
198
H.
MELAX
AND
T. S. LEESON
4. Discussion The intake of cholesterol in our experimental rats is similar to that used by other researchers such as Bajwa et al. [2], Besterman [3], Cambell et al. [5], Kritchevsky et al. [19], Moss and Benditt [27] and Vijayagopalan and Kurup [38], these researchers having described the effects of cholesterol feeding on various organs and tissues in rats and rabbits. Indeed the amount of cholesterol fed to the rats over 10 weeks is sufficient to induce atherosclerotic lesions. It now is well established that hypercholesterolemia is the main cause of atherosclerosis. Yamamoto and Yamamura [41] recently demonstrated that in rats, biosynthesis of cholesterol decreases with age and the blood cholesterol level rises. Consequently for the last few years, an increasing number of researchers have supported the idea that virtually all “aging diseases” start in the small blood vessels and the capillaries. The present study has shown that the cholesterol diet causes thickening of the basal laminae of the endothelium and degeneration of the myocardial cells in laboratory rats. Although the thickening of the vascular basal lamina in cholesterol fed animals and its significance has not been reported previously, the thickening of the capillary basal lamina in patients with diabetes mellitus as a general phenomenon has been well established [S, 321. It is also known that with increased age in humans and animals [40] the basal lamina of endothelium in general thickens. Oxygen transport in humans and animals decreases with age due to higher plasma concentration, [q, and presumably with the thickening of the basal laminae, metabolic exchange between the capillaries and the surrounding tissue is impaired. However, this hypothesis has yet to be proven. The origin and significance of the membrane-bound vacuoles in the extracellular space remains doubtful. It can be theorized, however, that the increased blood cholesterol level may produce changes in the endothelial plasma membrane resulting in partia1 loss of this membrane and vacuole formation. Nevertheless, these vacuoles may not be of endothelial origin and further studies are necessary to clarify their origin and significance. Our study appears to indicate that the mitochondrial changes were a major factor in the degeneration of the myocardium. Several investigations reported mitochondrial lesions in myocardial cells. Novi [30] described swelling of mitochondria induced by low irradiation doses of 500 R CO60 in rat myocardium. Lin and Huffy [20] in their report on cobalt induced myocardial lesions in rats stated that 10 h after an injection of cobalt nitrate, most of the mitochondria of the myocardial cells were swollen and showed fragmented cristae. Similar findings were reported by Alexander [I], Suzuki [37], Hall and Smith [12] and Rona [34]. However, none of these authors reported the formation of myelin structures within the mitochondria or in the sarcoplasm. Sacktor and Shimada [35] described “degenerative changes in the mitochondria from aging blowflies”. They reported on reorganization of the inner membrane of the mitochondria into myelin-like
PLATE 1. Normal. Electron micrograph taken from the interventricular septum demonstrating an intercalated disc (ID), mitochondria (MI), and extracellular space (E). x 15 000. PLATE 2. Four weeks cholesterol. Electron micrograph from the posterior wall of the right ventricle. It demonstrates the thickened basal lamina (BL) of the endothelial endocardium (EN), several membrane-bound bodies (arrows) in the subendothelial layer, and swollen mitochondria (MI) in adjacent myocardial cells. (L) lumen. x 15 000. [facing
page 1981
E
.
.
PLATE 3. Four weeks cholesterol. Electron micrograph from the papillary muscle. Numerous membrane bound bodies or vacuoles (arrows) are present in the extracellular space (E). (C) capillary; (M) myocardial cells. x 9500. PLATE 4. Four weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle. with highly vacuolated cytoplasm. (UN) unmyeIt demonstrates a myocardial foam cell (MFC) linated nerve fibres; (E) extracellular space; (M) myocardial cell. x 12 000.
PLATE 5. Four weeks cholesterol. Electron micrograph from the inter-ventricular septum showing a pycnotic nucleus (N), and numerous lipid droplets (LD). ID intercalated disc; (E) extracellular space. X 7500. PLATE 6. Four weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle, showing increased folding of an intercalated disc (ID) and an electron dense zone on the cytoplasmic aspects of opposed plasma membranes. (MI) mitochondria; (LD) lipid bodies; (E) extracellular space. x 8500.
PLATE 7. Four weeks cholesterol. Electron micrograph from the medial wall of the left atrium showing a small portion of a myocardial cell. The inner membrane structures of two mitochondria (MI) are being converted into myelin-like whorls (arrows). x 24 000. PLATE 8. Ten weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle demonstrating myelin body (MY). A few microtubules are present within it (arrows). (MI) mitochondria. x 18 000.
PLATE 9. Ten weeks cholesterol. Electron micrograph from the lateral wall of the right ventricle. Withi? the myelin body (MY) a row of microtubules (arrows) are present. x 14 000. PLATE 10. Ten weeks cholesterol. Electron micrograph from the lateral wall of left atrium demonstrating myelin structures (MY) among the myofibrils of a myocardial cell, and a large mass of monoparticulate glycogen which is present in the center of the whorl at the top. x 17 000.
PLATE 11. Ten weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle. It demonstrates a disintegrating intercalated disc (ID), some peculiar electron dense structures (arrows) within the thickened basal lamina of the capillary (C), and membrane-bound vacuoles (V) h the extracellular space (E). x 12 000. PLATE 12. Ten weeks cholesterol. Electron micrograph taken from the inter-ventricular septum close to the apex. Total disintegration of the intercalated disc (ID) seems to have occurred, and the adjacent myocardial cells have separated. (E) extracellular space. x 12 000.
MYOCARDIAL
DEGENERATION
IN
kAT$
FED
CHOLESTEROL
199
whorls, which they considered to be age-dependent degeneration of mitochondria. Earlier Pannese [31] found membranous whorls in mitochondria in the spinal ganglion neuroblast. He suggested that the whorls were related to formation of new mitochondria. However, our findings indicate that the conversion of mitochondrial inner membranes into myelin structures are degenerative changes, which confirms the observations of Sacktor and Shimada [35]. With an increased number of mitochondria being converted into myelin bodies, multifarious myelin structures occurred in the sarcoplasm. To date no such diverse structures within the myocardial cells have been reported. However, Meessen [ZZ] reported, in experimental hypertrophy in aortic stenosis, myelin structures among swollen mitochondria. He also reported the formation of tubules within the mitochondria in aortic stenosis which were similar in size to those we observed within the myelin structures of the myocardial cells. These findings indicate that the majority of the myelin structures are formed from degenerating mitochondria. However, the presence of microvesicles and glycogen particles commonly located in the center of a myelin whorl, indicate that a portion of the sarcoplasm had been included within a myelin body. This might occur when the outer membrane of the degenerating mitochondrion ruptures, allowing the surrounding sarcoplasm to enter and mix with the mitochondrial matrix followed by the enfolding of the cristae. In recent years ultrastructural studies of the myocardium from variety of species has shown that the myofibrils of the cardiac muscle cells are similar to those of the skeletal muscle containing thick (myosin) filaments and thin (actin) filaments. Nayler [28] in his report on the regulation of myocardial function, considers that the myocardial contraction involves generation of a “sliding force”, the same as or similar to what Huxley [14] has theorized for the functional aspects of striated muscle. In a cholesterol fed rat where dislocation of the myofilaments of the cardiac muscle cells occur, where a large number of mitochondria is being converted into myelin bodies, and with disintegration of the transverse tubular system and dilatation of the sarcoplasmic reticulum, cardiac function obviously will be impaired. In view of recent reports by Entman et al. [7], Gudbjarnason et aE. [IO], Kate [17], Nayler et al. [29] and others, regarding myocardial ATPase activity, calcium binding activity and related biochemical changes, one would conclude that in those rats which were fed on cholesterol diet for the longest period of time a gradual biochemical change did cause a considerable decrease in the functional ability of the cardial muscle cells. Our observations of the ultrastructural features of the intercalated discs in normal rat cardiac muscle were similar to those recently described in other mammalian heart muscle by Ferrans et al. [9], Kawamura and James [18] and McNutt [21]. The intercalated discs which are specialized intercellular junctions, consist of areas of close apposition of plasma membranes of adjacent cardiac muscle cells. In the normal rat, four types of structural arrangements of adjacent plasma membranes of intercalated discs are recognizable. (i) Nexus or peripheral tight
200
H.
MELAX
AND
T. S. LEESON
junction in which the space between the apposed plasma membranes is only 30 tf or less. The nexus is present at the entire periphery of the intercalated disc, being approximately half a micron in width; (ii) undifferentiated regions, in which the adjacent cell membranes display no special substructure, course parallel to each other, but are separated by a space of 160 to 210 A; (iii) desmosomes or maculae adherentes which are regions, ellipsoid in shape, where an electron-dense plaque, about 200 A thick, is subjacent to the cytoplasmic aspect of each of the two opposing membranes and (iv) myofibrillar insertion region which display a fine filamentous mat into which myofilaments attach. In a favourable plane of section, the intercalated discs displayed a moderately wavy appearance (Plate 1). In those rats which were kept on the cholesterol diet for a period of four weeks, an electron dense zone developed on the cytoplasmic side of the apposed plasma membranes of the intercalated discs (Plates 5 and 6). The plasma membranes of the intercalated discs showed a more wavy course compared to those in the normal myocardium. After ten weeks on the cholesterol diet, the intercalated discs showed further abnormalities or changes. The intercellular gap between apposed plasma membranes became irregular and enlarged (Plates 11 and 12). Cytoplasmic extensions from adjacent myocardial cells extended into the intercellular gap, and numerous membrane-bound bodies were present within it. It frequently appeared that total separation of adjacent myocardial cells had occurred. Ultrastructural changes of the intercalated discs have been reported by Sohal et al. [36] in exercised rat heart. Their report described an increased intercellular gap compared to that of the normal rat. However, they did not demonstrate the degree of separation as reported in this investigation. Presumably, the intercalated discs are involved in rapid intercellular ionic exchange due to their low electrical resistance [39]. With the degeneration occurring within the myocardial cells the ionic activity or exchange at the site of the intercalated discs probably decreases. The intercalated discs increase their complexity following cholesterol administration, and develop an electron dense zone or plaque immediately beneath the apposed plasma membranes, even after only four weeks on the cholesterol diet. The reason for this is not known. As the degeneration of the adjacent myocardial cells increases, adhesion between them decreases, and the intercalated disc disintegrates. Its function is lost and presumably myocardial conduction is decreased.
REFERENCES 1.
ALEXANDER, British
2.
Heart
C. S. Electron microscopicobservationsin alcoholic heart disease. Journal
29, 200-206
(1967).
BAJWA, G. S., MORRISON, L. M. & ERSHOFF, B. H. Induction of aortic
and
coronary
athero-arteriosclerosis in rats fed a hypervitaminosisD. cholesterol-containingdiet. Proceedings
of the Sociep for
Ex$erimmtal
BioloD
and Medicine
138, 975-985
(1971).
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3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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E. M. M. Effects of laminarin sulphate on experimental atherosclerosis and on serum lipids in rabbits during long-term intermittent cholesterol feeding. Atheroclerosis 12, 85-96 (1970). BOZNER, A., BARTA, E. & MRENA, E. Further investigations on the ultrastructure of myocardial nerve elements in rats, rabbits and dogs. Ex@erimental Mediciue and Surgery 24, 148-155 (1966). CmELL, D. J., DAY, A. J., SKINNER, S. L. & TUME, R. K. The effect of hypertension on the accumulation of lipids and the uptake of [sH] cholesterol by the aorta of normalfed and cholesterol-fed rabbits. Atherosclerosis 18, 301-319 (1973). CHISOLM, G. M., GAINER, J. L., STONER, G. E. & GAINER, J. V. Plasma proteins, oxygen transport and atherosclerosis. Atherosclerosis 15, 372-343 (1972). ENTMAN, M. L., ALLEN, J. C. & SCHWARTZ, A. Calcium-ouabain interaction in a ‘Lmicrosomal” membrane fraction containing Na f, K+-ATPase activity and calcium binding activity. Journal of Molecular and Cellular Cardiology 4, 435-441 (1972). FARID, N. R., WILKINSON, E., CONSTABLE, F. L. & ANDERSON, J. Basement membrane thickness of rectal capillaries in diabetes. Lancet 1, 837 (1973). FERRANS, V. J., ROBERTS, W. C., SHUGOLL, G. I., MASSUMI, R. A. & ALI, N. Plasma membranes extensions in intercalated discs of human myocardium and their relationship to partial dissociation of the discs. Journal of Molecular and Cellular Cardiology 5, 161-169 (1973). GUDBJARNASON, S., PURI, P. S. & MATHES, P. Biochemical changes in non-infarcted heart muscle following myocardial infarction. Journal of Molecular and Cellular Cardiolog 2, 253-276 (1971). HADEK, R. & TALSO, P. J. A study of nonmyelinated nerves in the rat and rabbit heart. Journal of Ultrastmcture Research 17,257-265 (1967). HALL, J. L. & SMITH, E. B. Cobalt heart disease. An electron microscopic and histochemical study in rabbit. Archive ofPathology 86,403412 (1968). HIBBS, R. G. & FERRANS, V. J. An ultrastructural and histochemical study of rat atria1 myocardium. American Journal of Anatomy 124,25 l-280 ( 1969). HUXLEY, H. E. The mechanism ofmuscular contraction. Science 164,1356-1366 (1969). JAMIESON, J. D. & PALADE, G. E. Specific granules in atria1 muscle cells. Journal of Cell Biology 23, 151-172 (1964). I(ARNOvSKY, J. J. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. (Abstract). Journal of Cell Biology 27, No. 2, 137A-138A (1965). KATZ, A. M. Contractile proteins of the heart. Physiological Reviews 50,63-158 (1970). KAWAMURA, K. & JAMES, T. N. Comparative ultrastructure of cellular junctions in working myocardium and the conduction system under normal and pathologic conditions. Journal of Molecular and Cellular Cardiology 3, 31-60 (1971). KRITCHEVSKY, D., KIM, H. K. & TEPPER, S. A. Cholesterol vehicle in experimental atherosclerosis. Atherosclerosis 15, 101-105 (1972). LIN, J. H. & DUFFY, J. L. Cobalt-induced myocardial lesions in rats. Labratory Investigation 23, 158-162 (1970). MCNUTT, N. S. Ultrastructure of intercellular junctions in adult and developing cardiac muscle. American Journal of Cardiology 25, 169-183 (1970). IV~ESSEN, H. Structural basis of myocardial hypertrophy. British Heart Journal 33 Supplement, 9499 (197 1). MELAX, H. & LEESON, T. S. Fine structure of the endocardium in adult rats. Cardiovascular Research 1, 349-355 (1967). &LAX, H. & LEESON, T. S. Fine structure of developing and adult intercalated discs in rat heart. Cardiovascular Research 3, 261-267 (1969). BESTERMAN,
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39. 40.
41.
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AND
T. S. LEESON
MELAX, H. & LEESON, T. S. Fine structure of the impulse-conducting system in rat heart. Canadian Journal of zoology 48, 837-839 (1970). MELAX, H. & LEESON, T. S. Electron microscope study of myocardial tissue space contents in rat heart. Cardiovascular Research 6, 89-94 (1972). Moss, N. S. & BENDITT, E. P. The ultrastructure of spontaneous and experimentally induced arterial lesions. Laboratory Investigation 23, 521-536 (1970). NAYLER, W. G. Regulation of myocardial function-a subcellular phenomenon. Journal of Molecular and Cellular Cardiology 5, 2 13-2 19 (1973). NAYLER, W. G., MCINNES, I., CHIPPERFFELD, D., CARSON, V. & KURTZ, J. B. Ventricular function and the calcium-accumulating activity of the sarcoplasmic reticulum. Journal of Molecular and Cellular Cardiology 1, 307-324 (1970). NOVI, A. N. Effects oflow irradiation doses on the ultrastructure of the rat myocardium. Virchows Archiv. Abteilung B.
and Cellular
Comparative in Adult
Cardiology
7, 195-202
Electron Microscope Rats Fed on Normal HAUKUR
Detartment
(1975)
MELAX
of Anatomy, (Received
AND
University
27 August
Studies of the Myocardium and Cholesterol Diets*
THOMAS
S. LEESON
of Alberta,
Edmonton,
1973, and accepd
18 March
Alberta,
Canada
1974)
H. MELAX AND T. S. LEESON. Comparative Electron Microscope Studies of the Myocardium in Adult Rats Fed on Normal and Cholesterol Diets. Journal of Molecular and Cellular Cardiology (1975) 7, 195-202. In young adult rats which were fed on cholesterol diet for four weeks, moderate morphological changes occurred within the myocardium, as compared to the myocardium in rats of the same age which were fed on normal rat diet only. After a period of ten weeks on the cholesterol diet, further changes or degenerations of the heart wall were observed. In general, the primary changes were thickening of the basal laminae of the endothelial layer of the endocardium and of the capillaries. In the extracellular spaces, numerous membrane-bound bodies or vacuoles were present. Within the myocardial cells, swelling of mitochondria occurred as well as their conversion into myelin bodies. Large numbers of formed myelin structures were present within the sarcoplasm which lead to a disruption in the normal relations of the myofibrils, the sarcoplasmic reticulum, and the transverse tubular system. Increased complexity (jigsawpattern) of the intercalated discs, and formation of an electron dense zone inside the apposed plasma membranes occurred. Eventually the intercellular gap of the intercalated discs became enlarged and irregular and finally adjacent myocardial cells became completely separated. WORDS: Cholesterol administration; Intercalated discs; Vacuoles.
KEY
Myocardial
degeneration;
Myelin
bodies;
1. Introduction In recent years ultrastructural studies have been carried out on the various parts of the heart in the normal rat, e.g. by Jamieson and Palade [15], Bozner et al. [4], Hadek and Talso [II], Hibbs and Ferrans [13] and Melax and Leeson [23-261. More recently some attention has been focused on the ultrastructural changes or degeneration which have resulted from excessive exercise or following drug administration Sohal et al. [36] revealed ultrastructural changes in the intercalated disc of exercised rats, and their report indicated a considerable increase in the intercellular space of the intercalated discs, or local swellings of that region. Lin and Duffy [ZO] described cobalt-induced myocardial lesions in rats. With the increase in heart disease, any information on morphological changes in the heart tissue induced or otherwise is of value. The present morphological observations and comparisons reveal evidence of direct damage to the myocardium in rats fed on a high cholesterol diet. * This work
was supported
by the Medical
Research
Council
of Canada
Grant
MA-4603.
196
H.
MELAX
AND
2. Materials
T. S. LEESON
and
Methods
Twelve juvenile male albino rats were selected from three litters fathered by the samemale. Each rat weighed about 180g. One third of the rats were fed on normal rat diet only. The others were fed on a mixture of normal rat diet containing pure cholesterol-the cholesterol being approximately l--+ to 2% of the total diet by weight. After four weeks, half of the rats were sacrificed. The rats in each group weighed about 210 g although those on the cholesterol diet were slightly heavier. Six weeks thereafter the remainder of the rats were sacrificed. The rats which were fed on normal rat diet had only slightly increased their weight in that period of time, while the ones fed on cholesterol diet for a total of ten weeks had gained considerable weight, and on the average they were 25 to 30 g heavier than those on normal diet. After sacrifice, the hearts were excised, dissectedinto several pieces, and small specimensfrom known regions were collected separately. The small blocks were fixed in 1% 0~04 adjusted to pH 7.4 with phosphate buffer. In some casesthe specimenswere fixed in a combined formaldehyde-glutaraldehyde fixative [16J followed by postfixation in 1o/o buffered 0~04. Epon 812 was used as embedding medium. The sectionswere variously stained with (1) an aqueoussolution of lead citrate [33], (2) 2% potassium permanganate in aqueous solution for 15 to 20 min, or (3) 3% uranyl acetate for 3 to 5 h. They were examined in a Philips EM100 or Philips EM200 electron microscope.
3. Results In the rats which were kept on norma rat diet, the myocardium showed no abnormalities or degenerations. Its ultrastructure was identical to the well-established ultrastructural pattern for the myocardium in the normal rat. However, in the rats which had been kept on the cholesterol diet for a period of four weeks,abnormalities within the heart wall were evident. Further, these abnormalities or degenerations which occurred within the myocardium were more advanced in those rats which were kept on the cholesterol diet for the longer period of time, i.e. ten weeks. The degeneration of the myocardium was evenly distributed, no specific areas or locations of the heart wall showing more excessivedegeneration than others. After four weeks on the cholesterol diet, the organelles of the myocardial cells showed displacement within the sarcoplasmand degenerationswere evident. Large numbers of mitochondria appeared swollen or vacuolated, while others displayed an unusually electron dense matrix., Mitochondria in groups or clusters often appeared to be densely packed between the myofibrils (Plates 2,5 and 7). Throughout the myocardium, mitochondria were found containing a myelin structure or
MYOCARDIAL
DEGENERATION
IN RATS
FED
CHOLESTEROL
197
structures (Plate 7). It appeared that the inner membrane structures of the mitochondria, i.e. cristae mitochondriales, did break up, and then converted into myelin-like whorls. Within the sarcoplasm of the myocardial cells lipid droplets in close association with the mitochondria and glycogen particles were developed (Plates 5 and 6). As these changes occurred the myofibrils were dislocated within the sarcoplasm, as well as the sarcoplasmic reticulum which also became dilated, and the relatively poorly developed transverse tubular system disintegrated. The nuclei which are spherical to ovoid in normal myocardial cells become heavily indented or even lobulated (Plate 5). After ten weeks on the cholesterol diet, most if not all myocardial cells had developed relatively large myelin structures within their sarcoplasm (Plates 8, 9 and lo), and further dislocation of the organelles occurred. The myelin structures, pleomorphic in shape and size, often appeared to be composed of several smaller myelin structures. Generally a myelin structure was composed of several membranous concentric rings enclosing a central core. The matrix of the center resembled the sarcoplasm with some glycogen particles and small vesicles present within it. Also present, within a group of myelin structures, were small tubules with a diameter of about 550 A (Plates 8 and 9). They were commonly found in parallel rows, and serial sections indicated that these are straight tubules several microns long. The extracellular content of the myocardial tissue space and the subendocardium of normal rat is known to consist of a rather electron lucent, homogeneous matrix, containing some native collagen fibrils as the only formed extracellular elements. However, in all those rats which were kept on the cholesterol diet a considerable number of membrane-bound bodies or vacuoles were present in the extracellular matrix (Plates 2, 3, 11 and 12). Serial sections indicated that these vacuoles were spherical in shape and of various sizes. Most of them contained an electron lucid homogeneous matrix, although a few also contained membranebound bodies. In those rats which were kept on the cholesterol diet, no obvious changes or degeneration were observed in the cellular components of the myocardial tissue space or the endocardium. However, an increase in number of the so-called myocardial foam cells (Plate 4) was noted [Xl. The endothelial cells of the capillaries and of the endocardium displayed a similar pattern in all cases, except for the basal laminae. In the normal rat, the capillaries and the endothelial endocardium displayed uniform homogeneous basal laminae of approximately 500650 A. In the experimental rats a thickening of the basal laminae occurred, measuring up to 2000 A or more (Plates 2 and 11). I n many instances electron dense bodies or structures were found within, or in close association with, the thickened basal laminae of the endothelium. In normal rats, the basal laminae of myocardial cells is identical structurally to that of the endothelium. A thickening of the basal laminae of cardiac muscle cells occurred in those rats fed on the cholesterol diet for 10 weeks, but this increase in thickness was lessthan that found in endothelial basal laminae.
198
H.
MELAX
AND
T. S. LEESON
4. Discussion The intake of cholesterol in our experimental rats is similar to that used by other researchers such as Bajwa et al. [2], Besterman [3], Cambell et al. [5], Kritchevsky et al. [19], Moss and Benditt [27] and Vijayagopalan and Kurup [38], these researchers having described the effects of cholesterol feeding on various organs and tissues in rats and rabbits. Indeed the amount of cholesterol fed to the rats over 10 weeks is sufficient to induce atherosclerotic lesions. It now is well established that hypercholesterolemia is the main cause of atherosclerosis. Yamamoto and Yamamura [41] recently demonstrated that in rats, biosynthesis of cholesterol decreases with age and the blood cholesterol level rises. Consequently for the last few years, an increasing number of researchers have supported the idea that virtually all “aging diseases” start in the small blood vessels and the capillaries. The present study has shown that the cholesterol diet causes thickening of the basal laminae of the endothelium and degeneration of the myocardial cells in laboratory rats. Although the thickening of the vascular basal lamina in cholesterol fed animals and its significance has not been reported previously, the thickening of the capillary basal lamina in patients with diabetes mellitus as a general phenomenon has been well established [S, 321. It is also known that with increased age in humans and animals [40] the basal lamina of endothelium in general thickens. Oxygen transport in humans and animals decreases with age due to higher plasma concentration, [q, and presumably with the thickening of the basal laminae, metabolic exchange between the capillaries and the surrounding tissue is impaired. However, this hypothesis has yet to be proven. The origin and significance of the membrane-bound vacuoles in the extracellular space remains doubtful. It can be theorized, however, that the increased blood cholesterol level may produce changes in the endothelial plasma membrane resulting in partia1 loss of this membrane and vacuole formation. Nevertheless, these vacuoles may not be of endothelial origin and further studies are necessary to clarify their origin and significance. Our study appears to indicate that the mitochondrial changes were a major factor in the degeneration of the myocardium. Several investigations reported mitochondrial lesions in myocardial cells. Novi [30] described swelling of mitochondria induced by low irradiation doses of 500 R CO60 in rat myocardium. Lin and Huffy [20] in their report on cobalt induced myocardial lesions in rats stated that 10 h after an injection of cobalt nitrate, most of the mitochondria of the myocardial cells were swollen and showed fragmented cristae. Similar findings were reported by Alexander [I], Suzuki [37], Hall and Smith [12] and Rona [34]. However, none of these authors reported the formation of myelin structures within the mitochondria or in the sarcoplasm. Sacktor and Shimada [35] described “degenerative changes in the mitochondria from aging blowflies”. They reported on reorganization of the inner membrane of the mitochondria into myelin-like
PLATE 1. Normal. Electron micrograph taken from the interventricular septum demonstrating an intercalated disc (ID), mitochondria (MI), and extracellular space (E). x 15 000. PLATE 2. Four weeks cholesterol. Electron micrograph from the posterior wall of the right ventricle. It demonstrates the thickened basal lamina (BL) of the endothelial endocardium (EN), several membrane-bound bodies (arrows) in the subendothelial layer, and swollen mitochondria (MI) in adjacent myocardial cells. (L) lumen. x 15 000. [facing
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PLATE 3. Four weeks cholesterol. Electron micrograph from the papillary muscle. Numerous membrane bound bodies or vacuoles (arrows) are present in the extracellular space (E). (C) capillary; (M) myocardial cells. x 9500. PLATE 4. Four weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle. with highly vacuolated cytoplasm. (UN) unmyeIt demonstrates a myocardial foam cell (MFC) linated nerve fibres; (E) extracellular space; (M) myocardial cell. x 12 000.
PLATE 5. Four weeks cholesterol. Electron micrograph from the inter-ventricular septum showing a pycnotic nucleus (N), and numerous lipid droplets (LD). ID intercalated disc; (E) extracellular space. X 7500. PLATE 6. Four weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle, showing increased folding of an intercalated disc (ID) and an electron dense zone on the cytoplasmic aspects of opposed plasma membranes. (MI) mitochondria; (LD) lipid bodies; (E) extracellular space. x 8500.
PLATE 7. Four weeks cholesterol. Electron micrograph from the medial wall of the left atrium showing a small portion of a myocardial cell. The inner membrane structures of two mitochondria (MI) are being converted into myelin-like whorls (arrows). x 24 000. PLATE 8. Ten weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle demonstrating myelin body (MY). A few microtubules are present within it (arrows). (MI) mitochondria. x 18 000.
PLATE 9. Ten weeks cholesterol. Electron micrograph from the lateral wall of the right ventricle. Withi? the myelin body (MY) a row of microtubules (arrows) are present. x 14 000. PLATE 10. Ten weeks cholesterol. Electron micrograph from the lateral wall of left atrium demonstrating myelin structures (MY) among the myofibrils of a myocardial cell, and a large mass of monoparticulate glycogen which is present in the center of the whorl at the top. x 17 000.
PLATE 11. Ten weeks cholesterol. Electron micrograph from the lateral wall of the left ventricle. It demonstrates a disintegrating intercalated disc (ID), some peculiar electron dense structures (arrows) within the thickened basal lamina of the capillary (C), and membrane-bound vacuoles (V) h the extracellular space (E). x 12 000. PLATE 12. Ten weeks cholesterol. Electron micrograph taken from the inter-ventricular septum close to the apex. Total disintegration of the intercalated disc (ID) seems to have occurred, and the adjacent myocardial cells have separated. (E) extracellular space. x 12 000.
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whorls, which they considered to be age-dependent degeneration of mitochondria. Earlier Pannese [31] found membranous whorls in mitochondria in the spinal ganglion neuroblast. He suggested that the whorls were related to formation of new mitochondria. However, our findings indicate that the conversion of mitochondrial inner membranes into myelin structures are degenerative changes, which confirms the observations of Sacktor and Shimada [35]. With an increased number of mitochondria being converted into myelin bodies, multifarious myelin structures occurred in the sarcoplasm. To date no such diverse structures within the myocardial cells have been reported. However, Meessen [ZZ] reported, in experimental hypertrophy in aortic stenosis, myelin structures among swollen mitochondria. He also reported the formation of tubules within the mitochondria in aortic stenosis which were similar in size to those we observed within the myelin structures of the myocardial cells. These findings indicate that the majority of the myelin structures are formed from degenerating mitochondria. However, the presence of microvesicles and glycogen particles commonly located in the center of a myelin whorl, indicate that a portion of the sarcoplasm had been included within a myelin body. This might occur when the outer membrane of the degenerating mitochondrion ruptures, allowing the surrounding sarcoplasm to enter and mix with the mitochondrial matrix followed by the enfolding of the cristae. In recent years ultrastructural studies of the myocardium from variety of species has shown that the myofibrils of the cardiac muscle cells are similar to those of the skeletal muscle containing thick (myosin) filaments and thin (actin) filaments. Nayler [28] in his report on the regulation of myocardial function, considers that the myocardial contraction involves generation of a “sliding force”, the same as or similar to what Huxley [14] has theorized for the functional aspects of striated muscle. In a cholesterol fed rat where dislocation of the myofilaments of the cardiac muscle cells occur, where a large number of mitochondria is being converted into myelin bodies, and with disintegration of the transverse tubular system and dilatation of the sarcoplasmic reticulum, cardiac function obviously will be impaired. In view of recent reports by Entman et al. [7], Gudbjarnason et aE. [IO], Kate [17], Nayler et al. [29] and others, regarding myocardial ATPase activity, calcium binding activity and related biochemical changes, one would conclude that in those rats which were fed on cholesterol diet for the longest period of time a gradual biochemical change did cause a considerable decrease in the functional ability of the cardial muscle cells. Our observations of the ultrastructural features of the intercalated discs in normal rat cardiac muscle were similar to those recently described in other mammalian heart muscle by Ferrans et al. [9], Kawamura and James [18] and McNutt [21]. The intercalated discs which are specialized intercellular junctions, consist of areas of close apposition of plasma membranes of adjacent cardiac muscle cells. In the normal rat, four types of structural arrangements of adjacent plasma membranes of intercalated discs are recognizable. (i) Nexus or peripheral tight
200
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junction in which the space between the apposed plasma membranes is only 30 tf or less. The nexus is present at the entire periphery of the intercalated disc, being approximately half a micron in width; (ii) undifferentiated regions, in which the adjacent cell membranes display no special substructure, course parallel to each other, but are separated by a space of 160 to 210 A; (iii) desmosomes or maculae adherentes which are regions, ellipsoid in shape, where an electron-dense plaque, about 200 A thick, is subjacent to the cytoplasmic aspect of each of the two opposing membranes and (iv) myofibrillar insertion region which display a fine filamentous mat into which myofilaments attach. In a favourable plane of section, the intercalated discs displayed a moderately wavy appearance (Plate 1). In those rats which were kept on the cholesterol diet for a period of four weeks, an electron dense zone developed on the cytoplasmic side of the apposed plasma membranes of the intercalated discs (Plates 5 and 6). The plasma membranes of the intercalated discs showed a more wavy course compared to those in the normal myocardium. After ten weeks on the cholesterol diet, the intercalated discs showed further abnormalities or changes. The intercellular gap between apposed plasma membranes became irregular and enlarged (Plates 11 and 12). Cytoplasmic extensions from adjacent myocardial cells extended into the intercellular gap, and numerous membrane-bound bodies were present within it. It frequently appeared that total separation of adjacent myocardial cells had occurred. Ultrastructural changes of the intercalated discs have been reported by Sohal et al. [36] in exercised rat heart. Their report described an increased intercellular gap compared to that of the normal rat. However, they did not demonstrate the degree of separation as reported in this investigation. Presumably, the intercalated discs are involved in rapid intercellular ionic exchange due to their low electrical resistance [39]. With the degeneration occurring within the myocardial cells the ionic activity or exchange at the site of the intercalated discs probably decreases. The intercalated discs increase their complexity following cholesterol administration, and develop an electron dense zone or plaque immediately beneath the apposed plasma membranes, even after only four weeks on the cholesterol diet. The reason for this is not known. As the degeneration of the adjacent myocardial cells increases, adhesion between them decreases, and the intercalated disc disintegrates. Its function is lost and presumably myocardial conduction is decreased.
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