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Comparative Ultrastructural Morphometry of the Relationship between the Sarcoplasmic Reticulum, Transverse Tubules and Myofibrils in Ventricular Myocytes of the Hamster and Bat Dennis E.E¹, Tagoe C.N.B¹, Yates R.D², Ayettey A.S¹ Department of Anatomy, University of Ghana Medical School,P.O.Box 4236, Accra,Ghana; and Department of Anatomy, Tulane University School of Medicine, New Orleans, Louisiana, U.S.A ABSTRACT Comparative ultrastructural morphometry shows that area of myofibrils covered by SR in left ventricular myocytes is 54% in the insect-eating bat (Pipistrellus pipistrellus), 40% in the nonhibernating hamster and 32% in the hamster in hibernating conditions. Similarly, the area of T-tubules covered by the SR is greater in the bat (23%) than in the non-hibernating hamster (14%) and the hibernating hamster (6%). Other parameters determined including volume densities of myofibrils, SR and T-tubules add to the evidence that mammalian cardiac cells are well structured for their different functional capacities, especially in regard to calcium storage for contractile activities. Keywords: Bat, Hamster, Sarcoplasmic reticulum, T-tubules, Ventricular myocytes. INTRODUCTION T-tubule in turn causes more calcium to be released from the SR that couples with the T-tubules. This SR calcium binds to troponin C to initiate contraction. On account of the role of the SR in excitation-contraction coupling, attempts have been made to determine its extent in mammalian cardiomyocytes. Forbes and van Niel [4], for example, have shown that SR to myofibrillar volume is approximately equal in ventricular myocytes of the mouse (0.16) and guinea pig (0.18). Percentage area of myofibrils covered by SR, however, is different in the two species, being 49% in the mouse and 43% in the guinea pig. In a recent study [5], it was noted that volumes of SR and T-tubules reduce significantly in hamsters subjected to hibernating conditions at a temperature of 5ºC. The sarcoplasmic reticulum [SR] is well known to be important in the processes that lead to excitation-contraction coupling in muscle [for e.g., 1] in relation to calcium storage and release. In mammalian cardiac cells, these tubules surround major cytoplasmic organelles such as myofibrils and mitochondria and also form junctional complexes with the surface sarcolemma as well as with its invaginated Ttubule portions. Mammalian cardiac myocytes depend on extracellular calcium for contraction [2] and T-tubules represent major pathways for entry of extracellular calcium in the interior of the myocyte for excitation-contraction coupling [3]. With each action potential, extracellular calcium is transferred to the cytosol through the T-tubule. The calcium released through the (1) volume. The other group of tissues was fixed in a mixture of aqueous OsO4 (one part 4% solution) and potassium ferrocyanide (two parts 0.8% solution) in sodium cacodylate buffer (one part 0.2 M solution, pH 7.6) for 1½ hours to enhance SR and T-tubule membranes for morphometry [4]. All post-fixed tissues were dehydrated in graded series of ethanol before embedding in Epon 812 resin. Thin sections were obtained with a Diatome diamond knife in a Reichert-Jung Ultracut E microtome and were picked on 200 mesh copper grids. These sections were stained with 0.5% saturated uranyl acetate in 50% ethanol for 1015 minutes and counterstained with lead citrate for 6-10 minutes. Stained sections were viewed in a JEOL 100CX II electron microscope at 406-KV. For stereology, four blocks of left ventricular tissue per heart were randomly selected. From one stained section of each block, one photomicrograph each was obtained from the right hand corner of three successive grid squares according to the sampling method of Weibel [8]. The point-counting method was used to determine volume fractions and surface densities of the subcellular organelles from the printed photomicrographs. To determine volume fraction (Vv) of SR, myofibrils and T-tubules, transparent double lattice of 1 cm and 0.5 cm square grids were used. The 1 cm lattice square grid was used for the determination of myofibrillar volume, and the 0.5 cm square grid for the SR and T-tubules. For determination of Vv of myofibrils, micrographs were taken at original magnification of 4,800 diameters and printed at 10,560 diameters. For Vv of SR and T-tubules, the original magnification was 10,000 diameters, with final prints made at 25,000 diameters. From point-counts, volume fractions were determined using the formula below: It is presumed from these studies that the degree of development of the SR should relate to the cardiac cycle as an adaptive feature. The present work is designed to establish this hypothesis using the bat, Pipistrellus pipistrellus that has a heart rate of about 1000 beats/min, the non-hibernating hamster with a heart rate of 300 beats/min and the hibernating hamster with a heart rate of about 5 beats/min [6]. MATERIALS AND METHODS Adult male insectivorous bats Pipistrellus pipistrellus (average weight-6g) and hamsters (average weight-180g) were used in this research. Five bats were captured in a dungeon at the Cape Coast Castle in Ghana, West Africa. Ten hamsters were obtained from the Tulane University Animal House. Five of the hamsters were kept in normal laboratory conditions at 2225ºC with humidity at 52%, while the other five were kept in a cold room at 5ºC. Photoperiod for the two groups of hamsters was maintained at 10 hours of light and 14 hours of darkness. The hamsters were kept in the cold till they adopted the characteristic curled hibernating posture [7] after 4 weeks. They were left in this state of stupor for another 6 weeks. All the animals were anesthetized with sodium pentobarbital at a dosage of 6gm/100gm body weight. They were thoracotomized and immediately perfused through the left ventricle with physiological saline for 1-3 minutes, followed by 3% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.4 for 10-15 minutes. The hearts were removed and stored overnight in fresh fixative at 4ºC, after which 1mm³ pieces of myocardial tissue were excised from the outer wall of the left ventricle. The pieces of tissue from each animal were separated into two groups after they had been washed free of excess fixative in three one-hourly changes of phosphate buffer. One group of tissue from each animal was postfixed in 1% OsO4 in phosphate buffer for 1½ hours for morphometric work involving determination of myofibrillar Vі/V cell = Pi/Pcell (where Vi is the volume of organelle, V cell the volume of cell, Pi, the number of points (2) Fig. 1-3. Electron micrographs of left ventricular myocytes showing general features: (1) Control hamster. The sarcomeres of the myofibrils are well aligned and T-tubules (T) are quite prominent. Arrows indicate SR and L lipid bodies. x20,000. (2) Experimental hamster. x20,000. (3) Bat. General features are similar to those of control and experimental hamsters x20,000 Fig. 4. Section of left ventricular myocyte of experimental hamster. Some of the small vesicles representing SR tubules (arrows) contain dense material. x25,000. (3) determined by number of intersections of grid lines on the organelle and P cell the total number of points covering the whole micrograph [9].) Surface densities of myofibrils, SR and T-tubules were estimated using a transparent 1 cm semicircular test grid [10]. The number of times test lines intersected the surface boundaries of each type of organelle and the number of points or semicircles covering the whole micrograph were determined. The number of points was then substituted in the equation: RESULTS In general, ventricular myocytes of the bat and of the two groups of hamsters have similar basic ultrastructure (Figs 1-3). However, lipid bodies are virtually absent in myocytes of the hibernating (experimental) hamsters (Fig. 2). The SR is made up of a network of branching and interconnecting tubules in the sarcoplasm. There are two main divisions of SR- those that form couplings with the sarcolemma (junctional SR or JSR) and the ones that surround myofibrils and mitochondria (free SR or FSR). The JSR couples with the surface sarcolemma and non-specialized parts of the intercalated disc (peripheral junctional SR or PJSR) and those of T-tubules (internal junctional SR or IJSR). Sv = 2 1/L (where I is the total number of intersections between organelle boundaries and test lines and L, the test line length. L=LT which is calculated thus): LT = PT.π/2d (where PT is the number of points of test frame covering the area of micrograph and d, the distance between two test points) [8]. The area of myofibrils covered by free SR (FSR) or the SR/myofibril ratio was estimated from micrographs of transverse and oblique sections of the myocytes. The same micrographs were also used to determine the extent of coverage of T-tubule surface by the internal junctional SR (IJSR) (IJSR/T-tubule ratio). A semicircular test grid was used for point counting. To determine the SR/myofibril ratio, (IFSR/Imyo), test line intersections with FSR (IFSR) as well as the test line intersections with remaining myofibrillar surface (Imyo) were counted. To determine the IJSR/T-tubule ratio, the number of test line intersections on IJSR-Ttubule junctions was substituted in the ratio, ½ Sv (IJSR)/Sv(t) [4]. This ratio was halved, because the relationship between the IJSR membrane and the T-tubule in cardiac muscle is dyadic (i.e. IJSR is applied to one side of the Ttubule). Data from stereological measurements were anlysed by Instat II statistical software using one-way ANOVA and Tukey-Kramer multiple comparison tests. Fig. 5. (4) Electron micrographs of bat left ventricular myocytes showing (a) peripheral junctional sarcoplasmic reticulum (PJSR) and (b) internal junctional sarcoplasmic reticulum (IJSR). Note linearly organized luminal granules. Arrows indicate junctional processes from outer membrane of JSR. (a) 90,000 x (b) 75,400 x The configuration of FSR tubules is similar in all three groups of animals (Figs. 1-3), except that in the hibernating hamster, some contained more electron-dense material (Fig. 4).Linearly organised luminal granules are encountered in IJSR and PJSR of myocytes of bat and hamster (Fig. 5). The sections of SR tubules at the junctional complexes also have feet-like processes that projected toward the sarcolemma (Fig. 5), with a junctional gap of about 20 nm. FSR tubules in the experimental hamster are wider in diameter (75 nm) than in the control hamster (46.2 nm) and in the bat (52.3 nm) (Fig. 6). Similarly, the IJSR tubules in the hibernating hamster (67 nm) are wider in comparison to those in the other animals studied (36.8 in control hamster and 38.8 nm in the bat) (Fig. 7). Fig. 7. Comparison of mean diameters of free sarcoplasmic reticulum (nm) ± S.E.M. in left ventricular myocytes of control (CH) and experimental (EH) hamsters and bat (PP). By one-way ANOVA: F 2 , 12 =5.5,p=0.0197 Stereologically, there were significant differences in SR, myofibrils and T-tubule volume fractions in ventricular myocytes of the three groups of animals studied. Volume fraction of SR was significantly higher in the bat (4%) and in the non-hibernating or control hamster (3.5%), than in the experimental hamster (2%). Comparative volumes of myofibrils were 49.0% in the bat, 60.6% in the control hamster and 51.5% in the experimental hamster. T-tubule volumes on the average, were 0.8% in the bat, 1.5% in the control hamster and 0.9% in the experimental hamster. These values are summarized in Tables 1 and 2. Surface densities of myofibrils, SR and T-tubules are given in Table 3. Myofibrillar surface density relates to myofibrillar volume, being greater in the hamsters than in the bat. Surface densities of total SR and IJSR are comparable in all groups, there being no significant differences. Free SR myofibril ratio was significantly lower in the experimental hamster (32%) than in the control hamster (40%) and in the bat (54%). Fig. 6. Comparison of mean diameters of free SR (nm) ± S.E.M. (nm) in left ventricular myocytes of control (CH) and experimental (EH) hamsters and bat (PP). By one-way ANOVA: F 2 , 12 = 11.8, p = 0.0014. By Tukey-Kramer test *p < 0.01 between EH and CH, and between EH and PP. There was no significant difference between control hamster and bat (5) (Fig. 8). IJST/T-tubule ratio in control hamster was about half what it was in the bat (Fig. 9). Fig. 8. Ratio of free SR in apposition to myofibrils (mean ± S.E.M.): comparison of control hamster (CH), experimental hamster (EH) and bat (PP). Fig. 9. Density of internal junctional SR on T-tubules (mean ± S.E.M.) in left ventricular myocytes of control (CH) and experimental (EH) hamsters and bat (PP) DISCUSSION It is evident from the present study that there are significant differences in SR volumes and in FSR/myofibril and IJSR/T-tubule ratios in the ventricular myocardium of the bat, the nonhibernating and hibernating hamster. The SR volume fraction is larger in the bat than in the two groups of hamster. This is not surprising as the bat, Pipistrellus pipistrellus, has a very high heart rate (about 1000 beats/min) [6] compared to the hibernating hamster (5 beats/min) [6]. Indeed, evidence from the results of Leak [11] and Yamamoto [12] support this view that volume of SR is related to the cardiac cycle. In cardiac myocytes of the boa constrictor, a reptile that has a heart rate of about 15 beats/min Leak [11] observed a sparse SR. Yamamoto [12] made a similar observation in the goldfish that has a heart rate of 25 beats/min. The significance in the volume fraction of SR becomes even more apparent when one relates it to the volume ratio of myofibrils and T-tubules. With low volume ratio of myofibrils in the bat than in the hamsters, the SR/myofibril ratio in (the bat) is therefore even much higher. Similarly, the low volume ratio of T-tubules in the bat compared with the non-hibernating hamsters gives a higher SR/T-tubule ratio in the bat, which is likely to equip the bat for higher functional cardiac activity. As expected, reduction in SR volume in the hibernating hamster matched reduction in myofibril and T-tubule volumes, as compared with the nonhibernating hamster. There were no differences, therefore, in SR/myofibril and SR/T-tubule ratios for the two groups of hamsters. The results of the present study also confirm that the T-tubules are generally wider in the animals investigated in the present study than in most mammals. The wide T-tubules reported in the bat and hamsters have been related to their ability to survive in extreme bradycardia [13]. The heart rate of the hamster is reduced from 300-500 beats/min to 5 beats/min when the animal is in stupor or hibernation [6]; that of the bat (Pipistrellus pipistrellus) which in normal activity ranges between 600-1000 beats/min, (6) Table 1: Volume fractions of myofibrils, SR and T-tubules of left ventricular myocytes of nonhibernating and hibernating hamsters and of bat (%mean±S.E.M.). Myofibrils Sarcoplasmic reticulum T-tubules Non hibernating hamster (CH) 60.63±1.93* 3.46±0.34 1.55±0.19 Hibernating hamster (EH) 51.49±2.81* 1.92±0.21 0.93±0.05 Bat (PP) 49.03±1.54* 3.98±0.55§ 0.81±0.03 For each group of animals n=5. Initial comparison is made using one-way ANOVA; further comparison is made using Tukey-Kramer’s test where significant differences indicated:*p<0.05 between CH and EH, p<0.01 between CH and PP; §p<0.05 between EH and PP, p<0.01 between EH and PP ‡p<0.001 between CH and EH, and between CH and PP. Tables 2: SR/myofibril, SR/T-tubule and T-tubule/myofibril volume ratios of non-hibernating and hibernating hamsters and of bat. SR/Myofibril SR:T/tubule T-tubule/Myofibril Non-hibernating hamster 1 : 17.5 1 : 0.45 1 : 29.2 Hibernating hamster 1 : 26.8 1 : 0.48 1 : 53.1 Bat 1 : 12.3 1 : 0.20 1 : 53.4 SR-sarcoplasmic reticulum Table 3: Surface densities (μm ֿ◌¹) of myofibrils, sarcoplasmic reticulum and T-tubule membranes in nonhibernating (CH) and hibernating (EH) hamsters and bat (PP) left ventricular myocytes (mean ±S.E.M.). Myofibrils SR IJSR T-tubules Non-hibernating hamster 1.26±0.07* 0.49±0.07 0.04±0.01 0.16±0.02‡ Hibernating hamster 1.08±0.07 0.35±0.07 0.01±0.00 0.12±0.00 Bat 0.56±0.06 0.31±0.03 0.03±0.00 0.08±0.01 Comparison by one-way ANOVA is followed by Tukey-Kramer test where significant differences indicated. *p<0.001 between PP and CH, and between PP and EH. ‡p<0.01 between PP and CH. (7) of myofibrillar surface by free SR (FSR) in ventricular myocytes of the bat add to the structural features that would facilitate reabsorption of calcium from the environs of myofibrils to induce relaxation after the contractile process. That this is likely an adaptation is supported by the work of Forbes and van Niel [4] in which myocardial cells of the mouse have higher FSR/myofibril ratio than in the guinea pig, the heart rate of the mouse being also much higher (600 beats/min) than in the guinea pig (230-300beats/min). An interesting observation is the wider SR tubules of ventricular myocytes of the experimental hamster than in either control hamster or bat myocytes. The functional significance of wide IJSR tubules in the experimental hamster is not easy to deduce but could suggest that more calcium is stored in the SR here, as myocytes of the hibernating mammal likely depend more on intracellular than extracellular calcium for initiation of the contractile process [18]. Wang & Lee [19] reported that accumulation of calcium by the SR increases two to three-fold in myocytes of hibernating ground squirrel, indicating greater amounts of calcium stored in these elements for contraction. This adaptation would also be necessary for the large stroke volume required of the heart of hibernators, even when cardiac output is reduced to 1/60 of normal level in such states [20]. The wider FSR tubules observed in myocytes of hibernating hamsters could also suggest greater accumulation of calcium from a more rapid uptake of that ion [19; 21]. In animals in which heart rate is significantly reduced in response to conditions such as diving or hibernation, this may be the mechanism by which more calcium is withdrawn from the cytoplasm for relaxation, especially as FSR/myofibril ratio is low. falls to about 30 beats/min in hibernation [6]. In the grey seal in which T-tubules are also wide (450 nm) [14], the heart rate drops from 120 beats/min to 4 beats/min during diving [15]. T-tubules as invaginations of the sarcolemma increase surface area of cell membrane available in myocyte for metabolic exchanges. In animals in which they feature prominently, metabolic exchanges in myocardial cells would be more efficient. This would protect cardiac cells with such features from arrhythmia, and ensure effective excitation-contraction coupling in extreme conditions [16], enabling efficient exchanges of calcium, sodium, and potassium and other ions required for excitationcontraction. That ventricular myocytes of the bat in this study have lower volume fraction of T-tubules compared to those of the control hamster, is surprising since demands on the cardiac cell in the bat are far greater than in the hamster. With a faster heart rate, myocytes of the bat should have a wider system of T-tubules compared to control and experimental hamsters. If T-tubules represent the major pathway for the entry of extracellular calcium into the cardiac cell [3], then, they should be more extensive in myocytes of the bat. This focuses more attention on the SR which is also important in calcium ion regulation and which is more preponderant in the bat. The SR to T-tubule and SR myofibrillar ratios in Table 2, indicate that the myocytes of the bat are better adapted for survival in extreme conditions. This observation is supported by Kawamura et al [17] who reported that myocytes of working ventricular cells of the little brown bat (Myotis lucifugus), with a heart rate of 540 beats/min (under anesthesia), have higher SR to myofibril ratio compared to myocytes of larger terrestrial mammals. As expected, proportion of IJSR in apposition to the T-tubule (IJSR/T-tubule ratio) is also higher in the bat than in control and experimental hamsters. In the bat, this would enable more rapid mobilization of calcium for initiation of contraction. The greater coverage ACKNOWLEDGMENT E. Dennis is grateful to the government of Ghana and Tulane University for financial (8) 11. Leak LV (1967) The ultrasturcture of myofibres in a reptilian heart: the boa constrictor. American Journal of Anatomy 120: 553-582 12. Yamamoto T (1967) Observations on the fine structure of the cardiac muscle cells in the goldfish (Carassius auratus). In Sano T, Mizuhira V, Matsuda K (eds). Electrophysiology and ultrastructure of the heart, pp. 1-13. Bunkodo Company Limited, Tokyo. 13. Ayettey AS, Navaratnam V (1981) The Ttubule system in the Specialised and general myocardium of the rat. Journal of Anatomy 127: 125-140. 14. Ayettey AS, Navaratnam V (1980) The fine structure of myocardial cells in the grey seal. Journal of Anatomy 131: 748. 15. Harrison RJ, Ridgeway SH (1976) Deep Diving in Mammals. Meadowfield Press, Durham. 16. Navaratnam V (1987) Heart Muscle: Ultrastructural Studies. Cambridge Press. 17. Kawamura K, Urthaler F, James, TN (1978) Fine structure of the conducting system and working myocardium in the little brown bat, Myotis lucifugus. In Kobayashi T, Ito Y, Rona G (eds). Recent advances in studies in cardiac structure and metabolism, pp. 81-91. University Park Press, Baltimore. 18. Kondo N, Shibata S (1984) Calcium source of excitation-contraction coupling in myocardium of non-hibernating and hibernating and hibernating chipmunks. Science 225: 641-643. 19. Wang LCH, Lee TF (1989) Perspectives in research: concepts and executions. In Malan A, Canguilhem B (rds). Living in the cold, pp509-519. Colloque INSERM/John Libbey Eurotext Limited. London. 20. Lyman CP (1982) In Lyman C, Willis JS, Malan NA, Wang LCH (eds). Hibernation and Torpor in mammals and birds, pp. 39-91 and 104-123. Academic Press, New York. 21. Belke DD, Pehowich DJ, Wang LCH (1987) seasonal variation in calcium uptake by cardiac sarcoplasmic reticulum in a hibernator, Richardson’s ground squirrel. Journal of Thermoregulatory Biology 12 (2): 53-56. assistance. We thank Dr. I-Li Chen formerly of Department of Anatomy, Tulane University Medical School for technical advice and Drs. F. Addai, University of Ghana Medical School and G. 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