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Changes in heart ultrastructure during development of Strigamia maritima leach (Myriapoda, Chilopoda, Geophilidae)
Int. J. InsectMorphoL &Embryo/., Vol. 13, No. 3, pp. 233 to 245, 1984.
Printed in Great Britain.
0020- 7322/84$3.00 + .00 © 1984PergamonPressLtd.
CHANGES IN HEART ULTRASTRUCTURE DURING DEVELOPMENT OF STRIGAMIA M A R I T I M A LEACH (MYRIAPODA, CHILOPODA, GEOPHILIDAE)* STEINAR OKLAND Cellular Cardiology Research Group, Zoological Laboratory, University of Bergen, Allegt. 41, 5000 Bergen, Norway (Accepted 18 January 1984)
A b s t r a c t - - T h e heart ultrastructure of 4 instars of Strigamia maritima (Myriapoda, Chilopoda, Geophilomorpha) (from 2 weeks to 5 years after hatching) is described and compared morphometrically. The single-layered, circular myofibers extend from middorsal to midventral regions, and are interconnected by short, interdigitating intercalated discs. The cardiac sarcomeres show distinct Z-, I-, and A- bands, but myofilaments do not form a well-ordered array. T-tubules originate from any part of the sarcolemma, forming a network of transverse and longitudinal tubules. The transverse tubules ramify in the heart of the foetus instar. The sarcoplasmic reticulum forms a loose sheath at Z-level, and participates in the formation of dyadic and triadic interior couplings. SR-tubules form peripheral couplings on both sides of the myocardium. Volume and length of the myofibers increase constantly during embryonic instars and the first 4 of 5 adult instars, accompanied with an increase in the volume fraction of contractile elements and mitochondria. New sarcomeres are formed abluminally and distally in the fibers, and sarcomeres increase in diameter. Myofibrils become better aligned, longitudinally in the fiber. The growth rate is reduced in the 4th adult instar, and the rough sarcoplasmic reticulum disappears in the 5th instar. Index descriptors (in addition to those in title): Sarcomerogenesis, cardiac sarcomere, T-tubules, sarcoplasmic reticulum.
INTRODUCTION FEW STUDIES exist on cardiac myogenesis in arthropods, and none at all on myriapod
heart development. Adult myocardium in myriapods has been described by Seifert and Rosenberg (1978) and Okland et al. (1982a) and as to cardiac myogenesis, some have dealt with insects (McCann and Tadkowski, 1980; Sedlak and Witten, 1976; Seifert and Schluter, 1983; Tadkowski and McCann, 1980). Okland et al. (1982a) examined the membrane systems and the sarcomere structure of the heart of adult Lithobius forficatus. It contains an extensive T-tubular system, with longitudinal tubules distending for several sarcomeres, dilating at Z-level of the parallel myofibrils. Interior couplings were mainly found at Z-level. Peripheral couplings were seen on both sides of the myocardium. The fenestrated SR sheaths were concentrated at Iand Z-levels of the sarcomeres. Tubules from this SR-sheath penetrated the continuous Z-disc. The cells contained an abundance of intercalated discs (ID), and nerve endings were reported. The aim of the present study is to describe the cardiac development in Strigamia maritima, and compare its ultrastructure with that of L. forfic'atus (Okland et al., 1982a). *This work was supported by grants from the Norwegian Research Council for Science and the Humanities. 233
234
STEINAR0KLAND
MATERIALS AND METHODS Live specimens of S. maritima were collected at sandy sea shores at Herdla near Bergen, Norway. On the west coast of Norway the eggs are laid in late June, and reared by the female for about 8 weeks. During this period, the larvae hatch as peripatoid (postembryonal) instars after 3 weeks, and pass to a foetus instar after 5 weeks, before passing to the 1st adult instar (adolescens 1 instar), and continue through 4 adult instars to adolescens V. Each adult instar lasts about a year. Most of the animals reproduce and subsequently die in adolescens IV (4 yr old), but a few survive to adolescens V. The following instars were collected and examined: foetus (just after molting, 2 weeks), adolescens IA (just after molting, 5 weeks), adolescens IB (just before molting, 1 yr), adolescens IV and adolescens V. The animals were decapitated, and the ventral parts removed. Some of the animals were cut transversely into sections, and others were stretched while being fixed. Transmission electron microscopy The tissues were fixed in situ for 3.5 hr in a modified Karnovsky fixative, in which distilled water was replaced
by a Ringer solution. After a short rinse in a phosphate buffered 4°7o sucrose solution, the tissues were postfixed for 1.5 hr in 1°70 OsO,, rinsed briefly in a compatible buffer, dehydrated through an acetone series and embedded in Epon 812. Sections, cut with a diamond knife on a Reichert Ultracut microtome, were stained for 1.5 hr in a 2°7o aqueous uranyl acetate solution, 5 rain in Reynolds lead citrate solution. The sections were examined in a Jeol 100CX, or a Jeol 100S transmission microscope at 80 kV in the Department of Electron Microscopy, The Faculty of Science, University of Bergen. The ruthenium red solution added to some fixatives was as described by Myklebust (1975). Scanning electron microscopy The tissue was fixed in situ for 3.5 hr, dehydrated through acetone, critical-point dried, coated with gold and
viewed using a Jeol JSM-35R microscope. GENERAL
DESCRIPTION
D e v e l o p m e n t is h e m i m e t a b o l o u s in Strigamia m a r i t i m a (i.e. s e g m e n t a t i o n is c o m p l e t e d b e f o r e hatching), a n d the structure o f the h e a r t wall is identical in all p a r t s o f the t u b u l a r heart. T h e c a r d i a c wall consists o f a thin, single-layered m y o c a r d i u m (Figs. 1, 12, 13), covered on b o t h sides by a thick, b r a n c h i n g b a s e m e n t m e m b r a n e (Figs. 6, 12, 13, 22). T h e l u m i n a l b a s e m e n t m e m b r a n e is the thicker o f the two (Figs. 13, 22). In the m a t e r i a l t r e a t e d with r u t h e n i u m red a n d t a n n i n , the outer layer o f the b a s e m e n t m e m b r a n e s c o n t a i n s electrondense g r a n u l a t i o n s (Figs. 6, 22). T h e r e is no e p i c a r d i u m or e n d o c a r d i u m . The m y o f i b e r s are circularly a r r a n g e d (Figs. 1, 12, 18). T h e y are slightly p o l a r i z e d , in t h a t m o s t o f the c o n t r a c t i l e m a t e r i a l is l o c a t e d t o w a r d s the l u m i n a l side, whereas the nucleus a n d m i t o c h o n d r i a are t o w a r d s the a b l u m i n a l side (Figs. 1, 10, 15). E a c h m y o f i b e r extends f r o m m i d d o r s a l to m i d v e n t r a l region, a n d t h e y are j o i n e d by i n t e r c a l a t e d discs (Figs. 1, 12, 13). T h e nucleus has a lateral p o s i t i o n , m i d w a y a l o n g the fiber (Fig. 1). T h e m y o f i b e r s are oval in transverse sections (Fig. 20). T h e h e a r t in all instars is c a p a b l e o f s t r o n g s u p e r c o n t r a c t i o n , resulting in a n e a r l y b l o c k e d l u m e n (Fig. 13, T a b l e 1). Contractile material
In m o s t places, the m y o f i b r i l s are closely p a c k e d in l o n g i t u d i n a l b u n d l e s (Figs. 6, 16, 25), b u t m a y be d i s o r g a n i z e d at the a b l u m i n a l side, near the ID (Fig. 8) a n d close to the nucleus (Fig. 15). T h e s a r c o m e r e s have distinct Z-, I- a n d A - b a n d s (Figs. 6, 22, 24). N o H- or M - b a n d s have been f o u n d . T h e Z - b a n d s m a y be c o n t i n u o u s (Figs. 6, 23, 25), or p e r f o r a t e d by SRtubules (Figs. 21, 22). T h e s a r c o m e r e s v a r y in length, with 3.81xm as the m a x i m u m (Table 1), a n d the thick m y o f i l a m e n t s have a core o f low electron density. T h e m y o f i l a m e n t s d o not f o r m a w e l l - o r d e r e d a r r a y (Figs. 20, 24), a n d each thick f i l a m e n t is s u r r o u n d e d by 6 to 12 thin f i l a m e n t s .
Cardiac Myogenesis in a Centipede
FiGs. 1 - 4 . Captions on p. 236.
235
236
STEINAR0KLAND
Sarcolemma The luminal and abluminal surface is s m o o t h in relaxed myofibers (Fig. 12), becoming increasingly convoluted in contracted fibers (Fig. 13). Three types o f myocardial cell borders can be distinguished: I. A narrow and convoluted type, partly filled with basement m e m b r a n e material (Figs. 5, 8, 10, 19). II. A wide lateral cell border, filled with basement m e m b r a n e material (Fig. 20). III. The end-to-end contact complex i.e. the middorsal and midventral intercalated discs (ID) (Figs. 1, 12, 13). The basement m e m b r a n e branches and fills the intercellular gap in the short, interdigitating ID (Figs. 8, 9). Fasciae adherentes are frequent in these areas.
The membrane systems The transverse tubular system (T-tubules).
This originates f r o m sarcolemmal invaginations. T-tubules invaginate f r o m all parts o f the sarcolemma (Figs. 2, 5, 9, 20). They are not restricted to any special sarcomeral band level. Longitudinal tubules (LTtubules) extend for several sarcomeres (Figs. 7, 11, 14). They invaginate directly f r o m the ID area (Fig. 9) and f r o m the convoluted cell border (Fig. 5). At or near the Z-level, LTtubules dilate (Figs. 7, 9, 14) and are continuous with the transversely running T-tubules (Tz-tubules) (Figs. 2, 4). Tz-tubules participate in the f o r m a t i o n of interior couplings (Figs. 4, 7, 23), and originate directly f r o m the sarcolemma (Figs. 2, 20, 24). M o r e than one LT-tubule m a y be seen between neighboring myofibrils (Fig. 23). Smooth sarcoplasmic reticulum (SSR). This is mostly accumulated as a loose fenestrated sheath a r o u n d the myofibrils at Z-level and parts o f the I-bands (Figs. 22, 23). SR-tubules penetrate the Z-disc (Figs. 21, 22), and extend also up to the sarcolemma to f o r m peripheral couplings (Fig. 15). Interior couplings are f o u n d both as dyadic (Figs. 4, 7, 11, 21, 23) or triadic couplings (Fig. 7) at Z-level o f the sarcomere, but interior and peripheral couplings are, in fact, f o u n d at all sarcomeral levels (Figs. 15, 24). Rough sarcop!asmic reticulum (RSR). This is mostly located abluminally, in all instars up to adolescens V (Figs. 3, 10, 17).
Innervation The dorsal ID (Figs. 1, 9, 12) and normally the ventral ID (Figs. 8, 12) are accompanied by ensheathed nerve bundles. The synaptic parts o f the axons are f o u n d in depressions in the myocardial surface (Fig. 16), and contain dense and clear vesicles. The synaptic membranes are not specialized. The synaptic gap is 10 nm wide.
FIG. 1. Transverse section of foetus heart. Two contracted myocardial cells are joint middorsally (arrow head) and midventrally (arrow) by intercalated discs. Dorsal ID is accompanied by a nerve bundle (N), and nuclei (n) in myofibers are found laterally, midway along fibers. Ep = epicardial cells; L = heart lumen, x 4000. FIG. 2. Foetus heart. Tz-tubules invaginate from sarcolemma (arrows) and ramify (asters) to several Z-band levels (dots). These Tz-tubules are continuous with LT-tubules (arrow head). L = heart lumen. Ruthenium red stained, x 28,000. FXG. 3. Foetus heart. Myofibers are slightly polarized, with nucleus (n), most of mitochondria (m), rough sarcoplasmic reticulum (asters), glycogen and ribosomes at abluminal side. L = heart lumen; z = Z-levels. × 14,000. FIG. 4. Longitudinal section of foetus heart, parallel to longitudinal surface through contractile layer, showing several parallel sarcomeres. Sarcomeres are separated by a LT-tubule (arrow heads) which dilate at Z-level (asters). LT-tubule is continuous with the Tz-tubules (arrows), which form interior couplings at Z-level (double arrow head). L = heart lumen; n = nucleus; z = Z-level. x 12,000.
237
Cardiac Myogenesis in a Centipede TABLE 1. COMPARISONOF VARIABLECHARACTERISTICSOF MYOCARDIALCELLS 1N FOUR INSTARS*
Area o f heart lumen measured in lam M a x i m u m area of one myocardial cell (~tm 2) Volume fraction of contractile materia Volume fraction of mitochondria Sarcomere diameter (Ixm)~/ Sarcomere length (~tm)
Foetus
Adolescens IA
Adolescens IB
Adolescens IV
Max 240 Min 120 130 43.1 6.4 0 . 2 5 - 0.43 mean 0.33 1.8 - 3.2
Max 1100 Min 170 190 46.1 7.8 0 . 2 7 - 0.65 mean 0.43 2.3 - 3.5
Max 1200 Min 25 700 54.3 14.7 0 . 5 - 1.1 mean 0.7 1.3 - 3.0
Max 3100 Min 110 820 54.9 13.3 0.75 - 1.4 mean 1.0 1.5 - 3.8
Nt 20 50 50 50
50
*The measurements for adolescens V are similar to adolescens IV. t N u m b e r s of measurements. ~/Mean diameter for comparison only.
FIG. 5. Foetus heart. Convoluted cell border (arrow heads) is strengthened by small desmosomes (circle), and LT-tuhules invaginate from sarcolemma (arrow). L = heart lumen; z = Z-level. x 21,000. FIG. 6. Foetus heart. Neighboring sarcomeres can often be separated only because they are not aligned (compare upper and lower Z-levels (asters)). Both luminal (arrow) and abluminal (arrow head) basement m e m b r a n e contain electrondense granulation. L = heart lumen. Ruthenium red stained. x 22,000.
FOETUS
INSTAR
The heart is fully formed, and most of the initial sarcomerogenesis is completed. There is more than a myofibril to the myofiber (Figs. 2, 6). A full set of bands is found in the
238
STEINAR 0KLAND
FIG. 7. Adolescens IA heart. T-tubules dilate at Z-levels (asters), and form dyadic (arrow head) and triadic (arrows) interior couplings with sarcoplasmic reticulum. L = heart lumen; n = nucleus. × 13,000. FIG. 8. Adolescens IA heart. Oblique section of ventral intercalated disc (ID), with accompanying nerve bundles (N). Parts of convoluted cell border (between arrow heads), bl = blood corpuscles; L = heart lumen; arrows = Z-levels. × 7000.
sarcomeres (Fig. 4), but the myofibrils are disorganized and may branch (Fig. 2). The basic architecture of the membrane systems has been established (Figs. 4, 5). Tz-tubules ramify, and make contact with several Z-levels (Fig. 2). The only intercellular junctions found are the fasciae adherentes in the ID, and small desmosomes in the convoluted cell border (Fig. 5). The cells have a relatively small amount of contractile material and mitochondria (Table 1). Non-contractile parts of the cells are mostly filled with RSR, ribosomes and glycogen (Fig. 3). ADOLESCENS
1A
INSTAR
(5WEEKS
AFTER
HATCHING)
The development during this 3-week period is related to cell growth (Table 1). the myofibers increase in length and volume, along with the contractile material, mitochondria, and RSR. Myofibrils are still disorganized (Fig. 11). Tz-tubules are not ramifying. ADOLESCENS
1B
1NSTAR
( I
Y R)
There is much increase in cell volume during the 1st winter (Table 1). The contractile material and mitochondria increase in relative volume (Table 1). Small nexuses appear in the ID. Though the contractile material improves in organization, the disorganized myofibrils are still seen close to the ID, and the nucleus (Fig. 15).
Cardiac Myogenesis in a Centipede
239
Fro. 9. Adolescens IA heart. Dorsal intercalated disc (ID) is always accompanied by ensheathed nerve bundles (N). LT-tubules (arrows) invaginate from ID, and dilate (arrow heads) at Z-levels (Z). L = heart lumen, x 11,000. FIG. 10. Adolescens IA heart. Non-contractile parts of myofibers are filled with rough sarcoplasmic reticulum and mitochondria (asters). Parts of a convoluted cell border (between arrows). L = heart lumen; Arrow heads = Z-levels. x 7000. FIG. 11. Adolescens IA heart. An LT-tubule (arrow heads) which extends for several sarcomeres, dilates (asters) at Z-levels (Z) and forms a dyadic interior coupling (arrow). x 13,000.
ADOLESCENS
4
INSTAR
There is a continuous increase in length and volume during the first 4 yr. Similarly, the volume fraction of myofilaments and mitochondria increases. The disorganized contractile material is now rudimentary. The convoluted cell borders are strengthened with many desmosomes and small nexuses (Fig. 19). Desmosomes are seen in the ID and in the lateral cell border (Fig. 20). ADOLESCENS
5
INSTAR
O n l y a s l i g h t i n c r e a s e i n cell v o l u m e is f o u n d , a n d R S R is n o t d e t e c t e d .
240
STEINAR 0KLAND
FIG. 12. Adolescens IB heart. Transverse section of heart in a partly contracted state. Two myofibers distend from middorsal ID (arrow) to midventral ID (arrow head). L = heart lumen; N = dorsal nerve bundle; n = ventral nerve bundle, x 3300. FIG. 13. Adolescens IB heart. Transverse section of heart in a supercontracted state. Arrow = dorsal ID; arrow head = ventral ID; asters = luminal basement membrane; dots = abluminal basement membrane; L = heart lumen; N = dorsal nerve bundle. × 3300.
Cardiac Myogenesis in a Centipede
FIGS. 14-- 17. Captions on p. 242.
241
242
STE1NAR OKLAND DISCUSSION
The heart in S. maritirna is fully formed, and most of the initial sarcomerogenesis is completed within 2 weeks after hatching. Nevertheless, these myocardial cells are still embryonic insofar as the contractile material is disorganized, and constitutes a relatively small portion of the total cell volume. Rough sarcoplasmic reticulum, abundant in these cells, is also an indication that sarcomerogenesis and growth are far from complete. The contractile layer is normally more than one sarcomere thick, and several sarcomeres wide in each cell. The membrane systems are formed, but the ramification of Tz-tubules to several Z-levels, is, in fact, closely similar to those of the posterior parts of the anostracan crustacean heart, regarded as embryonic by Okland et al. (1982b). The ramification of Tztubules disappears in later instars, indicating that the formation of a T-system, from sarcolemmal invaginations at Z-level in molluscs (Okland, 1982) and in crustacea (0kland et al., 1982b), may be similar in myriapods. This may be a general developmental pat.tern for myocardial cells. This foetal myocardium is loosely organized with thin walls and with wide intercellular gaps in the ID and in the lateral cell borders. The small desmosomes in the convoluted type of cell border is the only intercellular junction found. During the first 4 yr, the myocardial ceils increase both in length (Table 1, maximum area of heart lumen) and in total volume (Table 1, area of one myocardial cell). This increase in volume is prominent during the 1st winter. New sarcomeres are formed in the distal parts of the myofibers, and the contractile elements appear disorganized in these areas. New sarcomeres are also formed parallel to the already existing ones, and the abluminal parts are the most probable place, because contractile material is disorganized, and most of the RSR is located in this area. The myofibrils increase in diameter. The contractile elements increase both in organization and amount. This produces a more efficient heart, because the higher the percentage of contractile material in a muscle cell, the less "dead-weight" of non-contractile elements has to be moved during contraction (Katz, 1977). The intercellular junctions, and an increased percentage of mitochondrial volume fraction also indicate an increase in heart efficiency. The divergence in maximum sarcomere length (Table 1) must be regarded as an artifact, owing to the inability to arrest the myofibers in a relaxed state (i.e. stretching such small specimens). In Lithobiusforficatus (Okland et al., 1982a), no stretching was performed, but the heart size allowed us to pick out areas with maximum relaxation (i.e. maximum diameter of lumen). The maximum sarcomere length was 4.0gm. Morphometrical data are not available for L. forficatus (Okland et al., 1982a), but the myocardial ultrastructure is very similar to that in adolescens 4 instar. The basic
FIG. 14. Adolescens IB Heart. Dilations of LT-tubules (arrows) m a y be very long at places where neighboring sarcomeres are not aligned. Arrow heads = LT-tubule; L = heart lumen, x 11,000. FIG. 15. Adolescens IB heart. Sarcomeres m a y be disorganized (arrow head) at abluminal side. SRtubules form peripheral couplings on luminal (double arrow head) and abluminal (arrow) side. n = nucleus; z = Z-levels. R u t h e n i u m red stained, x 13,000. FIG. 16. Adolescens IB Heart. Surface section of a myofiber with m a n y parallel myofibrils, separated by LT-tubules (arrows). Arrow heads = Z-levels; Dots = LT-dilations; n = nerve endings, x 6000. FIo. 17. Adolescens IB Heart. Contractile material m a y still be disorganized, and non-contractile parts of myofibers are filled by mitochondria (m), ribosomes, glycogen and rough sarcoplasmic reticulum (asters). L = heart lumen; Z = Z-levels. × 13,000.
Cardiac Myogenesis in a Centipede
FIGS. 18 - 22. Captions on p. 245.
243
244
STEINAR 0KLAND
Flos. 23 - 2 5 . Captions on p. 245.
Cardiac Myogenesis in a Centipede
245
organization is identical for the contractile elements and the membrane systems with couplings. They have the same 3 types of cell borders. The myocardium in L. forficatus is thicker, and has more parallel sarcomeres. The SR-sheaths are more prominent in L.
forficatus. Despite a good organization and an increased efficiency of the heart during development in S. maritima, the heart wall is so thin that it is reasonable to assume from what is known from molluscs (Okland, 1982), that the somatic muscles participate, and may even play a major part in hemolymph circulation. Acknowledgement--I thank Egild Horneland for collecting the specimens of S. maritima. REFERENCES KATZ, A. M. 1977. Physiology of the Heart, Raven Press, New York. McCANN, F. V. and T. M. TADKOWSKI. 1980. Ultrastructure and electrical activity in embryonic and adult heart cells of the cricket. Comp. Biochem. Physiol. 66A: 447 - 54. MYKLEBUST, R. 1975. The ultrastructure of the myocardial cell in dragonfly Aeschna juncea L. Norwegian J. Zool. 23: 17-36. ~KLAND, S. 1982. The ultrastructure of the heart complex in Patella vulgata L. (Archaeogastropoda, Prosobranchia). J. Moll. Stud. 48:331-41. ~)KLAND, S., A. TJONNELAND, A. NYLUNDand I. CHRIST. 1982a. The membrane systems and the sarcomere in the heart of Lithobiusforficatus L. (Arthropoda, Chilopoda). Zool. Ariz. 208: 124-31. ~)KLAND, S., A. TJ~DNNELAND, L. N. LARSEN and A. NYLUND. 1982b. Heart ultrastructure in Branchinecta paludosa, Artemia salina, Branchipus schaefferi, and Streptocephalus sp. (Crustacea, Anostraca). Zoomorphology 101:71 - 81. SEDLAK,B. J. and J. WHITTEN. 1976. Changes in heart ultrastructure during development of the Flesh Fly, Sarcophaga bullata. Develop. Biol. 54: 308- 13. SEIFERT, G. and J. ROSENBERG. 1978. Feinstruktur der Herzwand des Doppelf0ssers Oxidus gracilis (Diplopoda: Paradoxosomatidae) und ailgemeine Betrachtungen zum Aufbau der Gefasse yon Tracheata und Onychophora. Entomol. Germ. 4: 224- 33. SEXFERT, G. and U. SCHLUTER. 1983. Die postembryonale Entwicklung der Herzwand yon Locusta migratoria (Orthoptera, Acrididae). Zool. Jahrb. Anat. 109: 565- 85. TADKOWSKI, T. M. and F. V. MCCANN. 1980. Ultrastructure and electrical activity in developing heart cells (Insec0. Develop. Biol. 74: 387- 400.
FIG. 18. Adolescens IV heart. Scanning micrograph from ventral side of heart, showing circular muscle cells (asters). Arrow heads = sarcolemmal foldings due to contraction. Bar indicate 1 mm. FIG. 19. Adolescens IV heart. Convoluted cell border with many desmosomes (asters) and small nexus (circle). z = Z-level. Ruthenium red stained. × 27,000. FIG. 20. Adolescens IV heart. Transverse section of a myofiber at A-band level, showing lack of SR between sarcomeres, and hence lack of sarcomeral separation. T-tubules invaginate from all parts of cell surface (arrows). Lateral cell border is very wide (between arrow heads) and is filled with basement membrane material. Aster = dyadic coupling; Double arrow head = desmosome in lateral gap; L = heart lumen. Ruthenium red stained, x 12,000. FIG. 21. Adolescens IV heart. Transverse section of sarcomeres at Z-band (Z) and I-band (1) levels. Z-level is perforated by SR-tubules (arrow heads), and an invaginating Tz-tubule (aster) forms a dyadic coupling (arrow). x 53,000. FIG. 22. Adolescens IV heart. Longitudinal section of a myofiber, showing SR-tubules (arrows) perforating Z-band level (Z). Luminal basement membrane is extremely thick (arrow head). Double arrow head = peripheral coupling; A = A-band level. Ruthenium red stained, x 15,000. FIc. 23. Adolescens IV heart. More than one LT-tubule may separate neighboring sarcomeres (one is indicated by arrows, other by arrow heads). Note SR at Z- and I-band levels (aster). Double arrow head = dyadic interior coupling; z = Z levels. Ruthenium red stained. × 16,000. FIG. 24. Adolescens IV heart. Transverse section of myofiber through Z- (Z), I- (i) and A- (A) band levels. Invaginating transverse tubule (arrow) forms a coupling (arrow head) at A-band level. x 37,000. FIG. 25. Adolescens IV heart. Longitudinal section of a myofiber. Parallel myofibrils are separated by LT-tubules (arrow head), which dilate (aster) at Z-level (Z). × 11,000.
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0020- 7322/84$3.00 + .00 © 1984PergamonPressLtd.
CHANGES IN HEART ULTRASTRUCTURE DURING DEVELOPMENT OF STRIGAMIA M A R I T I M A LEACH (MYRIAPODA, CHILOPODA, GEOPHILIDAE)* STEINAR OKLAND Cellular Cardiology Research Group, Zoological Laboratory, University of Bergen, Allegt. 41, 5000 Bergen, Norway (Accepted 18 January 1984)
A b s t r a c t - - T h e heart ultrastructure of 4 instars of Strigamia maritima (Myriapoda, Chilopoda, Geophilomorpha) (from 2 weeks to 5 years after hatching) is described and compared morphometrically. The single-layered, circular myofibers extend from middorsal to midventral regions, and are interconnected by short, interdigitating intercalated discs. The cardiac sarcomeres show distinct Z-, I-, and A- bands, but myofilaments do not form a well-ordered array. T-tubules originate from any part of the sarcolemma, forming a network of transverse and longitudinal tubules. The transverse tubules ramify in the heart of the foetus instar. The sarcoplasmic reticulum forms a loose sheath at Z-level, and participates in the formation of dyadic and triadic interior couplings. SR-tubules form peripheral couplings on both sides of the myocardium. Volume and length of the myofibers increase constantly during embryonic instars and the first 4 of 5 adult instars, accompanied with an increase in the volume fraction of contractile elements and mitochondria. New sarcomeres are formed abluminally and distally in the fibers, and sarcomeres increase in diameter. Myofibrils become better aligned, longitudinally in the fiber. The growth rate is reduced in the 4th adult instar, and the rough sarcoplasmic reticulum disappears in the 5th instar. Index descriptors (in addition to those in title): Sarcomerogenesis, cardiac sarcomere, T-tubules, sarcoplasmic reticulum.
INTRODUCTION FEW STUDIES exist on cardiac myogenesis in arthropods, and none at all on myriapod
heart development. Adult myocardium in myriapods has been described by Seifert and Rosenberg (1978) and Okland et al. (1982a) and as to cardiac myogenesis, some have dealt with insects (McCann and Tadkowski, 1980; Sedlak and Witten, 1976; Seifert and Schluter, 1983; Tadkowski and McCann, 1980). Okland et al. (1982a) examined the membrane systems and the sarcomere structure of the heart of adult Lithobius forficatus. It contains an extensive T-tubular system, with longitudinal tubules distending for several sarcomeres, dilating at Z-level of the parallel myofibrils. Interior couplings were mainly found at Z-level. Peripheral couplings were seen on both sides of the myocardium. The fenestrated SR sheaths were concentrated at Iand Z-levels of the sarcomeres. Tubules from this SR-sheath penetrated the continuous Z-disc. The cells contained an abundance of intercalated discs (ID), and nerve endings were reported. The aim of the present study is to describe the cardiac development in Strigamia maritima, and compare its ultrastructure with that of L. forfic'atus (Okland et al., 1982a). *This work was supported by grants from the Norwegian Research Council for Science and the Humanities. 233
234
STEINAR0KLAND
MATERIALS AND METHODS Live specimens of S. maritima were collected at sandy sea shores at Herdla near Bergen, Norway. On the west coast of Norway the eggs are laid in late June, and reared by the female for about 8 weeks. During this period, the larvae hatch as peripatoid (postembryonal) instars after 3 weeks, and pass to a foetus instar after 5 weeks, before passing to the 1st adult instar (adolescens 1 instar), and continue through 4 adult instars to adolescens V. Each adult instar lasts about a year. Most of the animals reproduce and subsequently die in adolescens IV (4 yr old), but a few survive to adolescens V. The following instars were collected and examined: foetus (just after molting, 2 weeks), adolescens IA (just after molting, 5 weeks), adolescens IB (just before molting, 1 yr), adolescens IV and adolescens V. The animals were decapitated, and the ventral parts removed. Some of the animals were cut transversely into sections, and others were stretched while being fixed. Transmission electron microscopy The tissues were fixed in situ for 3.5 hr in a modified Karnovsky fixative, in which distilled water was replaced
by a Ringer solution. After a short rinse in a phosphate buffered 4°7o sucrose solution, the tissues were postfixed for 1.5 hr in 1°70 OsO,, rinsed briefly in a compatible buffer, dehydrated through an acetone series and embedded in Epon 812. Sections, cut with a diamond knife on a Reichert Ultracut microtome, were stained for 1.5 hr in a 2°7o aqueous uranyl acetate solution, 5 rain in Reynolds lead citrate solution. The sections were examined in a Jeol 100CX, or a Jeol 100S transmission microscope at 80 kV in the Department of Electron Microscopy, The Faculty of Science, University of Bergen. The ruthenium red solution added to some fixatives was as described by Myklebust (1975). Scanning electron microscopy The tissue was fixed in situ for 3.5 hr, dehydrated through acetone, critical-point dried, coated with gold and
viewed using a Jeol JSM-35R microscope. GENERAL
DESCRIPTION
D e v e l o p m e n t is h e m i m e t a b o l o u s in Strigamia m a r i t i m a (i.e. s e g m e n t a t i o n is c o m p l e t e d b e f o r e hatching), a n d the structure o f the h e a r t wall is identical in all p a r t s o f the t u b u l a r heart. T h e c a r d i a c wall consists o f a thin, single-layered m y o c a r d i u m (Figs. 1, 12, 13), covered on b o t h sides by a thick, b r a n c h i n g b a s e m e n t m e m b r a n e (Figs. 6, 12, 13, 22). T h e l u m i n a l b a s e m e n t m e m b r a n e is the thicker o f the two (Figs. 13, 22). In the m a t e r i a l t r e a t e d with r u t h e n i u m red a n d t a n n i n , the outer layer o f the b a s e m e n t m e m b r a n e s c o n t a i n s electrondense g r a n u l a t i o n s (Figs. 6, 22). T h e r e is no e p i c a r d i u m or e n d o c a r d i u m . The m y o f i b e r s are circularly a r r a n g e d (Figs. 1, 12, 18). T h e y are slightly p o l a r i z e d , in t h a t m o s t o f the c o n t r a c t i l e m a t e r i a l is l o c a t e d t o w a r d s the l u m i n a l side, whereas the nucleus a n d m i t o c h o n d r i a are t o w a r d s the a b l u m i n a l side (Figs. 1, 10, 15). E a c h m y o f i b e r extends f r o m m i d d o r s a l to m i d v e n t r a l region, a n d t h e y are j o i n e d by i n t e r c a l a t e d discs (Figs. 1, 12, 13). T h e nucleus has a lateral p o s i t i o n , m i d w a y a l o n g the fiber (Fig. 1). T h e m y o f i b e r s are oval in transverse sections (Fig. 20). T h e h e a r t in all instars is c a p a b l e o f s t r o n g s u p e r c o n t r a c t i o n , resulting in a n e a r l y b l o c k e d l u m e n (Fig. 13, T a b l e 1). Contractile material
In m o s t places, the m y o f i b r i l s are closely p a c k e d in l o n g i t u d i n a l b u n d l e s (Figs. 6, 16, 25), b u t m a y be d i s o r g a n i z e d at the a b l u m i n a l side, near the ID (Fig. 8) a n d close to the nucleus (Fig. 15). T h e s a r c o m e r e s have distinct Z-, I- a n d A - b a n d s (Figs. 6, 22, 24). N o H- or M - b a n d s have been f o u n d . T h e Z - b a n d s m a y be c o n t i n u o u s (Figs. 6, 23, 25), or p e r f o r a t e d by SRtubules (Figs. 21, 22). T h e s a r c o m e r e s v a r y in length, with 3.81xm as the m a x i m u m (Table 1), a n d the thick m y o f i l a m e n t s have a core o f low electron density. T h e m y o f i l a m e n t s d o not f o r m a w e l l - o r d e r e d a r r a y (Figs. 20, 24), a n d each thick f i l a m e n t is s u r r o u n d e d by 6 to 12 thin f i l a m e n t s .
Cardiac Myogenesis in a Centipede
FiGs. 1 - 4 . Captions on p. 236.
235
236
STEINAR0KLAND
Sarcolemma The luminal and abluminal surface is s m o o t h in relaxed myofibers (Fig. 12), becoming increasingly convoluted in contracted fibers (Fig. 13). Three types o f myocardial cell borders can be distinguished: I. A narrow and convoluted type, partly filled with basement m e m b r a n e material (Figs. 5, 8, 10, 19). II. A wide lateral cell border, filled with basement m e m b r a n e material (Fig. 20). III. The end-to-end contact complex i.e. the middorsal and midventral intercalated discs (ID) (Figs. 1, 12, 13). The basement m e m b r a n e branches and fills the intercellular gap in the short, interdigitating ID (Figs. 8, 9). Fasciae adherentes are frequent in these areas.
The membrane systems The transverse tubular system (T-tubules).
This originates f r o m sarcolemmal invaginations. T-tubules invaginate f r o m all parts o f the sarcolemma (Figs. 2, 5, 9, 20). They are not restricted to any special sarcomeral band level. Longitudinal tubules (LTtubules) extend for several sarcomeres (Figs. 7, 11, 14). They invaginate directly f r o m the ID area (Fig. 9) and f r o m the convoluted cell border (Fig. 5). At or near the Z-level, LTtubules dilate (Figs. 7, 9, 14) and are continuous with the transversely running T-tubules (Tz-tubules) (Figs. 2, 4). Tz-tubules participate in the f o r m a t i o n of interior couplings (Figs. 4, 7, 23), and originate directly f r o m the sarcolemma (Figs. 2, 20, 24). M o r e than one LT-tubule m a y be seen between neighboring myofibrils (Fig. 23). Smooth sarcoplasmic reticulum (SSR). This is mostly accumulated as a loose fenestrated sheath a r o u n d the myofibrils at Z-level and parts o f the I-bands (Figs. 22, 23). SR-tubules penetrate the Z-disc (Figs. 21, 22), and extend also up to the sarcolemma to f o r m peripheral couplings (Fig. 15). Interior couplings are f o u n d both as dyadic (Figs. 4, 7, 11, 21, 23) or triadic couplings (Fig. 7) at Z-level o f the sarcomere, but interior and peripheral couplings are, in fact, f o u n d at all sarcomeral levels (Figs. 15, 24). Rough sarcop!asmic reticulum (RSR). This is mostly located abluminally, in all instars up to adolescens V (Figs. 3, 10, 17).
Innervation The dorsal ID (Figs. 1, 9, 12) and normally the ventral ID (Figs. 8, 12) are accompanied by ensheathed nerve bundles. The synaptic parts o f the axons are f o u n d in depressions in the myocardial surface (Fig. 16), and contain dense and clear vesicles. The synaptic membranes are not specialized. The synaptic gap is 10 nm wide.
FIG. 1. Transverse section of foetus heart. Two contracted myocardial cells are joint middorsally (arrow head) and midventrally (arrow) by intercalated discs. Dorsal ID is accompanied by a nerve bundle (N), and nuclei (n) in myofibers are found laterally, midway along fibers. Ep = epicardial cells; L = heart lumen, x 4000. FIG. 2. Foetus heart. Tz-tubules invaginate from sarcolemma (arrows) and ramify (asters) to several Z-band levels (dots). These Tz-tubules are continuous with LT-tubules (arrow head). L = heart lumen. Ruthenium red stained, x 28,000. FXG. 3. Foetus heart. Myofibers are slightly polarized, with nucleus (n), most of mitochondria (m), rough sarcoplasmic reticulum (asters), glycogen and ribosomes at abluminal side. L = heart lumen; z = Z-levels. × 14,000. FIG. 4. Longitudinal section of foetus heart, parallel to longitudinal surface through contractile layer, showing several parallel sarcomeres. Sarcomeres are separated by a LT-tubule (arrow heads) which dilate at Z-level (asters). LT-tubule is continuous with the Tz-tubules (arrows), which form interior couplings at Z-level (double arrow head). L = heart lumen; n = nucleus; z = Z-level. x 12,000.
237
Cardiac Myogenesis in a Centipede TABLE 1. COMPARISONOF VARIABLECHARACTERISTICSOF MYOCARDIALCELLS 1N FOUR INSTARS*
Area o f heart lumen measured in lam M a x i m u m area of one myocardial cell (~tm 2) Volume fraction of contractile materia Volume fraction of mitochondria Sarcomere diameter (Ixm)~/ Sarcomere length (~tm)
Foetus
Adolescens IA
Adolescens IB
Adolescens IV
Max 240 Min 120 130 43.1 6.4 0 . 2 5 - 0.43 mean 0.33 1.8 - 3.2
Max 1100 Min 170 190 46.1 7.8 0 . 2 7 - 0.65 mean 0.43 2.3 - 3.5
Max 1200 Min 25 700 54.3 14.7 0 . 5 - 1.1 mean 0.7 1.3 - 3.0
Max 3100 Min 110 820 54.9 13.3 0.75 - 1.4 mean 1.0 1.5 - 3.8
Nt 20 50 50 50
50
*The measurements for adolescens V are similar to adolescens IV. t N u m b e r s of measurements. ~/Mean diameter for comparison only.
FIG. 5. Foetus heart. Convoluted cell border (arrow heads) is strengthened by small desmosomes (circle), and LT-tuhules invaginate from sarcolemma (arrow). L = heart lumen; z = Z-level. x 21,000. FIG. 6. Foetus heart. Neighboring sarcomeres can often be separated only because they are not aligned (compare upper and lower Z-levels (asters)). Both luminal (arrow) and abluminal (arrow head) basement m e m b r a n e contain electrondense granulation. L = heart lumen. Ruthenium red stained. x 22,000.
FOETUS
INSTAR
The heart is fully formed, and most of the initial sarcomerogenesis is completed. There is more than a myofibril to the myofiber (Figs. 2, 6). A full set of bands is found in the
238
STEINAR 0KLAND
FIG. 7. Adolescens IA heart. T-tubules dilate at Z-levels (asters), and form dyadic (arrow head) and triadic (arrows) interior couplings with sarcoplasmic reticulum. L = heart lumen; n = nucleus. × 13,000. FIG. 8. Adolescens IA heart. Oblique section of ventral intercalated disc (ID), with accompanying nerve bundles (N). Parts of convoluted cell border (between arrow heads), bl = blood corpuscles; L = heart lumen; arrows = Z-levels. × 7000.
sarcomeres (Fig. 4), but the myofibrils are disorganized and may branch (Fig. 2). The basic architecture of the membrane systems has been established (Figs. 4, 5). Tz-tubules ramify, and make contact with several Z-levels (Fig. 2). The only intercellular junctions found are the fasciae adherentes in the ID, and small desmosomes in the convoluted cell border (Fig. 5). The cells have a relatively small amount of contractile material and mitochondria (Table 1). Non-contractile parts of the cells are mostly filled with RSR, ribosomes and glycogen (Fig. 3). ADOLESCENS
1A
INSTAR
(5WEEKS
AFTER
HATCHING)
The development during this 3-week period is related to cell growth (Table 1). the myofibers increase in length and volume, along with the contractile material, mitochondria, and RSR. Myofibrils are still disorganized (Fig. 11). Tz-tubules are not ramifying. ADOLESCENS
1B
1NSTAR
( I
Y R)
There is much increase in cell volume during the 1st winter (Table 1). The contractile material and mitochondria increase in relative volume (Table 1). Small nexuses appear in the ID. Though the contractile material improves in organization, the disorganized myofibrils are still seen close to the ID, and the nucleus (Fig. 15).
Cardiac Myogenesis in a Centipede
239
Fro. 9. Adolescens IA heart. Dorsal intercalated disc (ID) is always accompanied by ensheathed nerve bundles (N). LT-tubules (arrows) invaginate from ID, and dilate (arrow heads) at Z-levels (Z). L = heart lumen, x 11,000. FIG. 10. Adolescens IA heart. Non-contractile parts of myofibers are filled with rough sarcoplasmic reticulum and mitochondria (asters). Parts of a convoluted cell border (between arrows). L = heart lumen; Arrow heads = Z-levels. x 7000. FIG. 11. Adolescens IA heart. An LT-tubule (arrow heads) which extends for several sarcomeres, dilates (asters) at Z-levels (Z) and forms a dyadic interior coupling (arrow). x 13,000.
ADOLESCENS
4
INSTAR
There is a continuous increase in length and volume during the first 4 yr. Similarly, the volume fraction of myofilaments and mitochondria increases. The disorganized contractile material is now rudimentary. The convoluted cell borders are strengthened with many desmosomes and small nexuses (Fig. 19). Desmosomes are seen in the ID and in the lateral cell border (Fig. 20). ADOLESCENS
5
INSTAR
O n l y a s l i g h t i n c r e a s e i n cell v o l u m e is f o u n d , a n d R S R is n o t d e t e c t e d .
240
STEINAR 0KLAND
FIG. 12. Adolescens IB heart. Transverse section of heart in a partly contracted state. Two myofibers distend from middorsal ID (arrow) to midventral ID (arrow head). L = heart lumen; N = dorsal nerve bundle; n = ventral nerve bundle, x 3300. FIG. 13. Adolescens IB heart. Transverse section of heart in a supercontracted state. Arrow = dorsal ID; arrow head = ventral ID; asters = luminal basement membrane; dots = abluminal basement membrane; L = heart lumen; N = dorsal nerve bundle. × 3300.
Cardiac Myogenesis in a Centipede
FIGS. 14-- 17. Captions on p. 242.
241
242
STE1NAR OKLAND DISCUSSION
The heart in S. maritirna is fully formed, and most of the initial sarcomerogenesis is completed within 2 weeks after hatching. Nevertheless, these myocardial cells are still embryonic insofar as the contractile material is disorganized, and constitutes a relatively small portion of the total cell volume. Rough sarcoplasmic reticulum, abundant in these cells, is also an indication that sarcomerogenesis and growth are far from complete. The contractile layer is normally more than one sarcomere thick, and several sarcomeres wide in each cell. The membrane systems are formed, but the ramification of Tz-tubules to several Z-levels, is, in fact, closely similar to those of the posterior parts of the anostracan crustacean heart, regarded as embryonic by Okland et al. (1982b). The ramification of Tztubules disappears in later instars, indicating that the formation of a T-system, from sarcolemmal invaginations at Z-level in molluscs (Okland, 1982) and in crustacea (0kland et al., 1982b), may be similar in myriapods. This may be a general developmental pat.tern for myocardial cells. This foetal myocardium is loosely organized with thin walls and with wide intercellular gaps in the ID and in the lateral cell borders. The small desmosomes in the convoluted type of cell border is the only intercellular junction found. During the first 4 yr, the myocardial ceils increase both in length (Table 1, maximum area of heart lumen) and in total volume (Table 1, area of one myocardial cell). This increase in volume is prominent during the 1st winter. New sarcomeres are formed in the distal parts of the myofibers, and the contractile elements appear disorganized in these areas. New sarcomeres are also formed parallel to the already existing ones, and the abluminal parts are the most probable place, because contractile material is disorganized, and most of the RSR is located in this area. The myofibrils increase in diameter. The contractile elements increase both in organization and amount. This produces a more efficient heart, because the higher the percentage of contractile material in a muscle cell, the less "dead-weight" of non-contractile elements has to be moved during contraction (Katz, 1977). The intercellular junctions, and an increased percentage of mitochondrial volume fraction also indicate an increase in heart efficiency. The divergence in maximum sarcomere length (Table 1) must be regarded as an artifact, owing to the inability to arrest the myofibers in a relaxed state (i.e. stretching such small specimens). In Lithobiusforficatus (Okland et al., 1982a), no stretching was performed, but the heart size allowed us to pick out areas with maximum relaxation (i.e. maximum diameter of lumen). The maximum sarcomere length was 4.0gm. Morphometrical data are not available for L. forficatus (Okland et al., 1982a), but the myocardial ultrastructure is very similar to that in adolescens 4 instar. The basic
FIG. 14. Adolescens IB Heart. Dilations of LT-tubules (arrows) m a y be very long at places where neighboring sarcomeres are not aligned. Arrow heads = LT-tubule; L = heart lumen, x 11,000. FIG. 15. Adolescens IB heart. Sarcomeres m a y be disorganized (arrow head) at abluminal side. SRtubules form peripheral couplings on luminal (double arrow head) and abluminal (arrow) side. n = nucleus; z = Z-levels. R u t h e n i u m red stained, x 13,000. FIG. 16. Adolescens IB Heart. Surface section of a myofiber with m a n y parallel myofibrils, separated by LT-tubules (arrows). Arrow heads = Z-levels; Dots = LT-dilations; n = nerve endings, x 6000. FIo. 17. Adolescens IB Heart. Contractile material m a y still be disorganized, and non-contractile parts of myofibers are filled by mitochondria (m), ribosomes, glycogen and rough sarcoplasmic reticulum (asters). L = heart lumen; Z = Z-levels. × 13,000.
Cardiac Myogenesis in a Centipede
FIGS. 18 - 22. Captions on p. 245.
243
244
STEINAR 0KLAND
Flos. 23 - 2 5 . Captions on p. 245.
Cardiac Myogenesis in a Centipede
245
organization is identical for the contractile elements and the membrane systems with couplings. They have the same 3 types of cell borders. The myocardium in L. forficatus is thicker, and has more parallel sarcomeres. The SR-sheaths are more prominent in L.
forficatus. Despite a good organization and an increased efficiency of the heart during development in S. maritima, the heart wall is so thin that it is reasonable to assume from what is known from molluscs (Okland, 1982), that the somatic muscles participate, and may even play a major part in hemolymph circulation. Acknowledgement--I thank Egild Horneland for collecting the specimens of S. maritima. REFERENCES KATZ, A. M. 1977. Physiology of the Heart, Raven Press, New York. McCANN, F. V. and T. M. TADKOWSKI. 1980. Ultrastructure and electrical activity in embryonic and adult heart cells of the cricket. Comp. Biochem. Physiol. 66A: 447 - 54. MYKLEBUST, R. 1975. The ultrastructure of the myocardial cell in dragonfly Aeschna juncea L. Norwegian J. Zool. 23: 17-36. ~KLAND, S. 1982. The ultrastructure of the heart complex in Patella vulgata L. (Archaeogastropoda, Prosobranchia). J. Moll. Stud. 48:331-41. ~)KLAND, S., A. TJONNELAND, A. NYLUNDand I. CHRIST. 1982a. The membrane systems and the sarcomere in the heart of Lithobiusforficatus L. (Arthropoda, Chilopoda). Zool. Ariz. 208: 124-31. ~)KLAND, S., A. TJ~DNNELAND, L. N. LARSEN and A. NYLUND. 1982b. Heart ultrastructure in Branchinecta paludosa, Artemia salina, Branchipus schaefferi, and Streptocephalus sp. (Crustacea, Anostraca). Zoomorphology 101:71 - 81. SEDLAK,B. J. and J. WHITTEN. 1976. Changes in heart ultrastructure during development of the Flesh Fly, Sarcophaga bullata. Develop. Biol. 54: 308- 13. SEIFERT, G. and J. ROSENBERG. 1978. Feinstruktur der Herzwand des Doppelf0ssers Oxidus gracilis (Diplopoda: Paradoxosomatidae) und ailgemeine Betrachtungen zum Aufbau der Gefasse yon Tracheata und Onychophora. Entomol. Germ. 4: 224- 33. SEXFERT, G. and U. SCHLUTER. 1983. Die postembryonale Entwicklung der Herzwand yon Locusta migratoria (Orthoptera, Acrididae). Zool. Jahrb. Anat. 109: 565- 85. TADKOWSKI, T. M. and F. V. MCCANN. 1980. Ultrastructure and electrical activity in developing heart cells (Insec0. Develop. Biol. 74: 387- 400.
FIG. 18. Adolescens IV heart. Scanning micrograph from ventral side of heart, showing circular muscle cells (asters). Arrow heads = sarcolemmal foldings due to contraction. Bar indicate 1 mm. FIG. 19. Adolescens IV heart. Convoluted cell border with many desmosomes (asters) and small nexus (circle). z = Z-level. Ruthenium red stained. × 27,000. FIG. 20. Adolescens IV heart. Transverse section of a myofiber at A-band level, showing lack of SR between sarcomeres, and hence lack of sarcomeral separation. T-tubules invaginate from all parts of cell surface (arrows). Lateral cell border is very wide (between arrow heads) and is filled with basement membrane material. Aster = dyadic coupling; Double arrow head = desmosome in lateral gap; L = heart lumen. Ruthenium red stained, x 12,000. FIG. 21. Adolescens IV heart. Transverse section of sarcomeres at Z-band (Z) and I-band (1) levels. Z-level is perforated by SR-tubules (arrow heads), and an invaginating Tz-tubule (aster) forms a dyadic coupling (arrow). x 53,000. FIG. 22. Adolescens IV heart. Longitudinal section of a myofiber, showing SR-tubules (arrows) perforating Z-band level (Z). Luminal basement membrane is extremely thick (arrow head). Double arrow head = peripheral coupling; A = A-band level. Ruthenium red stained, x 15,000. FIc. 23. Adolescens IV heart. More than one LT-tubule may separate neighboring sarcomeres (one is indicated by arrows, other by arrow heads). Note SR at Z- and I-band levels (aster). Double arrow head = dyadic interior coupling; z = Z levels. Ruthenium red stained. × 16,000. FIG. 24. Adolescens IV heart. Transverse section of myofiber through Z- (Z), I- (i) and A- (A) band levels. Invaginating transverse tubule (arrow) forms a coupling (arrow head) at A-band level. x 37,000. FIG. 25. Adolescens IV heart. Longitudinal section of a myofiber. Parallel myofibrils are separated by LT-tubules (arrow head), which dilate (aster) at Z-level (Z). × 11,000.