Observations of the Ultrastructure of the Myocardium of the Common Lobster (Homarus americanus) Especially of the Myofibrils’

JOHN R. SMITH Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri

ABSTRACT The ultrastructure of the myocardial cells of the common lobster, Homarus americanus, was examined with the electron microscope. The contractile elements of the cells show the usual A, I and 2 band structures in longitudinally cut sections. Sarcomeres in slightly oblique section frequently exhibit distinct H zones at the midportions of the A bands. The fine filaments of the I band may be seen between the larger A band filaments in many areas of the sarcomeres, although in other areas there appears only to be filamentous “bridging” between the filaments of the A bands. Cross sections through the A band do not show a constant “array” of I and A filaments. The H zones, observed in these myocardial cells, appear to result from the separation of the finely filamentous material between the A rods at the middle of A bands when the sarcomeres are stretched. Notation is also made of the appear- ance of intercalated disks and the thickening and folding of the basement membranes of the sarcolemma at points of cellular angulation. Mitochondria are numerous in these cells, and there is an extensive sarcotubular system.

The mechanisms of the contraction of muscle have been of interest to anatomists and physiologists for many years. The ap- plication of the electron microscope to the study of cellular substructure has re- sulted in renewed interest in fundamental contractile mechanisms. The now-famous ultramicroscopic studies of Huxley (’57) on vertebrate striped muscle have pro- vided a possible explanation of contractile phenomena by the demonstration of many details of filamentous alignments and their possible function in cellular contraction. On the other hand, these anatomical ob- servations have not been completely ac- cepted by other observers (de Villafranca, ’61; de Villafranca and Philpott, ’61; Sjostrand and Anderson, ’56; Sjostrand and Anderson-Cedergren, ’57, ’59). More recently de Villafranca and Philpott (’61), in studies of the ultrastructure of skeletal muscle of certain arthropods, found that the filamentous arrangement was of differ- ent character, and that, on the basis of structure, shortening of muscle could not be fully accounted for by the precise “slid- ing mechanisms” of contraction proposed by Huxley.

During investigation of the “pacemaker ganglion” of the common lobster (Homa- rus americanus), the opportunity arose

for the study of the ultrastructure of the myocardium of this crustacean. Indeed, electron microscopic observation of this muscle disclosed a myofibrillar arrange- ment similar in some respects to that de- scribed by de Villafranca and Philpott (’61) in skeletal muscles of the horseshoe crab. This communication will offer a brief description of the fine structure of the lobster myocardium, especially of the contractile elements.

METHODS

Living lobsters (2 to 3 kg) were chilled one to two hours in the cold room before removal of the hearts. A “window” was incised in the dorsal carapace thereby ex- posing the heart. By careful removal of the segment of carapace the heart was exposed with the surrounding membranes intact. The pericardial membranes were cut away and the beating heart was dis- sected free and lifted into a container of “lobster heart f luid similar to that used by Cole (’41). It was usual to observe strong, rhythmic contractions of the organ in this salinous mixture. In order to pre- serve the pacemaker cells for separate

1This work was supported by grant H-519(C12) from The National Heart Institute of the National Institutes of Health, Bethesda, Maryland.

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study, a solution of 0.5% methylene blue in “heart f luid was injected into the ven- tricular cavity and was allowed to remain there for several minutes after the cessa- tion of cardiac beat. The heart was then opened by a longitudinal incision of the ventraI surface, and the small, tree-like pacemaker ganglion, stained intensely blue, was readily identified and excised for separate fixation ( Alexandrowicz, ’32- ’33). Small muscle bundles were then dis- sected from the myocardium under a dis- secting microscope. Some of the bundles were fixed to applicator sticks with insect pins under slight stretch (an estimated 10% of length after removal from the ventricle) and immediately fixed in Pa- lade’s solution, or in l% osmium tetroxide buffered with s-collidine (Bennet and Luft, ’59). The specimens were dehydrated in graded ethanol solutions with final embed- ding in butyl-methyl methacrylate (70-30 proportion respectively). A number of specimens, affixed to sticks, were placed in 50% glycerol at -20°C for 12-14 days. After slowly warming to room tempera- ture, they were washed in a solution of sodium and magnesium chloride (Hawkins and Smith, ’56). Fixation was carried out under stretch, as with the fresh muscle preparations, and taken to methacrylate embedding. All sections were cut on a Porter-Blum microtome, and were mounted on parlodion or formvar coated grids. Mounted specimens were then “stained by floating on 3% uranyl acetate solution. The grids were examined in an RCA EMU- 3C electron microscope. The resultant plate negatives were enlarged photographi- cally as desired, usually to five times.

RESULTS

General features of myocardial cells. The myocardial cells of these crustacea usually exhibit undulating outlines and voluminous quantities of mitochondria. The myofibrils are marked by the usual band patterns; the Z, A and I bands are readily apparent in longitudinally cut sec- tions. There seem to be distinct H bands in some sarcomeres, especially when the myofibrils are cut in somewhat oblique direction. The contractile structures are surrounded by a sea of mitochondria many of which are massed between the myo-

fibrils. Large aggregates of mitochondria occur beneath the sarcolemma and are arranged so densely as to appear in three or four layers. The mitochondria possess distinct double membranes, with many cristae. The disposition and conformity of these numerous mitochondria suggest that they are part of a very active mus- cular system (Mattison and Birch-Ander- sen, ’62).

The nuclei usually appear at the edges of cells. The nuclear membranes are double-layered and the nuclear chromatin content is moderately dense. One or two eccentrically placed nucleoli are usually present

The sarcolemmas of these cells are of some interest. Over most of the cellular periphery, the system consists of a fine inner membrane (plasma membrane) and a basement membrane that is thicker, and more irregular with numerous minute pro- jections. Over most of their extent, the sarcolemma is similar to those of muscles of other species (Bergman, ’60; Price, Weiss, Hatta, and Smith, ’55). The sarco- lemma1 envelope also is frequently thrown into conspicous folds at the edges of cells where there are clefts, or at areas of angu- lation (fig. 2). At these points particularly, the outer membrane of the sarcolemma may be undulated or festooned, often with finger-like projections, and apparently with several layers of membrane-like sheets. Many of these features of the cell mem- branes are present in the skeletal muscles of horseshoe crabs (de Villafranca and Philpott, ’61). The sarcolemmas of adja- cent cells (along the lateral borders) are superimposed upon each other so that only a thin, intervening space between the outer membranes is evident.

The contiguous ends of myocardial cells are often occupied by well-defined inter- calated disks. The structures are generally similar to such disks described in other invertebrate, or in vertebrate muscle (Price, Weiss, Hata, and Smith, ’55). At these points of contiguity, the disks occupy the position of a common Z band of the two cells. Outside of this common myo- fibrillar connection, the remaining adjoin- ing cellular parts exhibit a simple super- imposition of the sarcolemmal membranes. In the lobster heart muscle, therefore, the

ULTRASTRUCTURE OF THE LOBSTER HEART 393

Fig. 1 General appearance of myocardial cells of the lobster, showing an intercalated disk. The disk occupies the position of a 2 band (arrow) between adjoining cells, and is marked by the tortuous course and electron density of the membranes. Elsewhere, the adjoining membranes are superimposed, though they appear more dense than the membranes covering the exterior of the cells. The laterally placed nucleus ( N ) and large number of mitochondria ( m ) are readily appar- ent. Patency of the sarcoplasmic reticulum can be discerned at the disk and between the myofibrils. x 10,000.

394 JOHN R. SMITH

intercalated disk represents a cellular cou- pling through myofibrillar elements. The disks themselves are double-membraned structures which transsect the myofibrils in a tortuous, but uninterrupted course. In our specimens, the apposition of the membranes of the disks are strikingly dense. Irregularities of the disks are fre- quently occupied by vesicles of varying size.

In all micrographs of these myocardial cells, it is also notable that the sarco- plasmic reticulum appears to be extensive and highly expanded. In some sections a close relationship may be seen between the plasma membrane and tube-like struc- tures, which may be followed well into the interior of the cell (fig. 3) (de Villafranca and Philpott, ’61). The tubes may run in rather straight course, though not infre- quently they may run in circular courses embracing groups of myofibrils or organ- elles. Indeed, the patency of the sarco- plasmic reticulum may, in some instances, impart a strikingly perforated aspect to the cell (figs. 8 and 9). One is impressed that most of the reticular tubules run in transverse direction to the long axis of the myofibrils, although tubules may be found frequently that lie parallel to the contractile fibrils. Large vesicles, or highly dilated tubules, are located at the Z-I junctions; the precise “triad arrangement was not consistently observed in this muscle.

Myofibrils. Certain gross features of the myofibrils are common to the myo- cardial and skeletal muscles of vertebrate and invertebrate species. The 2, A and I bands are clearly marked in longitudinal sections of the cells. The Z bands are electron-dense, finely undulated structures extending across the sarcomere, frequently extending across interfibrillar spaces to the Z band of an adjacent myofibril (de Villa- franca and Philpott, ‘61). In fact, when our wecimens of the lobster mvocardium

osmium tetroxide. The interfilamentous spaces between the A band fibrils are about 180-200 A in width. The filaments appear to extend without interruption throughout the course of the A band (from one A-I junction to the other on the oppo- site end of the sarcomere). In some sec- tions these filaments demonstrate a hollow appearance (figs. 5 and 7). The I bands are made up of very fine filaments origi- nating at the Z band and continuing to the A band. At the A-I junction, the courses of the finer filaments are more difficult to follow. In the I band, itself, the filaments are disposed in a generally parallel fashion; however, the parallel dis- position is frequently broken by irregular spaces as if the fibrils were interlacing, or had been displaced by tubules of sarco- plasmic reticlum.

Under high magnifications (direct mag- nifications of 28,000 diameters or more) it was apparent in many sarcomeres that the I band filaments do, in fact, extend along the interfilamentous spaces of the A band, as shown in the accompanying micrographs. However, in the very thin sections at hand, the interdigitation of the I and A band filaments does not appear to be uniform. In many areas the smaller filamentous material appears as multitudi- nous cross-bands between the larger fila- ments so that the A band assumes a lat- ticed pattern. Transverse sections of the sarcomeres through the A bands usually show the larger filaments surrounded by the smaller I filaments. The usual cross sectional pattern suggests a “diamond” formed by bridges between each of the

Fig. 2 Edge of a myocardial cell showing the mitochondria encompassed by the sarcolemma. At points of indentation of the sarcolemma, the outer (basement) membrane may appear strik- ingly thickened and thrown into folds (arrows). x 20,000.

Fig. 3 General appearance of myocardial sar- comeres fixed under moderate stretch (see text).

were ‘‘fixed in chromic acid solutions, The A, I and 2 bands are evident. A lighter most of the myofilaments were destroyed, zone, transsecting the midportion of the A band

appears as the H band (arrow). At this magnifi- leaving the bands intact so that the cation the details of the H zone are difficult to ~~

Curious, almost fibrillar character of the observe. This micrograph also shows the apparent Z structure was evident. intimacy of the plasma membrane of the cell - . _. ~~

The A bands are composed of approxi- mately uniformly thick fikments of 150- 160 A diameter in specimens fixed in A band level. x 20,000.

to the tubules of the sarcoplasmic reticulum; many of the latter are in close relation to the Z bands of the sarcomeres, but occasionally at

Figures 2 and 3

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396 JOHN R. SMITH

Fig. 4 The two sarcomeres in this micrograph show the H zones (arrow) distinctly. The section was cut in slightly oblique plane. The H zone appears to result from less inter- fibrillar density at the middle of the A zone, although the thicker A band myofilaments traverse the zone in slightly differing plane. The muscle was fixed under slight stretch (see text). The dense material comprising the 2 band can be seen to extend laterally from the myofibril into areas of mitochondria and may be a part of, or closely associated with, the sarcoplasmic reticulum. X 40,000.

Fig. 5 Detail of a myocardial sarcomere at the level of the Z band. The Z band is distinct. In places this structure comprises a highly irregular, dense line to which the fine I band filaments attach (Knappeis and Carlsen, ’62) or pass through. The I band is fre- quently perforated by tubules of the sarcoplasmic reticulum. The fine (secondary) filaments of the I band can be followed in many areas into the interfilamentous spaces between the thicker primary filaments of the A band; in other areas of the A band, cross-bridging material is distinctly evident without the interposition of longitudinal secondary filament. x 110,000.

Figs. 6-7 Cross sections of myofibrils through the A bands. These sections show the apparent interconnections of the primary ( A ) filaments in many places giving the geo- metric configurations of squares. At higher magnification (fig. 7) the finer ( I ) filaments may be discerned between the larger ones in the proportion of 1:l. This arrangement ap- pears to produce an “array” of myofilaments suggesting the imbrications of finer and coarser squares. We have found no evidence for an array of four or five secondary filaments around each of the primary ones. Figure 6, X 55,000. Figure 7, x 80,000.

ULTRASTRUCTURE OF THE LOBSTER HEART 397

Figures 5 to 7

398 JOHN R. SMITH

Figs. 8-9 High magnification micrographs of two different sarcomeres at Z band level showing the disposition of the sarcoplasmic reticulum in relation to the Z substance. The tubular reticulum suggests the “triad” arrangement noted in vertebrate muscle specimens; in these specimens the triad arrangement is less clear. The tubular reticulum is found to be more numerous and more widely patent adjacent to the Z bands. The positions of the primary and secondary filaments, and the appearance of the interfilamentous cross-bridging substance is evident. Both X 110,000.

ULTRASTRUCTURE OF THE LOBSTER HEART 399

A fibrils giving the appearance of precise, interconnected rows of filaments. On the other hand, in other cross sectional areas one could observe the interposition of several secondary filaments between the larger ones, but not in hexagonal or other regular array. Other cross sections show little or no secondary fibrillar material between the primary filaments.

Under the conditions of slight tension and fixation used here, many of the sarco- meres exhibit a rarefied central band which grossly resembles an H band. In our experience, this band is accentuated in slightly oblique sections of sarcomeres. Examination of the band (figs. 3 and 4) suggests that the lighter zone results from loss of interfibrillar material between the A filaments as if the bridging material had been attenuated by the force of stretch. The phenomenon further sug- gests that the secondary fibrillar material is portioned between each half of the sarcomere, and can be separated (on sar- comeric elongation) at the central part of the A band. Occasionally (fig. 4 ) sec- ondary fibers were seen to enter the H band, but none were seen to cross it en- tirely. We could not discern, in these speci- mens, that interposition of the sarco- plasmic reticulum may have given rise to the lighter stripe at mid-sarcomere as re- cently suggested by de Villafranca and Philpott (‘61) for the muscles of horse- shoe crabs. Of the numerous sections made of the lobster myocardium, nothing resembling an M band has ever been found, nor was there thickening of the A filaments at mid-portion, which appears to make up the M band in vertebrate mus- cle. It seems certain that the M line struc- ture is entirely lacking in the heart muscle cells of lobsters.

DISCUSSION

On the basis of these observations of the myocardium of Homarus, the fine structure of the contractile elements is similar to the skeletal muscle of Limulus (de Villafranca, ’61; de Villafranca and Philpott, ’61). This myocardial tissue does differ from the crab skeletal muscle in the vast numbers of mitochondria that occupy the cardiac cells. However, the character- istics of the contractile fibrils are of special

interest. There is evidence that the fine filaments of the I bands are dispersed between the wider filaments of the A zone. A precise interfilamentous relation of the two does not appear to be consistent throughout the breadth of a sarcomere. In most of the cross sections of the muscle the I band threads seem to number one or more interspersed between the A fibers, producing a diamond-like geometric form. In other cross sectional areas the I fila- ments are not discernible, and the thicker A rods are joined by a cross-bridging ma- terial, which in longitudinal section prob- ably causes the latticed effect. It is not known whether the I band material is adherent to, or is an intimate part of, the A band substance. Nevertheless, the fact remains that it is in the most extended sarcomeres that the regular alignment of A and I filaments occurs (de Villafranca and Philpott, ’61). On the basis of these observations, it is at present not possible to decide whether a consistent “sliding” filament arrangement exists in the Homa- rus myocardium. While secondary fila- ments can be demonstrated, this muscle is sufficiently different from the classical pattern to raise doubts with regard to the “sliding” filament theory. In this way the observations are in agreement with those of de Villafranca and Philpott (’61) on Limulus muscles. Whatever the mechani- cal details of myocardial contraction may be, they are probably similar to that of the skeletal muscles of similar crustaceans.

A number of other details of these myo- cardial cells deserve comment. Among these are the peculiarities of the sarco- lemma. It has been pointed out that at areas where the cells abutt one another along longitudinal surfaces, the sarco- lemma of the contiguous cells frequently lie one upon the other in simple super- imposition. Well-defined intercalated disks may be found between the poles, or ex- tremities of the cells. The disks occur at the Z lines, as in the muscles of other we- cies (Price, Weiss, Hata, and Smith, ’55). Of particular interest also is the folding of the sarcolemma at certain points of ex- ternal angulation of the cells. The outer (basement) membrane of the sarcolemma may be grossly thickened and indented into conspicuous festoons. It is difficult to

400 JOHN R. SMITH

imagine the function of these modified membranes unless it is to cushion the motions of the cells in their various con- tacts.

The extensive sarcotubular system of this muscle tissue, together with the large number of mitochondria may be indicative of a muscle which must be persistently active (Mattison and Birch-Anderson, '62). The mitochondrial masses are laced with large sarcotubular channels which may be derived from imaginations of the plasma membranes and appear to be con- nected to the contractile elements them- selves. These facts confirm the generally held concepts that this tubular system is concerned with intracellular impulse con- duction, and, in the case of the crustacean heart, may also be concerned with the receipt and disposition of impulses from the neurogenic pacemaker.

ACKNOWLEDGMENT

The author is grateful to Dr. Roy Peter- son of the Department of Anatomy, Wash- ington University, for his many helpful comments.

LITERATURE CITED Alexandrowicz, J. S. 1932.1933 The innerva-

tion of the heart of the Crustacea. I. Decapoda. Quart. J. Micr. Sc., 75: 181-249.

Bennet, H. S., and J. H. Luft 1959 s-Collidine as a basis for buffering fixatives. J. Biophys. & Biochem. Cytol., 6: 113-114.

Bergman. R. A. 1960 A note on the fine struc- ture of the turtle heart. Bull. Johns Hopkins HOSP., 106: 46-54. 239 P.

Cole, W. H. 1941 A perfusing solution for the lobster (Homarus) heart and the effects of its constituent ions on the heart. J. Gen. Physiol.,

de Villafranca, G. W. 1961 The A and I band lengths in stretched or contracted horseshoe crab skeletal muscle. J. Ultrastructure Res.,

1961 The ultrastructure of striated muscle from Limulus polyphemus. Ibid., 5: 151-165.

Hawkins, L. W., and J. R. Smith 1956 Effect of certain cations on relaxation of glycerol- extracted heart muscle of frogs. Am. J. Physiol., 186: 304308.

Huxley, H. E. 1957 The double array of Ma- ments in cross-striated muscle. J. Biophys. and Biochem. Cytol., 3: 631-648.

Knappeis, G. G., and F. Carlsen 1962 The ultrastructure of the Z disc in skeletal muscle. J. Cell. Biol., 13: 323-335.

1962 On the fine structure of the mitochondria and its relation to the oxidative capacity in mus- cles in various invertebrates. J. Ultrastructure Res., 6: 205-228.

Price, K. D., J. M. Weiss, D. Hata and J. R. Smith 1955 Experimental needle biopsy of the myo- cardium of dogs with particular reference to histologic study by electron microscopy. J. Exp. Med., 101: 687-694.

Sjostrand, F. S., and E. Anderson 1956 The ultrastructure of skeletal muscle myofilaments at various conditions of shortening. Exp. Cell Res., 11: 49-96.

Sjostrand, F. S., and E. Anderson-Cedergren 1957 The ultrastructure of the skeletal muscle myoflaments at various states of shortening. J. Ultrastructure Res., I : 74-108.

The continuity of A- and I-band myofilaments in skeletal muscle. Ibid., 3:

25: 1-6.

5: 109-115. de Villafranca, G. W., and D. E. Philpott

Mattison, A. G. M., and A. Birch-Andersen

1959