Ultrastructure of the myocardial sarcolemma.

Circulation Research

JULY

1975

VOL. 37

NO. 1

An Official Journal of the American Heart Association

Brief Reviews Ultrastructure of the Myocardial Sarcolemma By N. Scott McNutt • The name sarcolemma is used in many electron microscopic studies to designate the 7.5-9-nm "unit" plasma membrane that encloses the cytoplasm of the muscle cell (1, 2), but this definition of the sarcolemma is incomplete as a description of the actual boundary between the cellular cytoplasm and the extracellular space. Two specific omissions exist in the sarcolemma definition. First, on the external aspect of the membrane, there is a specialized surface coat composed of fibrillar material, probably glycoprotein, bound to the plasma membrane. This surface coat (glycocalyx) covers most of the external aspect of the sarcolemma. It is 20-60 nm thick on mammalian sarcolemma and is rich in carbohydrates with acidic residues as shown by ruthenium red and colloidal iron staining procedures (3). Second, adjacent to the internal aspect of the membrane, there is a specialized layer of fibrillar cytoplasm that influences the overall properties of the boundary between the cytoplasm and the exterior. In cardiac muscle cells, this specialized cytoplasmic layer is prominent, particularly in regions of cell-to-cell adhesion (4). In addition, research on other cell types has indicated that a relatively inconspicuous fibrillar layer is bound to the inner surface of the plasma membrane and may be important in determining the distribution of surface sites on the exterior of the membrane (5, 6). It is a minor semantic point whether these specialized layers internal and external to plasma membrane are included in the definition of the sarcolemma, but these layers must be considered in terms of their influences on the unit membrane and the interface between the cell and the exterior as a whole. The importance of the surface coat is exemplified in the work of Langer and Frank (7), who have identified the surface coat layer on the myocardial cell as a site of ionic lanthanum bindFrom the Department of Pathology, University of California, San Francisco, and the Anatomic Pathology Service, Veterans Administration Hospital, San Francisco, California 94121.

ing. On the basis of the effect of lanthanum on calcium flux, they have suggested that the surface coat is a possible site for binding of the calcium necessary to link membrane depolarization to myofilament contraction (7). Such a role for the surface coat has not been proved definitely but is worthy of further experimentation. Interestingly, under certain conditions ionic lanthanum has been observed to form an electron-dense precipitate along the inner surface of the sarcolemma as well as in the surface coat material (8). It is not known whether electron-dense precipitates of lanthanum accurately reflect the localization of the cation binding sites that are important for the calcium involved in excitation-contraction coupling. Research on plasma membranes progresses most rapidly when simple isolation procedures give preparations with a high yield of relatively pure plasma membranes. Unfortunately, the complexity of the cardiac sarcolemma prevents easy isolation. However, encouraging efforts are being made toward this goal (9-12). Since certain aspects of research on the plasma membrane of noncardiac cells have important implications if the data apply to the myocardial sarcolemma as well, this review emphasizes selected recent data that apply to the basic structure and function of plasma membranes. For the purposes of discussion, the sarcolemma will be divided arbitrarily into those regions that are obviously specialized when they are viewed in electron micrographs and those regions that are not specialized in this regard and often are called "unspecialized" regions. Since unspecialized is undoubtedly a misnomer in a functional sense, the term general sarcolemma will be used instead. The sarcolemma has five categories of focal specializations that are present in the majority of cardiac muscle cells in the ventricular walls: (1) large invaginations that are tubular and constitute the T-system (Figs. 1 and 2), (2) small invaginations that are vesicular, i.e., caveolae and coated vesicles

Circulation Research, Vol. 37, July 1975

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McNUTT

FIGURE 1

Electron micrograph of a thin section at the periphery of a guinea pig cardiac muscle cell cut almost in cross section. This micrograph demonstrates the imagination of the sarcolemma to form a network of tubules called T-tubules (T). The surface coat (SC) is visible and extends into the T-tubules. A few vesicular inuaginations (V) of the sarcolemma are present. Cisternae of the sarcoplasmic reticulum (SR) can be seen in regions of attachment to the sarcolemma of the T-system. The filamentous layer (FL) subjacent to the sarcolemma is prominent particularly near the orifices of T-tubules. Bar = lii.

(Fig. 1), (3) regions of attachment with the sarcoplasmic reticulum (Fig. 1), (4) regions of cell-to-cell attachment, which are subclassified into desmosomes, nexuses ("gap junctions"), and fasciae adherentes (Figs. 3-5), and (5) regions of attachment of the contractile filament system to the membrane, i.e., fasciae adherentes and occasional sites of attachment of the Z band to the external

sarcolemma and the sarcolemma of the T-system (Fig. 1, 3, and 5). Variations in the extent of these five types of specialization constitute striking differences between the muscle cells (myocytes) in different locations within the heart as well as between myocytes of different species. Quantitative techniques for electron microscopy have great applicaCirculation Research, Vol. 37, July 1975


ULTRASTRUCTURE OF THE MYOCARDIAL SARCOLEMMA

PM

X

FIGURE 2

High-magnification electron micrograph of a thin section showing the sarcolemma at the orifice of a T-tubule (T) in cat ventricular myocardium. In addition to the trilaminar plasma membrane (PM), there is a prominent surface coat (SC) and a subplasmalemmal filament layer (FL). Bar

tion to the study of the sarcolemma and the regulatory mechanisms determining the amount of specialized regions (2, 13, 14). Since any brief review cannot illustrate, diagram, and discuss all of the aspects of the sarcolemma and its specializations, references are quoted to direct the reader both to primary studies and to other reviews. GENERAL SARCOLEMMA

In standard thin sections, the general sarcolemma appears, in cross section, to be formed of three layers: a central electron-lucent layer approximately 3 nm thick sandwiched between two electron-dense layers, each approximately 2.5 nm thick. The central electron-lucent zone is considered to be the hydrophobic interior of the lipid bilayer as indicated in the Davson-Danielli model of membrane structure (reviewed in 15). In standard thin sections, the electron-lucent zone rather characteristically appears unspecialized and uninterrupted; however, more information on the structure of this region has been obtained by methods for examination of frozen membranes. These methods have been named "freeze-etch," "freezefracture," or "freeze-cleave" techniques; all of these names often have been used interchangeably, resulting in some confusion in the minds of people who do not work actively in this field. In all of these

methods, cells are frozen and fractured. During fracturing, the cellular membranes either are broken across their full thickness or are preferentially cleaved along an internal plane (16), probably along the center of the lipid bilayer (17). Such membrane cleavage is considered likely because the hydrophobic "tails" of the phospholipid molecules in the bilayer would produce a mechanically weak plane within a frozen aqueous matrix. The data indicating that this type of cleavage occurs should not be overinterpreted, since the process of freezing itself may favor the organization of lipids as a bilayer whereas at physiological temperatures other configurations of the lipids may be in equilibrium with a bilayer (18-21). Despite possible arguments about the influence of freezing, the freezefracture method has proved very useful for studying the distribution of certain membrane proteins within the plane of the membrane. The proteins revealed by membrane cleavage apparently are molecules that protrude sufficiently into the lipid bilayer to result in a local deflection of the fracture plane thereby producing a bump or "particle" on the fracture face. Most of these particles are approximately 8-10 nm in diameter, although detailed size distributions have not been published for membrane particles. It is uncertain how deeply the proteins (as represented by the particles)



MclMUTT

FIGURE 3

Thin section of a portion of an intercalated disk attaching two cat ventricular myocytes. The major component of this cell-to-cell junctional region is a type of junction called the fascia adherens (FA). The plasma membranes at the fascia adherens are separated by a 20-30-nm interspace. In addition, actin filaments insert into a dense filamentous mat which appears to link the actin to the plasma membrane at the fascia adherens. Filaments in the filamentous mat often course parallel to the plane of the membrane (at arrow). At the nexus (N), the plasma membranes of adjacent cells are in contact. The nexus, sometimes called a gap junction, is thought to mediate electrical coupling. Bar = 0.5n. (Published with permission of J. Wiley & Sons, Inc., McNutt NS, Fawcett DW: Myocardial ultrastructure. In The Mammalian Myocardium, edited by GA Longer and AJ Brady. New York; J. Wiley & Sons, Inc., 1974.)

penetrate into the membrane, but it is likely that some of them span the full thickness of the bilayer. The concept that the particles represent membrane proteins with a portion exposed on the external surface is supported strongly by the results of Tillack et al. (22). They have observed that trypsinization of erythrocyte ghost membranes causes a clustering of the membrane particles. When trypsinized erythrocyte membranes are allowed to bind influenza virus, the virus causes depressions in the membrane which are localized to the regions of particle clusters. This finding suggests that at least some of the particles represent proteins that are exposed on the true surface and carry a receptor for influenza virus. To expose the true surface of the frozen cells, an additional step has to be added to the specimen preparation protocol after the fracturing step. This step, called etching, is essentially freeze-drying, i.e., vacuum sublimation of ice crystals within the specimen. The name freeze-etching is jargon for freeze-fracturing followed by vacuum sublimation of ice. The removal of small ice crystals from the

fracture face by sublimation allows the structures that are buried within the ice matrix to be visualized to better advantage. The etching step is unnecessary for demonstration of intramembrane particles but is necessary for the demonstration of the true surface of the membrane. At this point, additional details of nomenclature are required. Readers of papers dealing with freeze-etching of membranes will experience difficulty with the nomenclature, because it seems to be only partially standardized and in a state of flux. Since plasma membranes are cleaved along an interior plane, they are split into two lamellas, an outer lamella and an inner lamella. The cleavage thus does not reveal the true surface of the membrane but generates artificial surfaces, often called "faces" to distinguish them from true surfaces. The exposed face of the inner lamella is called the A face and the exposed face of the outer lamella is the B face, according to a convention that is gaining acceptance (but is not universal) (23). The cleavage process separates the membrane lamellas, leaving the inner lamella attached to the frozen cytoCirculation Research, Vol. 37, July 1975


FIGURE 4

Electron micrograph of a high-resolution freeze-etch replica of mouse cardiac muscle showing portions of the cleaved sarcolemma from two ventricular myocytes. Cleavage occurred in the interior of the membrane and left the outer-half membrane attached to the extracellular compartment, shown in this figure at a fascia adherens (FB). The exposed aspect of the outer-half membrane is called face B. Moreover, internal cleavage of the adjacent cell membrane has left its inner half attached to the cytoplasmic compartment. The exposed aspect of the inner-half membrane is called face A and can be seen in this illustration both in regions of the general sarcolemma (A) and in the fascia adherens (FA). This replica reveals a remarkable heterogeneity of particle sizes and shapes on the A face. Some of the particles appear as short segments of filaments (at arrow). Pits are also present in the face A where particles were removed in the cleavage process. On the face B of the membrane at the fascia adherens (FB), the face has numerous short grooves (at arrow) and pits but few particles. Such texturing of the face might be produced by cleaving short filaments and particles out of the membrane face. The tissue was fixed in 2% glutaraldehyde and replicated at - 105"C. The direction of shadowing is from the bottom of the micrograph which is printed as a positive. Bar = 0.25/1. Inset: Cleaved membranes at the nexus reveal an orderly packing of pits on the B face (NB) and particles on the A face (NA). Unfixed mouse ventricle replicated at -150°C. Direction of shadowing from bottom of picture. Bar = O.ln.


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McNUTT

FIGURE S

Freeze-cleave replica of mouse heart showing the sarcolemma at a fascia adherens. The membrane face B has many short segments of fine filaments (at small arrows). Some filaments pass from the cytoplasm to the interior of the membrane at the B face (at large arrows), indicating that the filamentous mat is strongly attached to the interior of the membrane. The mouse ventricle was fixed in 2% glutaraldehyde and was replicated at -150°C. Direction of shadowing is from the bottom of the picture. Bar = 0.25n.

plasm and the outer lamella attached to the extracellular compartment. To reveal the true surfaces of the membrane, the cells must be washed extensively and frozen in distilled water. Subsequent etching of cells causes the removal of the ice crystals abutting on the true membrane surfaces. The true cytoplasmic surface of the membrane may conveniently be labeled surface C, leaving the true exterior surface to be called surface D. When it is revealed by this etching technique, the true outer surface D of the plasma membrane of erythrocytes is remarkably smooth to the level of 5-nm resolution (24). The smoothness of the true surface may be an artifact of conventional and relatively slow freezing, since very rapid freezing

has been shown to give much improved preservation of the extended form of surface molecules in certain bacterial cells (25). Most of the particles protrude from the A face of the plasma membrane of erythrocytes (26) and cardiac muscle cells (4, 27). Rayns et al. (27) found 400-700 particles//*2 on the A face compared with 80-120 particles//^2 on the B face in the sarcolemma of guinea pig ventricular myocytes. The A face particles are a portion of a molecular structure that also is present at the true surface of the membrane (22, 28), but some distortion occurs during freeze-fracturing since there are not 400-700 depressions ("pits")/jit2 on the B face that correspond to the size of the A face particles (29). In Circulation Research, Vol. 37, July 1975



contrast, at certain specialized regions, (i.e., the nexus), A face particles are matched by B face pits (23). This point suggests that most A face intramembrane particles are different from those particles in specialized regions such as the nexus. The exact cause of the different fracturing properties of various types of intramembrane particles is unknown, but it allows some subclassification of the intramembrane particle population. It has been a great hope of many investigators that the freeze-etch technique would reveal differences between membranes that carry action potentials and membranes from electrically inactive mammalian cells. Unfortunately, this hope has yet to be realized. Recently, Rash and Ellisman (30) have studied the sarcolemma of rat skeletal muscle and have found that this electrically excitable membrane contains, on its A face, small particles stacked in orthogonal or "square" arrays with a 7-nm center-to-center spacing of the particles. In addition, when the fracturing process reveals the arrays on the A face, it leaves similar distinctive arrays of pits on the B face of the skeletal muscle sarcolemma. The fact that these particles in the arrays on the A face leave distinct pits in the B face indicates that these A face particles are different from the majority of A face particles. An interesting observation by Rash and Ellisman (30) is that, at the neuromuscular junction, there are no orthogonal arrays, whereas 0.5 mm from the neuromuscular junction the density of arrays reaches 50/^t2 sarcolemma. These investigators have pointed out that this distribution of orthogonal arrays correlates with the distribution of electrically excitable regions of the sarcolemma (30). A few similar distinctive orthogonal arrays of pits have been observed on the B face of the cardiac muscle sarcolemma (Fig. 6) (McNutt, unpublished observations). For these arrays to be correlated with electrical excitability, some explanation must be made for the fact that similar arrays were initially visualized in hepatocyte plasma membranes (31) and have been seen in intestinal epithelial cell membranes, where they were originally mistakenly identified as gap junctions (32, 33). These orthogonal arrays have also been observed on glial cell membranes (34). At present, it seems premature to correlate these orthogonal arrays with electrical excitability per se. Although freeze-etching shows striking similarity in the orthogonal arrays in liver and intestine membranes, the technique is capable of only 2-nm resolution, which is not sufficient to exclude the possibility of small differences in the molecules of the arrays in the various tissues.

Any membrane model must take into account the fact that, under certain conditions, surface markers and intramembrane particles have been shown to move within the plane of the membrane. This finding has led to the concept that the plasma membrane has a relatively fluid bilayer in which the integral membrane proteins may move laterally (35). Unrestrained lateral mobility of membrane proteins would be expected to lead to a random distribution of proteins in the membrane. Since many membranes, particularly the sarcolemma, do not have a random distribution of morphological (and functional) sites, then the question must be asked: what forces oppose the fluidity of membranes? In the case of erythrocyte membranes, the answer is becoming clear. Marchesi et al. (36) have characterized a filamentous protein associated with the inner surface of the erythrocyte membrane and named this protein spectrin. Spectrin is present only on the inner surface of this membrane (37). Recently, crucial experiments have been performed that demonstrate the importance of spectrin in stabilizing the plasma membrane. Nicolson and Painter (5) have found that antispectrin antibody causes clustering of spectrin on the cytoplasmic side of the membrane which in turn causes clustering of sialic acid groups on the extracellular side of the membrane, as detected by colloidal iron hydroxide binding. Furthermore, Elgsaeter and Branton (6) have shown that removal of spectrin from erythrocyte membranes allows intramembrane particle movement within the plane of the membrane. These data suggest that the distribution of cytoplasmic filaments may control the distribution of surface sites on the membrane. If spectrin were a protein restricted to the erythrocyte, it would be far less interesting. Recently, Tilney (38) has presented elegant evidence for the presence of a protein which is probably spectrin in sperm cell membranes and has shown that this spectrinlike protein binds actin. It remains to be determined whether spectrin is found in cardiac muscle and is bound to cardiac sarcolemma and whether spectrin could be a mechanical link between the actin filaments of the contractile apparatus and the sarcolemma. In freeze-etch replicas, filamentous material can be visualized on the B face of the cardiac sarcolemma (Fig. 5); occasional replicas demonstrate filaments extending from the B face into the cytoplasmic compartment. The chemical nature of these filaments is unknown. Filamentous material occasionally can be observed on the B face of erythrocyte membranes (39).



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FIGURE 6

High magnification of a freeze-cleaue replica of mouse sarcolemma face B. The membrane face has distinctive orthogonal arrays of pits {at arrows) with approximately a 7-nm center-to-center spacing. These face B pits probably complement similar arrays of face A particles that are not illustrated in this figure. The function of orthogonal arrays is unknown, but they have been found by Rash and Ellisman (30) to be concentrated in electrically excitable regions of skeletal muscle sarcolemma. Tissue was fixed in 2% glutaraldehyde and replicated at —150°C. Direction of shadowing is from bottom of picture. Bar = O.ln. T-SYSTEM

The sarcolemma invaginates to form an extensive tubular network that carries the extracellular space deeply into the volume of the cell (Fig. 1). This tubular network is called the T-system. Ttubules generally are transversely oriented in the cell and course near Z bands, but there are interconnections between T-tubules which are oriented longitudinally with respect to the myofilament

mass. In some species, the longitudinal connections are abundant (40). The T-system tubules are 50-300 nm in diameter and usually contain the prominent glycoprotein coat also found on the external, general sarcolemma (Figs. 1 and 2). The sarcolemma of the T-system has a distribution of intramembrane particles that does not appear to be different from that of the general sarcolemma (27). It is difficult to make this statement with great Circulation Research, Vol. 37, July 1975



certainty, however, since the curvature of T-system membranes makes it difficult to compare large areas of T-system membrane with general sarcolemma in freeze-etch preparations. It is not known whether orthogonal arrays are present on T-system sarcolemma. The amount of T-system sarcolemma is different in ordinary ventricular myocytes, atrial myocytes, and cells of the conductive system of the heart (reviewed in 41, 42). Page et al. (2, 13, 14) have estimated that T-system membrane represents approximately 20% of the total plasma membrane of rat ventricular myocytes based on a stereological study of standard thin sections. In addition, Page et al. (14) have shown that, during experimental cardiac hypertrophy, the area of T-system membrane increases so that the ratio of total sarcolemmal area to cell volume remains constant. Quantitative studies are difficult, since in some species it is easy to underestimate the amount of T-system membrane. Forssmann and Girardier (43) have shown that the extracellular tracer substance, horseradish peroxidase (molecular weight ~40,000), can pass from large-diameter T-tubules (100 nm in diameter) into small-diameter Ttubules (50-85 nm in diameter) in rat ventricular myocytes. The presence of small-diameter Ttubules is important for quantitative studies, since these tubules would probably be missed as belonging to the T-system in standard thin section preparations because they may not clearly exhibit the distinctive glycoprotein coat on the luminal surface of the tubule membrane. It is not clear from the study of Forssman and Girardier (43) whether small-diameter T-tubules are a quantitatively important or a minor component of the T-system. It appears to be a general finding that cells of the conducting system of mammalian hearts lack a T-system, a feature which, by decreasing membrane area, would lower total membrane capacitance and speed impulse conduction (43-46). Most atrial muscle cells lack a T-system (41, 43, 47), but a few atrial myocytes in some species do show T-tubules (43), thus raising the possibility that two populations of atrial myocytes are present in some species (45). Frog and chicken cardiac muscle lack a T-system in both the atria and the ventricles (45, 48). VESICULAR IMAGINATIONS

Little attention has been paid to the presence of small vesicles associated with the general sarcolemma as well as the sarcolemma of the T-system (1) (Fig. 1). These vesicles are approximately 50-80

nm in diameter and are of two types. One type, often called caveolae, has a smooth limiting membrane, a content of low electron density, and is thought to be similar to pinocytotic vesicles seen elsewhere that are involved with nutrient uptake. The second type has a membrane that is coated on its cytoplasmic surface by numerous small spikes or "bristles" (1, 47). These bristle-coated vesicles have a similar morphology to vesicles in oocytes that are involved in the uptake of proteins from the extracellular space. In fact, almost nothing is known about these two types of vesicles; for instance, it is not known to what extent they alter the total surface area of the sarcolemma, to what extent they actually are involved in transport, or what type of transport they perform or in what direction. Franzini-Armstrong and Dulhunty (49) have found that caveolae form a significant proportion of the sarcolemma (~25% excluding the Tsystem) in frog sartorius and semitendinosus muscle. It has been noted that there is a vesicular population in mammalian cardiac muscle that is localized to the sarcoplasmic reticulum near the Z band (1, 47). These vesicles are often connected to segments of the sarcoplasmic reticulum and have two distinctive characteristics: a cytoplasmic surface coated with small projections and an electrondense content in the lumen. It has been suggested that these vesicles, called coated dense vesicles, may be involved with transport between the sarcoplasmic reticulum and the sarcolemma, but the content and the direction of any such transport is unknown (1, 47). The elegant work of Ishikawa (50) on cultured skeletal muscle cells indicates that the sarcolemma of the T-system arises by progressive invagination of caveolae. It remains to be shown whether a similar mechanism operates in cardiac muscle cells for the formation of the T-system. SARCOLEMMA-SARCOPLASMIC RETICULUM ATTACHMENTS

In standard thin sections, a considerable amount of T-system sarcolemma is in close apposition with flattened saccules of the sarcoplasmic reticulum called subsarcolemmal cisternae (1) or junctional sarcoplasmic reticulum (44, 45) (Fig. 1). Similar appositions are present with the sarcolemma at the cell perimeter (45). At high magnification the region of apposition is characterized by 15-20-nm globular densities in the interspace between sarcolemma and reticulum. The cisterna is flattened and the lumen has an electron-dense content. In



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skeletal muscle, similar cisternae are apposed to the T-system sarcolemma, but these cisternae, called terminal cisternae, have a more voluminous lumen. These regions have been the focus of interest, because they are good candidates for regions of functional coupling between the sarcolemma and the sarcoplasmic reticulum. Depolarization of the T-system sarcolemma of skeletal muscle may cause the release of calcium stored in the terminal cisternae, thereby increasing intracellular calcium ion concentration and triggering contraction of the myofilament mass (reviewed in 51). The function of the cisternae in cardiac muscle may well be analogous (reviewed in 52). Another possible function of the cisternae is pointed out by the need for calcium taken up by the sarcoplasmic reticulum to be returned eventually to the extracellular space, since some extracellular calcium enters the cell with each action potential, particularly in cardiac muscle (reviewed in 52). Consequently, any specializations of the membranes at these attachment sites would be of particular interest. Recently, Franzini-Armstrong (53) has examined spider skeletal muscle by freeze-etching, since the cisternaesarcolemma attachments are rather broad and easily recognized in this muscle. These studies failed to show any specialization of the T-system sarcolemma but did demonstrate a unique roughening of the fracture faces of the sarcoplasmic reticulum membrane at the terminal cisternae. This roughening did not correspond to the pattern of organization of the globular densities between the sarcolemma and the cisternae. To date, the freeze-etch studies of cardiac T-system membranes have failed to reveal any specialization in the regions of attachment to the sarcoplasmic reticulum cisternae. CELL JUNCTIONS

The use of both standard thin sections and freeze-etch replicas has led to a considerable amount of information on cell junctions (reviewed in 54). In brief, standard thin sections have shown that the intercalated disks of cardiac muscle are regions of cell-to-cell attachment. The major component of the intercalated disks is the fascia adherens, a region where actin filaments are attached to the plasma membrane (Fig. 3). The actin filaments insert into electron-dense fibrillar material condensed on the cytoplasmic surface of the membrane. This dense material has been called the filamentous mat (4) and has a staining density similar to Z band material, with which it occasionally is seen to be continuous. The previous discus-

sion of actin-spectrin-membrane interactions raises the question whether the filamentous mat is composed of protein related to spectrin. Not only is the mechanism of attachment of actin to the membrane unknown, but also the mechanism of attachment of adjacent plasma membranes at the fascia adherens is unresolved. A 20-30-nm interspace separates the two membranes and contains a relatively amorphous material of low electron density in standard preparations. Freeze-etch preparations indicate that the membranes of the fascia adherens usually have fewer of the A face particles commonly found on the general sarcolemma. Highresolution freeze-etch replicas reveal a texturing of the fracture faces of the fascia adherens that would be produced by fine filaments attaching to the hydrophobic interior of the lipid bilayer (Figs. 4 and 5). These preparations do not adequately resolve whether such filaments enter the membrane interior from both cytoplasmic and extracellular surfaces of the membrane. The mechanism and the strength of attachment at the fascia adherens deserve consideration, since strong attachment is essential for transmission of mechanical tension throughout the tissue. Also, the properties of the fascia adherens should be considered in relation to the series elastic elements in the cardiac muscle. It is not known to what extent the fascia adherens is involved in producing the three to four times greater series elastic compliance observed in cardiac muscle compared with skeletal muscle (55). It has been suggested by Sonnenblick and Skelton (55) that fiber branching in cardiac muscle may be the cause of this difference in series elastic compliance. Although the width of the extracellular interspace at the fascia adherens has not been shown to vary with the degree of stretch of the muscle, stress produced by hypertonic medium has been reported to cause a 59% increase in the width of the junctional cleft in cat cardiac muscle (56). The nexus of cardiac muscle now is accepted widely as the structure responsible for the behavior of cardiac muscle as an electrical syncytium (reviewed in 23 and 54). At the nexus, thin sections reveal that the adjacent plasma membranes are very closely apposed (within 2 nm or less) and that membrane subunits connect the membranes of adjacent cells (23). Freeze-etch preparations show unique specialization in the nexus region (Fig. 4, inset). For a detailed analysis of the appearances of the nexus in freeze-etch replicas, the reader should consult more extensive primary papers (23, 57, 58) and reviews (54, 59, 60). At the nexus, face A particles are clustered often in a hexagonal array Circulation Research, Vol. 37. July 1975


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with a 9-10-nm center-to-center spacing. Face B has a similar array of small pits, indicating that this population of face A particles fractures in a different fashion than does the population of ordinary face A particles of the general sarcolemma. The nexus also electrically couples cells that do not show action potentials such as liver epithelial cells (60, 61). Hepatocyte nexuses (often called gap junctions) have been isolated by elegant techniques and have been partially characterized biochemically (62-64). Two polypeptides named connexin A and B have been isolated from purified nexuses and are thought to have molecular weights of approximately 10,000 and to be linked in the protein connexin by disulfide bonds (63). According to X-ray diffraction studies, the proteins penetrate the lipid layers of the membranes at the nexus (65). It is assumed that these proteins aggregate within the membrane to form cylindrical structures with a central pore 2.0-2.5 nm in diameter (64, 65) through which potassium ions may pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell, thereby carrying the electrical current (23). Quantitative thin-section electron microscopy has allowed estimation of the amount of nexus membrane area and calculation of an approximate specific resistance of nexus membrane (0.05 ohm cm2) (66). This junction presently is receiving further attention because of the possibility that larger molecules, such as nucleotides, may pass from cell to cell via nexuses. There have not been any differences noted between nexuses in electrically active and inactive tissues. However, recently Peracchia and Dulhunty (67) have suggested that the packing density of subunits in gap junctions in crayfish ganglia and rat stomach may differ in the coupled and uncoupled states. The desmosome also is a component of the regions of cell-to-cell attachment in ventricular cardiac muscle and is most evident in cells of the conduction system and in atrial cardiac muscle. At desmosomes, 10-nm cytoplasmic filaments (not actin) insert into dense plaques on the cytoplasmic surface of the membrane. The desmosome membranes of adjacent cells are separated by a 30-50-nm interspace containing fibrillar material that bridges between the membranes (41). Freezeetching has demonstrated that fibrillar material penetrates into the hydrophobic interior of the membrane at desmosomes but has yet to reveal the actual mechanism of cell-to-cell attachment (54). Further understanding of the function and the properties of desmosomes should follow the recent development of an isolation procedure for epithe-

Hal desmosomes (68) and partial chemical characterization of the isolated desmosomes (69). REGIONS OF SARCOLEMMA-ACTIN ATTACHMENT

The fascia adherens is the prime example of actin attached to the sarcolemma. In addition, there are regions in which actin filaments insert on the plasma membrane that are not cell-to-cell junctions. These regions are sites of attachment of Z bands to the sarcolemma and often resemble half of a fascia adherens. They are relatively frequent in developing ventricular cardiac muscle (4) but are also present in adult atrial myocytes and in the cells of the conducting system. In developing systems, these attachments are probably the precursors of fasciae adherentes. The dense material of the filamentous mat at these sites may also serve to anchor the orifices of the T-system tubules near the Z band in adult mammalian ventricular myocytes. Attachments between Z band and filamentous mat material are common in hearts of reptiles (70, 71). In summary, the sarcolemma is a plasma membrane with a number of complex focal specializations. Research on the sarcolemma is in need of methods for the isolation of general sarcolemma and its specialized regions in a relatively pure state and at high yields. A more detailed study of the sarcolemma not only will be interesting in terms of structure-function relationships in cardiac muscle but also will illuminate research on other cell membranes as well. For example, actin and myosin are being found in a wide variety of cells in which these proteins might be involved in cell motility and secretion (72). Actin and myosin are present in platelets (73) and even in isolated synaptosomes from brain (74). The isolation of intercalated disk membranes would give an opportunity to study large amounts of membrane involved in actinmembrane binding and may give clues as to how this interaction is mediated in cardiac muscle as well as in other cell types. References 1. FAWCETT DW, MCNUTT NS: Ultrastructure of the cat

myocardium: I. Ventricular papillary muscle. J Cell Biol 42:1-45, 1969 2. PAGE E, FOZZARD HA: Capacitive, resistive, and syncytial properties of heart muscle: Ultrastructural and physiological considerations. In Structure and Function of Muscle, vol 2, 2d ed, edited by GH Bourne. New York, Academic Press, 1973, pp 91-158 3. HOWSE HD, FERRANS VJ, HIBBS RG: Comparative histo-

chemical and electron microscopic study of the surface coatings of cardiac muscle cells. J Mol Cell Cardiol 1:158-168, 1970 4. MCNUTT NS: Ultrastructure of intercellular junctions in



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adult and developing cardiac muscle. Am J Cardiol 25:169-183, 1970 5. NICOLSON GL, PAINTER RG: Anionic sites of human erythrocyte membranes: II. Antispectrin-induced transmembrane aggregation of the binding sites for positively charged colloidal particles. J Cell Biol 59:395-406, 1973 6. ELGSAETEB A, BRANTON D: Intramembrane particle aggregation in erythrocyte ghosts: I. Effects of protein removal. J Cell Biol 63:1018-1030, 1974 7. LANGER GA, FRANK JS: Lanthanum in heart cell culture: Effect on calcium exchange correlated with its localization. J Cell Biol 54:441-455, 1972

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Ultrastructure of the myocardial sarcolemma. N S McNutt Circ Res. 1975;37:1-13 doi: 10.1161/01.RES.37.1.1 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1975 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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