Specific Granules in Atrial Muscle Cells

SPECIFIC GRANULES IN ATRIAL MUSCLE CELLS

J. D. JAMIESON, M.D., and G. E. PALADE, M.D.

From The Rockefeller Institute

ABSTRACT Large populations (up to 600/cell) of spherical, electron-opaque granules ~ 0.3 to 0.4/~ in diameter are characteristically found in muscle fibers of mammalian atria. They are absent in muscle fibers of the ventricles. The granules arc concentrated in the sarcoplasmic core and occur in lesser numbers in the sarcoplasmic layers between myofibrils and under the plasma membrane. Their intimate association with a central voluminous Golgi complex and the frequent occurrence of material reminiscent of the granular content within the cisternae of the Golgi complex suggest that the latter is involved in the formation of the atrial granules. Atrial granules are larger and more numerous in smaller species (rat, mouse), and generally smaller and less numerous in larger mammals (dog, cat, human); they are absent from the atrial fibers of very young fetuses (rat) but are present in those of newborn animals. A small population of bodies containing glycogen particles and remnants of the endoplasmic reticulum and mitochondria occurs in the sarcoplasmic cores of atrial as well as ventricular muscle fibers in the rat; they contain acid phosphatase and thus appear to be residual bodies of autolytic loci. Their frequency increases with the age of the animal. Typical lipofuscin pigment granules, which are known to contain acid phosphatase and are found in the sarcoplasmic cores in old animals (cat, dog and human), are presumed to arise by progressive aggregation and fusion of small residual bodies.

INTRODUCTION

The fine structure of mammalian ventricular atrial muscle cells from a wide variety of mam- myocardium has been described in several reports malian species, placing particular emphasis on (1-3) which have placed special emphasis on the their granules. The inquiry was conducted in organization of myofibrils and disposition of order to select a favorable material for the future sarcoplasmic reticulum in its muscle cells. Mam- chemical and functional characterization of these malian atrial myocardium has been less extensively granules. studied: only a few reports (4-10) on the fine structure of its muscle cells have appeared. The MATERIALS AND METHODS presence of a population of dense granules within atrial muscle cells was mentioned by some of Preparation for Electron Microscopy these authors and studied in more detail by Hearts of a variety of laboratory animals (rat, Bompiani et al. (8) in the rat heart. In 1961, Palade mouse, hamster, guinea pig, bat, cat, rabbit, dog,) (11) reported that the number of atrial granules and of the pig, ox, and human were used in this appeared to decrease in the rat upon reserpine study. Large animals were anesthetized with ether treatment and suggested that they may be intra- or intravenous Na pentobarbital; smaller animals cellular storage sites for catecholamines. Since were sacrificed by a blow to the head. The chests then, we have undertaken an extensive survey of were quickly opened and portions of the atria and

151 ventricles removed for fixation. In many cases, carried out according to the method of Wood and especially with smaller animals, the heart was per- Barrnett (17), fused and distended with fixative which was allowed to react in situ for several minutes to minimize con- Autoradiography traction and distortion of the muscle. Human left Osmium tetroxide-fixed tissues were embedded in atrial appendage was obtained during open heart methacrylate or Epon 812 and sections prepared for surgery performed on patients with mitral stenosis. light and electron microscopic autoradiography ac- In another instance, human material was obtained cording to the method of Caro (18). 3 hours postmortem. The specimens were fixed at N0 ° for 1 to 3 hours OBSERVATIONS in 1 per cent OsO4 buffered to pH 7.6 with 0.1 M K phosphate; in some instances, the tissue was pre- General Description of Atrial Muscle Cells fixed at 0 ° for 1 to 3 hours in 6.25 per cent glutaraldehyde in the same buffer (12), washed over- The general organization of these cells is similar night in the buffer with sucrose added to restore to that described by others in both atrial and ven- osmolarity, and finally fixed for 1 to 3 hours at 0 ° tricular muscle fibers (1, 2, 8). Of particular in 1 per cent OsO4 buffered to pH 7.6 with 0.1 interest for us were the structurcs comprising the M K phosphate. Following dehydration in the cold central sarcoplasmic column or core (Figs. 1 and in a graded series of ethanol solutions, the tissue 2). Centrally placed within this core is a single was embedded in Epon 812 according to the method of Luft (13), or in methacrylate cross-linked with 5 elongate nucleus with numerous large mitochon- per cent divinylbenzene (14). Sections were cut on dria packed around each of its poles. Adjacent to a Porter-Blum microtome equipped with a diamond one, and occasionally to both, of the poles of the knife, collected on naked copper grids, and stained nucleus, the sarcoplasmic core contains a large with 0.5 per cent aqueous uranyl acetate for 30 population of spherical, clcctron-opaque granules minutes followed by alkaline lead or with alkaline intimately interspersed among the elements of a lead solution alone (15). The sections were strength- voluminous Golgi complcx. Similar granulcs are ened by a thin coat of carbon and examined in an found in much smallcr numbers in other loci in the RCA EMU 2B electron microscope or a Siemens cell as discussed later. In addition to these predom- Elmiskop I equipped with a 50 ~t objective aperture inant structures, the sarcoplasmic core contains ele- and operated with a double condenser at 80 kv. ments of rough endoplasmic reticulum, accumula- Cytochemical Procedures tions of glycogen particles, a few lipid droplets, and, scattered among all thcsc components, a The Gomori reaction for the detection of acid phosphatase (16) was performed on 50 tt frozen variable number of polymorphic bodies which sections of rat atrium and ventricle fixed in the cold could be described as dense bodies, residual for 2 hours with 4 per cent glutaraldehyde in 0.05 bodies, or lipofuscin pigment granules. M Na cacodylate (pH 7.1) and subsequently washed The sarcoplasmic core is enveloped by bundles for 2 hours at 0 ° in buffer to which 8 per cent sucrose typical myofibrils which occupy the remainder of was added. The sections were incubated at 0 ° for 1 the cell volume. These bundles arc separated by hour in 3 per cent Na-C~-glycerophosphate in 0.1 M sarcoplasmic layers containing mitochondria, ele- Na acetate at pH 5.0; the reaction mixture contained ments of the sarcoplasmic rcticulum, atrial gran- 0.12 per cent PbNO3 to precipitate liberated in- ules, and glycogen particles. The volume of the organic phosphate as electron-opaque lead phos- cell occupied by the myofibrillar envelope varies phate. Following a brief wash in acetate buffer, the sections were fixed at 0 ° in 1 per cent OsO4 in 0,1 according to the region of the atrium examined, M Na cacodylate (pH 7.1) for 11~ hours. Routine tending to be small in cells of the nodal and con- dehydration and embedment in Epon 812 followed. ductile tissue and relatively large in the rest. The chromaiCfin reaction for the detection of Atrial muscle fibers are bounded by a usual norepinephrine and cpinephrine in thin section was plasma membrane covered externally by a base-

FIGURE 1 Longitudinally sectioned muscle cell illustrating general features of atrial muscle cell. Anterior wall of the right atrium (rat). The sarcoplasmie core contains a population of spherical granules (ag) , profiles of the Golgi complex (G), numerous mitochondria, an occasional residual body (rb), and a few lipid droplets (1). Sarcoplasmie layers between myofibrils contain an occasional granule. × 13,000.

152 THE JOURNAL OF CELL BIOLOGY " VOLUME 23, 1964 J. D. JAMIESON AND G. E. PALADE Atrial Granules 153 ment membrane; the intercellular junctions consist the material contained in the granule varies with of typical intercalated discs. the type of fixative, the embedding medium, and The structures in the sarcoplasmic core, specif- the staining procedures employed on the section. ically the atrial granules and their relationship to After OsO4 fixation, Epon embedment, and alka- neighboring elements, will be discussed in detail line lead staining, the content is electron-opaque below. As indicated, many mammalian species and finely granular. Embedment in methacrylate were examined but, as the heart of the rat was following OsO4 fixation tends to enhance this most extensively studied, the description and granularity, as does staining the tissue block with illustration will be based primarily on this material. potassium permanganate prior to embedding (Fig. 9). If glutaraldehyde fixation precedes Detailed Description of Atrial Granules osmium tetroxide fixation, the content of the granules is much less opaque and generally more 1. GENERAL INTRACELLULAR DISTRIBUTION homogeneous (@ Fig. 5). Finally, the content OF GRANULES becomes extremely opaque in sections stained with As already mentioned, most of the "specific" uranyl acetate. granules of the atrial muscle cells occur within the 3. RELATIONS OF SPECIFIC GRANULES TO sarcoplasmic core where they arc packed in large COMPONENTS IN THE CENTRAL COR~ masses (up to 600 per cell) (Figs. 2 and 3) adjacent to one pole of the nucleus. Occasionally a few a. GOLGI COMPLEX: The large masses of granules are found on the sides and adjacent to specific granules are intimately associated with a the other pole of the nucleus. Because of the spindle voluminous Golgi complex whose elements appear shape of the sarcoplasmic core, the granular mass scattered throughout the sarcoplasmic core. The occupies a roughly conical portion of the cell. complex consists mainly of flattcned, stacked, Specific granules are also scattered in small num- smooth-surfaced cisternac and associated small bers throughout the sarcoplasmic layers separating vesicles. Large vacuoles arc rare or absent. Favor- adjacent myofibrils (Fig. 4). Further, similar ably oriented sections show that the flattened granules are seen in sarcoplasmic pockets imme- cisternae are extensively fenestrated and have diately beneath the plasma membrane, again in festooned margins which suggest that small vesicles either bud from or coalesce with the association with other cell components. In this cistcrnal periphery (Fig. 8). Some of the vesicles location, they may occur singly or in relatively in continuity with or in the immediate vicinity of Jarge masses (Fig. 5). the stacked cisternac have thickened membranes ~. MORPHOLOGY OF GRANULES surrounded by a halo of condensed cytoplasmic matrix (Fig. 8) reminiscent of features described The atrial granules of the rat are spherical by us in connection with some atrial granules and structures ranging in diameter from 0.25 to 0.50 ~. by others in connection with certain pinocytic They are bound by a limiting membrane ~ 80 A vesicles (19, 20). thick (Fig. 6) of the unit membrane type whose In many instances, the cistcrnae of the Golgi electron-opaque leaflets appear symmetrical in complex arc filled with a fine granular material, OsO4-fixed, uranyl acetate-alkaline lead stained and local cisternal dilatations contain masses preparations. In many cases, the opaque leaflets comparable in dcnsity and texture with the con- are thickened and stain intensely on one side of tent of the specific granules (Fig. 9). These find- the granule, forming a "plate" which appears to ings, together with the fact that the limiting bc relatively rigid (Figs. 2 and 7). External to membranes of the elements of the Golgi complex the plate a layer of fuzzy material with a radial or and of the granules are similar in width and helical orientation is frequently seen (Fig. 7). At staining characteristics (Fig. 10), suggest that thc high magnification, the limiting mcmbrane ap- latter are formed within the complex. The as- pears to havc points of discontinuity which may sumption is also supported by the fact that ven- represent a preparative artifact. tricular muscle cells, which do not produce The opaque content of the granules is often comparable granules, have a simple Golgi com- separated from the limiting membrane by a thin plex which occupies little space in the correspond- light zone ~ 250 A wide (Fig. 7). The texture of ing sarcoplasmic cores (Fig. 2 a). The relatively

154 THE JOURNAL OF CELL BIOLOGY - VOLUME ~3, 1964 large volume of the Golgi complex in atrial muscle C. GI. YCOGEN PARTICLES AND LIPID DROP- cells is particularly striking in fetal and newborn LETS: Small (250 to 300 A) dense particles in animals in which the cisternal elements of the variable but normally large numbers are scat- complex are frequently distended. tered among the other elements of the core (Figs. b. ROUGH-SURFACED ENDOpLASMIC RETIC- 3, 12, 13). They are identified as glycogen par- ULUU: Rough-surfaced elements of the endo- ticles because of their morphological similarity to plasmic reticulum frequently occur in the central the B-glycogen particles studied by Drochmans sarcoplasm of the atrial muscle cells of dog, (21), their stainability with alkaline lead solutions guinea pig, and cat. ~I hey are less common in rat (22), 1 and their similarity to the particles described and mouse. Most of them are relatively large in the muscle cells of turtle atria by Fawcett and cisternae disposed usually in stacks of 6 to 10 Selby (23). Glycogen particles are particularly parallel elements and occasionally in more com- numerous in the atrial muscle ceils of dogs (Fig. plex circular or concentric patterns. As a rule, the 13), pigs, and guinea pigs where they frequently cisternae are flattened (small diameter ~'~ 40 form large compact masses. They are also m#) and do not contain any well defined material. numerous in atrial muscle cells of fetal and new- Typical ribosomes stud the outer cisternal surface, born animals (rats and mice). and grazing sections show that they are arranged Lipid droplets also occur usually in small on it in spiral, circular, or rosette-like patterns numbers among the other elements of the sarco- (Fig. 11). The rough-surfaced cisternae are plasmic core (Fig. 1). generally located near the Golgi complex but do f. RESIDUAL BODIES, LIPOFUSCIN GRANULES: not appear to have any preferential relationship At the periphery of the masses of specific to it or to the atrial granules. It should be noted granules or scattered among them, there is a small that similar elements are rarely seen in the sarco- population of irregularly shaped bodies. Each measures ~ 0.5 # in diameter and is limited by a plasmic core of ventricular muscle cells. unit membrane of symmetrical type whose dense c. MITOCHONDRIA: Most of the space in the leaflets appear thinner and less dense than those of sarcoplasmic core of atrial muscle cells is taken by the limiting membrane of the atrial granules large (diameter ~ 1.5 #) mitochondria, distrib- (Fig. 6). The content of most of these bodies is uted at random and forming an envelope around dense, homogeneous, and finely granular (Figs. the Golgi complex and the masses of specific I, 2, 3, 6), but some of them also contain recog- granules. Most mitochondrial profiles are circular nizable subcellular components such as mito- and show parallel, tightly packed, and occasionally chondria, glycogen particles, and elements of the concentrically arranged cristae. Quite often the endoplasmic reticulum altered to a varied extent. profiles have narrow tails with few or no cristae In this respect, these bodies resemble residual inside. In three dimensions they probably repre- bodies described by others (24, 25) in rat liver and sent long, fine, finger-like projections of the outer nerve cells. Such bodies are absent in newborn and possibly inner mitochondrial membranes. rats and mice, and, according to our survey, in- d. CENTRIOLES: One or two centrioles of crease in number and size with the age of the ani- typical structure (nine peripheral tubular doub- mal. In older animals, i.e. cat, dog (Fig. 13), and lets) are occasionally found in the immediate human, they come to occupy a relatively large vicinity of the nucleus, usually between it and the x It should be pointed out, however, that this staining Golgi complex (Fig. 12). reaction is not specific and not understood.

FIGURE ~ Longitudinally sectioned muscle fiber from anterior wall of right atrium (rat). Numerous atrial granules (ag), elements of the rough-surfaced endoplasmie reticulum (rer) and a few residual bodies (rb) are shown. G, Golgi complex; gl, glycogen particles; arrows indicate thickened "plates" on granules. Compare this figure with a corresponding section of ventricular muscle (Fig. e a). X 8~2,000.

FIGURE ~ a Longitudinally sectioned muscle fiber. Anterior wall of the right ventricle (rat). The sarcoplasmic core is devoid of "atrial" granules; it contains a small Golgi complex (/7) and numerous mitochondria with frequent intramitochondrial granules (rag). #/, glycogen particles; sr, elements of tile sareoplasmic reticulum. X 8~,000.

For figures, see following pages. 156 THE JOURNAL OF CELL BIOLOGY • VOLUME ~3, 1964 J. D. JAMIESON AND G. E. PALADE Atrial Granule8 157 FIGURE 3 Central core of atrial muscle fiber (rat) illustrating the extensive stacks of Golgi eisternae (G) associated with specific atrial granules (ag). Several granules show asymmetric thiekenings of their bounding membrane (arrows). A residual body containing membranous material and glycogen particles is marked rb. X 44,000. part of the sarcoplasmic core. In the same time, granules commonly occur individually or in small the morphology of their content seems to change groups in the sarcoplasmic ribbons between myo- gradually, for the largest bodies appear as con- fibrils. Here they are closely associated with mito- glomerates of dense granular material and less chondria, glycogen particles, and elements of the dense droplets of varied sizes (Fig. 13). Such large sarcoplasmic reticulum but do not bear any bodies are identical with the lipofuscin pigment systematic relationship to either the latter or to granules whose morphology and chemistry has the bands of the adjacent myofibrils (Fig. 4). been the object of a number of recent studies (26, Larger accumulations of atrial granules are often 27). seen in sarcoplasmic pockets between the plasma We did not find any indication that these bodies membrane and the most peripheral myofibrils, are related to atrial granules. In this respect, it where they are again randomly scattered among should be mentioned that residual bodies and lipo- mitochondria and profiles of the superficial sarco- fuscin granules occur with equal frequency in plasmic reticulum (Fig. 5). Occasionally, these atrial and ventricular muscle cells. peripheral accumulations are associated, as in the central sarcoplasm, with Golgi complexes (Fig. 4. REI~ATION OF GRANULES TO STRUCTURES 14). Although many of these peripheral granules ELSEWHERE IN THE CELL are separated from the plasma membrane by as Though most of the population of atrial granules little as 100 A, their bounding membranes were is located in the central sarcoplasm, a few similar never seen to fuse with the plasma membrane

158 THE JOUleNAL OF CELL BIOLOGY • VOLUME ~, 1964 FIGURE 4 Sarcoplasmic layer between superficial myofibrils in an atrial muscle cell (rat) ; it contains specific granules (ag), elements of the rough surfaced endoplasmic reticulum (rer), and glycogen particles (gl). The plasma membrane is marked pro. Arrow denotes ribosomes in a whorl on the surface of a cisterna. X 44,000. leading to the discharge of the granule contents the intracellular distribution of granules exist at by a process observed in many glandular cells. various stages of development. The youngest animal studied, a 10 mm rat fetus, possessed no 5. DISTRIBUTION THROUGHOUT THE granules in atrial fibers though the Golgi com- MYOCARDIUM plexes were prominent. In late fetal and neonatal In order to determine the distribution of the rats the sarcoplasmic core of atrial muscle cells granules throughout the rat myocardium, regions contains relatively large populations of granules from the anterior and posterior walls of both atria closely associated with prominent and distended and ventricles were sampled. In general, muscle Golgi cisternae. Similar granules also occur, as in cells from both right and left atria contain specific the adult, between bundles of myofibrils and in granules. The largest numbers were found in cells sarcoplasmic pockets beneath the plasma mem- from the anterior wall of the right atrium and the brane. In these young animals the granules are, smallest in cells near the sulcus terminalis. Similar on the average, one half the diameter of those of regional variations were found in the atria of other adults. species. In no instances were specific granules seen 7. VARIATION WITH SPECIES in ventricnlar muscle fibers (cf. Figs. 2 and 2 a). An estimate was made of the extent of the 6. AGE VARIATIONS granule population and the sizes of granules in the A survey was also carried out on atria of rats of atria of various species. The rat and other small various ages to determine whether differences in mammals (mouse, hamster, bat) possess as many

J. D. JAMIESON AND G. E. PALADE Atrial Granules 159 as 600 granules in the sarcoplasmic core of the in the atrium and ventricles became highly atrial fibers. With increasing size of the mammal labeled. (rabbit, cat, dog, human) the granule content per The Gomori test for acid phosphatase at the cell is generally smaller. electron microscopic level (16) was also carried out In the rat, the granules range in diameter from on rat hearts. The reaction product, lead phos- 0.2 # to 0.5 # (average diameter, 0.42 #). Measure- phate, was present in residual bodies (Figs. 15 and ments in other species are given in Table I. As a 16) but absent from adjacent atrial granules. first approximation, they show that atrial granules with the greatest average diameter are found in DISCUSSION small mammals while granules from larger mam- Granules identified as small mitochondria, dense mals tend to be smaller. microsomcs, microbodies, lipid droplets, or dichte Kb'rper have been casually mentioned in general surveys of the atrial muscle of guinea pig, ox TABLE I (Kisch, 1956, references 4 and 5), and man (Poche, Average and Maximum Diameters of Atrial Granules 1958, reference 6; Battig and Low, 1960, reference from Anterior Wall of Right Atrium I0). As corps denses they have been described in All material prefixed in glutaraldehyde, post- more detail in the conductile and contractile tis- fixed in OsO4, and embedded in Epon 812. sue of the right atrium of the rat by Bompiani et Average Maximum al. (8), who speculated that these granules are re- Specimen diameter diameter lated to the special excitability of the bundle of His. Our findings confirm the general morphology /~ /.t and intracellular distribution reported by these Rat (adult) 0.42 0.53 authors, except for the limiting membrane of the Rat (newborn) 0.20 0.27 granules which they found absent. Viragh and Mouse (adult) 0.38 0.45 Porte (7) observed similar granules in the atrial Mouse (fetal) 0.22 0.30 muscle of the rat, recognized that they were not Hamster 0.26 0.31 present in all myocardial fibers, and suggested Bat 0.17 0.20 that they evolve into lipofuscin granules, a view Guinea pig 0.23 0.26 which, as discussed later, is probably incorrect. Rabbit 0.23 0.26 In lower vertebrates, morphologically similar Cat 0.24 0.28 granules have been mentioned in the atrial muscle Dog 0.18 0.22 fibers of the eel (Couteaux and Laurent, 1957, Pig 0.22 0.26 Human 0.25 0.33 reference 28) and studied in detail in the atrial and ventricular fibers of cyclostomes (Ostlund et al., 1960, reference 29; Bloom et al., 1961, reference 30). The hearts of the latter also contain interstitial 8. ATTEMPTS TO DEFINE CHEMICALLY cells with a large number of granules of similar THE (JRANULES appearance. Cyclostome hearts, particularly their Several attempts to identify the nature of the atria, store catecholamines in concentration 100 granular content were carried out. Since reserpine times greater than mammalian atria (30). Granule treatment appears to deplete the rat atrium of its fractions containing up to 18.5 #g/gm catechola- granulcs, suggesting that the latter may contain mines have been isolated from such hearts and catecholamines, a chromaffin reaction for the de- found to account for 50 to 70 per cent of the total catecholamines of the original tissue (30). Cyclo- tection of epinephrine and norepinephrine in stome hearts also are remarkably sensitive to in thin sections (17) was applied to rat atrial muscle. vivo reserpine treatment which releases 70 to 85 It was negative in atrial granules, though positive per cent of their catecholamines (30). These re- in the granules of the rat adrenal medulla, known sults indicate that the catecholamines of cyclo- to contain epinephrine and norepinephrine. H 3- stome hearts are stored in granules, but do not dopamine, the immediate precursor of norepineph- identify the source of the catecholamine-containing rine, was not accumulated in the granules as granules. It could be the interstitial cells, the studied by autoradiography (18), though nerves muscle cells, or both.

160 THE JOURNAL OF CELL BIOLOGY • VOLUME ~8, 1964 In the present study, specific granules have been core, the presence in the Golgi cisternae of mate- found exclusively within atrial muscle fibers of all rial similar in appearance to the granule content, the species examined, though interspecies varia- and the fact that the unit membranes surrounding tions have been detected. In general, the atria of both the granules and the Golgi cisternae are small mammals contain many granules of rela- similar in width and staining properties, suggest tively large size (0.4 #), whereas both granule size that the granules form by progressive condensation and frequency tend to decrease with increasing of a cell product in the vesicles or cisternae of the

FIGUUE 5 Accumulation of specific granules at tile periphery of an atrial muscle fiber (rat). G, peripheral extension of Golgi complex. X 18,000. size of the animal. Some exceptions to the gener- Golgi complex. The relationship of granules to the ality of their occurrence are suggested by the other components of the sarcoplasmic core seems available literature: no comparable granules were of little functional significance: it is more likely a noted in the conductile tissue of steer (9) or sheep reflection of the necessity of packing tightly many (31) atria, and in the muscle cells of the turtle structures into a limited space. atrium (23). Age variations have also been en- The frequent occurrence of granules imme- countered: no granules have been found in the diately beneath the plasma membrane suggests functional, but not fully differentiated muscle cells that the muscle cell discharges its granules or their of young fetuses (10 mm rat); they were detected content through the membrane, but evidence sup- in late stages of fetal development, but were smaller porting this view has not been obtained. and less numerous than in adult animals. The presence of catecholamines in high concen- The intimate relationship of the atrial granules trations in granules isolated from cyclostome to the extensive Golgi complex in the sarcoplasmic hearts (30), the fact that these compounds are

J. D. JAMIESON AND G. E. PALADE At'rid Granules 161 more concentrated in the atria than in the ven- Wolfe et al. (34) have demonstrated by electron tricles of rats and other mammals (32), and the microscopic autoradiography that atrial granules finding that reserpine treatment, known from fail to accumulate injected H3-norepinephrine other studies (33) to deplete atria of catechol- though sympathetic nerves do. (e). Potter and amines, also reduces the population of atrial Axelrod (35) have shown that H3-norepinephrine granules in rats (9), suggested to us that mam- is concentrated in the microsomal fraction of rat malian atrial granules may contain catechol- hearts which we know not to contain granules. It

FIGURE 6 Unit membranes (apposed arrows) of atrial granules (ag) and a residual body (rb). The membrane bounding the latter stains less intensely than the limiting membrane of the granu]es. Note the heterogeneous content of the residual body. >( 100,000. amines. The evidence thus far obtained is against is unlikely that the atrial granules contain 5- this assumption: (a). Ventricular muscle contains hydroxytryptamine because this compound is catecholamines but its cells have no granules (this present only in small amounts in rat hearts (36), result, however, may be accounted for by stores of and because a labeled precursor of 5-hydroxy catecholamines in adrenergic nerve endings). (b). tryptamine, H3-5-hydroxytryptophan, is accumu- The chromaffin reaction failed to demonstrate lated in only small amounts in the heart (37). catecholamines in atrial granules. (c). Autoradio- Without excluding it, these findings render un- graphic studies failed to demonstrate any uptake likely the possibility that the granules contain of H3-dopamine, the immediate precursor of nore- either catecholamines or other monamines. Isola- pinephrine, into atrial granules though the nerves tion and purification of the granules from atrial of the heart as well as the granules of adrenal homogenates is required for the final identification medullary cells became specifically labeled, (d). of their content, yet mass isolation may prove

162 THE JOURNAL OF CELL BIOLOGY • VOLUME ~3, 1964 FIGURE 7 Atrial granules showing the symmetrical thickenings of their unit membrane which forms a "plate" (apposed arrows). A layer of fine fibrillar material is seen external to the plate of the upper granule. Its fibrils seem to be helically oriented. Right atrium (rat). )< 66,000.

FlOraE 8 Full-faced view of a fenestrated Golgi cisterna (G). Its margin shows interconnecting tubules and "budding" Golgi vesicles. The arrows point to the thickening of the membrane on certain "buds." A residual body is marked rb. )< 57,000.

J. D. JAMIESON AND G. E. PALADE Atrial Crranules 163 difficult because the granules are only a minor bounded bodies with a dense, heterogeneous con- component of atrial myocardium. tent which occupy a large volume of the cell. Such The pleomorphic dense bodies found in the bodies are identical with the lipofuscin pigment sarcoplasmic core of atrial and ventricular muscle granules described by Malkoff and Strehler (26) fibers of adult rats are similar to those already de- in electron micrographs of human heart. It has scribed in other cell types, especially in rat neurons been known for some time from light microscopic (25). We have identified these entities as residual studies (40) that the lipofuscin granules of human bodies because they frequently contain altered, myocardium contain acid phosphatase, and yet still recognizable subcellular components Strehler and Mildvan (27) have isolated there- (mitochondria, elements of the sarcoplasmic reticu- from a fraction of pigment granules rich in the lure, and glycogen particles). Such bodies are cur- same enzyme. This common enzymatic activity rently assumed to be foci of intracellular autolysis suggests that the large lipofuscin granules arise by (24). The assumption is supported by the fact that the progressive coalescence of the small residual they contain acid phosphatase, a well established bodies found in young animals. In this connection lysosomal enzyme (38). Though absent in muscle there is evidence that the amount of lipofuscin fibers of young rats and mice, residual bodies seem pigment increases linearly with age in the muscle to accumulate as the animal ages, as already ob- cells of human heart (41). served for other cell types (39). In old individuals The specific granules of rat atria were not found from other species (cat, dog, human), the sarco- to contain acid phosphatase as detected by the plasmic core of atrial and ventricular muscle cells Gomori test (16); accordingly, though closely contains large (2 to 3# in width) membrane- associated with the acid phosphatase-positive re-

Fmuan 9 Golgicisternae (G) containing material similar in appearance to the content of the atrial granules (arrow). Rat atrium. X 44,000.

164 ThE JOURNAL OF CELL BIOLOGY • VOLUME 23, 1964 FIGURE 10 Unit membranes (apposed arrows) of Golgi cisternae (G) and atrial granules (ag). Note that these membranes are similar in total thickness and in the staining properties of their leaflets. Rat atrium. >( 1~0,000. sidual bodies, they do not appear to be the pre- mammalian atrial muscle fibers is a rare example cursors of the latter as postulated by Viragh and of multiple cellular specialization. In this case, a Porte (7). Ventricular muscle cells, free of specific highly differentiated contractile cell appears to granules in all species studied, contain as many (if possess a second specialized function in its ability not more) residual bodies as do atrial muscle cells. to form and store a population of granules pre- These findings suggest that specific granules and sumably secretory in nature. residual bodies are different in origin and function. Finally, the presence of specific granules within Received for publication, December 23, 1963.

For References, see page 171.

J. D. JAm~so~ AND G. E. PaLADE Atrial Granules 165 FIGURE 11 Complex pattern of anastomosing, rough-surfaced cisternae of the endoplasrnic reticulum in the central core of an atrial muscle fiber (guinea pig). At arrows, the section grazes the cisternae and reveals the patterns formed by the ribosomes attached to their limiting membrane, rb, residual body. X 4~,000.

166 THE JOURNAL OF CELL BIOLOGY • VOLUME ~3, 1964 FIGtTRE 1~ Central core of atrial muscle fiber (guinea pig) illustrating a centriole (C) composed of 9 pairs of tubules. G, Golgi complex; gl, glycogen particles. )< 6~,000.

J. D. JAMIESON AND G. E. laAI~ADE Atrial Granules 167 FtGUaE 13 Lipofuscin pigment granule (lp) in the sarcoplasmic core of an atrial muscle fiber (dog). N, nucleus; gl, glycogen particles; mf, myofibrils. X 3~,000.

168 THE JOURNAL OF CELL BIOLOGY " VOLUME 23, 1964 FIGURE 14 Peripheral Golgi complex in an atrial muscle fiber (rat). rer, rough-surfa~d endoplasmic reticulum; gl, glycogen particles; ag, atrial granules. X 50,000.

J. D. JAMIESON AND G. E. PALADE Atrial Granules 169 170 THE JOITRNAL OF CEI,L BIOLOGY • VOLITME ~,~, 1964 REFERENCES I. STEN(~ER, R. J., and SPIRO, D., Structure of the 14. KUSHIDA, H., A new embedding method for cardiac muscle cell, Am. J. Med., 1961, 30,653. ultrathin sectioning using a methacrylate resin 2. MooRE, D. H., and RUSKA, H., Electron micro- with three dimensional polymer structure, J. scope study of mammalian cardiac muscle Electronmicroscopy, 1961, 10, 194. cells, J. Biophysic. and Biochem. Cytol., 1957, 3, 15. KARNOVSKY, M. J., Simple methods for "staining 261. ing with lead" at high pH in electron micro- 3. PORTER, K. R., and PALADE, G. E., Studies on scopy, J. Biophysic. and Biochem. Cytol., 1961, the endoplasmic reticulum. III. Its form and I1, 729. distribution in striated muscle cells, J. Bio- 16. MILLER, F., Acid phosphatase localization in physic, and Biochem. Cytol., 1957, 3, 269. renal protein absorption droplets, in 5th 4. KISCH, B., Electron microscopy of the atrium of International Congress for Electron Micros- the heart, Exp. Med. and Surg., 1956, 14, 99. copy, Philadelphia, 1962, (S. S. Breese, Jr., 5. KISCH, B., Electron microscopic investigation of editor), New York, Academic Press, Inc., the heart of cattle. I. The atrium of the heart 1962, Q-2. of cows, Exp. Med. and Surg., 1959, 17, 247. 17. WooD, J. G., and BARRNETT, R. J., Histo- 6. POCHE, R., Submikroskopische Beitr~ige zur Pa- chemical differentiation of epinephrine and thologic der Herzmuskelzelle bei Phosphorver- norepinephrine granules in the adrenal giftung, Hypertrophic, Atrophic und Kaliurn- medulla with the electron microscope, Anat. mangel, Virchow's Arch. path. Anat., 1958, 331, Rec., 1963, 145, 301. 165. 18. CARO, L. G., and VAN TUBEROEN, R. P., High 7. VIRAGI4, S., and PORTE, A., Le noeud de Keith resolution autoradiography. I. Methods, J. et Flack et les diff6rentes fibres auriculaires Cell Biol., 1962, 15, 173. du coeur de rat. ]~tude en microscopic optique 19. ROTH, W. F., and PORTER, K. R., Specialized et 6lectronique, Compt. rend. Acad. sc., 1960, sites on the cell surface for protein uptake, in 251, 2086. 5th International Congress for Electron Mi- 8. BOMPIANI, G. D., ROUILLER, C., and HATT, croscopy, Philadelphia, 1962, (S. S. Breese, P. Y., Le tissu de conduction du coeur chez Jr., editor), New York, Academic Press, Inc., le rat. l~tude au microscope 61ectronique, 1962, LL-4. Arch. Maladies Coeur. et Vaisseaux, 1959, 52, 20. FARQUHAR, M. G., WIsslo, S. L., and PALADE, 1257. G. E., Glomerular permeability. I. Ferritin 9. RttODIN, J. A. G., MISSIER, P., and REID, L. C., transfer across the normal glomerular capil- The structure of the specialized impulse- lary wall, J. Exp. Med., 1961, 113, 47. conducting system of the steer heart, Circula- 21. DROCHMANS, e., Morphologie du glycog~ne. tion, 1961, 24, 349. Etude au microscope 61ectronique de colora- 10. BATTIG, C. G., and Low, F. N., The ultra- tions n6gatives du glycog~ne particulaire, J. structure of human cardiac muscle and its Ultrastruct. Research, 1962, 6, 141. associated tissue space, Am. J. Anat., 1961, 22. REVEL, J. P., NAPOLITANO, L., and FAWCETT, 108, 199. D. W., Identification of glycogen in electron 11. PALADE, G. E., Secretory granules in the atrial micrographs of thin tissue sections, J. Bio- myocardium, Anat. Rec., 1961, 139, 262. physic, and Biochem. Cytol., 1960, 8, 575. 12. SABATINI, D. D., BENSCI4, K., and BARRNETT, 23. FAWCETT, D. W., and SELEY, C. C., Observa- R.J., Cytochemistry and electron microscopy. tions on the fine structure of the turtle atrium, The preservation of cellular ultrastructure J. Biophysic. and Biochern. Cytol., 1958, 4, 63. and enzymatic activity by aldehyde fixation, 24. ASHFORD, T. P., and PORTER, K. R., Cytoplasmic J. Cell Biol., 1963, 17, 19. components in hepatic lysosomes, J. Cell 13. LUFT, J. H., Improvements in epoxy resin em- Biol., 1962, 12, 198. bedding methods, J. Biophysic. and Biochem. 25. HERNDON, R. M., The fine structure of the Cytol., 1961, 9, 409. Purkinje cell, d. Cell Biol., 1963, 18, 167.

FIGURES 15 and 16 Sections of atrial muscle fibers (rat) reacted for acid phosphatase. Note the residual bodies (rb) which contain recognizable glycogen particles and are marked by dense crystals of reaction product (lead phosphate) (arrows). Adjacent atrial granules (ag) are free of reaction product and presumably lack acid phosphatase activity. Fig. 15, X 72,000; Fig. 16, X 70,000.

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172 THE JOURNAL OF CELL BIOLOGY • VOLUME ~3, 1964