jknal
of Molecular and Cellular Cardiology (1978)
Ultrastructural
10,449-459
Studies of Metabolically Adult Rat Heart Myocytes
Active
Isolated
E. C. CARLSON*, D. S. GROSSOt, S. A. ROMERO*, C. J. FRANGAKISt, C. V. BYUSf AND R. BRESSLEQ Defiartments of Anatomy*, Pharmmology~ and Internal Medic&f, College of Medicine, University of Arizona, Tucson, Arizona 85724, U.S.A. (Received 21 June 1977, accepted in revisedform 29 Ju& 1977) E. C. CARLWN, D. S. GROSSO,S. A. ROMERO, C. J. FRANGAKIS,C. V. Bws AND R. BRESSLER. Ultrastructural Studies of Metabolically Active Adult Rat Heart Myocytcs. Jouwzal of Molecular and Cellular Cardiology (1978) 10, 449-459. Myocytes from adult rat heart were isolated by a method recently developed by us in which hearts were pre-perfused with calcium-free phosphate-buffered saline medium containing collagenase and hyaluronidase. This was followed by incubation in the enzyme medium and cellular sedimentation through 3% Ficoll. Myocytes isolated in this manner were metabolically active and morphologically intact. In the present study, scanning and transmission electron microscopy of isolated myocytes showed long cylindrical cells with transverse microridges which corresponded directly with sarcomere lengths. Most cells appeared to be in a fully contracted state. Contractile elements and associated membranes, other intracellular compartments and sarcolemmae were indistinguishable from their in vim counterparts. Although myocytic basal laminae and other structurally identifiable surface coats were removed by our isolation procedure, sarcolemma e remained remarkably unaffected. Cyclic AMP assays in control and epinephrineor glucagon-stimulated cells strongly suggested that membranebound receptors were present and the functional integrity of the sarcolemmae was maintained in our preparations. KEY WORDS: Cyclic AMP.
Rat
heart;
Isolated
myocytes;
Ultrastructure;
Epinephrine;
Glucagon;
1. Introduction The development of a technique for the production of isolated adult heart myocytes which are morphologically and metabolically intact has been attempted by various investigators [3, 4, 6, 20, 23, 281. Such a method is valuable because cultures of isolated myocytes provide a potentially useful model for studies of this cell type in an environment which is free of neural and humoral influences. Moreover, this model provides an experimental tool for investigations of myocytic growth, differentiation, contraction, biochemical control mechanisms and pharmacological or electrical responses without regard for the modifying effects of extracellular matrix and non-muscular tissues present in vivo. We have recently developed a method [7] for the isolation of a uniform population of metabolically active myocytes from adult rat hearts which provides a yield 0022-2828/78/0501-0449
$02.00/O
0
1978 Academic
Press Inc.
(London)
Limited
450
E. C. CARLSON
ETAL.
(50 mg protein/g heart) and index of viability (90 to 95%) which is greater than those previously reported. Our preparations were virtually devoid of non-muscle cells and up to 80% of the myocytes spontaneously contracted. Furthermore, contraction could be halted (by reducing the temperature to 4°C) and resumed 24 to 48 h later by returning the cells to room temperature. In addition, we suggested that by scanning electron microscopy (SEM) , cu. 80% of all isolated myocytes were intact (based on topographical features). Sarcolemmae appeared undisturbed except for occasional blebs and microridges. In the present investigation, we provide a detailed ultrastructural analysis of myocytes isolated by our method. We demonstrate by both SEM and transmission electron microscopic (TEM) techniques that contractile elements, other intracellular compartments, and sarcolemmae of isolated myocytes maintain their morphological integrity. Moreover, our cyclic AMP assaysof control and hormone-stimulated myocytes demonstrate biochemically the functional integrity of myocytic cell membranes and associatedhormone receptors.
2. Materials
and Methods
Cell isolation
The isolation of myocytes from ventricular tissue of adult rats was performed as described previously [7]. Hearts from 150 to 200 g rats were perfused on a “Langendorlf” perfusion apparatus at 37°C in a retrograde manner through the aorta for 5 min to remove blood. The perfusion conditions were: rate, 30 ml/min; pressure, 60 mmHg; buffer, pH 7.4, containing 128 mu NaCl, 5.4 mM KCI, 2 mM NaHaPOd. HaO, 8 mM NasHPOd, 15 mM glucose, bubbled through with 95% OS/~% COa. The perfusate was changed and replaced with buffer containing 1 mg/ml collagenase (Type II, Worthington), 1 mg/ml hyaluronidase (Sigma), and 0.1 mg/ml BSA and perfused for 30 min. The ventricles were divided into pieces and placed in a clean siliconized 25 ml Erlenmeyer flask with 3 ml of enzyme-buffer solution and incubated at 37°C. After 15 min, 10 ml of cold buffer was added. The supernatant containing detached cells was decanted into a polyethylene centrifuge tube. Enzyme-buffer solution was added to the tissue fragments and the incubation procedure repeated. The cells were centrifuged immediately after decantation at 60 g for 3 min in a tabletop centrifuge. They were resuspendedand allowed to settle in 10 ml of buffer twice. The washed cells were layered atop 10 ml of 3% Ficoll in buffer (w/v) and centrifuged as above. Approximately 50 mg of cellular protein can be obtained from 1 g of heart tissue.The cells are free of contamination by blood cells, fibrous material and severely damaged heart cells. Viability as measured by trypan-blue exclusion is 90 to 95% and approximately 80% of the cells are contracting spontaneously.
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
451
Scanning electron microscopy (SEM)
Following isolation, isolated myocytes were fixed at room temperature in 30 to 50 ml of Karnovsky’s [II] paraformaldehyde-glutaraldehyde fixative buffered at pH 7.3 with 0.1 M sodium cacodylate. Double-sided cellophane tape was placed in the bottom of a small plastic beaker and the cells (still in fixative) were decanted into the beaker and allowed to adhere to the tape (usually overnight). Cells were carefully rinsed in 0.2 M cacodylate buffer before osmication (1 h in 2% cacodylate buffered 0~04). The cells were rinsed briefly in distilled water prior to dehydration in a graded seriesof acetones. This was followed by critical point drying in CO2 (DuPont-Sorvall critical point drying apparatus). The double-sided tape with adhering cells was mounted on aluminium specimen stubs, coated with evaporated carbon and gold (Denton DV-502 vacuum evaporator) and observed in an ETEC Autoscan scanning electron microscope at original magnifications of 175 to 1000 diameters. Some of the dried specimenswere disrupted with cellophane tape in an effort to remove the sarcolemma and render internal structures visible. These were recoated with carbon and gold prior to observation. Transmission
electron microscopy (TEM)
Pellets of cardiac cells were washed several times with phosphate-buffered saline and then immersed in cold (4°C) paraformaldehyde-glutaraldehyde fixative buffered at pH 7.3 with 0.2 M sodium cacodylate [IA. Fixation lasted 1 h after which the cells were rinsed with buffer and post-fixed 1 h at room temperature in cacodylate-buffered 2% osmium. The cellswere rinsed again in buffer, dehydrated in an ascending seriesof graded ethanols and propylene oxide prior to embedding in Epon-Araldite [l]. Epoxy blocks were cured overnight at 37°C and an additional 48 h at 60°C. One-micron-thick sections were cut on a DuPont-Sorvall MT-2 Ultramicrotome. These were stained with toluidine blue (1% in 1y. sodium borate) and observed by light microscopy for initial block orientation. Thin sections were cut using the same ultramicrotome equipped with a diamond knife. These sectionswere picked up on naked 300 mesh copper grids and stained with uranyl acetate (5% in absolute ethanol) and lead citrate [29]. Thin sections were observed in a Philips 300 transmission electron microscope at original magnifications of 3300 to 27 000 diameters. Cyclic AMP
Cyclic AMP was determined by measuring the activation of purified beefheart protein kinase [13]. Cells, 4.3 mg protein/ml, were incubated with or without hormones (or drug) for 2 min at 37°C in isolation medium. The reaction was stopped by addition of 0.4 N perchloric acid. Cyclic AMP was extracted into the
452
E. C. CARLSON
ETAL.
acid and purified by ion-exchange chromatography [16]. The purified cyclic AMP was quantitated by its ability to activate cyclic AMP-dependent protein kinase. Results were compared to a standard curve for cyclic AMP activation of protein kinase. Protein assay
Protein was measured by the method of Lowry et al. [15]. Bovine serum albumin was used asa standard. 3. Results Scanning electron microscopy (SEM)
When isolated adult rat myocytes are observed by SEM, they appear as long slender cells which average 20 to 25 pm in width and 100 to 125 l.mi in length (Plate 1). They exhibit numerous angular processeswhich could represent points of branching with adjacent myocytes in vivo. The ends of the cells are frequently rough and irregular suggestingpossibleinterdigitation with other cells in regions similar to intercalated discsdescribed by transmissionelectron microscopic (TEM) studies. The cells frequently display longitudinal ridges which extend more than half the length of the cell. A conspicuous and nearly constant feature of isolated myocytes is a seriesof transverse microridges ca. 1.5 to 2.0 pm in width. These run perpendicular to the longitudinal ridges and therefore to the long axis of the cell. They extend over the entire cellular surface imparting a courderoy appearance to the sarcolemma, the integrity of which is not disturbed. It appears as a clean, continuous, and competent encasementfor the cellular contents. When individual heart cells are teased apart with cellophane tape following critical point drying (Plate 2) numerous cytoplasmic structures are visualized. Individual myofibrils oriented parallel to the long axis of the cell are separated by this technique. Aggregates and long rows of mitochondria normally located between myofibrils are also recognizable. Undisturbed myofibrils are crossed at regular intervals (1.5 to 2.0 pm) by cylindrical structures which extend in register acrossthe cell. Separated myofibrils exhibit a similar periodicity suggesting that the cylindrical structures surround each individual myofibril at regular intervals. It is possible that these cross-striations represent transverse tubules of the myocardial T-tubule system. Transmission
electron microscopy ( TEM)
In an effort to derive the most morphological data from our TEM specimens, we endeavored to record photographically primarily those cells cut either parallel or normal to the long axis of the cell. At low magnifications, longitudinal sections through adult rat myocytes show numerous parallel (occasionally branched)
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
453
myofibrils. These are separated by rows of contiguous mitochondria which exhibit numerous angular cristae. Nuclei are centrally located and aggregates of mitochondria are present in cytoplasmic caps at nuclear poles. These areas are free of myofibriis and associated membranes. Each myofibril exhibits prominent sarcomeres delimited by Z-lines. Z-lines appear to be in register throughout the cell and are approximately 1.5 pm apart. Sarcolemmae appear as continuous membranes and are thrown into transverse microridges which run normal to the long axis of the cell. The ridges are separated by shallow but distinct troughs (1.5 pm apart) and these can be directly correlated with ridges and troughs identified by SEM (see Plate 1). The location of each trough corresponds precisely with the Zline of the subjacent myofibril. When myocytes cut in cross-section are observed at low magnification, the cylindrical shape of the cell is apparent (Plate 4). The centrally placed nucleus is likewise cylindrical and is surrounded by numerous mitochondria, myofibrils and their associated membrane systems. The T-tubule system of the myocardium has longitudinal elements (adjacent and parallel to myofibrils) which are particularly evident in cross-sections. Also, this latter orientation clearly demonstrates the excellent continuity of the sarcolemma. Only occasional blebs (which are not unexpected in tissues from which the basal lamina has been removed) mar an otherwise undisturbed cell membrane. Sarcolemmal invaginations are frequently seen where T-tubules are continuous with the extracellular space but most frequently myocardial cell membranes are subtended directly by myofibrils, mitochondria or subsarcolemmal cisternae of the sarcoplasmic reticulum. No extracellular materials are recognizable on the external surface of the sarcolemma. At higher magnifications, longitudinal sections through myocytes show that myofibrils vary in thickness along their length and are occasionally branched (Plate 5). They are surrounded along the length of each sarcomere by sarcoplasmic reticulum and at prominent Z-lines exhibit a single T-tubule. These latter structures are not as constant or pronounced as one would predict based on SEM observations (see Plate 2). Nevertheless, they are present in most preparations. The morphological integrity of the sarcolemma is clearly maintained and is invaginated occasionally by small micropinocytotic vesicles. Cross-sections through cytoplasmic areas similar to those seen in Plate 5 show numerous myofibrils surrounded by sarcoplasmic reticulum, longitudinal elements of the T-tubule system and mitochondria (Plate 6). A few lipid droplets are also observed. Mitochondria appear to occupy almost all of the cytoplasm not filled by contractile elements. Their cristae are highly indistinct when seen in cross-section (Plate 6) in contradistinction to the crisp, angular cristal plates observed when mitochondria are sectioned parallel to the long axis of the cell (Plate 5). This suggests that cross-sections yield enface views of the cristae and therefore most of them are oriented perpendicular to their adjacent myofibrils.
454
E. C. CARLSON
ETAL.
Individual sarcomeresof myocytes prepared by our method average about 1.5 pm between Z-lines. They consistently exhibit both thick and thin filaments throughout their length (Plate 7). Therefore, they do not demonstrate H-bands or I-bands which consist of only thick or only thin filaments respectively. An Mband composed of multiple transverse electron-dense lines is present in our specimens and is flanked on either side by an electron-lucent area. Both thick and thin filaments appear to extend laterally from M-bands to the substanceof the Z-lines. Glycogen granules are frequently associatedwith the myofilament lattice and/or the sarcoplasmic reticulum (Plate 8). High resolution TEM observations of cross-sectionsthrough myofibrils demonstrate both thick and thin filaments irrespective of the position along the sarcomere (Plate 9). The thick (150 to 160 A) filaments are hexagonally arranged around a seventh “central” thick filament. Thin (80 A) filaments are likewise hexagonally arranged around “central” thick filaments and appear to be equidistant from each other and from adjacent thick filaments. Occasionally, very fine projections from thick filaments are observed in close contact with the nearest thin filaments. TABLE
1. Effects of epinepbrine, glucagon and I-methyl-3-isobutylxantbine AMP levels in isolated cardiac myocytes*
Concentration (PM) 0 0.01 0.10 1.0 10.0
on cyclic
I-methyl-3isobutylxantbine Epinepbrine
Glucagon (pmol/cyclic AMP/mg
2.1 1.7 2.1
protein)
2.1 2.1 6.3 15.8 29.5
22.7
* Each value represents the mean of two determinations.
Hormonalstimulationof cyclic AMP levels In an effort to determine the integrity of the sarcolemma by biochemical means, cyclic AMP levels were determined in freshly prepared heart cells. Similar determinations
were carried
out in cells incubated
with
the hormones
epinephrine
and
glucagon and the phosphodiesteraseinhibitor 1-methyl-3-isobutylxanthine (MIX). The concentration of cyclic AMP, 2.1 pmol/mg protein (Table I), was found to be within the range previously reported for the perfused heart, 2 to 4 pmol/mg protein [12, 241, and ventricular tissue slices, 2.6 pmol/mg protein [Za. Epinephrine, glucagon and MIX all increased the levels of cyclic AMP in the cells by
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
455
approximately 14 times the amounts measured in controls at drug levels of 10 PM. Although glucagon and epinephrine induced the same stimulation of cyclic AMP production at a concentration of 10 PM, the threshold for glucagon appeared to be lower, less than 0.1 PM, as compared to 0.1 to 1.O PM for epinephrine. Glucagon has been reported to have a slightly greater effect on adenyl cyclase than epinephrine in the rat heart at concentrations between 0.01 and 1.0 PM [21]. 4. Discussion
Various procedures have been reported [3, 4, 6, 20, 23, 281 for the preparation of adult rat heart myocytes for in vitro studies. Most of these methods have been plagued with low yields and/or poor viability. Several investigators have illustrated their preparations with photomicrographs [4, 20, 281 or electron micrographs [3, 17] but, in general, careful morphological studies on isolated adult myocytes have not been carried out. In the present study, an ultrastructural analysis of metabolically active isolated adult rat myocytes was carried out. These cells were isolated by a technique [7] recently developed by us. Our method is superior to most others in that it provides : (a) a uniform population of myocytes with non-muscular cells virtually excluded, (b) a high yield of cells (50 mg protein/g heart), (c) an excellent index of viability (90 to 95% by trypan blue-exclusion), and (d) good longevity for biochemical experiments (75% or original lactate dehydrogenase activity is retained after 24 h) . Our previous report [7], showed that isolated myocytes were morphologically intact as illustrated by scanning electron microscopy (SEM). Sarcolemmae appeared undisturbed except for small microridges. The functional integrity of thesemembraneswas substantiated by biochemical data which showed that glucose uptake was stimulated by insulin in isolated cells. Therefore, preservation of insulin receptors and the integrity of myocytic sarcolemmaewas virtually assured. Other investigators [I, 20, 281 have shown by bright field or phase contrast photomicroscopy that isolated heart cells are cylindrical with abruptly angular ends. Most studies demonstrate an average length of 100 to 150 pm and widths of approximately 10 to 30 pm. The resolution of photomicrographs doesnot allow observation of good surface detail but most morphological studies of isolated cells show at least a hint of cross-striations. This finding is consistent with our SEM observations which show that most (if not all) isolated myocytes exhibit transverse microridges which extend over their entire length. The ridges are approximately 1.5 pm apart and are separated by shallow troughs. When the sarcolemma was mechanically removed and subjacent intracellular contents exposed, small cylindrical structures, which probably represent elements of the T-tubule system, extended in register (ca. 1.5 pm apart) acrossthe cell. These structures appeared to surround each individual myofibril since 1.5 pm periodicity could be demonstrated on disrupted myofibrils as well as those which remained in
456
E. C. CARLSON
ETAL.
situ. These results are consistent with a number of studies [Z, 28, 26, 271 which have demonstrated transverse and longitudinal elements of the T-tubule system in cryofractured myocardium in vivo. Most in vivo studies [Z, 18, 271 indicate that Ttubules are much wider spaced (up to 2.5 pm) than those seen in our investigation. This could be due to the physiological state of the muscle at the time of preparation. Our transmission electron microscopic (TEM) studies show conclusively that transverse sarcolemmal microridges seen in SEM correspond directly with the lengths of sarcomeres. Furthermore, the troughs between ridges are located precisely at the point of Z-lines. Since it is believed [19] that ca. 1.5 pm is the minimum length for sarcomeres, it is possible that most myocytes isolated by our procedure may be fully contracted at the time of fixation. This is not unreasonable since almost all isolated cells were contractile and the addition of glutaraldehyde, a know stimulator of protein cross-linking [22], to the incubation medium could result in rapid formation of actinomyosin cross-links. This is particularly true in light of the unrestrained nature of isolated cells. They are unbound by other cells, basal lamina or other components of the extracellular matrix and therefore are free to react as individual contractile units rather than in concert as in the whole organ in vivo. Intracellular contractile elements (myofibrils and associated membranes) were intact in nearly all of our preparations. Myofibrils were frequently branched, varied in width and were separated by rows of mitochondria. They showed sarcomerit segmentation which frequently crossed the entire cell in register. This is consistent with the normal morphology of myocardial cells in vivo [19]. The idea that most myocytes in our preparation were in a state of full contraction was corroborated by TEM observations of individual sarcomeres. Z-lines were approximately 1.5 pm apart and both thick and thin filaments appeared to extend this entire distance. H-zones and I-bands were not distinguishable and the entire sarcomere appeared to consist of a large A-band. This data was consistent with the morphology of classical mechanisms suggested for striated muscle contraction [ 101 and, since it is believed [19], that thick filaments are 1.5 pm in length, implied that the myofibrils were fully contracted. The appearance of M-bands and Llines (electron¢ areas on either side of the M-band) in our preparations was not unexpected because the dimension of this structural complex is not altered in different sarcomere lengths [ 191. Cross-sectional high-resolution microscopy further substantiated our idea of full contraction of myocytes because in each section cut normal to the direction of myofilaments, both thick (150 to 160 A) and thin (80 A) filaments were observed. These were hexagonally arranged around an arbitrary seventh “central” thick filament and were morphologically similar to those seen by other investigators in the non-H-zone portion of A-bands’. T-tubules surrounded myofibrils at points of Z-lines but were not as prominent as might have been predicted based on SEM of disrupted cells. The reasons for this inconsistency are not known but it is possible that tubules may collapse during
PLATE 1. Scanning electron micrograph of isolated adult rat myocyte. Cells typically appear as long slender cylinders and exhibit branching processes (*) which are parallel to the long axis of the cell. Irregular ends (ID) probably represent areas of intercalated discs. Longitudinal ridges (long arrows) and transverse microridges which average 1.5 [LM across (opposing arrows) are prominent features of the cell surface. x 1500. PLATE 2. Scanning electron micrograph of adult myocyte from which the sarcolemma has been removed. Underlying myofibrils (mf) are longitudinally arranged with intervening mitondondria (M). Elements of the T-tubule system are evident both in sihr (triple arrows) and associated with disrupted myofibrils (double arrows). Areas of branching (*) and intercalated discs (ID) are recognizable. x 1500. PLATE 3. Low power transmission electron micrograph of longitudinal section through isolated myocyte similar to that shown in Plate 1. The sarcolemma is thrown into transverse ridges (arrows) which measure IX. 1.5 pm (trough to trough). Individual myofibrils exhibit sarcomeres (S) whose length corresponds directly to the sarcolemmal microridges. M, mitochondrion; NU, nucleus, x 11300. PLATE 4. Low power transmission electron micrograph of cross-section through entire isolated myocyte. The central nucleus (NU) is surrounded by myofibrils (mf) cut in cross-section and numerous mitochondria (M). A few longitudinal components of the T-tubule system are recognizable. The sarcolemma is remarkably well-preserved and shows few blebs (b). Several sarcolemmal invaginations (arrows) could represent areas at which T-tubules may be continuous with the extracellular space. X 8300. PLATE 5. Higher magnification of isolated myocyte sectioned in a plane similar to that shown in Plate 3. Z-lines ofsarcomeres are cc. 1.5 ym apart and correspond directly with troughs of sarcolemmal transverse microridges (large arrows). The sarcolemma (S) is continuous and shows invaginations associated with micropinocytotic vesicles (small arrows). No basal lamina material or other surfaceassociated structures are present. Myofibrils (mf) vary in thickness and are occasionally branched. They are surrounded by sarcoplasmic reticulum (SR) along the length of each sarcomere and by Ttubules (T) at each Z-line (Z). Most mitochondria (M) are interspersed between myofibrils but some are located immediately beneath the sarcolemma (see Plate 4). x 23 500. PLATE 6. Higher magnification of isolated Plate 4. Myofibrils (mf) cut in cross-section mitochondria (M). Myofibrils are composed cross-section. E, lipid droplet; T, longitudinal
myocyte sectioned in a plane similar to that shown in are surrounded by sarcoplasmic reticulum (SR) and of hexagonally arranged myofilaments when cut in element of T-tubule system. x 38 000.
PLATE 7. Transmission electron micrograph showing detail of longitudinal section through single myofibril. Z-lines (Z) are associated with transverse tubules (T) of the T-tubule system. Individual sarcomeres exhibit thick (large arrows) and thin (small arrows) myofilaments throughout their length (A). A central dark band (m) runs transversely through the sarcomere and is flanked on either side by an electron-lucent area. M, mitochondrion. x 42 500. PLATE 8. Longitudinal section through (SR) to glycogen granules (G) between x 34500.
sarcomere myofibrils.
showing relationship M, mitochondrion;
of sarcoplasmic T, T-tubule;
PLATE 9. High resolution transmission electron micrograph showing cross-section subsarcolemmal myofibril. Both thick and thin filaments are present. Thick filaments (large are hexagonally arranged around an arbitrary central thick filament. Thin filaments (small are similarly hexagonally arranged around a central thick filament. M, mitochondrion; colemma; SSC, subsarcolemmal cisterna. X 71 000.
[facing
reticulum Z, Z-line. through arrows) arrows) S, sar-
page 456 ]
PLATES
1 and
2
PLATES
3 and 4
_- .--
-..---.-
PLATES
5 and 6
PLATES
7,8
and
9
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
457
TEM tissue preparations. Alternatively, they may not appear as conspicuous in TEM preparations because smaller cellular areas are observed in a single field. No continuity could be found between sarcoplasmic reticulum and the T-tubule system but occasional sarcolemmal invaginations suggested that T-tubules may be confluent with the extracelluiar space. Nuclei were centrally placed and were unremarkable. Cells were generally binucleate and cytoplasmic caps containing primarily mitochondria were located at each nuclear pole. Most mitochondria were arranged in rows between myofibrils and although some “exploded” mitochondria were observed, most were filled with crisp, angular cristae similar to those described by Berry et al. [3]. Comparisons of mitochondria cut normal and parallel to the long axis of the cell strongly suggested that most mitochondrial cristal plates were oriented normal to adjacent myofibrils. The significance of this finding is unknown but it is possible that such an arrangement provides more efficient diffusion of ATP and other metabolites to and from the adjacent myofibrils. Glycogen granules were frequently seen closely associated with intermyofibrillar mitochondria as well as within the myofilament lattice. Similar glycogen particles are present in myocardial cells in vivo [19] and are not easily depleted suggesting that the cells in our preparation do not suffer from glycogen starvation. Our TEM studies provide unequivocal data for excellent structural integrity of the sarcolemma. Only a few minor blebs (seen most easily in cross-sections) are seen and the membrane appears normal and competent. Its external surface is not lined by basal lamina or other ultrastructurally identifiable surface-associated substances. This probably accounts for the presence of microridges and blebs. Such unevenness is not uncommon in cells from which the basal lamina has been removed [9]. The sarcolemma is frequently invaginated (as described above), presumably to form confluences with the T-tubule system. It also shows numerous microinvaginations (micropinocytotic vesicles) which suggest active metabolic activity. These vesicles are often closely associated with subsarcolemmal cisternae of the sarcoplasmic reticulum and could represent a mechanism by which metabolites are passed to and from the protein synthesizing machinery of the cell. The integrity of the myocytic sarcolemma was further substantiated by our cyclic AMP assays of isolated cells. The cyclic AMP content of the isolated myocytes was found to be within the range reported for the perfused rat heart [12, 24 and cultured neonatal rat heart cells [S], 2 to 5 pmol/mg protein. Epinephrine, glucagon and the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine stimulated increases in the cyclic AMP content of the cells. The relative potencies of epinephrine and glucagon were similar to those previously reported for the rat heart, i.e. glucagon was effective at lower concentrations but the maximal response to both hormones was approximately the same [ 12, 211. Catecholamines and glucagon are believed to induce increases in cyclic AMP in the heart by interacting at separate receptor sites on the cell membrane [12, 24.
458
E. C. CARLSON
ETAL.
Since both were effective in elevating cyclic AMP in the isolated myocytes this is evidence that functional membrane-bound receptors are present for both types of hormones. The ability of epinephrine to raise cyclic AMP levels suggeststhat betareceptors are present on the myocytes as has been reported in other isolated heart cell preparations [5, 201. The present study showsthat adult rat myocytes isolated by our method [7] are morphologically intact and exhibit sarcolemmae, the structural and functional integrity of which is maintained. At present, the ability of these cells to: (a) grow and differentiate, (b) reaggregate, (c) perform synchronous metabolic and physical activities and (d) produce extracellular materials e.g. basal lamina, cell coat, etc, in vitro is unknown. These parameters of isolated myocytes are being further investigated. Acknowledgements
We wish to express our sincere thanks to MS Jerrolynn Campbell and Thomas Demlow for expert electron microscopic assistanceand to Connie Sontag for aid in the isolation of the cells. This work was funded in part by USPHS NIH Grant HL-17421-03S1 (E.C.), American Heart Association Grant 76-944 (E.C.) and USPHS NIH Grants HL-13636 (R.B.) and F32-HL-5361 (D.G.). REFERENCES 1. lhDERsoN, W. A. & ELLIS, R. A. Ultrastructure of TrrypanOsoma lewisi: flagellum, microtubules and kinetoplast. Journal of Protozoology 12, 483-499 (1965). 2. A~HRAF, M. & SYBERS, H. D. Scanning electron microscopy of ischemic heart. In Scanning Electron Microscopyll974, pp. 72 l-738. 0. Johari and I. Corvin, Eds. Chicago: 1.1-T. Research Institute (1974). 3. BERRY, M. N., FRIEND, D. S. & SCHEUER, J. Morphology and metabolism of intact muscle cells isolated from adult rat heart. Circulation &search 26, 679-687 (1970). 4. BLOOM, S. Spontaneous rhythmic contraction of separated heart muscle cells. Science 167, 1727-1729 (1970). 5. ERTEL, R. J., CLARKE, D. E., CHAO, J. C. & FRANKE, F. R. Autonomic receptor mechanisms in embryonic chick myocardial cell cultures. Journal of PharmacoLogy and Ex@Gnental nierapeutics 178, 73-80 (1971). GLICK, M. R., BURNS, A. H. & REDDY, W. J. Dispersion and isolation of beating cells from adult rat heart. Analytical Biochemistv 61,32-42 (1974). GROSSO, D. S., FRANGAKIS, C. J., CARLSON, E. C. & BRESSLER, R. Isolation and characterization of myocytes from the adult rat heart. (Submitted for publication.) HAMRY, I., RENAUD, J. F., SATO, E. & WALLACE, G. A. Calcium ions regulate cyclic AMP and beating in cultured heart cells. Jvature, 261,60-&l (1976). HAY, E. D. & DODSON, J. W. Secretion of collagen by cornea1 epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozenkilled lens. Journal of Cell Biology 57, 190-213 (1973). 10. HUXLEY, A. F. & HUXLEY, H. E. A discussion on the physical and chemical basis of muscular contraction. Proceedings of the Royal Socie@ (London), Series B 160,433 (1964).
ULTRASTRUCTURE 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
OF ISOLATED MYOCYTES
459
~OVSKY, M. J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. 3oumulof Cell Biology 27, 137a-138a ( 1965). KEELY, S. L., CORBIN, J. D. & PARK. C. R. Regulation of adenosine 3’,5’-monophosphate-dependent kinase. 3ouwzulof Biological Chemistry 250, 4832-4840 (1975). Kuo, J. & GREENCARD, P. An assay method for cyclic AMP and cyclic GMP based upon their abilities to activate cyclic AMP-dependent and cyclic GMP-dependent protein kinases. In Advances in Cyclic JVucieotide Research, Vol. 2, pp. 41-50. P. Greengard and G. A. Robison, Eds. New York: Raven Press. (1972). LEE, T. P., Kuo, J. F. & GRBENGARD, P. Regulation of myocardial cyclic AMP by isoproterenol, glucagon and acetylcholine. Biochemical and Biophysical Research Communications 45, 991-997 (1971). Loway, 0. H., ROSEBROUGH, N. J,, FARR, A. L. & IIANDALL, R. J. Protein measurement with the folin phenol reagent. 3ou~nul of Biological Chemistry 193,265-275 (1951). Lao, C. C. & GUIODTTI, A. Simultaneous isolation of adenosine 3’,5’-cyclic monophosphate and guanosine 3’,5’-cyclic monophosphate in small tissue samples. Analytical Biochemistry 59,63-68 (1974). MASSON-PEVET, M., JONCKWA, H. J. & DEBRUIJNE, J. Collagenase- and trypsindissociated heart cells: a comparative ultrastructural study. 3ournalof Molecular and Cellular Cardiology 8, 747-757 ( 1976). MCCWISTER, L. P., MUMAW, V. R. & MUNGER, B. L. Stereo ultrastructure of cardiac membrane systems in the rat heart. In Scanning Electron Microscofiyjl974, pp. 713-720. 0. Johari and I. Corvin, Eds. Chicago: I.I.T. Research Institute (1974). McNwrr, N. S. & FAWCET-I-, D. W. Myocardial ultrastructure. In The Mammalian Myocardium, pp. l-49. G. A. Langer and J. Brady, Eds. New York: J. Wiley and Sons (1974). MOUSTAFA, E., SKOMEDAL, T., OSNES, J.-B. & 0ye, I. Cyclic AMP formation and morphology of myocardial cells isolated from adult heart: effect of Gas+ and Mgs+. Biochimica et biojhysica acta 421,41 l-415 (1976). MURAD, F. & VAUGHAN, M. Effect of glucagon on rat heart adenyl cyclase. Biochemical Pharmacolog-y 18, 1053-1059 (1969). PEASE, D. C. Histological Techniques for Electron MicroscOpy, 2nd edt. New York and London : Academic Press ( 1964). PRETLOW, T. G., GLICK, M. R. & REDDY, W. J. Separation of beating cardiac myocytes from suspensions of heart cells. American 3ouml of Pathology 67, 2 15-224 (1972). ROBISON, G. A., BUTCHER, R. W., DYE, I., MORGAN, H. E. & SUTHERLAND, E. W. The effect of epinephrine on adenosine 3’,5’-monophosphate levels in the isolated perfused rat heart. Molecular Pharmacology 1, 168-177 (1965). SHELDON, C. A., FRIEDMAN, W. F. & SYBERS, H. D. Scanning electron microscopy of fetal and neonatal lamb cardiac cells. Journal of Molecular and Cellular Cardiology 8, 853-862 (1976). SYBERS, H. D. & ASHRAF, M. Preparation of cardiac muscle for SEM. In: Scanning Electron Microscopy/l973, pp. 341-348. 0. Johari and I. Corvin, Eds. Chicago: I.I.T. Research Institute (1973). SYBERS, H. D. & As-, M. Scanning electron microscopy of cardiac muscle. Laboratory Investigation 30,441-450 (1974). VAHOIJNY, G. V., WEI, R., STARK~EATHER, R. & DAVID, C. Preparation ofbeating heart cells from adult rats. Science 167, 1616-1618 (1970). VENABLE, J. H. & COGGESHALL, R. A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25,407-408 (1965).
of Molecular and Cellular Cardiology (1978)
Ultrastructural
10,449-459
Studies of Metabolically Adult Rat Heart Myocytes
Active
Isolated
E. C. CARLSON*, D. S. GROSSOt, S. A. ROMERO*, C. J. FRANGAKISt, C. V. BYUSf AND R. BRESSLEQ Defiartments of Anatomy*, Pharmmology~ and Internal Medic&f, College of Medicine, University of Arizona, Tucson, Arizona 85724, U.S.A. (Received 21 June 1977, accepted in revisedform 29 Ju& 1977) E. C. CARLWN, D. S. GROSSO,S. A. ROMERO, C. J. FRANGAKIS,C. V. Bws AND R. BRESSLER. Ultrastructural Studies of Metabolically Active Adult Rat Heart Myocytcs. Jouwzal of Molecular and Cellular Cardiology (1978) 10, 449-459. Myocytes from adult rat heart were isolated by a method recently developed by us in which hearts were pre-perfused with calcium-free phosphate-buffered saline medium containing collagenase and hyaluronidase. This was followed by incubation in the enzyme medium and cellular sedimentation through 3% Ficoll. Myocytes isolated in this manner were metabolically active and morphologically intact. In the present study, scanning and transmission electron microscopy of isolated myocytes showed long cylindrical cells with transverse microridges which corresponded directly with sarcomere lengths. Most cells appeared to be in a fully contracted state. Contractile elements and associated membranes, other intracellular compartments and sarcolemmae were indistinguishable from their in vim counterparts. Although myocytic basal laminae and other structurally identifiable surface coats were removed by our isolation procedure, sarcolemma e remained remarkably unaffected. Cyclic AMP assays in control and epinephrineor glucagon-stimulated cells strongly suggested that membranebound receptors were present and the functional integrity of the sarcolemmae was maintained in our preparations. KEY WORDS: Cyclic AMP.
Rat
heart;
Isolated
myocytes;
Ultrastructure;
Epinephrine;
Glucagon;
1. Introduction The development of a technique for the production of isolated adult heart myocytes which are morphologically and metabolically intact has been attempted by various investigators [3, 4, 6, 20, 23, 281. Such a method is valuable because cultures of isolated myocytes provide a potentially useful model for studies of this cell type in an environment which is free of neural and humoral influences. Moreover, this model provides an experimental tool for investigations of myocytic growth, differentiation, contraction, biochemical control mechanisms and pharmacological or electrical responses without regard for the modifying effects of extracellular matrix and non-muscular tissues present in vivo. We have recently developed a method [7] for the isolation of a uniform population of metabolically active myocytes from adult rat hearts which provides a yield 0022-2828/78/0501-0449
$02.00/O
0
1978 Academic
Press Inc.
(London)
Limited
450
E. C. CARLSON
ETAL.
(50 mg protein/g heart) and index of viability (90 to 95%) which is greater than those previously reported. Our preparations were virtually devoid of non-muscle cells and up to 80% of the myocytes spontaneously contracted. Furthermore, contraction could be halted (by reducing the temperature to 4°C) and resumed 24 to 48 h later by returning the cells to room temperature. In addition, we suggested that by scanning electron microscopy (SEM) , cu. 80% of all isolated myocytes were intact (based on topographical features). Sarcolemmae appeared undisturbed except for occasional blebs and microridges. In the present investigation, we provide a detailed ultrastructural analysis of myocytes isolated by our method. We demonstrate by both SEM and transmission electron microscopic (TEM) techniques that contractile elements, other intracellular compartments, and sarcolemmae of isolated myocytes maintain their morphological integrity. Moreover, our cyclic AMP assaysof control and hormone-stimulated myocytes demonstrate biochemically the functional integrity of myocytic cell membranes and associatedhormone receptors.
2. Materials
and Methods
Cell isolation
The isolation of myocytes from ventricular tissue of adult rats was performed as described previously [7]. Hearts from 150 to 200 g rats were perfused on a “Langendorlf” perfusion apparatus at 37°C in a retrograde manner through the aorta for 5 min to remove blood. The perfusion conditions were: rate, 30 ml/min; pressure, 60 mmHg; buffer, pH 7.4, containing 128 mu NaCl, 5.4 mM KCI, 2 mM NaHaPOd. HaO, 8 mM NasHPOd, 15 mM glucose, bubbled through with 95% OS/~% COa. The perfusate was changed and replaced with buffer containing 1 mg/ml collagenase (Type II, Worthington), 1 mg/ml hyaluronidase (Sigma), and 0.1 mg/ml BSA and perfused for 30 min. The ventricles were divided into pieces and placed in a clean siliconized 25 ml Erlenmeyer flask with 3 ml of enzyme-buffer solution and incubated at 37°C. After 15 min, 10 ml of cold buffer was added. The supernatant containing detached cells was decanted into a polyethylene centrifuge tube. Enzyme-buffer solution was added to the tissue fragments and the incubation procedure repeated. The cells were centrifuged immediately after decantation at 60 g for 3 min in a tabletop centrifuge. They were resuspendedand allowed to settle in 10 ml of buffer twice. The washed cells were layered atop 10 ml of 3% Ficoll in buffer (w/v) and centrifuged as above. Approximately 50 mg of cellular protein can be obtained from 1 g of heart tissue.The cells are free of contamination by blood cells, fibrous material and severely damaged heart cells. Viability as measured by trypan-blue exclusion is 90 to 95% and approximately 80% of the cells are contracting spontaneously.
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
451
Scanning electron microscopy (SEM)
Following isolation, isolated myocytes were fixed at room temperature in 30 to 50 ml of Karnovsky’s [II] paraformaldehyde-glutaraldehyde fixative buffered at pH 7.3 with 0.1 M sodium cacodylate. Double-sided cellophane tape was placed in the bottom of a small plastic beaker and the cells (still in fixative) were decanted into the beaker and allowed to adhere to the tape (usually overnight). Cells were carefully rinsed in 0.2 M cacodylate buffer before osmication (1 h in 2% cacodylate buffered 0~04). The cells were rinsed briefly in distilled water prior to dehydration in a graded seriesof acetones. This was followed by critical point drying in CO2 (DuPont-Sorvall critical point drying apparatus). The double-sided tape with adhering cells was mounted on aluminium specimen stubs, coated with evaporated carbon and gold (Denton DV-502 vacuum evaporator) and observed in an ETEC Autoscan scanning electron microscope at original magnifications of 175 to 1000 diameters. Some of the dried specimenswere disrupted with cellophane tape in an effort to remove the sarcolemma and render internal structures visible. These were recoated with carbon and gold prior to observation. Transmission
electron microscopy (TEM)
Pellets of cardiac cells were washed several times with phosphate-buffered saline and then immersed in cold (4°C) paraformaldehyde-glutaraldehyde fixative buffered at pH 7.3 with 0.2 M sodium cacodylate [IA. Fixation lasted 1 h after which the cells were rinsed with buffer and post-fixed 1 h at room temperature in cacodylate-buffered 2% osmium. The cellswere rinsed again in buffer, dehydrated in an ascending seriesof graded ethanols and propylene oxide prior to embedding in Epon-Araldite [l]. Epoxy blocks were cured overnight at 37°C and an additional 48 h at 60°C. One-micron-thick sections were cut on a DuPont-Sorvall MT-2 Ultramicrotome. These were stained with toluidine blue (1% in 1y. sodium borate) and observed by light microscopy for initial block orientation. Thin sections were cut using the same ultramicrotome equipped with a diamond knife. These sectionswere picked up on naked 300 mesh copper grids and stained with uranyl acetate (5% in absolute ethanol) and lead citrate [29]. Thin sections were observed in a Philips 300 transmission electron microscope at original magnifications of 3300 to 27 000 diameters. Cyclic AMP
Cyclic AMP was determined by measuring the activation of purified beefheart protein kinase [13]. Cells, 4.3 mg protein/ml, were incubated with or without hormones (or drug) for 2 min at 37°C in isolation medium. The reaction was stopped by addition of 0.4 N perchloric acid. Cyclic AMP was extracted into the
452
E. C. CARLSON
ETAL.
acid and purified by ion-exchange chromatography [16]. The purified cyclic AMP was quantitated by its ability to activate cyclic AMP-dependent protein kinase. Results were compared to a standard curve for cyclic AMP activation of protein kinase. Protein assay
Protein was measured by the method of Lowry et al. [15]. Bovine serum albumin was used asa standard. 3. Results Scanning electron microscopy (SEM)
When isolated adult rat myocytes are observed by SEM, they appear as long slender cells which average 20 to 25 pm in width and 100 to 125 l.mi in length (Plate 1). They exhibit numerous angular processeswhich could represent points of branching with adjacent myocytes in vivo. The ends of the cells are frequently rough and irregular suggestingpossibleinterdigitation with other cells in regions similar to intercalated discsdescribed by transmissionelectron microscopic (TEM) studies. The cells frequently display longitudinal ridges which extend more than half the length of the cell. A conspicuous and nearly constant feature of isolated myocytes is a seriesof transverse microridges ca. 1.5 to 2.0 pm in width. These run perpendicular to the longitudinal ridges and therefore to the long axis of the cell. They extend over the entire cellular surface imparting a courderoy appearance to the sarcolemma, the integrity of which is not disturbed. It appears as a clean, continuous, and competent encasementfor the cellular contents. When individual heart cells are teased apart with cellophane tape following critical point drying (Plate 2) numerous cytoplasmic structures are visualized. Individual myofibrils oriented parallel to the long axis of the cell are separated by this technique. Aggregates and long rows of mitochondria normally located between myofibrils are also recognizable. Undisturbed myofibrils are crossed at regular intervals (1.5 to 2.0 pm) by cylindrical structures which extend in register acrossthe cell. Separated myofibrils exhibit a similar periodicity suggesting that the cylindrical structures surround each individual myofibril at regular intervals. It is possible that these cross-striations represent transverse tubules of the myocardial T-tubule system. Transmission
electron microscopy ( TEM)
In an effort to derive the most morphological data from our TEM specimens, we endeavored to record photographically primarily those cells cut either parallel or normal to the long axis of the cell. At low magnifications, longitudinal sections through adult rat myocytes show numerous parallel (occasionally branched)
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
453
myofibrils. These are separated by rows of contiguous mitochondria which exhibit numerous angular cristae. Nuclei are centrally located and aggregates of mitochondria are present in cytoplasmic caps at nuclear poles. These areas are free of myofibriis and associated membranes. Each myofibril exhibits prominent sarcomeres delimited by Z-lines. Z-lines appear to be in register throughout the cell and are approximately 1.5 pm apart. Sarcolemmae appear as continuous membranes and are thrown into transverse microridges which run normal to the long axis of the cell. The ridges are separated by shallow but distinct troughs (1.5 pm apart) and these can be directly correlated with ridges and troughs identified by SEM (see Plate 1). The location of each trough corresponds precisely with the Zline of the subjacent myofibril. When myocytes cut in cross-section are observed at low magnification, the cylindrical shape of the cell is apparent (Plate 4). The centrally placed nucleus is likewise cylindrical and is surrounded by numerous mitochondria, myofibrils and their associated membrane systems. The T-tubule system of the myocardium has longitudinal elements (adjacent and parallel to myofibrils) which are particularly evident in cross-sections. Also, this latter orientation clearly demonstrates the excellent continuity of the sarcolemma. Only occasional blebs (which are not unexpected in tissues from which the basal lamina has been removed) mar an otherwise undisturbed cell membrane. Sarcolemmal invaginations are frequently seen where T-tubules are continuous with the extracellular space but most frequently myocardial cell membranes are subtended directly by myofibrils, mitochondria or subsarcolemmal cisternae of the sarcoplasmic reticulum. No extracellular materials are recognizable on the external surface of the sarcolemma. At higher magnifications, longitudinal sections through myocytes show that myofibrils vary in thickness along their length and are occasionally branched (Plate 5). They are surrounded along the length of each sarcomere by sarcoplasmic reticulum and at prominent Z-lines exhibit a single T-tubule. These latter structures are not as constant or pronounced as one would predict based on SEM observations (see Plate 2). Nevertheless, they are present in most preparations. The morphological integrity of the sarcolemma is clearly maintained and is invaginated occasionally by small micropinocytotic vesicles. Cross-sections through cytoplasmic areas similar to those seen in Plate 5 show numerous myofibrils surrounded by sarcoplasmic reticulum, longitudinal elements of the T-tubule system and mitochondria (Plate 6). A few lipid droplets are also observed. Mitochondria appear to occupy almost all of the cytoplasm not filled by contractile elements. Their cristae are highly indistinct when seen in cross-section (Plate 6) in contradistinction to the crisp, angular cristal plates observed when mitochondria are sectioned parallel to the long axis of the cell (Plate 5). This suggests that cross-sections yield enface views of the cristae and therefore most of them are oriented perpendicular to their adjacent myofibrils.
454
E. C. CARLSON
ETAL.
Individual sarcomeresof myocytes prepared by our method average about 1.5 pm between Z-lines. They consistently exhibit both thick and thin filaments throughout their length (Plate 7). Therefore, they do not demonstrate H-bands or I-bands which consist of only thick or only thin filaments respectively. An Mband composed of multiple transverse electron-dense lines is present in our specimens and is flanked on either side by an electron-lucent area. Both thick and thin filaments appear to extend laterally from M-bands to the substanceof the Z-lines. Glycogen granules are frequently associatedwith the myofilament lattice and/or the sarcoplasmic reticulum (Plate 8). High resolution TEM observations of cross-sectionsthrough myofibrils demonstrate both thick and thin filaments irrespective of the position along the sarcomere (Plate 9). The thick (150 to 160 A) filaments are hexagonally arranged around a seventh “central” thick filament. Thin (80 A) filaments are likewise hexagonally arranged around “central” thick filaments and appear to be equidistant from each other and from adjacent thick filaments. Occasionally, very fine projections from thick filaments are observed in close contact with the nearest thin filaments. TABLE
1. Effects of epinepbrine, glucagon and I-methyl-3-isobutylxantbine AMP levels in isolated cardiac myocytes*
Concentration (PM) 0 0.01 0.10 1.0 10.0
on cyclic
I-methyl-3isobutylxantbine Epinepbrine
Glucagon (pmol/cyclic AMP/mg
2.1 1.7 2.1
protein)
2.1 2.1 6.3 15.8 29.5
22.7
* Each value represents the mean of two determinations.
Hormonalstimulationof cyclic AMP levels In an effort to determine the integrity of the sarcolemma by biochemical means, cyclic AMP levels were determined in freshly prepared heart cells. Similar determinations
were carried
out in cells incubated
with
the hormones
epinephrine
and
glucagon and the phosphodiesteraseinhibitor 1-methyl-3-isobutylxanthine (MIX). The concentration of cyclic AMP, 2.1 pmol/mg protein (Table I), was found to be within the range previously reported for the perfused heart, 2 to 4 pmol/mg protein [12, 241, and ventricular tissue slices, 2.6 pmol/mg protein [Za. Epinephrine, glucagon and MIX all increased the levels of cyclic AMP in the cells by
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
455
approximately 14 times the amounts measured in controls at drug levels of 10 PM. Although glucagon and epinephrine induced the same stimulation of cyclic AMP production at a concentration of 10 PM, the threshold for glucagon appeared to be lower, less than 0.1 PM, as compared to 0.1 to 1.O PM for epinephrine. Glucagon has been reported to have a slightly greater effect on adenyl cyclase than epinephrine in the rat heart at concentrations between 0.01 and 1.0 PM [21]. 4. Discussion
Various procedures have been reported [3, 4, 6, 20, 23, 281 for the preparation of adult rat heart myocytes for in vitro studies. Most of these methods have been plagued with low yields and/or poor viability. Several investigators have illustrated their preparations with photomicrographs [4, 20, 281 or electron micrographs [3, 17] but, in general, careful morphological studies on isolated adult myocytes have not been carried out. In the present study, an ultrastructural analysis of metabolically active isolated adult rat myocytes was carried out. These cells were isolated by a technique [7] recently developed by us. Our method is superior to most others in that it provides : (a) a uniform population of myocytes with non-muscular cells virtually excluded, (b) a high yield of cells (50 mg protein/g heart), (c) an excellent index of viability (90 to 95% by trypan blue-exclusion), and (d) good longevity for biochemical experiments (75% or original lactate dehydrogenase activity is retained after 24 h) . Our previous report [7], showed that isolated myocytes were morphologically intact as illustrated by scanning electron microscopy (SEM). Sarcolemmae appeared undisturbed except for small microridges. The functional integrity of thesemembraneswas substantiated by biochemical data which showed that glucose uptake was stimulated by insulin in isolated cells. Therefore, preservation of insulin receptors and the integrity of myocytic sarcolemmaewas virtually assured. Other investigators [I, 20, 281 have shown by bright field or phase contrast photomicroscopy that isolated heart cells are cylindrical with abruptly angular ends. Most studies demonstrate an average length of 100 to 150 pm and widths of approximately 10 to 30 pm. The resolution of photomicrographs doesnot allow observation of good surface detail but most morphological studies of isolated cells show at least a hint of cross-striations. This finding is consistent with our SEM observations which show that most (if not all) isolated myocytes exhibit transverse microridges which extend over their entire length. The ridges are approximately 1.5 pm apart and are separated by shallow troughs. When the sarcolemma was mechanically removed and subjacent intracellular contents exposed, small cylindrical structures, which probably represent elements of the T-tubule system, extended in register (ca. 1.5 pm apart) acrossthe cell. These structures appeared to surround each individual myofibril since 1.5 pm periodicity could be demonstrated on disrupted myofibrils as well as those which remained in
456
E. C. CARLSON
ETAL.
situ. These results are consistent with a number of studies [Z, 28, 26, 271 which have demonstrated transverse and longitudinal elements of the T-tubule system in cryofractured myocardium in vivo. Most in vivo studies [Z, 18, 271 indicate that Ttubules are much wider spaced (up to 2.5 pm) than those seen in our investigation. This could be due to the physiological state of the muscle at the time of preparation. Our transmission electron microscopic (TEM) studies show conclusively that transverse sarcolemmal microridges seen in SEM correspond directly with the lengths of sarcomeres. Furthermore, the troughs between ridges are located precisely at the point of Z-lines. Since it is believed [19] that ca. 1.5 pm is the minimum length for sarcomeres, it is possible that most myocytes isolated by our procedure may be fully contracted at the time of fixation. This is not unreasonable since almost all isolated cells were contractile and the addition of glutaraldehyde, a know stimulator of protein cross-linking [22], to the incubation medium could result in rapid formation of actinomyosin cross-links. This is particularly true in light of the unrestrained nature of isolated cells. They are unbound by other cells, basal lamina or other components of the extracellular matrix and therefore are free to react as individual contractile units rather than in concert as in the whole organ in vivo. Intracellular contractile elements (myofibrils and associated membranes) were intact in nearly all of our preparations. Myofibrils were frequently branched, varied in width and were separated by rows of mitochondria. They showed sarcomerit segmentation which frequently crossed the entire cell in register. This is consistent with the normal morphology of myocardial cells in vivo [19]. The idea that most myocytes in our preparation were in a state of full contraction was corroborated by TEM observations of individual sarcomeres. Z-lines were approximately 1.5 pm apart and both thick and thin filaments appeared to extend this entire distance. H-zones and I-bands were not distinguishable and the entire sarcomere appeared to consist of a large A-band. This data was consistent with the morphology of classical mechanisms suggested for striated muscle contraction [ 101 and, since it is believed [19], that thick filaments are 1.5 pm in length, implied that the myofibrils were fully contracted. The appearance of M-bands and Llines (electron¢ areas on either side of the M-band) in our preparations was not unexpected because the dimension of this structural complex is not altered in different sarcomere lengths [ 191. Cross-sectional high-resolution microscopy further substantiated our idea of full contraction of myocytes because in each section cut normal to the direction of myofilaments, both thick (150 to 160 A) and thin (80 A) filaments were observed. These were hexagonally arranged around an arbitrary seventh “central” thick filament and were morphologically similar to those seen by other investigators in the non-H-zone portion of A-bands’. T-tubules surrounded myofibrils at points of Z-lines but were not as prominent as might have been predicted based on SEM of disrupted cells. The reasons for this inconsistency are not known but it is possible that tubules may collapse during
PLATE 1. Scanning electron micrograph of isolated adult rat myocyte. Cells typically appear as long slender cylinders and exhibit branching processes (*) which are parallel to the long axis of the cell. Irregular ends (ID) probably represent areas of intercalated discs. Longitudinal ridges (long arrows) and transverse microridges which average 1.5 [LM across (opposing arrows) are prominent features of the cell surface. x 1500. PLATE 2. Scanning electron micrograph of adult myocyte from which the sarcolemma has been removed. Underlying myofibrils (mf) are longitudinally arranged with intervening mitondondria (M). Elements of the T-tubule system are evident both in sihr (triple arrows) and associated with disrupted myofibrils (double arrows). Areas of branching (*) and intercalated discs (ID) are recognizable. x 1500. PLATE 3. Low power transmission electron micrograph of longitudinal section through isolated myocyte similar to that shown in Plate 1. The sarcolemma is thrown into transverse ridges (arrows) which measure IX. 1.5 pm (trough to trough). Individual myofibrils exhibit sarcomeres (S) whose length corresponds directly to the sarcolemmal microridges. M, mitochondrion; NU, nucleus, x 11300. PLATE 4. Low power transmission electron micrograph of cross-section through entire isolated myocyte. The central nucleus (NU) is surrounded by myofibrils (mf) cut in cross-section and numerous mitochondria (M). A few longitudinal components of the T-tubule system are recognizable. The sarcolemma is remarkably well-preserved and shows few blebs (b). Several sarcolemmal invaginations (arrows) could represent areas at which T-tubules may be continuous with the extracellular space. X 8300. PLATE 5. Higher magnification of isolated myocyte sectioned in a plane similar to that shown in Plate 3. Z-lines ofsarcomeres are cc. 1.5 ym apart and correspond directly with troughs of sarcolemmal transverse microridges (large arrows). The sarcolemma (S) is continuous and shows invaginations associated with micropinocytotic vesicles (small arrows). No basal lamina material or other surfaceassociated structures are present. Myofibrils (mf) vary in thickness and are occasionally branched. They are surrounded by sarcoplasmic reticulum (SR) along the length of each sarcomere and by Ttubules (T) at each Z-line (Z). Most mitochondria (M) are interspersed between myofibrils but some are located immediately beneath the sarcolemma (see Plate 4). x 23 500. PLATE 6. Higher magnification of isolated Plate 4. Myofibrils (mf) cut in cross-section mitochondria (M). Myofibrils are composed cross-section. E, lipid droplet; T, longitudinal
myocyte sectioned in a plane similar to that shown in are surrounded by sarcoplasmic reticulum (SR) and of hexagonally arranged myofilaments when cut in element of T-tubule system. x 38 000.
PLATE 7. Transmission electron micrograph showing detail of longitudinal section through single myofibril. Z-lines (Z) are associated with transverse tubules (T) of the T-tubule system. Individual sarcomeres exhibit thick (large arrows) and thin (small arrows) myofilaments throughout their length (A). A central dark band (m) runs transversely through the sarcomere and is flanked on either side by an electron-lucent area. M, mitochondrion. x 42 500. PLATE 8. Longitudinal section through (SR) to glycogen granules (G) between x 34500.
sarcomere myofibrils.
showing relationship M, mitochondrion;
of sarcoplasmic T, T-tubule;
PLATE 9. High resolution transmission electron micrograph showing cross-section subsarcolemmal myofibril. Both thick and thin filaments are present. Thick filaments (large are hexagonally arranged around an arbitrary central thick filament. Thin filaments (small are similarly hexagonally arranged around a central thick filament. M, mitochondrion; colemma; SSC, subsarcolemmal cisterna. X 71 000.
[facing
reticulum Z, Z-line. through arrows) arrows) S, sar-
page 456 ]
PLATES
1 and
2
PLATES
3 and 4
_- .--
-..---.-
PLATES
5 and 6
PLATES
7,8
and
9
ULTRASTRUCTURE
OF ISOLATED
MYOCYTES
457
TEM tissue preparations. Alternatively, they may not appear as conspicuous in TEM preparations because smaller cellular areas are observed in a single field. No continuity could be found between sarcoplasmic reticulum and the T-tubule system but occasional sarcolemmal invaginations suggested that T-tubules may be confluent with the extracelluiar space. Nuclei were centrally placed and were unremarkable. Cells were generally binucleate and cytoplasmic caps containing primarily mitochondria were located at each nuclear pole. Most mitochondria were arranged in rows between myofibrils and although some “exploded” mitochondria were observed, most were filled with crisp, angular cristae similar to those described by Berry et al. [3]. Comparisons of mitochondria cut normal and parallel to the long axis of the cell strongly suggested that most mitochondrial cristal plates were oriented normal to adjacent myofibrils. The significance of this finding is unknown but it is possible that such an arrangement provides more efficient diffusion of ATP and other metabolites to and from the adjacent myofibrils. Glycogen granules were frequently seen closely associated with intermyofibrillar mitochondria as well as within the myofilament lattice. Similar glycogen particles are present in myocardial cells in vivo [19] and are not easily depleted suggesting that the cells in our preparation do not suffer from glycogen starvation. Our TEM studies provide unequivocal data for excellent structural integrity of the sarcolemma. Only a few minor blebs (seen most easily in cross-sections) are seen and the membrane appears normal and competent. Its external surface is not lined by basal lamina or other ultrastructurally identifiable surface-associated substances. This probably accounts for the presence of microridges and blebs. Such unevenness is not uncommon in cells from which the basal lamina has been removed [9]. The sarcolemma is frequently invaginated (as described above), presumably to form confluences with the T-tubule system. It also shows numerous microinvaginations (micropinocytotic vesicles) which suggest active metabolic activity. These vesicles are often closely associated with subsarcolemmal cisternae of the sarcoplasmic reticulum and could represent a mechanism by which metabolites are passed to and from the protein synthesizing machinery of the cell. The integrity of the myocytic sarcolemma was further substantiated by our cyclic AMP assays of isolated cells. The cyclic AMP content of the isolated myocytes was found to be within the range reported for the perfused rat heart [12, 24 and cultured neonatal rat heart cells [S], 2 to 5 pmol/mg protein. Epinephrine, glucagon and the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine stimulated increases in the cyclic AMP content of the cells. The relative potencies of epinephrine and glucagon were similar to those previously reported for the rat heart, i.e. glucagon was effective at lower concentrations but the maximal response to both hormones was approximately the same [ 12, 211. Catecholamines and glucagon are believed to induce increases in cyclic AMP in the heart by interacting at separate receptor sites on the cell membrane [12, 24.
458
E. C. CARLSON
ETAL.
Since both were effective in elevating cyclic AMP in the isolated myocytes this is evidence that functional membrane-bound receptors are present for both types of hormones. The ability of epinephrine to raise cyclic AMP levels suggeststhat betareceptors are present on the myocytes as has been reported in other isolated heart cell preparations [5, 201. The present study showsthat adult rat myocytes isolated by our method [7] are morphologically intact and exhibit sarcolemmae, the structural and functional integrity of which is maintained. At present, the ability of these cells to: (a) grow and differentiate, (b) reaggregate, (c) perform synchronous metabolic and physical activities and (d) produce extracellular materials e.g. basal lamina, cell coat, etc, in vitro is unknown. These parameters of isolated myocytes are being further investigated. Acknowledgements
We wish to express our sincere thanks to MS Jerrolynn Campbell and Thomas Demlow for expert electron microscopic assistanceand to Connie Sontag for aid in the isolation of the cells. This work was funded in part by USPHS NIH Grant HL-17421-03S1 (E.C.), American Heart Association Grant 76-944 (E.C.) and USPHS NIH Grants HL-13636 (R.B.) and F32-HL-5361 (D.G.). REFERENCES 1. lhDERsoN, W. A. & ELLIS, R. A. Ultrastructure of TrrypanOsoma lewisi: flagellum, microtubules and kinetoplast. Journal of Protozoology 12, 483-499 (1965). 2. A~HRAF, M. & SYBERS, H. D. Scanning electron microscopy of ischemic heart. In Scanning Electron Microscopyll974, pp. 72 l-738. 0. Johari and I. Corvin, Eds. Chicago: 1.1-T. Research Institute (1974). 3. BERRY, M. N., FRIEND, D. S. & SCHEUER, J. Morphology and metabolism of intact muscle cells isolated from adult rat heart. Circulation &search 26, 679-687 (1970). 4. BLOOM, S. Spontaneous rhythmic contraction of separated heart muscle cells. Science 167, 1727-1729 (1970). 5. ERTEL, R. J., CLARKE, D. E., CHAO, J. C. & FRANKE, F. R. Autonomic receptor mechanisms in embryonic chick myocardial cell cultures. Journal of PharmacoLogy and Ex@Gnental nierapeutics 178, 73-80 (1971). GLICK, M. R., BURNS, A. H. & REDDY, W. J. Dispersion and isolation of beating cells from adult rat heart. Analytical Biochemistv 61,32-42 (1974). GROSSO, D. S., FRANGAKIS, C. J., CARLSON, E. C. & BRESSLER, R. Isolation and characterization of myocytes from the adult rat heart. (Submitted for publication.) HAMRY, I., RENAUD, J. F., SATO, E. & WALLACE, G. A. Calcium ions regulate cyclic AMP and beating in cultured heart cells. Jvature, 261,60-&l (1976). HAY, E. D. & DODSON, J. W. Secretion of collagen by cornea1 epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozenkilled lens. Journal of Cell Biology 57, 190-213 (1973). 10. HUXLEY, A. F. & HUXLEY, H. E. A discussion on the physical and chemical basis of muscular contraction. Proceedings of the Royal Socie@ (London), Series B 160,433 (1964).
ULTRASTRUCTURE 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
OF ISOLATED MYOCYTES
459
~OVSKY, M. J. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. 3oumulof Cell Biology 27, 137a-138a ( 1965). KEELY, S. L., CORBIN, J. D. & PARK. C. R. Regulation of adenosine 3’,5’-monophosphate-dependent kinase. 3ouwzulof Biological Chemistry 250, 4832-4840 (1975). Kuo, J. & GREENCARD, P. An assay method for cyclic AMP and cyclic GMP based upon their abilities to activate cyclic AMP-dependent and cyclic GMP-dependent protein kinases. In Advances in Cyclic JVucieotide Research, Vol. 2, pp. 41-50. P. Greengard and G. A. Robison, Eds. New York: Raven Press. (1972). LEE, T. P., Kuo, J. F. & GRBENGARD, P. Regulation of myocardial cyclic AMP by isoproterenol, glucagon and acetylcholine. Biochemical and Biophysical Research Communications 45, 991-997 (1971). Loway, 0. H., ROSEBROUGH, N. J,, FARR, A. L. & IIANDALL, R. J. Protein measurement with the folin phenol reagent. 3ou~nul of Biological Chemistry 193,265-275 (1951). Lao, C. C. & GUIODTTI, A. Simultaneous isolation of adenosine 3’,5’-cyclic monophosphate and guanosine 3’,5’-cyclic monophosphate in small tissue samples. Analytical Biochemistry 59,63-68 (1974). MASSON-PEVET, M., JONCKWA, H. J. & DEBRUIJNE, J. Collagenase- and trypsindissociated heart cells: a comparative ultrastructural study. 3ournalof Molecular and Cellular Cardiology 8, 747-757 ( 1976). MCCWISTER, L. P., MUMAW, V. R. & MUNGER, B. L. Stereo ultrastructure of cardiac membrane systems in the rat heart. In Scanning Electron Microscofiyjl974, pp. 713-720. 0. Johari and I. Corvin, Eds. Chicago: I.I.T. Research Institute (1974). McNwrr, N. S. & FAWCET-I-, D. W. Myocardial ultrastructure. In The Mammalian Myocardium, pp. l-49. G. A. Langer and J. Brady, Eds. New York: J. Wiley and Sons (1974). MOUSTAFA, E., SKOMEDAL, T., OSNES, J.-B. & 0ye, I. Cyclic AMP formation and morphology of myocardial cells isolated from adult heart: effect of Gas+ and Mgs+. Biochimica et biojhysica acta 421,41 l-415 (1976). MURAD, F. & VAUGHAN, M. Effect of glucagon on rat heart adenyl cyclase. Biochemical Pharmacolog-y 18, 1053-1059 (1969). PEASE, D. C. Histological Techniques for Electron MicroscOpy, 2nd edt. New York and London : Academic Press ( 1964). PRETLOW, T. G., GLICK, M. R. & REDDY, W. J. Separation of beating cardiac myocytes from suspensions of heart cells. American 3ouml of Pathology 67, 2 15-224 (1972). ROBISON, G. A., BUTCHER, R. W., DYE, I., MORGAN, H. E. & SUTHERLAND, E. W. The effect of epinephrine on adenosine 3’,5’-monophosphate levels in the isolated perfused rat heart. Molecular Pharmacology 1, 168-177 (1965). SHELDON, C. A., FRIEDMAN, W. F. & SYBERS, H. D. Scanning electron microscopy of fetal and neonatal lamb cardiac cells. Journal of Molecular and Cellular Cardiology 8, 853-862 (1976). SYBERS, H. D. & ASHRAF, M. Preparation of cardiac muscle for SEM. In: Scanning Electron Microscopy/l973, pp. 341-348. 0. Johari and I. Corvin, Eds. Chicago: I.I.T. Research Institute (1973). SYBERS, H. D. & As-, M. Scanning electron microscopy of cardiac muscle. Laboratory Investigation 30,441-450 (1974). VAHOIJNY, G. V., WEI, R., STARK~EATHER, R. & DAVID, C. Preparation ofbeating heart cells from adult rats. Science 167, 1616-1618 (1970). VENABLE, J. H. & COGGESHALL, R. A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25,407-408 (1965).
