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Correlation of ultrastructure and function in calcium-tolerant myocytes isolated from the adult rat heart
JOURNAL OF ULTRASTRUCTURE RESEARCH 81, 222--239 (1982)
Correlation of Ultrastructure and Function in Calcium-Tolerant Myocytes Isolated from the Adult Rat Heart N. J. SEVERS,* A. M. SLADE,* T. POWELL,? V. W. TWIST,? AND R. L. WARm~N$ *Department of Cardiac Medicine, Cardiothoracic Institute, 2 Beaumont Street, London WIN 2DX, England, and ?Department of Physics as Applied to Medicine, and $Institute of Nuclear Medicine, The Middlesex Hospital Medical School, Cleveland Street, London W1P 6DB, England Received March 2, 1982, and in revised form June 17, 1982 Thin-section and freeze-fracture techniques were applied to investigate the ultrastructure of myocytes isolated from the adult rat heart by in vitro perfusion with collagenase. A central feature of this preparation, which distinguishes it from most others, is that the structural and functional integrity of the individual myocytes is maintained in the presence of physiologic concentrations of extracellular calcium. Ultrastructural examination demonstrates that all the normal features of sarcolemmal structure, including the glycocalyx, are preserved in these isolated cells, and no irreversible damage is sustained as a consequence of separation of the intercalated discs. The structure of intracellular membranes and organelles also shows excellent preservation, indistinguishable from that reported in cells of the intact rat heart. A detailed description of the ultrastructure of the isolated heart cells is presented, together with data on their response to extracellular calcium and on their respiratory function. Emphasis is placed on interpreting the ultrastructural findings in relation to the functional properties demonstrated in the individual myocytes.
The importance of individual myocytes isolated from the adult heart as a model system for investigation of the cellular mechanisms underlying cardiac function is now being recognized. The plethora of dissociation techniques currently available claiming "normal" ultrastructure for isolated myocytes which do not, in fact, function satisfactorily in the presence of physiological concentrations of extracellular calcium (Berry et al., 1970; Farmer et al., 1977; Nag et al., 1977; Carlson et al., 1978; Fry el al., 1979; Moses and Kasten, 1979; Chiesi et al., 1981) has, however, caused considerable confusion as to the general suitability of preparations of isolated cells for experimental studies. In the laboratories of two of the present authors, a decade of experience on the optimum methods for cell isolation has now been gained (Gould and Powell, 1972; Powell and Twist, 1976a; Powell, 1979; Powell et al., 1980) and in
this time extensive experimental results on the functional characteristics of these isolated myocytes have been accumulated (reviewed in Dow et al., 1981a,b). As yet, however, no detailed description of the ultrastructure of this particular preparation of isolated cells has been reported. The aim of the present study was to fill this gap, using both thin-section and freeze-fracture techniques, and, more importantly, to correlate the ultrastructural findings with the known functional responses of the individual myocytes. Our results indicate that the cells isolated from the ventricles of adult rats by in vitro perfusion with collagenase do possess both ultrastructural and functional characteristics which closely resemble those of myocytes in the intact heart, thus providing an isolated cell preparation that is a valid model for a wide range of investigations concerned with myocardial homeostasis.
222 0022-5320/82/110222-18502.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ISOLATED MYOCYTES MATERIALS AND METHODS
Animals. Hearts were obtained from adult female Sprague-Dawley rats (bw 250-300 g) fed ad libitum on a standard chow diet. Cell preparation. Suspensions of purified cardiac myocytes were obtained by Langendorff perfusion of the hearts with crude collagenase in low calcium KrebsRinger bicarbonate buffer, as described in detail by Powell et al. (1980). The composition of the buffer was as follows: NaC1, 118.5 mM; NaHCO3, 14.5 raM; KC1, 2.6 mM; KH2PO4, 1.18 raM; MgSO4, 1.18 mM; glucose, 11.1 raM. Calcium was added as required from a stock solution of 1 M CaC12. The resulting cell suspension was preincubated at 37°C in gassed buffer containing 20 mg cm s bovine serum albumin for at least 30 min prior to any experimental procedures. This preincubation was carried out in either nominally calcium-free buffer or medium to which 0.5 mM calcium chloride had been added. All cell suspensions were gassed regularly with 95% OJ5% COs. Cells were fixed for electron microscopy or for counting by light microscopy by adding 1 vol cell suspension to 4 vol 2% (v/v) glutaraldehyde made up in V3 strength Krebs buffer. To determine cell number and purity, the fixed suspensions were centrifuged and washed, then counted in quadruplicate in an improved Neubauer hemocytometer. Calcium analysis. The levels of calcium in the solutions used for cell isolation and incubation were analyzed by emission flame spectrometry. We used a method by which each sample was evaporated from a tungsten filament into the fuel supply of an oxyhydrogen flame and the resulting light emission integrated by a photomultiplier-monochromator monitoring the appropriate spectrum line (422.67 nm with 0.11-nm band width) in the flame (Warren, 1980). By this technique, a solution containing 1 pM calcium could be measured with a standard deviation of _+5.4% and the limit of detection (calculated at three times the standard deviation of the background noise)was 0.02 vM, for sample volumes in the range 0.2--4 txl. Cellular respiration. Oxygen consumption of isolated cell preparations was measured by standard techniques using an oxygen electrode (Powell and Twist, 1975, 1976a). Stimulation of oxygen uptake by 10/xg cm -~ of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a measure of cellular respiratory control index (RCI). Results are given as mean _+ standard error of the mean, with the number of observations following in parentheses. Statistical analysis was carried out by a two-way analysis of variance when the data were appropriately symmetric (Sokal and Rohlf, 1969); otherwise group means were compared using Student's t test. Electron microscopy. The cell suspensions were fixed for 2 hr in the 2% glutaraldehyde solution at 20°C. For thin-section electron microscopy, samples were rinsed briefly in buffer and postfixed in 1% osmium tetroxide
buffered with 0.1 M sodium cacodylate (pH 7.3) for 2: hr at 4°C. After en bloc staining with saturated aqueous uranyl acetate for 1 hr at room temperature, the spec-imens were dehydrated in ethanol and embedded in Araldite via propylene oxide. Silver sections were prepared with a diamond knife using an LKB III ultramicrotome. For freeze-fracture, glutaraldehyde-fixed specimens were gradually infiltrated with glycerol (in Krebs-Ringer or cacodylate buffer) to a final concentration of 25 or 30% glycerol over a period of 45 rain. Droplets of concentrated cell suspension were mounted on Balzers specimen holders and rapidly frozen by plunging into liquid propane held at -180°C. Freeze-fracturing was carried out in a Balzers BAF 400T unit at specimen temperatures of - 100 to - 115°C and at a vacuum of 5 x 10-r mbar or better. Some specimens were etched briefly before replication. Replicas were cleaned in 40% chromic acid and rinsed twice in distilled water before mounting on uncoated grids. Thin sections and replicas were examined and images recorded using a Philips EM301 electron microscope. RESULTS
Calcium Analysis o f E x p e r i m e n t a l Solutions When Krebs solution, with calcium chlor i d e o m i t t e d , w a s m a d e u p in d o u b l e g l a s s d i s t i l l e d w a t e r ( c a l c i u m c o n c e n t r a t i o n <0.0,8 /xM), t h e t r a c e a m o u n t s o f c a l c i u m in t h e salts u s e d ( A n a l a r g r a d e ) r e s u l t e d in a final c a l c i u m c o n c e n t r a t i o n ([Ca]0) o f 8.8 _+ 0.5 /xM (n = 9). I n all i n c u b a t i o n s o l u t i o n s , b o v i n e s e r u m a l b u m i n w h e n a d d e d a t 2;0 mg/cm 3 increased Krebs solution calcium to 26.0 +_ 0.6 /xM (n = 9). B y c o n t r a s t , a t the end of the Langendorff perfusion during c e l l i s o l a t i o n , t h e p e r f u s i o n fluid c o n t a i n e d 35.5 + 0.8 /xM c a l c i u m (n = 61). T h u s Jin this p a p e r w h e n i n c u b a t i o n s o l u t i o n s a r e ref e r r e d t o as " n o m i n a l l y " o r " e s s e n t i a l l y " calcium-free, the calcium concentration of s u c h s o l u t i o n s is - 2 6 / a M .
Gross Morphology o f Isolated M y o c y t e s Examination of suspensions of isolated myocytes by light microscopy showed two distinct cell appearances; a population of rounded (damaged) cells, and a more num e r o u s r o d - s h a p e d f o r m ( F i g . 1). H i g h magnification inspection by light microsc o p y (Fig. 2) a n d l o w - p o w e r e l e c t r o n m i -
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croscopy (Fig. 3) revealed that the rodshaped cells displayed all the gross morphological features characteristic of cardiac muscle cells in the intact myocardium. U1trastructural examination at higher resolution by thin-section and freeze-fracture electron microscopy (Figs. 6-19 and 21-23, discussed in detail later) confirmed that these m y o c y t e s were indeed well preserved, closely resembling their counterparts in the intact heart.
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FIG. 4. Percentage viability of isolated myocytes Figure 4 shows the results of experiments incubated at 37°C in solutions containing differing in which, following preincubation in nomi- [Ca~+]0. nally calcium-free or 0.5 mM [Ca]0 media, 1-cm z aliquots of the isolated cell suspension were incubated at 37°C for 7 min in the effect was not seen in suspensions preinpresence of added calcium to give a final cubated with 0.5 mM [Ca]0. These results [Ca]0 in the range 0.5-10 raM. At the end indicate that the isolated cells are calcium of the incubation period the aliquots were tolerant when presented with an acute calLfixed and the numbers of rod-shaped and cium load. rounded cells counted. Suspensions exThe presence of 0.5 mM [Ca]0 also in,tposed only to calcium-free media contained proved longer term cell survival. In essen71.2 _ 1.4% (n = 16) rod-shaped cells, tially calcium-free Krebs-Ringer the prowhich was significantly higher (P < 0.05) portion of intact rod-shaped cells after 60 than for suspensions incubated in 0.5 min incubation at 37°C was 67.3 _+ 0.72%, mM [Ca]0 after calcium-free preincubation which decreased significantly (P < 0.00l) (67.3 _ 1.3%, n = 16), or for those incu- over the next 330 min to 28.7 _+ 17.8% (Fig. bated throughout in 0.5 mM [Ca]0 media 5). In contrast, although there was also a (67.3 _+ 1.4%, n = 16). Irrespective of significant decrease over the same time inpreincubation [Ca]0, there were no statisti- terval from 65.7 ___ 1.2% to 51.0 _ 6.7% for cal differences in the percentage of rod- cells incubated in solutions to which 0.5 n ~ shaped cells incubated with calcium in the CaC12 had been added, this decrease was range 0.5-5.0 raM. When the final incuba- less than that observed in the nominally caltion contained 10 mM [Ca]0, however, cells cium-free incubations. Under both incubapreincubated in calcium-free buffer showed tion conditions, RCI values were maindecreased percentage viability at 57.8 _+ tained for the first 5 hr of incubation at levels 1.6% (n = 16), which was significantly low- comparable to those to be shown later (Fig. er (P < 0.01) than at 5 mM calcium. This 20).
FIG. 1, Light microscope (bright field) survey view of an isolated myocyte preparation. Intact rod-shaped cells are easily distinguished from damaged cells that appear rounded and dark. x 100. FIG. 2, High-power light microscope view showing the regular structural details characteristic of intact rodshaped cells, x 800. FIG. 3. Survey electron micrograph of a longitudinal thin-section through an isolated rod-shaped myocyte. Myofilament bundles with their characteristic banded structure alternate between rows of mitochondria, as in cells of the intact heart. In this example, 64 sarcomeres can be counted along the length of the cell. x 1500.
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w i t h e x p o s u r e to c a l c i u m at 0.5 raM. E x p e r i m e n t a l l y , p r e p a r a t i o n s like t h o s e u s e d for the r e s u l t s d e p i c t e d in Fig. 5 a r e s t o r e d as " s t o c k s u s p e n s i o n s , " f r o m w h i c h samples m a y b e s e d i m e n t e d ( u n d e r g r a v i t y for 8 rain) at t i m e s f o r up to 6 h r a f t e r p r e p a r a t i o n to g i v e a p u r i t y o f - 7 0 % o v e r this period.
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FIG. 5. Long-term percentage viability for isolated myocytes incubated at 37°C in either a nominally calcium-free solution (O) or Krebs-Ringer to which 0.5 mM calcium chloride had been added (0). Numbers in parentheses indicate the number of experimental observations. *, P < 0.05; ***, P < 0.001,
A n o t h e r i n t e r e s t i n g f e a t u r e o f Fig. 5 is t h a t a l t h o u g h f r o m l to 5 hr a f t e r t h e 30rain p r e i n c u b a t i o n t h e r e is n o d i f f e r e n c e in t h e p e r c e n t a g e o f r o d - s h a p e d cells f o r t h e t w o c a l c i u m c o n c e n t r a t i o n s , t h e initial p e r c e n t a g e s a r e h i g h l y significantly d i f f e r e n t ( P < 0.001). In f a c t , t h e initial p e r c e n t a g e o f 62.5 _+ 1.2% (n = 16) a f t e r 30-rain incub a t i o n in 0.5 m M c a l c i u m s o l u t i o n is n o t significantly d i f f e r e n t f r o m the 60-rain v a l u e (65.7 _+ 1.2%), so t h a t o v e r this s e r i e s o f e x p e r i m e n t s t h e cells a r e " c a l c i u m intole r a n t " to t h e e x t e n t t h a t t h e r e is a m a x i m u m initial d e c r e a s e in p u r i t y o f a b o u t 5%
S o m e i n s i g h t into the s t r u c t u r a l b a s i s und e r l y i n g t h e s u r v i v a l o f i n t a c t cells a n d maintenance of their normal functional c h a r a c t e r i s t i c s is g a i n e d b y u l t r a s t r u c t u r a l e x a m i n a t i o n o f the s a r c o l e m m a . In transv e r s e thin s e c t i o n , the s a r c o l e m m a o f isol a t e d m y o c y t e s d i s p l a y s an i n t a c t t r i l a m i n a r u n i t - m e m b r a n e s t r u c t u r e , c o a t e d on its extracellular surface with a fuzzy glycocalyx (Fig. 6). F r e e z e - f r a c t u r e e l e c t r o n m i c r o s c o p y d i s c l o s e s i r r e g u l a r l y d i s t r i b u t e d intram e m b r a n e p a r t i c l e s w i t h a l o w e r d e n s i t y on t h e E f a c e t h a n on t h e P f a c e ( F i g s . 7 a n d 8). N e i t h e r p a r t i c l e a g g r e g a t i o n n o r s m o o t h m e m b r a n e r e g i o n s d e v o i d o f p a r t i c l e s (features indicative of membrane damage) are e v i d e n t . A l l t h e n o r m a l s a r c o l e m m a l spec i a l i z a t i o n s c h a r a c t e r i s t i c o f cells o f t h e int a c t h e a r t a r e f o u n d in t h e s e i s o l a t e d m y ocytes. Transverse tubules, bearing a g l y c o c a l y x c o n t i n u o u s w i t h t h a t o f the p e r i p h e r a l s a r c o l e m m a , p e n e t r a t e into t h e cell at t h e l e v e l o f the Z b a n d (Fig. 6). T h e tub u l e o p e n i n g s at t h e cell s u r f a c e a r e freq u e n t l y r e v e a l e d in p l a n a r f r e e z e - f r a c t u r e
Fins. 6-12. Ultrastructure of the general sarcolemma. Fio. 6. Specializations of the general sarcolemma as seen in thin section. The external surface of the plasma membrane is coated with a fuzzy glycocalyx (g). The long invagination at T marks the entrance to a transverse tubule, penetrating at the Z band (Z). c, caveolae; p, bristle-c0ated pit. x 80 000. FIG. 7. Freeze-fractured sarcolemma, E-face view. Relatively few intramembrane particles are present. × lO0 000. FIG. 8. Freeze-fractured sarcolemma, P-face view. Intramembrane particles are abundant compared with the previous figure and no bare patches of membrane are visible. The circular discontinuities represent the cross-fractured necks of caveolae, x 100 000. FIG. 9. Survey freeze-fracture view of the sarcolemma (P face) illustrating the characteristic regular arrangment of transverse tubule openings. This arrangement does not extend over the entire surface of the cell but is localized in occurrence. Deep invaginations of uninterrupted membrane are sometimes found at positions in which a transverse tubule opening would be predicted (example indicated with asterisk), x 25 000.
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FIG. 10. Survey thin-section view illustrating the variety of form exhibited by caveolae. The section plane is somewhat tangential to the cell surface in this example. C1 shows a triple type in which three caveolae unite to form a common neck; C~ is a long beaded chain of caveolae which extends deeply into the cytoplasm. × 36 000. FIG. 11. Caveolae sectioned transversly through the cell surface. Single forms of eaveolae are seen opening to the exterior in favorable section planes (eL C1 and C~). The complexity of multiple caveolae (C~) is only hinted at in thin section. CV, coated vesicle. × 70 000. FIG. 12. Freeze-fracture view of a chain of multiple caveolae fused end-on to one another. Branches are formed where additional chains are attached. S, sarcolemma (E-face view), x 80 000.
v i e w as o r d e r e d a r r a y s (Fig. 9), as previo u s l y r e p o r t e d b y s c a n n i n g e l e c t r o n mic r o s c o p y ( P o w e l l et al., 1978a). C a v e o l a e are s e e n as c i r c u l a r d i s c o n t i n u i t i e s (62 + 19-nm d i a m e t e r ) in e n f a c e v i e w s of the sarc o l e m m a , s c a t t e r e d at all levels of the sarc o m e r e s u r f a c e at a d e n s i t y of 6.0 -+ 1.8 /xm -2 (Figs. 8 a n d 9). T h e s e i n v a g i n a t i o n s d i s p l a y a striking v a r i e t y of f o r m a n d their
membrane characteristically reveals a d e a r t h o f P-face i n t r a m e m b r a n e particles c o m p a r e d with the s u r r o u n d i n g s a r c o l e m ma. T h e s i m p l e s t form of c a v e o l a c o n s i s t s of a single f l a s k - s h a p e d s a r c o l e m m a l inp o c k e t i n g (Figs. 6, 10, a n d ! 1) with a d e p t h a n d w i d t h r a n g i n g f r o m 40 to 140 n m (meas u r e d in t r a n s v e r s e t h i n s e c t i o n ) . M a n y c a v e o l a e are more c o m p l e x s t r u c t u r e s ,
ISOLATED MYOCYTES h o w e v e r ; two, t h r e e , or m o r e c a v e o l a r vesicles are f r e q u e n t l y f o u n d s h a r i n g a comm o n n e c k (Figs. 10 a n d 11), a n d m o r e exc e p t i o n a l l y , e x t e n d e d b r a n c h e d c h a i n s of f u s e d c a v e o l a e are s e e n (Figs. 10 a n d 12), similar to those r e p o r t e d in c o r o n a r y s m o o t h m u s c l e cells ( F o r b e s et al., 1979). O c c a sional b r i s t l e - c o a t e d pits a n d v e s i c l e s , dist i n c t f r o m the u s u a l f o r m s of c a v e o l a e , are also o b s e r v e d (Figs. 6 a n d 11). B e c a u s e i n the i n t a c t tissue c a r d i a c m u s cle cells are p h y s i c a l l y a n d m e t a b o l i c a l l y c o n n e c t e d to o n e a n o t h e r b y i n t e r c e l l u l a r j u n c t i o n s d i s p o s e d at f r e q u e n t sites a l o n g the i n t e r c a l a t e d disc, this r e g i o n of the sarc o l e m m a is p o t e n t i a l l y v u l n e r a b l e to d a m age d u r i n g cell s e p a r a t i o n . T h i n - s e c t i o n a n d freeze-fracture e l e c t r o n m i c r o s c o p y d e m o n strate that after cell i s o l a t i o n , the highly
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c o n v o l u t e d g e o m e t r y of the i n t e r c a l a t e d disc m e m b r a n e is, h o w e v e r , r e t a i n e d with-. o u t n o t i c e a b l e d i s r u p t i o n (Figs. 13 a n d 14). O c c a s i o n a l l y , sites of p o s s i b l e focal sarcol e m m a l d a m a g e are d e t e c t e d , b u t the state,' of p r e s e r v a t i o n of a d j a c e n t c y t o p l a s m i c : c o m p o n e n t s suggests t h a t in m o s t i n s t a n c e s t h e s e sites r e p r e s e n t debris o r i g i n a t i n g f r o m a d a m a g e d n e i g h b o r i n g cell. D e s m o s o m e s a n d fascia a d h e r e n t e s j u n c t i o n s are c l e a v e d a p a r t b y the e n z y m a t i c t r e a t m e n t , l e a v i n g h a l f - j u n c t i o n s at e a c h i n t e r c a l a t e d disc surface. B y c o n t r a s t , the gap j u n c t i o n c o m p o n e n t s of b o t h m e m b r a n e s are o f t e n ret a i n e d with j u s t o n e of the cells, r e v e a l i n g a n a t t a c h e d l o o p of m e m b r a n e , p r e s u m e d to h a v e b e e n t o r n f r o m a n a d j a c e n t cell dur'ing s e p a r a t i o n (Fig. 13). G a p j u n c t i o n m e m b r a n e is also o f t e n s e e n in the f o r m of cy-
FIGS. 13-15. Ultrastructure of the intercalated-disc sarcolemma. Fro. 13. Survey thin-section view of the intercalated disc region of the sarcolemma. The plasma membrane appears irregular but intact and adjacent cytoplasmic components are well preserved. Electron-dense zones on the cytoplasmic side of the membrane are the remnants of fascia adherentes and desmosomes retained with the cell. Other features of interest are indicated by 1, 2, and 3:1 probably represents a highly convoluted region of the membrane with adherent debris originating from an adjacent cell before isolation; 2 is the site of a gap junction with an attached loop of membrane; and 3 is an apparent vesicle of gap junction membrane (for an example at higher magnification, see Fig. 15). x 9000. FIG. 14. Freeze-fracture view of a portion of intercalated disc sarcolemma. The plasma membrane is intact and undamaged. Its highly convoluted nature is indicated by the portions of membrane observed apparently within the cytoplasm (asterisks) but which are in fact continuous with the surface sarcolemma, x 35 000. FIG. 15. Thin-section view of a "gap junction vesicle." Although this structure appears to be a discrete vesicle in the cytoplasm, serial sectioning showed that in this example at least, the "vesicle" membrane was in fact continuous with the surface sarcolemma, x 45 000. Fins. 16-19, Sarcoplasmic reticulum and mitochondrial ultrastructure. Fin. 16. The structural complexity of the sarcoplasmic reticulum (SR) is illustrated in this thin-section field. A network of tubules in the M region, the M rete (MR), is connected by longitudinal elements (L) to a z tubule (ZTI) seen here in longitudinal section. Examples of transversely sectioned z tubules, seen elsewhere, are indicated by ZTT. Junctional sarcoplasmic reticulum (JSR) is continuous with the free SR (arrow) and consists of flattened cisternae closely apposed to the sarcolemma. Peripheral junctional SR (JSRp) occurs in association with the surface sarcolemma, and interiorjunctional SR (JSR0 occurs against transverse-tubule membrane. The latter may take the form of dyads (D), which consist of a transverse tubule plus one JSR cisterna, or triads (T), which consist of a transverse tubule sandwiched between two JSR cisternae. The JSR lumen is characteristicaJtly bisected by an electron-dense line, and regularly spaced electron-dense feet project from the membrane facing the sarcolemma. Mitochondria (M) show well-preserved membranes and cristae. ML, M line; ZL, Z line; g, glycocalyx, x 50 000. FIG. 17. Freeze-fracture view of interior junctional SR/transverse-tubule association, with mitochondrial membranes above and below the complex. The portion of SR connected to the junctional SR has a cisternal rather than a tubular configuration (c). × 60 000. FIG. 18. Structural details of a transversely sectioned triad. T, transverse tubule; JSR, junctional SR; ZL, z line. × 100 000. Fin. 19. Length of tubular sarcoplasmic reticulum (SR) showing close association with mitochondria (M) as displayed by freeze-fracture. × 50 000.
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toplasmic vesicles (Fig. 15), though we have established by serial sectioning that the membranes of at least some of these apparently discrete vesicles are in fact continuous with the surface sarcolemma.
The Sarcoplasmic Reticulurn The intracellular membrane systems of the c e l l s - - b y virtue of their location--are less susceptible to damage during the isolation process, and their well-preserved ultrastructural appearance (Figs. 16-19, 21, and 22) is interpreted as a reflection of retention of their normal functional capacities. The junctional and free components of the sarcoplasmic reticulum (SR) are readily identified in isolated myocytes. As in the ceils of the intact myocardium, the free SR consists of a network (fete) in the M region of the sarcomere, linked by longitudinal tubules to the Z elements which may take the form of Z fetes and/or Z tubules (Fig. 16). Junctional SR is comprised of cisternae intercalated into the free SR network which form both peripheral couplings (against the peripheral sarcolemma) or interior couplings (against transverse tubules) in the Z region of the sarcomere (Figs. 16-18). In either situation, the junctional SR is closely apposed to the sarcolemma, leaving a gap of approximately 13 nm bridged by regularly spaced electron-dense structures. The lumen of transversely sectioned junctional SR is characteristically bisected by a line of electron-dense material. Interior couplings are seen both as dyads (Figs. 16 and 17) and as triads (Figs. 16 and 18). Freeze-fracture shows that the free SR occurs mainly in the form of tubules, 20-40 nm in diameter (Fig. 19), though these periodically broaden out to give a cisternal configuration (Fig. 17).
Mitochondrial Ultrastructure and Respiratory Control Figures 16-19 also demonstrate that the structure of mitochondrial inner and outer membranes, their cristae, and matrix remain well preserved as judged by thin-section and freeze-fracture. This is consistent
PREINCUBATION
[c~+'3o
40
[]
Ca++'FREE
r .o.+ 20
10
0
0 0+5 1.0 2.5 5.0 CALCIUM ADDED TO INCUBATION MEDIUM(rnM)
FIG. 20. Respiratory control indices (RCI) for cells exposed to differing [Ca]0 at 37°C. RCI values were estimated from stimulation of oxygen consumption after the addition of 10/zg/cm~ CCCP. Equivalent solvent volumes were run as blanks and calibration was achieved by conventional methods.
with the m a i n t e n a n c e of m i t o c h o n d r i a l function as reflected in the experimental measurements of RCI summarized in Fig. 20. Cells in media containing only trace amounts of calcium had an RCI of 16.7 + 2.4 (n = 6), which was not significantly different from that observed in cells exposed to 0.5 mM calcium (23.2 _+ 3.5, n = 6). Although myocytes preincubated in calciumfree media showed no change in RCI when [Ca]0 was increased in the range 1.0-5.0 raM, cells preincubated in 0.5 mM [Ca]0 showed a higher RCI (F = 24, P < 0.001), reaching a value of 35.2 _+ 1.9 (n = 4) at 2.5 mM [Ca]0. Thus mitochondrial function is maintained even when the cells are faced with high calcium concentrations. These experiments were carried out for up to 6 hr after cell preparation, with results similar to those in Fig. 20 obtained over this period.
The Nucleus The ultrastructural appearance of the nucleus is also a good indicator of cellular functional capacity, and isolated myocytes show no abnormal nuclear features. Most of the nuclear matrix contains diffuse chromatin, with a peripheral layer of hetero-
ISOLATED MYOCYTES
chromatin around the inner nuclear membrane and nucleolus (Fig. 21). The densities and distributions of nuclear pore complexes and intramembrane particles as seen in freeze-fractured nuclear membranes from isolated myocytes (Fig. 22) do not differ from those of cells from the intact heart.
2313 DISCUSSION
The ultrastructural features reported here for isolated myocytes in suspension compare very favorably with those reported previously for adult myocytes in intact myocardial tissue (McNutt and Fawcett, 1974.; Simpson et al., 1974; Sommer and JohnThe Contractile A p p a r a t u s son, 1979; Segretain et al., 1981). Although Myofibril structure appeared well pre- the morphology of isolated myocytes has served in thin-sectioned isolated myocytes, been documented in numerous previous inshowing the characteristic organization fa- vestigations (Berry et al., 1970; Nag et al., miliar from cells in the intact myocardium 1977; Carlson et al., 1978; Fry et al., 197q; (Fig. 23). The mean sarcomere length (+_SD) Moses and Kasten, 1979; Nag and Zak, measured as the distance between adjacent 1979; Chiesi et al., 1981), the cells in such Z bands in 67 cells cut in precise longitu- preparations showed pronounced "calcium dinal section was 1.64 +_ 0.21 t~m. Some intolerance," i.e., they went into marked variation in the state of relaxation and con- contracture in solutions containing microtraction was noted between individual cells, molar amounts of extracellular calcium. It and this was reflected in the clarity with has always been emphasized that cells prewhich the I and H bands could be dis- pared by our method do not exhibit this decerned. The cell in Fig. 23 is in a relatively ficiency and are stable in media containing relaxed condition; I and H bands are prom- physiological levels of calcium (Gould and inent, and the sarcomere length is 1.83 ~m. Powell, 1972; Powell and Twist, 1976a; This value is close to that recorded as an Powell et al., 1980). Recently, Kao et al. average for fresh or fixed cells not prepared (1980) have described the morphology of for electron microscopy (Nash et al., 1979; cells isolated from adult rat ventricle which Roos et al., 1982) but is toward the higher were also calcium tolerant, but the signifiend of the range measured in the present cance of the present paper is that we can study. Compared with the thin-section correlate the cellular ultrastructure reportmeasurements, higher values for sarcomere ed here with the functional characteristics length are also suggested from the spacing extensively investigated using the same of transverse tubules viewed in freeze-frac- preparation. ture (Severs and Powell, 1980). Because The thin-section and freeze-fracture evitransverse tubules do not always penetrate dence indicating an intact and structural]Ly exactly at the Z band level, this cannot be unaltered sarcolemma with associated glyconsidered as precise a method as direct cocalyx, is consistent with the experimenmeasurement between the bands. (Sarco- tal finding that individual myocytes tolerate lemmal indentations overlying the Z bands, impalement with one or two glass microas reported in myocytes from intact rabbit electrodes, even when the concentration of ventricle (Levin and Page, 1980) are not extracellular calcium is 5-10 raM, and have present in our isolated rat myocytes and so negative resting membrane potentials charcould not be used for measurements of sar- acteristic of those found in whole tissue comere length in freeze-fracture replicas.) (Powell et al., 1978b,c, 1980). FurtherOur results thus suggest that processing for more, these myocytes can generate conthin-sectioning (particularly dehydration) is ventional action potentials, indicating that responsible for some shrinkage of the con- active conductance mechanisms have been tractile apparatus, which is reflected in a preserved in the sarcolemma. This contendecrease of -10% in sarcomere length. tion has been confirmed by direct experi-
234
SEVERS ET AL.
ISOLATED MYOCYTES
235
FIG. 23. Typicalview of the contractile apparatus in an isolated myocyte. The presence of clearly defined I bands (I) indicates a relatively relaxed condition. Z, Z line; M, M line; A, A band. x 30 000.
mentation, which has shown that the cells have functional N a channels (Brown et al., 1980, 1981a,b) responsible for the rapid upstroke of the action potential, and also " s l o w channels" for conducting the Ca/Na current important in maintaining the plateau phase (Powell et al., 1981). In addition it has been demonstrated that functional sarcolemmal receptors are retained, as isoprenaline, noradrenaline or adrenaline pro-
duce reversible effects not only on the electrical activity of the cells (Powell et al., 1978b, 1981), but also on their production of Y,5'-cyclic adenosine monopho'sphate (Powell and Twist, 1976b). Our demonstration of intact, well-preserved interior and peripheral junctional SR, free SR, and mitochondria is consistent with m u l t i c o m p a r t m e n t efflux curves for calcium (Baker et al., 1981), together with tile
FIG. 21. Thin-section view of part of a nucleus and adjacent cytoplasm. No abnormal chromatin clumping or clearing of the nucleoplasm is evident. HC, heterochromatin; DC, diffuse chromatin; Nu, nucleolus; Np, nuclear pore; ZL, z line. x 50 000. Fro. 22. Freeze-fractured nucleus showing the P face of the inner nuclear membrane (INM) and the E face of the outer nuclear membrane (ONM). No abnormality in the density and distribution of nuclear pores or intramembrane particles is seen. x 35 000.
236
SEVERS ET AL.
observations that calcium uptake is dependent upon both the electric field across the sarcolemma and also intracellular stores (Fry et al., 1981). In contrast, potassium distribution within the cell is described largely by only one compartment (Powell and Twist, unpublished observations). That normal mitochondrial structure and function is preserved is strongly suggested by both the ultrastructural findings and the maintenance of good cellular respiratory control even in the face of a severe calcium challenge (which again indicates low sarcolemmal permeability to this ion). The quantitative experimental measurements on electrical characteristics, ionic c o m p a r t m e n t a t i o n , sarcolemmal responses, and respiratory activity have been obtained for up to 8 hr after cell isolation, even though myocytes were incubated during this period at 37°C in a simple oxygenated Krebs-Ringer bicarbonate buffer containing only glucose as substrate, and added bovine serum albumin. This raises the important question of how cells isolated in low calcium solutions and exposed to a dissociating enzyme (collagenase) can retain both the ultrastructural characteristics reported here (even in the presence of physiological extracellular calcium) together with functional responses typical of whole, intact myocardium. Such behavior contrasts markedly with the many reports in the literature describing enzymically dissociated myocytes that undergo irreversible contracture when exposed to micromolar amounts of calcium and which are, moreover, electrically inert in even nominally calcium-free solution (e.g., Rieser et al., 1979). After a comprehensive examination of the various isolation techniques employed (Dow et al., 1981a,b) we consider that a pivotal reason for the success of the method used here is that the isolation and incubation solutions always contain calcium, which at the end of the perfusion with enzyme is 30-40 p3/, and even in KrebsRinger solution with albumin is 20-30 p3/ (see results). This derives from endogenous
calcium, calcium present in and added with the collagenase, calcium present in albumin and salts, and calcium leached from the tissue during perfusion. Levels of calcium in this concentration range may well saturate high-affinity sarcolemmal binding sites and promote membrane integrity during both the isolation procedures and also in the incubations that are nominally calcium-free (Frank et al., 1976, 1977; Langer et al., 1976). Isolation of individual myocytes must inevitably involve disruption of the normal intercellular relationships existing in the multicellular tissue from which they are derived. The specializations of the intercalated disc sarcolemma responsible for intercellular adhesion (i.e., adherentes junctions), and transmission of the action potential and intercellular communication (i.e., gap junctions) are discrete structural units comprised of components associated with and built into two adjacent sarcolemmae. How these junctions become physically separated to allow production of viable cells, and the effects, if any, of such unavoidable structural alterations, are of central importance to the understanding and assessment of isolated myocyte function. Although adherentes junctions seem to be separated quite easily along an extracellular plane between the two membranes, freezefractured gap junctions in isolated cells often reveal the presence of components from both sarcolemmae (i.e., P-face and E-face views are seen in the same junction; Severs and Powell, 1980). This might suggest that each intact rod-shaped myocyte retains entire gap junction assemblies at the expense of permanent damage to its former neighbors. This interpretation, however, cannot easily be reconciled with the large numbers of gap junctions found at each intercalated disc (Matter, 1973) nor with yields of over 60% intact cells obtained in our best preparations immediately after isolation and before purification. We suggest therefore as a working hypothesis, that although some gap junctions are torn in toto with one cell from
ISOLATED MYOCYTES
its neighbor, separation may also occur by cleavage through the junctional plane, and minor membrane damage sustained during either process can be rapidly repaired (cf. Muir, 1967; Fry et al., 1979). A substantial number of cells nevertheless suffers extensive damage leading to irreversible injury and rounding up. Precise details of the repair mechanisms operating in the survivors and the eventual fate of their gap junctions require further investigation. High values for cell input resistances and resting membrane potentials (Powell et al., 1980) strongly suggest that those gap junctions detected in isolated cells have adopted an "open-circuit" configuration. The evidence presented here suggesting that the isolated rat myocytes have plasma membranes of normal ultrastructure in which caveolae are a prominent feature, has important implications in the calculation of specific membrane electrical characteristics. It has been demonstrated that the caveolae of these myocytes show a similar numerical density and distribution and have similar dimensions to those in the intact rat myocardium (Severs and Powell, 1980; Gabella, 1978). Should these membrane inpocketings prove to have electrical properties similar to those of the external surface sarcolemma and T-tubular membranes, then previous estimates used for myocyte surface area in the calculation of electrical characteristics (Powell et al., 1980) require revision. Following the nomenclature used by Stewart and Page (1978), let SEs and STT be the surface area (tzm 2) of the external sarcolemma (taking into account the periodic folded nature of the surface) and the T-tubular membranes, respectively. If VCE~.L (~m 3) is cell volume, then the data given in Table II of Stewart and Page (1978) can be used to calculate values (mean +__ SD, n = 4) of 0.31 _+ 0.01 ~m2//zm ~ for (SEs/ VCELL) and 0.16 _+ 0.03 /zm2//~m 3 for (STT/VcELt), derived from morphometric analysis of left ventricles of female Sprague-Dawley rats, corresponding in breed and weight (250-300 g) to the animals
237
used in the present experiments. The mean values of these ratios give an estimate of' 0.51 for (STa4SEs). Gabella (1978) has estimated that in male albino rats (250-300 g bw) the ratio (Sc/SEs) is about 0.27, where S c is total caveolar surface area (tzm2). From these figures, SEs + STT + Sc = 1.78SEs
(1)
Powell et al., (1980) noted that many my-ocytes could be described approximately by a rectangular block 100 ~zm × 20 /~m × 6 t~m, giving an estimate of 6000/xm 2 for SEs (which must be a conservative value, because no surface folding has been considered). Substituting for SEs on the right hand side of Eq. (1) yields a calculated total cell surface area of about 10 700 t~mz, which is 33% larger than the 8000/xm z used by Powell et al. (1980), taken from Page and McCallister (1973) for a 100-tzm-long rat ventricular cell (allowing for external surface sarcolemma, T system, and transverse boundary). Another estimate of total surface area can be obtained by assuming a mean myocyte volume of -25 000/zm 3 (Korecky and Rakusan, 1978; Page, 1978; Bishop and Drummond, 1979; Sommer and Johnson, 1979; Dow et al., 1981b) which implies that Sr.s is 7750/xm 2 if (SEs/Vc~Lt) is 0.31 /~m~//xm3. From Eq. (1), total surface area would now become almost 13 800/xm 2, i.e., over 72% larger than the quoted estimate of 8000/zm 2. A gross underestimation of myocyte surface area will result in calculated values of specific membrane capacitance (Cm, #.F cm -2) and peak sodium conductance (g~a, mS cm -2) which are too large, and values of specific membrane resistance (Rm, kl) cm 2) which are too small. Thus, values for Rm and Cm of 3.2 kl) cm 2 and 2.5 ~F cm -~ (Powell et al., 1980) and 25 mS cm -z for gNa (Brown et al., 1981) based on an average surface area of 8000/xm 2, should be revised to either 4.3 kf~ cm 2, 1.9 /xF cm -z, and 19 mS cm -z, respectively, for the new lower estimate of 10 700 /zm2, or to 5.5 k12 crn 2, 1.4 /zF cm -2, and 14 mS cm -2 if the upper
238
SEVERS ET AL.
estimate of 13 800 ~m ~ is used. The new estimates of Rm are nearer the value of 9.1 _+- 1.0 kl) cm 2 reported by Weidmann (1970) for trabecular muscle, and the values for Cm are compatible with specific capacitances of the order of 1 /~F cm -2, to be expected from the general organization and composition of plasma membranes of excitable tissues (Hodgkin and Huxley, 1952). As previously discussed by Levin and Page (1980), however, calculations of this type should be regarded with some caution. Whereas caveolar membrane area contributes to specific membrane capacitance, it may not necessarily do so to ionic conductance in view of the paucity of intramembraHe particles present. Furthermore, the surface area estimates will be markedly affected by the number of caveolae per neck, for which precise data are not yet available. The presence of multiple branched caveolae (Figs. 10 and 12), for example, could well lead to underestimated surface area values. In addition, account should be taken of the possibility that the frequency of caveolae in transverse tubule membrane could differ from that in the general plasma membrane. In conclusion, the results presented and discussed here further emphasize the similarity of ultrastructure and function between this preparation of isolated myocytes and their counterparts in the intact myocardium. It may be confidently predicted that use of this model system will continue to advance our understanding of myocardial function at the cellular level. This work was carried out with the aid of British Heart Foundation Grants 779 and 81/44 to N. J. S. and
80/25 and 81/82 to T. P.
REFERENCES BAKER, C. J., POWELL, T., TWIST, V. W., AND WILLIAMS, B. C. (1981) J. Physiol. 312, 36P. BERRy, M. N., FRIEND, D. S., AND SCHEUER,J. (1970) Circ. Res. 26, 679-687. BISHOP, S. P., AND DRUMMOND, J. L. (1979) J. Mol. Cell. Cardiol. 11,423-433.
BROWN, A. M., LEE, K. S., AND POWELL, T. (1980) J. Physiol. 301, 78-79P, BROWN, A. M., LEE, K. S., AND POWELL, T. (1981a) J. Physiol. 318, 455-477. BROWN, A. M., LEE, K. S., AND POWELL, Z. (1981b) J. Physiol. 318, 479-500. CARLSON, E. C., GROSSO, D. S., ROMERO, S. A., FRANGAKIS, C. J., BYUS, C. V., AND BRESSLER,R. (1978) J. Mol. Cell. Cardiol. 10, 449-459. CHIESI, M., Ho, M. M., INESI, G., SOMLYO, A. V., AND SOMLYO, A. P. (1981)J. CellBiol. 91,728-742. Dow, J. W., HARDING, N. G. L., AND POWELL~ T. (1981a) Cardiovasc. Res. 15, 483-514. Dow, J. W., HARDING, N. G. L., AND POWELL, T. (1981b) Cardiovasc. Res. 15, 549-579. FARMER, B. B., HARRIS, R. A., JOLLY, W. W., HATHAWAY, D. R., KATZBERG, A., WATANABE, A. M., WHITLOW, A. L., AND BESCH, H. R. (1977) Arch. Biochem. Biophys. 179, 545-558. FORBES, M. S,, RENNELS, M. L., AND NELSON, E. (1979) J. Ultrastruct. Res. 67, 325-339. FRANK, J. S., LANGER, G. A., NUDD, L. M., AND SERAYDARIAN, K. (1976) Fed. Proc. 35, 535P. FRANK, J. S., LANGER, G. A., NUDD, L. M., AND SERAYDARIAN, K. (1977) Circ. Res. 41, 702-714. FRY, C. H., POWELE, T., AND TWIST, V. W. (1981) J. Physiol. 315, 17-18P. FRY, D. M., SCALES,D., AND INESI, G, (1979)J. Mol. Cell. Cardiol. 11, 1151-1164. GABELLA, G. (1978) J. Ultrastruct. Res. 65, 135-147. GOULD, R. P., AND POWELL, T. (1972)J. Physiol. 225, 16-19P. HODGKtN, A. L., AND HUXLEY, A. F. (1952) J. Physiol. 117, 500-544. KAO, R. L., CHILISTMAN,E. W., LUH, S. L., KRAUHS, J. M., TYERS, G. F. O., AND WILLIAMS,E. H. (1980) Arch. Biochem. Biophys. 203, 587-599. KORECKY, B., AND RAKUSAN, K. (1978) Amer. J. Physiol. 234, H123-H128. LANGER, G. A., FRANK, J. S., NUDD, L. M., AND SERAYDARIAN, K. (1976) Science 193, 1013-1015. LEVIN, K. R., AND PAGE, E. (1980) Circ. Res. 46, 244-255. MATTER, A. (1973) J. Cell Biol. 56, 690-696. McNUTT, N. S., AND FAWCETT, D. W. (1974) in LANGER, G, A., AND BRADY, A. J. (Eds.), The Mammalian Myocardium, pp. 1-49, Wiley, N e w York. MOSES, R. L., AND KASTEN, F. H. (1979) J. Mol. Cell. Cardiol. 11, 161-172. MUIR, A. R. (1967) J. Anat. 101,239-261. NAG, A. C., FISCHMAN, D. A., AUMONT, M. C., AND ZAI% R. (1977) Tissue Cell 9, 419-438. NAG, A. C., AND ZAK, R. (1979) J. Anat. 129, 541559. NASH, G. B., TATHAM, P. E. R., POWELL, T., TWIST, V. W., SPELLER, R. D., AND LOVEROCR,L. T. (1979) Biochim. Biophys. Acta 587, 99-111. PAGE, E. (1978) Arner. J. Physiol. 235, C147-C158.
ISOLATED MYOCYTES PAGE, E., AND MCCALLISTER, L. P. (1973) J. Ultrastruct. Res. 43, 388-411. POWELL, T. (1979)J. Mol, Cell. Cardiol. 11, 511-513. POWELL, T., STEEN, E. M., TWIST, V. W., AND WOOLF, N. (1978a) J. Mol. Cell. Cardiol. 10, 287292. POWELL, T., TERRAR, D. A., ANt) TWIST, V. W. (1978b) J. Physiol. 284, 148P. POWELL, T., TERRAR, D. A., AND TWIST, V. W. (1978c) J. Physiol. 282, 23-24P. POWELE, T., TERRAR,D. A., AND TWIST, V. W. (1980) J. Physiol. 302, 131-153. POWELL, T., TERRAR,D. A., AND TWIST, V. W. (1981) J. Physiol. 319, 82-83P. POWELL, T., AND TWIST, V. W. (1975) J. Physiol. 247, 14-16P. POWELL, T., AND TWIST, V. W. (1976a) Biochem. Biophys. Res. Commun. 72, 327-333. POWEEL, T., AND TWIST, V. W. (1976b) Biochem. Biophys. Res. Commun. 72, 1218-1225. RIESER, G., SABBADINI,R., PAOLINI, P., FRY, D. M., AND INESI, G. (1979) Amer. J. Physiol. 236, C70.
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Roos, K. P., BRADY, A. J., AND TAN, S. T. (1982) Amer. J. Physiol. 242, H68-H78. SEGRETAIN, D., RAMBOURG, A., AND CLERMONTY, Y. (1981) Anat. Rec. 200, 139-151. SEVERS, N. J., AND POWELL, T. (1980) in BREDEROO,. P., AND DE PRIESTER, W. (Eds.), Electron Micros-copy, Vol. 2, pp. 134-135, Seventh European Congress on Electron Microscopy Foundation, Leiden. SIMPSON, F. O., RAYNS, D. G., AND LEDINGHAM, J. M. (1974)Advan, Cardiol. 12, 15-33. SOKAL, R. R., AND ROHLF, F. J. (1969) Biometry, Freeman, San Francisco. SOMMER, J. R., AND JOHNSON, E. A. (1979) in BERNE, R. M., SPEREEAKIS,N., AND GERGER, S. R. (Eds.), Handbook of Physiology, Section 2, The Cardiovascular System, Vol. 1., The Heart, 113-186. STEWART, J. M., AND PAGE, E. (1978) J. Ultrastruct. Res. 65, 119-134. WARREN, R. L. (1980) Analyst 105, 227-233. WEIDMANN, S. (1970) J. Physiol. 210, 1041-1054.
Correlation of Ultrastructure and Function in Calcium-Tolerant Myocytes Isolated from the Adult Rat Heart N. J. SEVERS,* A. M. SLADE,* T. POWELL,? V. W. TWIST,? AND R. L. WARm~N$ *Department of Cardiac Medicine, Cardiothoracic Institute, 2 Beaumont Street, London WIN 2DX, England, and ?Department of Physics as Applied to Medicine, and $Institute of Nuclear Medicine, The Middlesex Hospital Medical School, Cleveland Street, London W1P 6DB, England Received March 2, 1982, and in revised form June 17, 1982 Thin-section and freeze-fracture techniques were applied to investigate the ultrastructure of myocytes isolated from the adult rat heart by in vitro perfusion with collagenase. A central feature of this preparation, which distinguishes it from most others, is that the structural and functional integrity of the individual myocytes is maintained in the presence of physiologic concentrations of extracellular calcium. Ultrastructural examination demonstrates that all the normal features of sarcolemmal structure, including the glycocalyx, are preserved in these isolated cells, and no irreversible damage is sustained as a consequence of separation of the intercalated discs. The structure of intracellular membranes and organelles also shows excellent preservation, indistinguishable from that reported in cells of the intact rat heart. A detailed description of the ultrastructure of the isolated heart cells is presented, together with data on their response to extracellular calcium and on their respiratory function. Emphasis is placed on interpreting the ultrastructural findings in relation to the functional properties demonstrated in the individual myocytes.
The importance of individual myocytes isolated from the adult heart as a model system for investigation of the cellular mechanisms underlying cardiac function is now being recognized. The plethora of dissociation techniques currently available claiming "normal" ultrastructure for isolated myocytes which do not, in fact, function satisfactorily in the presence of physiological concentrations of extracellular calcium (Berry et al., 1970; Farmer et al., 1977; Nag et al., 1977; Carlson et al., 1978; Fry el al., 1979; Moses and Kasten, 1979; Chiesi et al., 1981) has, however, caused considerable confusion as to the general suitability of preparations of isolated cells for experimental studies. In the laboratories of two of the present authors, a decade of experience on the optimum methods for cell isolation has now been gained (Gould and Powell, 1972; Powell and Twist, 1976a; Powell, 1979; Powell et al., 1980) and in
this time extensive experimental results on the functional characteristics of these isolated myocytes have been accumulated (reviewed in Dow et al., 1981a,b). As yet, however, no detailed description of the ultrastructure of this particular preparation of isolated cells has been reported. The aim of the present study was to fill this gap, using both thin-section and freeze-fracture techniques, and, more importantly, to correlate the ultrastructural findings with the known functional responses of the individual myocytes. Our results indicate that the cells isolated from the ventricles of adult rats by in vitro perfusion with collagenase do possess both ultrastructural and functional characteristics which closely resemble those of myocytes in the intact heart, thus providing an isolated cell preparation that is a valid model for a wide range of investigations concerned with myocardial homeostasis.
222 0022-5320/82/110222-18502.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
223
ISOLATED MYOCYTES MATERIALS AND METHODS
Animals. Hearts were obtained from adult female Sprague-Dawley rats (bw 250-300 g) fed ad libitum on a standard chow diet. Cell preparation. Suspensions of purified cardiac myocytes were obtained by Langendorff perfusion of the hearts with crude collagenase in low calcium KrebsRinger bicarbonate buffer, as described in detail by Powell et al. (1980). The composition of the buffer was as follows: NaC1, 118.5 mM; NaHCO3, 14.5 raM; KC1, 2.6 mM; KH2PO4, 1.18 raM; MgSO4, 1.18 mM; glucose, 11.1 raM. Calcium was added as required from a stock solution of 1 M CaC12. The resulting cell suspension was preincubated at 37°C in gassed buffer containing 20 mg cm s bovine serum albumin for at least 30 min prior to any experimental procedures. This preincubation was carried out in either nominally calcium-free buffer or medium to which 0.5 mM calcium chloride had been added. All cell suspensions were gassed regularly with 95% OJ5% COs. Cells were fixed for electron microscopy or for counting by light microscopy by adding 1 vol cell suspension to 4 vol 2% (v/v) glutaraldehyde made up in V3 strength Krebs buffer. To determine cell number and purity, the fixed suspensions were centrifuged and washed, then counted in quadruplicate in an improved Neubauer hemocytometer. Calcium analysis. The levels of calcium in the solutions used for cell isolation and incubation were analyzed by emission flame spectrometry. We used a method by which each sample was evaporated from a tungsten filament into the fuel supply of an oxyhydrogen flame and the resulting light emission integrated by a photomultiplier-monochromator monitoring the appropriate spectrum line (422.67 nm with 0.11-nm band width) in the flame (Warren, 1980). By this technique, a solution containing 1 pM calcium could be measured with a standard deviation of _+5.4% and the limit of detection (calculated at three times the standard deviation of the background noise)was 0.02 vM, for sample volumes in the range 0.2--4 txl. Cellular respiration. Oxygen consumption of isolated cell preparations was measured by standard techniques using an oxygen electrode (Powell and Twist, 1975, 1976a). Stimulation of oxygen uptake by 10/xg cm -~ of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a measure of cellular respiratory control index (RCI). Results are given as mean _+ standard error of the mean, with the number of observations following in parentheses. Statistical analysis was carried out by a two-way analysis of variance when the data were appropriately symmetric (Sokal and Rohlf, 1969); otherwise group means were compared using Student's t test. Electron microscopy. The cell suspensions were fixed for 2 hr in the 2% glutaraldehyde solution at 20°C. For thin-section electron microscopy, samples were rinsed briefly in buffer and postfixed in 1% osmium tetroxide
buffered with 0.1 M sodium cacodylate (pH 7.3) for 2: hr at 4°C. After en bloc staining with saturated aqueous uranyl acetate for 1 hr at room temperature, the spec-imens were dehydrated in ethanol and embedded in Araldite via propylene oxide. Silver sections were prepared with a diamond knife using an LKB III ultramicrotome. For freeze-fracture, glutaraldehyde-fixed specimens were gradually infiltrated with glycerol (in Krebs-Ringer or cacodylate buffer) to a final concentration of 25 or 30% glycerol over a period of 45 rain. Droplets of concentrated cell suspension were mounted on Balzers specimen holders and rapidly frozen by plunging into liquid propane held at -180°C. Freeze-fracturing was carried out in a Balzers BAF 400T unit at specimen temperatures of - 100 to - 115°C and at a vacuum of 5 x 10-r mbar or better. Some specimens were etched briefly before replication. Replicas were cleaned in 40% chromic acid and rinsed twice in distilled water before mounting on uncoated grids. Thin sections and replicas were examined and images recorded using a Philips EM301 electron microscope. RESULTS
Calcium Analysis o f E x p e r i m e n t a l Solutions When Krebs solution, with calcium chlor i d e o m i t t e d , w a s m a d e u p in d o u b l e g l a s s d i s t i l l e d w a t e r ( c a l c i u m c o n c e n t r a t i o n <0.0,8 /xM), t h e t r a c e a m o u n t s o f c a l c i u m in t h e salts u s e d ( A n a l a r g r a d e ) r e s u l t e d in a final c a l c i u m c o n c e n t r a t i o n ([Ca]0) o f 8.8 _+ 0.5 /xM (n = 9). I n all i n c u b a t i o n s o l u t i o n s , b o v i n e s e r u m a l b u m i n w h e n a d d e d a t 2;0 mg/cm 3 increased Krebs solution calcium to 26.0 +_ 0.6 /xM (n = 9). B y c o n t r a s t , a t the end of the Langendorff perfusion during c e l l i s o l a t i o n , t h e p e r f u s i o n fluid c o n t a i n e d 35.5 + 0.8 /xM c a l c i u m (n = 61). T h u s Jin this p a p e r w h e n i n c u b a t i o n s o l u t i o n s a r e ref e r r e d t o as " n o m i n a l l y " o r " e s s e n t i a l l y " calcium-free, the calcium concentration of s u c h s o l u t i o n s is - 2 6 / a M .
Gross Morphology o f Isolated M y o c y t e s Examination of suspensions of isolated myocytes by light microscopy showed two distinct cell appearances; a population of rounded (damaged) cells, and a more num e r o u s r o d - s h a p e d f o r m ( F i g . 1). H i g h magnification inspection by light microsc o p y (Fig. 2) a n d l o w - p o w e r e l e c t r o n m i -
224
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croscopy (Fig. 3) revealed that the rodshaped cells displayed all the gross morphological features characteristic of cardiac muscle cells in the intact myocardium. U1trastructural examination at higher resolution by thin-section and freeze-fracture electron microscopy (Figs. 6-19 and 21-23, discussed in detail later) confirmed that these m y o c y t e s were indeed well preserved, closely resembling their counterparts in the intact heart.
PREINCUBATION [Ca++]o 80
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Extracellular Calcium and Cell Survival
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CALCIUM ADDEDTO INCUBATION MEDIUM(mM)
FIG. 4. Percentage viability of isolated myocytes Figure 4 shows the results of experiments incubated at 37°C in solutions containing differing in which, following preincubation in nomi- [Ca~+]0. nally calcium-free or 0.5 mM [Ca]0 media, 1-cm z aliquots of the isolated cell suspension were incubated at 37°C for 7 min in the effect was not seen in suspensions preinpresence of added calcium to give a final cubated with 0.5 mM [Ca]0. These results [Ca]0 in the range 0.5-10 raM. At the end indicate that the isolated cells are calcium of the incubation period the aliquots were tolerant when presented with an acute calLfixed and the numbers of rod-shaped and cium load. rounded cells counted. Suspensions exThe presence of 0.5 mM [Ca]0 also in,tposed only to calcium-free media contained proved longer term cell survival. In essen71.2 _ 1.4% (n = 16) rod-shaped cells, tially calcium-free Krebs-Ringer the prowhich was significantly higher (P < 0.05) portion of intact rod-shaped cells after 60 than for suspensions incubated in 0.5 min incubation at 37°C was 67.3 _+ 0.72%, mM [Ca]0 after calcium-free preincubation which decreased significantly (P < 0.00l) (67.3 _ 1.3%, n = 16), or for those incu- over the next 330 min to 28.7 _+ 17.8% (Fig. bated throughout in 0.5 mM [Ca]0 media 5). In contrast, although there was also a (67.3 _+ 1.4%, n = 16). Irrespective of significant decrease over the same time inpreincubation [Ca]0, there were no statisti- terval from 65.7 ___ 1.2% to 51.0 _ 6.7% for cal differences in the percentage of rod- cells incubated in solutions to which 0.5 n ~ shaped cells incubated with calcium in the CaC12 had been added, this decrease was range 0.5-5.0 raM. When the final incuba- less than that observed in the nominally caltion contained 10 mM [Ca]0, however, cells cium-free incubations. Under both incubapreincubated in calcium-free buffer showed tion conditions, RCI values were maindecreased percentage viability at 57.8 _+ tained for the first 5 hr of incubation at levels 1.6% (n = 16), which was significantly low- comparable to those to be shown later (Fig. er (P < 0.01) than at 5 mM calcium. This 20).
FIG. 1, Light microscope (bright field) survey view of an isolated myocyte preparation. Intact rod-shaped cells are easily distinguished from damaged cells that appear rounded and dark. x 100. FIG. 2, High-power light microscope view showing the regular structural details characteristic of intact rodshaped cells, x 800. FIG. 3. Survey electron micrograph of a longitudinal thin-section through an isolated rod-shaped myocyte. Myofilament bundles with their characteristic banded structure alternate between rows of mitochondria, as in cells of the intact heart. In this example, 64 sarcomeres can be counted along the length of the cell. x 1500.
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w i t h e x p o s u r e to c a l c i u m at 0.5 raM. E x p e r i m e n t a l l y , p r e p a r a t i o n s like t h o s e u s e d for the r e s u l t s d e p i c t e d in Fig. 5 a r e s t o r e d as " s t o c k s u s p e n s i o n s , " f r o m w h i c h samples m a y b e s e d i m e n t e d ( u n d e r g r a v i t y for 8 rain) at t i m e s f o r up to 6 h r a f t e r p r e p a r a t i o n to g i v e a p u r i t y o f - 7 0 % o v e r this period.
(b)
FIG. 5. Long-term percentage viability for isolated myocytes incubated at 37°C in either a nominally calcium-free solution (O) or Krebs-Ringer to which 0.5 mM calcium chloride had been added (0). Numbers in parentheses indicate the number of experimental observations. *, P < 0.05; ***, P < 0.001,
A n o t h e r i n t e r e s t i n g f e a t u r e o f Fig. 5 is t h a t a l t h o u g h f r o m l to 5 hr a f t e r t h e 30rain p r e i n c u b a t i o n t h e r e is n o d i f f e r e n c e in t h e p e r c e n t a g e o f r o d - s h a p e d cells f o r t h e t w o c a l c i u m c o n c e n t r a t i o n s , t h e initial p e r c e n t a g e s a r e h i g h l y significantly d i f f e r e n t ( P < 0.001). In f a c t , t h e initial p e r c e n t a g e o f 62.5 _+ 1.2% (n = 16) a f t e r 30-rain incub a t i o n in 0.5 m M c a l c i u m s o l u t i o n is n o t significantly d i f f e r e n t f r o m the 60-rain v a l u e (65.7 _+ 1.2%), so t h a t o v e r this s e r i e s o f e x p e r i m e n t s t h e cells a r e " c a l c i u m intole r a n t " to t h e e x t e n t t h a t t h e r e is a m a x i m u m initial d e c r e a s e in p u r i t y o f a b o u t 5%
S o m e i n s i g h t into the s t r u c t u r a l b a s i s und e r l y i n g t h e s u r v i v a l o f i n t a c t cells a n d maintenance of their normal functional c h a r a c t e r i s t i c s is g a i n e d b y u l t r a s t r u c t u r a l e x a m i n a t i o n o f the s a r c o l e m m a . In transv e r s e thin s e c t i o n , the s a r c o l e m m a o f isol a t e d m y o c y t e s d i s p l a y s an i n t a c t t r i l a m i n a r u n i t - m e m b r a n e s t r u c t u r e , c o a t e d on its extracellular surface with a fuzzy glycocalyx (Fig. 6). F r e e z e - f r a c t u r e e l e c t r o n m i c r o s c o p y d i s c l o s e s i r r e g u l a r l y d i s t r i b u t e d intram e m b r a n e p a r t i c l e s w i t h a l o w e r d e n s i t y on t h e E f a c e t h a n on t h e P f a c e ( F i g s . 7 a n d 8). N e i t h e r p a r t i c l e a g g r e g a t i o n n o r s m o o t h m e m b r a n e r e g i o n s d e v o i d o f p a r t i c l e s (features indicative of membrane damage) are e v i d e n t . A l l t h e n o r m a l s a r c o l e m m a l spec i a l i z a t i o n s c h a r a c t e r i s t i c o f cells o f t h e int a c t h e a r t a r e f o u n d in t h e s e i s o l a t e d m y ocytes. Transverse tubules, bearing a g l y c o c a l y x c o n t i n u o u s w i t h t h a t o f the p e r i p h e r a l s a r c o l e m m a , p e n e t r a t e into t h e cell at t h e l e v e l o f the Z b a n d (Fig. 6). T h e tub u l e o p e n i n g s at t h e cell s u r f a c e a r e freq u e n t l y r e v e a l e d in p l a n a r f r e e z e - f r a c t u r e
Fins. 6-12. Ultrastructure of the general sarcolemma. Fio. 6. Specializations of the general sarcolemma as seen in thin section. The external surface of the plasma membrane is coated with a fuzzy glycocalyx (g). The long invagination at T marks the entrance to a transverse tubule, penetrating at the Z band (Z). c, caveolae; p, bristle-c0ated pit. x 80 000. FIG. 7. Freeze-fractured sarcolemma, E-face view. Relatively few intramembrane particles are present. × lO0 000. FIG. 8. Freeze-fractured sarcolemma, P-face view. Intramembrane particles are abundant compared with the previous figure and no bare patches of membrane are visible. The circular discontinuities represent the cross-fractured necks of caveolae, x 100 000. FIG. 9. Survey freeze-fracture view of the sarcolemma (P face) illustrating the characteristic regular arrangment of transverse tubule openings. This arrangement does not extend over the entire surface of the cell but is localized in occurrence. Deep invaginations of uninterrupted membrane are sometimes found at positions in which a transverse tubule opening would be predicted (example indicated with asterisk), x 25 000.
ISOLATED MYOCYTES
227
228
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FIG. 10. Survey thin-section view illustrating the variety of form exhibited by caveolae. The section plane is somewhat tangential to the cell surface in this example. C1 shows a triple type in which three caveolae unite to form a common neck; C~ is a long beaded chain of caveolae which extends deeply into the cytoplasm. × 36 000. FIG. 11. Caveolae sectioned transversly through the cell surface. Single forms of eaveolae are seen opening to the exterior in favorable section planes (eL C1 and C~). The complexity of multiple caveolae (C~) is only hinted at in thin section. CV, coated vesicle. × 70 000. FIG. 12. Freeze-fracture view of a chain of multiple caveolae fused end-on to one another. Branches are formed where additional chains are attached. S, sarcolemma (E-face view), x 80 000.
v i e w as o r d e r e d a r r a y s (Fig. 9), as previo u s l y r e p o r t e d b y s c a n n i n g e l e c t r o n mic r o s c o p y ( P o w e l l et al., 1978a). C a v e o l a e are s e e n as c i r c u l a r d i s c o n t i n u i t i e s (62 + 19-nm d i a m e t e r ) in e n f a c e v i e w s of the sarc o l e m m a , s c a t t e r e d at all levels of the sarc o m e r e s u r f a c e at a d e n s i t y of 6.0 -+ 1.8 /xm -2 (Figs. 8 a n d 9). T h e s e i n v a g i n a t i o n s d i s p l a y a striking v a r i e t y of f o r m a n d their
membrane characteristically reveals a d e a r t h o f P-face i n t r a m e m b r a n e particles c o m p a r e d with the s u r r o u n d i n g s a r c o l e m ma. T h e s i m p l e s t form of c a v e o l a c o n s i s t s of a single f l a s k - s h a p e d s a r c o l e m m a l inp o c k e t i n g (Figs. 6, 10, a n d ! 1) with a d e p t h a n d w i d t h r a n g i n g f r o m 40 to 140 n m (meas u r e d in t r a n s v e r s e t h i n s e c t i o n ) . M a n y c a v e o l a e are more c o m p l e x s t r u c t u r e s ,
ISOLATED MYOCYTES h o w e v e r ; two, t h r e e , or m o r e c a v e o l a r vesicles are f r e q u e n t l y f o u n d s h a r i n g a comm o n n e c k (Figs. 10 a n d 11), a n d m o r e exc e p t i o n a l l y , e x t e n d e d b r a n c h e d c h a i n s of f u s e d c a v e o l a e are s e e n (Figs. 10 a n d 12), similar to those r e p o r t e d in c o r o n a r y s m o o t h m u s c l e cells ( F o r b e s et al., 1979). O c c a sional b r i s t l e - c o a t e d pits a n d v e s i c l e s , dist i n c t f r o m the u s u a l f o r m s of c a v e o l a e , are also o b s e r v e d (Figs. 6 a n d 11). B e c a u s e i n the i n t a c t tissue c a r d i a c m u s cle cells are p h y s i c a l l y a n d m e t a b o l i c a l l y c o n n e c t e d to o n e a n o t h e r b y i n t e r c e l l u l a r j u n c t i o n s d i s p o s e d at f r e q u e n t sites a l o n g the i n t e r c a l a t e d disc, this r e g i o n of the sarc o l e m m a is p o t e n t i a l l y v u l n e r a b l e to d a m age d u r i n g cell s e p a r a t i o n . T h i n - s e c t i o n a n d freeze-fracture e l e c t r o n m i c r o s c o p y d e m o n strate that after cell i s o l a t i o n , the highly
229
c o n v o l u t e d g e o m e t r y of the i n t e r c a l a t e d disc m e m b r a n e is, h o w e v e r , r e t a i n e d with-. o u t n o t i c e a b l e d i s r u p t i o n (Figs. 13 a n d 14). O c c a s i o n a l l y , sites of p o s s i b l e focal sarcol e m m a l d a m a g e are d e t e c t e d , b u t the state,' of p r e s e r v a t i o n of a d j a c e n t c y t o p l a s m i c : c o m p o n e n t s suggests t h a t in m o s t i n s t a n c e s t h e s e sites r e p r e s e n t debris o r i g i n a t i n g f r o m a d a m a g e d n e i g h b o r i n g cell. D e s m o s o m e s a n d fascia a d h e r e n t e s j u n c t i o n s are c l e a v e d a p a r t b y the e n z y m a t i c t r e a t m e n t , l e a v i n g h a l f - j u n c t i o n s at e a c h i n t e r c a l a t e d disc surface. B y c o n t r a s t , the gap j u n c t i o n c o m p o n e n t s of b o t h m e m b r a n e s are o f t e n ret a i n e d with j u s t o n e of the cells, r e v e a l i n g a n a t t a c h e d l o o p of m e m b r a n e , p r e s u m e d to h a v e b e e n t o r n f r o m a n a d j a c e n t cell dur'ing s e p a r a t i o n (Fig. 13). G a p j u n c t i o n m e m b r a n e is also o f t e n s e e n in the f o r m of cy-
FIGS. 13-15. Ultrastructure of the intercalated-disc sarcolemma. Fro. 13. Survey thin-section view of the intercalated disc region of the sarcolemma. The plasma membrane appears irregular but intact and adjacent cytoplasmic components are well preserved. Electron-dense zones on the cytoplasmic side of the membrane are the remnants of fascia adherentes and desmosomes retained with the cell. Other features of interest are indicated by 1, 2, and 3:1 probably represents a highly convoluted region of the membrane with adherent debris originating from an adjacent cell before isolation; 2 is the site of a gap junction with an attached loop of membrane; and 3 is an apparent vesicle of gap junction membrane (for an example at higher magnification, see Fig. 15). x 9000. FIG. 14. Freeze-fracture view of a portion of intercalated disc sarcolemma. The plasma membrane is intact and undamaged. Its highly convoluted nature is indicated by the portions of membrane observed apparently within the cytoplasm (asterisks) but which are in fact continuous with the surface sarcolemma, x 35 000. FIG. 15. Thin-section view of a "gap junction vesicle." Although this structure appears to be a discrete vesicle in the cytoplasm, serial sectioning showed that in this example at least, the "vesicle" membrane was in fact continuous with the surface sarcolemma, x 45 000. Fins. 16-19, Sarcoplasmic reticulum and mitochondrial ultrastructure. Fin. 16. The structural complexity of the sarcoplasmic reticulum (SR) is illustrated in this thin-section field. A network of tubules in the M region, the M rete (MR), is connected by longitudinal elements (L) to a z tubule (ZTI) seen here in longitudinal section. Examples of transversely sectioned z tubules, seen elsewhere, are indicated by ZTT. Junctional sarcoplasmic reticulum (JSR) is continuous with the free SR (arrow) and consists of flattened cisternae closely apposed to the sarcolemma. Peripheral junctional SR (JSRp) occurs in association with the surface sarcolemma, and interiorjunctional SR (JSR0 occurs against transverse-tubule membrane. The latter may take the form of dyads (D), which consist of a transverse tubule plus one JSR cisterna, or triads (T), which consist of a transverse tubule sandwiched between two JSR cisternae. The JSR lumen is characteristicaJtly bisected by an electron-dense line, and regularly spaced electron-dense feet project from the membrane facing the sarcolemma. Mitochondria (M) show well-preserved membranes and cristae. ML, M line; ZL, Z line; g, glycocalyx, x 50 000. FIG. 17. Freeze-fracture view of interior junctional SR/transverse-tubule association, with mitochondrial membranes above and below the complex. The portion of SR connected to the junctional SR has a cisternal rather than a tubular configuration (c). × 60 000. FIG. 18. Structural details of a transversely sectioned triad. T, transverse tubule; JSR, junctional SR; ZL, z line. × 100 000. Fin. 19. Length of tubular sarcoplasmic reticulum (SR) showing close association with mitochondria (M) as displayed by freeze-fracture. × 50 000.
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toplasmic vesicles (Fig. 15), though we have established by serial sectioning that the membranes of at least some of these apparently discrete vesicles are in fact continuous with the surface sarcolemma.
The Sarcoplasmic Reticulurn The intracellular membrane systems of the c e l l s - - b y virtue of their location--are less susceptible to damage during the isolation process, and their well-preserved ultrastructural appearance (Figs. 16-19, 21, and 22) is interpreted as a reflection of retention of their normal functional capacities. The junctional and free components of the sarcoplasmic reticulum (SR) are readily identified in isolated myocytes. As in the ceils of the intact myocardium, the free SR consists of a network (fete) in the M region of the sarcomere, linked by longitudinal tubules to the Z elements which may take the form of Z fetes and/or Z tubules (Fig. 16). Junctional SR is comprised of cisternae intercalated into the free SR network which form both peripheral couplings (against the peripheral sarcolemma) or interior couplings (against transverse tubules) in the Z region of the sarcomere (Figs. 16-18). In either situation, the junctional SR is closely apposed to the sarcolemma, leaving a gap of approximately 13 nm bridged by regularly spaced electron-dense structures. The lumen of transversely sectioned junctional SR is characteristically bisected by a line of electron-dense material. Interior couplings are seen both as dyads (Figs. 16 and 17) and as triads (Figs. 16 and 18). Freeze-fracture shows that the free SR occurs mainly in the form of tubules, 20-40 nm in diameter (Fig. 19), though these periodically broaden out to give a cisternal configuration (Fig. 17).
Mitochondrial Ultrastructure and Respiratory Control Figures 16-19 also demonstrate that the structure of mitochondrial inner and outer membranes, their cristae, and matrix remain well preserved as judged by thin-section and freeze-fracture. This is consistent
PREINCUBATION
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Ca++'FREE
r .o.+ 20
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0 0+5 1.0 2.5 5.0 CALCIUM ADDED TO INCUBATION MEDIUM(rnM)
FIG. 20. Respiratory control indices (RCI) for cells exposed to differing [Ca]0 at 37°C. RCI values were estimated from stimulation of oxygen consumption after the addition of 10/zg/cm~ CCCP. Equivalent solvent volumes were run as blanks and calibration was achieved by conventional methods.
with the m a i n t e n a n c e of m i t o c h o n d r i a l function as reflected in the experimental measurements of RCI summarized in Fig. 20. Cells in media containing only trace amounts of calcium had an RCI of 16.7 + 2.4 (n = 6), which was not significantly different from that observed in cells exposed to 0.5 mM calcium (23.2 _+ 3.5, n = 6). Although myocytes preincubated in calciumfree media showed no change in RCI when [Ca]0 was increased in the range 1.0-5.0 raM, cells preincubated in 0.5 mM [Ca]0 showed a higher RCI (F = 24, P < 0.001), reaching a value of 35.2 _+ 1.9 (n = 4) at 2.5 mM [Ca]0. Thus mitochondrial function is maintained even when the cells are faced with high calcium concentrations. These experiments were carried out for up to 6 hr after cell preparation, with results similar to those in Fig. 20 obtained over this period.
The Nucleus The ultrastructural appearance of the nucleus is also a good indicator of cellular functional capacity, and isolated myocytes show no abnormal nuclear features. Most of the nuclear matrix contains diffuse chromatin, with a peripheral layer of hetero-
ISOLATED MYOCYTES
chromatin around the inner nuclear membrane and nucleolus (Fig. 21). The densities and distributions of nuclear pore complexes and intramembrane particles as seen in freeze-fractured nuclear membranes from isolated myocytes (Fig. 22) do not differ from those of cells from the intact heart.
2313 DISCUSSION
The ultrastructural features reported here for isolated myocytes in suspension compare very favorably with those reported previously for adult myocytes in intact myocardial tissue (McNutt and Fawcett, 1974.; Simpson et al., 1974; Sommer and JohnThe Contractile A p p a r a t u s son, 1979; Segretain et al., 1981). Although Myofibril structure appeared well pre- the morphology of isolated myocytes has served in thin-sectioned isolated myocytes, been documented in numerous previous inshowing the characteristic organization fa- vestigations (Berry et al., 1970; Nag et al., miliar from cells in the intact myocardium 1977; Carlson et al., 1978; Fry et al., 197q; (Fig. 23). The mean sarcomere length (+_SD) Moses and Kasten, 1979; Nag and Zak, measured as the distance between adjacent 1979; Chiesi et al., 1981), the cells in such Z bands in 67 cells cut in precise longitu- preparations showed pronounced "calcium dinal section was 1.64 +_ 0.21 t~m. Some intolerance," i.e., they went into marked variation in the state of relaxation and con- contracture in solutions containing microtraction was noted between individual cells, molar amounts of extracellular calcium. It and this was reflected in the clarity with has always been emphasized that cells prewhich the I and H bands could be dis- pared by our method do not exhibit this decerned. The cell in Fig. 23 is in a relatively ficiency and are stable in media containing relaxed condition; I and H bands are prom- physiological levels of calcium (Gould and inent, and the sarcomere length is 1.83 ~m. Powell, 1972; Powell and Twist, 1976a; This value is close to that recorded as an Powell et al., 1980). Recently, Kao et al. average for fresh or fixed cells not prepared (1980) have described the morphology of for electron microscopy (Nash et al., 1979; cells isolated from adult rat ventricle which Roos et al., 1982) but is toward the higher were also calcium tolerant, but the signifiend of the range measured in the present cance of the present paper is that we can study. Compared with the thin-section correlate the cellular ultrastructure reportmeasurements, higher values for sarcomere ed here with the functional characteristics length are also suggested from the spacing extensively investigated using the same of transverse tubules viewed in freeze-frac- preparation. ture (Severs and Powell, 1980). Because The thin-section and freeze-fracture evitransverse tubules do not always penetrate dence indicating an intact and structural]Ly exactly at the Z band level, this cannot be unaltered sarcolemma with associated glyconsidered as precise a method as direct cocalyx, is consistent with the experimenmeasurement between the bands. (Sarco- tal finding that individual myocytes tolerate lemmal indentations overlying the Z bands, impalement with one or two glass microas reported in myocytes from intact rabbit electrodes, even when the concentration of ventricle (Levin and Page, 1980) are not extracellular calcium is 5-10 raM, and have present in our isolated rat myocytes and so negative resting membrane potentials charcould not be used for measurements of sar- acteristic of those found in whole tissue comere length in freeze-fracture replicas.) (Powell et al., 1978b,c, 1980). FurtherOur results thus suggest that processing for more, these myocytes can generate conthin-sectioning (particularly dehydration) is ventional action potentials, indicating that responsible for some shrinkage of the con- active conductance mechanisms have been tractile apparatus, which is reflected in a preserved in the sarcolemma. This contendecrease of -10% in sarcomere length. tion has been confirmed by direct experi-
234
SEVERS ET AL.
ISOLATED MYOCYTES
235
FIG. 23. Typicalview of the contractile apparatus in an isolated myocyte. The presence of clearly defined I bands (I) indicates a relatively relaxed condition. Z, Z line; M, M line; A, A band. x 30 000.
mentation, which has shown that the cells have functional N a channels (Brown et al., 1980, 1981a,b) responsible for the rapid upstroke of the action potential, and also " s l o w channels" for conducting the Ca/Na current important in maintaining the plateau phase (Powell et al., 1981). In addition it has been demonstrated that functional sarcolemmal receptors are retained, as isoprenaline, noradrenaline or adrenaline pro-
duce reversible effects not only on the electrical activity of the cells (Powell et al., 1978b, 1981), but also on their production of Y,5'-cyclic adenosine monopho'sphate (Powell and Twist, 1976b). Our demonstration of intact, well-preserved interior and peripheral junctional SR, free SR, and mitochondria is consistent with m u l t i c o m p a r t m e n t efflux curves for calcium (Baker et al., 1981), together with tile
FIG. 21. Thin-section view of part of a nucleus and adjacent cytoplasm. No abnormal chromatin clumping or clearing of the nucleoplasm is evident. HC, heterochromatin; DC, diffuse chromatin; Nu, nucleolus; Np, nuclear pore; ZL, z line. x 50 000. Fro. 22. Freeze-fractured nucleus showing the P face of the inner nuclear membrane (INM) and the E face of the outer nuclear membrane (ONM). No abnormality in the density and distribution of nuclear pores or intramembrane particles is seen. x 35 000.
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SEVERS ET AL.
observations that calcium uptake is dependent upon both the electric field across the sarcolemma and also intracellular stores (Fry et al., 1981). In contrast, potassium distribution within the cell is described largely by only one compartment (Powell and Twist, unpublished observations). That normal mitochondrial structure and function is preserved is strongly suggested by both the ultrastructural findings and the maintenance of good cellular respiratory control even in the face of a severe calcium challenge (which again indicates low sarcolemmal permeability to this ion). The quantitative experimental measurements on electrical characteristics, ionic c o m p a r t m e n t a t i o n , sarcolemmal responses, and respiratory activity have been obtained for up to 8 hr after cell isolation, even though myocytes were incubated during this period at 37°C in a simple oxygenated Krebs-Ringer bicarbonate buffer containing only glucose as substrate, and added bovine serum albumin. This raises the important question of how cells isolated in low calcium solutions and exposed to a dissociating enzyme (collagenase) can retain both the ultrastructural characteristics reported here (even in the presence of physiological extracellular calcium) together with functional responses typical of whole, intact myocardium. Such behavior contrasts markedly with the many reports in the literature describing enzymically dissociated myocytes that undergo irreversible contracture when exposed to micromolar amounts of calcium and which are, moreover, electrically inert in even nominally calcium-free solution (e.g., Rieser et al., 1979). After a comprehensive examination of the various isolation techniques employed (Dow et al., 1981a,b) we consider that a pivotal reason for the success of the method used here is that the isolation and incubation solutions always contain calcium, which at the end of the perfusion with enzyme is 30-40 p3/, and even in KrebsRinger solution with albumin is 20-30 p3/ (see results). This derives from endogenous
calcium, calcium present in and added with the collagenase, calcium present in albumin and salts, and calcium leached from the tissue during perfusion. Levels of calcium in this concentration range may well saturate high-affinity sarcolemmal binding sites and promote membrane integrity during both the isolation procedures and also in the incubations that are nominally calcium-free (Frank et al., 1976, 1977; Langer et al., 1976). Isolation of individual myocytes must inevitably involve disruption of the normal intercellular relationships existing in the multicellular tissue from which they are derived. The specializations of the intercalated disc sarcolemma responsible for intercellular adhesion (i.e., adherentes junctions), and transmission of the action potential and intercellular communication (i.e., gap junctions) are discrete structural units comprised of components associated with and built into two adjacent sarcolemmae. How these junctions become physically separated to allow production of viable cells, and the effects, if any, of such unavoidable structural alterations, are of central importance to the understanding and assessment of isolated myocyte function. Although adherentes junctions seem to be separated quite easily along an extracellular plane between the two membranes, freezefractured gap junctions in isolated cells often reveal the presence of components from both sarcolemmae (i.e., P-face and E-face views are seen in the same junction; Severs and Powell, 1980). This might suggest that each intact rod-shaped myocyte retains entire gap junction assemblies at the expense of permanent damage to its former neighbors. This interpretation, however, cannot easily be reconciled with the large numbers of gap junctions found at each intercalated disc (Matter, 1973) nor with yields of over 60% intact cells obtained in our best preparations immediately after isolation and before purification. We suggest therefore as a working hypothesis, that although some gap junctions are torn in toto with one cell from
ISOLATED MYOCYTES
its neighbor, separation may also occur by cleavage through the junctional plane, and minor membrane damage sustained during either process can be rapidly repaired (cf. Muir, 1967; Fry et al., 1979). A substantial number of cells nevertheless suffers extensive damage leading to irreversible injury and rounding up. Precise details of the repair mechanisms operating in the survivors and the eventual fate of their gap junctions require further investigation. High values for cell input resistances and resting membrane potentials (Powell et al., 1980) strongly suggest that those gap junctions detected in isolated cells have adopted an "open-circuit" configuration. The evidence presented here suggesting that the isolated rat myocytes have plasma membranes of normal ultrastructure in which caveolae are a prominent feature, has important implications in the calculation of specific membrane electrical characteristics. It has been demonstrated that the caveolae of these myocytes show a similar numerical density and distribution and have similar dimensions to those in the intact rat myocardium (Severs and Powell, 1980; Gabella, 1978). Should these membrane inpocketings prove to have electrical properties similar to those of the external surface sarcolemma and T-tubular membranes, then previous estimates used for myocyte surface area in the calculation of electrical characteristics (Powell et al., 1980) require revision. Following the nomenclature used by Stewart and Page (1978), let SEs and STT be the surface area (tzm 2) of the external sarcolemma (taking into account the periodic folded nature of the surface) and the T-tubular membranes, respectively. If VCE~.L (~m 3) is cell volume, then the data given in Table II of Stewart and Page (1978) can be used to calculate values (mean +__ SD, n = 4) of 0.31 _+ 0.01 ~m2//zm ~ for (SEs/ VCELL) and 0.16 _+ 0.03 /zm2//~m 3 for (STT/VcELt), derived from morphometric analysis of left ventricles of female Sprague-Dawley rats, corresponding in breed and weight (250-300 g) to the animals
237
used in the present experiments. The mean values of these ratios give an estimate of' 0.51 for (STa4SEs). Gabella (1978) has estimated that in male albino rats (250-300 g bw) the ratio (Sc/SEs) is about 0.27, where S c is total caveolar surface area (tzm2). From these figures, SEs + STT + Sc = 1.78SEs
(1)
Powell et al., (1980) noted that many my-ocytes could be described approximately by a rectangular block 100 ~zm × 20 /~m × 6 t~m, giving an estimate of 6000/xm 2 for SEs (which must be a conservative value, because no surface folding has been considered). Substituting for SEs on the right hand side of Eq. (1) yields a calculated total cell surface area of about 10 700 t~mz, which is 33% larger than the 8000/xm z used by Powell et al. (1980), taken from Page and McCallister (1973) for a 100-tzm-long rat ventricular cell (allowing for external surface sarcolemma, T system, and transverse boundary). Another estimate of total surface area can be obtained by assuming a mean myocyte volume of -25 000/zm 3 (Korecky and Rakusan, 1978; Page, 1978; Bishop and Drummond, 1979; Sommer and Johnson, 1979; Dow et al., 1981b) which implies that Sr.s is 7750/xm 2 if (SEs/Vc~Lt) is 0.31 /~m~//xm3. From Eq. (1), total surface area would now become almost 13 800/xm 2, i.e., over 72% larger than the quoted estimate of 8000/zm 2. A gross underestimation of myocyte surface area will result in calculated values of specific membrane capacitance (Cm, #.F cm -2) and peak sodium conductance (g~a, mS cm -2) which are too large, and values of specific membrane resistance (Rm, kl) cm 2) which are too small. Thus, values for Rm and Cm of 3.2 kl) cm 2 and 2.5 ~F cm -~ (Powell et al., 1980) and 25 mS cm -z for gNa (Brown et al., 1981) based on an average surface area of 8000/xm 2, should be revised to either 4.3 kf~ cm 2, 1.9 /xF cm -z, and 19 mS cm -z, respectively, for the new lower estimate of 10 700 /zm2, or to 5.5 k12 crn 2, 1.4 /zF cm -2, and 14 mS cm -2 if the upper
238
SEVERS ET AL.
estimate of 13 800 ~m ~ is used. The new estimates of Rm are nearer the value of 9.1 _+- 1.0 kl) cm 2 reported by Weidmann (1970) for trabecular muscle, and the values for Cm are compatible with specific capacitances of the order of 1 /~F cm -2, to be expected from the general organization and composition of plasma membranes of excitable tissues (Hodgkin and Huxley, 1952). As previously discussed by Levin and Page (1980), however, calculations of this type should be regarded with some caution. Whereas caveolar membrane area contributes to specific membrane capacitance, it may not necessarily do so to ionic conductance in view of the paucity of intramembraHe particles present. Furthermore, the surface area estimates will be markedly affected by the number of caveolae per neck, for which precise data are not yet available. The presence of multiple branched caveolae (Figs. 10 and 12), for example, could well lead to underestimated surface area values. In addition, account should be taken of the possibility that the frequency of caveolae in transverse tubule membrane could differ from that in the general plasma membrane. In conclusion, the results presented and discussed here further emphasize the similarity of ultrastructure and function between this preparation of isolated myocytes and their counterparts in the intact myocardium. It may be confidently predicted that use of this model system will continue to advance our understanding of myocardial function at the cellular level. This work was carried out with the aid of British Heart Foundation Grants 779 and 81/44 to N. J. S. and
80/25 and 81/82 to T. P.
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