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Morphological analysis of contracting and quiescent adult rabbit cardiac myocytes in long-term culture.

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THE ANATOMICAL RECORD 227:285-299 (1990)
Morphological Analysis of Contracting and
Quiescent Adult Rabbit Cardiac Myocytes in
Long-Term CuIture
MARLENE L. DECKER, DAVID G. SIMPSON, MONICA BEHNKE,
MELISSA G . COOK, AND ROBERT S. DECKER
Departments of Medicine (Cardiology) (M.L.D., M.B., M.G.C., R.S.D.), Cell Biology and
Anatomy (D.G.S., R.S.D.) and Feinberg Cardiovascular Research Institute (R.S.D.),
Northwestern University Medical School, Chicago, Illinois
ABSTRACT
Isolated rabbit ventricular cardiac myocytes adapt readily to primary culture. As the myocytes spread and flatten over the culture substratum, the
myofibrillar apparatus retains a “rod-like” orientation. Development of contractile
activity is crucial in the maintenance of the integrity of the myofibrillar apparatus
during prolonged culture. Myocytes that fail to beat display morphological indications of atrophy; conversely, myocytes that commence beating show no such morphological signs of myofibrillar disorganization. The subcellular organization of
other elements of the contractile apparatus, including the transverse tubular system and the sarcoplasmic reticulum, retain their structural relationship with the
myofibrils in beating myocytes but not in quiescent cells. Cultured adult myocytes
represent a n important model to investigate the influence of mechanical factors on
the organization and maintenance of the adult cardiac phenotype.
The isolation and culture of adult cardiac myocytes
represents a n increasingly popular approach to investigate directly phenomena that regulate myocyte behavior under precisely defined conditions (Jacobson
and Piper, 1986; Liebermann et al., 1987). Such a
model system offers distinct advantages in analyzing
the influence of neurohumeral and mechanical factors
on myocyte structure and function which cannot be
readily retrieved from perfused or intact hearts. Previous reports have demonstrated that the majority of
freshly isolated calcium tolerant, adult r a t cardiac myocytes gradually rounded-up when placed in primary
culture (Jacobson, 1977; Claycomb and Palazzo, 1980;
Schwarzfeld and Jacobson, 1981; Moses and Claycomb,
1982b; Nag et al., 1983; Eppenberger et al., 1988; Piper
et al., 1988). As the myocytes “rounded,” they lost
many of their myotypic features, including elements of
the intercalated disc, organized myofibrils, and their
accompanying sarcoplasmic reticulum; the transversetubular system (T-tubules), and numerous mitochondria, many of which were apparently blebbed and released from the sarcolemmal surface (Moses and
Claycomb, 1982b, 1984; Nag et al., 1983). Although
many of these rounded myocytes subsequently attached to a substratum (Piper e t al., 19881, commenced
beating (Bugaisky and Zak, 1989; Spahr et al., 19891,
and reacquired a morphology that closely resembled
the adult myocyte from which they were derived
(Moses and Claycomb, 1982a, 1984; Nag et al., 19831,
the question persisted-how favorably did these “redifferentiated” (Jacobson and Piper, 1986) myocytes compare with their in vivo counterparts (Jacobson and
Piper, 1986; Bugaisky and Zak, 1989; Eppenberger et
al., 1988)?
Although considerable effort has been expended de0 1990 WILEY-LISS, INC.
scribing the structural alterations that accompany the
culture of “redifferentiating” r a t cardiac myocytes
(Jacobson, 1977; Claycomb and Palazzo, 1980;
Schwarzfeld and Jacobson, 1981; Nag e t al., 1983;
Moses and Claycomb, 1982a,b, 1984, 1985; Jacobson
and Piper e t al., 19861, few attempts have been made to
compare and contrast such observations with myocytes
isolated and cultured from other species (Cooper et al.,
1986; Haddad e t al., 1988a), nor has the adaptation of
the “redifferentiated” myocyte been compared to the
“rapidly attached” heart cell (Jacobson and Piper,
1986). Two aspects of in vitro rat myocyte behavior
required further comparison with myocytes cultured
from other laboratory animals. First, most rat myocytes rounded-up during the first week of culture, resulting in disruption of the contractile apparatus (Nag
et al., 1983; Moses and Claycomb, 1982a,b, 1984; Eppenberger et al., 1988; Piper et al., 1988; Bugaisky and
Zak, 1989; Spahr et al., 1989), regardless of the nature
of the culture environment (Piper et al., 1988). Second,
as rat myocytes entered the second week of in vitro life,
they commenced beating spontaneously (Bugaisky and
Zak, 1989). Neither feline (Cooper et al., 1986; Decker
et al., 1989a,b) nor rabbit (Haddad et al., 1988a) cardiac myocytes displayed these properties; rather, they
maintained a rod-shaped configuration while spreading over the culture substratum and exhibited no contractile activity except when cultured a t high densities.
Received June 30, 1989; accepted November 2, 1989.
Address reprint requests to Marlene L. Decker, Department of Medicine (Cardiology), Searle 2-575, Northwestern University Medical
School, 303 E Chicago Ave., Chicago, IL 60611.
M.L. DECKER ET AL.
286
The principal objective of the present investigation was
to describe the morphological changes that developed
in the contractile apparatus when rabbit myocytes
were maintained at a low density in a quiescent state
and to compare and contrast these observations with
those obtained from high density, synchronously beating myocyte preparations. Our observations were focused on interpreting the evolving changes in cytoarchitecture and the changing relationships between the
organelles that constitute the contractile apparatus of
the heart cell in quiescent and contractile myocytes.
The principal goal of this investigation was to evaluate
how mechanical factors influenced the maintenance of
the adult phenotype in vitro.
MATERIALS AND METHODS
Isolation and Culture of Adult Rabbit Myocytes
and/or transmission or scanning electron microscopy
(Decker et al., 1988a,b). The organization of the myofibrillar apparatus was also evaluated by staining
briefly fixed coverslips with rhodamine-conjugated
phalloidin (Molecular Probes, Junction City, OR),
which specifically binds to f-actin (Wulf et al., 1979) or
with a monoclonal (CM-52) anti-myosin antibody developed by Clark et al. (1982). Coverslips were
mounted with Aquamount for viewing with a Leitz Orthoplan fluorescence microscope equipped with fluorescein and rhodamine optics. The nature and distribution of attachment sites was assessed on live cells
with interference reflection microscopy a s described
previously (Decker et al., 1988a).
Petri dishes were briefly rinsed in serum-free MEM
and preserved in situ according to the transmission
electron microscope (TEM) protocols briefly outlined
below (Decker et al., 1988a). Routine preparations
were fixed with 4% glutaraldehyde buffered in 0.1 M
sodium cacodylate buffer (pH 7.4) for 2-4 hours, rinsed
in three changes of 0.1 M cacodylate buffer plus 7.5%
sucrose, postfixed in 2% osmium tetroxide for 1 hour,
rinsed again and then en bloc stained in aqueous uranyl acetate (0.5%) to enhance membrane contrast.
Other cultures were treated with 2% tannic acid or
osmium ferrocyanide (0.8%)to stain the glycocalyx and
intensify myofibrillar contrast or to stain the sarcoplasmic reticulum (Decker et al., 1988a). The preparations were dehydrated and then infiltrated with 50150
mixture of ethanol and Medcast (Ted Pella, Inc., Tustin, CA); the dishes were then drained of epoxy and a
thin layer of fresh plastic added. At this juncture, Beem
capsules were inserted into the epoxy and the plastic
was polymerized for 18 hours at 55°C. The dishes were
then removed and the capsules filled with resin and
polymerized for a n additional 2 days a t 55°C. En face,
transverse and sagittal thin sections (Decker et al.,
1988a) were stained with uranyl acetate and lead citrate and viewed with a JEOL 100 CX electron microscope.
Myocytes plated onto coverslips were employed to
study changes in cell surface topography with the scanning electron microscope (SEMI. Coverslips were
rinsed twice in serum-free medium and fixed in 4%
glutaraldehyde for 1 hour a t room temperature. The
cells were then rinsed in 0.1 M cacodylate buffer plus
7.5% sucrose, postfixed in buffered 1% OsO,, and stabilized with 1% tannic acid and 2.5% uranyl acetate.
The samples were then dehydrated, critically point
dried, sputter coated with gold, and viewed in a JEOL
35CF scanning electron microscope operating at 10 kV
(Decker et al., 1988a).
Ventricular cardiac myocytes were enzymatically
isolated from 1.8 kg adult male New Zealand white
rabbits by a protocol previously described in considerable detail (Haddad et al., 1988a). Rabbits were briefly
heparinized (500 U/kg body wt.), anesthetized with sodium pentobarbital (30 mglkg body wt.), and the hearts
removed and mounted onto a Langendorff perfusion
apparatus in a sterile laminar flow hood. Hearts were
perfused briefly with nominally Ca2+ free KrebsRinger bicarbonate (KRB) buffer containing 15 mM
HEPES (pH 7.35) followed by KRB supplemented with
80 U/ml class I1 collagenase (Worthington Biochemical, Freehold, NJ), 0.5 mgiml testicular hyaluronidase
(Sigma Chemical Co., St. Louis, MO), and 20 pM Ca2+
(Powell et al., 1980) a t 37°C in a 5% co,/95%02 atmosphere. When the heart became flaccid (about 30-45
min), it was removed, teased apart in KRB plus 1%
bovine serum albumin (BSA, ICN Immunobiologics,
Lisle, IL), filtered through a 550 pm nylon mesh to
eliminate connective tissue debris and centrifuged (50g
for 90 seconds) to remove rounded, non-viable cells.
The pelleted myocytes were resuspended in KRB plus
2% BSA, re-centrifuged, and the pellet finally diluted
with Eagle’s minimum essential medium (MEM) with
Earle’s salts (Northwestern University Media Center)
supplemented with 5% fetal calf serum and antibiotics.
Myocytes were plated onto 35 mm petri dishes
(Corning, NY) or coverslips previously coated with
laminin (20 g/ml) at either a low (1x lo4 cellsldish) or
high (1x 10 cells/dish) density. Proliferation of nonmyocytic cells was inhibited by adding 10 pM cytosine1-p- -D-arabinofuranoside (Sigma Chem. Co., St. Louis,
MO) to the culture medium (Haddad e t al., 1988a). Cultures were incubated a t 37°C in a humidified atmosphere of 5%co,/95% air, and the culture medium was
exchanged every other day. The quality of the cultures
RESULTS
was monitored by measuring adenosine triphosphate
(ATP) and creatine phosphate (CrP) content, the reProperties of Freshly Attached Myocytes
lease of creatine kinase (CK) and lactate dehydrogenase (LDH) enzyme activity into the culture medium
Calcium tolerant myocytes adhere rapidly to a lamiand the ability of the myocytes to exclude trypan blue nin-coated substratum (Fig. 1).Such freshly prepared
a s described previously (Haddad et al., 1988a).
myocyte cultures exclude trypan blue, release little
LDH activity into the culture medium, and possess
Light and Electron Microscopy
“normal” levels of CrP and ATP in the appropriate proCoverslips and petri dishes were selected after 1hour portion (Table 1). Adherent myocytes also appear to be
and 1 , 4 , 7 , 1 4 ,and 28 days of culture and prepared for modestly loaded when compared to age-matched coundifferential interference (DIC), interference reflection terparts maintained unattached in suspension. Mea(IR), phase contrast (PC), fluorescence microscopy, surements of intrasarcomeric dimensions obtained
P
CONTRACTION AND MYOFIBRILLAR STRUCTURE
287
Figs. 1-6. The morphology of freshly attached (1 hr) cardiac myocytes is illustrated. Cells adhere randomly, but frequently appear in
close contact (arrows) with one another (Fig. 1).Phase contrast (Fig.
2) and IRM (Fig. 3) images demonstrate the close contacts (arrows)
that develop along the length of the basal myocyte surface (Fig. 3),
running parallel to Z-lines (Fig. 2). Minute adhesion plaques (Figs. 2,
5) also appear at the distal ends (large arrowhead) of the cells. Such
cells display a dimpled cell surface (bracketed arrows) (Fig. 4)with
small blebs (arrowheads) emerging between the costameres (Fig. 5).
Thin-sections (Fig. 6) reveal that these blebs contain “normal” mitochondria (m); also illustrated are the internalized elements (arrows)
of the intercalated disc. Sarcomeres are preserved in a modestly contracted state. Z-lines; I-band; A-band. Fig. 1, X 450; Figs. 2,3, X 1,200;
Fig. 4, X 840; Fig. 5, X 5075; Fig. 6, X 12,000.
from suspended and attached cells reveals that a significant reduction (6.4*0.1%)in Z-2 widths is apparent
in adherent myocytes when both preparations are preserved identically. The rapid development of minute
attachment plaques (Figs. 2 , 5 ) at the distal ends of the
myocyte and the appearance of close contacts that parallel the Z-lines (Figs. 2 , 3 ) coincide with apparent tension development as evidenced by a reduction in I-band
288
M.L. DECKER ET AL.
TABLE 1. Changes in high energy phosphate content and cell viability during primary culture of adult rabbit
cardiac myocytes
LDH
Preparation
Intact Heart
Freshly isolated myocytes
1-Hr myocyte culture
1-Day myocyte culture
7-Day myocyte culture
14-Day myocyte culture
Non-Beating
28-Day myocyte culture
Non-Beating
14-Day myocyte culture
Beating
28-Day myocyte culture
Beating
Trypan Blue
%Rounds3
ND
15.6 2 2.4
4.9 ? 0.4
1.7 t 0.3
1.4 ? 0.2
Exclusion’
ND
74.2 t 4.9
95.3 t 0.4
98.7 t 0.6
98.1 ? 0.5
1.6 2 0.2
1.5 t 0.1
98.4 t 0.4
7.9 t 1.1
25.6 t 2.0
1.5 k 0.3
1.5 t 0.2
98.7 2 0.4
3.4 t 0.2
40.2 t 4.5
26.6 2 1.2
1.5 t 0.2
2.5 t 0.4
95.4 t 0.2
4.8
42.7 t 2.4
29.4 2 1.9
1.5 k 0.3
2.1 t 0.4
96.3 t 0.4
6.4 t 1.5
CrP
ATP
CrPIATP
45.5 2 1.8
34.6 k 4.1
47.3 3.8
45.7 t 3.1
46.8 t 4.1
28.4 5 1.3
20.7 t 3.0
29.7 k 1.7
34.4 5 1.3
28.1 t 1.9
1.6 t 0.1
1.7 t 0.4
1.6 t 0.1
1.3 2 0.1
1.7 2 0.1
39.1 t 3.5
24.7
2.2
38.7
2.5
*
2
2
Release’
ND
34.2 t 6.3
5.1 2 0.7
5.7 t 0.5
6.4 2 0.8
2
2.1
‘Values are means ? S.E. of 10-15 replicate cultures that are exposed to 10 uM Ara-C or the mean ? S.E. of five whole hearts or myocyte
preparations. Adenosine triphosphate (ATP) and creatine phosphate (CrP) are expressed in nanomoles per mg. protein. Lactate dehydrogenase
(LDH) activity is expressed as % of total LDH released into the culture medium after a 2 hr. incubation in fresh medium. % of cells that do not
stain with trypan blue after a 2 hr. incubation in fresh medium.
ND Not determined
‘Values are means ? S.E. for 5 cultures exposed to lOuM Ara-C.
Values represent mean number of round cells as % of total counted cells for 5 cultures.
width and, therefore, in sarcomere length (Fig. 6). SEM
images also depict the existence of randomly scattered
protrusions on the sarcolemmal surface that develop
between adjacent costameres as myocytes adhere to the
substratum (Figs. 4, 5 ) . Thin sections reveal that mitochondria present in such blebs appear indistinguishable from those that intermingle among neighboring
myofibrils (Fig. 6).
Morphology of Cultured Myocytes
After attachment to laminin, rod-shaped myocytes
gradually spread into a highly flattened configuration.
Although this dramatic morphological transformation
accompanies their adaptation into primary culture,
neither ATP or CrP levels nor the ratio of these highenergy metabolites display any significant reduction
(Table 1). Moreover, the release of CK and LDH activity is negligible after 1day and greater than 95% of the
cultured myocytes exclude trypan blue during the
course of culture. Furthermore, unlike rat preparations
that display significant cell rounding, regardless of the
extracellular matrix molecules that the cells are cultured on, fewer than 10% of the rabbit myocytes round
during prolonged culture (Table 1).
Day 1 quiescent myocyte cultures
Within 24 hours of plating, the distal ends of the
adherent myocytes display well-developed adhesion
plaques when observed with interference reflection microscopy (IRM) and SEM (Figs. 9-11). IRM further reveals the continued presence of close contacts along the
length of the myocyte, which are oriented perpendicular to the long axis of the cell (Fig. 9). The terminal
edges of these distal plaques further demonstrate close
apposition with the substratum and the contour of the
cell surface suggests the development of subsarcolemma1 actin stress fibers (Fig. 11). Thin sections of this
region reveal that many of the Z-lines are disrupted
and that resident myofibrils lose their classical sarcomeric organization (Fig. 12). Proximal to the adhesion
sites (Fig. 12), the structural organization of the myocyte closely resembles that of the freshly isolated cell
with the myofibrillar apparatus retaining its registry
and T-tubules assuming their normal morphological
relationships. Fluorescence microscopy further illustrates that the organization of the myofibrillar apparatus, as visualized with rhodamine phalloidin or
monoclonal anti-myosin antibodies, appears normal
(Figs. 7, 8). The mean length of these sarcomeres measures 1.78k0.05 pm ( n = 351, similar to values obtained
24 hours earlier, implying that the heart cells remain
“loaded.” Lastly, at sites where adjacent myocytes are
closely apposed to one another, no evidence could be
garnered for the reassembly of specialized intercellular
junctions at this time (Fig. 12).
Day 7 quiescent myocyte cultures
Although rabbit myocytes retain their rod-shape
through the first week of culture, the distal ends of the
cells transform into fan-shaped plaques (Fig. 13-16)
that can be easily identified as adhesion plaques by
interference reflection microscopy (Fig. 15).Such zones
of contact disclose the presence of close contacts; focal
contacts of the variety that characterize cardiac fibroblasts are not a prominent feature in the contact zones
of quiescent myocytes. Lateral cell processes, likewise,
develop and spread over the substratum at this time
(Fig. 16). By day 7 in culture, the sarcolemmal surface
of the myocyte has acquired a relatively smooth texture with only faint impressions of the subjacent myofibrillar apparatus apparent in SEM images (Fig. 16)
and only a few sarcolemmal blebs of the variety encountered at day 1are visible.
Significant alterations in the structure and organization of the contractile apparatus are apparent in
myocytes maintained for 7 days in vitro. Fluorescent
distribution of myofibrillar proteins reveals that contractile elements are restricted primarily to the cylindrical portion of the myocyte where their registry is
disrupted (Figs. 13, 14). Furthermore, such myocytes
CONTRACTION AND MYOFIBRILLAR STRUCTURE
Figs. 7-1 2. The changes in myocyte structure after one day in vitro.
Actin (Fig. 7) and myosin (Fig. 8 ) staining reveals a well ordered
rnyofibrillar apparatus (M) which is confirmed in thin-sections (Fig.
12). IRM and SEM reveal that distal attachment plaques (arrowheads, Figs. 9,10,11) enlarge and close contacts are extensive (arrow,
Fig. 9) along the length of the rnyocyte. In the plaque regions (Fig. 12),
myofibrillar order is disrupted (arrows) although thick and thin fila-
289
ments do project into the plaque and can be observed just below the
cell surface in SEM profiles (arrows, Fig. 11). The cell surface is also
decorated with a few blebs (arrows) similar to those seen earlier (Fig.
10). Areas of close contact also develop between adjacent cells (arrowheads) but no junctions are apparent. (Fig. 12). Figs, 7, $, x 650; Fig.
9, x 1,200; Fig. 10, x 975; Fig. 11, X 3,300; Fig. 12, X 10,600.
display sarcomeres whose Z-lines range from being the appearance of cytoplasmic “wedges” that develop
partially intact to almost nonexistent (Figs. 17, 18). amongst the contractile elements. Such regions exhibit
Myofibrillar register is progressively interrupted by parallel arrays of rough endoplasmic reticulum and nu-
290
M.L. DECKER ET AL.
Figs. 13-20. Fluorescent, IRM, SEM and thin-section profiles of
myocytes cultured for 7 days. In such myocytes (Fig. 16) both actin
(Fig. 13) and myosin (Fig. 14) lose their normal registry in more
proximal portions of the cell. Large fan-shaped distal adhesion
plaques are well developed (Figs. 15, 16).Close contact zones (arrows)
are only apparent in the periphery of the plaque while contact sites
along the length of the myocyte (arrowhead) appear to be receeding
and are being replaced by punctate focal contacts (Fig. 15). E n face
(Fig. 17, 18) and transverse (Figs. 19, 20) sections obtained from the
rod-shaped portion (Figs. 17,181; the rod-plaque junction (Fig. 19) and
the plaque area, proper (Fig. 20) from myocytes like the one illus-
trated in Fig. 16, reveal major changes in myofibril (M) structure and
T-tubular organization. Some 2-lines are disrupted (open arrowhead)
while others are nearly intact (arrowhead) (Figs. 17, 18). T-tubules
retain their association with Z-lines and junctional sarcoplasmic reticulum (Fig. 181, but in the peripheral plaques, sarcolemmal invaginations (arrows) are apparent but not closely associated with sarcoplasmic reticulum or myofibrils (Fig. 19, 20). Parallel arrays of rough
endoplasmic retriculum (RER) also appear at 7 days as the cytoplasmic compartment increases in size (Fig. 17). c, caveolae. Figs. 13, 14,
x 600; Fig. 15, x 1,500; Fig. 16, x 1,500; Fig. 17, x 31,000; Fig. 18,
x 25,000; Fig. 19, x 20,000; Fig. 20, x 25,300.
CONTRACTION AND MYOFIBRILLAR STRUCTURE
291
merous polysomal profiles that are not normally en- display numerous caveolae (Fig. 24). Peripheral T-tucountered in adult myocytes (Figs, 17).
bules also enlarge dramatically (Fig. 23) and become
As the myocyte spreads, total reliance on frontal or decorated with caveolae (Figs. 24,25). These T-tubules
en face images provides insufficient morphological in- are frequently found associated with elements of juncformation on the evolving subcellular reorganization; tional sarcoplasmic reticulum (Figs. 25, 26) and
therefore, sagittal and transverse thin sections are em- aligned with myofibrillar Z-lines (Fig. 25). T-tubules
ployed to clarify cell-substrate interactions; to evaluate and their neighboring junctional sarcoplasmic reticuthe structure of the myocyte processes, and, in partic- lum are also frequently observed but not always closely
ular, to evaluate myofibrillar integrity. In sagittal sec- associated with neighboring myofibrils (Fig. 26). Nontions taken from the distal portions of a myocyte in the junctional sarcoplasmic reticulum retains its plexiform
region of a n attachment plaque (Fig. 161, myofibrils morphology in these contracting heart cells (Fig. 23,
rather abruptly lose their sarcomeric organization and inset).
are reduced to arrays composed of thick and thin filaments, which in most instances, lack well defined ZQuiescent Myocytes
material (Fig. 20). In areas of transition where the adNon-beating myocytes cultured for 2 weeks are
hesion plaque is contiguous with the cylindrical spread
extensively and are characterized by a n overt
portion of the myocyte (Fig. 16), myofibrils display a
disruption
of the contractile apparatus when compared
significant degree of disorder (Fig. 19). The sarco- to paired contracting
myocyte preparations (See Figs.
lemma reveal few caveolae on the apical and basal sur- 22, 28). Myofibrillar register is almost completely abfaces (Figs. 19, 20), unlike their in vivo counterparts. sent in these cells, with the remaining f-actin and myT-tubules retain their normal configuration and asso- osin assuming a variety of unusual configurations
ciation with intact myofibrils (Fig. 18); however, in (Figs. 28, 29). Perhaps the most striking alteration in
spread zones “T-tubule-like” invaginations develop on the contractile apparatus is a pronounced condensation
the basolateral surface of the sarcolemma (Figs. 19, of actin that frequently develops in the perinuclear re20). T-tubules can be positively identified by the pres- gions of most myocytes and the appearance of minute
ence of their accompanying junctional sarcoplasmic re- myofibrils which approach the dimensions of stress fiticulum. Non-junctional sarcoplasmic reticulum con- bers (Fig. 28). Some of these fibers possess myosin, but
tinues to exhibit its “honeycomb” pattern when found the vast majority of this immunofluorescently detectin association with a n “intact” myofibril, but in regions able protein exists in a rather diffuse pattern throughwhere orderly myofibrillar structure is lost, the sarco- out the cytoplasm of the myocyte (Fig. 29). Relatively
plasmic reticulum is transformed into a branching ar- “normal” myofibrils displaying uniformly spaced, Zray typical of smooth endoplasmic reticulum which in- lines (Fig. 27) are often observed in electron microtermingles with rough endoplasmic reticulum (Fig. graphs along with abnormal myofibrils with irregu17).
larly spaced and distorted Z-lines (Figs. 27, 30). The
abnormal myofibrils reveal few, if any, myosin thick
Day 14 myocyte cultures
filaments amongst the actin thin filaments (Fig. 30).
By days 10-12 in culture, a few densely plated cul- The honeycomb organization of the sarcoplasmic retictures of myocytes develop spontaneous contractile ac- ulum is also radically altered, with it assuming a mortivity. The cultures commence beating synchronously phology reminiscent of tubular smooth endoplasmic reduring this interval with individual myocytes contract- ticulum in areas devoid of intact myofibrils, but
ing a t a rate of 150 beats per minute. Such cultures displaying disorganized myofibrillar elements (Fig.
retain their contractile properties for well over a 32). The T-tubules in such regions exhibit a complex
month. The acquisition of contractile function is accom- branching pattern, but they do remain closely associpanied by no change in high energy phosphate metab- ated with elements of the junctional sarcoplasmic reolism or loss of viability (Table 1);conversely, other ticulum (Fig. 32). That portion of the sarcoplasmic
paired myocyte cultures where a significant number of reticulum associated with the remaining intact myomyocytes have detached remain quiescent and fail to fibrils retains its plexiform morphology (Fig. 31).
beat even when maintained indefinitely.
Day 28 myocyte cultures
Contractile Myocytes
Contractile rnyocytes. Synchronously contracting 4A beating myocyte can be immediately distinguished
from a non-beating cell by the organization of its myo- week-old cells display a linearly ordered myofibrillar apfibrillar apparatus. Furthermore, such cells retain a paratus (Figs. 33,34); moreover, ultrastructural images of
rod-like configuration not unlike that seen in 7-day-old the myofibrils reveal no obvious differences in their orgamyocytes except that the fan-like adhesion plaque is nization or structure when compared to the myofibrils
transformed into well developed, branching processes present in 14-day-old beating cells (cf. Fig. 23 with Fig.
(Fig. 21). The contractile apparatus retains its integ- 34). These myofibrils do, however, project well into the
rity in the central core of the cell and myofibrils can be processes of these flattened myocytes (Fig. 33). Adjacent
observed penetrating into such processes (Fig. 22). The cells in such preparations display normal intercalated
myofibrils of these beating cells display no evidence of discs (Fig. 34) in which preferential attachment of myothe “disrupted” Z-lines (Fig. 23, inset) that are such a fibrils can be demonstrated. All such myocytes are binuprominent feature of 7-day old quiescent myocytes cleate (Fig. 341, and no evidence for karyokinesis is de(Figs. 17, 18). The basal sarcolemmal invaginations tectable in rabbit myocyte cultures. The only other
present a t 7 days appear to develop into large dilated notable structural changes apparent in the beating myocavitations of the basal cell surface (Figs. 23,241 which cytes are an apparent increase in polyribosome and rough
292
M.L. DECKER ET AL.
Figs. 21-26. are SEM, fluorescent and TEM images of contractile
myocytes cultured for 14 days. Beating cells spread extensively but
still retain a central rod-shaped segment (Fig. 21). Numerous phalloidin-positive myofibrils are apparent and many project into cell processes (Fig. 22). E n face images reveal myofibrils (M) with intact
2-lines (Figs. 23, inset; 25); T-tubules (T) appear to develop as basolateral invaginations (arrowheads) of the sarcolemma (Figs. 23, 24).
The glycocalyx of these T-tubules (arrowheads) stains with tannic
acid demonstrating their continuity with the cell surface (Figs. 23,
25); such T-tubules are associated with junctional components of the
sarcoplasmic reticulum (arrows) (Figs. 25, 26). The sarcoplasmic reticulum (SR) retains its plexiform pattern around myofibrils (Fig. 23,
inset). Saggital sections also show apical and basal caveolae (c) (Fig.
24) and basal T-tubules (TI;
coated vesicles (cv) are also visible (Fig.
24). D, desmosome. Fig. 21, x 600; Fig. 22, X 750; Fig. 23, X 10,000;
Fig. 23 inset, x 10,000. Fig. 24, x 25,700; Fig. 25, x 35,500; Fig. 26,
x 62,500.
CONTRACTION AND MYOFIBRILLAR STRUCTURE
Figs. 27-32. depict the organization of the contractile apparatus in
quiescent myocytes cultured for 14 days. Myofibrils (M) are only
rarely well developed (arrow) when viewed in en face sections (Fig.
27). Phalloidin- (Fig. 28) and myosin- (Fig. 29) stained cells illustrate
a profound disruption of myofibrils with only a few small fibrils
clearly displaying both proteins (arrows). Much of the actin condenses
(Fig. 28) while myosin is distributed in a diffuse-reticular pattern
(Fig. 29). Many of the myofibrils (Fig. 30) reveal few thick filaments
293
and the Z-lines are distorted (arrows). Other myofibrils appear normal and display a plexiform sarcoplasmic reticulum (*) encircling
these myofibrils (Fig. 31). Myofibrillar breakdown is associated with
a disruption in sarcoplasmic reticulum (SR) order, but T-tubules still
maintain their close association with junctional elements of the sarcoplasmic reticulum (arrow) (Fig. 32). ID, intercalated disc, c, caveolae. Fig. 27, x 4,500; Figs. 28, 29, x 500; Fig. 30, x 21,600; Fig. 31,
x 34,000; Fig. 32, x 31,000.
294
M.L. DECKER ET AL.
Figs. 33 and 34.The distribution of myofibrils in 28-day cultured
contractile myocytes. Transverse sections reveal that myofibrils (MI
penetrate into the peripheral processes of two interdigitating cells
(Fig. 33). En face images (Fig. 34) of the perinuclear (N) region of the
cell depict well ordered myofibrils (M). Lysosomes (L) are also prevalent in such cells. ID, intercalated disc. Fig. 33, x 15,000; Fig. 34,
x 8,000.
endoplasmic reticulum content and the presence of numerous lysosomal residual bodies (Fig. 34).
Quiescent myocytes. Myofibrils, such as they are, become distributed in a complex branching pattern throughout the myoplasm with many projecting into the spread
processes of the flattened, non-beating myocytes (Fig. 35).
At this juncture much of the condensed f-actin deposits
present in 14-day-oldmyocytes (Fig. 28) are replaced by a
lacy network of minute myofibrils. Unlike stress fibers,
which are difficult to visualize in the non-beating cells,
these minute myofibrils disclose a regular periodicity
(Fig. 35). Myosin distribution remains essentially unchanged from that observed in 14-day-old quiescent myocytes (See Fig. 291, with much of the protein present in the
cytoplasm and not associated with the minute myofibrils.
Such structures lack well-defined Z-lines, display little ev-
CONTRACTION AND MYOFIBRILLAR STRUCTURE
Figs. 35-37. A disorganized contractile apparatus in 28-day old
quiescent cultured myocytes. Phalloidin-positive cells reveal virtually
no myofibrillar registry, rather branching actin filaments displaying
a regular periodicity (arrowheads) are apparent (Fig. 35). TEM show
these structures (arrows) to be myofibrils (Fig. 36). T-tubules are oc-
295
casionally encountered; the sarcoplasmic reticulum (SR) exhibits a
plexiform morphology when observed with myofibrils (M) but most
often appears in a reticular pattern (arrows; Fig. 37) in non-beating
cells. m, mitochondria. Fig. 35, X 2,000; Fig. 36, x 3,500; Fig. 37,
x 33,100.
296
M.L. DECKER ET AL.
idence of their previous orderly register, and, in most instances, reveal a paucity of thick filaments (Fig. 36). Ttubules and the sarcoplasmic reticulum remain evident
in non-beating myocytes in spite of the marked reorganization in the contractile apparatus. T-tubules and their
adjacent junctional sarcoplasmic reticulum and the honeycomb morphology characteristic of non-junctional sarcoplasmic reticulum can be observed in association with
the modified myofibrils (Fig. 37). However, the sarcoplasmic reticulum assumes a reticular pattern in regions
where myofibrillar disruption is apparent (Fig. 37). As in
contractile cells, numerous polyribosomes are readily apparent in the sarcoplasm (Fig. 37).
DISCUSSION
Previous results from this laboratory demonstrate
that quiescent cultures of adult rabbit cardiac myocytes can be established on laminin-coated surfaces
that are suitable for combined biochemical and morphological study (Haddad et al., 1988a,b; Decker et al.,
1988a,b). Both beating and non-beating myocyte preparations maintain high CrP/ATP ratios, release negligible amounts of cytoplasmic enzyme activity, and exclude trypan blue, testifying to the consistent quality of
such cultures (Table 1).Adult rabbit myocytes retain
their rod-shaped morphology and gradually flatten
without passing through a n intervening round phase
(Haddad et al., 1988a; Decker et al., 1988a,b) and the
accompanying disruption of contractile elements that
is characteristic of most cultured adult rat myocytes
(Moses and Claycomb, 1982b; Nag et al., 1983; Jacobson and Piper, 1986; Piper et al., 1988; Eppenberger et
al., 1988); nevertheless, a more subtle form of subcellular remodeling is evident a s adult rabbit myocytes
adapt to culture. If freshly isolated myocytes are plated
at high density (1x lo6 cellsi35 mm petri dish), then
approximately 20% of the cultures commence beating
synchronously between day 10 and 12 of culture. Although we can only speculate on the mechanism responsible for the initiation of contraction, only cultures
that have re-established cell-cell contact and assembled a n intercalated disc at this juncture (Decker et al.,
1989a) beat synchronously. Since approximately 50%
of the adult rabbit heart cells detach from the culture
vessel during the first week of culture (Haddad et al.,
1988a1, it is conceivable that in high-density cultures
some surviving pacemaker cells may establish contact
with neighboring ventricular myocytes provoking contractile automaticity (Meier et al., 1986; Jacobson et
al., 1988b). Conversely, in low-density cultures i t is
likely that solitary rabbit ventricular cells normally
remain quiescent because they can maintain high
transmembrane resting potentials (Powell et al., 1980;
Bkaily et al., 1984; Meier et al., 1986). The present
investigation describes the subcellular reorganization
that accompanies the adaptation of adult rabbit myocytes to in vitro life and clearly demonstrates that myocytes which fail to develop contractile function display
“myofibrillar atrophy” and ultimately reconfigure
those remaining structural components of the contractile apparatus to assume a pattern vaguely reminiscent of embryonic myofibrils (Fischman, 1967). Those
myocytes that commence beating retain many of the
morphological features which characterize their in vivo
progenitors.
The progressive disruption of the contractile apparabus represents the major structural alteration that
evolves in the quiescent myocyte during the first 2
weeks of culture. During the first week of culture,
fluorescent images of phalloidin- and anti-myosinlabeled myocytes illustrate some loss of myofibrillar
order that may be correlated with the development of
minor discontinuities within the Z-lines of some
sarcomeres. A similar pattern of Z-line dissolution is
apparent in the myofibrils of quiescent rat myocytes
cultured for similar lengths of time (Jacobson and
Piper, 1986; Schwartz et al., 1985); however, nonbeating feline myocytes are reported not to disclose
such features (Cooper e t al., 1986). Since the development of such Z-line abnormalities is frequently
associated with disuse atrophy in skeletal muscle
(Jacobson and Piper, 1986), the present observations
imply that non-beating, but adherent rabbit and rat
cardiac myocytes display myofibrillar atrophy. Although myocytes isolated from each of these species
would appear to be “loaded’ (Cooper et al., 1986) to the
same degree (i.e., attached to a laminin- or fetal calf
serum-treated substrata), the feline contractile apparatus apparently responds more favorably to this “in
vitro load” than do its r a t or rabbit counterparts,
disclosing little morphological sign of atrophic myofibrils for nearly 2 weeks (Cooper et al., 1986). The
mechanism(s) that might mediate the disruption of
myofibrillar organization are only now being investigated, but several experiments clearly indicate that
the synthesis of myofibrillar proteins is almost
completely suppressed in non-beating rabbit myocytes
(Haddad et al., 198813; Decker et al., 1989b). Since the
disappearance of intact myofibrils appears to proceed
relatively rapidly, the degradation of contractile
proteins may not be altered significantly in quiescent
myocytes (Decker et al., 1989b). Future experiments
designed to measure the rate of actin and myosin
degradation will be required to verify this hypothesis.
The present observations support the contention that
synthesis of contractile and, perhaps, cytoskeletal
proteins a s well (Simpson et al., 1988) may be
regulated to a large extent by mechanical factors and
that the myofibrillar proteins are degraded andlor
disassembled in the absence of a “significant” in vitro
load (Cooper et al., 1986; Mann et al., 1989) and/or
spontaneous contractility (McDermott and Morgan,
1989).
The disruption of the contractile apparatus proceeds
unabated a s non-beating myocytes continue to spread
and flatten a s culture is prolonged. At 2 weeks, fluorescence (Figs. 28, 29) and thin-section images (Figs.
27, 30) reveal a n apparent disintegration of myofibrillar elements with few “intact” myofibrils recognizable
in most myocytes. These myofibrils, such as they are,
reveal a paucity of thick filaments and aberrant “Zlines” (Fig. 30). At this juncture, myosin assumes primarily a diffuse perinuclear distribution with only a
modest amount of co-localization with actin-positive
fibrils (Figs. 28, 29). Ultimately, the myofibrillar apparatus becomes reordered into a n interdigitating pattern of “minute-myofibrils” (Fig. 35) that appear to replace the normal, repeating registry of the mature
contractile apparatus. This structural reorganization is
accompanied by a marked decline in the myofibrillar
CONTRACTION AND MYOFIBRILLAR STRUCTURE
volume density in these quiescent myocytes (Decker et
al., 1989a)-whether such myocytes retain a contractile potential awaits further investigation.
The fate of the T-tubule system and the organization
of the sarcoplasmic reticulum also seems linked with
changes in the organization of the contractile apparatus in non-beating myocytes. Through the first week of
culture, little change could be documented in either the
structure or the distribution of T-tubules or the sarcoplasmic reticulum. The former retained its close association with Z-lines and junctional sarcoplasmic reticulum and the latter encircled myofibrils, displaying its
classical plexiform morphology (Fawcett and McNutt,
1969; Forbes and Speralakis, 1977). However, as these
quiescent myocytes gradually spread and lose their
myofibrils, the T-tubules are less frequently encountered, and the spatial relationships that T-tubules and
the sarcoplasmic reticulum normally enjoy with myofibrils are lost in areas where myofibrillar disruption is
apparent. T-tubules are no longer closely associated
with Z-lines in many instances (Moses and Claycomb,
1982a) and the plexiform morphology that typifies the
non-junctional sarcoplasmic reticulum is transformed
into a tubulo-vesicular configuration somewhat reminiscent of smooth endoplasmic reticulum seen in steroid secreting cells (Fig. 32).
One structural association remains constant throughout this period of myofibrillar atrophy, and that is the
continued presence of junctional sarcoplasmic reticulum closely apposed to T-tubules (Fig. 32). Such results
imply that in the absence of mechanical activity, the
structural relationships between the myofibrillar apparatus and T-tubules and the sarcoplasmic reticulum
are lost. Whether these “atrophied” myocytes retain
the potential to contract if stimulated appropriately
will be the focus of future experiments. It should be
emphasized, however, that factors other than contractility may modify the organization of the T-tubule system and the sarcoplasmic reticulum. For example, the
extensive cell spreading that transpires in vitro (Claycomb and Palazzo, 1980; Haddad et al., 1988a; Jacobson et al., 1988a,b; Piper et al., 1988) provokes a dramatic change in the surface to volume ratio of these
cells. Since the depolarization of the sarcolemma and
its T-tubule extensions promotes the synchronous release of calcium ions from the sarcoplasmic reticulum
and other sources, the increase in surfacelvolume ratio
that accompanies myoc t e spreading may effectively
reduce the distance Ca’+ ions must diffuse to elicit
excitation-contraction coupling in cultured heart cells
(Langer et al., 1979, 1987), thereby obviating the need
for a n extensively developed T-system and wellordered sarcoplasmic reticulum. Delcarpio and associates (Delcarpio et al., 1986) also report that extensively
spread adult rat myocytes contain fewer T-tubules
than freshly isolated cells (Decker et al., 1989a).
Nevertheless, when rabbit myocytes are cultured a t
high density, some of the preparations commence beating synchronously between day 10 and 12 of culture.
Such contractile activity promotes the retention of myofibrils in myocytes examined at 14 and 28 days of culture; moreover, no indication of Z-line fragmentation
can be demonstrated in these contracting myocytes
(Fig. 23). The morphological relationships between Ttubules, the sarcoplasmic reticulum, and myofibrils
297
also appear to be stabilized by beating. T-tubules are
frequently encountered adjacent to myofibrillar Z-lines
or a s branching baso-lateral invaginations of the sarcolemmal surface similar to those reported by other
investigators (Moses and Claycomb, 1982a, 1985;
Langer et al., 1987). The sarcoplasmic reticulum retains its honeycomb configuration with little evidence
of reorganization similar to t h a t observed in the nonbeating myocytes. Clearly, contractile activity represents the principal trophic influence that maintains
the organization of the myofibrillar apparatus and its
associated membranous components in cultured adult
rabbit myocytes.
The results of the present investigation demonstrate
that a unique subcellular remodeling of adult cardiac
myocytes attends long-term culture and that in the absence of contractile function, myocytes display morphological signs of marked myofibrillar atrophy. Several
investigations support the argument that the mechanical load placed upon heart cells may well be of primary
importance in the maintenance of the structural and
functional properties of cardiac myocytes. For example,
transection of the chordae tendinae of feline right ventricular papillary muscle induces rapid and progressive
atrophy of the cardiac myocytes (Cooper and Tomanek,
1982). This atrophic response is characterized by a significant reduction in myofibrillar volume density, a
concomitant decline in both actin and myosin content,
and a depression in the force generation of the unloaded papillary muscle (Thompson et al., 1984; Kent
et al., 1985). A novel feature of this model is that the
unloaded papillary muscle continues to contract in synchrony with the right ventricle (Cooper and Tomanek,
1982), implying that changes in load and not beating
per se, control the composition and organization of the
contractile apparatus. When the papillary muscle is
reloaded, it quickly regains its normal structure and
function (Thompson et al., 1984). The morphological
dissolution of papillary myofibrils (Tomanek and Cooper, 1981) associated with the atrophy in this model
closely resembles the events described in the present
study where the focal loss of Z-line material and disruption of myofibrillar organization is accompanied by
a major increase in the myocytic cytoplasmic ground
substance. Recent observations further illustrate that
cultured beating neonatal myocytes grow larger when
attached to a substratum than corresponding unattached beating cells (Marino et al., 1987). Moreover,
attached contractile myocytes apparently retain their
myofibrillar organization, whereas unattached cells
display a poorly ordered contractile apparatus. Such
results further implicate “load” as the principal factor
that regulates the composition and structure of the contractile apparatus in the isolated heart cell.
In this regard, Cooper’s recent results also bear importantly on the issue of “load” (Cooper et al., 1986).
Non-beating feline adult myocytes attached to laminin
and cultured in serum-free conditions reveal only minor reductions in contractile protein content and minimal myofibrillar disruption when maintained in vitro
for 2 weeks. Cooper et al. (1986) suggests that attachment, itself, induces a load significant enough to inhibit atrophy. The results in the present study, however, demonstrate that rabbit myocytes attached to a
laminin substratum clearly exhibit atrophy. Further-
M.L. DECKER ET AL.
298
more, the present observations suggest that attachment is not sufficient, in and of itself, to prevent the
atrophy and reorganization of the contractile apparatus in cultured rabbit myocytes. Two caveats must,
however, be mentioned in light of the present observations. Cooper’s study is conducted in the absence of
serum, and the present investigation employs this supplement, which is recognized to enhance cell spreading,
but also ensures long-term survival of cultured cardiac
myocytes (Borg and Terracio, 1988; Jacobson et al.,
1988a; Piper et al., 1988). Nevertheless, under the
present culture conditions, significant evidence of myofibrillar atrophy [i.e., Z-line disruption and loss of contractile units (Decker et al., 1989a)l precedes significant cell spreading; therefore, disruption of the
contractile apparatus does not appear to be closely
linked with cell spreading per se. Secondly, Cooper’s
group (Cooper et al., 1986) employed significantly
higher concentrations of laminin for cell attachment
than those used in the present study (Haddad et al.,
1988a); whether these differences influence myocyte
spreading or the composition of the contractile apparatus awaits further study. The results of these investigations suggest that attachment, alone, is insufficient
to maintain a “normal” myofibrillar apparatus and
that a n isometric load created in the beating myocyte
appears to be a primary stimulus for supporting a fully
developed contractile apparatus.
ACKNOWLEDGMENTS
The present study was supported by public health
service grants HL 33616 and HL 19648 and the Feinberg Cardiovascular Research Institute of Northwestern University Medical School.
The authors wish to thank Dr. William A. Clark for
providing our laboratory a sample of the CM-52 monoclonal anti-myosin antibody.
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