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Membrane systems of guinea pig myocardiumUltrastructure and morphometric studies.

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THE ANATOMICAL RECORD 222:362-379 (1988)
Membrane Systems of Guinea Pig Myocardium:
UItrastructure and Morphometric Studies
Electron Microscopy Laboratory, Department of Physiology, University of Virginia
School of Medicine, Charlottesuille, Virginia
The structure and quantitative contribution of membrane systems
(transverse-axial tubular system [TATS] and sarcoplasmicreticulum [SR])have been
investigated in the heart of the adult guinea pig. Although previous quantitative
studies have been made of guinea pig myocardium, this is the first such study that
has utilized tissue in which membrane system elements were clearly identified by
selective staining (in this case by the osmium-ferrocyanide [OsFeCN] postfixation
method). Both membrane systems are highly developed in ventricular cells, but a
TATS is essentially absent from atrial myocytes. The ventricular TATS consists
principally of large-bore elements which may be oriented transversely, axially, or
obliquely, making numerous anastomoses with one another to form a highly interconnected system of extracellular spaces that penetrate to all myoplasmic depths of
the ventricular cell. The cell coat that lines the lumina of these tubules is structured,
containing fibrillar structures that run along the length of the tubule. The volume
fraction (V,) of the ventricular TATS is low (2.5-3.2%), in consideration of the
qualitative prominence of the TATS in these cells.
The relative total population of sarcoplasmic reticulum is higher in the atria (V,
of 10-11%) than in the ventricles (Vv of ca. 8%).In all guinea pig myocytes, several
major structural divisions of SR can be discerned, which include network SR, junctional SR, corbular SR, and cisternal SR. Junctional SR (J-SR) in the atrial cells is
limited almost exclusively to peripheral saccules of junctional SR (PJSR), whereas
both interior J-SR and P J S R are present in the ventricle. Two distinct morphological
types of PJSR appear in atrial cells, including both flattened and distended saccules,
the latter resembling PJSR of lower vertebrate heart. Spheroidal bodies of SR with
opaque contents (corbular SR) are prominent at or near Z-line levels of the sarcomeres of atrial and ventricular cells. Cisternal SR is likely a subset of network SR,
but some examples appear related to rough endoplasmic reticulum. An overall
impression obtained from this study is that guinea pig atria are composed of structurally primitive cells, whereas the ventricular cardiac muscle cells are more highly
developed entities.
In the parlance of our times, the term guinea pig has
come to denote an experimental subject. Nowhere today
is this more true than in the field of heart research, in
which (for example) isolated atrial muscle cells from
guinea pig have been found to be ideal subjects for
electrophysiological studies with modern techniques
such as patch-clamping (Bechem et al., 1983).
In addition, the study of myocardial ultrastructure has
long depended on the guinea pig as a source of material.
Early electron microscopy of the intercalated disc demonstrated extensive gap junctions in guinea pig heart
(Sjostrand et al., 1958; Dewey and Barr, 1964).The welldeveloped system of transverse tubules in guinea pig
ventricle has been studied both in thin sections (Simpson, 1965) and in freeze-fracture replicas (Rayns et al.,
1967, 1968). Further investigation revealed that a Tsystem is virtually nonexistent in the atrial cells of
guinea pig heart, however (Sperelakis and Rubio, 19711,
and that transverse-axial tubular system (TATS) devel0 1988 ALAN R. LISS, INC.
opment in guinea pig ventricle is qualitatively complete
by the time of birth (Forbes and Sperelakis, 1976). ReAbbreuiatiom
Interior junctional sarcoplasmic reticulum
junctional sarcoplasmic reticulum
network SR network sarcoplasmic reticuluin
ferrocyanide-reduced osmium; "osmium ferrocyanide"
peripheral junctional sarcoplasmic reticulum
sarcoplasmic reticulum
surface density; surface area of cell component per unit
volume of cell, expressed in units of pm"/pm3 or
transverse-axial tubular system
volume fraction; volume of cell component per unit
volume of cell, expressed as a percentage
Received March 9, 1988;accepted May 24, 1988.
Address reprint requests to Michael S. Forbes, Ph.D., Electron Microscopy Laboratory, Box 449,Department of Physiology, University
of Virginia School of Medicine, Charlottesville, VA 22908.
cently the guinea pig atrioventricular node and bundle-of particular interest in functional studies-have
been described in great detail in terms of the qualitatively differing structure of sarcoplasmic reticulum (SR)
among the five anatomically discernible regions in that
area (Tomita and Ferrans, 1987).
Despite this intense interest in its function and structure, only a limited amount of quantitative work has
been devoted to the guinea pig heart (Denoit and Coraboeuf, 1965; Hirakow and Gotoh, 1980).Neither of these
earlier studies utilized tissue in which selective contrasting of membrane system elements (SR and T tubules) had been carried out. Our previous studies of
mouse heart (Forbes e t al., 1984, 1985) have demonstrated that stereological measurement of routinely prepared heart leads to considerable underestimation of
membrane system volume fractions (V), and surface
densities (SV).We undertook the present study of guinea
pig heart because of the important role this tissue has
played to date in cardiac research. The stereological
data generated by this investigation are analyzed together with equivalent parameters obtained from our
previous study of mouse myocardial sarcoplasmic reticulum (Forbes et al., 1985);this is part of a n ongoing set
of comparative stereological studies of mammalian
hearts which differ from one another in physiological
properties such as ambient rate of heartbeat. The use of
selectively stained tissue has in addition emphasized
some interesting qualitative aspects of the membrane
systems of guinea pig myocardial cells, and these are
noted as well in this communication.
and dehydrated in a graded series of ethanol solutions
(70%, 95%, loo%), passed through propylene oxide, and
gradually infiltrated with PolyBed 812 resin (Polysciences, Inc., Warrington, PA).
Since our original study of selective staining of muscle
membrane systems by means of postfixation in ferrocyanide-reduced osmium solution (OsFeCN Forbes et
al., 19771, we have found three factors to be of crucial
importance during this type of tissue processing. First,
postfixation in OsFeCN should be carried out without
agitation; use of a rotator at this stage has the undesirable effect of producing a low degree of SR delineation,
to the point that no selective staining a t all may be
found in many blocks. Second, it should be kept in mind
that the osmium-ferrocyanide-cacodylate solution is not
truly buffered, as pointed out by Neiss (1984); as made
up, its intrinsic pH is generally in the range of 10.010.2, In our hands, postfixation at this alkaline pH tends
to produce tracing of the TATS, with or without a degree
of SR staining, whereas deliberate establishment of a
postfixative solution pH of ca. 7.6 gives a more general
delineation of the SR. The third crucial step is infiltration. Difficulties in securing adequate embedment of
OsFeCN-treated tissues have been reported previously
(Hoshino et al., 1976); we have found satisfactory infiltration to be promoted by a gradual introduction of the
epoxy resin into the tissue through intermediary solutions of propylene oxide and resin used in the following
approximate schedule:
Tissue Preparation
2: 1propylene oxide:PolyiBed, overnight;
1 : l propylene oxide:Poly/Bed, 8 h r the next day;
1:2 propylene oxide:Poly/Bed, overnight;
pure PolyBed, 6 h r under vacuum.
Adult specimens of the Hartley strain of guinea pig
(either sex) were used for these studies. Each animal
was anesthetized with intraperitoneal injections of pentobarbital and its chest cavity opened to expose the
heart. Whole-body perfusion was accomplished by insertion of a cannula into the tip of the left ventricle and
injection of 30-40 ml of fixative solution, which left the
circulatory system through a small incision in the right
atrium. The primary fixative employed in all cases consisted of a n aqueous solution containing 3% glutaraldehyde, 3% dextrose, 3% dextran (81,500 avg. MW), and
50 mM CaC12 (pH 7.4-7.6). Following the period of perfusion (ca. 3 rnin), each heart was removed in toto and
immersed in fresh fixative solution for 30-45 min, following which time small pieces were removed to vials of
fixative and left a n additional 60 min. The tissue samples were then washed overnight in aqueous 3% dextrose-3% dextran-50 mM CaC12 (pH 7.4-7.6).
Samples were subsequently processed by one of three
regimens: 1) “conventional” preparation; 2) selective
staining of sarcoplasmic reticulum or TATS; or 3) freezefracture. Solutions used for processing procedures 1)and
2) differed only in the postfixative. Tissue for procedure
1)was postfixed 2 h r in a n aqueous solution of 2% OsO4
in 0.1 M sodium cacodylate, pH 7.2-7.4. In procedure 21,
SR staining was accomplished by immersion at room
temperature for 2 hr in 2% OsO4 and 0.8% potassium
ferrocyanide in 0.1 M sodium cacodylate, pH 7.6 (TATS
tracing was effected at a higher pH [see below]). Tissues
for both procedures 1)and 2) were then stained en bloc
for 30 rnin in saturated aqueous uranyl acetate solution,
In contrast to the postfixation step, all dehydration
and infiltration steps up to the placement of tissues
under vacuum should be carried out on a rotating device. For final curing, tissues are transferred to a 60°C
oven (a vacuum oven is not necessary) and left there for
2 days.
All resin-embedded tissues were sectioned with diamond knives mounted in Sorvall MT-2 or LKB Ultrotome-I11ultramicrotomes. Thin (50-70 nm) sections were
collected on bare or Formvar-coated copper-mesh grids
and stained sequentially with uranyl acetate (saturated
solution i n 50% acetone: 2 min) and 0.4% alkaline lead
citrate (Venable and Coggeshall, 1965: 1 min). Thicker
sections (up to 2 pm) of selectively stained heart were
collected and allowed to dry down on Formvar-coated
grids (Forbes and Sperelakis, 1980a). Such sections were
either examined unstained or stained for 2 min with
lead citrate solution.
For freeze-fracture (procedure 31, glutaraldehyde-fixed
tissue samples were gradually infiltrated with glycerol
in dextrose-dextran solution up to a final glycerol concentration of 30%. The samples were frozen in liquid
Freon 22, fractured at -100°C in a Balzers BAI? 300
freeze-fracture apparatus, and allowed to etch for 2 min
prior to coating with a 2-2.5 nm layer of platinum a t a
shadowing angle of 39”, followed by application of a
stabilizing layer of carbon. The platinum-carbon replicas were digested away from the underlying tissue in
bleach solution, and collected on hexagonal-mesh copper
Most specimens were examined and photographed in
a Zeiss EM 10CA transmission electron microscope operated at accelerating voltages of 60, 80 or 100 keV.
Selected thick sections of TATS-traced ventricle were
investigated with a Zeiss EM902 equipped with a Castaing-Henry type energy-filtering spectrometer, operated at 80 keV; “energy filtering electron microscopy”
(EFEM) has been demonstrated to be quite effective in
resolving fine structure in thick sections, particularly
those of specimens treated with selective staining
(Peachey et al., 1987).
Stereological Measurements
Volume fraction (Vv) and surface density (SV)
Measurements of VV and Sv of the TATS and SR were
carried out in a manner essentially identical with that
previously described (Forbes et al., 1984,1985).Samples
used for stereology came from tissues prepared by the
variation on method 2) which produced staining of the
SR. The regions sampled were right and left atria (exclusive of any regions containing conducting tissue), right
and left ventricular wall, and right and left ventricular
papillary muscles. Preliminary evaluation of ventricular wall by means of a nested analysis of variance (Shay,
1975)indicated that a total of six animals was sufficient
for adequate quantitation (at a confidence level of 90%
with a sensitivity of 0.1%)of SR categories of relatively
low incidence (e.g., peripheral junctional SR). Th‘in sections were prepared from two blocks of each region of
interest (e.g., right ventricular wall), collected on Formvar-coated 150-mesh copper grids, and micrographs
taken randomly of these sections (according to methods
described in Forbes et al., 1985). VV values were obtained by point counts of micrographs printed at 14,500x
magnification with a cross-hatch test lattice pattern
photographically superimposed (scale interval of 0.63
SVvalues were determined by printing, for each block,
five of the micrographs used for Vv determinations at a
magnification of 20,200 x together with a semicircular
test overlay (in order to minimize the effects of any
preferential orientation [anisotropy] of the SR: Weibel,
VV values were also obtained in ventricular wall and
papillary muscle for other cell components, including
myofibrils, mitochondria, the TATS (in the guinea pig
these large tubular elements are identifiable without
selective staining), and nuclei. For atrial cells, all the
components listed above were measured, as well as specific atrial granules, elements of the Golgi apparatus
(including saccules and small vesicles), and lysosomes
(the latter comprising multivesicular bodies, peroxisomes, and lipofuscin).SVvalues for the TATS were also
obtained for all regions. Pairwise comparison of atrial
or ventricular parameters was made by means of the
Mann-Whitney test, and significant differences were
judged to exist when P < 0.05.
Myofibril-associatedsarcoplasmic reticulum
In a previous study (Forbes et al., 1985), we carried
out measurements of surface density of total SR and
network SR, and compared these figures with surface
density of myofibrils. Data used for this relationship
were obtained from micrographs (printed at 25,000 x
with semicircle test overlays) of transverse or near-
transverse thin sections of SR-stained myocardium, in
which myofibrillar surfaces could be distinguished not
only by dint of their own substance, but also because of
the closely apposed mitochondria and opacified tubules
of sarcoplasmic reticulum. The relationship between SR
and myofibril surface densities reduces t o the simple
ratio of intersections, (I (sR)/I(myofibril)or 1(N-SR)&m ofibril).
The quotient approximates the amount of myofiirillar
surface covered by SR tubules. For a more stringent
appraisal of this parameter, we recounted both the SV
data from mouse right ventricular wall and guinea pig
right and left papillary muscle, counting only the actual
surface intersections of total SR and network SR with
myofibrillar surfaces, and then divided each of these
figures by the number of myofibrillar intersections obtained in the previous measurements.
Another parameter of interest is the relationship between interior junctional SR and the TATS. One way of
expressing the contribution of interior couplings formed
by JSR-TATS apposition is to quantify the area of the
TATS surface which is covered by interior JSR. This was
obtained for each of the ventricular regions by dividing
&~JSR) by Sv ATS), and then halving this value (since
in general onyy one face of the IJSR abuts a TATS
membrane: Forbes et al., 1985).
The most notable structural difference between the
“working” myocardial cells of the atrial and ventricular
regions of guinea pig heart is found in their overall
degree of differentiation. In general, the atrial muscle
cells are visibly thinner. The myofibrillar architecture
of atrial cells is less strongly defined, furthermore, and
their mitochondria1 complement is less prominent.
Closer qualitative inspection and quantitative measurement of the ultrastructure of the two cell types has
underscored this general impression, and uncovered additional differences, notably between their membrane
Atrial Cardiomyocytes
Filling of the system of extracellular fluid spaces with
osmium precipitate, in combination with the preparaFigs. 1-8. Left atrium of guinea pig heart.
Fig. 1. Thick (ca. 130 nm) longitudinal section of several muscle cells.
The sarcolemmal borders (SL) in this field are delineated by osmium
precipitate, and for the most part display only small vesicular inpouchings (caveolae); a single distinct invagination is seen at the arrow. Nu,
muscle cell nucleus. ~ 7 4 4 0 .
Fig. 2. Detail of the transverse tubule (TT) in Figure 1. Aside from
single caveolar vesicles (C), such short tubules are the sole sort of
sarcolemmal invagination found in guinea pig atrial cells. x 25,000.
Fig. 3. Longitudinal, 150-nm-thicksection of SR-stained atrial muscle cell. A variety of sarcoplasmic reticulum configurations is evident.
The predominant category, network SR (N-SR), in association with
myofibrillar surfaces displays a sarcomere-specific pattern of tubules
that are heavily concentrated over the A-bands. A spheroid of corbular
SR (C-SR) is anastomosed with network SR, this association distinguishing it from the specific atrial granules (SAG) that are found near
the nucleus and scattered throughout the cell. Distended segments of
SR (“cisternal” SR: Cs-SR) are intercalated with network SR, and a
large cisternal mass of ribosome-decorated, rough endoplasmic reticulum (RER) is located in the nuclear pole myoplasmic region. The Golgi
apparatus (GA) is usually not stained by osmium-ferrocyanide postfixation. x 16,000.
tion of thick sections, provides a “negative” view of the
sarcolemmal surface of guinea pig atrial cells and indicates a near absence of transverse tubular invaginations
as well as a paucity of surface-connected caveolar vesicles (Fig. 1).The few tubular incursions that can be
found are quite short, extending only to the level of the
outermost myofibrils (Figs. 1,2).
A wide variety of configurations of the sarcoplasmic
reticulum is evident in atrial cells (Figs. 3-8). In
OsFeCN-treated material, opacified profiles of SR tubules and cisternae can be distinguished from elements
of the Golgi apparatus, which rarely fill with osmium
precipitate. Sections through the nuclear pole regions of
atrial cells typically contain profiles of the Golgi, accumulations of mitochondria, and collections of specific
atrial granules (Fig. 3). Prominent as well in these regions are examples of rough endoplasmic reticulum,
often massed into a localized collection of parallel cisternae; such cisternae are frequently resolved in grazing
sections as extensive sheetlike structures (Fig. 3).
The network (“free,” “longitudinal”) SR displays a
variety of patterns, including meshworks applied beneath the surface sarcolemma (Fig. 4)and on the myofibrillar surfaces (Figs. 3-5). Connected with these coarse
tubular retes there also appear simple longitudinal tubules (Figs. 3-5) and perforated cisternae (“fenestrated
collars”), these last SR elements located predominantly
over the middle levels (pseudo-H zones) of the sarcomeres (Figs. 3,5).
Two divisions of the SR, corbular SR and Z tubules,
are located a t or near the Z-line levels of atrial sarcomeres. Corbular SR elements are rounded or ovoid bodies, ca. 120 nm in average diameter (ovoid examples
have a major diameter of up to 200 nm); they are anastomosed with the network SR, and often occur in clusters (Figs. 5, 6). Z tubules are elements of the network
SR which form partial or complete collars around the Zline material; this encircling configuration is most obvious in transverse sections (Fig. 6).
The absence of a system of transverse tubules from
guinea pig atrial cells precludes, by definition, the existence of a significant population of interior junctional
SR (IJSR), that specialized SR division which forms complex appositions (couplings) with transverse and axial
tubules. Virtually all atrial couplings, therefore, are
formed between the surface sarcolemma and the peripheral J-SR. In grazing thick sections that pass longitudinally along the sarcolemma (Fig. 4), the peripheral J-SR
is evident a s distended, platelike bodies that are apposed
to the inner sarcolemmal surface and interlinked with
the subsarcolemmal tubular meshwork of network SR.
Two distinct types of peripheral J-SR-as distinguished by their shape-exist in working atrial cells.
Both forms of peripheral J-SR can coexist in the same
cell, adjacent to one another along the sarcolemmal border, and examples of either type can be traced to connections with network SR. One type is the “conventional”
sort of J-SR, each example a flattened saccule containing
a linear array of intrasaccular ‘Ijunctional granules”
(Fig. 7); the second comprises cisternae that are markedly distended in their vertical plane (Figs. 6, 8) and
filled with lightly opaque granular contents similar in
appearance to junctional granules in the attenuate peripheral J-SR (Fig. 8). In both types of couplings, bridging junctional processes (“pillars”) can be discerned in
the space between the J-SR and sarcolemmal membrane
leaflets (Figs. 7, 8).
Estimates of relative frequencies (made from counts
on the prints used for stereological data) indicate an
approximate ratio between the flattened and distended
varieties of peripheral junctional SR of 1:2 in both the
right and left atria; that is, in guinea pig atrial working
muscle cells, the engorged variety of’ PJSR is the predominant one.
Ventricular Cardiornyocytes
Transverse tubules
Early studies of guinea pig ventricular myocardial
cells established that the large-diameter invaginations
from the sarcolemma are not merely a collection of
transversely oriented tubules, but rather a n intricate
and interconnected array of transversely, longitudinally, and obliquely oriented elements, which has come
to be known as a “transverse-axial tubular system”
(now more conveniently abbreviated to TATS). The anastomosis of TATS elements into latticeworks is particularly evident in thick sections (0.25 pm or greater) of
OsFeCN-traced tissues (Figs. 9-11). It becomes especially apparent through examination of stereoscopic
micrograph pairs that the TATS is not a simple
architectural entity consisting of strictly transversely
running tubules joined at precise right angles to absolutely vertical (“axial”) tubules. In particular, it can be
noted that the axial tubules frequently display arclike
profiles (Figs. 9, 10).
Substantial TATS luminal interconnection exists for
great distances within the cell, both in the longitudinal
(Fig. 10) and transverse axes (Fig. 11). Adding to the
complexity of the guinea pig ventricular TATS is the
frequent presence of “doublets,” usually composed of
pairs of transverse tubules (Figs. 12, 15). Such doublets
are usually not derived from branching of transverse
Fig. 4. Thick (200-nm) longitudinal section. Toward the right side of
the field, close to the surface of a n atrial cardiac muscle cell, an
extensive array of network SR tubules blends in numerous places with
expanded saceules of peripheral junctional SR (PJ-SR) that form couplings with the inner surface of the sarcolemma. Proximity of this SR
to the cell surface is indicated by caveolar profiles (C) interspersed
with the meshes of the network SR. At deeper levels of the cell,
network SR forms simple longitudinal tubules (LT) that lie in myofibrillar clefts. x 20,000.
Fig. 5. Another field in the same section. This SR array contains
spherules of corbular SR (C-SR)that are restricted to Z-line levels of
the underlying sarcomeres, longitudinal N-SR segments (LT), distended cisternal SR (Cs-SR), and a perforated “fenestrated collar”
region (FC). ~ 3 5 , 5 0 0 .
Fig. 6. Transverse, 100-nm-thick section through SR-stained atrial
cell. The SR is most prominent at the levels of the Z discs (Z), where it
forms closely adherent uninterrupted stretches of so-called Z tubules
(ZT). Corbular SR (C-SR) is also present at these levels. The simple
cylindrical form of the longitudinal N-SR tubules (LT) is confirmed by
their appearance in cross section. Expanded bodies of peripheral junctional SR (PJ-SR) abut the inner sarcolemma. ~36,000.
Figs. 7, 8. Stereoscopic micrograph pairs of the two different forms
of peripheral junctional SR in guinea pig atrial cells. Though the
flattened saccule in Figure 7 is the more conventional category in
appearance, the distended cisterna in Figure 8 represents the predominant type of PJ-SR. Strands of opaque material (junctional processes)
bridge the myoplasmic gap between the SR and sarcolemmal surfaces;
in some places these can be resolved into linear “pillars” (between
arrows). Stereo angles: Figure 7, 10”; Figure 8, 5”. Both figures
x 110,000.
tubules, but rather (as best seen in freeze-fracture replicas) from the close but separate origin of T tubules from
the surface sarcolemma (Fig. 12).
TATS elements display substantial variation in their
profiles, from round to flattened ovoid shapes; their diameters may be as small as 40 nm in extreme cases, but
generally fall within the range of 200-360 nm. This
compares well with previous estimates (310 nm: Rubio
and Sperelakis, 1971; 340 nm: Rayns et al., 1975).
Though cursory inspection of the TATS gives the
impression of a highly ordered system, the geometric
regularity of the TATS is in fact limited. This can be
shown both by inspection of thick longitudinal sections
(Fig. 9) and freeze-fracture replicas of the surface sarcolemma (Figs. 12-14). Although large areas appear in
which T-tubule ostia are present in regularly spaced
arrays-at or near the Z-line levels of the underlying
myofibrils (Fig. 12)-many areas of the ventricular myocardial surface display irregular patterns of ostial location. There are regions in which discernible T-tubule
openings are missing from many points in the surface
lattice (Fig. 13), or in which ostia are altogether absent
(Fig. 14).Given the large diameters of both the ostia (up
to 600 nm) and the corresponding tubules, the origination points of the T tubules can readily be distinguished
from the smaller surface-connected sarcolemmal invaginations, the caveolae (Figs. 12-14). Caveolae are not so
prominent in guinea pig ventricle as they are in some
other mammalian hearts (e.g., mouse), but appear in
limited numbers attached to surface sarcolemma and to
the TATS elements themselves (Figs. 15, 23). Caveolae
of guinea pig heart are almost always found as single
entities, in contrast to the multiunit “caveolar chains”
found frequently in mouse (Forbes et al., 1984).
Thin sections of conventionally stained tissue reveal
the complexity of the TATS luminal contents, which
frequently can be resolved into fibrillar structures of
12-14-nm diameter (Figs. 17, 18).These fibrils in longitudinal sections seem to correspond for the most part to
structures located toward the centers of the lumina (Fig.
17). In addition, smaller (ca. 9 nm), roughly circular
profiles can be made out, which possibly represent condensed regions of the cell coat that lines the margins of
the tubules (Figs. 17,18).
Sarcoplasmic reticulum
The sarcoplasmic reticulum in ventricular cells is more
highly organized than that of the atrial cells. The network SR tends to appear in more closely arranged, sarcomere-specificpatterns when apposed to the myofibrils
than when it is located in a subsarcolemmal position
(Fig. 19). Guinea pig ventricular peripheral junctional
SR is best visualized in en face sections of the cell surface (Fig. 19). Interior J-SR is far more abundant, forming numerous appositions with the TATS (Figs. 15-20,
23). Junctional SR in ventricle, unlike atrium, consists
solely of flattened saccules that contain linearly arranged junctional granules. Pillarlike structures are evident among the junctional processes that connect the JSR and TATS membranes (Fig. 16).
Possibly because of the great size of the TATS elements, interior J-SR in guinea pig ventricle form couplings that are only partial enwrapments around
transverse or axial tubules. The form of these couplings
is best appreciated in thick SR-stained sections (Fig. 20)
or-with thin sections-in fortuitous grazing planes
through the TATS surface (Fig. 23).
Another notable SR component in ventricular cells is
cisternal SR. Each example of cisternal SR is a distended
region intercalated with the regular small tubules of
network SR, and not necessarily restricted to a specific
sarcomere level (Figs. 20, 21); the surfaces of cisternal
SR are frequently studded with ribosomes (Fig. 21). Fenestrated segments of SR (Figs. 20,26) also appear t o be
a type of cisternal SR; these are rare, but when found
tend to lie either at Z-line levels or over the pseudo-H
zones of the sarcomeres. There is no ultrastructural evidence, in cisternal SR, of junctional granules or surface
projections which might identify these segments as examples of the “extended junctional SR” as described in
other mammalian hearts (Dolber and Sommer, 1980;
Scales, 1981).
Corbular SR bulbules are a notable feature of guinea
pig ventricle; they are found attached primarily to the
perimyofibrillar network SR, but not to the subsarcolemma1SR arrays (Figs. 19,ZO).Examples of ventricular
corbular SR are similar in size and structure to their
atrial counterparts, but are not strictly limited in location to the Z-line levels (Figs. 19,201.Structural similarities between corbular SR and the definitive junctional
SR include intraluminal granular densities and opaque
projections from the limiting membranes, the latter of
which resemble the junctional processes of couplings
(Fig. 22).
Z tubules are prominent components of guinea pig
ventricular SR (Figs. 23-26). As is the case in atrial
cells, the close relationship between the SR and the Zline material is best demonstrated in transverse sections
(Figs. 23, 24). Examination of longitudinal sections of
SR-stained material, on the other hand, reveals a number of different SR configurations at the Z-line levels
which correspond to “Z tubules” (Figs. 25,26).
Stereological Observations
In general, our stereological measurements of guinea
pig heart indicate that the contributions of the various
cell constituents are quite similar within the major heart
regions (right atrium compared to left atrium, right
ventricle compared to left ventricle) (Tables 1-3). Between the atria, in fact, the only statistically significant
difference which can be discerned is in the myofibrillar
volume fraction, which is greater in the right atrium
than in the left (Table 1). These measurements also
confirm the qualitative observations which previously
had indicated that neither a T system nor a significant
Figs. 9-1 1. Thick sections through TATS-traced myocardial cells in
guinea pig right ventricular wall.
Fig. 9. Half-micrometer-thick longitudinal section. The transverse
tubules (TT) and longitudinally oriented (axial I tubules (AxT) are continuous at numerous points, thus forming an extensive interconnected
tubular latticework. x 11,000.
Fig. 10. In this field (0.5-pm-thicksection) the continuity of the TATS
can be traced for a distance that extends for seven sarcomere lengths.
Stereo angle, 20”. ~ 6 , 5 0 0 .
Fig. 11. Quarter-micrometer-thick section which demonstrates the
interconnected TATS in the transverse plane of the cell. Distended
regions appear at various places in the latticework, particularly at the
branching points (arrow). x 12,500.
population of interior junctional SR is present in guinea
pig atrial myocytes.
Cells from the different ventricular regions sampled
are likewise quite similar in their overall constitution
(Tables 2, 3). Here small yet statistically significant
differences are seen only in the VV(TATS)
between right
ventricular wall and right papillary (Table a), and in
and &(TATS) in some ventricular regions (as detailed in Table 3).
The relationship between surface densities of interior
junctional SR and the TATS of guinea pig ventricular
myocardial cells is detailed in Table 4; this demonstrates that between 10.5%and 14%of the TATS surface
is occupied by couplings. This is somewhat lower on
average than equivalent values calculated for mouse
ventricular cells (13-16%) in our previous studies (Forbes
et al., 1984; Forbes and Sperelakis, 1987).
When the question of coverage of the myofibrils by
tubules of sarcoplasmic reticulum was investigated, it
was found (Table 5) that the relative amount of myofibrillar surface directly apposed to network SR is similar
in right and left papillary of guinea pig. This amount
(ca. 43.5% for network SR) is, however, significantly
lower than the value measured for mouse ventricular
wall (ca. 49%).
Comparison of Guinea Pig Atrial and Ventricular Cells
It is established that in vertebrates the primitive heart
tube differentiates in a craniocaudal direction; the result of this is that the ventricle forms first and initiates
the heart beat, with the atrial musculature and cells of
the atrioventricular conducting system becoming defined and electrophysiologically functional at a later
time during development. This delay in development
may account for the less highly structured architecture
of the guinea pig atrial muscle cells. Both the volume
and cross-sectional area of guinea pig atrial cells are
smaller than those of the ventricular cells, furthermore
(Campbell et al., 1987). In functional terms, furthermore, mammalian atria are now proposed to constitute
endocrine organs, in view of their production and secretion of vasoactive peptides such as atrial natriuretic
factor and cardiodilatin (Forssmann et al., 1984).
The appearance in adult guinea pig heart of "dilated"
examples of peripheral junctional SR in the atrial working cells and nodal conducting cells (but not in the ventricular working cells) suggests a difference in the
relative degree of differentiation of the major myocardial regions. The distended peripheral junctional SR of
guinea pig is particularly reminiscent of myocardial
junctional SR of lower vertebrate heart (lizard heart:
Forbes and Sperelakis, 1974; salamander myocardium:
Anderson et al., 1976). In mammalian heart, distended
peripheral PJSR seems limited in general to the more
primitive regions of the heart, including the conducting
system (Rybicka, 1977; Tranum-Jensen, 1978; Tomita
and Ferrans, 19871, and now is seen to exist in atrial
working cells as well. Though appearing in the same
cells with the more conventional flattened JSR saccules,
the distended variety is actually twice as populous in
guinea pig atrium. The primitive nature of the distended JSR is further underscored by its transient appearance in embryonic guinea pig ventricle, there also
coexisting with the more conventional flattened JSR
saccules (unpublished observations).
TABLE 1. Summary of volume fractions of myocardial cell
components in guinea pig atrial muscle cells'
Right atrium
Left atrium
9.97 f 0.80
8.98 f 0.61
10.92 k 0.67
9.96 + 0.56
0.51 f 0.14
0.32 + 0.09
0.15 + 0.02
45.27 2.04*
17.25 f 0.61
0.10 f 0.02
4.42 f 1.09
0.13 i 0.06
0.34 & 0.11
0.12 f 0.04
0.40 f 0.06
0.30 k 0.06
0.26 0.06
41.14 1.01*
18.54 2 0.88
0.06 0.03
3.15 0.29
0.21 f 0.05
0.32 0.04
0.12 0.03
'SR, sarcoplasmic reticulum; N-SR, network SR; IJSR, interior
junctional SR; PJSR, peripheral junctional SR; CSR, corbular SR;
CsSR, cisternal SR; myofib, myofibrils; mito, mitochondria; TATS,
transverse-axial tubular system; nuc, nucleus; SAG, specific atrial
granules; GA, Golgi apparatus; lys, lysosomes.
* P < 0.05 in pairwise comparison with Mann-Whitney test.
Thus in terms of morphological features of its atrial
cells (namely, sarcomere structure, the virtual lack of
the TATS, and appearance of the peripheral JSR), one
can view the guinea pig heart as being composed of two
distinct yet joined entities: a relatively primitive portion, comprising the conducting system and the atrial
working tissue, and a highly differentiated portion, represented by the components of the ventricular chambers
of the heart. It should be recognized that this argument
is a generalization, in consideration of the transitional
cell forms which have been described in other hearts
(Martinez-Palomoet al., 1970), the presence of conducting tissue (the Purkinje system) within the ventricles,
the admixture of atrial and ventricular myosin isozymecontaining cells in the ventricular tissue (Sartore et al.,
1981), and the physiological superiority of atrial tissue
in some respects (Urthaler et al., 1975).
A feature common to both atrial and ventricular cells
of guinea pig is the presence of distended portions of
nonjunctional SR; similar structures have been termed
"cisternal" SR by Scales (1981). In many cases a substantial subset of cisternal SR is the myocardial cell's
complement of "rough" endoplasmic reticulum, shown
by Slade and Severs (1985) to be prominent in rabbit
heart. Even though rough endoplasmic reticulum is
Figs. 12-16. Longitudinal freeze-fracture replicas of guinea pig right
ventricular wall.
Fig. 12. Stereoscopic micrograph pair (10" stereo angle). In this Pface replica there appears, at the 2-line levels, a fairly regular array of
transverse tubule apertures (examples shown a t TT). Double T-tubule
apertures (D) are commonly found. ~ 8 , 0 0 0 .
Fig. 13. Stereo pair of sarcolemmal E-face (stereo angle of 10"). In
contrast t o the field shown in Figure 12, the array of T-tubule apertures is neither regularly spaced into a lattice nor strictly located at Zline levels. In fact, many points at which a T-tubule opening would be
expected to appear are devoid of any such profile. ~ 7 , 0 0 0 .
Fig. 14. Expanse of E-face sarcolemmal leaflet (longitudinal axis of
myocardial cell runs horizontally in this micrograph). Although the Zline levels of the myofibrils can readily be made out in this replica, the
sarcolemma is virtually devoid of T-tubule apertures, and is instead
decorated with numerous caveolar inpouchings (C). x 13,000.
TABLE 2. Summary of volume fractions of cell components in guinea pig ventricular
muscle cells
ventr. wall
ventr. wall
8.27 f 0.49
7.40 0.41
0.45 f 0.07
0.15 f 0.03
0.17 f 0.01
0.16 & 0.04
44.91 f 1.13
24.95 & 0.68
2.71 f 0.18"
2.94 i 0.52
8.17 f 0.43
7.35 i 0.33
0.41 f 0.05
0.12 k 0.03
0.15 k 0.03
0.16 i 0.03
45.54 & 0.98
25.71 i 0.75
2.53 0.18
2.58 k 0.25
8.25 i 0.31
7.46 f 0.28
0.35 f 0.05
0.12 0.02
0.17 f 0.01
0.15 0.02
45.08 k 1.87
25.45 f 0.77
3.23 i 0.09"
3.20 i 0.63
7.95 i 0.47
7.11 f 0.44
0.37 f 0.05
0.11 0.01
0.18 0.02
0.18 f 0.03
43.88 i 1.42
25.64 f 0.67
3.06 k 0.35
2.11 k 0.15
VVC myafi h 1
:kP< 0.05 between right VW and right papillary i n TATS parameter; no other statistically significant
differences exist among ventricular wall and papillary parameters a s measured by Mann-Whitney test.
TABLE 3. Summary of surface density values in atria and ventricles of guinea pig
L. atr.
Rt. atr.
2.187 f 0.212
SV(N-SR) 1.987 i 0.201
L. vw
Rt. VW
f 0.221
f 0.189
f 0.003
2.000 f 0.102
2.064 i 0.158
1.797 i 0.085
1.873 i 0.148
0.109 f 0.009"""
0.110 i 0.015
0.028 f 0.003** 0.015 f 0.003""
0.037 f 0.005
0.031 0.004
0.028 f 0.007
0.036 i 0.013
0.439 & 0.019
0.396 f 0.040
Rt. papillary
Left papillary
1.947 f 0.083
1.747 f 0.080
0.110 0.007"
0.035 f 0.004""
0.025 5 0.004
0.031 0.006
0,477 k 0.020""
1.941 & 0.143
1.773 f 0.133
0.080 i 0.011*,**"
0.020 f 0.003""
0.032 + 0.004
0.037 0,011
0.376 & 0.018""
0.003 f 0.002
0.079 i 0.008
0.084 f 0.017
f 0.013
i 0.017
SV(C~SR) 0.035 k 0.013
SV(TATS) 0.018 i 0.008 0.009 i 0.004
* P < 0.05 i n pairwise comparison.
"*P < 0.05 for t h e following pairs: right ventricular wall vs. left ventricular wall; right papillary vs. left papillary; right ventricular wall vs.
left papillary; left ventricular wall vs. right papillary.
***P < 0.05 (left VW vs. left papillary).
TABLE 4. Density of interior junctional sarcoplasmic reticulum saccules on
transverse-axial tubules in guinea pig ventricular myocardial cells'
ventr. wall
0.124 i 0.015
ventr. wall
0.141 f 0.011"
0.116 i 0.009
0.105 i 0.010"
'Derived from the equation SV,I.TSRJSV(TATS~~.
< 0.05 (left ventricular wall versus left papillary).
qualitatively more evident in the perinuclear cytoplasm son et al., 1973), each example so called because of its
of guinea pig atrial cells, the total volume fraction of all strict and close adherence to the perimeter of a myoficisternal SR is quite similar throughout the heart (ranging from 0.15% to 0.26%). For the purposes of this comFig. 15. In this replica, a portion of the TATS latticework is exposed,
munication, the distinction made between network SR encompassing both axial tubules (AxT) and a doublet (D) composed of
and cisternal SR should be considered valid in structural two parallel transverse tubules in close apposition. Caveolalike bodies
terms alone, since no physiological differences between (C) are fused with the TATS membranes. The plane of fracture also
through a flattened saccule of interior junctional SR (IJ-SR)
the two have as yet been determined, nor has the signif- tpasses
h a t forms a coupling with a n axial tubule. ~31,500.
icance of myocardial rough endoplasmic reticulumFig. 16. Detail of the coupling in Figure 15. The interior of the
often joined with smooth-surfaced SR tubules-been established. The question raised by Slade and Severs (1985) junctional SR is exposed (IJ-SR), and particles can be discerned in the
space between the SR and axial tubule (AxT). Some of these particles
as to the tubular or cisternal conformation of rough form
a thin pillarlike body (arrow). X 111,000.
endoplasmic reticulum in mammalian heart is readily
Fig. 17. Thin section of a transverse tubule i n right ventricular wall.
answered-in the guinea pig at least-by the inspection
Interior junctional SR (IJ-SR) is apposed to t h e tubule (cf. Figs. 15, 16).
of thick, OsFeCN-stained sections, in which numerous Within
t h e tubule is a distinct surface coat (arrows) which interconexamples of extensive cisternae of rough endoplasmic nects with
fibrillar bodies (F)oriented along t h e long axis of the tubule.
reticulum can be found.
Distinct circular profiles ("1 appear i n a n adjacent T tubule caught in
cross section. ~ 6 1 , 0 0 0 .
Z tubules
A particular structural specialization long associated
with the SR of guinea pig heart is the "Z tubule" (Simp-
Fig. 18. Stereoscopic micrograph pair (20" stereo angle) of a transverse tubule in thin section. The electron-opaque portion of the surface
coat is separated from t h e tubule membrane by a space of ca. 20 nm,
and some of t h e opaque substance is collected into circular profiles
(arrows), implying a fibrillar substructure. ~86,000.
TABLE 5. Ratio of apposed surfaces of sarcoplasmic reticulum and myofibrils:
comparison of mouse and guinea pig ventricular muscle’
Mouse right
ventricular wall
Guinea pig
right papillary
Guinea pig
left papillary
49.60 k 2.10*
48.90 & 2.10*
44.52 f 1.76
43.35 i 1.75
44.55 f 1.83
43.46 i 1.84
‘Measured in transversely sectioned cells; n = 10 for mouse, n = 6 for guinea pig. Values
given as means k S.E.M.
* P < 0.05 as determined in pairwise comparison with Mann-Whitney test.
brillar Z disc. Some morphological evidence has suggested that Z tubules are physically connected to the Zline substance (Forbes and Sperelakis, 1980b, 1987). Z
tubules are far more clearly discernible in transversely
cut sections that pass through the Z-line levels of the
sarcomeres. In such views, they appear as a partial or
complete girdling ring around the myofibril. As reported
by Simpson et al. (19731, guinea pig Z tubules are very
difficult to detect in longitudinal thin sections. The use
of thick sections of SR-stained material is therefore more
efficacious in the visualization of longitudinal SR organization at the Z bands. Although simple slim tubules
can on rare occasions be found over Z bands, it appears
that for the most part in the guinea pig-as in the mouse
(Forbes et al., 1985)-discrete Z tubules as seen in cross
sections are largely an illusory optical phenomenon that
is the result of the coalescence of a melange of tubular,
cisternal, and fenestrated elements of network SR components at or near the Z-lines.
Comparison of guinea pig and mouse heart
When comparing the results of our quantitative measurements of guinea pig (the present study) and mouse
heart (Forbes et al., 1984, 1985; Forbes and Sperelakis,
1987),one must keep in mind the difference between the
total proportionality (volume fraction: see Table 2) versus the proportional location of the SR (Table 5). The
first parameter merely indicates the total average fraction of the cell volume that is occupied by SR, and thus
certain parameters (as measured in ventricular tissue)
such as VV(SRflV(myofibril) are similar between the two
species (ca. 0.16 in mouse, 0.18 in guinea pig). A small
but significant difference is found, however, between the
actual amounts of SR apposed directly to the myofibrillar surfaces in the two hearts, with the greater coverage
found in mouse ventricle (ca. 50% in mouse, 45% in
guinea pig [total SR]; 49% in mouse, 43-44% in guinea
pig [network SR]). This indicates that in mouse ventricle
there is a greater amount of network SR that is strategically located in the immediate proximity of the contractile mechanism of the myocardial cell. It remains for
further comparative and quantitative studies of additional mammalian species to determine whether structural parameters such as this one can be generally and
positively correlated with such physiological parameters
as speed of shortening of the muscle cell (as roughly
indicated by average heart rate of a particular species’
One correlation of this sort has been postulated to
exist between the relative incidence of specialized SR
segments and the velocity of sarcomere shortening (Nassar et al., 1987). These workers noted that in isolated
neonatal (3-week) rabbit ventricular cells, corbular SR
is more prominent, and sarcomere shortening velocity
lower, in comparison to the situation in isolated adult
rabbit ventricular cells. Corbular SR is thought-on the
basis of its calsequestrin content (Jorgensen et al., 1985)
and its ultrastructural features (junctional granules and
junctional processes: Dolber and Sommer, 1980, 1984)to be a form of junctional SR, specifically the mammalian equivalent of “extended” junctional SR (in bird
heart these saccules presumably take the place of interior junctional SR: Jewett et al., 1971).
Nassar and coworkers conclude that the rate andor
amount of activator calcium release to the myofibrils
from corbular SR stands to be lower than that from the
definitive junctional SR which is in contact with the
surface sarcolemma or TATS, through which is relayed
the electrical or electrochemical signal that elicits calcium release. Many workers believe that calcium release can occur only across the “junctional face
membrane” (that surface of the junctional SR which
directly abuts the sarcolemma or TATS membrane) of
the coupling (e.g.,Costello et al., 1986),and that the flow
of calcium across network SR membranes is unidirectional: from the myoplasm into the SR lumen. In intuitive terms, this indicates that the efficiency of the
contraction of myofibrils is lower than that of their relaxation; alternative hypotheses have been advanced
which consider the possibility of the network SR’s parFig. 19. Survey micrograph of a 200-nm-thick longitudinal section
through SR-stained right ventricular wall. The plane of section captures myoplasmic regions both at the cell surface (bottom of Geld) and
deeper within the cell (toward the top of the field). The network SR (NSR) displays different patterns according to its location: subsarcolemma1 N-SR is a simple meshwork that intcrdigitates with caveolar
profiles (C) and anastomoses with peripheral junctional SR saccules
(PJ-SR).The N-SR surrounding the myofibrils observes a tighter, sarcomrre-oriented confieuration. and its comaonent tubules meree with
bulblike bodies of corbular SR <C-SRjand sa’ccules of interior jugctional
SR (IJ-SR). X20,OOO.
Fig. 20. Stained SR array in a longitudinal 200-nm-thick section of
right ventricular wall. Fused with the N-SR are numerous examples
of corbular SR (C-SRj,not all of which are strictly aligned with 2 bands
of the underlying myofibrils. Perforated cistctrnae C‘fenestrated collars”: FC) are located over both Z-line and A-band levels of the under^
lying sarcomeres, and simple distensions of the SR (cisternal SR: CsSR) also are present in connection with the M-SR, as are saccules of
interior junctional SR (IJ-SR) that form coupiings with a transverse
tubule (TT). ~ 3 2 , 5 0 0 .
Fig. 21. Cisternal SR seen in a thin longihdinal section of right
ventricular wall. This example of distended SR is decorated with granular bodies, which may be either ribosomes or 0-particles of glycogen.
x 39,000.
Fig. 22. Stereoscopic pair of a body of corbular SR, whose connection
with N-SR can be seen (top of field). The corbule membrane surface is
studded with projections of opaque material which in side view (arrows) resemble the junctional processes seen in couplings (cf. Fig. 171,
and in en face view (double-headed arrow) appear circular. 10” stereo
angle. x 141,500.
ticipation in both phases of the excitation-contraction
cycle (e.g., Forbes et al., 1985). However, if calcium indeed can only exit the SR across the faces of the various
categories of junctional SR, then the contribution of
corbular SR, along with its location, must be given consideration. Even though the excitation signal, which
evokes calcium release from the corbular SR, may be
delayed by its being transmitted through a circuitous
route (from the interior junctional SR to the network
SR, thence to the corbular SR), each of the numerous
bulbules of corbular SR represents a nearly spherical
face across which calcium can be liberated. In guinea
pig ventricle, furthermore, corbular SR accounts for 2227% of the total junctional SR, and can exist at sarcomere levels other than the 2 lines (to which many of the
definitive interior JSR saccules, coupled with transverse
tubules, are relegated). The existence of an extensive
population of corbular SR could therefore be viewed as
a compensatory mechanism through which a more widespread release of activator calcium could be accomplished. This distribution of release sites would be
augmented to some degree by the presence of couplings
along axial segments of the TATS, as well as by the
participation of the units of peripheral junctional SR,
which are not positioned according to specific sarcomere
It is interesting in terms of the comparison of different
species and heart regions to note that the incidence of
corbular SR in mouse ventricle is very low. Though we
have previously described corbular SR in mouse ventricle, in the context of its gravitation to the Z-line levels
of the ventricular myofibrils (Forbes and Sperelakis,
1980b), its quantitative occurrence in comparison to
other SR divisions is negligible (Forbes et al., 1985;
Forbes and Sperelakis, 1987).In contrast, corbular SR is
both a qualitatively and quantitatively prominent division of the sarcoplasmic reticulum in both atria and
ventricles of guinea pig heart. Furthermore, corbular
SR is quite prominent in the atrial cells of mouse heart
(Forbes et al., 1985),and its volume contribution appears
to be of the same general magnitude (40-50% of the
entire junctional SR) as that in guinea pig atrium (unpublished observations).
Symmetry of the guinea pig TATS
A particularly useful technique for evaluating the geometric regularity of the guinea pig ventricular TATS is
the inspection of E-face sarcolemmal replicas. In these
replicas, the T-tubule apertures are clearly displayed as
broken-off stumps which protrude from their origin at
the sarcolemma toward the cell interior, whereas caveolae appear as single smooth-surfaced hemispheres. Our
overall impression from our inspection of such replicas
is that the arrays of ventricular T-tubule openings in
guinea pigs are far from regular and geometric. Large
areas of the sarcolemma may lack these openings (Fig.
14);such defects in the TATS surface arrays are reflected
deep in the cell, where substantial volumes of myoplasm
may lack a population of TATS elements. These observations can be reconciled with our stereological findings
that the volume fraction of the guinea pig ventricular
TATS is rather small (ranging from ca. 2.5-3.2%, depending on the ventricular region). Our measurements
for VV(TATS)
are also in general agreement with those of
Denoit and Coraboeuf (1965)(3.24% in adult guinea pig
right papillary) and Hirakow et al. (1980) (1.7-2.1% in
2-week postnatal ventricle). Previous qualitative examinations of thin sections (Sperelakis and Rubio, 1971)or
freeze-fracture replicas alone (Rayns et al., 1967, 1968)
have indeed suggested a far more substantial contribution of this membrane system element; in all likelihood
this has been a result of the natural tendency to concentrate upon the most orderly TATS formations that come
to view and to generalize from them.
Comparison with previous stereological studies
As pointed out in the preceding section, our measurements of volume fractions of the TATS are comparable
to those found in previous studies (Denoit and Coraboeuf, 1965; Hirakow and Gotoh, 1980). An additional
study of guinea pig left atrium was published by Frank
et al. (1975). The major disparity between the present
study and that of Frank et al. is the volume fraction of
network SR (9.96%versus 1.7%),whereas the measurements for peripheral JSR are quite similar (0.4% and
0.5%, respectively). We have pointed out previously that
a study based on selectively stained material tends to
weight the measurement toward the side of the contrasted constituents (the SR, in the present case), particularly in the case of a cell constituent which would
otherwise be difficult to resolve against the background
of more structured components such as the myofibrils
and mitochondria. In conventionally prepared and
stained thin sections of heart, network SR is just this
sort of constituent; accordingly, in stereological studies
of such preparations its volume contribution has been
substantially underestimated. On the other hand, an
entity such as a saccule of junctional SR, which presents
a characteristic shape and complement of junctional processes, is readily recognized in either conventionally or
selectively stained material; therefore, it is not surprising that our study is in agreement with that of Frank et
al. (1975) in terms of VV(~JSR)
but differs substantially
for VV(NSR).The same sort of argument holds true for
measurements of the TATS, and again our values are
similar to those previously published (see above). It was
initially puzzling, therefore, t o compare our figures for
volume fractions of the two major myocardial cell comFig. 23. Thin transverse section of SR-stained right ventricular wall.
The plane of section passes at or near the 2-line level of many of the
myofibrils in this field (Z); profiles of elements of the TATS are therefore prominent, some of which have associated caveolar projections (C).
Interior junctional SR (IJ-SR), seen en face, forms a partial collar
around a portion of a T tubule. Adjacent to the Z-band material are
closely adherent tubules of SR (Z tubules: ZT). X39,OOO.
Fig. 24. Transverse thin section of SR-stained right ventricular wall.
A Z tubule forms a complete encirclement of a small myofibril at the
level of its Z band. This Z tubule branches at several points (arrows)to
lead into other tubules of network SR. AxT, axial tubule of the TATS.
Flg. 25. Longitudinal thick (ca. 160 nm) section through the myofibrillar surface of a cell in right ventricular wall. Superimposed on the
Z line is a simple profile of network SR, apparcntly corresponding to a
Z tubule of the sort seen in transverse sections (cf. Figs. 23, 24).
Fig. 26. Same section and orientation as in Figure 25. Network SR
is coalesced over four successive Z lines (arrows). Though from this
vantage point the SR varies in conformation from incidental N-SR
tubules to fenestrated cisternal elements, transverse sections taken at
any of these levels would yield profiles of “ 2 t,ubules,” since superposition of the stained SR components would mask their complex vertical
organization. X 35,500.
ponents-myofibrils and mitochondria-with those of
Hirakow and Gotoh (1980), which substantially exceeded ours in the cases of VV(myofibril)of both atria and
ventricles, and VV(mitochon&ia) for ventricular tissue. An
extreme example is found in the case of the myofibrillar
complement of left atrium (our value, 41.14%, as compared to 65.5% in the study by Hirakow and Gotoh). One
possible source of difference is the age of the animals;
Hirakow and Gotoh have presented a table of age-related changes in developing guinea pig heart, ending
with the 2-week postnatal animal (our results come from
the fully adult heart). Although much of the qualitative
development of guinea pig heart seems to have been
completed by the time of birth (Toth and Schiebler, 1967;
Forbes and Sperelakis, 1976), it may be that further
quantitative changes occur as the animal matures. One
indication of this is seen in the fact that the results of
Hirakow and Gotoh on the younger animals fail to reveal the difference in VV(myofibril) between the right and
left atria that our study has shown in fully adult guinea
As mentioned before, a stereological study of SRstained heart may yield overestimated values for SR
components, particularly the network SR. This is a n
example of the “Holmes effect,” as a result of which
structures occupying only a fraction of the section thickness appear to exist at all levels of the section (because
of the artifact of optical projection). Thus profiles of
network SR stand out in OsFeCN-treated cells and-in
measurement of Vv and SVvalues-are counted at the
expense of underlying structures. Therefore, at the same
time the SR is overestimated, myofibrils and mitochondria are subjected to some degree of underestimation.
Even to consider every SR count of our study to represent a lost myofibril count (which obviously is not the
case) would not account totally for the differences we
have noted above, however.
One critical advantage in making a quantitative study
of specifically stained material is that of recognition. In
comparing studies of SR-stained myocardial embedments versus conventionally prepared heart, it becomes
apparent that this recognition factor applies not only to
the SR, but also-by default-to adjacent structures (most
often myofibrils and mitochondria). Simply put, it is far
easier to determine whether a lattice point or surface
intersection lies on a myofibril or on the juxtaposed SR
when the S R can be clearly identified. Therefore, in lowmagnification electron micrographs of conventionally
prepared tissue, the SR encircling a myofibril is virtually invisible-even if it is not superimposed on the
myofibril, as is the case in transverse sections of the
cell-and thus can be mistakenly scored as part of that
myofibril. We believe, then, that the problem of structure discrimination that is intrinsic to the examination
of conventionally prepared tissue likely accounts in substantial part for what we consider to be the overestimated volume fractions of SR-associated structures (such
as myofibrils) in studies such as that of Hirakow and
Gotoh (1980) on guinea pig and of Bossen et al. (1978) on
mouse heart (cf. Forbes et al., 1985).
This research was supported by a grant from the Public Health Service (HL 28329). Most of the tissues used
in this study were generously provided by Dr. Luiz Belardinelli (Department of Internal Medicine, University
of Virginia School of Medicine) and Mrs. Susan I. PurdyRamos (Department of Physiology, IJniversity of Virginia School of Medicine).
Freeze-fracture replicas were prepared by Ms. Bonnie
Sheppard of the Central Electron Microscope Facility of
the University of Virginia School of Medicine. We are
grateful as well to Ms. Lisa Kremer for her skillful
preparation of the electron micrographs used for the
stereology studies reported in this communication. Special thanks go to Mr. Guenter Lamprecht of Carl Zeiss,
Inc., Thornwood, New York, for providing access to the
Zeiss EM 902 instrument, and to Mr. Jean-Marc Theler
(Department of Physiology, University of Virginia School
of Medicine) for valuable discussions concerning aspects
of this research.
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