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The transverse-axial tubular system (tats) of mouse myocardium Its morphology in the developing and adult animal.

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The Transverse-Axial Tubular System (TATS) of Mouse
Myocardium: Its Morphology in the Developing and Adult Animal
Department of Physiology, university of Virginia School of Medicine,
Charlottesville, Virginia 22908
Invaginations of the sarcolemma that generate the transverseaxial tubular system (TATS) of the ventricular myocardial cells have begun to
develop in the mouse by the time of birth. The formation of the TATS appears
to be derived from the repetitive generation of caveolae, which forms “beaded
tubules”. Beaded tubules are retained in the adult, in which they frequently
present a spiraled topography. Development of the TATS progresses so rapidly
that complex systems are already present in the cardiac muscle cells of young
mice; by 10-14 days of age, the ultrastructure is essentially identical to that of
the adult. The mouse myocardial TATS is composed of anastomosed elements
that are directed transversely and axially (longitudinally).Many tubules have
an oblique orientation, however, and most elements of the TATS are highly
pleiomorphic. In this respect the TATS of the mouse heart is relatively primitive in appearance in comparison with the more ordered TATS latticeworks
typical of the ventricular cells of other mammals. Stereological analysis of the
mouse TATS indicates that the volume fraction (VV)and surface density (SV)
are considerably greater than previously reported (3.24%and 0.5028 pm-’,
respectively). The most complex ramifications of the TATS are embodied in the
subsarcolemmal caveolar system and the deeper tubulovesicular “labyrinths”,
both of which can be found in early postnatal and adult ventricular cells. In
atrial cells, TATS development is initiated several days later than in the
ventricular cells. The TATS of adult atrial myocardial cells is less prominent
than the ventricular TATS and consists largely of axial elements; the incidence
of the TATS, furthermore, is more pronounced in the left than in the right
The mouse heart is often used to present
typical examples of myocardial cell ultrastructure. Nevertheless, pronounced differences exist among the cardiac muscle cells of
different species. In particular, the transverse-axial tubular system (TATS), a collection of membrane-bound cavities that
pervade all levels of mammalian myocardial
cells, is particularly elaborate in mouse
heart. In addition, the mouse TATS presents
a phylogenetically primitive appearance in
terms of its components, compared t o the
more uniform, “finished” morphology displayed by myocardial TATSs of species such
as guinea pig, dog, and monkey (Sperelakis
and Rubio, 1971; Forbes and Sperelakis,
1983, 1984).
0 1984 ALAN R. LISS, INC.
Several published micrographs of mouse
ventricle, prepared by high-voltage electron
microscopy (Sommer and Waugh, 1976; Sommer and Johnson, 1979; Yamada and Ishikawa, 1981), indicate a predominantly
transverse orientation for the elements of the
TATS, thus underscoring the concept of the
“T” (transverse) tubular system long ascribed to cardiac and skeletal muscle cells
alike. A significant contribution of longituN. Sperelakis is now at the Department of Physiology, College
of Medicine, University of Cincinnati, Cincinnati, OH 45267.
Address reprint requests to Michael S. Forbes, Ph.D., Department of Physiology, Electron Microscopy Laboratory, University
of Virginia School of Medicine,Jordan Medical Education Building, Charlottesville, VA 22908.
Received November 28, 1983. Accepted February 16,1984.
dinally aligned (axial) tubules to the TATS is
found in guinea pig ventricular myocytes
(Rubio and Sperelakis, 1971; Sperelakis and
Rubio, 1971). In addition, a substantial axial
component of the TATS has been reported for
mouse ventricular cells (Forbes and Sperelakis, 1977, 1980a, 1983).
The present communication is addressed
specifically to the description of TATS anatomy in the various regions of normal mouse
heart, including the relationship of the components of the TATS to the surface sarcolemma and to the surface-connected caveolae.
In addition, the development of the TATS has
been examined. We have found that the TATS
is considerably more complex, its volume and
surface area contributions far greater, and
its clevelopment far earlier than heretofore
Electron microscopy
Young (newborn to 17-day-old) and adult
mice of the C57BL and ICR strains were
anesthetized with intraperitoneal injections
of pentobaxbital, thoracotomized, and wholebody perfused with glutaraldehyde fixative
(3%glutaraldehyde made up in a solution of
3% dextran [79,000 M.W.] and 3% dextrose,
with or without 50 mM CaC12 added, adjusted to pH 7.6). To demonstrate the extracellular spaces of the heart, most tissues were
postfixed for 2 hr in a solution consisting of
2% osmium tetroxide and 0.8% potassium
ferrocyanide in 0.1 M sodium cacodylate, pH
7.6 (Forbes et al., 1977). For conventional
preparation of tissue for thin sectioning, osmication was carried out for 2 hr with 2%
osmium tetroxide in 0.1 M sodium cacodylate
(pH 7.2). Samples from adult animals were
stained en bloc for 30 min in saturated
aqueous uranyl acetate solution; most tissues taken from neonatal animals were prepared by a regimen which omitted this step,
in view of its documented removal of pooled
glycogen Wye and Fischman, 1970.).
All tissues intended for thin and thick sectioning were dehydrated in ethanol solutions, passed through propylene oxide, and
embedded in Epon 812 or PolyiBed 812 epoxy
resin (Polysciences, Inc., Warrington, PA).
Thin (50-80 nm) and thick (250 nm-2 pm)
sections were cut with diamond knives on
Sorvall MT-2 ultramicrotomes, and mounted
on bare or Formvar-coated 150-mesh copper
grids. The thin sections were stained sequentially with saturated uranyl acetate in 50%
acetone (2 min) and 0.4% lead citrate (1min),
whereas thick sections were left unstained
(see Forbes and Sperelakis, 1980b, 1983, for
details of the preparation of thick sections for
“pseudo high-voltage electron microscopy”).
For freeze-fracture replication, glutaraldehyde-fixed tissues were washed in dextrosedextran vehicle and gradually infiltrated
with glycerol to a final 30% glycerol concentration. The glycerinated tissues were frozen
in liquid Freon 22, fractured at - 100°C in a
Balzers BAF 300 freeze-fracture apparatus,
and allowed to etch for 2 min before coating
with platinum (to a depth of 2.0-2.5 nm, a t a
shadowing angle of 45”)and carbon. The replicas were freed from the underlying tissues
by digestion in bleach solution, and then collected on bare copper grids.
Specimens were examined and photographed in a transmission electron microscope (either Zeiss EM-9A, Philips EM 200,
or JEOL 100s) operated a t a n accelerating
voltage of 60 keV.
For estimation of the contribution of the
TATS to the ventricular myocardium of adult
mouse heart, hearts from ten male ICR mice,
49 days of age, were prepared so that the
system of extracellular spaces was selectively opacified, according to the procedure
described above. A single thin section was
examined from each of four randomly oriented tissue blocks from the right ventricular wall of each mouse heart; from each
section ten micrographs were prepared a t a
final magnification of x 14,500. On each section, a n area in the upper left-hand corner of
each grid square that contained suitably
traced myocardial cells was photographed. If
the particular section did not provide a s a i cient number of micrographs, we collected
data next from the upper right corners, then
the lower right corners, and then the centers
of the grid squares. The micrographs were
printed together with a grid lattice overlay
having a scale spacing of 0.65 pm. Pointcounting was used to establish the VV (volume fraction) of the TATS relative to the
volume of the myocardial cells (including the
Another important stereological parameter measured was the quantity of surface
area contributed by the TATS. The SV~ATS)
(surface density) was determined by use of a
semicircular test grid overlay (this, in addition to the use of randomly oriented sections,
eliminates the effects of anisotropy [Weibel,
1969; Bossen et al., 19781)printed onto micrographs having a final magnification of x
25,000 (6.Bossen et al., 1978). In this instance, photographically enlarged copies of
many of the same micrographs utilized for
VV determinations were prepared and analyzed. The SVwas determined from 200 micrographs, representing the collection of ten
negatives from each of two blocks of the ventricular tissues of ten mice. The number of
line intersections with TATS membrane profiles constituted the value Ii. The total test
length (LT)that fell inside myocardial cells
was measured with a Microplan I1 digital
planimeter (Laboratory Computer Systems,
Inc., Cambridge, MA) and converted into
units of micrometers. The Sv value was
therefore com uted in units of pm2 TATS
membranelpmf: myocardial cell volume by
the following formula (Weibel, 1969, 1973):
sv= 2 . riLT.
The transverse-axial tubular system
(TATS)consists of the interconnected threedimensional latticework of transverse (T) tubules and axial tubules, the latter directed
more or less longitudinally (parallel to the
long axis of the cell). The lumina of all components of the TATS are open to the extracellular fluid space, and so can be marked by
their filling with opaque substances, such as
the elemental osmium precipitate employed
in this study. The outer cell membrane of
myocardial cells-that portion of the “skin”
of the cells that does not make deep divergence into the myoplasm-is termed the surface sarcolemma. The flask- or alveolarshaped vesicular invaginations that emanate
from the surface sarcolemma, and whose lumina also admit extracellular fluid, are referred to as caueolae (“little caves”).
The TATS in adult myocardium
Ventricular cells
Thin sections of mouse ventricular myocardium usually allow the visualization only of
isolated profiles of the TATS. In processed
tissue fragments properly exposed to a n extracellular tracer material, such profiles frequently appear in survey micrographs as
opaque dots located at or near the Z lines of
the nearest myofibrils; less often they assume the form of longitudinally or obliquely
directed tubular bbdies (Fig. 1). Substantially thicker slices of the same specimens
confirm the existence of a transverse component of the TATS but also make evident numerous axial tubules that interconnect
adjacent transverse tubules (Figs. 2, 3, 7). In
longitudinal view, thick sections of the opacified TATS demonstrate that it forms a
framework whose elements occupy the myoplasmic spaces among the rods or pleiomorphic columns of contractile filaments (the
myofibrils or myofibrillar masses) (Figs. 3,7).
The numerous “axial” tubules of the mouse
TATS may run longitudinally or obliquely
with respect to the cell axis (Figs. 2 , 3 , 7 ) and
are frequently but not always thinner than
their transversely oriented counterparts
(Figs. 2-4, 7). The diameters of both axial
and transverse tubules vary along their respective lengths. The TATS is characterized
by distended pockets a t the points of T-axial
intersection, which usually occur at the Z
lines (Figs. 2-4, 7); similar dilatations also
form adjacent to the A- and I-bands of the
sarcomeres (Figs. 3,5). The pervasive nature
of the TATS is obvious in transverse sections
of the myocardial cell, and its pleiomorphism
in this plane is evident as well (Figs. 5, 6).
Values for the volume fraction and surface
density of the TATS in cells of the right ventricular wall are given in Table 1.
A common pattern of the TATS is one in
which its most peripheral components are
composed of transverse tubules, the system
thence ramifying toward the center of the
cell, a t various points and in different directions, to form a profuse anastomotic tangle
(Figs. 2, 3, 7). Many of the transverse elements originate at surface sarcolemmal levels that lie nearly opposite the Z lines of the
outermost myofibrils (Figs. 7-9). This is not
a specific site of emanation, however, since T
tubules may form from sarcolemmal regions
that appose other segments of the sarcomere
and then veer, along their approximately
transverse course, into alignment with the
nearest Z lines.
The sarcoplasmic reticulum (SR) enwraps
the myofibrils in a collection of tubules, saccules, bulbules, and cisternae. This SR complex is continuous across and around the
elements of the TATS, and the junctional saccules form couplings of variable configuration with the TATS (Figs. 8-10). The TATS
component of couplings may be severely flattened (Fig. 8), but in some instances remains
relatively unchanged in diameter where it
Fig. 3. Stereoscopic pair of electron micrographs of a
thick (ca. 2 pm) section made longitudinally through
OsFeCN-traced TATS in myocardial cell of mouse right
ventricular wall. The banding pattern of the myofibriis
can be made out in these micrographs; although trans-
verse TATS elements are aligned with the Z lines of the
sarcomeres, a considerable portion of the tubules in this
field are longitudinally oriented. Large dilatations are
common along both the transverse and axial tubules.
Stereo angle = 12". ~6,000.
Fig. 1. Thin (70-nm) longitudinal section of mouse
right ventricular myocardium treated with osmium-ferrocyanide (OsFeCN) postfixation to fill the system of
extracellular spaces, which includes the transverse-axial
tubular system (TATS). TATS elements appear in thin
section for the most part as punctate profiles located
near the Z-line levels of myofibrils, thus apparently representing transversely oriented tubules (").
The thick-
ness of this section captures only a few longitudinally
directed elements of the TATS (axial tubules: AxT).
Fig. 2. Section, ca. 0.5 pm thick, cut successive to the
thin section shown in Figure 1. The TATS is revealed as
interconnected, pleiomorphic tubules, many of which run
axially or obliquely with respect to the long axis of the
myocardial cell. ~6,500.
Fig. 4. Freeze-fracture replica. Plane of fracture passes
longitudinally through this myocardial cell (left ventricular wall). Both transverse ('IT) and axial tubules (AxT:
both oblique and truly longitudinal) appear in relief
(only their E faces are represented in this field), along
with their interconnections at several points. The irregular contours of the TATS are evident in the beaded
segments of the axial tubules and in distensions in regions of T-axial anastomosis (arrows). Two caveolar evaginations (C) from one T tubule have been broken off at
their necks. Other recognizable myocardialcell components include network SR (N-SR), junctional SR (J-SR),
and mitochondria N).x68,OOO.
Fig. 5. Thick (0.5 pm) section of transversely cut,
TATS-traced myocardial cell in right ventricular wall.
An extensive interconnection between transverse elements of the TATS can be traced for great distances. One
connection between the TATS and the surface sat-colemma is seen (arrow); subsarcolemmal “beaded tubules” (BT) are also part of the TATS. The numerous
dilatations (*) of the TATS also display corrugated surfaces. X25,500.
Fig. 6. Stereo micrograph pair of 1-pm-thick transverse section of right ventricular wall. The profile of a
nucleus RJ) is seen below some of the TATS elements,
the rest of which are rather evenly distributed within
the myoplasm. Stereo angle = 12”. ~ 5 , 0 0 0 .
TABLE 1. Stereological measurements ofthe transuerse-axial tubular
system in the myocardial cells of right ventricular wall of4Bday-old
male ICR mice
Volume fractiona = Vv
Surface densityb = Sv
+ S.E.
3.24 + 0.10
0.5028 + 0.0126
"Volumeof TATS relative to total myocardial cell volume.
bSurfacearea of TATS membrane per unit volume of myocardial cell cytoplasm.
comes into contact with the junctional SR
(Figs. 9, 10).
The openings of T tubules a t the level of
the surface sarcolemma in many instances
are only 49-55 nm in diameter (Figs. 8, 9).
As a result, a regular array of recognizable
T-tubule apertures is seldom present in replicas of the surface sarcolemma (Fig. 12).
Large-bore transverse tubules (up to 300 nm
diameter) occur in some cells, but their apparently correspondent ostia-although
clearly distinguishable (Figs. 11, 13)-are of
widely differing size and shape and do not
always overlie the Z lines of the subsarcolemma1 myofibrils (Fig. 11).
In the majority of mouse ventricular myocytes, the surface openings of T tubules are
not distinguishable from caveolar apertures
(Fig. 12). Such surface invaginations appear
on the sarcolemma a t any point over the
underlying contractile material (Fig. 12). Adjacent myocytes may display a striking disparity in the relative degree of caveolar
population, as well as in number of definitive
T-tubule apertures.
caveolar openings range from 46-62 nm in
diameter in freeze-fracture replicas, whereas
the caveolar bodies themselves open up to
70-90 nm (measured in thin sections). Caveolae are found both singly and in alveolar
or racemose clusters composed of three, four,
or more individual caveolae that fuse a t a
point below the cell surface and open to the
extracellular space through a single neck
(Fig. 14). The clusters of caveolae also lead
into reticular formations whose elements
may display either beaded profiles or evencontoured tubules (Fig. 14). Junctional SR
saccules may form couplings with the subsarcolemmal caveolar complex (Fig. 15). When
replicated, the caveolae display small numbers of intramembranous particles (IMPS)on
their P faces as compared to the P face of the
surface sarcolemma proper (Fig. 16).
A distinct resemblance exists between the
subsarcolemmal ramifications and the Zuby
rinths, proliferated portions of the TATS that
may incorporate vesicular, tubular, and lamellar elements. Labyrinths may be found
at any depth within the ventricular myocardial cell (Forbes and Sperelakis, 1973, 1977,
Thin and thick sections exemplify the sinuosity and corrugation of extensive segments
of the TATS (Figs. 2, 3, 5-9). Stretches of
small-diameter tubules having a beaded or
spiral appearance are characteristic of both
transverse and axial constituents (Fig. 5).
The topography of these segments is best
appreciated in freeze-fracture replicas (Figs.
Fig. 7. Stereo pair of longitudinal section (ca. 2 pm)
through right ventricular myocardial wall. At the lefthand side, a regular array of T tubules (TT)is seen to
project into the cell from the surface sarcolemma. The
system ramifies profusely deeper within the cell to form
a latticework of interconnected tubules, many of which
display beaded or saccular profiles. Stereo angle = 12".
Fig. 8. Right ventricular wall cell. Thin section of
OsFeCN-traced transverse tubule, whose course can be
followed from the origin (arrow) at the surface sarcolemma (this ostium is approximately 49 nm in diameter),
through a convoluted "beaded" segment, and into a flattened portion, only 30 nm in thickness, associated with
junctional SR saccules (J-SR) to form an interior coupling which is juxtaposed to the Z line (Z) of the nearest
myofibril. ~81,500.
Fig. 9. Thin section of untraced myocardial cell in
right ventricular myocardium, in which the transverse
tubule opens to the extracellular fluid space via a 50-nrn
ostium (arrow) and follows a course toward the 2-level of
the most peripheral myofibril. The T tubule is 106 nm
thick at its widest point, and it participates in a multipartite coupling that consists of three junctional SR (JSR) profiles and an additional T-tubule profile. X 107,000.
Fig. 10. Coupling in right ventricular cell, composed
of a n unflattened T tubule completely encircled by junctional SR. ~77,500.
4,17, 18).In these “spiral tubules”, the contours of the E faces (that class of unit membrane leaflets closest to the lumen and
extracellular fluid) appear as twisted rods
having varying degrees of symmetry along
their lengths (Figs. 4, 17). These membrane
faces do not exhibit a noticeably different
distribution of IMPS than is present on the E
faces of tubules having greater diameters and
more even contours (Fig. 17). However, the P
faces of the spiral tubule segments, which
resemble a n interconnected series of slanted
caveolar profiles, bear considerably fewer
IMPS than do the P faces of larger TATS
elements (Fig. 18).
Atrial cells
It is established that mammalian atrial
myocardial cells are thinner and shorterand hence considerably less voluminousthan the ventricular muscle cells (Fig. 19). In
addition, the TATS content of atrial cells is
distinctly lower than that observed in ventricular myocytes. Muscle cells of the right
atrium are typified by considerable proliferation of caveolae in the subsarcolemmal cytoplasm, but few tubules bear extracellular
fluid to levels beyond 1-2 pm of the surface
sarcolemma (Fig. 19). In contrast, the TATS
of the left atrium, although sparse in comparison with the ventricular myocardial cells,
is in the adult animal substantially further
developed than that of the right atrium and
Fig. 11. Freeze-fracture replica of the P face of the
surface sarcolemma of a myocardial cell in the left ventricular wall. The long axis of the cell is parallel with
the horizontal axis of the micrograph. A number of large
depressions, which represent the openings of transverse
tubules W), are evident; not all of these are located
directly over the Z-line levels of the underlying myofibrils (Z). The T-tubule ostia vary considerably in their
shape. Smaller openings, most of which probably represent caveolae (C), are scattered over the sarcolemmal
surface. ~ 2 5 , 5 0 0 .
Fig. 12. E face of sarcolemma of another left ventricular myocardial cell. Numerous caveolae (C) appear in
this field but are not limited in occurrence to any particular sarcolemmal regions (Z-line levels are indicated).
The fracturing process has broken off many of the surface sarcolemmal invaginations P),and it is not clear
which if any of these are the openings of T tubules.
Fig. 13. T-tubule opening on the sarcolemmal E face
of a cell adjacent to the one shown in Figure 12. Here
the T tubule can be clearly distinguished from caveolae
(C). ~ 6 5 , 0 0 0 .
consists largely of axial components (Fig. 20).
In atrial cells, the beaded and often distended silhouettes of axial and transverse
tubules display their evolution from caveolae
(Fig. 20).
Developing myocardial cells
During the first several days of postpartum
life of the mouse, the individual ventricular
myocardial cells exhibit a highly variable
degree of caveolar presence immediately beneath their surface sarcolemmata (Fig. 21).
Through the ages of 0-3 days, numerous ventricular cells possess smooth sarcolemmal
profiles; scattered among these cells, however, are cells whose borders are distinguished by pockets of caveolation (Figs. 21,
22). These caveolae may exist as individual
profiles just beneath the sarcolemma (Fig.
21). Beaded tubules (Figs. 21-24) appear as
well, both as individual simple tubules and
as complex racemose arrays from which several caveolar projections radiate (Fig. 22). The
specific levels of caveolation may face the Z
bands of the outermost myofibrils but, like T
tubules in the adult cell, do not observe a
strict sarcomere-related origin. The connections between caveolar systems and the surface sarcolemma may be thin corridors (Fig.
23); but more often they are openings which
have diameters similar to those of caveolae
(Fig. 221, like the ostia of many T tubules of
adult myocardium (Figs. €49).
The interaction of the forming TATS and
the elements of the SR is evident in the neonatal animal. Perimyofibrillar networks of
SR are well developed in the newborn animal
(Forbes and Sperelakis, 19831, and certain
differentiated portions of this system-the
junctional SR saccules-appear in close contact with the pleiomorphic profiles of groups
of fused caveolae (Fig. 25). These caveolar
clusters are destined to constitute the definitive TATS, including labyrinths (Figs. 25,26).
Atrial cells in newborn mice are thin, but
already contain specific atrial granules. They
exhibit numerous caveolae a t their sarcolemma1 borders, but few deep incursions are
present in muscle cells of either atrium at
this stage. By 5 to 7 days of age, a few TATS
elements are beginning to appear in cells of
both atria.
After 10-14 days of postnatal development,
the muscle cells of the various regions of
mouse heart display a fully formed sarcomeric banding pattern, associated in the ventricular regions with arrays of transverse
tubules that appear at the Z-line levels. Axial tubules and labyrinths are encountered
as well in ventricular cells, and the atrial
cells exhibit small numbers of TATS profiles
and couplings. Hence, the ultrastructure of
the hearts at this age is qualitatively indistinguishable from that of the fully adult
Formation of the TATS
A considerable amount of evidence has implicated surface-connected caveolae as cell
components which are vital t o the format‘ion
of transverse and axial tubules, both in skeletal muscle (Ezerman and Ishikawa, 1967;
Ishikawa, 1968; Kelly, 1971) and myocardial
cells (Forbes and Sperelakis, 1973; Ishikawa
and Yamada, 1975; Forbes and Sperelakis,
1976).The TATS of adult mouse myocardium
is a particularly convincing example of this
relationship, since its typical components
consist primarily of beaded and spiraled seg-
Fig. 14. Grazing thin section that passes along the
sarcolemma of a myocardial cell in right ventricular
wall. The subsarcolemmal myoplasm contains numerous
racemose and tubular bodies, many of which are obviously derived from caveolae. Lucent, 50-nm-diameter
circular profiles in two of the bodies (3indicate the
caveolar neck, which opens to the extracellular fluid
space at the level of the cell surface (cf. Figures 11-13).
Fig. 15. OsFeCN-traced left ventricular wall. A saccule of junctional SR (J-SR) forms a coupling with a
subsarcolemmal collection of fused caveolae. ~89,000.
Fig. 16. Freeze-fracture replica of the left ventricular
wall in which the plane of fracture has revealed the P
face of the surface sarcolemma (SL) and then diverted
downward into the myoplasm. A single caveolar opening
is seen at the cell surface (arrow), and six caveolar profiles are evident just beneath the sarcolemma. The E
face of one caveola (CE) displays a smooth surface, and
only a few intramembranous particles appear on the
caveolar P faces (Cp). ~94,000.
Fig. 17. Freeze-fracture replica of axial tubule in left
ventricular wall cell. The topography of this tubule includes a segment of smooth contour (at the left) and a
narrow, spiralled length (at right). The E face of this
tubule contains few intramembranous particles.
Fig. 18. P face of an axial tubule in left ventricular
wall. The larger, distended luminal portion bears numerous intramembranous particles (IMP) in a distribution similar to that seen in the sarcolemma (Fig. 16);but
the spiraled segment, which resembles a series of fused
caveolae, displays a considerably lower density of such
particles. x 102,500.
ments. The low incidence of intramembranous particles within these segments is also
typical of the structure of subsarcolemmal
The considerable ramification of the subsurface caveolae to form complex racemose
structures and beaded tubules, which lie just
beneath the sarcolemma, indicate that all
caveola-based bodies might be considered as
part of the TATS. The extensive labyrinthine
proliferations of the TATS, which are continuous with transverse and axial tubules
(Forbes and Sperelakis, 1973), occur at various depths within mouse ventricular myocardial cells and closely resemble, on a grand
scale, the subsarcolemmal caveolar complexes. A further parallel is evident among
all the various subcategories of the TATS;
namely, the saccules of junctional SR form
definitive couplings with transverse and axial tubules (Forbes and Sperelakis, 19771,
labyrinths (Forbes and Sperelakis, 19731, and
subsarcolemmal caveolar systems alike
(Forbes and Sperelakis, 1982). It is possible
that many of the caveolar complexes are not
blind-ended structures, but may form a continuum with the deeper transverse tubules,
axial tubules, and labyrinths.
In addition to being the means of generation of T tubules in developing skeletal muscle fibers, caveolae are retained in the adult
fibers in the form of short beaded segments,
interposed between the surface sarcolemma
and the smooth-contouredtransverse tubules
(Zampighi et al., 1975). This might impart
some flexibility to the T-tubule segments
nearest the fiber surface, thus protecting
their integrity during contraction (Forbes and
Sperelakis, 198Ob). Similarly, the coiled
TATS segments of mouse heart could act as
reservoirs of membrane which expand and
flex during the cycles of cell shortening and
The TATS of mouse heart exhibits a plasticity not commonly encountered in other
mammalian myocardial cells. The retention
of the archetypal form may allow caveolation
to proceed along many vectors, thus creating
exotic configurations such as those seen in
labyrinths (Forbes and Sperelakis, 1973,
1977, 1983).The TATS participates as well in
such unusual structures as “T-tubule desmosomes”, in which desmosomal plaques face
one another across the lumen of a TATS element, thus forming internal, reflexive junctions (Myklebust and Jensen, 1978; Forbes
and Sperelakis, 1980a). The SR is particu-
larly plastic as well. Multipartite couplings
are prominent constituents of mouse heart,
usually manifested in thin sections as multiple alternations of J-SR saccules and TATS
profiles (Forbes and Sperelakis, 19771, but
more likely being the product of interdigitated S-and C-shaped membrane system elements (Forbes and Sperelakis, 1980a, 1983).
The incidence of longitudinal and oblique
(axial) tubules, and their contribution to the
TATS, is high in the mouse heart. This is not
in agreement with reports by Sommer and
Waugh (1976) and Sommer and Johnson
(1979). These authors suggested that‘ many
profiles of presumed axial tubules result from
the plane of section passing through folds of
the surface sarcolemma or through limited
longitudinal excursions of transverse tubules
(Waugh and Sommer, 1975). This latter caveat has been eliminated from consideration
in our study of mouse heart, because of the
use of thick TATS-traced sections.
Axial tubules are likely derived from caveolation which occurs a t right angles to previously formed transverse tubules. Much of
the sarcolemma a t the cell ends is occupied
by the insertions of myofibrils and by the
intermembranous junctions (e.g., Forbes and
Sperelakis, 1980a, 1984). Although some axial elements can be seen to emanate from the
cell ends in some developing and adult myocardial cells, for the most part only restricted
spaces are available, in the regions of cell-tocell apposition, to accommodate the openings
of axial tubules.
atrium possess T tubules, whereas some of
the right atrial myocytes lack a T system.
This opinion is based on the finding that the
VV of the TATS is similar in the two atria,
but that values for the VV and SVof interior
junctional SR-which forms couplings with
the TATS-are twice a s great in the left
atrium as in the right atrium. Our own examinations of regions of mouse atria whose
extracellular spaces had been traced out by
OsFeCN postfixation have found a definite
disparity in the distribution of the TATS between the right and left atria, thus supporting the interpretation of Bossen and
colleagues. Bossen et al. (1981)and Sommer
(1982) consider the two muscle-cell populations of mouse right atrium to represent: 1)
working myocytes (those possessing the
TATS), and 2) vestigial or transitional cells
that lack the TATS and are related more
closely to the atrioventricular conducting
system (AVCS) (concentrated in the right
atrium). Many cardiac muscle cells of murine
right atrium appear to be devoid of transverse and axial tubules; the absence of the
TATS, however, is a doubtful criterion for the
identification of AVCS cells (Osculati and
Garibaldi, 1974; Osculati et al., 1978; Rybicka, 1977; Forbes and Sperelakis, 1984).
Furthermore, the TATS is absent from muscle cells of both right and left atria of guinea
pig heart (Sperelakis and Rubio, 1971).
Without doubt, then, the TATS of mouse
atrial myocytes is far less notable in incidence and complexity than its ventricular
counterpart. Furthermore, the concept of a
The TATS in atrial cells
“T system”, composed of membranous incurBossen et al. (1981)have drawn the conclu- sions which have a predominantly transsion, based on morphometric determinations, verse orientation, is vitiated for atrial tissue,
that most muscle cells of the mouse left both by the present investigation and by
Forssmann and Girardier’s (1970) study of
the rat. When present, the mouse atrial TATS
nevertheless is composed of intricately anastomosed bodies composed of caveola-derived
Fig. 19. Right atrial cells in oblique longitudinal sec- units, as is clearly the case for the ventricution; extracellular spaces have been traced out with
OsFeCN postfixation. Only a few TATS elements (TT) lar TATS (Forbes and Sperelakis, 1973, 1976,
1977, 1982, 1983; Forbes et al., 1977; Ishiappear deep within the cells, although there is profuse
caveolation (C) at the sarcolemmal border. x 10,500.
kawa and Yamada, 1975; Yamada and Ishikawa, 1981). Pleiomorphism of the TATS
Fig. 20. Thick longitudinal section (ca. 0.3 pm)
similar t o that of the ventricular myocytes is
through myocardial cells of mouse left atrium. Though
also typical in atrial muscle cells. It appears,
the TATS complement is less extensive than that of the
ventricular cells (cf. Figures 2, 3, 7), it is better devel- therefore, that the same generative mechaoped than that of the right atrium (Fig. 19). It consists of
nism for the TATS is in force in all regions of
prominent axial (AxT) elements and some transverse
the mouse heart, although the onset of TATS
components (’IT),all of which display corrugated and
development appears to occur later in the
distended segments similar to those of the ventricular
TATS. X23,500.
atrial than in the ventricular cells.
Fig. 21. Survey view of a transverse section through
OsFeCN-traced right ventricular wall from a 3-day-old
mouse. Long stretches of surface sarcolemma are devoid
of caveolae, but at some points caveolar chains (arrows)
and racemose vesicular complexes (*) project from the
sarcolemma into the myoplasm. x 16,500.
Volume and surface area contribution of the
There is a fourfold difference between the
VV values derived for the TATS of mouse
ventricular cells by Bossen et al. (1978) and
by us (respective values of 0.81% and 3.24%).
It seems likely that this disparity has resulted from the preparation of tissue samples
in the two studies. The clear identification of
TATS elements is of paramount importance;
the properties of OsFeCN postfixation (Forbes
et al., 1977)have made our regimen ideal for
this purpose. The procedure utilized by Bossen and colleagues may be less effective for
two reasons. First, the lanthanum hydroxide
colloid used to trace the extracellular fluid
space can be washed out during the postfixation and dehydration steps (Revel and Karnovsky, 1967). Compounding the difficulty of
accurate identification of the finer ramifications of the TATS is the fact that Bossen et
al. (1978) examined sections from both lanthanum-impregnated and conventionally
prepared (lanthanum-free) blocks of tissue
and used this admixture for the generation
of stereological data, Given our VV determinations, it is not surprising that our value
for the surface density (Sv)of mouse ventricular TATS is also considerably greater
(0.5028 pm-’, as opposed to 0.2234 pm-l
found by Bossen and colleagues), indicating
a 2.25-fold larger surface area for the mouse
TATS than previously reported.
The large Vv and SVvalues of mouse heart
TATS that we have reported are not particularly surprising in consideration of the qualitative view of this system which is readily
obtained through the use of such techniques
as selective staining and thick-section electron microscopy. The VVnATS)determined for
certain other mammals (rat,cat, pig) achieves
values of approximately 1-2% (Denoit and
Coraboeuf, 1965; Pager, 1971; Page and
McCallister, 1973; Mall et al., 1978; Hirakow
et al., 1980; Singh et al., 1982, Gotoh, 1983).
The guinea pig ventricular myocardial cell is
notable in that its TATS is far more regular
in distribution, contour, and average diameter of its elements than that of the mouse
(Rubio and Sperelakis, 1971; Sperelakis and
Rubio, 1971; Forbes and Sperelakis, 1983).In
this way, the guinea pig TATS may represent
one of the most advanced stages in development of the TATS, whereas the mouse TATS
is among the most primitive of such systems.
Values given to date for the VV(TATS)of
guinea pig ventricular cells vary widely. Hir-
akow and Gotoh (1980) have obtained a value
of 1.7-2.1% in the 2-week-old animal in
guinea pig (the TATS begins its development
during the embryonic period Forbes and
Sperelakis, 1976). A study of the fully adult
animal has determined the volume contribution to be 3.34% (Denoit and Coraboeuf,
1965), and other estimates range from 7.7%
to 15% (Rubio and Sperelakis, 1971; Sperelakis and Rubio, 1971). The presence of the
TATS, a n “excitatory network” (Hoyle, 1965)
having substantial volume and surface area,
is compatible with the properties of the
mouse heart, most notably its high rate of
contraction (up to 600 beatdmin; Geddes,
1970).In consideration, however, of the lower
heart rate of the guinea pig (ca. 300 beats/
min), the comparative morphometric values
thus far reported for mouse and guinea pig
TATSs do not support a n absolute correspondence between relative TATS population
and physiological properties (e.g., speed of
shortening of myocardial cells). Other parameters, such as average diameter of TATS
elements, volume and surface contributions
of couplings, and over-all geometry of muscle
cells and cell bundles, are likely to play a
part in the physiological performance of different hearts.
Function of the TATS
We have demonstrated that the transverseaxial tubular system of mouse ventricle is a n
intricate and pervasive entity, although it
lacks the geometric precision and large luminal diameters of the latticeworks which
predominate in myocardial cells of many
other mammals. It has recently been argued
that the role of all such membrane-limited
extensions of the extracellular fluid space in
heart is not obvious (Sommer and Waugh,
1976; Sommer and Johnson, 1979). Although
the majority of mammalian hearts possess
the TATS, the thinner, less voluminous cardiomyocytes of lower vertebrates generally
lack it. The greater volume and diameter of
mammalian heart cells may account for the
need for the TATS to provide the optimum
diffusion distance requisite to the even distribution of excitation and the simultaneous
initiation of contraction at all depths of the
myocardial cell.
The voltage- and time-dependent Ca-Na
slow channels of the myocardial cells are
probably located in the TATS membrane as
well as in the surface sarcolemma. Activation of these cation channels during the cardiac action potential would allow a n influx of
Ca+ ions down their electrochemical gradient into the myoplasm and would serve to
couple contraction with excitation. Thus, the
TATS would promote the influx of C a f + a t
multiple levels within each myocardial cell,
thereby shortening the diffusion distance and
time required to activate the contractile
The great ramification of the mammalian
myocardial TATS may be necessary to provide ready access by all regions of each individual “working” cardiac muscle cell to
electrically excitable membrane and extracellular fluid (e.g., Sperelakis et al., 1974).In
this regard, a notable role of the TATS might
be the maintenance of a constant surface:volume ratio, especially under conditions of induced hypertrophy (Page and
McCallister, 1974; Anversa et al., 1979; Tomanek, 1979). This capability of the TATS to
provide membrane augmentation in response to cellular enlargement may also be
of use in the natural volume increase that
accompanies maturation and aging of myocardial cells.
The work reported here was supported by
grants awarded to Dr. Forbes by the American Heart Association (78-753) and the Na-
Figs. 22-26. Thin sections of newborn mouse right
ventricular wall, treated with OsFeCN postfixation to
opacify the system of extracellular spaces and all channels open to it within the myocardial cells.
Fig. 22. Several caveolar complexes, presumably
forming transverse tubules, are filled with osmium precipitate. The connection of the rightmost complex to the
surface sarcolemma can be discerned (arrow). X 35,000.
Fig. 23. A collection of approximately six fused caveolar elements is open to the extracellular space through
a narrow channel (arrow). ~ 8 1 , 5 0 0 .
Fig. 24. A transverse tubule including beaded and
spiraled profiles extends deep into the myocardial cell.
Fig. 25. A complex caveolar body that partially surrounds a forming saccule of junctional SR (J-SR) whose
lumen contains wisps of opaque material (junctional
granules). In the gap between the J-SR and the forming
T tubule, opaque projections (arrows)are visible; these
appear to be junctional processes. x 104,500.
Fig. 26. Complicated caveolar assemblage (*) that resembles a “labyrinth” (tubulovesicular proliferation of
TATS elements) such as that seen in adult heart. Deeper
within the myocardial cell, a smooth-surfaced T tubule
forms a coupling with a saccule of junctional SR (JSR). ~ 8 5 , 0 0 0 .
tional Institutes of Health (HL-28329 and
Research Career Development Award 5 KO4
HL-00550)and by NIH grant HL-18711to Dr.
Sperelakis. Freeze-fracture replicas were
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Sheppard (Central Electron Microscope Facility of the University of Virginia School of
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