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J. Cell Sri. II, 179-203 (1972)
Printed in Great Britain
STRUCTURES OF PHYSIOLOGICAL INTEREST
IN THE FROG HEART VENTRICLE
SALLY G. PAGE AND R. NIEDERGERKE
Biophysics Department, University College London, Govier Street, London WCiE 6BT,
England
SUMMARY
Two structures of physiological interest in frog heart ventricles have been examined in
detail: (a) the layer of endothelial cells which encloses each bundle of heart fibres, and (b) the
sarcoplasmic reticulum (SR) inside the heart fibres. Some additional observations on fibre sizes
and types have been made.
Movement across the endothelial cell layer of molecules (molecular or ionic size < 12-5 nm)
occurs through narrow clefts separating each endothelial cell from its neighbour. This conclusion results from experiments made with the extracellular markers ferritin and horseradish
peroxidase.
A diffusion equation describing the movement of solutes into and out of the fibre bundle has
been derived using several geometrical parameters, such as the length and width of the clefts and
the size of the extracellular aqueous space inside the bundle, all of which were determined from
electron micrographs of the tissue.
The theoretical solution for a stepwise change of external calcium concentration gives a halftime of 23 s (± c 8 s, s.D. of 13 bundles) for diffusion equilibrium at the surface of the heart
fibres; this value, however, is likely to be an overestimate, by some 20-30%, on account of
several systematic errors which are described.
The sarcoplasmic reticulum in heart fibres consists of a network of thin tubules which
partially encircle the myofibrils at Z-line level and also form occasional longitudinal connexions.
Branches extend to peripheral regions of the cell and terminate in close apposition to the inner
surface of the cell membrane. The volume of the SR is estimated to be approximately 0-5 % of
the myofibrillar volume of the cells.
Cross-sectional areas of heart fibres (and also their shapes) vary considerably, from less than
2 to more than 100 fim1 (average 174 /4m1). Fibres of large size and small surface/volume ratio
contain many fewer myofibrils and more glycogen granules than fibres of the same size but
larger surface/volume ratio.
Physiological implications of these results are discussed.
INTRODUCTION
Much work has been devoted to the fine structure of heart muscle fibres in various
lower vertebrates (e.g. fish: Kilarski, 19640,6; amphibia: Lindner, 1957; Scheyer,
i960; Naylor & Merrillees, 1964; Staley & Benson, 1968; Sommer & Johnson, 1969;
Gros & Schrevel, 1970; Baldwin, 1970; and certain reptilia: Fawcett & Selby, 1958;
Slautterback, 1963; Leak, 1967; Forbes & Sperelakis, 1971), and several features
common to these tissues have become apparent. For example, the heart cells of these
animals are small and devoid of a T- (transverse tubular) system, and the sarcoplasmic
reticulum (SR) is sparse by comparison with that in most skeletal muscle cells. During
recent work on the function of the frog heart (Niedergerke, Page & Talbot, 1969;
180
S. G. Page and R. Niedergerke
Chapman & Niedergerke, 1970) some further points of structural detail have come to
light requiring closer investigation, which has been undertaken in the present work.
One of the structures examined is the sheath of endothelial cells which surrounds
individual heart fibre bundles or trabeculae (cf. Ecker & Wiedersheim, 1896), and our
attention has been focused on the nature and size of the pathways, present in this
sheath, for the movement of ions and other solutes between the ventricular cavity and
the surface of the heart cells. On the basis of histological data which we obtained a
diffusion equation has been set up describing the theoretical time course of such ion
movements, in particular that for calcium. From the comparison of this writh the
experimentally determined time course of the tension responses to changes of external
calcium concentration (Chapman & Niedergerke, 1970) essential information for the
interpretation of these responses is obtained.
Another subject of study has been the structure of the sarcoplasmic reticulum of
frog ventricle fibres. Our intention here was to assess the precise distribution and
volume of this organelle within the cells and so obtain an estimate of the intracellular
storage capacity of this tissue for calcium, the activator of contraction.
Finally, some observations on fibre sizes and types have been made. These are relevant to the understanding of impulse conduction in this tissue and, in addition,
provide data on certain parameters used for physiological measurements.
METHODS
Ventricles from the frog heart (Ranapipiens) were attached to glass cannulae inserted through
the atrial-ventricular orifice, and the aortic trunk together with the aortic valves was removed to
allow rapid perfusion of the ventricle. For routine fixation, the ventricles were first perfused
with a Ringer's fluid containing io~° g/ml tetrodotoxin, which prevented contraction during the
subsequent perfusion with the fixative. In the course of this work, various methods of fixation
were used (usually at 20-22 °C, but occasionally at 4 °C): (i) fixation in a solution of 1 % OsO 4
and 005 M phosphate buffer (pH 70-7-4) for 0-5 h; (ii) fixation for 2-3 h in a solution of 5 %
glutaraldehyde (also buffered with 0 0 5 M phosphate buffer at p H 7-0-7-4), followed by a
period (several hours) of washing of the tissue in a 005 M phosphate buffer solution and a final
0 5 - h period of fixation in a solution of 1 % OsO 4 and 005 M phosphate buffer (pH 7-0—7-4);
(lii) a 2-4 h period of fixation in a fluid containing 2-5 % glutaraldehyde and 2 % paraformaldehyde (Karnovsky, 1967) and 0-08 M cacodylate buffer (pH 7-2-7-4), followed by washing for
several hours in a 0 1 M cacodylate buffer fluid and a final 0'5-h fixation in a solution of 1 %
OsO 4 and either phosphate (005 M, pH 7-0-7-4) or veronal-acetate (0075 M> pH 7-2-7-4)
buffer. Of these methods, (iii) was found to give the most consistent preservation of the sarcoplasmic reticulum, and was therefore used for the quantitative examination of this structure;
nevertheless, most features of the SR to be described were also observed in tissue fixed by
methods (i) and (ii).
After fixation in the case of (i), or after the first fixation period in (ii) and (iii), the ventricles
were detached from the cannulae and cut in half by a longitudinal incision from base to tip.
Short lengths (1-2 mm) of bundles were then dissected from regions in which adjacent bundles
were roughly parallel and in which little branching occurred. Many of these bundles, taken from
the wall of the ventricular subcavities, had been oriented, approximately, in a longitudinal
direction within the ventricle (i.e. from its base to tip), and they are therefore representative of
the bundles studied by Chapman & Niedergerke (1970) in experiments with which the results
of the present work are to be compared. Other bundles, however, were from different regions of
the ventricle, but a systematic study of any of their properties relating to orientation or location
has not been made. The isolated bundles were dehydrated in an ethanol series (in the case of
methods (ii) and (iii) after the final fixation in OsO 4 ), then soaked in propylene oxide and
Fine structure of frog heart cells
181
finally embedded in Araldite (CIBA). In some instances, the tissue was stained before dehydration in a 0 5 or 2 % uranyl acetate solution which was buffered either with maleate (Karnovsky,
1967) or with veronal-acetate (Farquhar & Palade, 1965). However, since this 'en bloc' staining
extracted the glycogen granules from the myocardial cells, it was used mainly when examining
the endothelial sheath.
Some ventricles were perfused before fixation (by methods (i) or (ii) above) with a Ringer's
fluid containing the extracellular marker ferritin ( 0 1 - 0 2 mg/ml). For others, the perfusion fluid
contained 1-2 mg/ml horseradish peroxidase, and subsequent fixation was with 1% paraformaldehyde, 1-5 % glutaraldehyde (o-i M cacodylate buffer, pH 7-2-7-4). In the latter case
the isolated bundles were subsequently rinsed, first in o-i M cacodylate buffer fluid and then in
distilled water, before a 10—20 min incubation period in a medium containing 005 % 3,3'diaminobenzidine tetrahydrochloride, o-oi % H a O 2 and 0-05 M Tris maleate buffer (pH 76)
(Karnovsky, 1967). After incubation the bundles were again rinsed in water and finally fixed in
1% OsO< (method (i)).
In experiments designed to identify intracellular structures which might be capable of storing
calcium ions, the ventricles (cannulated as above) were first treated in one of two ways to make
cell membranes permeable to the ions of the incubation medium: (a) by perfusion for 15-20
min with a 1 mM calcium Ringer's fluid containing 2 mM diaminoethanetetra-acetic acid
(Thomas, i960); (b) by exposure, at 4 °C, to a glycerol-water (1:1) mixture for 13-18 h and
then for 10 min to a solution containing 30 mM KC1, 5 mM MgCl 2 and 30 mM potassium phosphate buffer (pH 6-5) (Pease, Jenden & Howell, 1965). After either treatment, the ventricle was
incubated for 1-1-5 h in a medium from which calcium uptake has been obtained with other
muscles (Pease et al. 1965; S. Page, 1969): a solution of 5 mM adenosine triphosphate, 6 mM
creatine phosphate, 5 mM potassium oxalate, 2 mM ethyleneglycol bis(aminoethylether)-7v^iVvtetra-acetic acid, o-8 mM CaCl s and 30 mM Tris-maleate buffer (pH 64). The subsequent fixation was according to method (i), except that the fixative was saturated with calcium oxalate.
Finally, the tissue was dehydrated in 100% ethanol and propylene oxide, and embedded in
Araldite.
Sections were cut with glass or diamond knives on a Porter-Blum microtome and usually
stained, first at 40-50 °C with a saturated solution in 5 0 % ethanol of uranyl acetate and afterwards with lead hydroxide (Karnovsky, 1961) or lead citrate (Venable & Coggeshall, 1965). The
sections were viewed in a Siemens Elmiskop I microscope. For many of the measurements of
bundle cross-sectional areas io-/*m thick sections were cut with a glass knife, and afterwards
viewed with a phase-contrast microscope.
For the purpose of calibrating the electron microscope when the precise magnification factor
was required, a diffraction grating replica was photographed immediately after the tissue section
had been examined, i.e. without an intervening change in lens current. At low magnification,
< 3000 times, image distortion was reduced by working with the intermediate lens current
switched off. Cross-sectional areas (of the subendothelial space, and of fibres and bundles) were
determined either by planimetry or by cutting out and weighing appropriate areas of the print
(see Results section for more detail). Length measurements, such as the circumference of fibres
or fibre bundles, were made on the prints with a curvimetre map-reader. It should be mentioned
that the values of the various parameters so determined were from bundles whose fibres had
sarcomeres of about 2-2-2-5 Z4111) a n d had been fixed, therefore, in a slightly stretched condition.
RESULTS
As is well known (e.g. Ecker & Wiedersheim, 1896), muscle cells of frog heart
ventricles are arranged in bundles, or trabeculae, surrounded by a layer of endothelial
cells. These bundles branch and interconnect in a complex network to form the wall of
the ventricle and the divisions between the various cavities inside it (Gompertz, 1884).
The bundle size varies considerably, as is shown by the histogram of the distribution
of the cross-sectional area of 91 such bundles in Fig. 1. Appreciable variation also
exists in the outline of bundle cross-sections, of which only the smallest are approxi-
S. G. Page ad R. Niedergerke
182
20
16
ii 12
JO
i—11 :—in . n n;—!
4
8
12
16
20
24
Cross-sectional area, 103 //m 2
iu •
28
B
8 -i
4>
is
6 •
<"d
h?
J
pi
2
4
0 20
rn
_n
4 -
ri
8
12
16
20
24
Cross-sectional area, 103 /im 2
40
60
80
Radius, /im
28
100
Fig. i. Distribution of cross-sectional areas of 91 bundles. Histograms show, A, number
of bundles per class (class division 1000 /tfn8), B, % of total area occupied by bundles
in each class against cross-sectional area; subsidiary abscissa, radius of cylindrical
bundles of the same cross-sectional area as the bundles examined. Data uncorrected for
effects of shrinkage during histological preparation of the tissue.
mately circular, the others being ribbon- or horse-shoe-shaped, or more irregular still.
One result of this irregularity is that the shortest bundle diameter rarely exceeds 50-60
/im, and the effective diffusion distance between the periphery and the innermost cells
is therefore relatively short for all bundles. (Note, for comparison, that the diameter of
cylindrical bundles of the same cross-sectional areas would range up to more than
180/im, Fig. 1.)
The first point to be discussed concerns the way in which solutes move between the
ventricular cavity and the subendothelial space within the bundle. This movement,
across the sheath of endothelial cells, could be by one of the two routes which have
been proposed for the movement of solutes across muscle capillary walls (Chambers &
Zweifach, 1947; Palade, 1953; Landis & Pappenheimer, 1963), i.e. either simple
diffusion within the aqueous spaces of the narrow clefts separating adjacent cells (Figs.
5, 6), or special transport through the cells by means of the small intracellular vesicles
which could be imagined to shuttle to and fro between opposite sides of these cells.
These alternatives were examined in 2 series of experiments. In the first series
ventricles were exposed for various periods to Ringer's fluid containing horseradish
Fine structure of frog heart cells
183
peroxidase as an extracellular marker. It was found that after a period as short as 1 min
the clefts between endothelial cells were filled with the end-product of the subsequent
histochemical reaction (cf. Fig. 8). The end-product could also be detected in the
subendothelial space and in some of the 'pinocytotic' vesicles of peripheral muscle
cells, but only very rarely in the vesicles of the endothelial cells, where it was seen to a
noticeable extent (usually in large vesicles) only after prolonged exposure to the
labelled fluid (^ 1 h). In the other series of experiments, ventricles were perfused with
Ringer's fluid containing ferritin for periods of 1 h or longer. After such times, ferritin
particles were contained in the clefts between the endothelial cells, though not in all
of them, and also, to varying extents, in the subendothelial space. The number of
vesicles inside the endothelial cells filled with ferritin was quite small. It is of interest
to mention here that in similar experiments with muscle capillaries, horseradish peroxidase was found to pass only relatively slowly through the clefts (Karnovsky, 1967)
and ferritin not at all (Bruns & Palade, 1968).
From these findings it is clear that, in the ventricle, the movement across the endothelial layer of molecules smaller than the 2 labels studied (particle diameter of ferritin
in wet crystals, 12-2 ± o-6 nm, Harrison, 1963; of horseradish peroxidase, approximately
5 nm, Karnovsky, 1967) is mainly via the clefts between, rather than the vesicles
within, the cells. Based on this result, an analysis of the time course of diffusion of
solutes between ventricular cavity and subendothelial space has been made, the particular case considered being that which arises from sudden changes in concentration of
a solute in the ventricular cavity. F01 simplicity, the fluids on either side of the endothelial cell layer are assumed to be well mixed.
Consider the following model (Fig. 2): a unicellular layer of endothelial cells which
are all equal in size and rectangular, of length y and width x, and separated by clefts of
width w; w is taken to be constant throughout the course of a cleft and to be the same
for each cleft; s is the (highly variable) length of the path of the cleft across the endothelial layer, and / its length along this layer. The flux, miy of a solute through cleft i is
given by
where cn is the concentration of the solute in the ventricular cavity and c that in the
subendothelial space, D is its diffusion coefficient within the cleft. The total flux, M,
through all clefts in unit length of a given bundle is
^ - .
(2)
y
(See legend to Fig. 2B; the assumption that the circumferential and longitudinal clefts
have the same path length si is discussed below, page 186.)
The differential equation describing the rate of concentration change within unit
length of the bundle is then
184
S. G. Page and R. Niedergerke
nr
*
nnr
Fig. 2. Model for calculating the diffusion delay across layer of endothelial cells, A,
schematic representation of a cleft between 2 adjacent endothelial cells, zv, width of
cleft; s, its path length across, /, its length along the layer of endothelial cells, B, hypothetical arrangement of endothelial layer: surface view of cells each of length y and
width x. For convenience of analysis, the cleft path length, st, across the layer is taken to
be constant along the half perimeter (x + y) of a given cell. The length (/) of cleft of this
path length (st) is then (x + y)ly per unit bundle length.
where a is the subendothelial space per unit length of bundle, comprising the aqueous
space both underneath the endothelial cells and that extending between the heart
fibres. The solution of equation (3) for the case of a concentration step in the ventricular cavity is an exponential with time constant T, which, in turn, is given by
D
ay
1
(4)
To obtain T for any one bundle, measurements of (i) w, (ii) x and y, (iii) a, and (iv) si
were made, of which values of (i) and (ii) were constant for all bundles (cf. Discussion
below), in contrast to those of (iii) and (iv), which varied and had to be determined
separately for each bundle.
(i) The cleft width w. The measured value of this, from cross-sections of bundles
fixed in aldehyde + OsO4 (by methods ii or iii), was 11 -o nm (mean of 31 clefts, 5 ventricles) in tissue that had not been stained before embedding, and 9-0 nm (12 clefts, 3
ventricles) after block staining with uranyl acetate. Both these values are smaller than
expected from the finding that ferritin particles (diameter 12-2 nm) passed through the
clefts in the living tissue; they are probably underestimated as a result of shrinkage of
the preparation during fixation and dehydration. Consequently, the appropriate value
for w was taken to be 12-5 nm, although the true value may, of course, be still greater.
(ii) Width (x) and length (y) of the endothelial cells: x was determined in crosssections of 11 bundles from the measured circumference of each bundle and the
number of clefts around it, and came to an average value of 27-0 /im (139 cells, S.E.
5-7); y, obtained in an analogous fashion from longitudinal sections, was 38-0/(111
(average from 6 bundles, 48 cells, S.E. 2-2). Neither x nor y was dependent on the
bundle size.
(iii) Subendothelial space, a. This was determined from cross-sections through the
bundles, on the assumption that its size is constant along the bundle length considered.
The major part, consisting of the space between the endothelium cells and the muscle
fibres, together with occasional broad gaps between the fibres, was evaluated by cutting
Fine structure of frog heart cells
185
Table 1. Measured parameters of heart fibre bundles and values of
calculated from them
no.
Crosssectional
area, /an 1
(i)
(2)
Bundle
Subendothelial
No. of
space,
clefts in
Min. and
% of bundle bundle cross- max. values
of st, /Jm
volume
section, n
(3)
(4)
I
200
7-9
2
210
I3-3
2
2
3
503
980
5-6
3
126
5
15-5
11 9
6
4
5
6
7
8
9
IO
ii*
12
13
14
15
1390
1790
23SO
2 550
3080
3140
3200
3290
5220
8500
11 800
16
16850
17
28600
11
124
9
io-8
17
90
11
no
15
199
10
io-8
12
124
17
29
5-4
7-4
6-6
44
49
97
104
104
(average of
bundles 4-17,
excluding n # )
(5)
-Yn
si
(6)
tj, 8
(7)
0-5- s-o
11
0-4- 1-5
o-6- 12
i-i-n-8
0-5- 63
o-6-io-o
i-8- 8-3
1-1-15-7
07-10-6
0 7 - 9-9
is
03
0-4
1-2
o-3S
o-43
2'5
2-2
1-6
1-4-167
0-5- 60
11—11-4
1 0 - 6-8
0-5-12-4
i-o- 9-2
0-1-13-6
065
o-53
o-34
0-47
o-45
0-38
2-3
2-O
036
7'3
0-64
0-38
o-33
o-43
0-38
067
3'9
I'5
2-1
4-0
1-9
1-9
2-4
i-6
2-3±o-8
(average, ±s.D.,
of bundles 4-17,
excluding n * )
• Bundle 11 has been excluded from the evaluation of the mean values in cols. 3 and 7; its
exceptionally large subendothelial space is likely to be due to some artifact or to tissue injury
during the preparative procedures.
out and weighing these regions from micrographs of each bundle. Its value, expressed
as a percentage of bundle volume, ranged from about 4 to 14% (average from 13
bundles, 9-4%), with a slight tendency to decrease with increasing bundle size (col. 3,
Table 1). An additional but small contribution is made by the narrow gaps which
separate individual muscle fibres in regions of close apposition of the cells, where the
basement membrane is absent (Figs. 15-18; see also Staley & Benson, 1968). This was
taken as 1 % of the bundle volume, a figure based on a measured gap width of 30 nm,
and the total gap length, which was actually measured in the case of 2 bundles, and in
the others estimated from a simple geometrical model of the fibre arrangement within
the bundle.
(iv) The path length, si: of every cleft around the bundle circumference was measured
in bundle cross-sections. Its value varied considerably, even for the clefts of a given
bundle, from as little as o-i to 16 /tm (col. 5, Table 1).
Equation (4), after insertion of the measured parameters just described, could, of
course, serve to calculate the time course of equilibration around muscle cells of any
186
S. G. Page and R. Niedergerke
diffusing substance whose diameter is small compared with the width of the endothelial cleft (provided the substance is not being taken up to any large extent by cells
and other structures); the particular example chosen is that of the calcium ion for
which the time course of the twitch response to a stepwise concentration change is
known (Chapman & Niedergerke, 1970). For the diffusion coefficient, D, a value of
8 x io~ 6 cm2 s ' 1 (Wang, 1953) is taken, assuming the cleft space to contain no other
material but aqueous fluid, and the result is expressed in terms of the values of
^( = 0-693 x r) calculated for each individual bundle (col. 7, Table 1). As is seen, the
average value of t^ in 13 bundles (nos. 4 to 17, excluding ir, see footnote to Table 1)
comes to 2-3 s (± o-8, S.D.). This result, which is of considerable interest for the interpretation of the kinetics of calcium action in the heart, will be discussed on page 191
below.
Two subsidiary points arising from Table 1 should be mentioned. (1) Inspection of
the data obtained from bundles 4-17 indicates that values of U are not in any consistent
way related to bundle size, over cross-sectional areas from 980 to 28600 fim2, a range
which comprises some 9 8 % of the total bundle population (estimated from 91
bundles, see Fig. I B ) . This suggests that the estimated average of ^ is, indeed, a
measure of the diffusion time for the ventricle as a whole. As to the origin of this constancy in t±, it was found, by analysing the relation between bundle size and circumference (and the related number of clefts in bundle cross-sections), that these 2
parameters vary approximately in proportion to each other: a condition, clearly, for
diffusion times to remain constant. (2) However, in bundles of very small size (crosssectional areas < 1000 /tm2), values of t^ are much shorter than the average, which can
be attributed to the high proportion of clefts with low values of s and, thus, short
mean diffusion paths (cf. large values of (i/n) £(i/$4) for bundles 1-3 in col. 6, Table 1).
Errors and assumptions
(1) Assumption of a constant cleft width, w. In 57 of the 98 clefts examined to test this
assumption (i.e. clefts with clearly defined membrane contours for most of their length), w was,
indeed, reasonably constant (e.g. Figs. 5, 6); in the others, however, the cleft narrowed for a
length of about 20-50 nm in the radial direction (Fig. 7), and for up to 0-5 fim in the longitudinal
direction (the latter value being established from serial cross-sections). The extent of the
narrowing, determined in tissue stained in the blocks, was such as to reduce iv to about 3—4 nm.
However, it is clear that even if the cleft were to be entirely occluded in these regions, the major
part of the cleft space would still remain open to the diffusing particles. For this reason, any
correction of ij on account of this impediment to diffusion is quite small, amounting to an
increase by only 2 % , or less, as can be shown by means of geometrical models along lines
similar to those previously applied by Lassen & Trap-Jensen (1970) to the analysis of diffusion
rates in the endothelial wall of muscle capillaries.
(2) Assumption of identical path length, s, for clefts extending longitudinally and circumferentially along the bundle surface. This was tested by comparing the mean values of s( obtained
from a number of different bundles in either cross-sections or longitudinal sections of the tissues.
The 2 means, 3-2 /an ( ± 2 5 , S.D. of 249 clefts) and 4-0/tm ( ± 1 8 , S.D. of 31 clefts) respectively,
were not significantly different on the i-test (P = 0 1 ) .
(3) The simple model in Fig. 2B of endothelial cells with rectangular outlines cannot be
expected to hold strictly, since cell boundaries are more likely to run an oblique or zig-zag
course along the length and breadth of the bundle surface. The effect of such obliquities would
be to increase both x and y above the values taken in calculating tj, whereas values of s would be
overestimated in this calculation since, obviously, the shortest diffusion path through the clefts
Fine structure of frog heart cells
187
is in a direction perpendicular to y or x, rather than in the plane of the section in which it had
been determined. In assessing the errors which arise in this way, the courses of longitudinal
clefts have been followed in series of cross-sections of bundles at 5-/wn intervals and in further
subdivisions of i-fim intervals. As a measure of the obliquity, the angles were determined
between the lines along the bundle surface connecting the midpoints of individual clefts and
either (a) the vertical, in the case of the sections 5 /tm apart, or (A) the line through the 5-/MT1
interval points, in the case of the sections separated by 1 /an. The average angle so obtained was
in (a) 150 ( ± 180, S.D. of 23), and of probably similar magnitude in (b) as far as could be judged
by means of the present, not very precise method. Assuming the course of circumferential clefts
to be of similar obliquity to that of the longitudinal ones, the errors in x, y and s were obtained
from simple geometrical principles, their combined effect being to reduce the calculated value of
<l by about 25 %.
(4) Of the remaining uncertainties, the most important is due to shrinkage of the preparation
during fixation and dehydration and the resulting distortion of some of the above parameters.
An estimate of the error involved has not been attempted because of the arbitrariness of the
assumptions which would have to be made.
The sarcoplasmic reticulum
When viewed in either longitudinal or transverse sections the sarcoplasmic reticulum
(SR) seems to consist of a number of small vesicles and tubules situated in the vicinity
of the myofibrils and, to a lesser extent, in other parts of the cells (e.g. Lindner, 1957;
Scheyer, i960; Naylor & Merrillees, 1964; Staley & Benson, 1968; Sommer &
Johnson, 1969; Baldwin, 1970). In re-examining this structure 2 questions were considered in detail: whether these vesicles and tubules are connected with each other in
any consistent pattern; and how great a volume they occupy within the cells. The
results are best described with reference to the schematic diagram in Fig. 3 of an
inside view of a portion of a heart fibre containing 2 myofibrils together with elements
of the SR. As is shown, the SR is a loose network of fine tubules extending between
adjacent myofibrils and also in regions beneath the cell surface. The cross-section of
these tubules is approximately circular, their diameter varying between about 35 and
60 nm. The major proportion of the tubules are transversely oriented at Z-line level
and form a continuous system, as suggested by several observations. In longitudinal
sections, under favourable conditions such as obtained in Figs. 9 and 10, some of the
tubules could be followed for the whole width of a myofibril and others from the edge
of a myofibril to that of its neighbour; yet others extended from areas close to a
myofibril to the inner surface of the cell membrane (Figs. 9, 12, 13) or into the cell
interior. In cross-sections cut at Z-line level, the tubules would lie juxtaposed to a
myofibril, encircling it some way before passing to a different region of the cell (Figs.
15-19). To estimate the extent of distribution of these tubules a count was made, in
longitudinal sections, of their profiles at either end, or both, of the Z-lines. The result,
a total of 728 profiles per 609 Z-lines, suggests that the tubules surround some 60 % of
the myofibrillar circumference.
As to the less-frequent longitudinal tubules, considerable lengths (1-2 /tm) of them
were found, on occasion, in the interfibrillar spaces (Figs. 9-11), but more often only
short lengths were to be seen. However, it was shown by means of serial sections that
these, too, were segments of a continuous structure, though sectioned obliquely. Thus,
individual tubules running the whole length of a sarcomere could be pieced together
i88
S. G. Page and R. Niedergerke
Fig. 3. Schematic representation of the organization of the SR in frog heart fibres.
Fibre interior showing sections of the cell membrane and 2 myofibrils together with
tubules and terminals of the SR. A large proportion of the tubules partially surround
the myofibrils at Z-line level; connected with these are others oriented in a roughly
longitudinal direction. Portions of both tubules arefilledwith dense material (arrows).
At the cell periphery branches of the SR tubules terminate, usually at I-band level, in
disk-like structures closely apposed to the inner surface of the cell membrane.
from consecutive (longitudinal) sections. The frequency of occurrence of these
tubules was obtained (in longitudinal sections) by determining the cumulative length
of tubular portions adjacent to a number (260) of different sarcomeres. The result, a
tubular length equivalent to 6% of the total length of all sarcomeres examined,
suggests that, on average, every second sarcomere of each myofibril is accompanied by
one such tubule. (This is a simple geometrical consequence of the use of sections
approximately 100 nm thick, and an average myofibrillar diameter taken as 1 /im.)
In regions of the fibre periphery, tubules of the SR approach the cell membrane,
where they bend to lie closely apposed to the inner membrane surface for up to 1 /tm
before terminating. In forming these terminals the tubules usually broaden, parallel
with the cell surface, into disk-like structures. The frequency of the terminals is
considerable, as is illustrated by the finding that (in longitudinal sections) the profile
of such a terminal occurs in association with an average of 75 % of the sarcomeres of
superficial myofibrils. A clear predominance of location exists for the region of the
I-band, which contains more than three-quarters of the terminals found. It is also of
interest that, in a few, perhaps geometrically favourable, sections the narrow gap
Fine structure of frog heart cells
189
(10-20 nm) between internal membrane surface and tubular terminal was bridged by
small bars of dense material at a repeat distance of approximately 20-25 nm> quite
similar to the structures described in both the triads of skeletal muscles (Revel, 1962;
Franzini-Armstrong, 1970) and the couplings or dyads of mammalian cardiac fibres
(Johnson & Sommer, 1967; Fawcett & McNutt, 1969).
More detailed points of interest may be summarized as follows:
The volume of the SR. If this is expressed as a percentage of the volume of an individual myofibril (average diameter 1 /tm) associated with the SR, its size comes to
about 0-5 %, which is made up, approximately, of (a) 0-2 % for the transverse tubules,
(b) o-i % for the longitudinal tubules, and (c) 0-2 % for the terminals. These estimates
were obtained in the cases of (a) and (b) from the above figures of the frequency of the
2 types of tubule and an average diameter of the tubules of 50 nm, and of (c) by taking
the average length of the terminals to be 12-5 % of the circumference of the myofibrils
and measured values for their height and width of 0-25 /tm and 40 nm, respectively.
Results with extracellular markers and those obtained by use of histochemicalprocedures.
In experiments in which ventricles had been perfused before fixation with fluids containing the extracellular markers ferritin or horseradish peroxidase, neither of these
markers was detected within the tubules described above (e.g. Fig. 8), indicating that
the tubules are, indeed, elements of the SR rather than of a T-system. In another
series, the histochemical procedures outlined in the Methods sections were used to
investigate whether the SR is able to accumulate calcium and store it subsequently as
calcium oxalate within its lumen. The results of these experiments, also, were negative
in failing to show any oxalate precipitate within the cells. Although the idea of the SR
in this tissue storing calcium therefore remains unsupported, the possibility of the
existence of such a mechanism cannot be ruled out, since the SR might differ in some
other, perhaps trivial, aspect from the SR in those skeletal muscles in which calcium
accumulation was demonstrated with the present method (Pease et al. 1965; Page,
1969). For example, the cardiac SR might be more susceptible to damage during the
preparative procedures and so lose the ability to transport calcium more readily than
the SR of skeletal muscle cells.
Enclosures of dense material. As may be seen in Fig. 10, and as is known from earlier
work (Naylor & Merrillees, 1964; Sommer & Johnson, 1969), some of the vesicles and
tubules of the SR are filled with an electron-dense material which is usually surrounded by a light zone. The proportion of filled tubules was approximately 5 % of the
total SR, as determined by the same quantitative procedures as used for estimating the
extent of the distribution of the SR.
Properties of ventricular muscle fibres related to fibre size
Frog heart cells, which have an average diameter of about 5 /tm (see, for example,
Marceau, 1904), are thin compared with skeletal muscle fibres (average diameter in
frog sartorius, 84 /im: Mayeda, 1890), and their size varies considerably. To illustrate
this variation in size, cross-sectional areas of 190 fibres, all from the same bundle, have
been plotted in the histogram of Fig. 4. The average area is 17-4/im2, with a range
extending from below 2 up to 85 /im2, and in other bundles up to more than 100 /(ml
S. G. Page and R. Niedergerke
190
50
40
t 30
I 20
10
20
40
60
80
Cross-sectional area, /1m2
12
_
8
-=
4
20
40
60
80
Cross-sectional area,
01
2
3
4
Radius, ur
Fig. 4. Distribution of cross-sectional areas of 190 fibres from a single bundle. Histograms show A, number of fibres per class (class division 5 /im1), and B, % of total area
occupied by fibres in each class against fibre cross-sectional area; subsidiary abscissa,
radius of cylindrical fibres of the same cross-sectional area as that of the fibres
examined.
Also indicated in Fig. 4 are the radii of cylindrical fibres of the same cross-section, to
provide a measure of the fibre width; but it should be mentioned that the majority of
fibres are elliptical or more complex in cross-sectional outline (e.g. Fig. 21).
Several physiological properties are critically dependent on fibre size and its
variation, and it is useful, therefore, to summarize these properties while discussing
the results illustrated in Fig. 4 and also in Figs. 20 and 21. When microelectrodes are
used to measure resting and action potentials, the number of successful cell penetrations by the electrode, i.e. those yielding stable resting potentials, is quite small, and
even the potentials (of — 83 mV) obtained after successful penetrations are probably
underestimated (by some 10 mV) due to the shunt of the membrane ' battery' resulting
from the hole in the cell surface made by the electrode (Niedergerke & Orkand, 1966).
The depolarizing effect of this membrane shunt is related to the fibre size (see Katz &
Thesleff, 1957). More precisely, the amount of the depolarization due to the shunt
increases with the magnitude of the cellular input impedance which, in turn, is proportional to (a.u)~i, a and u being the cross-sectional area and circumference of the
fibre, respectively. In most fibres the product (a.u) is small, the average being 410
/«n3, but it reaches values of 6000 /tm3, and more, in a few fibres (in 7 of 2000 fibres
Fine structure of frog heart cells
191
examined, corresponding to a fibre volume of 3 % of the total). Most likely, stable high
resting potentials are obtained only in these large fibres. It should, however, be added
that another factor probably contributing to stable resting potentials resides in the
syncytial connexions of the tissue, through which each cell helps to maintain the
resting potential of its neighbours.
Another parameter of physiological importance, affected by variation in cell size, is
the ratio s/v of the surface over the volume of the fibres. Both the conduction velocity
and the kinetics of tracer ion fluxes depend on this ratio which, in ventricle fibres, has
an average value of 1-27/tm"1. (Note that the relevant figure used for calculating
tracer ion fluxes, of I-I or ro/im" 1 (Niedergerke, 19636, and present study), is a
weighted mean which takes into account the volume of tissue occupied by each fibre.)
A point of interest is that in the upper range of fibre size considerably smaller sjv
values than this are to be found, i.e. values < o-6 /6m"1 in 6 out of 200 fibres examined.
Since the conduction velocity of the excitatory impulse is likely to increase in proportion with (s/v)~i (Hodgkin, 1954), it is clear that fibres in this range conduct impulses
more rapidly, by a factor of ^1-5, than those of average size. On this basis, it is
expected that impulses conducted over long distances inside the heart are carried
mainly by these large fibres. As the cross-section of one such fibre shows (see Fig. 20;
s/v value of large fibre, 0-49 /(m"1), its myofibrillar content is small relative to the
content of glycogen granules. This is in contrast to the situation in large fibres of
ribbon-shaped cross-section (Fig. 21) which have a relatively high density of myofibrils; but their sjv values are near average and conduction by these fibres should
therefore not be rapid. It will also be noted that the fibre in Fig. 20 with the low
myofibrillar but high glycogen content resembles a Purkinje fibre of large mammals,
though differing from the latter in failing to occur in association with other similar
fibres in separate bundles or, even, clearly defined groups. Nevertheless, the histological similarity associated with a predicted similarity in function is remarkable, if as
yet unexplained.
DISCUSSION
Two results of this work are relevant for understanding the function of the frog
heart: (1) the estimate, based on electron-microscopic data, of the time course of
diffusion of solutes between ventricular cavity and heart cells, and (2) the size and
distribution of the sarcoplasmic reticulum inside heart cells. With regard to (1), taking
as a measure of the diffusion rate the half-time, t^, with which calcium ions equilibrate
at the surface of the heart cells, we obtained a value of approximately 2 s for this parameter after an assumed step change of concentration in the ventricular cavity. (See
p. 186 for the various uncertainties to which this estimate is subject.) For a comparison of this result with the physiological time course of calcium action, experiments
concerned with the kinetics of tension responses to a sudden change of calcium concentration might be recalled (Chapman & Niedergerke, 1970). These responses
usually consisted of an initial rapid and a later slow phase of tension change, and for
the initial phase a half-time as low as 3 s was obtained (see fig. 8 of that paper). The
192
S. G. Page and R. Niedergerke
closeness of this value to the one above clearly supports the idea proposed earlier that
the rapid tension changes are related to a process immediately dependent on the
change of calcium concentration at the surface of the heart cells. However, the difference between the 2 values is probably also real, and may be greater still, since our
present figure of 2 s is likely to be overestimated, being based on a value for the cleft
width (12-5 run) just wide enough to allow passage of ferritin molecules (a wider cleft
width would give more rapid diffusion rates and thus lower values for t^). Indeed, a
difference between the time courses under comparison is expected for 2 reasons: the
mixing of solutes in the ventricular cavity and especially in the subendothelial space is
unlikely to occur instantaneously; and the presence of substantial numbers of adsorption sites for calcium on tissue surfaces tends to slow the diffusion rate of this ion
(Niedergerke, 1957), an effect which has been neglected in the present treatment for
lack of sufficient information on the density of these sites.
With regard to the structure of the sarcoplasmic reticulum, our findings suggest
that it is more extensively developed in frog heart cells than has previously been
thought (e.g. Staley & Benson, 1968); a certain regularity in its organization has also
been demonstrated. Indeed, in several respects the arrangement of the SR is similar to
that in mammalian and avian hearts, in which some elements of the tubular network
are also oriented at Z-line level surrounding the myofibrils, while others make contact
with the inner surface of the cell membrane in structures variously termed ' flattened
sacs' (Simpson & Rayns, 1968), 'couplings' (Sommer & Johnson, 1968, 1969), or
'subsarcolemmal cisternae' (Fawcett & McNutt, 1969). The main differences are in
the greater extent of that part of the SR which in mammalian and avian fibres surrounds the myofibrils at levels other than the Z-line, and the presence, at least in the
case of mammalian ventricular fibres, of terminals situated at the intracellular surface
of the T-tubules. On account of both these factors, therefore, the volume of SR associated with each myofibril is probably much smaller in the frog than in the mammalian heart, as is borne out by a comparison of the appropriate quantitative data.
Thus, in rat ventricle, the volume of the SR has recently been reported to be about
3'5 % °f t n e c e ^ volume (E. Page, McCallister & Power, 1971), or 7-3 % of the myofibrillar volume, and is therefore some 15 times greater than the corresponding figure
(°'5 %) m t n e fr°g ventricle. A still greater difference exists between frog heart and
skeletal muscle cells; for example, the volume of the SR in sartorius fibres (of 9-14 %
the myofibrillar volume (S. Page, 1964; Peachey, 1965)) is 20- to 30-fold that in the heart
fibres. Since there are good reasons for believing that the SR in both skeletal muscle
and rat ventricle fibres provides a cellular store for calcium which is responsible, on
release, for activation of contraction, it is clear, assuming the SR in the frog heart to
have the same function, that a much smaller capacity for this purpose exists inside these
heart cells. This conclusion (see also Niedergerke, 1963 a) serves to support the hypothesis that the major source of activator calcium in this tissue is derived from the
external medium by inward flow of calcium ions during each action potential (Niedergerke & Orkand, 1966). However, it must be admitted that the argument is still
indirect and, even, that the evidence itself may be questioned. For example, it could be
argued that the ability of the SR to store calcium is exceptionally high in the frog heart
Fine structure of frog heart cells
193
and so provides large enough quantities of activator. Also, the possibility is not
entirely excluded that living frog heart cells may, in fact, contain elements of SR
which readily disintegrate during fixation of the tissue. Clearly, different or more
advanced techniques are required to help answer these questions conclusively.
We are grateful for the technical assistance of Miss C. Tate. The work was supported by a
grant from the British Heart Foundation.
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STALEY, N. A. & BENSON, E. S. (1968). The ultrastructure of frog ventricular cardiac muscle and
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THOMAS, L. J. (i960). Ouabain contracture of frog heart: Ca44 movements and effect of
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[Received 24 November 1971)
Fine structure of frog heart cells
V :
*
•
/
• • :
/
?r,'j
' *
\
Figs. 5-7. For legend see p. 196.
13-2
196
S. G. Page and R. Niedergerke
Figs. 5-7. Illustration of 3 different types of cleft in endothelial layer. Fig. 5, cleft of
relatively short, Fig. 6, of long path length, both, however, of constant width; Fig. 7,
cleft with constriction at its entrance. Arrows mark entrance into, or exit from, clefts;
double-headed arrow, fenestration in the wall lining the cleft (very rare). Figs. 5 and 6
are sections stained with uranyl acetate and lead; x 50000. Fig. 7, tissue stained with
uranyl acetate in the block, followed by uranyl acetate and lead staining of the sections,
x 140000.
Fig. 8. Section through portion of endothelial cell layer and adjacent region of a fibre
bundle. Tissue exposed to peroxidase and later incubated for histochemical reaction.
Note the electron-dense end-product of the reaction inside the cleft, in the subendothelial space, and in a thin layer at the surface of the endothelial cells, but its
absence within the muscle fibres (arrows indicate terminals of the SR). Sections
stained with uranyl acetate, x 52000.
Fig. 9. Elements of the SR in a longitudinal section through a muscle cell. Tubules
oriented in transverse direction (r.h.s. arrows) extending across the whole width of a
myofibril whose surface has been grazed by the section. The interfibrillar gap, below,
contains tubules oriented in both transverse (l.h.s. arrow) and longitudinal (doubleheaded arrows) directions. Section stained with uranyl acetate and lead, x 37000.
Fine structure of frog heart cells
•4
* ^ * * •*•«•*
•
-
••
I
'
!
,
•
.
•
•
'
197
198
S. G. Page and R. Niedergerke
Figs. 10-14. Longitudinal sections of heart fibres illustrating various structural aspects
of the SR. All sections stained with uranyl acetate and lead.
Fig. 10. Portions of 2 fibres separated by intercellular gap (arrow head). Upper
fibre: terminal (te) in close apposition to surface membrane and a tubule (tr) extending
from the surface towards the centre of the fibre. Lower fibre: tubules in both transverse (tr) and longitudinal (It) directions and connexion between 2 such tubules
(double-headed arrow); dense material in some of the longitudinal tubules (arrows),
x 32000.
Fig. 11. Interfibrillar space containing segments of longitudinal tubules (arrows),
x 34000.
Fig. 12. Terminal (te) of SR with tubule attached to it (tr). x 50000.
Fig. 13. As in Fig. 12; also showing connexion between 2 tubules (tr and It).
x 50000.
Fig. 14. Another terminal (te), whose associated tubule is not in the plane of section
(frequent occurrence), x 50000.
Fine structure of frog heart cells
r \
tr
12
14
2OO
S. G. Page and R.
Niedergerke
Figs. 15-19. Cross-sections of heart muscle fibres, all near Z-line level (Z), further
illustrating features of the SR. Note, also, the absence of basement membrane when
intercellular gap (arrow heads) is narrow and its presence in wide gaps (asterisks). All
sections stained with uranyl acetate and lead.
Fig. 15. Several terminals (te) in apposition to the cell membrane, x 40000.
Figs. 16, 17. Tubules (arrows) around periphery of myofibrils, connected to terminals (te). x 40000.
Figs. 18, 19. Tubules (arrows) around periphery of myofibrils, some with branches
to cell surface (especially in Fig. 19). x 40000.
Fine structure of frog heart cells
zor
te
202
S. G. Page and R. Niedergerke
Figs. 20, 21. Comparison of fibre of approximately circular cross-section with others of
more complex shape. Sections stained with uranyl acetate and lead. Note different
magnifications in the 2 figures.
Fig. 20. Cross-section of large cylindrical fibre containing only few myofibrils but
considerable amounts of glycogen. (Note, also, the small surrounding fibres whose
myofibrillar content is high by comparison.) x 11 500.
Fig. 21. Several ribbon-shaped fibres in cross-section (cell boundaries have been
picked out with ink for clarity). Note that relative myofibrillar density of these fibres is
higher than that of the large fibre in Fig. 20, except in the regions in which there is a
marked increase in cell width (asterisk), x 4500.
Fine structure of frog heart cells
203
5//m