STRUCTURES OF PHYSIOLOGICAL INTEREST IN THE FROG HEART ... · Fine structure of frog heart cells 181 finally embedded in Araldite (CIBA). In some instances, the tissue was stained

J. Cell Sri. II, 179-203 (1972)Printed in Great Britain

STRUCTURES OF PHYSIOLOGICAL INTERESTIN THE FROG HEART VENTRICLE

SALLY G. PAGE AND R. NIEDERGERKEBiophysics Department, University College London, Govier Street, London WCiE 6BT,England

SUMMARY

Two structures of physiological interest in frog heart ventricles have been examined indetail: (a) the layer of endothelial cells which encloses each bundle of heart fibres, and (b) thesarcoplasmic reticulum (SR) inside the heart fibres. Some additional observations on fibre sizesand 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 con-clusion results from experiments made with the extracellular markers ferritin and horseradishperoxidase.

A diffusion equation describing the movement of solutes into and out of the fibre bundle hasbeen derived using several geometrical parameters, such as the length and width of the clefts andthe size of the extracellular aqueous space inside the bundle, all of which were determined fromelectron micrographs of the tissue.

The theoretical solution for a stepwise change of external calcium concentration gives a half-time of 23 s (± c8 s, s.D. of 13 bundles) for diffusion equilibrium at the surface of the heartfibres; this value, however, is likely to be an overestimate, by some 20-30%, on account ofseveral systematic errors which are described.

The sarcoplasmic reticulum in heart fibres consists of a network of thin tubules whichpartially 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 innersurface of the cell membrane. The volume of the SR is estimated to be approximately 0-5 % ofthe myofibrillar volume of the cells.

Cross-sectional areas of heart fibres (and also their shapes) vary considerably, from less than2 to more than 100 fim1 (average 174 /4m1). Fibres of large size and small surface/volume ratiocontain many fewer myofibrils and more glycogen granules than fibres of the same size butlarger 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 variouslower 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 featurescommon to these tissues have become apparent. For example, the heart cells of theseanimals are small and devoid of a T- (transverse tubular) system, and the sarcoplasmicreticulum (SR) is sparse by comparison with that in most skeletal muscle cells. Duringrecent 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 tolight requiring closer investigation, which has been undertaken in the present work.

One of the structures examined is the sheath of endothelial cells which surroundsindividual heart fibre bundles or trabeculae (cf. Ecker & Wiedersheim, 1896), and ourattention has been focused on the nature and size of the pathways, present in thissheath, for the movement of ions and other solutes between the ventricular cavity andthe surface of the heart cells. On the basis of histological data which we obtained adiffusion equation has been set up describing the theoretical time course of such ionmovements, in particular that for calcium. From the comparison of this writh theexperimentally determined time course of the tension responses to changes of externalcalcium concentration (Chapman & Niedergerke, 1970) essential information for theinterpretation of these responses is obtained.

Another subject of study has been the structure of the sarcoplasmic reticulum offrog ventricle fibres. Our intention here was to assess the precise distribution andvolume of this organelle within the cells and so obtain an estimate of the intracellularstorage capacity of this tissue for calcium, the activator of contraction.

Finally, some observations on fibre sizes and types have been made. These are rele-vant 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 throughthe atrial-ventricular orifice, and the aortic trunk together with the aortic valves was removed toallow rapid perfusion of the ventricle. For routine fixation, the ventricles were first perfusedwith a Ringer's fluid containing io~° g/ml tetrodotoxin, which prevented contraction during thesubsequent perfusion with the fixative. In the course of this work, various methods of fixationwere used (usually at 20-22 °C, but occasionally at 4 °C): (i) fixation in a solution of 1 % OsO4

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 005 M phosphate buffer at pH 7-0-7-4), followed by aperiod (several hours) of washing of the tissue in a 005 M phosphate buffer solution and a final05-h period of fixation in a solution of 1 % OsO4 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 % paraformalde-hyde (Karnovsky, 1967) and 0-08 M cacodylate buffer (pH 7-2-7-4), followed by washing forseveral hours in a 0 1 M cacodylate buffer fluid and a final 0'5-h fixation in a solution of 1 %OsO4 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 sarco-plasmic 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 bymethods (i) and (ii).

After fixation in the case of (i), or after the first fixation period in (ii) and (iii), the ventricleswere 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 bundleswere roughly parallel and in which little branching occurred. Many of these bundles, taken fromthe wall of the ventricular subcavities, had been oriented, approximately, in a longitudinaldirection within the ventricle (i.e. from its base to tip), and they are therefore representative ofthe bundles studied by Chapman & Niedergerke (1970) in experiments with which the resultsof the present work are to be compared. Other bundles, however, were from different regions ofthe ventricle, but a systematic study of any of their properties relating to orientation or locationhas not been made. The isolated bundles were dehydrated in an ethanol series (in the case ofmethods (ii) and (iii) after the final fixation in OsO4), 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 dehydra-tion in a 05 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' stainingextracted the glycogen granules from the myocardial cells, it was used mainly when examiningthe endothelial sheath.

Some ventricles were perfused before fixation (by methods (i) or (ii) above) with a Ringer'sfluid containing the extracellular marker ferritin ( 0 1 - 0 2 mg/ml). For others, the perfusion fluidcontained 1-2 mg/ml horseradish peroxidase, and subsequent fixation was with 1% para-formaldehyde, 1-5 % glutaraldehyde (o-i M cacodylate buffer, pH 7-2-7-4). In the latter casethe isolated bundles were subsequently rinsed, first in o-i M cacodylate buffer fluid and then indistilled water, before a 10—20 min incubation period in a medium containing 005 % 3,3'-diaminobenzidine tetrahydrochloride, o-oi % HaO2 and 0-05 M Tris maleate buffer (pH 76)(Karnovsky, 1967). After incubation the bundles were again rinsed in water and finally fixed in1% OsO< (method (i)).

In experiments designed to identify intracellular structures which might be capable of storingcalcium ions, the ventricles (cannulated as above) were first treated in one of two ways to makecell membranes permeable to the ions of the incubation medium: (a) by perfusion for 15-20min 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 andthen for 10 min to a solution containing 30 mM KC1, 5 mM MgCl2 and 30 mM potassium phos-phate buffer (pH 6-5) (Pease, Jenden & Howell, 1965). After either treatment, the ventricle wasincubated for 1-1-5 h in a medium from which calcium uptake has been obtained with othermuscles (Pease et al. 1965; S. Page, 1969): a solution of 5 mM adenosine triphosphate, 6 mMcreatine phosphate, 5 mM potassium oxalate, 2 mM ethyleneglycol bis(aminoethylether)-7v^iVv-tetra-acetic acid, o-8 mM CaCls and 30 mM Tris-maleate buffer (pH 64). The subsequent fixa-tion 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 inAraldite.

Sections were cut with glass or diamond knives on a Porter-Blum microtome and usuallystained, first at 40-50 °C with a saturated solution in 50% ethanol of uranyl acetate and after-wards with lead hydroxide (Karnovsky, 1961) or lead citrate (Venable & Coggeshall, 1965). Thesections were viewed in a Siemens Elmiskop I microscope. For many of the measurements ofbundle cross-sectional areas io-/*m thick sections were cut with a glass knife, and afterwardsviewed with a phase-contrast microscope.

For the purpose of calibrating the electron microscope when the precise magnification factorwas required, a diffraction grating replica was photographed immediately after the tissue sectionhad 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 currentswitched off. Cross-sectional areas (of the subendothelial space, and of fibres and bundles) weredetermined 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 fibresor fibre bundles, were made on the prints with a curvimetre map-reader. It should be mentionedthat the values of the various parameters so determined were from bundles whose fibres hadsarcomeres 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 heartventricles are arranged in bundles, or trabeculae, surrounded by a layer of endothelialcells. These bundles branch and interconnect in a complex network to form the wall ofthe 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 distributionof the cross-sectional area of 91 such bundles in Fig. 1. Appreciable variation alsoexists in the outline of bundle cross-sections, of which only the smallest are approxi-

182 S. G. Page ad R. Niedergerke20

16

ii 12JO

i—11 :—in .n n;—!4 8 12 16 20 24 28

Cross-sectional area, 103 //m2

iu •

8 -

4>

is 6 •

<"d

h? 4 -

2

J- i

r

B

pi

_nnr i

4 8 12 16 20 24 28

Cross-sectional area, 103 /im2

0 20 40 60 80Radius, /im

100

Fig. i. Distribution of cross-sectional areas of 91 bundles. Histograms show, A, numberof bundles per class (class division 1000 /tfn8), B, % of total area occupied by bundlesin each class against cross-sectional area; subsidiary abscissa, radius of cylindricalbundles of the same cross-sectional area as the bundles examined. Data uncorrected foreffects 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 cellsis therefore relatively short for all bundles. (Note, for comparison, that the diameter ofcylindrical bundles of the same cross-sectional areas would range up to more than180/im, Fig. 1.)

The first point to be discussed concerns the way in which solutes move between theventricular 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 havebeen proposed for the movement of solutes across muscle capillary walls (Chambers &Zweifach, 1947; Palade, 1953; Landis & Pappenheimer, 1963), i.e. either simplediffusion 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 vesicleswhich 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 seriesventricles were exposed for various periods to Ringer's fluid containing horseradish


peroxidase as an extracellular marker. It was found that after a period as short as 1 minthe clefts between endothelial cells were filled with the end-product of the subsequenthistochemical reaction (cf. Fig. 8). The end-product could also be detected in thesubendothelial space and in some of the 'pinocytotic' vesicles of peripheral musclecells, but only very rarely in the vesicles of the endothelial cells, where it was seen to anoticeable extent (usually in large vesicles) only after prolonged exposure to thelabelled fluid (^ 1 h). In the other series of experiments, ventricles were perfused withRinger's fluid containing ferritin for periods of 1 h or longer. After such times, ferritinparticles were contained in the clefts between the endothelial cells, though not in allof them, and also, to varying extents, in the subendothelial space. The number ofvesicles inside the endothelial cells filled with ferritin was quite small. It is of interestto mention here that in similar experiments with muscle capillaries, horseradish per-oxidase 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 endo-thelial layer of molecules smaller than the 2 labels studied (particle diameter of ferritinin wet crystals, 12-2 ± o-6 nm, Harrison, 1963; of horseradish peroxidase, approximately5 nm, Karnovsky, 1967) is mainly via the clefts between, rather than the vesicleswithin, the cells. Based on this result, an analysis of the time course of diffusion ofsolutes between ventricular cavity and subendothelial space has been made, the parti-cular case considered being that which arises from sudden changes in concentration ofa solute in the ventricular cavity. F01 simplicity, the fluids on either side of the endo-thelial cell layer are assumed to be well mixed.

Consider the following model (Fig. 2): a unicellular layer of endothelial cells whichare all equal in size and rectangular, of length y and width x, and separated by clefts ofwidth w; w is taken to be constant throughout the course of a cleft and to be the samefor each cleft; s is the (highly variable) length of the path of the cleft across the endo-thelial layer, and / its length along this layer. The flux, miy of a solute through cleft i isgiven by

where cn is the concentration of the solute in the ventricular cavity and c that in thesubendothelial 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 cleftshave the same path length si is discussed below, page 186.)

The differential equation describing the rate of concentration change within unitlength of the bundle is then


* nrnnrFig. 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 ofcleft; s, its path length across, /, its length along the layer of endothelial cells, B, hypo-thetical arrangement of endothelial layer: surface view of cells each of length y andwidth x. For convenience of analysis, the cleft path length, st, across the layer is taken tobe constant along the half perimeter (x + y) of a given cell. The length (/) of cleft of thispath length (st) is then (x + y)ly per unit bundle length.

where a is the subendothelial space per unit length of bundle, comprising the aqueousspace both underneath the endothelial cells and that extending between the heartfibres. The solution of equation (3) for the case of a concentration step in the ventri-cular cavity is an exponential with time constant T, which, in turn, is given by

ayD 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. Discussionbelow), in contrast to those of (iii) and (iv), which varied and had to be determinedseparately for each bundle.

(i) The cleft width w. The measured value of this, from cross-sections of bundlesfixed in aldehyde + OsO4 (by methods ii or iii), was 11 -o nm (mean of 31 clefts, 5 ven-tricles) in tissue that had not been stained before embedding, and 9-0 nm (12 clefts, 3ventricles) after block staining with uranyl acetate. Both these values are smaller thanexpected from the finding that ferritin particles (diameter 12-2 nm) passed through theclefts in the living tissue; they are probably underestimated as a result of shrinkage ofthe preparation during fixation and dehydration. Consequently, the appropriate valuefor 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 cross-sections of 11 bundles from the measured circumference of each bundle and thenumber 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 thebundle size.

(iii) Subendothelial space, a. This was determined from cross-sections through thebundles, 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 musclefibres, together with occasional broad gaps between the fibres, was evaluated by cutting


Table 1. Measured parameters of heart fibre bundles and values ofcalculated from them

Bundleno.( i )

I

2

3

456789

IO

i i *

1 2

13

14

1516

17

Cross-sectionalarea, /an1

( 2 )

2 0 0

2 1 0

503

9801390179023SO2 550308031403200329052208500

11 8001685028600

Sub-endothelial

space,% of bundle

volume(3)

7-9I3-35-6

1 2 6

15-511 91 2 4

io-89 0

n o1 9 9

io-81 2 4

5-47-46-69 7

1 0 4

(average ofbundles 4-17,

excluding n # )

No. ofclefts in

bundle cross-section, n

(4)

2

2

3

56

1 1

9171 1

151 0

1 2

17

29

4449

104

Min. andmax. values

of st, /Jm(5)

0-5- s-o0-4- 1-5o-6- 12i-i-n-80-5- 63o-6-io-oi-8- 8-31-1-15-707-10-60 7 - 9-91-4-1670-5- 601 1 — 1 1 - 4

1 0 - 6-80-5-12-4i-o- 9-20-1-13-6

- Y -n si

(6)

1 1

i s1-2

o-430 6 5

o-53o-340-47o-450-380 3 60-640-38o-33o-430-380 6 7

tj, 8(7)

0 30-4

o-3S2 '52-2

1-6

3'9I ' 5

2-32-O

7'32 - 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 fromexceptionally large subendothelial spaceduring the preparative procedures.

the evaluation of the mean values in cols. 3 and 7; itsis likely to be due to some artifact or to tissue injury

out and weighing these regions from micrographs of each bundle. Its value, expressedas a percentage of bundle volume, ranged from about 4 to 14% (average from 13bundles, 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 whichseparate individual muscle fibres in regions of close apposition of the cells, where thebasement membrane is absent (Figs. 15-18; see also Staley & Benson, 1968). This wastaken 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 inthe others estimated from a simple geometrical model of the fibre arrangement withinthe bundle.

(iv) The path length, si: of every cleft around the bundle circumference was measuredin bundle cross-sections. Its value varied considerably, even for the clefts of a givenbundle, from as little as o-i to 16 /tm (col. 5, Table 1).

Equation (4), after insertion of the measured parameters just described, could, ofcourse, serve to calculate the time course of equilibration around muscle cells of any


diffusing substance whose diameter is small compared with the width of the endo-thelial cleft (provided the substance is not being taken up to any large extent by cellsand other structures); the particular example chosen is that of the calcium ion forwhich the time course of the twitch response to a stepwise concentration change isknown (Chapman & Niedergerke, 1970). For the diffusion coefficient, D, a value of8 x io~6 cm2 s ' 1 (Wang, 1953) is taken, assuming the cleft space to contain no othermaterial 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, theaverage 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 inter-pretation of the kinetics of calcium action in the heart, will be discussed on page 191below.

Two subsidiary points arising from Table 1 should be mentioned. (1) Inspection ofthe data obtained from bundles 4-17 indicates that values of U are not in any consistentway related to bundle size, over cross-sectional areas from 980 to 28600 fim2, a rangewhich comprises some 98% of the total bundle population (estimated from 91bundles, see Fig. IB). This suggests that the estimated average of ^ is, indeed, ameasure of the diffusion time for the ventricle as a whole. As to the origin of this con-stancy in t±, it was found, by analysing the relation between bundle size and circum-ference (and the related number of clefts in bundle cross-sections), that these 2parameters vary approximately in proportion to each other: a condition, clearly, fordiffusion times to remain constant. (2) However, in bundles of very small size (cross-sectional areas < 1000 /tm2), values of t^ are much shorter than the average, which canbe attributed to the high proportion of clefts with low values of s and, thus, shortmean 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 thisassumption (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 alength of about 20-50 nm in the radial direction (Fig. 7), and for up to 0-5 fim in the longitudinaldirection (the latter value being established from serial cross-sections). The extent of thenarrowing, 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 majorpart of the cleft space would still remain open to the diffusing particles. For this reason, anycorrection of ij on account of this impediment to diffusion is quite small, amounting to anincrease by only 2%, or less, as can be shown by means of geometrical models along linessimilar to those previously applied by Lassen & Trap-Jensen (1970) to the analysis of diffusionrates in the endothelial wall of muscle capillaries.

(2) Assumption of identical path length, s, for clefts extending longitudinally and circum-ferentially along the bundle surface. This was tested by comparing the mean values of s( obtainedfrom a number of different bundles in either cross-sections or longitudinal sections of the tissues.The 2 means, 3-2 /an (± 25 , 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 = 01).

(3) The simple model in Fig. 2B of endothelial cells with rectangular outlines cannot beexpected to hold strictly, since cell boundaries are more likely to run an oblique or zig-zagcourse along the length and breadth of the bundle surface. The effect of such obliquities wouldbe to increase both x and y above the values taken in calculating tj, whereas values of s would beoverestimated in this calculation since, obviously, the shortest diffusion path through the clefts


is in a direction perpendicular to y or x, rather than in the plane of the section in which it hadbeen determined. In assessing the errors which arise in this way, the courses of longitudinalclefts have been followed in series of cross-sections of bundles at 5-/wn intervals and in furthersubdivisions of i-fim intervals. As a measure of the obliquity, the angles were determinedbetween the lines along the bundle surface connecting the midpoints of individual clefts andeither (a) the vertical, in the case of the sections 5 /tm apart, or (A) the line through the 5-/MT1interval points, in the case of the sections separated by 1 /an. The average angle so obtained wasin (a) 150 ( ± 180, S.D. of 23), and of probably similar magnitude in (b) as far as could be judgedby means of the present, not very precise method. Assuming the course of circumferential cleftsto be of similar obliquity to that of the longitudinal ones, the errors in x, y and s were obtainedfrom 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 preparationduring 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 theassumptions 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 vicinityof 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 con-sidered in detail: whether these vesicles and tubules are connected with each other inany consistent pattern; and how great a volume they occupy within the cells. Theresults are best described with reference to the schematic diagram in Fig. 3 of aninside view of a portion of a heart fibre containing 2 myofibrils together with elementsof the SR. As is shown, the SR is a loose network of fine tubules extending betweenadjacent myofibrils and also in regions beneath the cell surface. The cross-section ofthese tubules is approximately circular, their diameter varying between about 35 and60 nm. The major proportion of the tubules are transversely oriented at Z-line leveland form a continuous system, as suggested by several observations. In longitudinalsections, under favourable conditions such as obtained in Figs. 9 and 10, some of thetubules could be followed for the whole width of a myofibril and others from the edgeof a myofibril to that of its neighbour; yet others extended from areas close to amyofibril to the inner surface of the cell membrane (Figs. 9, 12, 13) or into the cellinterior. In cross-sections cut at Z-line level, the tubules would lie juxtaposed to amyofibril, 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, inlongitudinal 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 % ofthe myofibrillar circumference.

As to the less-frequent longitudinal tubules, considerable lengths (1-2 /tm) of themwere found, on occasion, in the interfibrillar spaces (Figs. 9-11), but more often onlyshort lengths were to be seen. However, it was shown by means of serial sections thatthese, 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 withtubules and terminals of the SR. A large proportion of the tubules partially surroundthe myofibrils at Z-line level; connected with these are others oriented in a roughlylongitudinal direction. Portions of both tubules are filled with dense material (arrows).At the cell periphery branches of the SR tubules terminate, usually at I-band level, indisk-like structures closely apposed to the inner surface of the cell membrane.

from consecutive (longitudinal) sections. The frequency of occurrence of thesetubules was obtained (in longitudinal sections) by determining the cumulative lengthof tubular portions adjacent to a number (260) of different sarcomeres. The result, atubular length equivalent to 6% of the total length of all sarcomeres examined,suggests that, on average, every second sarcomere of each myofibril is accompanied byone such tubule. (This is a simple geometrical consequence of the use of sectionsapproximately 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 /tmbefore terminating. In forming these terminals the tubules usually broaden, parallelwith the cell surface, into disk-like structures. The frequency of the terminals isconsiderable, as is illustrated by the finding that (in longitudinal sections) the profileof such a terminal occurs in association with an average of 75 % of the sarcomeres ofsuperficial myofibrils. A clear predominance of location exists for the region of theI-band, which contains more than three-quarters of the terminals found. It is also ofinterest that, in a few, perhaps geometrically favourable, sections the narrow gap


(10-20 nm) between internal membrane surface and tubular terminal was bridged bysmall bars of dense material at a repeat distance of approximately 20-25 nm> quitesimilar 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 indi-

vidual myofibril (average diameter 1 /tm) associated with the SR, its size comes toabout 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 estimateswere obtained in the cases of (a) and (b) from the above figures of the frequency of the2 types of tubule and an average diameter of the tubules of 50 nm, and of (c) by takingthe average length of the terminals to be 12-5 % of the circumference of the myofibrilsand 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 con-taining the extracellular markers ferritin or horseradish peroxidase, neither of thesemarkers was detected within the tubules described above (e.g. Fig. 8), indicating thatthe tubules are, indeed, elements of the SR rather than of a T-system. In anotherseries, the histochemical procedures outlined in the Methods sections were used toinvestigate whether the SR is able to accumulate calcium and store it subsequently ascalcium oxalate within its lumen. The results of these experiments, also, were negativein failing to show any oxalate precipitate within the cells. Although the idea of the SRin this tissue storing calcium therefore remains unsupported, the possibility of theexistence of such a mechanism cannot be ruled out, since the SR might differ in someother, perhaps trivial, aspect from the SR in those skeletal muscles in which calciumaccumulation was demonstrated with the present method (Pease et al. 1965; Page,1969). For example, the cardiac SR might be more susceptible to damage during thepreparative procedures and so lose the ability to transport calcium more readily thanthe SR of skeletal muscle cells.

Enclosures of dense material. As may be seen in Fig. 10, and as is known from earlierwork (Naylor & Merrillees, 1964; Sommer & Johnson, 1969), some of the vesicles andtubules of the SR are filled with an electron-dense material which is usually sur-rounded by a light zone. The proportion of filled tubules was approximately 5 % of thetotal SR, as determined by the same quantitative procedures as used for estimating theextent 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 infrog sartorius, 84 /im: Mayeda, 1890), and their size varies considerably. To illustratethis variation in size, cross-sectional areas of 190 fibres, all from the same bundle, havebeen plotted in the histogram of Fig. 4. The average area is 17-4/im2, with a rangeextending from below 2 up to 85 /im2, and in other bundles up to more than 100 /(ml


50

40

t 30

I 20

10

12

_ 8

-= 4

20 40 60 80

Cross-sectional area, /1m2

20 40 60

Cross-sectional area,

80

01 2 3 4

Radius, ur

Fig. 4. Distribution of cross-sectional areas of 190 fibres from a single bundle. Histo-grams show A, number of fibres per class (class division 5 /im1), and B, % of total areaoccupied 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 fibresexamined.

Also indicated in Fig. 4 are the radii of cylindrical fibres of the same cross-section, toprovide a measure of the fibre width; but it should be mentioned that the majority offibres are elliptical or more complex in cross-sectional outline (e.g. Fig. 21).

Several physiological properties are critically dependent on fibre size and itsvariation, and it is useful, therefore, to summarize these properties while discussingthe results illustrated in Fig. 4 and also in Figs. 20 and 21. When microelectrodes areused to measure resting and action potentials, the number of successful cell penetra-tions by the electrode, i.e. those yielding stable resting potentials, is quite small, andeven the potentials (of — 83 mV) obtained after successful penetrations are probablyunderestimated (by some 10 mV) due to the shunt of the membrane ' battery' resultingfrom 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 shuntincreases with the magnitude of the cellular input impedance which, in turn, is pro-portional to (a.u)~i, a and u being the cross-sectional area and circumference of thefibre, 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


examined, corresponding to a fibre volume of 3 % of the total). Most likely, stable highresting potentials are obtained only in these large fibres. It should, however, be addedthat another factor probably contributing to stable resting potentials resides in thesyncytial connexions of the tissue, through which each cell helps to maintain theresting potential of its neighbours.

Another parameter of physiological importance, affected by variation in cell size, isthe ratio s/v of the surface over the volume of the fibres. Both the conduction velocityand the kinetics of tracer ion fluxes depend on this ratio which, in ventricle fibres, hasan average value of 1-27/tm"1. (Note that the relevant figure used for calculatingtracer ion fluxes, of I-I or ro/im"1 (Niedergerke, 19636, and present study), is aweighted 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 sjvvalues 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 propor-tion with (s/v)~i (Hodgkin, 1954), it is clear that fibres in this range conduct impulsesmore rapidly, by a factor of ^1-5, than those of average size. On this basis, it isexpected that impulses conducted over long distances inside the heart are carriedmainly 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 thecontent of glycogen granules. This is in contrast to the situation in large fibres ofribbon-shaped cross-section (Fig. 21) which have a relatively high density of myo-fibrils; but their sjv values are near average and conduction by these fibres shouldtherefore not be rapid. It will also be noted that the fibre in Fig. 20 with the lowmyofibrillar but high glycogen content resembles a Purkinje fibre of large mammals,though differing from the latter in failing to occur in association with other similarfibres in separate bundles or, even, clearly defined groups. Nevertheless, the histo-logical similarity associated with a predicted similarity in function is remarkable, if asyet unexplained.

DISCUSSION

Two results of this work are relevant for understanding the function of the frogheart: (1) the estimate, based on electron-microscopic data, of the time course ofdiffusion of solutes between ventricular cavity and heart cells, and (2) the size anddistribution of the sarcoplasmic reticulum inside heart cells. With regard to (1), takingas a measure of the diffusion rate the half-time, t^, with which calcium ions equilibrateat the surface of the heart cells, we obtained a value of approximately 2 s for this para-meter after an assumed step change of concentration in the ventricular cavity. (Seep. 186 for the various uncertainties to which this estimate is subject.) For a com-parison of this result with the physiological time course of calcium action, experimentsconcerned with the kinetics of tension responses to a sudden change of calcium con-centration might be recalled (Chapman & Niedergerke, 1970). These responsesusually consisted of an initial rapid and a later slow phase of tension change, and forthe initial phase a half-time as low as 3 s was obtained (see fig. 8 of that paper). The


closeness of this value to the one above clearly supports the idea proposed earlier thatthe rapid tension changes are related to a process immediately dependent on thechange of calcium concentration at the surface of the heart cells. However, the differ-ence between the 2 values is probably also real, and may be greater still, since ourpresent figure of 2 s is likely to be overestimated, being based on a value for the cleftwidth (12-5 run) just wide enough to allow passage of ferritin molecules (a wider cleftwidth would give more rapid diffusion rates and thus lower values for t^). Indeed, adifference between the time courses under comparison is expected for 2 reasons: themixing of solutes in the ventricular cavity and especially in the subendothelial space isunlikely to occur instantaneously; and the presence of substantial numbers of adsorp-tion 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 forlack of sufficient information on the density of these sites.

With regard to the structure of the sarcoplasmic reticulum, our findings suggestthat it is more extensively developed in frog heart cells than has previously beenthought (e.g. Staley & Benson, 1968); a certain regularity in its organization has alsobeen demonstrated. Indeed, in several respects the arrangement of the SR is similar tothat in mammalian and avian hearts, in which some elements of the tubular networkare also oriented at Z-line level surrounding the myofibrils, while others make contactwith the inner surface of the cell membrane in structures variously termed ' flattenedsacs' (Simpson & Rayns, 1968), 'couplings' (Sommer & Johnson, 1968, 1969), or'subsarcolemmal cisternae' (Fawcett & McNutt, 1969). The main differences are inthe greater extent of that part of the SR which in mammalian and avian fibres sur-rounds the myofibrils at levels other than the Z-line, and the presence, at least in thecase of mammalian ventricular fibres, of terminals situated at the intracellular surfaceof the T-tubules. On account of both these factors, therefore, the volume of SR asso-ciated with each myofibril is probably much smaller in the frog than in the mam-malian 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 about3'5 % °ftne c e^ volume (E. Page, McCallister & Power, 1971), or 7-3 % of the myo-fibrillar 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 andskeletal 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 heartfibres. Since there are good reasons for believing that the SR in both skeletal muscleand rat ventricle fibres provides a cellular store for calcium which is responsible, onrelease, for activation of contraction, it is clear, assuming the SR in the frog heart tohave the same function, that a much smaller capacity for this purpose exists inside theseheart cells. This conclusion (see also Niedergerke, 1963 a) serves to support the hypo-thesis that the major source of activator calcium in this tissue is derived from theexternal medium by inward flow of calcium ions during each action potential (Nieder-gerke & Orkand, 1966). However, it must be admitted that the argument is stillindirect and, even, that the evidence itself may be questioned. For example, it could beargued that the ability of the SR to store calcium is exceptionally high in the frog heart


and so provides large enough quantities of activator. Also, the possibility is notentirely excluded that living frog heart cells may, in fact, contain elements of SRwhich readily disintegrate during fixation of the tissue. Clearly, different or moreadvanced 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 agrant from the British Heart Foundation.

REFERENCES

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BRUNS, R. R. & PALADE, G. E. (1968). Studies on blood capillaries. II. Transport of ferritinmolecules across the wall of muscle capillaries. J. Cell Biol. 37, 277-299.

CHAMBERS, R. & ZWEIFACH, B. W. (1947). Intercellular cement and capillary permeability.Physiol. Rev. 27, 436-463.

CHAPMAN, R. A. & NIEDERGERKE, R. (1970). Effects of calcium on the contraction of the hypo-dynamic frog heart. J. Physiol., hand. 211, 389-421.

ECKER, A. & WIEDERSHEIM, R. (1896). Anatomie des Frosclies, vol. 2, pp. 247-270. Braunschweig:Friedrich Vieweg und Sohn.

FARQUHAR, M. G. & PALADE, G. E. (1965). Cell junctions in amphibian skin. J. Cell Biol. 26,263-291.

FAWCETT, D. W. & MCNUTT, N. S. (1969). The ultrastructure of the cat myocardium. I.Ventricular papillary muscle. J. Cell Biol. 42, 1-45.

FAWCETT, D. W. & SELBY, C. C. (1958). Observations on the fine structure of the turtle atrium.J. biophys. biochem. Cytol. 4, 63-72.

FORBES, M. S. & SPERELAKIS, N. (1971). Ultrastructure of lizard ventricular muscle. J. Ultra-struct. Res. 34, 439-451.

FRANZINI-ARMSTRONC, C. (1970). Studies of the triad. I. Structure of the junction in frogtwitch fibers. J. Cell Biol. 47, 488-499.

GOMPERTZ, C. (1884). Ueber Herz und Blutkreislauf bei nackten Amphibien. Arch. Anat.Physiol. pp. 242-260.

GROS, D. & SCHREVEL, J. (1970). Ultrastructure compared du muscle cardiaque ventriculaire del'Ambystome et de sa larve, l'Axolotl. J. Microscopie 9, 765-784.

HARRISON, P. M. (1963). The structure of apoferritin: molecular size, shape and symmetryfrom X-ray data. J. molec. Biol. 6, 404-422.

HODGKIN, A. L. (1954). A note on conduction velocity. J. Physiol., Lond. 125, 221-224.JOHNSON, E. A. & SOMMER, J. R. (1967). A strand of cardiac muscle. Its ultrastructure and the

electrophysiological implications of its geometry. J. Cell Biol. 33, 103-129.KARNOVSKY, M.J . (1961). Simple methods for 'staining with lead' at high pH in electron

microscopy. J. biopltys. biochem. Cytol. 11, 729-732.KARNOVSKY, M. J. (1967). The ultrastructural basis of capillary permeability studied with

peroxidase as a tracer. J. CM Biol. 35, 213-236.KATZ, B. &THESLEFF, S. (1957). On the factors which determine the amplitude of the 'miniature

end-plate potential'. J. Physiol., Land. 137, 267-278.KlLARSKl, W. (1964a). The organization of the cardiac muscle cell of the lamprey (Petromyzon

marinus L.). Ada biol. cracov. (S6rie zool.) 7, 75-87.KILARSKI, W. (19646). Observations on the myocardium of the cardiac chamber of the sand-eel

(Ammodytes tobianus L.). Acta biol. cracov. (SeVie zool.) 7, 235-240.LANDIS, E. M. & PAPPENHEIMER, J. R. (1963). Exchange of substances through the capillary

walls. In Handbook of Physiology, Circulation section 2, vol. 2 (ed. W. F. Hamilton & P. Dow),pp. 961-1034. Washington, D.C.: American Physiological Society.

LASSEN, N. A. & TRAP-JENSEN, J. (1970). Estimation of the fraction of the inter-endothelial slitwhich must be open in order to account for the observed transcapillary exchange of smallhydrophilic molecules in skeletal muscle in man. In Capillary Permeability (ed. C. Crone &N. A. Lassen), pp. 647-653. Copenhagen: Munksgaard, Scandinavian University Books.

13 C E L I I


LEAK, L. V. (1967). The ultrastructure of myofibers in a reptilian heart: the boa constrictor.Am. J. Anat. 120, 553-582.

LINDNER, E. (1957). Die submikroskopische Morphologie des Herzmuskels. Z. Zellforsch.nrikrosk. Anat. 45, 702-746.

MARCEAU, F. (1904). Recherches sur la structure et le deVeloppement compared des fibrescardiaques dans la se>ie des vert6bres. Annls Set. nat., 8* sdrie, Zoologie, 19, 191-365.

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NAYLOR, W. G. & MERRILLEES, N. C. R. (1964). Some observations on the fine structure andmetabolic activity of normal and glycerinated ventricular muscle of toad. J. Cell Biol. 22,533-55°-

NIEDERGERKE, R. (1957). The rate of action of calcium ions on the contraction of the heart. J.Physiol., Lond. 138, 506-515.

NIEDERGERKE, R. (1963 a). Movements of Ca in frog heart ventricles at rest and during con-tractures. J. Physiol., Lond. 167, 515-550.

NIEDERGERKE, R. (19636). Movements of Ca in beating ventricles of the frog heart. J. Physiol.,Lond. 167, 551-580.

NIEDERGERKE, R. & ORKAND, R. K. (1966). The dual effect of calcium on the action potential ofthe frog's heart. J. Physiol., Lond. 184, 291-311.

NIEDERGERKE, R., PAGE, S. & TALBOT, M. S. (1969). Determination of calcium movements inheart ventricles of the frog. J. Physiol., Lond. 202, 58-60P.

PAGE, E., MCCALLISTER, L. P. & POWER, B. (1971). Stereological measurements of cardiac ultra-structures implicated in excitation-contraction coupling. Proc. natn. Acad. Sci. U.S.A. 68,1465—1466.

PAGE, S. (1964). The organization of the sarcoplasmic reticulum in frog muscle. J. Physiol.,Lond. 175, 10-11P.

PAGE, S. G. (1969). Structure and some contractile properties of fast and slow muscles of thechicken. J. Physiol., Lond. 205, 131-145.

PALADE, G. E. (1953). Fine structure of blood capillaries. J. appl. Physics 24, 1424.PEACHEY, L. D. (1965). The sarcoplasmic reticulum and transverse tubules of the frog's

sartorius. J. Cell Biol. 25, 209—231.PEASE, D. C , JENDEN, D. J. & HOWELL, J. N. (1965). Calcium uptake in glycerol-extracted

rabbit psoas muscle fiber. II. Electron microscopic localization of uptake sites. J. cell. camp.Physiol. 65, 141-153.

REVEL, J. P. (1962). The sarcoplasmic reticulum of the bat cricothyroid muscle. J. Cell Biol. 12,571-588.

SCHEYER, S. C. (i960). Fibrillar and membranal relationships in frog ventricular muscle. Anat.Rec. 136, 273.

SIMPSON, F. O. & RAYNS, D. G. (1968). The relationship between the transverse tubular systemand other tubules at the Z disk levels of myocardial cells in the ferret. Am. J. Anat. 122,193-207.

SLAUTTERBACK, D. B. (1963). The sarcoplasmic reticulum in a turtle heart. J. Cell Biol. 19, 66 A.SOMMER, J. S. & JOHNSON, E. A. (1968). Cardiac muscle. A comparative study of Purkinje

fibers and ventricular fibers. J. Cell Biol. 36, 497-526.SOMMER, J. S. & JOHNSON, E. A. (1969). Cardiac muscle. A comparative ultrastructural study

with special reference to frog and chicken hearts. Z. Zellforsch. mikrosk. Anat. 98, 437-468.STALEY, N. A. & BENSON, E. S. (1968). The ultrastructure of frog ventricular cardiac muscle and

its relationship to mechanisms of excitation-contraction coupling. J. Cell Biol. 38, 99-114.THOMAS, L. J. (i960). Ouabain contracture of frog heart: Ca44 movements and effect of

EDTA. Am. J. Physiol. 199, 146-150.VENABLE, J. H. & COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron

microscopy. J. Cell Biol. 25, 407-408.AVANG, J. H. (1953). Tracer diffusion in liquids. IV. Self diffusion of calcium ion and chloride

ion in aqueous calcium chloride solutions. ,7. Am. client. Soc. 75, 1769-1772.

[Received 24 November 1971)

Fine structure of frog heart cells

V : * • /

• • : /

?r,'j

' *

\

Figs. 5-7. For legend see p. 196.

13-2


Figs. 5-7. Illustration of 3 different types of cleft in endothelial layer. Fig. 5, cleft ofrelatively 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 6are sections stained with uranyl acetate and lead; x 50000. Fig. 7, tissue stained withuranyl 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 fibrebundle. Tissue exposed to peroxidase and later incubated for histochemical reaction.Note the electron-dense end-product of the reaction inside the cleft, in the sub-endothelial space, and in a thin layer at the surface of the endothelial cells, but itsabsence within the muscle fibres (arrows indicate terminals of the SR). Sectionsstained with uranyl acetate, x 52000.

Fig. 9. Elements of the SR in a longitudinal section through a muscle cell. Tubulesoriented in transverse direction (r.h.s. arrows) extending across the whole width of amyofibril whose surface has been grazed by the section. The interfibrillar gap, below,contains tubules oriented in both transverse (l.h.s. arrow) and longitudinal (double-headed arrows) directions. Section stained with uranyl acetate and lead, x 37000.


•4 * ^ * * • * • « • *• - • •

I

' , • . • • '

!


Figs. 10-14. Longitudinal sections of heart fibres illustrating various structural aspectsof the SR. All sections stained with uranyl acetate and lead.

Fig. 10. Portions of 2 fibres separated by intercellular gap (arrow head). Upperfibre: terminal (te) in close apposition to surface membrane and a tubule (tr) extendingfrom the surface towards the centre of the fibre. Lower fibre: tubules in both trans-verse (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 \

12

tr

14

2OO S. G. Page and R. Niedergerke

Figs. 15-19. Cross-sections of heart muscle fibres, all near Z-line level (Z), furtherillustrating features of the SR. Note, also, the absence of basement membrane whenintercellular gap (arrow heads) is narrow and its presence in wide gaps (asterisks). Allsections 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 termi-nals (te). x 40000.

Figs. 18, 19. Tubules (arrows) around periphery of myofibrils, some with branchesto cell surface (especially in Fig. 19). x 40000.

Fine structure of frog heart cells zor

te


Figs. 20, 21. Comparison of fibre of approximately circular cross-section with others ofmore complex shape. Sections stained with uranyl acetate and lead. Note differentmagnifications in the 2 figures.

Fig. 20. Cross-section of large cylindrical fibre containing only few myofibrils butconsiderable amounts of glycogen. (Note, also, the small surrounding fibres whosemyofibrillar content is high by comparison.) x 11 500.

Fig. 21. Several ribbon-shaped fibres in cross-section (cell boundaries have beenpicked out with ink for clarity). Note that relative myofibrillar density of these fibres ishigher than that of the large fibre in Fig. 20, except in the regions in which there is amarked increase in cell width (asterisk), x 4500.


5//m

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STRUCTURES OF PHYSIOLOGICAL INTEREST IN THE FROG HEART ... · Fine structure of frog heart cells 181 finally embedded in Araldite (CIBA). In some instances, the tissue was stained