Download Comparative Ultrastructural Morphometry Of The Relationship

Comparative Ultrastructural Morphometry of the Relationship
between the Sarcoplasmic Reticulum, Transverse Tubules and
Myofibrils in Ventricular Myocytes of the Hamster and Bat
Dennis E.E¹, Tagoe C.N.B¹, Yates R.D², Ayettey A.S¹
Department of Anatomy, University of Ghana Medical School,P.O.Box 4236, Accra,Ghana; and
Department of Anatomy, Tulane University School of Medicine, New Orleans, Louisiana, U.S.A
ABSTRACT
Comparative ultrastructural morphometry shows that area of myofibrils covered by SR in left
ventricular myocytes is 54% in the insect-eating bat (Pipistrellus pipistrellus), 40% in the nonhibernating hamster and 32% in the hamster in hibernating conditions. Similarly, the area of T-tubules
covered by the SR is greater in the bat (23%) than in the non-hibernating hamster (14%) and the
hibernating hamster (6%). Other parameters determined including volume densities of myofibrils, SR
and T-tubules add to the evidence that mammalian cardiac cells are well structured for their different
functional capacities, especially in regard to calcium storage for contractile activities.
Keywords: Bat, Hamster, Sarcoplasmic reticulum, T-tubules, Ventricular myocytes.
INTRODUCTION
T-tubule in turn causes more calcium to be
released from the SR that couples with the
T-tubules. This SR calcium binds to troponin C
to initiate contraction.
On account of the role of the SR in
excitation-contraction coupling, attempts have
been made to determine its extent in mammalian
cardiomyocytes. Forbes and van Niel [4], for
example, have shown that SR to myofibrillar
volume is approximately equal in ventricular
myocytes of the mouse (0.16) and guinea pig
(0.18). Percentage area of myofibrils covered
by SR, however, is different in the two species,
being 49% in the mouse and 43% in the guinea
pig. In a recent study [5], it was noted that
volumes of SR and T-tubules reduce
significantly in hamsters subjected to
hibernating conditions at a temperature of 5ºC.
The sarcoplasmic reticulum [SR] is well
known to be important in the processes that lead
to excitation-contraction coupling in muscle
[for e.g., 1] in relation to calcium storage and
release. In mammalian cardiac cells, these
tubules surround major cytoplasmic organelles
such as myofibrils and mitochondria and also
form junctional complexes with the surface
sarcolemma as well as with its invaginated Ttubule portions.
Mammalian cardiac myocytes depend on
extracellular calcium for contraction [2] and
T-tubules represent major pathways for entry of
extracellular calcium in the interior of the
myocyte for excitation-contraction coupling [3].
With each action potential, extracellular calcium
is transferred to the cytosol through the
T-tubule. The calcium released through the
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volume. The other group of tissues was fixed in
a mixture of aqueous OsO4 (one part 4%
solution) and potassium ferrocyanide (two parts
0.8% solution) in sodium cacodylate buffer (one
part 0.2 M solution, pH 7.6) for 1½ hours to
enhance SR and T-tubule membranes for
morphometry [4]. All post-fixed tissues were
dehydrated in graded series of ethanol before
embedding in Epon 812 resin.
Thin sections were obtained with a Diatome
diamond knife in a Reichert-Jung Ultracut E
microtome and were picked on 200 mesh copper
grids. These sections were stained with 0.5%
saturated uranyl acetate in 50% ethanol for 1015 minutes and counterstained with lead citrate
for 6-10 minutes. Stained sections were viewed
in a JEOL 100CX II electron microscope at 406-KV.
For stereology, four blocks of left ventricular
tissue per heart were randomly selected. From
one stained section of each block, one
photomicrograph each was obtained from the
right hand corner of three successive grid
squares according to the sampling method of
Weibel [8]. The point-counting method was
used to determine volume fractions and surface
densities of the subcellular organelles from the
printed photomicrographs.
To determine volume fraction (Vv) of SR,
myofibrils and T-tubules, transparent double
lattice of 1 cm and 0.5 cm square grids were
used. The 1 cm lattice square grid was used for
the determination of myofibrillar volume, and
the 0.5 cm square grid for the SR and T-tubules.
For determination of Vv of myofibrils,
micrographs were taken at original magnification of 4,800 diameters and printed at 10,560
diameters. For Vv of SR and T-tubules, the
original magnification was 10,000 diameters,
with final prints made at 25,000 diameters.
From point-counts, volume fractions were
determined using the formula below:
It is presumed from these studies that the
degree of development of the SR should relate
to the cardiac cycle as an adaptive feature. The
present work is designed to establish this
hypothesis using the bat, Pipistrellus pipistrellus
that has a heart rate of about 1000 beats/min, the
non-hibernating hamster with a heart rate of 300
beats/min and the hibernating hamster with a
heart rate of about 5 beats/min [6].
MATERIALS AND METHODS
Adult male insectivorous bats Pipistrellus
pipistrellus (average weight-6g) and hamsters
(average weight-180g) were used in this
research.
Five bats were captured in a dungeon at the
Cape Coast Castle in Ghana, West Africa. Ten
hamsters were obtained from the Tulane
University Animal House. Five of the hamsters
were kept in normal laboratory conditions at 2225ºC with humidity at 52%, while the other five
were kept in a cold room at 5ºC. Photoperiod
for the two groups of hamsters was maintained
at 10 hours of light and 14 hours of darkness.
The hamsters were kept in the cold till they
adopted the characteristic curled hibernating
posture [7] after 4 weeks. They were left in this
state of stupor for another 6 weeks.
All the animals were anesthetized with
sodium pentobarbital at a dosage of 6gm/100gm
body weight. They were thoracotomized and
immediately perfused through the left ventricle
with physiological saline for 1-3 minutes,
followed by 3% glutaraldehyde in 0.1 M sodium
phosphate buffer at pH 7.4 for 10-15 minutes.
The hearts were removed and stored overnight
in fresh fixative at 4ºC, after which 1mm³ pieces
of myocardial tissue were excised from the
outer wall of the left ventricle. The pieces of
tissue from each animal were separated into two
groups after they had been washed free of
excess fixative in three one-hourly changes of
phosphate buffer. One group of tissue from
each animal was postfixed in 1% OsO4 in
phosphate buffer for 1½ hours for morphometric
work involving determination of myofibrillar
Vі/V cell = Pi/Pcell
(where Vi is the volume of organelle, V cell the
volume of cell, Pi, the number of points
(2)
Fig. 1-3. Electron micrographs of left ventricular myocytes showing general features: (1) Control hamster. The sarcomeres
of the myofibrils are well aligned and T-tubules (T) are quite prominent. Arrows indicate SR and L lipid bodies.
x20,000. (2) Experimental hamster. x20,000. (3) Bat. General features are similar to those of control and
experimental hamsters x20,000
Fig. 4.
Section of left ventricular myocyte of experimental hamster. Some of the small vesicles representing SR tubules
(arrows) contain dense material. x25,000.
(3)
determined by number of intersections of grid
lines on the organelle and P cell the total number
of points covering the whole micrograph [9].)
Surface densities of myofibrils, SR and
T-tubules were estimated using a transparent 1
cm semicircular test grid [10]. The number of
times test lines intersected the surface
boundaries of each type of organelle and the
number of points or semicircles covering the
whole micrograph were determined.
The
number of points was then substituted in the
equation:
RESULTS
In general, ventricular myocytes of the bat
and of the two groups of hamsters have similar
basic ultrastructure (Figs 1-3). However, lipid
bodies are virtually absent in myocytes of the
hibernating (experimental) hamsters (Fig. 2).
The SR is made up of a network of branching
and interconnecting tubules in the sarcoplasm.
There are two main divisions of SR- those that
form couplings with the sarcolemma (junctional
SR or JSR) and the ones that surround
myofibrils and mitochondria (free SR or FSR).
The JSR couples with the surface sarcolemma
and non-specialized parts of the intercalated disc
(peripheral junctional SR or PJSR) and those of
T-tubules (internal junctional SR or IJSR).
Sv = 2 1/L
(where I is the total number of intersections
between organelle boundaries and test lines and
L, the test line length. L=LT which is calculated
thus):
LT = PT.π/2d
(where PT is the number of points of test frame
covering the area of micrograph and d, the
distance between two test points) [8].
The area of myofibrils covered by free SR
(FSR) or the SR/myofibril ratio was estimated
from micrographs of transverse and oblique
sections of the myocytes. The same micrographs were also used to determine the extent of
coverage of T-tubule surface by the internal
junctional SR (IJSR) (IJSR/T-tubule ratio). A
semicircular test grid was used for point
counting. To determine the SR/myofibril ratio,
(IFSR/Imyo), test line intersections with FSR
(IFSR) as well as the test line intersections with
remaining myofibrillar surface (Imyo) were
counted.
To determine the IJSR/T-tubule ratio, the
number of test line intersections on IJSR-Ttubule junctions was substituted in the ratio, ½
Sv (IJSR)/Sv(t) [4]. This ratio was halved,
because the relationship between the IJSR
membrane and the T-tubule in cardiac muscle is
dyadic (i.e. IJSR is applied to one side of the Ttubule).
Data from stereological measurements were
anlysed by Instat II statistical software using
one-way ANOVA and Tukey-Kramer multiple
comparison tests.
Fig. 5.
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Electron micrographs of bat left ventricular
myocytes showing (a) peripheral junctional
sarcoplasmic reticulum (PJSR) and (b) internal
junctional sarcoplasmic reticulum (IJSR). Note
linearly organized luminal granules. Arrows
indicate junctional processes from outer
membrane of JSR. (a) 90,000 x (b) 75,400 x
The configuration of FSR tubules is similar
in all three groups of animals (Figs. 1-3), except
that in the hibernating hamster, some contained
more electron-dense material (Fig. 4).Linearly
organised luminal granules are encountered in
IJSR and PJSR of myocytes of bat and hamster
(Fig. 5). The sections of SR tubules at the
junctional complexes also have feet-like
processes that projected toward the sarcolemma
(Fig. 5), with a junctional gap of about 20 nm.
FSR tubules in the experimental hamster are
wider in diameter (75 nm) than in the control
hamster (46.2 nm) and in the bat (52.3 nm)
(Fig. 6). Similarly, the IJSR tubules in the
hibernating hamster (67 nm) are wider in
comparison to those in the other animals studied
(36.8 in control hamster and 38.8 nm in the bat)
(Fig. 7).
Fig. 7.
Comparison of mean diameters of free
sarcoplasmic reticulum (nm) ± S.E.M. in left
ventricular myocytes of control (CH) and
experimental (EH) hamsters and bat (PP). By
one-way ANOVA: F 2 , 12 =5.5,p=0.0197
Stereologically, there were significant
differences in SR, myofibrils and T-tubule
volume fractions in ventricular myocytes of the
three groups of animals studied.
Volume
fraction of SR was significantly higher in the
bat (4%) and in the non-hibernating or control
hamster (3.5%), than in the experimental
hamster (2%).
Comparative volumes of
myofibrils were 49.0% in the bat, 60.6% in the
control hamster and 51.5% in the experimental
hamster. T-tubule volumes on the average,
were 0.8% in the bat, 1.5% in the control
hamster and 0.9% in the experimental hamster.
These values are summarized in Tables 1 and 2.
Surface densities of myofibrils, SR and
T-tubules are given in Table 3. Myofibrillar
surface density relates to myofibrillar volume,
being greater in the hamsters than in the bat.
Surface densities of total SR and IJSR are
comparable in all groups, there being no
significant differences.
Free SR myofibril ratio was significantly
lower in the experimental hamster (32%) than in
the control hamster (40%) and in the bat (54%).
Fig. 6. Comparison of mean diameters of free SR (nm) ±
S.E.M. (nm) in left ventricular myocytes of
control (CH) and experimental (EH) hamsters
and bat (PP). By one-way ANOVA: F 2 , 12 = 11.8,
p = 0.0014. By Tukey-Kramer test *p < 0.01
between EH and CH, and between EH and PP.
There was no significant difference between
control hamster and bat
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(Fig. 8). IJST/T-tubule ratio in control hamster
was about half what it was in the bat (Fig. 9).
Fig. 8.
Ratio of free SR in apposition to myofibrils
(mean ± S.E.M.): comparison of control hamster
(CH), experimental hamster (EH) and bat (PP).
Fig. 9.
Density of internal junctional SR on T-tubules
(mean ± S.E.M.) in left ventricular myocytes of
control (CH) and experimental (EH) hamsters
and bat (PP)
DISCUSSION
It is evident from the present study that there
are significant differences in SR volumes and in
FSR/myofibril and IJSR/T-tubule ratios in the
ventricular myocardium of the bat, the nonhibernating and hibernating hamster. The SR
volume fraction is larger in the bat than in the
two groups of hamster. This is not surprising as
the bat, Pipistrellus pipistrellus, has a very high
heart rate (about 1000 beats/min) [6] compared
to the hibernating hamster (5 beats/min) [6].
Indeed, evidence from the results of Leak [11]
and Yamamoto [12] support this view that
volume of SR is related to the cardiac cycle. In
cardiac myocytes of the boa constrictor, a reptile
that has a heart rate of about 15 beats/min Leak
[11] observed a sparse SR. Yamamoto [12]
made a similar observation in the goldfish that
has a heart rate of 25 beats/min.
The significance in the volume fraction of
SR becomes even more apparent when one
relates it to the volume ratio of myofibrils and
T-tubules. With low volume ratio of myofibrils
in the bat than in the hamsters, the SR/myofibril
ratio in (the bat) is therefore even much higher.
Similarly, the low volume ratio of T-tubules in
the bat compared with the non-hibernating
hamsters gives a higher SR/T-tubule ratio in the
bat, which is likely to equip the bat for higher
functional cardiac activity.
As expected,
reduction in SR volume in the hibernating
hamster matched reduction in myofibril and
T-tubule volumes, as compared with the nonhibernating hamster. There were no differences,
therefore, in SR/myofibril and SR/T-tubule
ratios for the two groups of hamsters.
The results of the present study also confirm
that the T-tubules are generally wider in the
animals investigated in the present study than in
most mammals. The wide T-tubules reported in
the bat and hamsters have been related to their
ability to survive in extreme bradycardia [13].
The heart rate of the hamster is reduced from
300-500 beats/min to 5 beats/min when the
animal is in stupor or hibernation [6]; that of the
bat (Pipistrellus pipistrellus) which in normal
activity ranges between 600-1000 beats/min,
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Table 1: Volume fractions of myofibrils, SR and T-tubules of left ventricular myocytes of nonhibernating and hibernating hamsters and of bat (%mean±S.E.M.).
Myofibrils
Sarcoplasmic
reticulum
T-tubules
Non hibernating
hamster (CH)
60.63±1.93*
3.46±0.34
1.55±0.19
Hibernating
hamster (EH)
51.49±2.81*
1.92±0.21
0.93±0.05
Bat (PP)
49.03±1.54*
3.98±0.55§
0.81±0.03
For each group of animals n=5. Initial comparison is made using one-way ANOVA; further comparison
is made using Tukey-Kramer’s test where significant differences indicated:*p<0.05 between CH and EH,
p<0.01 between CH and PP;
§p<0.05 between EH and PP, p<0.01 between EH and PP
‡p<0.001 between CH and EH, and between CH and PP.
Tables 2: SR/myofibril, SR/T-tubule and T-tubule/myofibril volume ratios of non-hibernating and
hibernating hamsters and of bat.
SR/Myofibril
SR:T/tubule
T-tubule/Myofibril
Non-hibernating hamster
1 : 17.5
1 : 0.45
1 : 29.2
Hibernating hamster
1 : 26.8
1 : 0.48
1 : 53.1
Bat
1 : 12.3
1 : 0.20
1 : 53.4
SR-sarcoplasmic reticulum
Table 3: Surface densities (μm ֿ◌¹) of myofibrils, sarcoplasmic reticulum and T-tubule membranes in nonhibernating (CH) and hibernating (EH) hamsters and bat (PP) left ventricular myocytes (mean ±S.E.M.).
Myofibrils
SR
IJSR
T-tubules
Non-hibernating
hamster
1.26±0.07*
0.49±0.07
0.04±0.01
0.16±0.02‡
Hibernating
hamster
1.08±0.07
0.35±0.07
0.01±0.00
0.12±0.00
Bat
0.56±0.06
0.31±0.03
0.03±0.00
0.08±0.01
Comparison by one-way ANOVA is followed by Tukey-Kramer test where significant differences indicated.
*p<0.001 between PP and CH, and between PP and EH.
‡p<0.01 between PP and CH.
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of myofibrillar surface by free SR (FSR) in
ventricular myocytes of the bat add to the
structural features that would facilitate
reabsorption of calcium from the environs of
myofibrils to induce relaxation after the
contractile process. That this is likely an
adaptation is supported by the work of Forbes
and van Niel [4] in which myocardial cells of
the mouse have higher FSR/myofibril ratio than
in the guinea pig, the heart rate of the mouse
being also much higher (600 beats/min) than in
the guinea pig (230-300beats/min).
An interesting observation is the wider SR
tubules of ventricular myocytes of the
experimental hamster than in either control
hamster or bat myocytes.
The functional
significance of wide IJSR tubules in the
experimental hamster is not easy to deduce but
could suggest that more calcium is stored in the
SR here, as myocytes of the hibernating
mammal likely depend more on intracellular
than extracellular calcium for initiation of the
contractile process [18]. Wang & Lee [19]
reported that accumulation of calcium by the SR
increases two to three-fold in myocytes of
hibernating ground squirrel, indicating greater
amounts of calcium stored in these elements for
contraction. This adaptation would also be
necessary for the large stroke volume required
of the heart of hibernators, even when cardiac
output is reduced to 1/60 of normal level in such
states [20]. The wider FSR tubules observed in
myocytes of hibernating hamsters could also
suggest greater accumulation of calcium from a
more rapid uptake of that ion [19; 21]. In
animals in which heart rate is significantly
reduced in response to conditions such as diving
or hibernation, this may be the mechanism by
which more calcium is withdrawn from the
cytoplasm for relaxation, especially as
FSR/myofibril ratio is low.
falls to about 30 beats/min in hibernation [6]. In
the grey seal in which T-tubules are also wide
(450 nm) [14], the heart rate drops from 120
beats/min to 4 beats/min during diving [15].
T-tubules as invaginations of the sarcolemma
increase surface area of cell membrane available
in myocyte for metabolic exchanges. In animals
in which they feature prominently, metabolic
exchanges in myocardial cells would be more
efficient. This would protect cardiac cells with
such features from arrhythmia, and ensure
effective excitation-contraction coupling in
extreme conditions [16], enabling efficient
exchanges of calcium, sodium, and potassium
and other ions required for excitationcontraction.
That ventricular myocytes of the bat in this
study have lower volume fraction of T-tubules
compared to those of the control hamster, is
surprising since demands on the cardiac cell in
the bat are far greater than in the hamster. With
a faster heart rate, myocytes of the bat should
have a wider system of T-tubules compared to
control and experimental hamsters. If T-tubules
represent the major pathway for the entry of
extracellular calcium into the cardiac cell [3],
then, they should be more extensive in myocytes
of the bat. This focuses more attention on the
SR which is also important in calcium ion
regulation and which is more preponderant in
the bat.
The SR to T-tubule and SR myofibrillar
ratios in Table 2, indicate that the myocytes of
the bat are better adapted for survival in extreme
conditions. This observation is supported by
Kawamura et al [17] who reported that
myocytes of working ventricular cells of the
little brown bat (Myotis lucifugus), with a heart
rate of 540 beats/min (under anesthesia), have
higher SR to myofibril ratio compared to
myocytes of larger terrestrial mammals.
As expected, proportion of IJSR in
apposition to the T-tubule (IJSR/T-tubule ratio)
is also higher in the bat than in control and
experimental hamsters. In the bat, this would
enable more rapid mobilization of calcium for
initiation of contraction. The greater coverage
ACKNOWLEDGMENT
E. Dennis is grateful to the government of
Ghana and Tulane University for financial
(8)
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(1987)
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assistance. We thank Dr. I-Li Chen formerly of
Department of Anatomy, Tulane University
Medical School for technical advice and Drs. F.
Addai, University of Ghana Medical School and
G. Armah, Noguchi Memorial Institute for
Medical Research, University of Ghana, for
their support.
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