Download Relation of Ultrastructure to Function in the Intact Heart: Sarcomere

Relation of Ultrastructure to Function
in the Intact Heart: Sarcomere Structure
Relative to Pressure Volume Curves
of Intact Left Ventricles of Dog and Cat*
By Henry M. Spotnitz, B.A., Edmund H. Sonnenblick, M.D., and
David Spiro, M.D., Ph.D.
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• The sarcomere is the basic ultrastructural
unit of contraction in striated muscle.1 Huxley
and Hanson 2 have shown that the alternating
band patterns of the sarcomere in skeletal
muscle derive from the longitudinal disposition of two sets of interdigitating contractile
protein filaments. On the basis of these and
other studies, the sliding filament hypothesis
for muscle contraction was proposed. 3 - 4 The
ultrastructure of heart muscle has also been
examined and its sarcomeres shown to be similar in structure to those of skeletal muscle.3
More recently sarcomere structure has been
studied in relation to the length-tension curve
of both skeletalli"s and cardiac7- ° muscle, and
despite previous objections," it has been
demonstrated that the sliding filament hypothesis- explains the band pattern changes at
varying muscle lengths as well as the relation
between active tension and muscle length
for both types of striated muscle. 7 - 8 The relevance, however, of sarcomere length-tension
relations for linear samples of myocardium
(papillary muscle) to physiological performance of the intact left ventricle has not
From the Department of Pathology, College of
Physicians and Surgeons of Columbia University,
New York, New York, and the Cardiology Branch,
National Heart Institute, Bethesda, Maryland.
Supported in part by a General Research Support
Crant from the U. S. Public Health Service and
Grant H-5906 and Health Research Council Crant
U-1075 from the City of New York.
Presented in part before the American Heart
Association October 23, 1964, Atlantic City, New
Jersey. Circulation 30, suppl. Ill: 163, 1964.
*This paper is number I from a continuing study.
Accepted for publication June 28, 1965.
Orculalion Research, Vol. XVIII, January 1966
been reported. The purpose of these experiments was, therefore, to examine sarcomere
length as a function of filling pressure and
volume under passive conditions in the intact
mammalian left ventricle.
Methods
Fresh hearts were used after removal from 27
dogs and 10 cats anesthetized with sodium pentobarbital (25 mg/kg) and sacrificed acutely. The
mitral orifice of the hearts from dogs was sealed
with a clamp, and the right ventricle was opened
widely. A Gregg cannula was inserted into the
left main coronary artery and the aorta was
sealed around the proximal portion of the cannula. For studies including the range of normal
left ventricular end diastolic pressure (0 to 15
mm Hg), two cannulas with plastic guards to
prevent leakage were introduced through the interventricular septum into the cavity of the left
ventricle. Intraventricular pressure was measured with a Statham P23G pressure transducer
attached to one cannula and was recorded on a
Sanborn oscillograph. The second cannula was
utilized for the introduction and withdrawal of
fluid increments. When passive intraventricular
pressures beyond the physiological range were to
be investigated (15 to 55 mm Hg), the canine
ventricles were prepared with an intraventricular
balloon attached to a large bore cannula which
was placed through and sealed into the mitral
valve orifice. The balloon employed was large so
that stretching of its wall and development of
tension were avoided over the volumes explored.
Four or five curves relating pressure and volume
were obtained for each left ventricle to ensure
reproducibility. Satisfactory ventricular sealing,
demonstrated by the quantitative recovery of the
fluid introduced into the chamber, was established
as a criterion for the acceptability of each preparation. These procedures were completed within
fifteen to thirty minutes after the excision of the
heart from the animal.
Fixation for electron microscopy followed ad49
50
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justment of intraventricular conditions to a desired position on the pressure-volume curve. The
preparation was perfused with fixative through
the previously cannulated left coronary artery,
employing glutaraldehyde, 6.25%, in phosphate
buffer, pH 7.6. Previous studies have shown that
this fixative does not change resting muscle tension9 and does not significantly alter filament
dimensions or sarcomere length.10 The ventricles
from cats, after similar preparation, were fixed
by immersion and replacement of the intraventricular fluid volume with fixative. A fluid exchange device, consisting of a pair of matched
syringes placed back-to-back, assured that the
fluid removed from the ventricle was replaced
simultaneously with an identical volume of glutaraldehyde. Fixation continued for three hours.
The left ventricular weight was then determined
after removing the atria at the atrioventricular
groove and separating the entire free wall of the
right ventricle. Slices of the left ventricular wall
were obtained and washed in cold phosphate
buffer for 24 hours. With the aid of a dissecting
microscope, these slices were trimmed to thin
plates, less than 0.5 mm in thickness, cut parallel
to the planes containing the muscle fibers, and
fixed for an additional three hours in buffered
osmium tetroxide. Tissues were then dehydrated
in alcohol and' embedded in Araldite. Details of
the process of fixation and tissue preparation for
electron microscopy have been described elsewhere." Care was taken in sectioning by orienting
muscle fiber direction parallel to the knife edge 11
to avoid compression artifacts. Postfixation staining of thin sections with potassium permanganate1or lead citrate i;! ' 14 was used to heighten contrast. Left ventricles from both the dog and the
cat were sampled from the inner, middle, and
outer muscle layers in the region of the anterior
descending branch of the perfused left coronary
artery. 15 ' lc In addition, portions of the interventricular septum and papillary muscles were
obtained from the left ventricles of cat heart.
Thin sections were examined in a carefully
calibrated RCA EMU-3 electron microscope
operated at a single magnification (tap 6) with
care to normalize microscope lenses at 50 kv. Five
or six micrographs at an initial magnification of
8,000 were obtained for each sample, with about
forty sarcomeres per field. In the several samples
from each of the 37 hearts, sarcomere structure
was examined and the average sarcomere length
and band widths were determined, employing 25
or 30 measurements per sample. Sarcomere length
was taken as the center-to-center distance between
adjacent Z-lines. The variation in sarcomere
length between adjacent fields was generally less
than 0.05 /A. The data were treated statistically
and sarcomere length was plotted relative to ven-
SPOTNITZ, SONNENBLICK, SPIRO
tricular pressure and volume. Phase contrast microscopy of thick sections embedded in Araldite
and optical microscopy of hematoxylin and eosin
sections were also employed as an aid to tissue
orientation and as a check for sampling errors.
Results
Data were obtained for 27 canine left ventricles averaging 96.4 ± 2.6 (SE) g in weight
from dogs weighing an average of 17.1 kg
(range: 13 to 28 kg). The combined weight
of right and left ventricles in this series
averaged 125 g.
A representative example of a passive
pressure-volume curve for the canine left ventricle is illustrated in figure 1. Afillingpressure
of 0 mm Hg corresponds in this instance to
an intraventricular volume of 12 cc. Intraventricular pressure rises at an increasing rate
as a function of filling volume, reaching 12
mm Hg with an intraventricular volume of 47
cc. Further increases in volume are associated
with gradual stiffening of the ventricular wall,
. LEFT VENTRICLE
( DOG, 17kg. ,»ll ]
Typical pressure-volume curve of the canine left
ventricle. At 12 mm Hg filling pressure, left ventricular volume is 47 cc. At 0 mm Hg, left ventricular
volume is 12 cc. Note that negative pressures are
required to approach zero volume.
Circulation Research, Vol. XVIII, January 1966
CARDIAC ULTRASTRUCTURE AND FUNCTION
51
such that small increments in volume cause
increasingly marked pressure elevations. As
illustrated, negative pressure is required to remove all fluid from the ventricle. Average intraventricular volume at a pressure of 0 mm
Hg for the 27 canine ventricles was 12.5 ± 0.9
(SE) CC. At a pressure level of 12 mm Hg, intraventricular volume averaged 53 ± 3.6 cc
(fig. 2 and table 1).
Figure 3 shows the appearance of a typical
low-power field for the canine left ventricle as
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30
40
50
VOLUME-CC
FIGURE 2
Left ventricular filling pressure plotted against mean
intraventricular volume averaged for 27 canine left
ventricles, mean left ventricular weight 96.4 g.
Standard error indicated by brackets. See table 1.
observed in the electron microscope. The excellent preservation of all cytoplasmic structures and uniform distribution of sarcomere
lengths, with dilatation of the capillaries,
suggest that perfusion of the coronary arteries
with glutaraldehyde produces rapid, uniform
fixation. The overall tissue preservation was
excellent. This has been found to hold true
even in hearts fixed as long as one hour after
sacrificefof the animal.
The relationship of passive filling pressure
to sarcomere length for the middle or sinospiral layer of myocardium in the canine left
ventricle is illustrated in figure 4. Each of the
twenty-seven points is derived from a separate heart, and the best approximate curve is
drawn between these points. Average sarcomere^ length increases gradually from 1.92 ±
0.05 [x. at an intraventricular pressure of'O^mm
Hg (fig. 5) to 2.25±0.04 yu, at 12 mm Hg
(fig. 6). Beyond this point, sarcomere length
changes less with increasing pressure. Thus,
at an intraventricular pressure of 35 mm Hg,
observed sarcomere length approximates 2.35
/A. Some scatter of experimental data relative
to the plotted line in figure 4 exists at all
filling pressures, but decreases with higher
pressures, as indicated in table 2. Figures 7
TABLE 1
Canine Left Ventricles: Mean Pressure-Volume Relations for 27 Specimens, With Calculated
Dimensions for a Derived Spherical Model
R'i
p*
mm
Vol
Hg
0
2*
3*
3*
5
10
12
15
20
30 + *
30 + *
R:;
h
Ri
R«
cc
mm
mm
mm
mm
12.5 ± 0.9f
25.2
30.0
32.5
36.2 ± 2.5
49.3 ± 3.6
53
57.2 ± 4.5
62.0 ± 4.5
75.6
98.6
144
182
193
198
205
227
233
239
246
262
287
220
244
252
256
261
277
281
285
291
.304
32.3
296
307
311
313
317
327
329
332
336
345
359
152
125
118
115
112
100
96
95
90
83
72
Ratios
R'2
R.
R=
Rr,
1.00
1.26
1.34
1.37
1.42
1.58
1.62
1.66
1.71
1.82
1.99
1.00
1.11
1.15
1.17
1.19
1.26
1.28
1.30
1.32
1.38
1.47
1.00
1.04
1.05
1.06
1.07
1.10
1.11
1.12
1.14
1.17
1.21
*P: transmural pressure in mm Hg. Vol: intraventricular volume, cc. Rj: calculated radius
to inner surface of ventricular wall, mm. R 3 : calculated radius to outer surface of ventricular
wall, mm. R.,: calculated mean radius, mm. h: calculated wall thickness, mm.
fsE of mean.
^Indicates estimated value.
Mean animal weight 17.1 kg, mean left ventricular weight 96.4 g.
Circulation Research, Vol. XVHI, January 1966
52
SPOTNITZ, SONNENBUCK, SPIRO
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FIGURE 3
Survey electron micrograph
pressure and filling volume
capillaries are widely patent
length. Paired arrows at the
and capillaries hy C.
of inner layer of canine left ventricle fixed at 1 nun Hg filling
of 24 cc. All cellular components are well preserved and the
as a result of the perfusion fixation. Sarcomeres measure 2.1 ix in
level of the Z line delimit a sarcomere. Nuclei (ire indicated }>y .V
and 8 show similar relationships of sareomere
length to filling pressure in the outer (subepieardial) layer and the inner (subendocardial) layer. Sareomere length appears to
change most in relation to intraventricular
pressure or volume in the innermost regions
of the ventricular wall (fig. 9). Over the
range studied in the canine left ventricles,
sarcomeres average 0.04 /x longer in the inner
layer of myocardium than in the middle layer,
and these in turn average 0.05 /LA longer than
those in the outer layer. Applying statistical
analysis for significance of paired data, t values
of 1.94 and 2.25 support these differences in
sarcomere length as significant at 0.07 and 0.04
levels respectively.
Figure 10 relates .sareomere length to intraventricular volume; the values correspond to
those plotted in figure 4 for sarcomere length
related to iiitraventricular pressure. The
dashed line in figure 10 represents for the
"middle layer" of the wall, the theoretical
relation of the changes in volume in a sphere
of uniform wall thickness to the changes in
circumference. The weight of the wall in the
model is taken to he 96.4 g with a density of
1.00 g/ce. Calculated data for ventricular
radii and wall thickness, used in preparing the
theoretical plot, are presented in table 1. The
theoretical and experimental curves diverge
clearly in the region encompassing sarcomere lengths greater than 2.2 /x, sarcomere
length changes being smaller in this range
than anticipated from the theoretical curve
( fix 10). For sarcomere lengths less than 2.2
fj.. and intraventricular volumes less than 40
cc. the observed and theoretical curves are
correlated more closely. Despite efforts to
V.it
Will.
].,<
53
CARDIAC ULTRASTRUCTURE AND FUNCTION
empty the left ventricle completely, and despite resulting negative intraventricular pressures, sarcomeres less than 1.85 /x in length
have not been observed generally in the nonactivated ventricle.
The sarcomere lengths observed at varying
filling presures in the ventricles from cats are
Sorcomere Length vs. L.V.
Riling Pressure ( Dog)
[middle layer J
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L.V.
PRESSURE
mmHg
180
4i*
190* 200
«'«4>
2.10
2 20
230
240
2.50
SARCOMERE LENGTH — MICRONS
FIGURE 4
Left ventricular pressure vs. sarcomere length for the
middle layer of the canine left ventricle with the best
approximate curve drawn between the points. At 12
mm Hg pressure sarcomere length is approximately
2.2 11. At zero pressure, sarcomeres approach 1.9
fi in length.
TABLE 2
Data Scatter and Filling Pressure: Deviation of Experimental Data from Interpolated Relation of Sarcomere
Length and Left Ventricle Pressure, 27 Canine Hearts
(fig- 4)
LV
pressure
mm Hg
0- 0.5
0.5- 5.0
5-12
12-22
22-30
30-55
Hearts
sampled
Average sarcomere
length
no.
4
5
4
4
4
5
Circulation Research, Vol. XVlll,
Average
deviation
/i
1.93
2.10
2.20
2.28
2.33
2.38
January 1966
0.06
0.08
0.04
0.02
0.04
0.01
illustrated in figure 11. In the ten cats studied,
left ventricular weight averaged 6.4 g. Intraventricular volume at 10 mm Hg averaged 4.0
cc. Despite a more than tenfold difference in
ventricular volume and weight, a similar curve
relating sarcomere length to left ventricular
filling pressure pertains for both the cat and
the dog. In both species sarcomere lengths of
2.2 fi are associated with filling pressures of
10 to 12 mm Hg.
Sarcomere band pattern changes are a function of sarcomere length in the myocardium of
both the cat and dog. While A-band width
remains constant at 1.5 /JL, I-band width
varies linearly with the length of the sarcomere
(figs. 5 and 6). A central dark band termed
the M-line traverses the A-band and is closely
flanked by two light areas termed the L (or
para-M)-lines. This M-L complex is constant
in width at all sarcomere lengths. In sarcomeres less than 2.2 to 2.0 fi in length, however,
the L (or para-M)-lines are less clearly defined (fig. 5) than those observed in longer
sarcomeres (fig. 6). In sarcomeres measuring
2.30 to 2.40 /A, H zones may or may not be
present (figs. 12, 13, 14). However, in sarcomeres measuring more than 2.4 //,, H zones
are generally seen. Reference to figures 4 to
6 indicates that sarcomere lengths associated
with the appearance of H zones are observed
with ventricular filling pressures in excess of
15 to 20 mm Hg. The distribution of data in
figure 15 indicates generally that H-zone
width increases with sarcomere length.
Discussion
The pressure-volume curves as presented
here for left ventricles from dogs resemble
those previously reported.17"20 The present
methods have allowed the delineation of reproducible passive pressure-volume curves
with fixation of each ventricle for electron
microscopic analysis at a known point along
its curve. Reproducibility is indicated by the
superimposition of successive curves from the
same heart and the quantitative recovery of
added fluid. The constancy of observed data
also indicates that under the conditions described heart muscle does not develop
SPOTNITZ, SONNENBLICK, SPIRO
54
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Janujry
Rl-siatib. Vol. Xl'lll.
Ci'ibhti'm
55
CARDIAC ULTRASTRUCTURE AND FUNCTION
rigor in the first half hour after removal
from the animal, consistent with findings
reported by others.-" Stress relaxation21 was
found to be a factor in pressure-volume relations of the left ventricle only if filling pressure exceeded 15 mm Hg. Even in this circumstance, a tendency to return to the original
curve was observed if the ventricle was
allowed to recover at a lower pressure-volume
point.
Previous studies of the isolated cat papillary
muscle have shown that sarcomere length,
band patterns, and actively developed tension
are functions of overall muscle length7' 9 and
are in accord with a sliding filament model
for muscle contraction. A reproducible curve
for sarcomere length vs. active tension has
been derived from these data. Active tension
is zero at sarcomere lengths of approximately
1.5 /J. and rises along the ascending portion of
the curve to a maximum at initial sarcomere
lengths of 2.18 to 2.24 /A. With further increments in muscle and sarcomere length, the
descending limb of the active tension curve
appears, and developed tension declines. Also,
in accordance with the sliding filament model,
progressive penetration of thin actin filaments
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'." 7
50
45
Sarcomere Length vs.L.V.
Pressure (Dog)
[outer laye
Sarcomere Length vs. L.V.
Pressure (Dog)
[inner layer]
/
40
/
/
35
•
4
•
30
L.V. PRESSURE
30
*
• . 'I
/
L V. PRESSURE
mmHg
25
25
m
I
•
20
•
15
•
/•
10
•
/
/
5
•
•
•
(tig71
180
*I9O
200
2.10
2 20
2 30
240
SARCOMERE LENGTH — MICRONS
250
SARCOMERE LENGTH — MICRONS
FIGURE 8
FIGURE 7
Left ventricular pressure vs. sarcomere length in the
outer layer of the canine left ventricle.
FIGURE
^
S
(top,
Craph similar to figures 4 and 7 except that left
ventricular pressure is plotted here vs. sarcomere
length in the inner layer of the canine left ventricle.
facing
page)
Electron micrograph of outer layer of canine left ventricle fixed at a filling pressure of 0 mm Hg
and a filling volume of 11 cc. Sarcomeres average 1.94 ± 0.04 n in length, l-bands and A-hands
as well as Z, M, and L(or para-M)-lines are indicated.
FIGURE 6
(bottom, facing
page)
Electron micrograph of outer layer of canine left ventricle fixed at 12 min Hg filling pressure
and 52 cc filling volume. Sarcomeres are extended further than those in figure 5, measuring
an average of 2.22 ± 0.05 n, and show proportional increments in 1-band length. The L(or
para-M)-lines are clearly defined and are more electron lucent than those of figure 5.'
Circulation Research,
Vol. XVlll,
January 1966
SPOTNITZ, SONNENBLICK, SPIRO
56
into the M-L complex occurs at sarcomere
lengths less than 2.2 to 2.0 /Lt, with the appearance of A-contraction bands due to a double
overlap of thin filaments at sarcomere lengths
of less than 1.8 /JL.
The present results, including an A-band
of constant width and I-band width proportional to sarcomere length, are in full accord
with the sliding mechanism. The relative
darkening of the L (or para-M)-lines at the
shortest sarcomere lengths observed in this
study, again reflects the passage of thin actin
filaments into the center of the sarcomere.
However, no A-contraction bands were observed, since sareomeres shorter than 1.85 fJ.
were not encountered. Further evidence for
the sliding model is afforded by the presence
of H zones22''"' which are consistently observed in sareomeres measuring more than
2.4 ix. These H zones are created by the
progressive withdrawal of thin actin filaments
from the A-band at long sarcomere lengths,
but it is difficult to ascertain a linear relationship between H-zone width and sarcomere
length. There is no simple explanation for the
fact that H zones are not consistently present
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50
Sarcomere Length vs. L.V.
Pressure (Dog)
[ 3 principal muscle layers]
45
40
Middle
layer
• Inner layer
• Middle layer
A Outer layer
35
30
L.v. PRESSURE
mmHg
25
20
I 5
10
5 -
0 1.80
1.90
2.00
£10
2.20
2.30
2.40
SARCOMERE LENGTH — MICRONS
2.50
FIGURE 9
Composite of figures 4, 7, and 8 showing the sarcomere length pressure relationships for the
three principal muscle layers (outer, middle, inner) of the canine left ventricle. Note that the
changes in sarcomere length over the plotted range in filling pressure appear to be greatest in
the inner layer, least in the outer layer. At pressures greater than 5 mm Hg, the sareomeres are
largest in the inner layer and shortest in the outer layer (see text).
Circulation Research, Vol. XVIII, January 1966
CARDIAC ULTRASTRUCTURE AND FUNCTION
57
end diastolic filling pressure and reaches a
maximum as ventricular filling pressure approximates the upper limit of the normal
range. These observations serve to support
the view that the normal left ventricle functions only along the ascending portion of the
length tension curve, when end diastolic sarcomere lengths measure 2.2 fi or less.27
The fundamental importance of the relation of sarcomere length to passive left ventricular filling pressure is reflected in the
constancy of this relation for the dog and the
cat, despite a tenfold difference in ventricular
mass and volume. A mathematical basis for
the generality of this relationship, independent
of absolute chamber size, is given by a modified Laplace equation, which expresses intramural stress as a function of intraventricular
100
So rcomere Length vs. L.V.
Volume (Oog)
( middle layer )
90
/ •
/
/
/
•
80
/
•
70
L.v.
VOLUME
6 0
50
/
/
/
•
/
/
/
40
• IS'
• y
30
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20
10
1
,)ig,0)
1.8
1
1.9
m
i
1
20
1
1—i—i
2.1
2.2
i
i
2.3
1
i
2.4
i
i
2.5
SARCOMERE LENGTH — MICRONS
FIGURE 10
Experimental volume vs. sarcomere length curve for
the middle layer of the canine left ventricle (solid
line) is compared to a theoretical curve for the middle
layer of a spherical model of total mass identical to
the average for the canine left ventricles studied
(dashed line, see table 1). Theoretical curve is chosen
to intersect the experimental curve at a point where
sarcomere length is 1.9 p and filling volume is 12 cc.
in sarcomeres measuring 2.3 to 2.4 JJL, nor is
it apparent why the upper limit of observed
sarcomere length, as noted previously,7 is
approximately 2.6 /x.
In figure 11, the relation of sarcomere length
to actively developed tension in the cat papillary muscle" is correlated with the curve for
sarcomere length vs. filling pressure for the
middle layer of the left ventricle of both the
cat and the dog. The upper limit of normal
for left ventricular end diastolic pressure in
the intact heart is approximately 12 mm
pjg.21,24-2« t n j s corresponds to a sarcomere
length of 2.2 /x in the middle layer of the
ventricular wall for both animals. This same
sarcomere length also corresponds to the
apex of the active length-tension curve of the
isolated papillary muscle. Thus, the tension
developed by the left ventricle during active
contraction (systole) increases with increased
Circulation Research, Vol. XVIII, January 1966
Lefl Ventricle
• Dog
6
Si
s
5 S
I
I
2DO
2.10
220
£30
2.40
SARCOMERE LENGTH — MICRONS
FIGURE 11
Curve of active tension vs. sarcomere length for isolated cat papillary muscle us superimposed on the
curve of left ventricular pressure vs. sarcomere length
incorporating data obtained for the intact left ventricle
of both the dog and the cat. The apex of the active
length tension curve for papillary muscle corresponds
to a sarcomere length of 2.2 n, a sarcomere length
observed as well in the middle layer with a filling
pressure of 12 mm Hg in the intact left ventricle of
both animals.
58
SPOTNITZ, SONNENBLICK, SPIRO
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CirtuUnon RciurJ-,
Vol. XVIII,
Jjnuur; 1 <JfiC
59
CARDIAC ULTRASTRUCTURE AND FUNCTION
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FIGURE 14
Electron micrograph of tissue from the middle layer of canine left ventricle filled to 40 mm Hg
pressure with an intraventriculur volume of 68 cc. Average sarcomere length is 2.3! ± 0.02 y..
No 11 zones are observed as is often the case in sarcomcres of 2.3 to 2.4 i*. The various sarcomere bands, including the \ lines (S), are identified.
pressure. If the ventricle is treated as an elasticsphere of uniform wall thickness, tangential
stress is related to transmnral filling pressure
hv this modified formula:
s,-
RP
211
(1)
where S, is tangential stress (force per unit
area), R is the mean radius of the ventricle,
F is transmural pressure, and It is wall thickness.-"' For any two given left ventricles,
regardless of absolute size, tangential stresses
in the wall will be approximately equal at a
FIGURE 12 'top, facing page)
Electron micrograph of middle layer of the canine left ventricle filled to a pressure of 35 mm
Hg at a volume of 5H cc. At this high pressure at sarcomercs average 2.35 ±_ 0.07 /J. in length
and exhibit demonstrable II zones III) with irregular margins. The various other sarcomere
bands are identified.
FIGURE 13 I bottom, facing page
Higher magnification from the same tissue shown in figure 12. The absolute band measurements
are less than those of figure 12 since it is necessary to section the muscle in a direction perpendicular to the long axis of the fibers in order to demonstrate the tico types of filaments. The
thin act in filaments (act) terminate at the margins of the 11 zone ill). The myosin filaments
(my) extend the entire length of the A-barul including the H zone. IS. is the interstitial apace
between two adjacent heart muscle cells.
(iruAjtum Ren;ir,h. \',,l. Will. J,,rlr,.,r;
SPOTNITZ, SONNENBLICK, SPIRO
/
.75
.70 -
/ •
.65
/
.60
/#
.55
?* • •*
a.
x 50
2 .45
2
/
1
.30
.25
/
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/
.20
/
.15
.10 -
/
/
/
.05
/
/
•
K
-
/
-/ ^*' /y -'
/
/
-
expression reaches a maximum when R = a,
indicating that the distribution of stress is not
uniform, increasing to a maximum at the inner
surface of the chamber wall and decreasing
toward the outer surface.
In line with this analysis, the present study
has shown that at a given ventricular filling
pressure sarcomeres are longest in the inner
layers of the left ventricular wall, decreasing
in length toward the more superficial layers.
This trend, confirmed by statistical methods,
is suggested as well by the superimposition of
2.50
nn
°°
2.1
"*•'"
2.2
2.3
2.4
2.5
2.6
2.7
SARCOMERE LENGTH y.
FIGURE 15
Relation of sarcomere length to H zone width in
sarcomeres from canine left ventricles filled to high
intraventricular pressures. Dashed lines indicate the
anticipated relation suggested by the sliding filament
hypothesis with thin actin filaments measuring 0.9,
1.0, or 1.1 ix in length.
given transmural pressure if the ratio of radius
to wall thickness is a constant. Thus, if R/h
= k, equation 1 reduces to
(2)
St = %kP = k'P
Therefore, if the relative dimensions of the left
ventricle are all increased or decreased proportionately in hearts of varying sizes, sarcomere length will remain a constant function
of filling pressure, provided that the passive
sarcomere length-resting tension curve for elements of the myocardium is unchanged. A
more exact treatment has been given by Timoshenko29 for elastic spheres of uniform wall
thickness,
_ (Pa») (2R» + b»)
{
(2R*)
(68-o3)
'
where a is the radius to the inside of the wall,
b is the radius to the outside of the wall.
Other variables are as described above. This
i(
1.9
2.0
2.1
2.2
2.3
2.4
2.5
Hij.161
SARCOMERE LENGTH-MICRONS
FIGURE 16
Normalized fixation volume vs. sarcomere length. Relation of sarcomere length in the middle layer of
the canine left ventricle to fixation volume normalized
as a decimal fraction of the filling volume for each
preparation corresponding to an intraventricular pressure of 10 mm Hg. Several cubic functions which intersect the experimental curve at arbitrary points are
illustrated. A cubic function describes the relation of
circumference (c) changes to volume (v) changes in
a sphere or in a symmetrically expanding ellipsoid of
revolution or cylinder if sections are examined in
planes perpendicular to the long axis. Marked divergence between the slopes of the theoretical and experimental curves is evident at large filling volumes.
A similar divergence is shown in figure 11.
Circulation Research, Vol. Will.
January 1966
61
CARDIAC ULTRASTRUCTURE AND FUNCTION
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tracings in figure 9. This gradient, which is
particularly evident at elevated filling pressures, reflects the maximization of tangential
stresses at the inner surface of the ventricular
wall (equation 3). It also reflects the geometric
relation noted by others30"3- that any given
volume change results in maximal relative
changes in circumference in those regions of
the wall with the shortest radius (table 1).
The present experimental findings are consistent with the concept that the ratio of ventricular radius to wall thickness (R/h) in
individual left ventricles is adjusted to produce
a relation of sarcomere length to transmural
pressure independent of absolute size of the
ventricle. As a result, transmural filling pressure represents a more general and reproducible index of sarcomere length (figs. 4 and 11)
than either absolute or normalized filling volume (figs. 10 and 16). This finding, consistent
with the Laplace equation, is even more noteworthy in the face of deviations from theory.
Thus, structural factors, such as the content
and disposition of connective tissue in the
chamber wall, dictate that normal ventricles
of similar size and weight may have dissimilar
pressure-volume relations (fig. 17), while sarcomere lengths remain comparable at comparable filling pressures. A further complication is introduced by the true shape of the left
ventricle which is complex and is not given
accurately by any simple geometric model,
spherical or otherwise.33"30 Nonetheless, experiments have indicated that variation in wall
thickness compensates for the varying radii
of curvature in different myocardial segments
so that tangential stresses remain essentially constant throughout/"':i8 Thus, although
marked differences may be seen in wall thickness, sarcomere measurements are comparable in all regions of the wall of any given
left ventricle, some allowances being made for
statistical variation and the differential distribution of stress in the several myocardial
layers.
The relation of cross-sectional stresses to
sarcomere length in the wall of the intact
ventricle is more complex than that observed
in papillary muscle. Resting tension in papilCirculation Research, Vol. XV111, January 1966
80
70
60
o
2
50
UJ
2
40
O
>'
30
20
.O.O67X+1210
60
"''' 7>
80
100
120
140
160
VENTRICULAR WEIGHT IN GRAMS
180
FIGURE 17
Distensibility vs. chamber weight. Filling volumes in
the canine left ventricle at intraventricular pressure
levels of 0 and 10 mm Hg are illustrated as functions
of left ventricular weight. Although a statistical method has been used to derive the indicated regression
lines, it is apparent that considerable variation in
pressure-volume relations may occur for normal left
ventricles of similar absolute weight.
lary muscle approaches zero at sarcomere
lengths of 1.5 fi. The structure of the left
ventricular wall, however, is such that sarcomere lengths of 1.87 to 1.95 (JL are observed
in the various muscle layers (figs. 4, 5, 7, 8)
when intraventricular pressure (and, therefore, tangential wall stress) is zero. The difference between papillary muscle and the intact
ventricle is, in all probability, a consequence
of differences in three-dimensional structure.
In the papillary muscle, principal fiber direction is parallel to the long axis of the muscle
while the left ventricular wall is an intricate
system in which the long axes of fibers in
the various layers are arranged obliquely with
respect to one another.10'10 Thus, while forces
sufficient to extend sarcomeres beyond 1.5 (JL
may be exerted on individual contractile
62
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units, the three dimensional arrangement of
muscle fibers in the intact wall results in
cancellation of such forces, resultant overall
tension summating to zero.
This suggested stressing of the ventricular
wall is in accord with the observation reported
above, that even when negative pressures are
employed to extract all fluid from the left
ventricular cavity, sarcomeres shorter than
1.85 /JL are not observed in any of the principal
layers of myocardium. The observed rate of
change of sarcomere length relative to passive
filling volume in approaching this lower limit
of sarcomere length, moreover, is not theoretically adequate for ejection of physiological
stroke volumes from the actively contracting
canine left ventricle, as indicated in table 1.
Indeed, observations in beating hearts that
have been fixed in systole have shown that
sarcomere lengths as short as 1.5 to 1.6 /A
may be .observed in the wall of the actively
contracting ventricle.30 With the augmented
tension available in activated muscle, the lower
limit of sarcomere length is shorter than that
observed under passive conditions. Therefore,
the general shape of the curve relating sarcomere length to intraventricular volume differs
for activated and nonactivated heart muscle.
In fact, the geometry of active systolic contraction has been found to be significantly different
from that of diastolic filling.35 Also consistent
with this concept are the findings of Hort and
Linzbach30'40 who have utilized optical interferometric methods and observed, in many
species, very short sarcomeres in hearts during
rigor. Hort has reported for preparations of
the canine left ventricle in rigor (average residual volume 6 cc) that sarcomere length
averages 1.6 fi in the middle layer.10 Locker
previously had reported the observation of
varying degrees of sarcomere shortening with
the onset of rigor in skeletal muscle.41
The ventricular model employed in evaluation of the present results is defined in table 1
as a simple sphere of uniform wall thickness.
For purposes of calculation, the wall was
assumed to displace a volume of 96.4 cc,
equivalent to the average weight of the canine
ventricles used in the present study. In addi-
SPOTNITZ, SONNENBLICK, SPIRO
tion to predicting the expected degree of
change in sarcomere length for any given volume change, the model illustrates clearly the
very different degrees of shortening required
in the various myocardial layers. Thus, a
stroke volume of 23 cc, representing 43% of
an end diastolic volume of 53 cc* would
require changes in circumferences of 17, 10,
and 5.5% in the inner, middle,-and outer layers,
respectively. Experimentally observed changes
in external cardiac dimensions during systole43
have thus been smaller than predicted in calculations neglecting the effect of the thickness
of the left ventricular wall.27
The wall thicknesses calculated for the model
agree well with experimental data. Calculated
thicknesses of 9.6 mm at 53 cc left ventricular
volume, and 11.8 mm at 30 cc volume, may
be compared to experimental values of 8.4
mm and 10.3 mm recently reported for minimal diastolic and maximal systolic wall thickness respectively in the canine left ventricle.44
In weighing the left ventricles employed
in this study, nonmural musculature (trabeculae carneae, papillary muscle) was not distinguished from mural myocardium. Wall
thickness, as calculated for the model is augmented by this additional nonmural muscle,
and this accounts partially for differences
between theoretical and experimental values
for mean wall thickness.
The use of a spherical model is adequate
for the approximate evaluation required here
and is subject to relatively simple mathematical
treatment. Others have found that an ellipsoid
of revolution is a more accurate description
of the shape of the left ventricle.33' 30 Symmetrical expansion of such a model for the left
ventricle would lead to the same relation between changes of circumference and volume
as that derived from the spherical model,
when considering sections including fibers of
the middle layer which form circular rings
'Stroke volume in the dog has been determined by
both angiographic and dye dilution methods to be
approximately 20 cc for a 17-kg animal. Values for
end systolic and end diastolic volumes vary, however, and have been estimated at 30 to 10 cc and
50 to 30 cc respectively.31. «
Circulation Jiesearch. Vol. XV111, January 1966
CARDIAC ULTRASTRUCTURE AND FUNCTION
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of muscle in planes perpendicular to the long
axis of the ventricle.15'10 The same relation
obtains also when considering the ventricle
as a cylinder, again allowing symmetrical
expansion of all dimensions and considering
only cross sections including the "ring fibers"
of the middle layer. As has been noted,
systolic contraction involves, in all likelihood,
a somewhat different geometric relationship.35
Figure 10 shows that the experimental
curve for sarcomere length vs. intraventricular
volume diverges from a theoretical curve,
based on the spherical model defined in table
1, for changes in ventricular circumference
relative to chamber volume. Figure 16 illustrates that this divergence is not decreased
significantly by normalizing the volume data
in order to compensate for variations in
absolute ventricular size. The use of alternate
theoretical curves, similarly, has little effect.
Confirmation for the observation of relatively
short sarcomers, 2.3 to 2.4 n in length, in
overdistended left ventricles is available in
the work of Hort.10
Certain relevant complexities of left ventricular geometry must be considered. The presence of papillary muscles and trabeculae
carneae in the ventricular lumen reduces
effective space33'45 and may make volume
changes appear disproportionately large. Thus,
if 8 cc of the chamber volume were occupied
by incompressible muscle, a twofold expansion
in chamber size from 10 to 20 cc would appear
to be a sixfold change from 2 to 12 cc. A
similar effect would appear were collapse and
buckling of the inner region of the ventricular
wall to occur, converting smooth concentric
layers of sarcomeres to involuted and serrated
rings. Such infoldings at the inner surface of
the ventricle can reduce the effective circumference without corresponding decreases in
sarcomere length. Another form of collapse,
appearing as a transition in shape from an
ellipsoid to a sphere, similarly would change
internal volume but not circumference. A
minor degree of such transition of ventricular
form from the ellipsoidal toward the spherical
has been noted in passive filling.35 The significance of these mechanisms, however, in the
Circulation Research, Vol. XVIII, January 1966
63
divergence of theoretical and experimental
curves for sarcomere length vs. intraventricular
volume is questionable. This divergence is most
prominent at filling volumes of such great
magnitude (greater than 53 cc at 12 mm Hg
pressure for left ventricles in the present
study) that the relative volume occupied by
nonmural musculature becomes insignificant,
and the importance of wall collapse, infoldings, or changes in ventricular shape appears
similarly reduced. At smaller chamber volumes, however, these mechanisms may become
effective, serving in varying degrees to permit
maximum ventricular emptying with minimal
changes in sarcomere length.
The observation that changes of sarcomere
length are apparently incommensurate with
linear changes in the dimensions of the ventricular wall resembles observations in studies
of the sarcomere length-tension curve for
papillary muscle.9 These studies have shown
that high intramuscular tension is required to
extend sarcomeres beyond 2.3 fi and that
under conditions of such high stress expansion
of sarcomeres ceases to be proportional to
changes in overall muscle length. The association of this finding with high levels of stress
in both the papillary muscle and the intact
ventricular wall is consistent with possible
structural distortion resulting in relative slippage of myocardial elements. As an analogy
to one type of distortion considered for the
intact ventricle, if a large coil spring is
twisted back upon itself, the circumference,
and thus the enclosed volume, will increase
while the length of wire forming the spring
remains unchanged. In this connection, gross
torsional motion of the left ventricular wall
during overdistention has not been observed,
although a few degrees of such motion have
been noted for the left ventricle of the beating
heart during systole.35'39 Furthermore, major
rearrangements of shell-like masses of myocardium in the left ventricle have been
opposed on the basis of observations recently
reported by Hort.1G Thus it would appear
that structural distortion, if occurring, can be
observed only at microscopic levels. The failure to observe marked changes in the con-
64
SPOTNITZ, SONNENBLICK, SPIRO
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figuration of the intercalated disc under load
suggests that within a given myocardial fiber,
at least, relative sarcomere position remains
constant. Slippage of columns offibers,however, might account for the apparent failure of
sarcomere expansion. Thus, while gross rearrangement of whole shells of muscle is
unlikely in the intact ventricle, slippage of
columns of fibers within those shells, relative
to one another, may occur.
The finding that the apex of the sarcomere
length-tension curve is reached with ventricular transmural pressures of 10 to 15 mm
Hg, and its relevance to the Frank-Starling
principle,46-47 has been discussed recently.27
Ventricular filling pressures significantly
greater than 15 mm Hg cause sarcomeres to
expand beyond 2.3 /u, resulting in partial
withdrawal of actin filaments from the Abands with the appearance of an H zone. This
places the myocardium at a relative disadvantage by forcing it onto the descending limb of
the active length-tension curve, and may contribute to myocardial failure.
Consideration of the modified Laplace
formula (equation 1) reveals the effect of
pathological hypertrophy or ventricular dilatation on the relations discussed above. In the
hypertrophied heart, abnormally high filling
pressures may exist without extending sarcomeres to the point at which myocardial performance is compromised. But in the dilated
ventricle, even ordinary filling pressures may
extend sarcomeres beyond normal limits.
pressure is observed to be a more general
and reproducible index of sarcomere length
than absolute or normalized filling volume. H
zones are often present in the sarcomeres of
the ventricular wall with filling pressures
greater than 15 mm Hg and sarcomeres
greater than 2.3 [L in length. These findings
are discussed in relation to previous studies
of papillary muscle and in relation to mathematical models for the left ventricle. The
present results indicate that the normal left
ventricle functions along the ascending portion
of the length-tension curve, where the end
diastolic sarcomere lengths are 2.2 /u, or less.
Acknowledgment
The excellent technical assistance of Miss Kay
Barthelmess, Michael Ann Callahan, and Mr. Moshe
Rosen, and the photographic aid of Mr. Lewis W.
Koster are gratefully acknowledged, with additional
thanks to Miss Dorothy Fennell for typing the manuscript.
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Circulation Research, Vol. XVIII, January 1966
Relation of Ultrastructure to Function in the Intact Heart: Sarcomere Structure Relative to
Pressure Volume Curves of Intact Left Ventricles of Dog and Cat
Henry M. Spotnitz, Edmund H. Sonnenblick and David Spiro
Downloaded from http://circres.ahajournals.org/ by guest on May 7, 2017
Circ Res. 1966;18:49-66
doi: 10.1161/01.RES.18.1.49
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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