Download Electron Microscopic Studies of Skeletal and

A M . ZOOLOGIST, ll:54S-5">7 (1971).
Electron Microscopic Studies of Skeletal and Cardiac Muscle
of a Benthic Fish. I. Myofibrillar Structure in Resting
and Contracted Muscle
LAWRENCE HERMAN
Department of Pathology, State University of Neiv York Downstate
Medical Center, Brooklyn, New York 11203
AND
PAUL DREIZEN
Department of Medicine and Program in Biophysics, State University of
Neiu York, Downstale Medical Center, Brooklyn, New York 11203
SYNOI'SIS. Electron microscopic studies are reported on glycerinated skeletal and cardiac
muscle of a benthic fish, Coryphaenoides species. In white skeletal muscle, the
sarcomeres have a resting length of approximately 1.8 p., with thick filaments 1.4 ^ and
thin filaments 0.75 fi in length. These dimensions are somewhat shorter than filament
lengths of oilier vertebrate muscles, possibly due to the elfect of volume increase
during assembly of thick and thin filaments at high hydrostatic pressure. During
ATl'-induced contraction of Coryphaenoides muscle from sarcomere lengths of 1.8 ^ to
1.6 jx, there is a characteristic interdigitation of thick and thin filamenLs, with decrease
in I band length and no change in length of thick or thin filaments. Howe\er, in
sarcomeres contracted to lengths of 1.5 y. to 1.2 p., there is a slight shortening of the A
band, apparently due to shortening of thick filaments, that occurs despite the presence
of residual ] band in the same sarcomeres. There is no obvious crumpling or distortion
of thick filaments during contraction to sarcomere lengths as low as 1.0 /i, but
filament organization undergoes extensive disarray at sarcomere lengths approaching
0.7 fi. Although effects from heterogeneity of filament length cannot be excluded with
certainty, the present evidence does suggest that contraction ot Coryphaenoideb muscle
from 1.6 fi to 1.0 fi sarcomere lengih is accompanied by shoitening of thick filaments
consequent to a structural change within the thick filament core.
„
., , ,
.
.
,.
Detailed electron microscopic studies
.,,
, TT i mrr- TT I , n ™
(Hanson and Huxley, 1955: Huxley, 1960,
,V.O. ,
,,-,,,
,
1963) have established that vertebrate stri,'
.
. . . .
ated muscle contains an interlocking array
. ..,
. , n,
, ,? . ;
of thick (myosin) filaments and thin (acv ;
/
i
tin) hlaments, with cross-bridges extending
&
' . ,. .
, ,
, ,. , „.b
at periodic intervals along each thick hla1
5
...
, r „
,
ment. It is widely accepted, following the
original proposal oi A. F. Huxley and
Niedergerke (19o4) and H. E. Huxley and
Hanson (1954), that contraction involves
a sliding together of thick and thin fila.,
,
. l
, .,
ments without change in cfilament
length,
, ,
.. ° r
.
, °
and that contractile force is generated by
. . .
.
..
.
. .. '
cyclic interaction ol myosin cross-bridges
,
• •
,
,.
,. ,
and actin sites. According to this theory,
,
, ••
contraction involves a net linear move,•
, .,
,
. . . . . . , .
ment ot cross-bridges along individual thin
.
.,., ° „ ° , ,
rl
hlaments, without structural change in
^ Jeast ^ n d e r
l h e ihick
fi,amcm
h iol icall
significant conditions (A.
J,_ ; Hux f eyj {95^ H E H u x ] e y >
^
This work was supported in part by research
grants from the National Institutes of Health (CA-
19G9). However, some objections have
|-, een raised to a simple linear scheme (see
06801 and AM-06165), the Health Research Coun-
„
INTRODUCTION
-
1 r»'-i\
1
1
»•
1 1
cil of Xew York Cit>, and the New York Heart Drcizcn, 19/1), and an alternative model
Association. P.D. is a Career Scientist of the Health has been proposed, according to which the
Research Council of Xew York City. We grate- cros s-bridge interactions result in torsionfully acknowledge the technical assistance of Mr.
. . .
„.
Alan I.ieberman and Miss Catherine Ales.
543 al Winding
ofr the thick filament core
FIG. 1. Longitudinal si.Ltions of Coryphacnaides
white skeletal muscle. (A) Sarcomeres at rest
length (1.8 /j), with characteristic Z lines, I
bands, A bands, and central M lines. Glycogen
bodies are scattered throughout; and remnants of
tlie sartoplasmic reticulum (destroyed during glyc-
trol extraction) lie between fibril, x 28,400 (B)
Sarcomeres o£ different length within the same
fiber. Upper region contains sarcomeres of 1.4 ^
length, with narrow I bands; lower region contains
sarromeres of 1.1 <r. lenith, with widening of '/.
lines. >< 24,000.
MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH
545
FIG. 2. Longitudinal sections o£ Coryphaenoid.es
white skeletal muscle, showing sarcomeres of length
1.7 n (A) and 1.8 ^ (B). Note the decrease in A
band length from 1.38 ^ in the relaxed sarcomere
(A) to 1.10 in in the partially contracted sarcomere
(B). The I bands persist throughout, so that A
band shortening can not be attributed to passive
buckling of thick filaments against Z lines. X 70,000.
(Dreizen and Gershman, 1970).
These general features are derived largely from studies of rabbit and frog skeletal
muscles, but similar structural features are
found in skeletal and cardiac muscles of
other vertebrates (Hoyle, 1969), including
those of fish (Franzini-Armstrong and
Porter, 1964; Bergmann, 1964; Kilarski,
1967; Nishihara, 1967). This communication describes electron microscopic studies
of control and ATP-contracted glycerinated skeletal and cardiac muscle of a
benthic fish, Coryphaenoid.es species, whose
natural environment includes conditions
of temperature close to 0°C and pressure
about 3,000 psi.
waters near the Galapagos Islands, during
the 1970 expedition of the Alpha Helix
(see Phleger, 1971). Glycerinated muscle
was prepared essentially by the method of
Szent-Gyorgyi (1951), as modified by
Huxley (1963). Elongated strips of white
muscle from the dorsal and lateral trunk,
red muscle from the subcutaneous lateral
line region, and cardiac muscle (ventricle)
were excised, tied to polyethylene rods,
and stored in 50% glycerol-0.0067M
K2HPO4, pH 7.2, at 4°C. The glycerol
solutions were changed at one and two
days, and the muscle strips were stored at
—20°C for periods of three to four months.
Portions of glycerinated muscle were
transferred to a solution containing 15%
glycerol-85% buffer (0.1M KC1, lmn MgCl2,
0.0067M K2HPO4, pH 7) and allowed to
stand for 2-3 hours at 4°C. The muscle was
shredded and then homogenized for about
MATERIALS AND METHODS
CUycerinalcd muscle preparations. The
Coryphaenoid.es fish were captured at a
depth of 2,200 meters in the off-shore
546
LAWRENCE HERMAN AND PAUL DREIZEN
three minutes using a Sorvall Omnimixer
at 4°C. Muscle fibers in different stages of
contraction were obtained by adding ATP
(at 4 mM, 1 HIM, and 0.4 HIM) in 15% glycerol-85% buffer (as above) to an equivalent volume of homogenized muscle fibers
in suspension. The fibers were examined
with a Reichert polarizing microscope
(Xenon source, at 5400 A) for possible
changes in striation spacing and pattern of
birefringence.
Electron microscopy. After incubation in
ATP solutions for 30 minutes, the suspension of glycerinated muscle fibers was fixed
with 8 volumes of glu tar aldehyde (Polysciences Inc., Rydal, Pa) buffered with 0.1M
sodium cacodylate at pH 7.4, for one
hour, according to the method of Sabatini
et al. (1963). The fixed muscle homogenate was centrifuged at 15,000 rpm for
15 minutes, using a Beckman SW-39L
swinging bucket rotor; the pellet was
washed three times in 0.2M sucrose-O.lM sodium cacodylate, pH 7.4, for ten minutes.
All procedures were done at 4°C, and the
samples were stored at 4°C until dehydration in a graded series of alcohols, followed
by embedding in Epon (Luft, 1961).
Ultra-thin sections were cut on LKB
Ultratome I, mounted on uncoated Athene
grids, stained with methanolic uranyl acetate (Stempak and Ward, 1964), and examined in a Philips 300 electron microscope. Electron micrographs were taken at
3400, 8700, 27,000, and 65,000 magnifications and photographically enlarged subsequently.
RESULTS
White skeletal muscle. The general morphology of white skeletal muscle of Coryphaenoides appears similar to comparable
muscle of other fish and mammals. Irregularly shaped bundles of myofibrils are separated by clusters of sarcoplasmic reticulum, from which the soluble components
were extracted during prolonged storage in
glycerol. On longitudinal section, the sarcomere.s show characteristic Z lines, I
bands, A hands, and tontral H-zones that
are bisected by M lines (Fig. 1A). The
resting length of a sarcomere is approximately 1.8 fx,; however, many fibers contain shorter sarcomeres and occasional
fibers contain longer sarcomeres. This
heterogeneity probably arises from differences in contractile activity prior to death
of the fish, but structural variation among
the myofibrils cannot be excluded.
The sarcomeres are composed of interdigitating thick and thin filaments. At sarcomere lengths close to rest length, the
thick filaments are approximately 1.4 JU, in
length and the thin filaments are approximately 0.75 /i in length (from center of Z
line to free end of the filament). Both sets
of filaments are well visualized in crosssections of myofibrils in the region of the
A band, where six thin filaments are
arranged in an hexagonal array about each
thick filament, with an average of two
thin filaments per thick filament, as in other vertebrate skeletal muscle (Hanson and
Huxley, 1955; Hoyle, 1969). Thick filaments are approximately 110 A and thin
filaments are about 40-45 A in diameter.
Although cross-bridges may be seen as
poorly defined projections of thick filaments in cross section, the cross-bridges are
more readily visualized in longitudinal
section as periodic densities along the axis
of the thick filament or as knobby protuberances that extend towards the thin filaments (Figs. 2, 3).
Observations with the polarizing microscope show that addition of ATP results in
shortening of glycerinated fibers, with decrease of striation interval from roughly
1.8 /j. to as low as 0.8 ju.. Although the
extent of shortening is dependent in general on the concentration of ATP, there is
considerable variation in the periodicity of
striations within different fiber bundles,
ranging from maximal shortening in small
fibrils and near the surface of large fibers
to little, if any, shortening within the interior of large fibers. The variation in striation pattern presumably arises from a gradient in ATP concentration due to incomplete diffusion of ATP into the muscle
fibers under the given experimental conditions. Electron micrographs show compara-
MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH
547
3H
FIG. 3. Contraction series for
Coryphaenoides
white skeletal muscle. Sarcomeres are shown at
lengths of 1.7 y (A, B), 1.6 y (C), 1.5 y. (D), 1.4
y, (E, F, G), 1.2 y, (H), 1.1 y (I), 1.0 y (J). 0-9
^ (K), and 0.7 y, (L). These sarcomeres are
representative of the large sample of sarcomeres
used for the measurements summarized in Fig. 4.
Note the slight but progressive decrease in
length of A band from 1.38 y, (B and C) to 1.31 y
(D, E, F) to 1.26 y, (G), although I band is
present in each sarcomere. Thick and thin filaments retain their parallel, ordered array during
shortening to 1.0 y. sarcomere length, but prominent contraction bands form and filaments undergo extensive disarray during contraction below 1.0 ix. Approximate location of Z lines are
noted in 3L. x 72,000.
548
LAWRENCE HERMAN AND PAUL DREIZEN
31
ble variation in the extent of contraction
within different fibrils of the same fiber
bundle, as shown, for example, in Fig. IB.
The morphological changes which occur
during ATP-induced contraction are illustrated in longitudinal sections of white
skeletal muscle at sarcomere lengths from
1.7 /* to 0.7 fx. (Figs. 2, .•)). Shortening of sarcomeres from rest length to about 1.4 t, is
characterized by interdigitation of thick
and thin filaments as Z lines are brought
closer together and I bands are progressively shortened (Fig. 3A-G). However,
there appears to be slight shortening of the
A band in sarcomeres having some residual I band (Figs. 2B, 3D-G). The I band is
completely obliterated during contraction
to and below sarcomere lengths of 1.2 p
(Fig. 311). In sarcomeres contracted to
lengths as low as 1.0 //,, there is no appar-
MYOFJBRILLAR STRUCTURE OF A BENTHIC FISH
549
extensive disarray of filament organization
(Fig. 3L).
The effect of ATP-induced contraction
•4
fon filament lengths of Goryphaenoidcs
white skeletal muscle is summarized quantitatively in Figure 4. The data are plotted without respect to average ATP con•30.4
centration (0.4 HIM, 1.0 mil, and 4 HIM) in
the fiber suspension, since a distribution of
sarcomere lengths is obtained at each concentration of ATP. During shortening of
sarcomeres from 1.8 /*. to 1.6 p, there is
linear decrease of I band length, without
significant change in lengths of thick or
thin filaments, whereas contraction of sar5-1.4
comeres from 1.5 fi to 1.2 p is accompanied
by some shortening of the A band. This
change is presumably due to shortening of
thick filaments, in view of die presence of
residual I band and the absence of gross
distortion of thick filaments. The I band is
1.0
obliterated during contraction to 1.2 p
1.6
18
1.2
1.4
sarcomere length, and shortening of sarSARCOMERE LENGTH
comeres below 1.2 fx, is accompanied by an
equivalent decrease in net length of A
FIG. 4. Lengths of A band, I band, and thin filaband. Thin filaments do not undergo any
ments plotted against sarcomere length for Coryphaenoides white skeletal muscle in the presence
significant change in length during conof ATP. Mean values (± standard deviation) are
traction of sarcomeres from 1.8 /x to 1.0 ju..
shown. Thin filaments are measured from
middle of Z line to free end of thin filament; I
Red skeletal muscle. The overall morband length refers to total sarcomere length less
phology
of red skeletal muscle is similar to
A band length, and includes some contribution
from Z line protein.
that of white skeletal muscle, with
myofibrils separated by clusters of sarcoent crumpling or folding of thick filaments plasmic reticulum (Fig. 5). Red skeletal
in directions perpendicular to filament muscle contains large, moderately dense
axis, although an increased density in the particles that are interspersed between or
region of the Z line may possibly represent on myofilaments, without any particular
some piling up of the terminal ends of localization within a sarcomere. These parthick filaments (Fig. 3I-J). There is no ticles appear as irregularly shaped bodies
evidence for penetration of thick filaments or as solid or doughnut shaped spheroids
through the Z line into adjacent sar- and probably represent the glycogen-rich
comeres, as described in barnacle muscle stores characteristic of red skeletal muscle,
by Hoyle et al. (1965). An interdigitation although the extent and size of these deof thin filaments is evident during contrac- posits greatly exceeds that shown in prevition to 1.0 n sarcomere length, but entire ous studies of fish muscle (Franzinithin filaments are not readily discernible at Armstrong and Porter, 1964; Bergmann,
lengths below 1.0 /*. Shortening of sar- 1964; Kilarski, 1967; Nishihara, 1967).
Most fibers of red skeletal muscle concomeres to 1.0 ju. and less is accompanied
by widening of the Z lines and a partial tain sarcomeres with short I bands or no 1
disruption of the parallel aiTangement of bands at all, although occasional fibers are
filaments (Fig. 3K); shortening to sar- seen with sarcomeres as long as 1.9 p.. In
comere lengths of 0.7 /x is accompanied by the longer sarcomeres, filament dimensions
t-n
L2
550
LAWRENCE HERMAN AND PAUL DREIZEN
Hi;, b. CoryphaenoUles red skeletal muscle. Filaments contain randomly dispersed, large, round
to oval particles of glycogen. Z lines are promi-
nent; I bands are lacking; and A bands display
poorly defined M lines, x 24,000.
are approximately the same as in comparable sarcomeres of white skeletal muscle.
Unlike white muscle, red muscle does not
contain well-defined M lines, but shows an
occasional darkening of the A band mid-
way between Z lines.
Although red skeletal muscle appears to
have undergone extensive contraction during or prior to extraction with glycerol, the
fibers undergo further shortening in the
MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH
FIG. 6. Contracted sarcomeres of Coryphaenoides
red skeletal muscle, at lengths of 1.2 p. (A), 1.1 M
(B), 0.9 fi (C), 0.9 n (D), 0.7 M (E), and 0.6 M
551
(F). Approximate locations of Z lines are noted
in 6E and F. x72,O0O.
552
LAWRENCE HERMAN AND PAUL DREIZEN
70
lit;. 7. Contracted sarcomeres of Goryphaenoid.es
cardiac muscle, at lengths of 1.3 p, (A), 1.1 p. (B),
1.0 n (C), and 0.7 p (D). Note occasional penetra-
tion of thick filaments through Z line into adjacent
sarcomeres in (D); region of Z lines is marked.
X 72,000.
presence of ATP to sarcomere lengths of
Cardiac muscle. The myofibrils of Cory1.1 ix to 0.7 i*. (Fig. 6). As in white skeletal phaenoides cardiac muscle show wellmuscle, there is widening and increased defined Z lines, thick filaments, and thin
density of the Z line at sarcomere lengths filaments, but no evidence of M line matebelow 1.1 /j. (Fig. 6A-D), and disarray of rial. Prolonged extraction of cardiac musthe filament structure at sarcomere lengths cle with glycerol results in destruction of
most of the mitochondria and sarcoplasmic
approaching 0.7 //, (Fig. 6E, F).
MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH
reticulum; however, their persisting remnants indicate a relatively high ratio of
these organelles to myofibrils in Coryphaenoides cardiac muscle. In contrast, in
other vertebrate heart muscles, the myofibrils constitute the bulk of the muscle,
with minimal amounts of mitochondria
and sarcoplasmic reticulum. Glycogen
bodies are scattered throughout sarcomeres of Coryphaenoides heart muscle,
but to a lesser extent than in red skeletal
muscle.
As in red skeletal muscle, most sarcomeres of cardiac muscle have already
shortened without addition of ATP, although occasional fibers contain sarcomeres with length about 1.8 /x. In the
presence of ATP, the sarcomeres of cardiac
muscle shorten to lengths below 1.1 ^
(Fig. 7A-D). Contraction to this extent
results in the formation of contraction
bands which are less prominent than those
formed at comparable sarcomere lengths
in red or white skeletal muscle. This may
be in part related to penetration of some
thick filaments through the Z line, as evident in sarcomeres shortened to 0.7 /j.
length (Fig. 7D).
DISCUSSION
Myofilaments of resting muscle. White
skeletal muscle of Coryphaenoides contains
sarcomeres of rest length ~1.8 ^, with
thick filaments of 1.4 ^ length and thin
filaments of 0.75 p length. These dimensions are relatively short in comparison
with those of the most extensively studied
vertebrate muscles, namely, rabbit psoas
muscle and frog skeletal muscle, which
contain sarcomeres of rest length 2.2 /*, and
2.5 ^, respectively, with thick filaments of
1.6 ix length and thin filaments of 1.0 ju,
length (Page and Huxley, 1963). Similar
sarcomere dimensions are described in a
number of different fish muscles (Kilarski,
1967), although relatively short sarcomeres
(~1.6 fj.) have been reported in trunk
muscle of black Mollies
(FranziniArmstrong and Porter, 1964). These measurements were obtained on muscle prepa-
553
rations fixed with glutaraldehyde, which
better preserves true filament dimensions
(Page and Huxley, 1963; FranziniArmstrong and Porter, 1964), as compared
with earlier studies of osmium-fixed specimens which are characterized by shrinkage
of filaments and poorer morphological
preservation.
Previous studies on frog skeletal muscle
show morphological differences between
the myofibrils of fast (white) fibers and
slow (red) fibers, notably, greater sarcomere lengths in red fibers (although filament lengths are approximately the same
in red and white fibers), an absence of
well-defined M line in red fibers, and
widening of thick filaments at their junction with the Z line (Page, 1965) . Similar
morphological differences are found in red
and white skeletal fibers of Coryphaenoides, although most of the red
skeletal fibers and cardiac fibers appear to
have contracted below resting sarcomere
length, presumably due to terminal contractile activity of these muscles prior to
death of the fish or to subsequent release
of products from the extensive sarcoplasmic reticulum of these muscles.
It seems unlikely that the relatively
short lengths of Coryphaenoides myofilaments represent artifacts due to tissue
shrinkage, since specimens of glycerinated
rabbit skeletal muscle prepared in this laboratory under identical conditions as
Coryphaenoides muscle yielded sarcomeres
whose filament dimensions are in keeping
with the prior studies of Page and Huxley
(1963). Moreover, the intersections of A
and I bands and of H and M zones are
sharply demarcated in Coryphaenoides
muscle, whereas greatly shrunken tissue
would be likely to have poorly defined intersections. The well-defined organization
of sarcomeres and myofilaments in white
skeletal muscle of Coryphaenoides indicates that ascent of the fish from 2,200
meters depth to the ocean surface does not
have drastic effect on myofibrillar architecture.
There is some evidence that the short
filament lengths of Coryphaenoides mus-
554
LAWRENCE HERMAN AND PAUL DREIZEN
cles may reflect the high pressures encoun- TABLE 1. Effect of volume change on association of
actin and myosin filaments at $,000 psi.*
tered under benthic conditions. Previous
studies on rabbit skeletal proteins have
Myosin
Actin
shown that the assembly of F-actin from
&V
85
cc/mole
300 cc/mole
G-actin (Ikkai and Ooi, 1966) and myosin Kp/K°
0.26
0.06
lp
o
filaments
from
myosin
monomers (AJ -A^ )/monomcr 0.4 kcaJ/mole 1.54 kcal/mole
(Josephs and Harrington, 1968) is inhib* Calculations aro based on the equations,
ited by high hydrostatic pressure. Thermodynamic analyses indicate a volume AV=-RTl / d In K \) and AF = -RTlnK.
change, AF, of 84 cc/mole for actin (Ikkai
\
dp
JT
and Ooi, 1966) and 300 cc/mole for myo- Superscripts p and o refer to pressure of 3,000 psi
1
sin (Josephs and Harrington, 1968) dur- and atmosphere, respectively.
ing association into filaments. According to rabbit skeletal muscle. The effect of prespresent concepts of protein structure, a sure might be minimized if the peptide
volume increase during association of pro- residues implicated in filament stabilizatein monomers is largely due to the trans- tion were to involve stronger intermolecufer of water from tightly organized clusters lar forces in Coryphaenoides muscle than
about hydrophobic or polar sites in mon- in rabbit skeletal muscle, or if the filaomeric protein to more loosely organized ments were to pack more closely with
water in free solution as protein sites un- greater overlap per monomer. However,
dergo interaction during polymerization preliminary measurements on Cory(Kauzmann, 1959). The effects of salt and phaenoides white skeletal muscle indicate
temperature on the assembly of actin and cross-bridge periodicities that are roughly
myosin filaments are consistent with a pre- comparable with those found in rabbit
dominant involvement of hydrophobic skeletal muscle.
groups in actin filaments (Ikkai and Ooi,
Structural changes during contraction.
1966) and polar groups in myosin fila- An essential basis for the sliding filament
ments (Josephs and Harrington, 1968) . In theory of muscular contraction is the eviboth cases, the effect of a positive A.V dence from electron microscopy of rabbit
would be to shift the equilibrium from and frog skeletal muscle that the lengths of
filament towards monomer, with increase thick
and thin filaments remain
in association free energy, AF, of 0.42 unchanged during forced extension of muskcal/mole for actin and 1.54 kcal/mole for cle and during contraction to about 70%
myosin during elevation of pressure from of rest length (Hanson and Huxley, 1955;
atmospheric pressure to 3,000 psi (Table Page and Huxley, 1963). Yet vertebrate
skeletal muscle may shorten from 70% of
rest
length to about 30% of rest length
Comparable volume changes (at least
(Huxley
and Hanson, 1954; Hanson and
qualitatively) might be expected during
Huxley,
1955),
and contraction to this exfilament formation in Coryphaenoides
tent
would
necessitate
concomitant shortmuscle. Thus, under benthic conditions
of
the
A
band.
In explanation of
ening
(3,000 psi), the equilibrium would tend to
this
phenomenon,
Huxley
(1960, 1965)
shift from filament towards monomer, rehas
suggested
that
the
terminal
ends of
sulting in the assembly of thick and thin
thick
filaments
are
crumpled
as
they
are
filaments with fewer monomers per filadriven
against
the
Z
line,
although
no
dement in Coryphaenoides muscles than in
tailed evidence seems to have been
iThis value is based on a 468.000 molecular presented on this point. During maximal
weight for rabbit skeletal myosin (Dreizen et al., contraction of skeletal muscle (30-40% of
1967; Gershman et al., 1969), and is somewhat less rest length), there is disruption of the filathan the AV value of 350-400 cc/mole that was
originally estimated by Josephs and Harrington ment lattice and formation of a dense con(1968), based on a molecular weight of 540,000 to
traction band in the region of the Z line
600,000 for mvosin.
MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH
(Hanson and Huxley, 1955; Sjostrand and
Jagendorf-Elfvis, 1967). This phenomenon, termed supercontraction, is attained in glycerinated muscle fibers at high
ATP concentrations and in fresh muscle
fibers following repeated tetanic stimulation (Gordon et ah, 1966) and rapid cooling in the presence of caffeine (Fujii and
Sakai, 1969). Supercontraction does not
seem to represent a physiological event
(Huxley, 1960, 1965; Gordon et ah, 1966).
Coryphaenoides skeletal muscle contains sarcomeres with resting length (1.8
n) only slightly greater than A band
length (1.4 p), and shortening of A bands
occupies a prominent role during ATPinduced contraction. Thus, during contraction of white skeletal fibers (Fig. 4), the
length of A band remains constant at sarcomere lengths from 1.8 ix to 1.6 ^, that is,
about 85% of rest length. During further
contraction from 1.5 jx to 1.2 /* sarcomere
length, the A bands undergo slight shortening, despite the persistence of residual I
band. This finding is in contrast with earlier studies on rabbit and frog skeletal
muscle in which A bands remain constant
in length until obliteration of the I band
(A. F. Huxlev and Niedergerke, 1954; H.
E. Huxley and Hanson, 1954; Hanson and
H. E. Huxley, 1955; H. E. Huxley, 1960).
The slight shortening of A bands in Coryphaenoides muscle does not seem attributable to shrinkage of thick filaments during
fixation, since tissue shrinkage is minimal
with glutaraldehyde fixation of muscle
(Page and Huxley, 1963; FranziniArmstrong and Porter, 1964) ; nor can the
decrease in A band length be attributed to
differential shrinkage of thick filaments,
since thick filaments of varying length are
obtained in the same fibers. Although
heterogeneity in thick filament dimensions
cannot be excluded on the present evidence, this degree of heterogeneity has not
been described in other vertebrate muscles, where filament length appears to be
determined within narrow limits by physical-chemical factors (see above) or possibly specific protein components (as yet
unspecified). Moreover, thin filament
555
length remains constant in Coryphaenoides
fibers, unlike Crustacean fibers, where
thick and thin filaments exhibit concomitant differences in length (FranziniArmstrong, 1970).
These considerations would suggest that
thick filaments undergo slight shortening
under conditions in which the Z lines have
not yet impinged upon the terminal ends
of the thick filaments.2 Furthermore, during contraction of sarcomeres from 1.2 ju. to
below 1.0 n in length, the thick filaments
extend almost to the middle of the Z line,
without obvious folding or crumpling
against the Z line, and without other gross
structural distortion of the filament lattice
or individual thick filaments. These results
are difficult to reconcile with any explanation of A band shortening that is based
simply on passive buckling of thick filaments against the Z line. Instead, the
present evidence on Coryphaenoides muscle would suggest that the thick filament
core may undergo some kind of structural
change during shortening of sarcomeres
below 1.5 ft length. This interpretation
does not, of course, question the wellestablished evidence that contractile force
is generated by cyclic interactions of myosin cross-bridges with thin filament sites
(Hanson and H. E. Huxley, 1955; A. F.
Huxley, 1957; H. E. Huxley, 1960, 1969).
However, the present interpretation would
suggest that cross-bridge interactions have
two consequences; a sliding together of
thick and thin filaments, and a structural
change within the thick filament core
which may lead to shortening of the thick
filament.
If this interpretation be correct, then the
conventional sliding filament model requires some modification, at least for Coryphaenoides muscle. In this respect, several
2 In studies on crab muscle, evidence has been
reported that ATP-induced contraction of Limulus striated muscle is accompanied by shortening
of A bands at sarcomere lengths where I bands
have not yet been obliterated (DeVillafranca and
Marschhaus, 1963). However, this interpretation
has been disputed by Franzini-Armstrong (1970),
who has noted the marked variability of filament
dimensions in single fibers of the crab Portunus
Depurator.
556
LAWRENCE HERMAN AND PAUL DREIZEN
recently proposed mechanisms for the contraction of vertebrate skeletal muscle are
of interest, as follows:
(1) Phase transition, that is, a melting of
myofilaments during contraction of muscle, as proposed by Mandelkern et al.
(1959). As noted by Hill (1968), however,
any such theory would be inconsistent
with the unequivocal experimental observation that isometric force is proportional
with the number of overlapping sites between myosin cross-bridges and thin filament sites (Gordon et al., 1966).
(2) Torsional model. Based on reinterpretation of X-ray diffraction, polarization,
and energetic evidence on the contraction
of vertebrate striated muscle, Dreizen and
Gershman (1970) have proposed the hypothesis that myosin cross-bridges interact
with thin filament sites along a helical
path, resulting in torsional winding of the
thick-filament core within a rigid thinfilament lattice. According to this model,
contraction might proceed initially without significant change in thick filament
length, but further contraction would be
accompanied by shortening of thick filaments. A torsional movement of the thick
filament core would be manifest at the
molecular level by altering interactions
among the myosin molecules and breaking
a-helical bonds within the light meromyosin part of individual myosin molecules.
According to a torsional model, supercontraction would represent the limiting case
in which the light meromyosin ends of the
myosin molecules have undergone a helixcoil phase transition within the thick filament core.
Although definitive interpretation is not
possible on the available evidence, it is
notable that the present findings on Coryphaenoides skeletal muscle would be consistent with a torsional model for contraction. A direct test of this interpretation
would involve demonstration of changes in
cross-bridge periodicity during contraction
of skeletal muscle, and this matter will be
explored in subsequent papers on Coryphaenoides muscle and other striated muscles.
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