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Contraction of glycerinated embryonic myoblasts.

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CONTRACTION O F GLYCERINATED
EMBRYONIC MYOBLASTS
HOWARD HOLTZER* AND J O A N ABBOTT
Department of Anatomy, School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania
INTRODUCTION
Though several recent reviews (e.g., Hoyle, '57 ; Dubuisson,
'54; Barer, '48) state that embryonic muscle can contract
before cross-striated myofibrils appear, the experimental literature on this point is by no means clear. DeRenyi and
Hogue (,31, ' 3 5 ) reported that skeletal muscle fibers from
4-to 11-day chick embryos, grown in tissue culture, contracted,
even though striated myofibrils could not be detected when
the fibers were examined histologically. Friedheim ('31) observed contractions in similar material, but only in those
fibers in which cross-striated myofibrils could be demonstrated
under monochromatic polarized light.
Copenhaver ('39)' Goss ('40) and Olivo, Petralia, and
Ricamo ('48) reported that embryonic cardiac muscle of
salamander, rat and chick contracted before striated myofibrils were demonstrable. The earliest age at which spontaneous contractions have been observed (Sabin, '20 ; Patten
and Kramer, '33) in cardiac muscle of the chick is 28-30
hours (9-10 somite stage). M. Lewis ( '19) , however, showed
figures of cardiac myoblasts from chick embryos at this
stage with well-differentiated cross-striated myofibrils. Furthermore, Lewis stressed that the staining of cross-striated
myofibrils is capricious ; many fixatives and stains obscure
and distort the cross-striations, and successful visualization
of the cross-striations depends on whether or not the muscle
This investigation was supported by Research Grants B-493 and B-1692 from
the National Institute of Neurological Diseases and Blindness of the National
Institutes of Health, United States Public Health Service.
* Scholar i n Cancer Research of the American Cancer Society, Inc.
417
415
H O W A R D HOLTZER AND J O A N ABBOTT
is properly extended. According to Lewis, cardiac myoblasts from a 30-hour chick embryo display the typical adult
sarcomere pattern of A, I, and Z bands. Yet, according to
Hibbs ( '56), who depended on electron microscopy, fully
differentiated myofibrils were first found in 72 hour material.
Lewis' observations that striations are present in cardiac
myoblasts at 30 hours have been confirmed by the fluorescent
antibody technique (Holtzer, Marshall and Finck, '57 ;Holtzer,
Abbott and Cavanaugh, '58).
From this brief review it is apparent that the evidence is
uiisatisfactory for the idea that skeletal o r cardiac myoblasts
contract before they acquire cross-striated fibrils. The difficulties seem to be of two sorts. The first is the uncertainty
of methods for detecting or measuring contractility. The
second is the difficulty which Lewis clearly defined, and which
IIibbs ' studies illustrate, that of detecting cross-striations
in early myoblasts.
The fluorescent antibody method has been shown to reveal
striations in myoblasts of chick skeletal muscle a t very early
stages, before they could be detected by iron hematoxylin
staining or by phase contrast microscopy (Holtzer, Marshall
and Finck, '57). I n the same work, it was reported that
glycerinated myoblasts would contract on treatment with
ATP. Because the two techniques, one for revealing crossstriations, the other for detecting contractility, seem more
sensitive than the methods which have been used previously,
we have reexamined the question of the relation of cross-striations to contractility in embryonic muscle.
MATERIALS AND METHODS
For testing contractility, the response of myoblast models
to A T P was used (Szent-Gyorgyi, '49). Whole chick embryos, removed from the yolk, were extracted for two days
in 50% glycerol a t 0". The glycerol was changed and the
material placed in the deep freeze (-20") for no more than
two days before being tested. The disodium salt of ATP
; p H 6.8).
was prepared in Simms solution (4 X
CONTRACTIOX O F MYOBLA4STS
419
To determine whether isotonic eontraction of the myoblasts might be masked because of resistance imposed by
associations with other cells, three different test situations
were used:
(1) Test for contraction of myoblasts in situ. The glycerol
extracted trunk was split down the mid-line and one half
placed in 1 m l of Simms solution, the other in 1 m l of the
A T P solution. After 5 minutes both halves were washed in
25% glycerol for 5 minutes and each half immersed overnight
in 0.5 ml of fluorescein-labelled antimyosin (for further details
see Holtzer, Marshall and Finck, '57). Each half was then
washed several times in 25% glycerol (phosphate buffer
M/30; p H 7.4) and inspected as a whole mount under the
fluorescence microscope. Whether the myoblasts, their relationships with surrounding cells intact, had contracted, was
easily determined by comparing the half treated with ATP
with the untreated half.
( 2 ) Test for contraction of myoblasts in small clusters.
The glycerol extracted trunk was divided transversely into
cervical, brachial and thoracic regions. Each of these regions
was placed on a slide in a drop of 25% glycerol and teased
under a dissecting microscope so as t o yield clusters of 5
to 15 laterally adhering myoblasts. The ends of these myoblasts in packets were free. A cover slip was placed over
the material and the effect of introducing A T P under the
cover slip observed with the phase contrast microscope.
(3) Test for contraction of isolated myoblasts. The myoblasts were prepared as in ( 2 ) except that individually isolated
myoblasts were tested. These myoblasts were completely free
of all neighboring cells.
The quantitative estimates of the degree of contraction
of the myoblasts, particularly those in clusters and those
isolated, are only approximate. Because even older myoblasts, if they adhere to slide or cover slip, fail to develop
enough tension to shorten when exposed to ATP, only freely
suspended myoblasts were tested. Under these conditions the
420
HOWARD HOLTZER A N D J O A N ABBOTT
myoblasts were often curved, making exact measurements of
length difficult. Furthermore, the changes in refractive index
of the mounting medium incidental to the addition of ATP
tended to obscure cellular detail. For these reasons only
contractions of 25% o r more (down to 75% or less of rest
length) were considered valid.
The chick embryos were staged according to the tables
of Hamburger and Hamilton ( '51).
RESULTS
With antimyosin-stained material, myoblasts with crossstriated myofibrils are observed under the fluorescence microscope in the anterior cervical myotomes as early as stage 14
or 15 (50-55 hours). Not until stage 16 or 17 (52-65 hours)
are comparable myofibrils found in the brachial regions,
while they do not develop in the thoracic region until stage
18 or 19 (65-72 hours). By stage 17 o r 18 there are over a
thousand mono-nucleated myoblasts in the cervical region.
These have well differentiated cross-striations as revealed by
fluorescence microscopy. However, when an unstained and
teased cervical myotome from stage 19 is viewed under the
phase microscope only an occasional cross-striated myofibril
can be seen. This same relative lag between the time when
a fibril can be visualized as cross-striated with antibody
treated material, as compared with the time striations can
be detected by other means, holds for the brachial and thoracic
regions. The principal change in all myoblasts from the time
the first fine cross-striation is detected until the 5th day is
an increase in the length and width of the myofibril and an
increase in the number of myofibrils per myoblast. While
precise measurements have not been made, it is our impression
that there is a progressive increase in the myofibrilar sarcoplasm ratio. This occurs without any appreciable increase
in the girth of the myoblast (Holtzer and Marshall, '58).
Table 1 summarizes the response of glycerol extracted
myoblasts to A T P under three different conditions of restraint. These results indicate that: (1) the differentiation
Clust.
Isol.
X
14 (50-53 hre.)
X
-.
0
0
__
15 (50-55 h i s . )
-~ -
0
0
X
0
0
+++ +++ +++
+ +++ +++
0
++-+ +++
0
+ ++
0
+
0
In situ
CERVICAL
16 (51-56 hru.)
17 (52-64 hrs.)
18 (65-69 hrs.)
19 (68-72 hrs.)
20 (70-72 hrs.)
23 (84-96 hrs.)
STAGE
~
Clust.
C I I I A L_
.B R A_
Isol.
_
_
X
X
X
s
X
0
0
0
0
++
I n situ
_
X
~.
_
X
X
X
0
0
0
_ _ _ __
0
0
0
+++ +++ +++
0
++ +++
0
+ ++
0
+
0
In situ
~
Isol.
x
X
X
X
X
0
0
0
X
0
0
0
+++ +++
+
++
Clust.
THORACIC
and 0 denote contraction of 75%, 5076, 25% and less than 25%. The area above the horizontal line in each
column indicates the stages when cross-striated niyofibrils are observed with labelled antibody. X indicates myoblasts less than
80 p i n total length; such myoblasts cannot be identified with certainty under the phase microscope. The initial length of the
test myoblasts ranged from over 300 y i n older myoblasts t o around 80 y i n the less mature myoblasts. Each entry is based on
the average of 5 separate trials.
+++, ++, +,
TABLE 1
Degree of contraction o f glycerol myoblast models i n h c e d b y A T P
422
H O W A R D HOLTZER A N D J O A N ABBOTT
of myoblasts with striated myofibrils in a cephalocaudal sequence is reflected in the stage when myoblasts contract and
in the extent to which they contract, and ( 2 ) there may be a
brief period in the development of a myoblast when a crossstriated myofibril is present, but when the cell itself does
not contract to any appreciable degree. It should be stressed
that estimates of no contraction refer to the absence of
pronounced shortening -the myoblasts did not contract more
than 25%. It is quite possible that many such cells underwent
some change in length, but due to the limitations of the measuring technique such changes were not recorded.
The following features of the reaction of the myoblasts
to A T P suggest that the shortening is mediated by contraction of the myofibrils and not due to general syneresis. (1)
Cross-striated myofibrils exhibiting the relaxed sarcomere
pattern of broad A bands can be observed uiider the phase
microscope in the older myoblasts. After treatment with
ATP the striated pattern is that typical of the contracted
state (Hanson and Huxley, '55; Hodge, '55). (2) The first
formed myofibril runs along one side of the myoblast. I n
the contracted state, particularly in the isolated condition,
the myoblasts a r e generally C-shaped ; the myofibril running
along the concave surface (see fig. 7 in Holtzer, Marshall
and Finck, '57). (3) Shortening occurs primarily in the
longitudinal axis. I€ the changes induced by A T P were clue
primarily to syneresis, shortening in all dimensions might
he expected. Nevertheless, the fact that all myoblasts decrease
in girth approximately 10-15% after exposure to A T P , including those t h a t fail to contract, might indicate some dehydration. (4) After the ATP has reached the myoblast,
the contraction is completed in one minute.
In a previous paper (Holtzer, Marshall and Finck, '57)
it was reported that isotonic contractions of brachial myohlasts could not be elicited in myoblasts younger than stage
20. F r o m table 1 it will be seen that slightly younger brachial
myolilasts (stage 1s-19) mill also contract. This discrepancy
CONTRACTION O F MYOBLASTS
423
is attributed to the following: (1) Brief storage of the
extracted myoblasts in the deep freeze enhances the degree of
contraction; this procedure was not routinely followed f o r the
earlier work, and (2) More care was exercised to insure that
the myoblasts did not adhere t o glass slide or cover slip.
The myoblasts in clusters are aligned in parallel with
varying numbers of small mono-nucleated cells interspersed
between them. As these small cells do not bind the antibody
there is no way of determining whether they are presumptive
myoblasts or presumptive connective tissue cells. Also unknown is whether at these stages (stages 1 6 2 3 ) the myoblasts are enmeshed in a collagen network. Thus, the nature
and strength of the forces binding the myoblasts laterally one
to the other are uncertain. That lateral adhesive forces do
affect contraction is indicated by the fact that clusters of
young myoblasts do not shorten, though these same myoblasts
shorten maximally when isolated (table 1). Since the clusters
measure no more than 50-100~in width and are less than
2 0 in
~ thickness, it is highly unlikely that the diffusion
of the nucleotide is a limiting factor (see calculations of
Weber, '52). Therefore, while small clusters of myoblasts
freely suspended in glycerol are contracting isotonically, they
are in all probability also contracting against a load. The
load under these circumstances is the lateral adhesive forces
binding the myoblasts together.
These same considerations suggest that myoblasts iiz situ
are subject to additional resistance to contraction by virtue
of their associations with the myosepta. The apical growth
tips of the elongated myoblasts terminate in and around the
small cells of the myoseptal region. The nature of the association between myoblasts and myoseptal cells is unknown.
That the tips of the myoblasts do adhere to the cells of the
myosepta is shown by the fact that myoblasts in intact trunks
cannot shorten maximally until stage 23. Presumably the
binding between the tips of the myoblasts and the cells of
the myosepta prevents shortening at earlier stages. The
occurrence of maximal contraction irz situ by stage 23 sug-
424
H O W A R D H O L T Z E R A N D J O A N ABBOTT
gests that as the cross-striated myofibrils become more prominent with age there is an increase in the tension the
myoblasts can develop. It is tempting to speculate that the
binding of the tips of the myoblasts by myoseptal cells plays
a role in the orderly alignment and conspicuous elongation
of the muscle cells during normal morphogenesis.
DISCUSSION
The observations of DeRenyi and Hogue (,31, '34) that
skeletal muscle contracted before striated fibrils formed, mere
made on fibers from chick embryos as old as 11 days. The
earlier work of the Lewises ( '15, '17) demonstrated that this
material possessed cross-striations. It should be noted though
that when the Lewises performed their experiments, the notion that myofibrils were responsible for contraction T V ~ S
not yet generally accepted. F o r the most part muscle from
these older chick embryos (6-11 days) no longer consists
primarily of mono-nucleated myoblasts, but is largely composed of multi-nucleated myotubes. When stained with labelled antimyosin this advanced muscle is found to be rich
in striated fibrils and hen these myotubes are extracted
with glycerol and exposed to ATP, they invariably contract
to around 25% of rest length (Holtzrr and Marshall, 5 8 ) .
The earliest spontaneous movements i ~ ,vitro of trunk
muscles occur in the cliick in the cervical area during the
late 4th day (Kao, '32; Orr and TVindle, '34). Szepsenwol
('48) reported that direct electrical stimulation failed to
elicit contractions in chick cervical muscles younger than
85-90 hours. Furthermore the extent of contraction induced
by A T P in mature muscle models greatly exceeds that evoked
by stimulating a muscle via its nerve o r even directly. Taken
together these observations suggest that the contractions described in this paper probably represent the earliest stages at
which myoblasts are capable of contraoting.
Our finding that even after striated fibrils have first formed,
a myoblast may not contract is in harmony with the observations of Wigglesworth ('57) on the differentiating muscles
CONTRACTION O F MYOBLASTS
425
of the insect, Rhodnius. According to Wigglesworth, transection of the sternal muscles stimulates the cut muscles to
contract down to 10-15% of their initial length. If, during
the periods of myogenesis between molts, these muscles are
cut a t a stage shortly after the appearance of conspicuous
birefringent fibrils, the muscles only shorten to 90-957. of
their initial length. I n later stages of development as more
myofibrils form, cutting of the muscle results in successively
stronger contractions.
When a freely suspended myoblast contracts some energy
is probably expended t o overcome the internal viscosity of
the relatively large mass of sarcoplasm. I t would be of interest to know if the first-formed, striated myofibrils are
intrinsically incapable of contracting, due possibly to the
absence o r incomplete activity of one of their components,
or if failure to contract reflects the inability of the myofibrils
to overcome the resistance of the sarcoplasm.
Binding of the antimyosin by the myofibril tells us more
than that cross-striations are present: it indicates the presence of myosin. Work to be published elsewhere, using labelled antiactin, indicates the presence of actin in these early
myofibrils. The question then is raised as to whether the
ability to contract is dependent upon the formation of crossstriations per se, or whether it is dependent upon the presence
of actomyosin. Glycerinated mature fibers, threads prepared
from actomyosin extracted as the complex from muscle, and
those prepared by combining the purified proteins, all contract to the same extent in the absence o i a load (T'eber
and Portzehl, '52). The distribution then of the contractile
proteins into cross-bands is not essential for contraction.
These considerations, and the fact that smooth muscle contracts, lead us t o suspect that the critical factor is the
aggregation and quantity of the contractile proteins. What
is important is that when the contractile proteins are able
to cause a myoblast to contract, they are present in sufficient
concentrations to be visualized cytologically as components
of a cross-striated myofibril. Prior to the appearance of the
426
HOWARD HOLTZER AND J O A N ABBOTT
cross-striations the myoblast has not yet synthesized and
organized the proteins into a system capable of contracting
the cell.
The notion that quick contractions may be due t o the
striated myofibrils and that slow contractions may be due
to the sarcoplasm, was first advanced by Bottazzi in 1897.
To our knowledge there is little experimental evidence to
support this view. It may be that the living sarcoplasm is
contractile to the extent that amoeboid cells, or nerve cells
(Lewis, '50) o r glial cells (Pomerat, '51) display a t y p e of
contractility. Our experiments were not designed to detect
this type of contraction. Further speculation on the relationships among general protoplasmic contraction, the contraction of glycerinated fibroblasts and mitotic spindles
(Hoffman-Berling, '54a,b), and muscle contraction would be
unprofitable until more is known of the mechanisms of any
one of these.
SUMMARY
1. Myoblast models treated with ATP do not contract before cross-striations can be detected with fluorescein labelled
antimyosin.
2. Myoblast models treated with ATP do contract shortly
after the first cross-striated myofibrils can be visualized with
fluorescein labelled antimyosin.
3. There is a direct relationship between the resistance a
contracting myoblast can overcome, and the degree of development of the cross-striated myofibrillar apparatus.
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M. 1954 Muscular Contraction. Charles C Thomas, Springfield.
CONTRACTION O F MYOBLASTS
427
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HOWARD HOLTZER AND JOAN ABBOTT
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