J. Embryol. exp. Morph. 77, 1-14 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
An analysis of contractile proteins in developing
chick heart by SDS polyacrylamide gel
electrophoresis and electron microscopy
BySOO-SIANG LIM 1 , M. NICOLA WOODROOFE AND
LARRY F. LEMANSKI
From the Department of Anatomy and the Muscle Biology Laboratory,
University of Wisconsin, Madison
SUMMARY
Chick heart development was studied using transmission electron microscopy and SDSpolyacrylamide gel electrophoresis in combination with densitometry. Myosin heavy chain,
or-actinin, actin and tropomyosin accumulations were analysed in developing hearts from
preheartbeat stage 9 (Hamburger-Hamilton staging series) through 2 days after hatching. At
the preheartbeat stage, electron microscopy revealed a significant number of thin filaments
scattered throughout the cytoplasm of the myoblasts; however, very few thickfilamentswere
seen. There was no obvious association between the twofilamenttypes. SDS-polyacrylamide
tube gels of heart muscle homogenates demonstrated the presence of allfiveprpteins in hearts
at the preheartbeat stage. Further analyses of the proteins by gel densitometry indicated that
both actin and myosin accumulated rapidly during heart development while a-actinin and
tropomyosin levels remained relatively static. Our results show that detectable quantities of
myosin heavy chain, a-actinin, actin and tropomyosin accumulate in myocardial cells prior to
the appearance of myofibrils and initiation of the contractile function.
INTRODUCTION
The now classical description of the myofibril as an interdigitating hexagonal
array of thick and thin filaments (Huxley & Hanson, 1954) and the subsequent
localization of the various proteins by biochemical and immunohistochemical
methods has provided a means, though limited, by which the electron microscopist can identify and follow the aggregation and organization of these contractile proteins into the adult sarcomere structure (ref. reviews by Hermann,
Hey wood & Marchok, 1970; Fischman, 1970). Nevertheless, considerable
disagreement exists regarding the temporal relationships of the various filaments
and filament aggregates at both the ultrastructural (Allen & Pepe, 1965;
Fischman, 1967, 1970; Hay, 1963; Heuson-Stiennon, 1965) and biochemical
levels (Heywood & Rich, 1968; Masaki & Yoshizaki, 1972; Potter & Hermann,
1970; Roy, Sreter & Sarkar, 1979).
1
Author's address (for reprints): Molecular Biology Laboratory, University of Wisconsin,
Madison, Wisconsin 53706, U.S.A.
2
S. LIM, M. N. WOODROOFE AND L. F. LEMANSKI
Certain biochemical events associated with protein synthesis and assembly
have been described in some detail. Of the several myofibrillar proteins, myosin
and actin have been the most extensively monitored in synchronous populations
of differentiating skeletal muscle cells (Emerson & Beckner, 1975; Emerson,
1977). These investigations have demonstrated that synthesis of both myosin and
actin increase dramatically during and shortly after myoblast fusion in skeletal
muscle cell cultures. In more recent studies, Devlin & Emerson (1978) showed
that the synthesis of myosin heavy chain, two myosin light chains, two subunits
of troponin, and two subunits of tropomyosin are initiated simultaneously at the
time of myoblast fusion. Synthesis of only one myofibrillar protein, a-actin, is
detected in cultures of dividing myoblasts. Isotope dilution studies (Allen,
Stromer, Goll & Robson, 1978, 1979) also show an increase in tropomyosin
during and after the initiation of muscle cell fusion.
The bulk of biochemical studies on contractile proteins in developing muscle
have concentrated on skeletal muscle cells in culture. To date, no comparable
studies have been made on developing cardiac muscle tissue. Before we can
begin to understand myofibrillogenesis in cardiac muscle, it will be necessary to
know the relative quantities of proteins present at different stages of development. In skeletal muscle, myofibrillar protein synthesis is highly coincident with
myotube formation. Since developing cardiac muscle cells do not fuse and since
myofibrillogenesis has not been examined in detail in developing heart, we
undertook the present study to correlate the ultrastructural features of myofibril
formation with the accumulation patterns of the major myofibrillar proteins.
Myosin, actin, tropomyosin and a-actinin were chosen because they represent
the primary components of the thick filament, thin filament, calcium regulatory
complex and Z-line respectively.
Our results show that all four proteins are present in the heart tube at the
preheartbeat stage even though morphologically, no organized myofibrils are
seen. With further development, a marked increase in the accumulation of actin
and myosin are noted, corresponding to the increase in number and organization
of thin and thick filaments. However, the levels of accumulation of a-actinin and
tropomyosin remain relatively constant throughout development.
MATERIALS AND METHODS
Procurement of tissues
Fertilized White Leghorn eggs were incubated at 38 °C for 26 h to 21 days. The
Hamburger-Hamilton staging system for chick embryos was used. Hearts from
embryos ranging from Hamburger-Hamilton stage 9 (26 h of incubation) through
stage 44 (18 days of incubation) and 2-day-old chicks were investigated.
Contractile protein standards
Contractile proteins extracted and purified by published methods were used
Contractile proteins in developing heart
3
as standards in the electrophoresis experiments. These included: 1) chicken
skeletal muscle myosin (Richards, Chung, Menzel & Olcott, 1967); 2) chicken
skeletal muscle actin (Spudich & Watt, 1971); 3) porcine skeletal muscle
a-actinin (Goll, Suzuki, Temple & Holmes, 1972); 4) chicken heart tropomyosin
(Eisenberg & Kielly, 1974; Bailey, 1948); 5) chicken skeletal muscle
tropomyosin (Eisenberg & Kielly, 1974; Bailey, 1948); and 6) chicken skeletal
muscle myofibrils (Etlinger, Zak & Fischman, 1976).
Electron microscopy
Embryonic hearts were fixed by perfusion with 2 % formaldehyde, 2-5%
glutaraldehyde and 0-1 % Picric acid buffered to pH7-3 with 0-15M-phosphate
buffer (modified from Ito & Karnovsky, 1968). Drawn glass capillary tubing was
used for perfusion of the stage-9 and stage-15 embryos, while a syringe with a
small gauge needle was used in the older embryos. After perfusion fixation, small
pieces of the myocardium were then immersed in the fixative for an additional
2h, postfixed in 1 % OsCU, embedded in Epon and routinely processed for
transmission electron microscopy. Thin sections were mounted on copper grids,
double stained with lead citrate and uranyl acetate and viewed on Phillips 200 or
Hitachi HU-11D electron microscopes at accelerating voltages of 60 or 75 kV.
SDS-polyacrylamide gel electrophoresis
Embryonic and adult heart tissues were dissected in quantity and prepared for
analysis using the stacking gel method of Laemmli (1970). Tissue samples included: a) embryonic hearts from Hamburger-Hamilton stage 9 (26-30 h of
incubation), stage 15 (51-56 h of incubation), stage 28-30 (6 days of incubation),
stage 38 (12 days of incubation) and stage 44 (18 days of incubation); b)
ventricular and atrial heart regions of newly hatched 2-day-old chicks; c) adult
chicken myofibrils from cardiac and skeletal muscle prepared by the method of
Etlinger et al. (1976); d) purified protein standards; and e) non-muscle control
using brain from 12-day embryo and 2-day-old chicks.
Particular care was taken in the dissection of the newly fused heart tubes of
stage-9 embryos. The heart tube, including the prospective atria, was very carefully removed by a cut just cephalad to the conus region followed by cuts caudal
to the sinus venosus. A hair loop was used to remove any non-myocardial tissue.
About 175 hearts were required for each tube gel.
After dissection, all tissues were homogenized at 0 °C in 0-1 M-Tris-HCl buffer
pH7-4 to which the proteolytic inhibitor phenyl methyl sulphonyl fluoride was
added to a final concentration of 1-0 mM. The samples were assayed for protein
concentration (BIORAD method) and suspended in a 10 % (w/v) glycerol, 5 %
(v/v) beta-mercaptoethanol, 2-3% (w/v) SDS and 0-0625 M-Tris-HCl, pH6-8
and heated at 100 °C for 5 min.
Samples with 35-50 /il of protein were layered on the tops of 10 cm x 5 mm
tube gels containing 10 % acrylamide and electrophoresed at 1-5 milliamp./gel.
4
S. LIM, M. N. WOODROOFE AND L. F. LEMANSKI
To ensure linearity between O.D. and protein quantity, the same samples were
run at two or three different concentrations. Optimal linearity of samples with
the best resolution was achieved when 42 /ig of protein was loaded. Coelectrophoresis with actin, myosin, a-actinin and tropomyosin standards as well
as mixtures of standards and unknowns were used to identify the various protein
bands in the unknowns. The latter was found to be the more effective method
of identifying the protein bands in tube gels. When the gels were scanned, the
enlarged peak (due to addition of the specific purified protein standard) could be
easily determined. The gels were stained routinely with 0-05 % Coomassie
brilliant blue R250. Graphic traces were made using a Gilford Spectrophotometer Model 250 (Gilford Instruments, Oberlin, Ohio) equipped with a gelscanner attachment at an absorbance wavelength of 550 nm. From the gel scans,
ratios of each of the contractile proteins to total protein and to actin were determined by use of a tracing device linked to a Numonics digitizer interfaced with
a HP9815S programmable calculator.
RESULTS
Electron microscopy
At stage 9, after the fusion of its paired primordia, the heart is a nearly straight
tube. Electron micrographs (Fig. 1) reveal that the cytoplasm of cardiac
myoblasts contain free ribosomes, glycogen and lipid droplets. Thin (6nm) filaments are often seen though thick (15 nm) filaments are rare.
Spontaneous contractions begin in the chick heart at stage 10 (nine to ten
somites) on the right side of the ventricle. By stage 15, the heart is beating
rhythmically and electron microscopy reveals a marked increase in the cytoplasmic complexity of developing myocardial cells (Fig. 2). Numerous myofibrils at
various stages of assembly are scattered throughout the cytoplasm; although the
highly differentiated myofibrils with distinct A bands, I bands and Z-lines tend
to be located in the peripheries of the cells. Granular endoplasmic reticulum,
free ribosomes and Golgi complexes are apparent and adjacent cell membranes
are interspersed with desmosomes and other regions of electron density.
Fig. 1. Transmission electron micrograph of a stage-9 embryo (seven somites).
Intercellular spaces (is) sometimes separate adjacent myoblasts. The granular appearance of the cytoplasm is due to the abundance of free ribosomes and glycogen.
Mitochondria (m), rough endoplasmic reticulum, and lipid droplets (li) often are
seen. At this preheartbeat stage, no formed sarcomeres are visible, although thin
(6 nm)filaments(arrowheads) are scattered throughout the cytoplasm. X4000; Scale
bar =
Fig. 2. Transmission electron micrograph of stage-15 embryonic myocardium. The
heart is beating rhythmically by this time. Myofibrils at various stages of assembly are
seen in the cytoplasm; some complete with Z-lines (Z). Desmosomes (d) and fascia
adherentes (fa) and other regions of increased electron density are often present at
adjacent cell membranes, x 16000; Scale bar =
Contractile proteins in developing heart
1
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Figs 1 & 2
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6
S. LIM, M. N. WOODROOFE AND L. F . LEMANSKI
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Contractile proteins in developing heart
Table 1. Per cent ofmyosin heavy chain, alpha-actinin, actin and tropomyosin in
the developing chick heart
Percent of total protein
Age
Tissue
Fused hearttubes
Stage 15
Whole heart
6 days
Embryo
ventricle
72 days
Embryo
ventricle
18 days
Embryo
ventricle
2 days postNewlyhatches
hatched chick
ventricle
Stage 9
Myosin
a-Actinin
Actin
Tropomyosin
1-84 ±0-5
1-14 ±0-3
8-53 ±0-8
1-0 ±0-3
3-34 ±0-8
1-3 ±0-2
10-52 ±1-0
1-76 ±0-6
7-03 ±0-7
1-60 ±0-3
12-96 ±1-0
1-48 ±0-2
9-4 ±1-0
1-55 ±0-5
15-53 ±1-0
2-38 ±0-7
10-32 ±0-9
1-71 ±0-3
16-63 ±1-2
2-14 ±0-4
16-92 ±0-8
2-04 ±0-5
16-94 ±0-8
1-95 ±0-3
In this table, values for percent total protein are expressed as the mean ± standard deviation . Five SDS-electrophoresis gels of embryonic heart tissues were analysed for each developmental stage except stage 9 where three were evaluated. Statistical comparisons were made
using the Student's t-test.
After six days of incubation, myofibrils several sarcomeres in length align
parallel to each other, with the Z-lines in register (Fig. 3). As myofibrillogenesis
progresses, the number, size, and complexity of the myofibrils increase such that
at 18 days of incubation, the cytoplasm of the myocardial cells is filled with
organized myofibrils (Fig. 4).
SDS poly aery lamide gel electrophoresis
All heart tissues examined contain proteins which co-migrate with myosin
heavy chain (200 000 daltons), actin (45 000 daltons), a-actinin (100 000 daltons),
and tropomyosin (34 000 daltons) standards on SDS polyacrylamide gels (Fig. 5).
Densitometric scans of the gels (Fig. 6) are analysed to determine the percentage
of each specific contractile protein relative to the total protein content in the
heart. A summary of findings is given on Table 1 and Fig. 7.
Fig. 3. Transmission electron micrograph of 6-day embryonic ventricular myocardium. Myofibrils are more numerous at this stage and some are in parallel arrays,
with their Z-lines (Z) in register. x8000; Scale bar = 2/an.
Fig. 4. Transmission electron micrograph of 18-day embryonic ventricular myocardium. The number of myofibrils has increased significantly and now occupy most of
the sarcoplasm, with clear regions mostly at the ends of the nucleus (n). The myocardium contains fully developed myofibrils complete with distinct A-bands, I-bands,
Z-lines, and M-lines. xll 000; Scale bar =
9 9
~t
w*^^^^^
Fig. 5. SDS polyacrylamide gels of heart homogenates and protein standards include: (a) preheartbeat stage-9 hearts; (b) stage15 hearts; (c) 6-day ventricles; (d) 12-day ventricles; (e) 18-day ventricles; (f) ventricle from 2-day-old chick; (g) adult chicken
ventricular myofibrils; (h) tropomyosin standard; (i) actin standard; (j) a-actinin standard; (k) myosin heavy chain standard. The
specific proteins that were examined include: M, myosin heavy chain; a-A, a-actinin; A, actin; T, tropomyosin.
a-A
M
W
I-1
o
W
o
o
o
w
o
o
oo
Contractile proteins in developing heart
st.9
12-d
Fig. 6. Our estimation of the approximate percentage of total protein represented
by myosin heavy chain (M), a-actinin (a-A), actin (A) and tropomyosin (T) is based
on the gel scans. Total protein content is represented by the area under the entire gel
scan, determined by a tracing device linked to a programmable computer to integrate
the area covered. Peak areas are defined by perpendicular lines dropped to the baseline.
Myosin heavy chain comprises 1-84% of the total protein in stage-9 hearts
(preheartbeat). A steady increase in percent accumulation of this protein continues throughout development. By 18 days, myosin HC accumulation has increased five-fold to represent 10-32% of the total protein. At two days after
hatching, there is a sharp rise in myosin content to 16-78 % of total protein.
Similarly, actin (45 000 daltons) represents 8-53 % of the total protein in stage9 hearts. This actin component showed steady and consistent increases in accumulation with development. At 2 days post-hatching, actin comprises about
16-94 % of the total protein in the chick heart.
As expected, a-actinin (1-14%) and tropomyosin (1-0%) accumulations at
stage 9 are smaller than either actin or myosin. Although both a-actinin and
tropomyosin showed relatively small increases numerically, their accumulation
doubled between stage 9 and 2 days after hatching.
Analyses of the gel scans for non-muscle control tissue (brain) indicated that
even as late as 2 days posthatching, the actin and myosin contents still appeared
10
S. LIM, M. N . WOODROOFE AND L. F . LEMANSKI
20
18
16
14
c
g
12
a
~3
o 10
Preheart beat
Post heartbeat
10
12
14
16
18
20 ildph 2dph
Hatching
Days of incubation
Fig. 7. Graph showing protein accumulation at the different stages of heart development. • actin; O myosin; • tropomyosin; # a-actinin.
to be slightly less than the levels in precontractile hearts; furthermore, no relative
increases in the contractile proteins were noted in the brain tissue with advancing
embryonic development.
DISCUSSION
Our results show significant increases in actin and myosin accumulation
throughout development while a-actinin and tropomyosin levels remain relatively constant. SDS polyacrylamide gels of the precontractile heart indicate the
presence of the four proteins although electron microscopy reveals only a few
thick and mostly thin filaments scattered in the cytoplasm. No thick-thin filament arrangement or Z-lines are seen. In general, the appearance of the sarcomere is taken as a direct measure of macromolecular assembly. However, at
the preheartbeat stage, no direct morphological correlates can be made. Obviously, the mere presence of these proteins is not sufficient for the formation of
the sarcomere apparatus. Both quantitative and qualitative changes may be
needed before these proteins can eventually assemble into an identifiable
myofibril. For example, a critical concentration may be needed before
polymerization of these individual proteins can occur. Alternatively, an inhibitor
Contractile proteins in developing heart
11
to polymerization for actin could be present at this time. Since there are defined
stoichiometric relationships among the proteins in the myofibril (Potter, 1974),
one expects that its assembly might depend upon changes in relative quantities
of the protein components.
Of the proteins investigated, actin represents the most abundant myofibrillar
protein throughout development. Although our one-dimensional gel analyses of
actin do not distinguish between muscle and non-muscle actin, our twodimensional gel analyses of the same contractile proteins in the chick heart show
the presence of both a-actin, a muscle-specific actin isozyme, and /3-actin, a 'nonmuscle' type actin isozyme in equivalent amounts (Woodroofe, Lim-Spiker &
Lemanski, 1980). This finding was also confirmed by Wiens & Spooner (1981).
a-Actin becomes the dominant actin form as development progresses.
In skeletal muscle cell cultures, several reports indicate a rapid burst in protein
synthesis related to myoblast fusion. Myosin synthesis has been reported to occur
either during cell fusion (Devlin & Emerson, 1978; Emerson & Beckner, 1975;
Young, Goll & Stromer, 1975; Paterson & Strohman, 1972), or shortly after cell
fusion (Coleman & Coleman, 1972). Actin synthesis also increases rapidly in
fused cells (Paterson, Roberts & Yaffe, 1974; Rubinstein, Chi & Holtzer, 1974;
Rubinstein etal. 1976; Garrels, 1979a,b). Similar relationships exist for a-actinin
synthesis (Allen etal. 1979) and tropomyosin synthesis (Allen etal. 1978; Allen
etal. 1979; Carmon, Neuman & Yaffe, 1978; Garrels, 1979a,b) which also reflect
the same abrupt change during the final phase of skeletal muscle differentiation.
Actin and myosin in the heart do not accumulate in rapid 'bursts' but rather show
steady increases in protein accumulation throughout development.
Since significant quantities of the myofibrillar proteins have already
accumulated in the heart cells prior to sarcomere formation, it appears that only
small increases in quantities of the proteins from the preheartbeat stage 9 to the
newly postheartbeat stage 15 are necessary for myofibril formation to occur.
Application of the Student's t-test to the data (Table 1) reveals that between
stage 9 and stage 15 only myosin (P < 0-01) and actin (P < 0-05) show statistically
significant increases and even these increases are very small. This is not surprising since heart function is precocious in vertebrate embryos. The interrelated
events to cytodifferentiation and establishment of myocardial architecture occur
very rapidly to produce a functional organ early in embryonic life. In less than
40 h of incubation, precardiac mesoderm has undergone re-organization, from
a sheet of undifferentiated cells to a tubular heart whose cells exhibit a high
degree of specialization. Thus, it appears that contractile proteins must
accumulate in myocardial cells in significant quantities well in advance of a
heartbeat.
It is obvious that one-dimensional SDS-polyacrylamide gel electrophoresis
does not discriminate between muscle and non-muscle forms of these proteins.
We believe, however, that it is the muscle-specific forms of these proteins which
contribute to the increase in myofibrillar organization and complexity of
12
S. LIM, M. N. WOODROOFE AND L. F. LEMANSKI
developing cardiac muscle since the contractile protein accumulation in the nonmuscle tissue (brain), even at the most advanced stage, are less than in preheartbeat heart tubes. Furthermore, non-muscle tissue does not show significant increases in contractile proteins during development. Similar results have been
reported in non-muscle tissues in the axolotl (Ambystoma mexicanum) system
(Lemanski, 1976; Lemanski, Mooseker, Peachey & Iyengar, 1976).
In conclusion, our results show that significant quantities of myosin heavy
chain, a-actinin, actin and tropomyosin are present in preheartbeat cardiomyocytes prior to the appearance of myofibrils. Increased protein accumulation
during heart development is reflected in a corresponding increase in myofibrillar
organization and complexity.
The authors gratefully acknowledge Dr Allen Clark for the use of his computer, Dr Edward
Schultz for help with the statistical analyses, Mr Robert Schlotthauer for preparing the graphs
and Ms Sue Leonard for secretarial assistance. This work was supported by a postdoctoral
fellowship from the American Heart Association of Wisconsin and by an NIH Traineeship
(T32-HD-7118) to S.S.L., by a Muscular Dystrophy Association Postdoctoral Fellowship to
M.N.W., and by NIH grant HL 22550, a Basic Research Grant from the National Foundation
March of Dimes, and an American Heart Association Grant-in-Aid to L.F.L. This work was
done during the tenure of an American Heart Association Established Investigatorship Award
to L.F.L.
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123-135.
WOODROOFE,
{Accepted 31 May 1983)