Determinants of the contractile properties in the embryonic
chicken gizzard and aorta
Ozgur
Ogut1 and
Frank V.
Brozovich1,2
Departments of 1 Physiology and Biophysics and
2 Medicine (Cardiology), Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106-4970
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ABSTRACT |
Smooth muscle is generally grouped into two
classes of differing contractile properties. Tonic smooth muscles show
slow rates of force activation and relaxation and slow speeds of
shortening (Vmax) but force maintenance, whereas
phasic smooth muscles show poor force maintenance but have fast
Vmax and rapid rates of force activation and
relaxation. We characterized the development of gizzard and aortic
smooth muscle in embryonic chicks to identify the cellular determinants
that define phasic (gizzard) and tonic (aortic) contractile properties.
Early during development, tonic contractile properties are the default
for both tissues. The gizzard develops phasic contractile properties
between embryonic days (ED) 12 and
20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of
force activation correlates with expression of the acidic isoform of
the 17-kDa essential myosin light chain (MLC17a). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM.
J Muscle Res Cell Motil 18: 103-110, 1997) have
postulated that myosin heavy chain (MHC) isoform expression is a
determinant for Vmax in intact tissues. In the
current study, differences in Vmax did not
correlate with previously published differences in MHC or
MLC17a isoforms. Rather, Vmax was
increased with thiophosphorylation of the 20-kDa regulatory myosin
light chain (MLC20) in the gizzard, suggesting that a
significant internal load exists. Furthermore, Vmax in the gizzard increased during postnatal
development without changes in MHC or MLC17 isoforms.
Although the rate of MLC20 phosphorylation was similar at
ED 20, the rate of MLC20 dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with
expression of the M130 isoform of the myosin binding subunit in the
myosin light chain phosphatase (MLCP) holoenzyme. These results
indicate that unique MLCP and MLC17 isoform expression marks the phasic contractile phenotype.
force; 17-kilodalton myosin light chain; phasic contractile
properties; tonic smooth muscle; development
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INTRODUCTION |
SMOOTH MUSCLE
CONTRACTION controls the tone of many involuntary muscles, such
as those lining arteries and the walls of the stomach. On the basis of
their contraction profiles in response to K+ depolarization
(43), smooth muscles are divided into two general contractile phenotypes: tonic and phasic. Tonic smooth muscles demonstrate slow rates of force activation and relaxation, lower maximum speeds of shortening (Vmax) as well as
force maintenance, whereas phasic smooth muscles display rapid rates of
force activation and relaxation and have a high
Vmax but poor force maintenance (reviewed in
Ref. 15). In both smooth muscle types, phosphorylation of
the 20-kDa regulatory myosin light chains (MLC20) by
Ca2+-calmodulin-activated myosin light chain kinase (MLCK)
promotes actomyosin cycling to produce force. Although previous studies have suggested that isoform expression of myosin and actin
filament-associated proteins are molecular determinants of the
contractile properties of smooth muscle (46), the
molecular determinants of the unique contractile properties of phasic
versus tonic smooth muscle phenotypes remain unclear.
Proteins controlling the activation of the myosin ATPase activity,
namely MLCK and myosin light chain phosphatase (MLCP), have been
suggested as candidates that define contractile properties in phasic
and tonic smooth muscles (12, 23). These proteins control
MLC20 phosphorylation and therefore the level of
activation. However, the unique rates of force activation in phasic and
tonic smooth muscles are not simply explained by differences in
Ca2+ transients or rates of MLC20
phosphorylation because phasic tissues maintain higher rates of force
activation when skinned or when the MLC20 is
thiophosphorylated to dissociate force activation from MLCK and MLCP
kinetics (22). Nonetheless, maximum shortening velocity
(Vmax) in smooth muscle is related to the level
of activation as defined by MLC20 phosphorylation (1,
10). Furthermore, it has been proposed that MLCP may play a role
in the regulation of relaxation because protein kinase C activation and
subsequent MLCP inhibition decreases the rate of MLC20
dephosphorylation and force relaxation (25, 34),
parameters thought to reflect a decrease in MLCP catalytic activity.
Moreover, the MLCP holoenzyme may exist as isoforms, which differ in
the expression of two unique myosin binding subunits (M130/M133; Ref.
41). Therefore, in addition to contractile protein isoform
diversity, the control of actomyosin kinetics in smooth muscle may also
rely on factors modulating MLC20 phosphorylation.
The smooth muscle myosin heavy chain (MHC) is a single gene with two
pairs of alternatively spliced exons. Two isoforms, named SM1 and SM2,
differ by expression of a 43- or 9-amino acid COOH-terminus, respectively (5). Their contribution to contractility
remains controversial (2, 27, 36). In addition,
alternative splicing of an exon near the NH2-terminal 25- to 50-kDa junction of the myosin head accounts for a 7-amino acid
insert in visceral SM1 and SM2 MHCs (28). MHC isoform
diversity at the NH2-terminus has been hypothesized as a
determinant of the tonic and phasic contractile properties in smooth
muscle (28, 30, 46). Additionally, the 17-kDa essential
myosin light chains (MLC17) associated with the neck region
of MHCs also show isoform diversity. Two splice variants of
MLC17 have been found in smooth muscle, and they differ by
inclusion (MLC17b) or exclusion (MLC17a) of a
39-bp exon (38). Studies have shown a correlation between
increased MLC17b expression and decreased actin activated
Mg2+-ATPase activity of myosin (18) as well as
decreased Vmax in intact tissues
(31).
We assumed that the major factors controlling the contractile
properties of developing gizzard and aortic smooth muscle would be the
myosin isoforms (MHC and MLC17) or proteins controlling myosin activation through MLC20 phosphorylation (MLCK and
MLCP). Because MHC and MLC17 isoforms switch during
development of the embryonic chicken aorta and gizzard
(7), we hypothesized that changes in protein isoform
expression throughout development would be reflected by changes in the
contractile phenotype. Therefore, we investigated MHC,
MLC20, MLC17, MLCK, MLCP, and thin
filament-associated proteins to determine the markers for phasic and
tonic contractile properties. In the aorta and gizzard, the tonic
phenotype was the default during early embryonic development.
Approximately 1 wk before hatching, the phasic phenotype developed in
the gizzard. We demonstrate that the MLC17 and MLCP
isoforms are molecular markers for the phasic contractile phenotype.
Expression of MLC17a defined the rapid rate of force
activation, whereas expression of a gizzard-specific MLCP isoform was a
determinant for the rapid rate of relaxation, both hallmarks of phasic
smooth muscles.
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MATERIALS AND METHODS |
Identification of regulatory protein isoforms in the embryonic
chicken.
Fertilized chicken eggs were incubated at 37°C in humidified air and
collected at embryonic days (ED) 10,
12, 14, 16, 18, and
20. The gizzard and thoracic aortas from five or more
embryos were dissected in Ca2+-free physiological saline
solution (in mM: 140 NaCl, 4.7 KCl, 0.3 Na2HPO4, 2 MOPS, 0.5 EGTA, 0.2 EDTA, 1.2 MgCl2, 5.6 glucose, final pH adjusted to 7.4 with 1 N KOH).
The tissues were immediately homogenized in SDS-PAGE buffer, heated to
80°C for 5 min, and clarified by centrifugation at 14,000 g in a microcentrifuge. The samples were resolved by either
12% SDS-PAGE with an acrylamide-to-bisacrylamide ratio of 29:1
(calponin, MLC20) or 14% SDS-PAGE with an
acrylamide-to-bisacrylamide ratio of 180:1 (MLCK, myosin binding
subunit of MLCP). To ensure comparable sample loading, the samples were
normalized according to the G-actin band in SDS-PAGE. Once sample
loadings were comparable, the SDS-PAGE-resolved proteins were
transferred to 0.22-µm nitrocellulose membrane in a transfer buffer
(100 mM glycine, 10 mM Tris, pH 8.6, 20% methanol) and blocked at room
temperature for 3 h using Tris-buffered saline (TBS; 150 mM NaCl,
50 mM Tris · HCl, pH 7.4) containing 1% BSA. Blocked
nitrocellulose membranes were incubated overnight at 4°C in TBS-0.1%
BSA with monoclonal antibodies (MAbs) against calponin (CP1;
26), MLC20 (My-21; Sigma Immunochemicals), MLCK (LKH3 and
LKH18; Ref. 6), or MLCP myosin binding subunit (Covance).
For MLCK identification, a purified smooth muscle MLCK protein sample
was used as a control. The membranes were washed three times for 10 min
in TBS containing either 0.05% Tween 20 or 0.5% Triton
X-100-0.05% SDS and then rinsed with TBS. The membranes were
subsequently incubated with TBS-0.1% BSA containing alkaline phosphatase-conjugated goat anti-mouse IgG or IgM second antibody for
50 min at room temperature. After membranes were washed after second
antibody incubation, the color reaction was developed in alkaline
phosphatase substrate buffer (in mM: 110 NaCl, 5 MgCl2, 100 Tris, final pH 9.5) containing 0.04% (wt/vol) nitro blue tetrazolium and 0.02% (wt/vol) 5-bromo-4-chloro-3-indolylphosphate.
Tissue strips from ED 12, 16, and 20 aorta and gizzard were also freshly dissected and fixed in buffered
Formalin solution for paraffin embedding. The tissues were sectioned at
7 µm and stained with hematoxylin and eosin according to standard protocols.
Force of contraction of developing chicken gizzard and aorta.
Embryonic chicken gizzard and aorta strips were excised from embryos at
various stages of development, skinned, and allowed to contract in high
Ca2+ (0.1 mM free Ca2+) and relax in low
Ca2+ (1 nM free Ca2+) solutions. The solutions
were mixed according to a computer program that calculated the amount
of stock solutions required for a given set of free ion concentrations
(3). The binding constants for the ionic species present
were corrected for temperature and ionic strength. The strips were
clipped between two aluminum foil T clips (11) and skinned
at room temperature for 15 (gizzard) or 30 min (aorta) in a pCa9
solution [in mM: 5 Na2ATP, 5 EGTA, 25 potassium methane
sulfonate, pH 7, 6.9 MgCl2 (1 mM free Mg2+), 25 creatine phosphate, 2 glutathione, and 400 µM
-escin, final pH to
7 with 1 N KOH]. The times used for skinning provided optimal Ca2+ activated force per cross section for the
preparations; longer times compromised the force response. After
skinning, the tissues were transferred to pCa9 relaxing solution [in
mM: 5.4 Na2ATP, 5 EGTA, 71 potassium methane sulfonate, 7.1 MgCl2 (1 free Mg2+), 25 creatine phosphate, 25 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
and 12 µM CaCl2, final pH to 7 with 1 N KOH] and
mounted on the stage of a computer-controlled mechanics workstation.
One end of the tissue strip was hooked to a length driver (Physik Instrumente, Waldbron, Germany) and the other to a force transducer (Akers AE 801 Sensonor, Horten, Norway). The strips were stretched to
1.3 times their resting length (or to the length for maximal force
development) while in relaxing solution. Contractions were initiated by
transferring the strips to a well containing pCa4 activating solution
[in mM: 60 potassium methane sulfonate, pH 7, 5 EGTA, 5.3 CaCl2, 6.98 MgCl2 (1 free Mg2+),
5.56 Na2ATP, 25 creatine phosphate, 25 BES, final pH to 7 with 1 N KOH]. Previous studies (7) have demonstrated
that tissue from the developing aorta and gizzard is predominantly
smooth muscle cells. Force generated was normalized to force per
cross-sectional area (mN/cm2) by measuring the dimensions
of the tissues and assuming that the tissues were rectangular. Tissue
strips were of comparable sizes on given days of development and were
typically 250 and 1,000 µm long, 80-150 µm wide, and
80-150 µm thick. Results are reported as the average ± SE.
All experiments were done at room temperature.
To measure the rate of force activation or the rate of relaxation,
tissue strips were skinned and allowed to contract in the pCa4
activating solution until force reached a steady plateau and then
allowed to relax by transferring the tissues to pCa9 relaxing solution.
The contraction profiles were stored on a digital oscilloscope (Nicolet
310). To determine a rate, the force activation and relaxation profiles
were fit to a single exponential. Results are reported as the
average ± SE. Differences were deemed significant if
P < 0.05 by Student's t-test.
Experiments were also done wherein MLC20 was
thiophosphorylated to assure maximal activation of myosin heads during
contraction (20). To thiophosphorylate MLC20,
skinned tissues were incubated for 5 min in an activating solution
wherein 5.56 mM (5 mM free) ATP-
-S (Calbiochem) was substituted for
ATP and creatine phosphate. The tissues were then transferred into pCa9
relaxing solution containing ATP and force developed. The rates of
force activation for thiophosphorylated tissues were determined as
described above.
Vmax in developing chicken gizzard and aorta.
The unloaded speed of shortening of the skinned tissues was determined
from the force versus velocity relationship (19). A
computer program based on Smith and Barsotti (42) was
written in LabView (National Instruments, Dallas, TX) to control length for constant loads. Force and length signals were sampled and controlled using an analog-to-digital-digital-to-analog board (National
Instruments PCI-MIO-16XE-10, 100 × 103 samples/s, 16 bit). After activation of the gizzard and aorta strips, the load on the
tissues was decreased to any set percentage of the isometric force. The
tissues were maintained at a fraction of the initial isometric load
(1-90%) by a program monitoring tension (200-300 samples/s)
and rapidly adjusting (shortening) length to provide constant load. A
force clamp from an initial tone to any desired new load was achieved
within 10 (300 samples/s) to 30 ms (200 samples/s). The shortening
profiles of the tissues were saved, and the speed of shortening at
various loads was calculated from the tangent to the length versus time
trace at 150 ms after the step change in load. The unloaded speed of
shortening was calculated by fitting the force versus velocity
relationship to the Hill equation (19) or taken as the
speed of shortening at 1% of initial isometric load (42).
The speeds of shortening were normalized to muscle lengths per second
(ML/s). For each tissue, at least three separate measurements were
taken at each load tested; n represents the number of
tissues tested. However, the Vmax for smooth
muscles before ED 12 could not be reliably determined
because of the low forces of Ca2+-activated contractions.
Results are presented as the average ± SE.
Analysis of MLC20 phosphorylation in developing
chicken gizzard and aorta.
To determine the extent of phosphorylation of MLC20, a
glycerol-urea-PAGE procedure (17) was adapted to resolve
the phosphorylated and unphosphorylated forms of MLC20.
Strips of chicken gizzard or aorta were skinned as before, washed, and
resuspended in the relaxing buffer. To measure the rate of
phosphorylation of MLC20, tissue strips were incubated for
0, 30, 60, 120, and 300 s in pCa4 activating solution. Activation
of the strips was terminated by adding excess 15% (vol/vol)
trichloroacetic acid in acetone. The tissues were frozen in liquid
nitrogen for 30 min. After denaturation, the tissue strips were washed
twice with acetone, dried using a Speed-Vac evaporator and homogenized
in PAGE sample buffer containing 6 M urea, 20 mM glycine, 22 mM
Tris · HCl, pH 8.6, and 1 mM EDTA. Glycerol (5% vol/vol) and
bromophenol blue (0.1% wt/vol) were added to the samples before
loading. The samples were resolved by a 19:1 (acrylamide-bisacrylamide)
10% polyacrylamide gel containing 40% (vol/vol) glycerol and 4 M urea
and polymerized in a gel buffer containing 20 mM glycine and 22 mM
Tris · HCl, pH 8.6. The gel was prerun in the electrophoresis
buffer (20 mM glycine, 22 mM Tris · HCl, pH 8.6, 1 mM
thioglycolic acid) for 1 h at 200 V before loading of the samples.
The samples were resolved overnight at 200 V or roughly 2 h after
the bromophenol blue dye front had exited the gel. Resolved proteins
were transferred to nitrocellulose membrane, and the phosphorylated and
unphosphorylated MLC20 were identified by immunoblotting as
described earlier. The percentage of MLC20 phosphorylated
was calculated by determining the ratio of the phosphorylated
MLC20 band versus the total MLC20
(phosphorylated + unphosphorylated forms) identified by the mAb.
The rate of dephosphorylation of MLC20 was measured in a
fashion similar to phosphorylation. After the tissue strips were skinned, all were activated for 300 s in the activation buffer. After activation, the strips were allowed to relax in a
Ca2+-free rigor solution without ATP or creatine phosphate
(173 mM potassium methane sulfonate, pH 7, 5 mM EGTA, 12 µM
CaCl2, 1.16 mM MgCl2, 25 mM BES, final pH to 7 with 1 N KOH) for 0, 30, 60, 120, and 300 s. The dephosphorylation
was terminated by denaturing the tissue strips in 15% trichloroacetic
acid in acetone. The tissues were prepared for glycerol-urea-PAGE as
before. For phosphorylation and dephosphorylation experiments, results
are expressed as the average ± SE of three paired experiments.
The experimental data were well fit to a single exponential.
Tissue strips were also activated with ATP-
-S as described
before, and the extent of thiophosphorylation was determined by glycerol-urea-PAGE. Thiophosphorylation of MLC20 was near
100% in samples of aorta (86.9 ± 6.6%, n = 3)
and gizzard (89.6 ± 9.3%, n = 3) incubated for
300 s in thiophosphorylation solution. To ensure that tissues were
thiophosphorylated as intended, glycerol-urea-PAGE was also performed
on samples that were incubated in the dephosphorylation buffer for
300 s after thiophosphorylation. Because thiophosphorylated MLC20 is not a substrate for MLCP, the high percentage of
thiophosphorylated MLC20 should remain. This was confirmed
by the persistence of the phosphorylated form of MLC20 in
glycerol-urea-PAGE analysis of both the aorta (90.2 ± 5.0%;
n = 3) and the gizzard (89.7 ± 9.5%,
n = 3) after 300 s incubation in the
Ca2+-free rigor solution.
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RESULTS |
Rapid rate of force activation in the developing gizzard.
Representative contraction profiles for embryonic aorta and gizzard at
various stages demonstrate the development of the contractile phenotypes of these tissues (Fig. 1).
Both smooth muscle tissues show a slow rate of force activation early
during embryonic development (ED 8-12). The faster
contractile properties of the gizzard strips become apparent by
ED 16, with clearly increased rates of force activation and
relaxation compared with the aorta (Table
1 and see Table 6). The rates of
activation between ED 12 gizzard and aorta were not
different (P > 0.05). Although the rates of force activation were not significantly different between ED 16 and 20 gizzard, they were always significantly faster than
either ED 16 or 20 aorta (P < 0.05). The rates of force activation increased with development in the
gizzard and were significantly faster at ED 20 versus
ED 12 (P < 0.05). Thiophosphorylation of
MLC20 significantly increased the rates of force activation
in both ED 20 aorta (0.018 ± 0.003 s
1) and gizzard (0.060 ± 0.009 s
1) versus Ca2+ activation (P < 0.05). However, the rate of force activation for ED 20 gizzard remained threefold faster than for ED 20 aorta with
or without thiophosphorylation.

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Fig. 1.
Contraction profiles of developing embryonic chick aorta and
gizzard strips. Representative tracings of skinned embryonic
days (ED) 8, 12,
16, and 20 chicken aorta and gizzard strips after
Ca2+ activation. Early in development, both the aorta and
gizzard show tonic contractile properties marked by a slow rate of
force activation. The rapid rate of force activation in the gizzard
emerges by ED 16, implying that the determinants of tonic
and phasic contractile properties are established. A similar trend is
seen for the rates of force relaxation, as ED 16 gizzard
shows faster rates of relaxation compared with the aorta (Table 5).
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Orientation of cells in the developing aorta and gizzard.
To determine whether changes in the orientation of the cells were
occurring with development, ED 12, 16, and
20 aorta and gizzard sections were stained with hematoxylin
and eosin (Fig. 2). With development in
both tissues, an increase in cell density was apparent. In the aorta,
there was no change in the orientation of the cells with development.
In the gizzard, however, the orientation of cells changed by ~20°
between ED 12 and 16, with cells changing from
parallel to the axis of the strip at ED 12 to 20° off the axis at ED 16. No subsequent change in cell orientation was
observed from ED 16 to 20 gizzard.

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Fig. 2.
The orientation of cells in embryonic aorta and gizzard tissues.
Tissue samples from ED 12, 16, and 20 aorta and gizzard were sectioned and stained with hematoxylin and eosin
to determine the orientation of the cells. During development of the
aorta, no change in the orientation of the cells was observed. During
development of the gizzard, a change of ~20° in the orientation of
the cells is evident from ED 12 to 16. No change
in orientation was seen between ED 16 and 20 gizzard. Bars denote 10 µm.
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Protein isoform expression in the developing aorta and gizzard.
The expression of multiple regulatory protein isoforms is a common
theme in developing muscle. Representative smooth muscle-specific markers were chosen, and their isoform expression throughout
development of the embryonic chicken gizzard and aorta was monitored
(Fig. 3). Calponin, an actin
filament-associated marker of smooth muscle, was present throughout
development of the gizzard and aorta as a single isoform. Using MAb
MY-21 (Sigma), we identified a single isoform of MLC20
expressed throughout development of the aorta and gizzard. Comparison
of the expression of the M130/M133 isoforms of the myosin binding
subunit of MLCP (41) indicated that only M133 was
expressed throughout development of the aorta. On the other hand, a
developmental isoform switch of M130 for M133 was observed in the
developing gizzard, with M130 expression beginning at ED 20.
This is consistent with upregulation of M130 expression to become the
dominant myosin binding subunit isoform expressed in adult chicken
gizzard (41). The smooth muscle MLCK isoform was the
abundant MLCK isoform expressed in both the gizzard and aorta during
embryonic development. Because the Western blots only monitored isoform
expression, we could not make any conclusions with respect to changes
in protein content with development.

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Fig. 3.
The expression of regulatory protein isoforms during
development. With the use of specific monoclonal antibodies, the
expression pattern of the myosin binding subunit of myosin light chain
phosphatase (MLCP), myosin light chain kinase (MLCK), h1-calponin, and
20-kDa myosin light chain (MLC20) were determined in the
developing gizzard and aorta. All proteins showed expression of a
single protein isoform throughout development except for MLCP. The
gizzard showed expression of the M130 myosin binding subunit beginning
between ED 18 and 20, with M130 being the
dominant isoform expressed at ED 20.
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Force of contraction of the developing gizzard and aorta.
We measured the force per cross-sectional area in developing aorta and
gizzard to determine whether a correlation could be made with myosin
isoform expression. Although the force per cross-sectional area
increased with development for both embryonic aorta and gizzard (Table
2), ED 20 gizzard was
significantly higher (P < 0.05) than ED 20 aorta, with or without thiophosphorylation of the myosin regulatory
light chain. The increases in force per cross section may be achieved
by differences in actin/myosin content or tissue content of smooth
muscle.
Vmax in embryonic chicken gizzard and aorta.
To monitor changes in Vmax with development of
the smooth muscle contractile phenotypes, the
Vmax for embryonic aorta and gizzard strips was
determined at three stages of development. The
Vmax for the developing aorta was higher than
for the developing gizzard at all stages (Table
3). This was surprising given that adult
phasic tissues have faster speeds of shortening than tonic tissues
(15). Thiophosphorylation of MLC20 with
ATP-
-S resulted in no significant change of
Vmax for ED 20 aorta but a doubling in the gizzard (0.038 ± 0.004 vs. 0.084 ± 0.018 ML/s).
Additional experiments with Ca2+-activated skinned gizzard
strips from 1-wk-old chicks indicated that Vmax
increased after hatching (0.115 ± 0.013 ML/s, n = 3). Therefore, Vmax continues to increase
significantly after birth, given that adult chicken gizzard strips have
a Vmax of ~0.3 ML/s (1).
Force-velocity curves of thiophosphorylated fibers showed that the
shortening velocity as a percentage of Vmax
(Fig. 4B) achieved by the
gizzard was higher at most loads compared with the aorta. The extent of
MLC20 thiophosphorylation in the preparations was
comparable (Table 4).

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Fig. 4.
The force versus velocity relationship in
thiophosphorylated ED 20 aorta and gizzard. After
thiophosphorylation of skinned ED 20 aorta and gizzard
strips, the force versus velocity relationship was determined by
decreasing the load on the strips to various levels and measuring the
speed of shortening. For a given tissue, either the absolute speeds of
shortening [muscle length (ML)/s] at various loads were reported
(A) or speeds of shortening were normalized by dividing by
the maximum speed of shortening (Vmax) of the
tissue and expressing the speed as a fraction of the
Vmax (B). A: the force
versus velocity relationship from single ED 20 aorta and
gizzard fibers. Data points are the average ± SE for 3 measurements at each load. B: pooled data for ED
20 aorta (n = 8) and ED 20 gizzard
(n = 11) strips. The pooled data were fit to the Hill
equation. Note that the gizzard displays a flatter relationship,
indicating that a higher absolute Vmax is
achieved at loads 40% of maximum (A) and that a greater
percentage of maximum speed is achieved for isotonic contractions above
zero load (B).
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Time course of MLC20 phosphorylation and
dephosphorylation.
To assess whether phosphorylation-dephosphorylation of
MLC20 played a significant role in the rates of force
activation and relaxation, we determined the time to reach half-maximal
phosphorylation and dephosphorylation in skinned ED 16 and
20 aorta and gizzard. We chose ED 16 and
20 because phasic and tonic contractile phenotypes appeared
to be well established at these days of development. The levels of
MLC20 phosphorylation in the gizzard and aorta after 0 or
300 s of activation were not significantly different (Fig. 5). Both ED 20 aorta and
gizzard showed similar levels of maximum MLC20
phosphorylation (aorta, 47.3 ± 4.0%, n = 7;
gizzard, 44.2 ± 3.3%, n = 7), which were not
different from ED 16 aorta (42.6 ± 5.0%,
n = 8) or gizzard (42.7 ± 4.6%,
n = 7), indicating that the maximum level of activation
does not change between ED 16 and 20. Likewise, for
dephosphorylation experiments, we found no difference between the level
of MLC20 phosphorylation in the gizzard and aorta after 0 or 300 s of dephosphorylation (Fig. 6).

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Fig. 5.
The phosphorylation of MLC20 in ED
20 aorta and gizzard. With the use of skinned ED 20 aorta and gizzard strips, the time course of MLC20
phosphorylation was followed by separation of the phosphorylated and
unphosphorylated MLC20 by glycerol-urea-PAGE. There was no
difference in the time course of phosphorylation or maximal level of
MLC20 phosphorylation in the tonic and phasic tissues.
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Fig. 6.
Dephosphorylation of MLC20 in ED
20 gizzard is faster than ED 20 aorta. After activation
of the skinned tissues for 5 min, the time course of dephosphorylation
of MLC20 was followed by incubating the tissue strips in a
Ca2+-free rigor solution. Dephosphorylation of
MLC20 was significantly faster in ED 20 gizzard
compared with ED 20 aorta (P < 0.05). The
solid line shows a fit to the data by a single exponential with a rate
of 0.051 ± 0.016 s 1 in the gizzard and 0.021 ± 0.003 s 1 in the aorta.
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Our experiments characterizing the time course of MLC20
phosphorylation in ED 20 aorta and gizzard showed no
significant difference between the two tissues. However,
MLC20 phosphorylation was near maximal at 30 s, which
was the first time point measured. Therefore, calculation of the time
for half-maximal phosphorylation of MLC20 would very likely
be an underestimate of the actual time course and may mask differences
in the rates of MLC20 phosphorylation. Nonetheless, maximum
force in the skinned smooth muscles was achieved well after 30 s
(Fig. 1), indicating that maximum MLC20 phosphorylation preceded maximum force. Therefore, the time course of MLC20
phosphorylation would not be rate limiting for the rate of force
activation in ED 20 aorta and gizzard.
For the dephosphorylation of MLC20, no differences were
observed for the rates of dephosphorylation between ED 16 aorta (0.016 ± 0.004 s
1) and gizzard (0.022 ± 0.008 s
1). For ED 20 aorta, the rate of
dephosphorylation of MLC20 (0.021 ± 0.003 s
1) was not significantly different from ED 16 aorta or gizzard. On the other hand, the rate of MLC20
dephosphorylation for ED 20 gizzard (0.051 ± 0.016 s
1) increased significantly versus ED 16 gizzard (P < 0.05) and was faster than the rate of
MLC20 dephosphorylation measured in ED 16 or
20 aorta (Table 5).
Rate of force relaxation in embryonic chicken smooth muscles.
We measured the rates of force relaxation for skinned embryonic gizzard
and aorta strips at ED 12, 16, and
20 (Table 6) to determine
whether relaxation could be correlated with differences in the rate of
dephosphorylation of MLC20. For ED 12,
16, and 20 aorta, rates of relaxation were
0.001 ± 0.001 (n = 3), 0.004 ± 0.001 (n = 5), and 0.005 ± 0.002 s
1
(n = 7), respectively. These were not different from
the rate of relaxation of ED 12 gizzard (0.003 ± 0.002 s
1, n = 3). However, the rates of
relaxation for ED 16 gizzard (0.019 ± 0.002 s
1, n = 8) and ED 20 gizzard
(0.030 ± 0.004 s
1, n = 8) were
significantly faster compared with the aorta (P < 0.05). Furthermore, the rate of relaxation in ED 20 gizzard was significantly faster (P < 0.05) than in ED
16 gizzard.
 |
DISCUSSION |
Regulatory protein isoform heterogeneity during development.
Through development, regulatory proteins may be expressed as multiple
isoforms. During chicken development, MHC and MLC17 expression both show default tonic phenotypes at early embryonic stages, with expression of MLC17b and noninsert containing
MHC (7). However, in the gizzard, polymerase chain
reaction and Western blot analyses have shown that expression of the
7-amino acid insert containing MHCs dominates expression in SM1 and SM2 MHC isoforms from ED 10 (>70%) to adult (~100%; 7, 28),
whereas the aorta did not express the 7-amino acid insert in SM1 and
SM2 myosins. Furthermore, MLC17a expression is upregulated
with development to hatching in the gizzard (>50% at ED
12, >70% at ED 16, and ~100% for adult; 7, 28),
whereas MLC17b is predominant throughout aorta development
(>60% from ED 12 to adult; 7, 28). Therefore, in the
gizzard, MHC and MLC17 isoform maturation correlates with development of the phasic phenotype.
We detected expression of only the smooth muscle-specific MLCK isoform
in both the developing gizzard and aorta (Fig. 3), suggesting that
isoform switching of MLCK does not impart phasic or tonic contractile
properties. It has been demonstrated that two splice variants of the
myosin binding subunit of MLCP, designated M130 and M133
(41), are expressed in the chicken gizzard. Monoclonal antibody detection of the myosin binding subunits indicated that M130
expression begins between ED 18 and 20 in the
gizzard (Fig. 3), consistent with M130 being the dominant isoform
expressed in the adult (41). On the other hand, the M133
subunit was constitutively expressed in the developing aorta.
Therefore, expression of a gizzard-specific MLCP holoenzyme may
contribute to the differences in phasic versus tonic contractile properties.
Kinetics of MLC20 phosphorylation in embryonic gizzard
and aorta.
Although MLC20 phosphorylation controls smooth muscle force
production, the increased rate of force activation in the gizzard is
not well explained by MLCK activity. Phosphorylation of
MLC20 in both aorta and gizzard preceded maximal force
activation (see Figs. 1 and 5). In addition, both tissues expressed
identical MLCK isoforms (Fig. 3) and reached the same maximum level of
MLC20 phosphorylation (Fig. 5). However, between ED
20 gizzard and aorta, we detected a significant difference in the
time course of MLC20 dephosphorylation (Fig. 6). If MLCP
activity is constitutive in the presence and absence of
Ca2+, this implies that steady-state MLCK activity was
higher in ED 20 gizzard than in the aorta. This difference
was not resolved on the time scale of our in vitro phosphorylation
experiments. However, the difference between MLCK activity for the
aorta and gizzard can be determined assuming that the steady-state
level of MLC20 phosphorylation is defined by MLCK and MLCP
activities as described by MLCK/(MLCK + MLCP). Because
steady-state MLC20 phosphorylation levels were not
significantly different between ED 20 aorta and gizzard
(~45%) and assuming that the rates of dephosphorylation are
accurate, the MLCK activity in the aorta would calculate to 41% of
that in the gizzard. This implies that the rates of phosphorylation
were underestimated. Although thiophosphorylation of MLC20
in ED 20 aorta and gizzard increased the rates of force activation compared with phosphorylation of MLC20, an
approximately threefold difference remained (Table 1) in the rates of
force activation in these tissues. Therefore, MLC20
phosphorylation was not a limiting factor in the slow rate of force
activation in the aorta, indicating that with phasic muscle the rapid
rate of force activation is not explained by increased MLCK activity alone. Thus although the steady-state MLCK activity may be higher in
the gizzard, its contribution to the differences in the rates of
Ca2+-activated force appears to be small. Furthermore,
differences in the rates of force activation between aorta and gizzard
are unlikely to be due to diffusional effects. Activation of
thiophosphorylated gizzard fibers (wherein ATP diffusion is limiting)
showed that diffusion can sustain rates <0.060
s
1. Therefore, the differences in the rates of
force activation in Ca2+-activated gizzard and aorta fibers
(wherein rates were <0.02 s
1) would not be due to
diffusion. In fact, thiophosphorylated phasic and tonic tissues
activated by photolysis of caged ATP still show significant differences
in the rates of force activation (21). With flash
photolysis, diffusion is not a factor and differences in the rates of
force activation point to unique kinetics of the actomyosin interaction
in these tissues. In addition, the differences in the rates of force
activation for Ca2+-activated smooth muscle strips was not
due to differences in calmodulin content (or loss) after skinning,
because thiophosphorylation achieved near complete levels of
MLC20 phosphorylation in both ED 20 aorta and
gizzard (Table 4) while preserving the differences in the rates of
force activation (Table 1). We also considered the possibility that
changes in maximum force could be due to variations in the orientation
of sarcomere equivalents. However, because force is related to the
cosine of the angle between the axis of tissue preparation and the
orientation of the cells, large changes in angle (>60°) would be
required to affect maximum force by twofold. The data in Fig. 2
demonstrate that changes in cell orientation were not an issue with the
developing aorta. In the gizzard, the ~20° change in orientation
between ED 12 and 16 would result in the
underestimation of the measured force at ED 16 and 20 by ~6%. This suggests that the increase in force with
development is due to more smooth muscle cells per cross section; this
is supported by data that the force per stiffness is not significantly different with development (data not shown). However, the orientation of cells is a factor only for the force measured, not the rate of force
activation. The data indicate that the primary determinant for the
rapid rate of force activation in the gizzard is at the level of the
actin and myosin filaments. Because the MLC20
phosphorylation measurements (Fig. 5) were done on skinned preparations
activated directly with Ca2+, we excluded possible
modulation of MLCK activity in the aorta and gizzard through second
messenger pathways (44), but not through
Ca2+-activated kinases. However, thiophosphorylated fibers
maintained differences in the rates of force activation, downplaying
the importance of Ca2+-activated kinases (other than MLCK),
unless a kinase was specifically coupled to the contractile apparatus
in only gizzard or only aortic smooth muscle. Therefore, differences in
the rates of force activation between gizzard and aortic smooth muscle
remain in the absence of tissue-specific agonist-dependent pathways
that differentially modulate MLCK/MLCP activity through phosphorylation
(9).
Rates of MLCP dephosphorylation in developing gizzard and aorta.
There was a significant difference in the rates of MLC20
dephosphorylation between ED 20 gizzard and ED 16 gizzard or ED 16 and 20 aorta (Table 5; Fig. 6).
The increased rate of MLC20 dephosphorylation in ED
20 gizzard correlated with expression of a unique isoform of the
myosin binding subunit of MLCP (M130; 16, 41) that was not expressed in
the aorta (Fig. 3). There was no difference in the rates of
dephosphorylation in ED 16 aorta and gizzard versus ED
20 aorta (Table 4), wherein all smooth muscle tissues expressed M133. Therefore, our results suggest that the MLCP holoenzyme containing the M130 myosin binding subunit correlates with the increased rate of MLC20 dephosphorylation in ED
20 gizzard, either through increased activity of this MLCP
holoenzyme or an increase in the MLCP-to-myosin ratio. Coupled with
data characterizing the rates of force relaxation, two conclusions may
be drawn. First, we demonstrated that the rate of relaxation in the
gizzard is faster than in the aorta at both ED 16 and
20. At ED 16, both tissues express the MLCP
holoenzyme with the M133 subunit and show similar time courses for
MLC20 dephosphorylation. Therefore, the differences in the
rates of force relaxation between ED 16 aorta and gizzard
(Table 6) are best attributed to changes in the kinetics of the myosin
isoforms in the cross-bridge cycle, likely due to inclusion (gizzard)
or exclusion (aorta) of the 7-amino acid insert in the MHC or different
MLC17 isoform expression in the two tissues. With laser
trap experiments, it has been demonstrated that heavy meromyosin
composed of the noninsert (aortic) form of MHC has an attachment time
on actin roughly twice as long as the insert (gizzard) form
(30). This would agree with our results that relaxation in
ED 16 gizzard is faster than in ED 16 aorta, although the tissues show similar rates of MLC20
dephosphorylation. Nonetheless, the increase in the rate of relaxation
of ED 20 versus 16 gizzard indicates that
expression of MLCP holoenzymes containing the M130 myosin binding
subunit correlates with increases in the rate of MLC20
dephosphorylation and thus the rate of force relaxation, whether by
increased MLCP isoform activity or increased MLCP-to-myosin ratio.
Unloaded speed of shortening in developing chicken gizzard and
aorta.
Previous reports have demonstrated that increased MLC17a
content may account for faster speeds of shortening in smooth muscle tissues (31). Increasing the proportion of
MLC17a expression by exchange of MLC17 isoforms
in trifluoperazine-treated smooth muscle was also shown to increase the
shortening velocity (35), although contraction in these
tissues was independent of Ca2+ or
MLC20 phosphorylation. On the other hand, in in vitro
motility assays, exchange of MLC17 isoforms had no effect
on the speed of actin filament movement by myosins (28,
40). Although the embryonic gizzard expressed a higher
proportion of MLC17a than the aorta (7), in
our study, the gizzard had slower speeds of shortening under all
conditions tested (Table 3). Therefore, MLC17a content was
not a factor in defining a faster Vmax in these tissues. It remains a possibility that changes in content or isoform of
calponin and caldesmon (37) may contribute to the
mechanical parameters measured in these experiments.
Interestingly, after thiophosphorylation of MLC20, there
was a significant increase in the speed of shortening in the gizzard, without a significant change in the aorta. This implies that there is a
higher internal load on ED 20 gizzard cells, which is
overcome by maximal (~90%) phosphorylation of MLC20 or
by increasing the level of activation of the preparation. This result
is similar to cardiac muscle wherein shortening velocity is dependent
on the level of Ca2+ activation (33). In fact,
the Vmax of Ca2+-activated gizzard
strips increases 1 wk after hatching, approaching the
Vmax of ~0.3 ML/s measured from adult chicken
gizzard strips (1). The postnatal increases in
Vmax occur independent of changes in the level
of MLC20 phosphorylation from ED 20 (this study) to adult (1) or differences in MHC/MLC17
isoform expression, because myosin expression has already matured to
the adult patterns by ED 20. Experiments characterizing
isolated myosins in in vitro motility assays (28, 40) have
indicated that intestinal myosins propel actin filaments faster, along
with having a higher actin-activated Mg2+-ATPase activity,
although care must be taken when interpreting data from in vitro
motility assays (13). With the optical trap, the MHC
determined the kinetics of cross-bridge attachment (30). Nonetheless, our data indicate that because of a significant internal load, the level of activation is a major factor in determining Vmax (1, 10). The presence of an
internal load in single smooth muscle cells has been shown previously
to affect the extent of shortening (47). In the case of
ED 20 gizzard, the internal load was overcome by increasing
the level of MLC20 phosphorylation from 45 to 90% in
thiophosphorylated fibers, presumably increasing the number of actively
cycling, force-generating myosin heads. In fact, the increase in
Vmax of the gizzard with development from
hatching to birth and after birth takes place in the absence of changes
in myosin isoforms or MLC20 phosphorylation. One possible explanation for the increase is a decrease in the internal load on
gizzard myosins with maturation, either defined by changes in the
intracellular or extracellular matrix properties. This is also
supported by the significant increase of Vmax
with development of the aorta wherein there is a modest change in
MLC17 isoform expression but no changes in MHC isoform
expression or MLC20 phosphorylation. Thus changes in
internal load and thus the level of activation or MLC20
phosphorylation appears to be the major determinant of Vmax in this model.
Role of MLC17 isoforms in determining tonic and phasic
contractile properties.
It is well known that the 20-kDa regulatory myosin light chains play a
defining role in controlling actomyosin interaction in smooth muscle.
However, the function of the 17-kDa essential myosin light chain has
not been as well characterized. The essential myosin light chains line
the neck region of the MHC dimers, possibly acting as a scaffold for
this extended helix (39). In this respect, the interaction
of different MLC17 isoforms with the neck region of MHC
dimers may result in changes in the stiffness of this lever arm. The
rapid rate of force activation in the gizzard due to MLC17a
expression may be explained if this isoform provided added stiffness to
the myosin lever arm.
If MLC17 is a determinant in regulating the actomyosin
cross bridge's ability to bear a load, then we would expect that speed of shortening would be higher with MLC17a than
MLC17b at or near physiological loads, as shown in Fig. 4.
In fact, the gizzard, which expresses predominantly MLC17a,
is able to develop a higher relative percentage of its
Vmax at all loads compared with the aorta, which
expresses predominantly MLC17b. This is coupled by higher
absolute isotonic shortening velocities at loads
40% of isometric
level, supporting the conclusion that MLC17 is a major determinant of the speed of contraction under physiological loads. In
addition, MHC isoform contribution to the rate of force activation is
not significant. This is supported by previous experiments demonstrating that endothelin-1 treatment of gizzard smooth muscle cells increased MLC17b isoform expression, did not change
MHC isoform expression, but decreased the rate of force activation (7). Furthermore, transfection of single aortic smooth
muscle cells with eukaryotic plasmids expressing MLC17a
increases the rate of force activation, whereas transfection of gizzard
smooth muscle cells with MLC17b decreases the rates of
force activation, both in the absence of MHC isoform changes
(24).
In summary, we show that MLC17 and MLCP are molecular
markers for the phasic contractile phenotype of smooth muscle. The
MLC17 isoforms regulate the rate of force activation and
expression of myosin binding subunit isoforms of the MLCP holoenzyme
modulate the rate of force relaxation, two hallmarks for tonic and
phasic contractile properties.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Guay-haur Shue for writing the LabView Force Clamp
program and Albert Rhee and Carie Trbovich for assistance. We thank Dr.
Jian-Ping Jin for the anti-calponin mAb and Drs. Mitsuo Ikebe and
Steven Fisher for the anti-MLCK mAbs. We also thank Drs. Michiko
Watanabe and Midori Hitomi for histochemical staining of tissue sections.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-44181 and an American Heart Association grant-in-aid.
Address for reprint requests and other correspondence: F. V. Brozovich, Dept. of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH
44106-4970 (E-mail: fxb9{at}po.cwru.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 January 2000; accepted in final form 4 August 2000.
 |
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