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219
Smooth and Skeletal Muscle Actin
Are Mechanically Indistinguishable
in the In Vitro Motility Assay
David E. Harris and David M. Warshaw
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Smooth muscle produces as much stress as skeletal muscle with less myosin. To determine if the actin
isoforms specific to smooth muscle contribute to the enhanced force generation, the motility of actin
filaments from smooth and skeletal muscle were compared in an in vitro assay in which single
fluorescently labeled actin filaments slide over a myosin-coated coverslip. No difference was observed
between the velocity of smooth versus skeletal muscle actin filaments over either smooth or skeletal muscle
myosin over a large range of assay conditions (changes in pH, ionic strength, and [ATP]). Similarly, no
difference was observed between the two actins when the filaments moved under load over mixtures of
phosphorylated smooth and skeletal muscle myosin. Thus, it appears that the actin isoforms of smooth
and skeletal muscle are mechanically indistinguishable in the motility assay and that smooth muscle's
enhanced force generation may originate within the myosin molecule specific to smooth muscle.
(Circulation Research 1993;72:219-224)
KEY WoRDs * actin * smooth muscle * motility * protein isoform
T he basic mechanism of smooth muscle contraction' and the biochemical steps of the smooth
muscle actomyosin ATPase cycle2 are thought
to be qualitatively similar to those of skeletal muscle.
Nevertheless, smooth muscle's mechanical properties
distinguish it from skeletal muscle. One obvious difference is smooth muscle's slower maximum velocity of
shortening. More interestingly, both vascular and visceral smooth muscle generate as much force per crosssectional area as skeletal muscle with only 20% as much
myosin.3-5 This apparent enhancement in smooth muscle myosin's force production may simply reflect greater
numbers of crossbridges working effectively in parallel.
Such a mechanical advantage could result from longer
(i.e., 2.2-gm) myosin filaments6 or a more parallel
arrangement of contractile units within the smooth
muscle cell.1 At the actomyosin level, however, differences in kinetics of the smooth muscle crossbridge cycle
could result in crossbridges spending a higher fraction
of their cycle in the active force-generating state (i.e.,
higher duty cycle) under isometric conditions.1
The distinct mechanical properties of smooth and
skeletal muscle have been primarily explained on the
basis of differences in the myosin molecule, with little
attention given to the role of actin. Interest in actin's
contribution to the force-generating event has gained
momentum now that the primary structure of actin can
From the Department of Physiology and Biophysics, University
of Vermont, Burlington.
Supported by National Institutes of Health grants AR-34872
and HL-45161 (D.M.W.). D.M.W. is an Established Investigator
of the American Heart Association.
Address for correspondence: Dr. D.E. Harris, Department of
Physiology and Biophysics, University of Vermont, Burlington,
VT 05405.
Received August 3, 1992; accepted October 6, 1992.
be correlated to its tertiary structure, derived from
actin:DNAse I cocrystals.7 G-actin is a bilobed structure consisting of large and small domains, with the
myosin binding site contained within the small domain.
Given that a single genetically engineered amino acid
change to actin, at its putative myosin binding site, can
result in altered crossbridge kinetics in Drosophila flight
muscle,8 actin's contribution to force generation must
be considered. This is important, since smooth and
skeletal muscle express different actin isoforms, which,
in part, may account for smooth muscle's enhanced
force-generating ability.
There are two smooth muscle actin isoforms (asmooth muscle actin and y-smooth muscle actin), one
or both of which are found in every smooth muscle
tissue studied.9-11 The a-smooth muscle actin isoform
predominates in vascular tissue, whereas visceral
smooth muscle tissue expresses y-smooth muscle actin.
Extensive amino acid homology exists between the
smooth and skeletal muscle actins, with only six differences between chicken gizzard (y-smooth muscle) and
chicken pectoralis (a-skeletal muscle) actin.i2 However,
two of these changes occur within the cluster of acidic
residues at the N-terminal end of actin (residues 1 and
3), believed to be part of the myosin binding site.13 In
vitro motility can be altered in actins with reduced
acidity in this cluster, including wild-type yeast actin14
and mutated Dictyostelium actin.15 Thus, it is possible
that the naturally occurring amino acid differences
between smooth and skeletal muscle actin, which include the absence of the initial aspartic acid found in
skeletal muscle actin, may contribute to smooth muscle's unique mechanical properties.
The goal of this study was to determine if, in an in
vitro motility assay in which single fluorescently labeled
220
Circulation Research Vol 72, No 1 January 1993
actin filaments were observed sliding over a myosincoated coverslip,16 the characteristics of actin motility
were dependent on actin isoform. Specifically, we
wished to determine if the motility of smooth muscle
actin differed from that of skeletal muscle actin over
either phosphorylated smooth or skeletal muscle myosin over a wide range of assay conditions. The assay
conditions studied were chosen so that direct comparisons could be made to the extensive body of literature
on skeletal muscle actin motility in vitro.
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Materials and Methods
Contractile Protein Isolation and Preparation
Chicken skeletal pectoralis muscle myosin and turkey
gizzard smooth muscle myosin were prepared as previously described.17 Smooth muscle myosin was 100%
thiophosphorylated by the addition of myosin light
chain kinase, calmodulin, CaCI2, and ATP-y-S. Skeletal
muscle actin was isolated from chicken pectoralis acetone powder.18 Smooth muscle actin was purified from
chicken gizzard acetone powder as described,19 except
that, before the final polymerization, size fractionation
was carried out with a Sephacryl 300 column (Pharmacia). The purity of all protein-containing fractions was
assessed on a 12% polyacrylamide gel, and the purest
fraction was retained for polymerization and use. Both
smooth and skeletal muscle actin were stored in filamentous form at 4°C and fluorescently labeled with
tetramethylrhodamine phalloidin (Sigma Chemical Co.,
St. Louis, Mo.).17
Actin purity and composition were determined by
one- and two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), as previously described.20 One-dimensional SDS-PAGE of both
smooth and skeletal muscle actin showed a single major
protein band with minor contaminants visible in both
preparations only when the actin was overloaded. Twodimensional electrophoresis revealed that the smooth
muscle actin focused overwhelmingly as a single species
(data not shown). This is in agreement with previous
determinations that chicken gizzard actin is 80%
y-smooth muscle actin with minor contributions from
the nonmuscle actin species.10
In Vitro Motility Assay
The in vitro motility assay was carried out essentially
as described previously.172' Briefly, flow-through chambers were constructed from a nitrocellulose-coated coverslip and a glass microscope slide. Then the following
solutions were introduced into the chamber: 30 gl
myosin (250 ,ug/ml) in a 300 mM KCl buffer, which
allowed the myosin to adhere to the nitrocellulose
without forming filaments (i.e., in monomeric form); 60
gl bovine serum albumin (0.5 mg/ml) in the same buffer
to wash out unbound myosin and block any uncovered
nitrocellulose; 60 ,ul fluorescently labeled actin (0.5
,ug/ml) in ATP-free assay buffer; 60 ,l ATP-free assay
buffer to remove unbound actin; and 90 gl assay buffer
to initiate motility. Under standard conditions, the
assay buffer contained 25 mM KCI, 25 mM imidazole, 1
mM EGTA, 4 mM MgCl2, 1 mM dithiothreitol, 0.5%
methylcellulose, and 1 mM ATP, pH 7.4, with an
enzymatic oxygen scavenger system (0.1 mg/ml glucose
oxidase, 0.018 mg/ml catalase, and 2.3 mg/ml glucose)
added to retard photobleaching. The content of this
buffer was adjusted depending on the variable being
examined (e.g., changes in [KCl], pH, and [ATP]). Where
required, smooth and skeletal muscle myosin were mixed
before introduction into the flow-through chamber. Temperature was controlled at 30°C by an objective heater
(Vermont Technologies, Burlington, Vt.).
Fluorescently labeled actin filaments were viewed
through an inverted microscope equipped for epifluorescence.'7 Intensified video images were digitized and
analyzed for actin filament velocity by computer.22
Statistics
All velocity measurements were obtained from at
least 10 actin filaments and are presented as mean+SD.
Since the velocities that contribute to each mean value
were all obtained from the actin population in a single
flow-through chamber, mean velocities were considered
an n of one for subsequent statistical analysis.
The effect of actin filament type as a function of assay
condition (Figures 2-5) was determined by fitting velocity data for each filament type by polynomial regression (order of polynomial determined by analysis of
variance [ANOVA]) and then comparing the quality of
the regressions by F test. To allow comparison to
previously published data in which no difference
(p>0.05) was found between actin filament types, data
were combined and fit to the most appropriate polynomial as determined by ANOVA. Curve fitting was
performed with the TABLECURVE and SIGMAPLOT software packages (Jandel Scientific, Corte Madera, Calif.).
Results
Smooth and Skeletal Muscle Actin Filament Motility
With Changes in Assay Condition
Under standard assay conditions (see "Materials and
Methods"), skeletal and smooth muscle actin filament
velocities over monomeric skeletal muscle myosin were
not significantly different (p>0.05), as was the case with
monomeric smooth muscle myosin (Figure 1). As previously reported for filamentous myosin,17 the velocity
of actin filament motion over monomeric skeletal muscle myosin was approximately an order of magnitude
faster than over monomeric phosphorylated smooth
muscle myosin.
To more rigorously probe for differences between
myosin's interaction with smooth and skeletal muscle
actin, the motilities of the two actin filament types were
compared over a wide range of assay conditions. Starting from standard assay conditions, each of the following parameters was varied independently: pH, [ATP],
and [KCl]. These comparisons were performed over
both smooth and skeletal muscle monomeric myosin.
When ionic strength was varied by changing [KCI],
both smooth and skeletal muscle actin filament velocities increased with increasing ionic strength ([KCI],
12.5-75 mM) when observed over either phosphorylated smooth muscle (Figure 2A) or skeletal muscle
(Figure 2B) myosin. No significant difference (p>0.05)
was observed between the ionic strength dependence of
skeletal versus smooth muscle actin motility over either
smooth or skeletal muscle myosin.
Harris and Warshaw Effect of Actin Isoform on Motility
Smooth Muscle Myosin
221
Skeletal Muscle Myosin
0.8
0
0iW1
FIGURE 1. Bar graphs showing the velocity of
smooth muscle versus skeletal muscle actin filaments with smooth muscle myosin (left panel)
and skeletal muscle myosin (right panel). Data
are mean + SD of six independent experiments.
The comparison between actins was made by t
test.
0
-J
uJ
Skeletal
Actin
Skeletal
Actin
Smooth
Actin
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Smooth and skeletal muscle actin motility reacted
similarly to changes in pH when measured over either
smooth or skeletal muscle myosin. Actin filament velocity on both phosphorylated smooth (Figure 3A) and
skeletal (Figure 3B) muscle myosin reached a broad
peak between pH 7.0 and pH 8.0.
As [ATP] was reduced, actin filament velocity slowed
for both skeletal and phosphorylated smooth muscle
myosin (Figure 4). Since no difference was observed
A
Smooth
Actin
between the velocity of skeletal versus smooth muscle
actin over either smooth or skeletal muscle myosin
(p>0.05), actin filament types were combined for subsequent analysis. Double reciprocal (Lineweaver-Burk)
analysis showed that the ATP-dependent Km of actin
filament velocity on smooth muscle myosin (46 ,uM) was
not significantly different from that on skeletal muscle
myosin (53 ,uM).
The ionic strength, pH, and [ATP] dependence of
actin filament velocity over both phosphorylated
A
1.5 r
0.9 F
1.0 [
S
hi
0
E
0.6 F
-j
m
0
0
_i
W
0
_3
W 0.3 h
0.5 F
Smooth Muscle Myosin
0.0
0
25
B
50
75
(mM)
[KCI]
A
l
0.0
6.0
100
t
6.5
7.0
osi
8.0
7.5
pH
B
9r
y
Smooth Muscle Myosin
9r
0
E 6
E6
C.)
0
_1
W
0
-J3
W
A
3F
Skeletal Muscle Myosin
Skeletal Muscle Myosin
Dv
0
0
25
50
75
[KCII (mM)
100
FIGURE 2. Graphs showing smooth muscle actin filament
(A) and skeletal muscle actin filament (o) velocity as a
function of [KCl] with monomeric phosphorylated smooth
muscle myosin (panel A) and skeletal muscle myosin (panel
B) at pH 7.4, 1 mMATP, and 300C. Data are mean +SD.
Solid lines are polynomialfits to data from both actin filament
types: for panel A, y= -0.00014x2+0.02x+0.27 (r2=0.92);
for panel B, y=0. 02x+5.08 (r2=0. 94).
6.0
6.5
7.5
7.0
8.0
8.5
pH
FIGURE 3. Graphs showing smooth muscle actin filament (A)
and skeletal muscle actin filament (o) velocity as a function of
pH with monomeric phosphorylated smooth muscle myosin
(panel A) and skeletal muscle myosin (panel B) at 25 mMKCl,
1 mMATP, and 300C. Data are mean ±SD. The solid lines are
polynomial fits to data from both actin filament types: forpanel
A, y=-0.33x2+5.0x-18.03 (r2=0.94); for panel B,
y=-2.55x2+38.89x-141.63 (r2=0.86).
Circulation Research Vol 72, No 1 January 1993
222
6r
0)
E
SMOOTH PHOSPHORYLATED MYOSIN (%)
/
Smooth Muscle /
Myosin
///
/
/
4
100
0
w
60
40
20
0
20
40
60
80
100
/
//
a///
0.75
W /
0
80
1.00
Er:
/
¢
0.50
2
/^/
/
,/
A
W 0.25
/=:==-'
/
/
-50
Skeletal Muscle
Myosin
0
50
100
1/[ATP] (l/mM)
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FIGURE 4. Double reciprocal plot showing the effect of
[ATP] on actin filament velocity for smooth muscle actin (A)
and skeletal muscle actin (o) at 25 mM KCI, pH 7.4, and
30°C with both monomeric phosphorylated smooth and skeletal muscle myosin. Solid lines are best-fit linear regression
with 95% confidence limits as dashed lines. The comparison
of the [A TP] needed for half-maximum actin filament velocity
(ATP-dependent Km) for smooth versus skeletal myosin was
made by noting that the 95% confidence limits of the linear
regressions for the Lineweaver-Burk plots of the two myosins
overlap at the x axis. The linear fits are as follows: for smooth
muscle myosin, y=0.055x+1. 188 (r2=0.98); for skeletal muscle myosin, y=O.009x+0. 17 (r2=0. 96).
smooth and skeletal muscle myosin monomers reported
here (Figures 2-4) are in agreement with previous
results from the motility assay.16 '7,23,24
Smooth and Skeletal Muscle Actin Filament Motility
With Monomeric Myosin Mixtures
Actin filament velocity over mixtures of myosins having
different cycling rates may be determined by mechanical
interactions between myosins.l7,2526 If so, comparison of
smooth and skeletal muscle actin filament motility over
mixtures of monomeric phosphorylated smooth and skeletal muscle myosins may reveal differences between the
two actins under loaded conditions.
When increasing amounts of monomeric phosphory-
lated smooth muscle myosin were mixed with skeletal
muscle myosin, the velocity of both smooth and skeletal
muscle actin filament velocity dropped precipitously
(Figure 5). By increasing the proportion of smooth
muscle myosin to 32%, actin filament velocity was
reduced by 50% of the velocity for skeletal muscle
myosin alone. No significant difference (p>0.05) was
observed between smooth and skeletal muscle actin
velocities in this experiment.
Discussion
Actin's role in muscle contraction has been viewed
generally as both a cofactor to enhance myosin ATPase
activity and a passive mechanical structure that transmits force generated by myosin to the ends of the
contractile unit (i.e., sarcomere in striated muscle).
However, a series of recent studies on nonmammalian
actin isoforms'4 and actins altered by enzymatic cleav-
0.00
L
0
SKELETAL MYOSIN (%)
FIGURE 5. Smooth muscle actin filament (A) and skeletal
muscle actin filament (o) velocities (V) plotted against monomeric myosin mixtures of phosphorylated smooth and
skeletal muscle myosin at 25 mM KCl, pH 7. 4, 1 mM A TP,
and 30°C. Velocities are mean values normalized to maximum value (Vmax) for each data set. Error bars are omitted
for clarity. Solid line is fit to data sets from both actins. Vmax
is 7.21 um/sec for smooth muscle actin and 7.43 gm/sec for
skeletal muscle actin. The equation of the line is as follows:
y=O.00009X2-O. 00057x+0.095 (r 2=O. 96).
age,27 chemical cross-linking,28 and genetic engineering8s15 suggests that actin may play a significant role in
the chemomechanical transduction process (see Morel
and Mereh29 for review). In fact, models in which
actomyosin force generation originates within the actin
filament itself have been proposed.30 Is it possible that
smooth muscle's enhanced force-generating capacity is
related in part to the smooth muscle-specific actin
isoform?
Previous biochemical studies, designed to address this
question, have shown that both smooth and skeletal
muscle actin are equally capable of activating skeletal
myosin ATPase activity.3' However, actomyosin ATPase
measurements in solution merely reflect the enzymatic
properties rather than the mechanical characteristics of
the actomyosin interaction. Therefore, the in vitro motility
assay provides a unique opportunity to assess the mechanical consequences of actin isoform on the actomyosin
interaction through alterations in actin filament motility.
At least three studies using the in vitro motility assay
have attempted to characterize whether the velocity of
actin motility is related to either the actin or the myosin
isoform.i624,32 Kron and Spudich'6 demonstrated that
Dictyostelium and rabbit skeletal muscle actin filament
velocity are similar over both Dictyostelium and rabbit
skeletal muscle myosin. Similarly, Umemoto and Sellers24 and Okagaki et a132 found no difference between
the velocity of gizzard and skeletal muscle actin over
smooth muscle myosin. These investigators concluded
that actin filament motility was independent of actin
isoform and that'the velocity of movement was governed
by the myosin isoform. However, this conclusion was
drawn in each case from a very limited experimental
protocol performed at only one ionic strength, pH, and
Harris and Warshaw Effect of Actin Isoform on Motility
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[ATP]. Given that both muscle fiber mechanics and in
vitro actin filament motility are exquisitely sensitive to
the assay conditions, we believe that a comparison of
smooth versus skeletal muscle actin motility over a far
wider range of assay conditions was necessary before a
definitive conclusion could be drawn about the effect of
actin isoform on the actomyosin interaction in vitro.
The results of the present study suggest that, over the
wide ranges of pH and [KCl] investigated in which actin
filaments are presumed to be under no load, the
y-smooth muscle and a-skeletal muscle actin isoforms
are mechanically indistinguishable over both smooth
and skeletal muscle myosin. Even though the two actin
isoforms reacted similarly to changes in assay conditions, actin isoform may still affect actin filament motility when actin filaments experience a load. We approached this problem by studying the motility of actin
filaments with mixtures of smooth and skeletal muscle
myosin.
Mechanical interactions between myosin species occur
in the motility assay whenever two myosin populations
having different cycling rates attach to the same actin
filament.172526 The resultant actin filament velocity is
dependent on the force-generating capacity of the faster
cycling myosin and the magnitude of the load created by
the slower cycling species as it is negatively strained by the
faster cycling crossbridges.17,21 Therefore, actin filaments
moving over a myosin mixture must be experiencing the
opposing forces created by the mechanical interactions
between the two myosin species.
Our laboratory has previously characterized the dependence of actin filament velocity on the proportion of
phosphorylated smooth and skeletal muscle myosin.17 In
these earlier studies in which skeletal muscle actin was
used, actin filament velocity for an equal mixture of
smooth and skeletal muscle myosin was more similar to
the velocity over smooth muscle myosin alone. If actin
isoform is crucial to force production, then one would
predict that the shape of the relation between actin
filament velocity and myosin mixture content might shift if
the smooth muscle actin were used. However, no difference was observed between smooth versus skeletal muscle
actin filament velocity over mixtures of smooth and skeletal muscle myosin (Figure 5). Once again, it appears that
actin isoforms do not alter or modulate the force produced
by the actomyosin interaction. This conclusion is supported by the finding that no difference was observed
between the velocities of smooth and skeletal muscle actin
filaments with either smooth or skeletal muscle myosin at
low [ATP] (Figure 4), where slowing is presumably caused
by the load imposed by rigor bridges. Therefore, differences in myosin isoforms between smooth and skeletal
muscle may be the predominate cause for smooth muscle's
enhanced stress production. These results argue against
models proposing that force generation originates in the
actin filament.30
The fact that actin isoforms are mechanically indistinguishable in the motility assay may relate to the
conserved nature of actin. In fact, the naturally occurring amino acid substitutions that do exist between
chicken a-skeletal and chicken y-smooth muscle actin
are quite conservative with respect to amino acid charge
and polarity.12 Therefore, the present results may not be
too surprising. It still remains to be determined
whether, under truly isometric conditions, changes in
223
actin isoform can modulate actomyosin force production. Future studies that include isometric force measurements on single actin filaments may reveal a mechanical difference between smooth and skeletal muscle
actin.33 Alternatively, differences between actin isoforms may only be revealed when a more native smooth
muscle thin filament, containing tropomyosin and possibly caldesmon and calponin, is reconstituted.
Acknowledgments
We would like to thank Steven Work for the computer
programs used to track actin filament motion, Janet Woodcock-Mitchell and John Mitchell for running the SDS-PAGE,
Gary Badger and Joe Carpenter for assistance with the
statistical analysis, and Kathy Trybus for providing many of the
proteins and for her comments and contributions to an earlier
version of this manuscript.
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Smooth and skeletal muscle actin are mechanically indistinguishable in the in vitro
motility assay.
D E Harris and D M Warshaw
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Circ Res. 1993;72:219-224
doi: 10.1161/01.RES.72.1.219
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1993 American Heart Association, Inc. All rights reserved.
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