Impaired vascular contractility and blood pressure
homeostasis in the smooth muscle ␣-actin null mouse
LISA A. SCHILDMEYER,* RENEE BRAUN,* GEORGE TAFFET,† MARIELLA DEBIASI,‡
ALAN E. BURNS,† ALLAN BRADLEY,§ AND ROBERT J. SCHWARTZ*,1
1
Department of Molecular and Cellular Biology, †Department of Medicine, Section of Cardiovascular
Sciences, ‡Department of Molecular Physiology and Biophysics and §Howard Hughes Medical
Institute, Department of Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
The smooth muscle (SM) ␣-actin gene
activated during the early stages of embryonic cardiovascular development is switched off in late stage
heart tissue and replaced by cardiac and skeletal
␣-actins. SM ␣-actin also appears during vascular
development, but becomes the single most abundant
protein in adult vascular smooth muscle cells. Tissuespecific expression of SM ␣-actin is thought to be
required for the principal force-generating capacity
of the vascular smooth muscle cell. We wanted to
determine whether SM ␣-actin gene expression actually relates to an actin isoform’s function. Analysis of
SM ␣-actin null mice indicated that SM ␣-actin is not
required for the formation of the cardiovascular
system. Also, SM ␣-actin null mice appeared to have
no difficulty feeding or reproducing. Survival in the
absence of SM ␣-actin may result from other actin
isoforms partially substituting for this isoform. In
fact, skeletal ␣-actin gene, an actin isoform not
usually expressed in vascular smooth muscle, was
activated in the aortas of these SM ␣-actin null mice.
However, even with a modest increase in skeletal
␣-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM
␣-actin-defective mice. This study supports the concept that SM ␣-actin has a central role in regulating
vascular contractility and blood pressure homeostasis, but is not required for the formation of the
cardiovascular system.—Schildmeyer, L. A., Braun, R.,
Taffet, G., DeBiasi, M., Burns, A. E., Bradley, A., and
Schwartz, R. J. Impaired vascular contractility and
blood pressure homeostasis in the smooth muscle
␣-actin null mouse. FASEB J. 14, 2213–2220 (2000)
ABSTRACT
Key Words: SM ␣-actin gene 䡠 homologous recombination
䡠 vascular tone
Actins, members of a highly conserved contractile
protein multigene family, are differentially expressed during the ontogeny of an organism (1–5).
The high degree of sequence conservation between
the actin proteins suggests that this multigene family
arose by duplication and subsequent divergence
0892-6638/00/0014-2213/$02.25 © FASEB
from a common ancestral gene (1, 6). In the course
of these events, certain regulatory and structural
features of the actin loci diversified to produce the
specialized actin genes observed today. In birds and
mammals, actins are represented by at least six
different isoforms, including skeletal and cardiac
␣-actins, vascular and visceral ␣ and ␥ smooth muscle
actins, and the ubiquitous ␤- and ␥-cytoplasmic actins (2–5). These actins are highly homologous,
differing from each other by less than 5% of their
amino acid sequence and encoded by separate unlinked genes (2, 3). Tissue-specific expression of
actin isoforms, regulated expression of isoforms during muscle differentiation, and coexpression of multiple isoforms in various cell types suggest that actin
isoforms may have specialized functions (5– 8). For
example, the cytoplasmic actins, including ␤ and ␥,
are enriched in nonmuscle cells where they participate in the formation of the cytoskeletal apparatus
and microfilaments that function in cell motility,
endocytosis, and related processes (1). In contrast,
the striated cardiac and skeletal ␣-actins and smooth
muscle ␣- and ␥-actins compose contractile sarcomeres and smooth muscle myofibrils of warmblooded vertebrate muscle tissues (6 –10).
The smooth muscle (SM) ␣-actin gene is activated
during the early stages of embryonic heart development (11–15) The sequential activation of SM ␣-actin and cardiac ␣-actin gene activity follows the
formation of the definitive heart tube. Since the
earliest myocardial filament bundles are reminiscent
of the organization in smooth muscle, it was suggested that SM ␣-actin might be important in
supporting the early contractile functions of the
developing heart when myofibrillogenesis is just beginning (11). SM ␣-actin is down-regulated during
late stage cardiac morphogenesis, being replaced by
cardiac and skeletal ␣-actins (11–13). In contrast, SM
␣-actin also appears early during vascular development, but is retained to become the single most
1
Correspondence: Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030,
USA. E-mail: [email protected]
2213
abundant protein in adult vascular smooth muscle
cells. Skeletal and cardiac ␣-actin transcripts are not
detected during normal vasculogenesis (11), and SM
␣-actin is thought to be required for the principal
force-generating capacity of the vascular smooth
muscle cell (8, 9)
Recent studies support the contention that individual actin isoforms are involved with specific cellular contractile functions (16 –20). Lloyd et al. (18)
showed that the level of the nonmuscle ␤- and
␥-actin protein may determine a feedback-regulatory
response. Also, the up-regulation of skeletal muscle
␣-actin gene and SM ␣-actin gene activity in hearts of
cardiac ␣-actin null mice supports a cellular sensing
mechanism that might respond to changes in sarcomeric contractility and/or the absence of cardiac
␣-actin by eliciting compensatory gene activity (19).
We wanted to determine whether SM ␣-actin gene
expression relates to an actin isoform’s specialized
function or is simply the manifestation of programmatic gene regulation of the actin multigene family.
This study defines the role of SM ␣-actin in the
cardiovascular system by using gene targeting in
mouse embryonic stem cells to inactivate SM ␣-actin
gene transcription.
NEN, Wilmington, Del.), and UV cross-linked to the membrane. A probe corresponding to the 3⬘UTR of the mouse SM
␣-actin gene (22; ⬃160 bp fragment) was labeled by random
priming. Probes for the cardiac ␣-actin gene (27; ⬃140 bp
fragment) and the skeletal ␣-actin gene (27; ⬃200 bp fragment) were labeled as riboprobes. Blots were hybridized
16 –18 h at either 45°C for the random primed probes or 68°C
for the riboprobes in a solution containing 50% formamide,
5⫻ SSPE, 5⫻ Denhardt’s solution, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% ficoll, 1% sodium
dodecyl sulfate (SDS), 10% dextran, and 200 mg/ml sheared
salmon sperm. Blots were exposed with intensifying screens to
X-ray film (Kodak X-Omat AR) at ⫺70°C for 1 to 7 days.
Protein Western blot analysis
MATERIALS AND METHODS
As described above, powdered tissues were extracted (⬃10 ml
of extraction solution per 1 g of powdered tissue) in a
solution of 25 mM Na2HPO4, 1% ␤-mercaptoethanol, and
protease inhibitors at 4°C for 1–2 h. SDS was added to a final
concentration of 1%. The samples were incubated at 37°C for
20 min and clarified by centrifugation at 14,000 g for 5 min.
Total protein concentration was determined by Bradford
assay. Protein samples (10 ␮g per well) were electrophoresed
on 10% acrylamide-1% SDS gels with a 2.5% acrylamide-1%
stacking gel and then transferred to Immobilon-P transfer
membrane (Millipore, Bedford, Mass.). Blots were probed
with mAb 1A412 (28; Sigma, St. Louis, Mo.) specific for SM
␣-actin and mAb 5C5 (29; Sigma), specific for cardiac and
skeletal ␣-actins, and reacted with a horseradish peroxidase
secondary antibody detected by chemiluminescence with the
ECL Western Blotting Analysis System (Amersham, Arlington
Heights, Ill.) and exposed to X-ray film (Kodak X-Omat AR).
SM ␣-actin gene targeting
Immunohistochemistry
Mouse SM ␣-actin genomic DNA clones were isolated from a
129 SVJ mouse genomic library (Stratagene, San Diego,
Calif.). The targeting vector was constructed by inserting a
Pol2NeobpA cassette (21) at the ⫹1 site of the SM ␣-actin
gene and consisted of an upstream 2.1 kb EcoRV/SmaI
fragment and a downstream 3.5 kb SmaI/XbaI fragment of the
SM ␣-actin gene (22). The targeting plasmid vector was cut at
a unique site before electroporation into AB2.2 ES cells. DNA
isolated from G418-resistant clone was digested with XbaI and
screened using the mini Southern procedure (23). Digestion
of wild-type (wt) DNA gave a fragment of ⬃10 kb, whereas
digestion of DNA from targeted clones gave the expected 10
kb wt fragment, as well as a 5.5 kb fragment from the targeted
allele. A chimeric male mouse capable of germline transmission was obtained by injecting targeted cells into 3.5-day-old
blastocysts from C57 mice. The male chimera was mated to
produce offspring carrying the mutated allele.
Tissues were fixed in zinc formalin, paraffin embedded, and
sectioned at 5 ␮m. Tissue sections were then stained with a
combination of trichrome and elastica stains to detect muscle,
connective, epithelial, and elastic tissues. Tissue sections were
also stained using the SM ␣-actin monoclonal antibody 1A4
(28; dilution 1:1000) visualized by DAB.
RNA Northern blot analysis
Mice were killed by CO2 asphyxiation. Tissues of interest were
isolated, rinsed in phosphate buffered standard saline (0.9%
NaCl, 050 M NaPO4, pH 7.2), blotted, frozen in liquid N2, and
crushed using a percussion mortar cooled with liquid N2.
Tissues used for RNA isolation were sections of the aorta that
extended from the aortic arch to the diaphragm, the ventricles of the heart, the lower part of the large intestine, skeletal
muscle from the upper portion of the hind limb, and the
uterus. Total RNA was extracted from the powdered tissues
using methods previously described (24 –26). RNA samples
were electrophoresed on 1.0% agarose, 2.2% formaldehyde
gels, blotted onto Genescreen nylon membrane (DuPont,
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Transmission electron microscopy
Aortas were fixed for 2 h in phosphate-buffered saline (PBS)
containing 2.5% glutaraldehyde, transferred to PBS containing 1% tannic acid (5 min), and then postfixed in PBS
containing 1% osmium tetroxide (60 min). This was followed
by en bloc staining with aqueous uranyl acetate (60 min),
dehydration through ethanol, and embedding in LX 112
resin (Polysciences, Inc., Warrington, Pa.). Ultrathin sections
were stained with uranyl acetate and lead citrate prior to
viewing on a JEOL 200C⫻ electron microscope.
Vascular contractility
Aortas were carefully excised from killed mice, immediately
placed in ice-cold Krebs-Ringer solution, and dissected free of
surrounding tissue. A section of the thoracic/abdominal
aorta (extending from a region just below the aortic arch to
the diaphragm) was cut into 3 approximately equal segments
of 2–3 mm each. The aortic ring sections were mounted in 13
ml tissue baths containing a Krebs-Ringer bicarbonate solution (110 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.2 mM
KH2PO4, 25 mM NaHCO3, 11.1 mM glucose, and 2.5 mM
CaCl2) at 37°C and bubbled with a mixture of 95% O2 and
The FASEB Journal
SCHILDMEYER ET AL.
5% CO2. Isometric tension recordings were made using a
Brush Mark 200 chart recorder. Aortic ring sections were
equilibrated under tension for at least 1 h before experiments
were begun. Ability to contract was evaluated at different
initial tensions (0.75 g, 1.0 g, and 1.25 g) using two concentrations of KCl (80 mM and 120 mM) to initiate aortic
contraction.
Blood pressure measurement
Systolic blood pressure measurements were made in conscious nice by a noninvasive tail cuff method (30; IITC Inc.
Life Science, Woodland, Calif.). Three weeks prior to taking
pressure measurements, mice were trained by gentle handling and warming to 30°C. Baseline pressure measurements
were obtained by taking five consecutive measurements each
day for 7 to 10 days for each mouse. Blood pressure measurements were also taken after infusion of sodium nitroprusside
(0.5 mg/ml) for 15 min via the tail artery. Blood pressure
measurements were continuously monitored over an hour
after withdrawal of drug.
Tail artery blood flow
Mice were anesthetized with an intraperitoneal injection of a
combination anesthetic (Ketamine, Xylazine, Acepromazine). A pulsed-Doppler (20 MHz) flow probe placed around
the tail at least 1.5 cm from the base of the tail recorded blood
flow through the caudal artery as used in the rat (31). A
temperature probe inserted into the rectum measured core
body temperature. A 60 W incandescent light was used to
warm the animal. The outputs of the flow probe were
directed into a spectrum analyzer, which performed fast
Fourier transforms on the data. Anesthetized mice were first
warmed to a core basal body temperature of 38°C and the
Doppler flow probe was positioned to obtain maximum flow
signals through the caudal artery. Mice were then cooled to
⬍34°C and allowed to remain ⬃15 min at this temperature.
The mice were warmed again; at approximately every increase
of 0.5° in basal body temperature, the blood flow through the
tail artery was recorded for 1 s, providing a relative measure of
blood flow.
RESULTS
SM ␣-actin null mutant mice
This study defines the role of SM ␣-actin in the
cardiovascular system by using gene targeting in
mouse embryonic stem cells to inactivate SM ␣-actin gene transcription. To block the accumulation
of SM ␣-actin transcripts, a Pol II promoter neomycin hybrid gene was inserted into the SM ␣-actin
gene locus at the ⫹1 cap site. Electroporation of
the targeting vector (Fig. 1a, b) into AB2.2 cells
resulted in a targeting frequency 1 out of 12
positive homologous recombinants per G418-resistant clones. A male chimeric mouse obtained from
a targeted 129 strain was mated with C57 females,
resulting in seven heterozygous breeding pairs.
Intercrosses between these SM ␣-actin heterozygous mice yielded a number of offspring that
approximated the predicted Mendelian ratio of
SMOOTH MUSCLE ␣-ACTIN
Figure 1. Targeted disruption of the SM ␣-actin gene allows
for activation of skeletal ␣-actin gene expression in the aortas
of homozygous SM ␣-actin null mice. a) The targeting vector
is represented. Abbreviations: ‘HIII’-HindIII,‘RI’-EcoRI,‘RV’EcoRV. In the endogenous allele, the ⫹1 cap site of the SM
␣-actin gene is indicated by an arrow. A positive targeting
event inserts the Pol2NeobpA cassette between the SM ␣-actin
promoter and coding region, disrupting transcription (a3). b)
Genomic DNA from wt and targeted ES cells was analyzed by
Southern blot. Digestion of wt DNA by XbaI results in a 10 kb
fragment, shown in lane 1. Insertion of the Pol2Neo into the
endogenous SM ␣-actin results in a 10 kb fragment, as well as
a 5.5 kb fragment, shown in lane 2. c) Northern blots show the
absence of SM ␣-actin mRNA in aorta, intestine, and uterus
RNA samples of a ⫺/⫺ SM ␣-actin null mice. Note the
appearance of skeletal ␣-actin message in the aorta mRNA
from SM ␣-actin null mice, whereas no change occurred in
the distribution of cardiac ␣-actin mRNA. d) Western blots
show the absence of SM ␣-actin protein in the aorta, intestine
and uterus protein extracts and the appearance of sarcomeric
␣-actin protein extracts of the aorta from SM ␣-actin null
mice.
1:2: 1 for wt, ⫾, and ⫺/⫺ mice and about an equal
number of males and females, as shown in Table 1.
Mice identified as null mutants (SM ␣-actin ⫺/⫺)
for the SM ␣-actin gene by Southern blot analysis
presented no obvious phenotype on gross examination and were fertile, reaching adulthood without succumbing to abnormalities resulting from
the absence of this protein.
2215
TABLE 1. Genotypes of back crosses between SM ␣-actin
heterozygous breeding pairs of mice yielded expected Mendelian
ratios of offspringa
Absence of vascular contractility and lowered basal
blood pressure in SM ␣-actin null mice
Number of
offspring
SM ␣-actin mAb cross-reactivity marked the medial
layers of normal aorta, carotid artery, and cardiac
arteriole (Fig. 2a, c, e) but not ⫺/⫺ vessels (Fig. 2b,
114
Ratio of offspring
Females
Males
wt
⫹/⫺
⫺/⫺
63
0.45
51
0.55
21
0.8
60
2.1
32
1.1
a
This table presents the number of offspring by their sex and
genotype determined by DNA blotting, as shown in Fig. 1, from 7
breeding pairs of SM ␣-actin (⫹/⫺) heterozygous mice.
Skeletal ␣-actin up-regulated in SM ␣-actindefective vessels
The expression of muscle ␣-actin genes in SM
␣-actin null mice was evaluated by RNA Northern
blots using 3⬘ untranslated region (3⬘ UTR) labeled probes specific for each actin isoform. As
shown in Fig. 1c, robust SM ␣-actin expression is
demonstrated by the appearance of 1.7 kb RNA
species in wild-type aorta, intestine, and uterus
tissues and expressed to a lesser extent in wt
cardiac and skeletal tissue. In comparison, the
accumulation of SM ␣-actin RNA transcripts are
blocked by neo⫹ insertion mutagenesis in tissues
from the SM ␣-actin null mice. Expression of the
cardiac ␣-actin gene was not influenced by the SM
␣-actin-defective gene, as shown by its comparable
mRNA levels in wt and mutant striated muscle
tissues (Fig. 1c). Although cardiac and skeletal
␣-actin genes are silent in normal vascular smooth
muscle tissues, skeletal ␣-actin RNA unexpectedly
was up-regulated in the majority of SM ␣-actin
⫺/⫺ aortas examined (Fig. 1c).
SM ␣-actin was immunodetected by Western
blots of electrophoresed protein extracts of wt
aorta, uterus, and intestine reacted with a wellcharacterized SM ␣-actin mAb (Fig. 1d ). Reduced
amounts of SM ␣-actin were detected in any wt
cardiac and skeletal muscle protein extracts. Under identical assay conditions, SM ␣-actin immunoreactive species were not detected in extracts of
muscle tissues taken from SM ␣-actin null mutants,
thus confirming the absence of SM ␣-actin gene
expression in SM ␣-actin null mice. Striated ␣-actins from cardiac and skeletal muscle tissue samples of both wt and mutant mice reacted strongly
with the sarcomeric ␣-actin mAb (Fig. 1d), but wt
aortic extracts did not demonstrate the presence
of striated ␣-actin gene expression in wt smooth
muscle tissues. However, aortic samples from SM
␣-actin null mice (Fig. 1d) exhibit sarcomeric
␣-actin reactive protein, substantiating the novel
observation of up-regulation of skeletal ␣-actin
gene activity seen in the Northern blots.
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Figure 2. Immunohistochemical analysis reveals defective
vasculature of the SM ␣-actin null mice. Cross sections stained
with SM ␣-actin mAb of aorta (a, b), carotids (c, d), and
arterioles in a section of cardiac muscle (e, f ) of wt (a, c, e)
and ⫺/⫺SM ␣-actin (b, d, f ) mice show positive SM ␣-actin
staining as black only in the wt vessels. Sections of wt (g) and
⫺/⫺ SM ␣-actin (h) vessels stained with a combination of
silver stain, which stains elastica black, and trichrome stain,
which stains muscle red and connective tissue blue, show both
elastic and muscle tissue components. The elastic layers of the
wt aorta (a, g) appear ruffled whereas the mutant aorta (b, h)
lacks this ruffled appearance. Ruffling is also seen in the wt
carotid (c) but not in the SM ␣-actin null carotid (d). The
sizing bar in h for the light micrographs (a– h) corresponds to
100 mm. Electron micrographs show dense myofilaments in
the smooth muscle layer of the wt aorta (i), whereas myofilaments are less dense and disorganized in the mutant aorta
( j ). The arrows point to myosin thick filaments that were less
frequently observed in the mutant aorta, consistent with the
interpretation that structural differences exist in the organization of myofilaments between the wt and mutant vessels.
Sizing bars in panels i and j correspond to 0.5 mm.
The FASEB Journal
SCHILDMEYER ET AL.
d, f). Aortic tissue sections stained with a combination of trichrome and elastica (Figs. 2g, h) showed
the presence of muscle (red) and elastic (black)
components in both wt and SM ␣-actin ⫺/⫺ tissue.
The elastic layers of the wt aorta have a contracted,
ruffled-like appearance (Fig. 2g) whereas the comparably stained mutant aorta appears unruffled and
flat (Fig. 2h), as also seen in the antibody-stained
sections (Fig. 2a–f ). At higher resolution, electron
microscopy revealed disorganization and reduction
of myofilamentous arrays in the SM ␣-actin ⫺/⫺
aortic tissue sections (Fig. 2j, see arrows) in comparison to wt tissue sections (Fig. 2i). The appearance of
the ⫺/⫺ vessels led us to examine the likelihood of
impaired biological function.
Vascular contractility was used to evaluate the
functional consequences of the SM ␣-actin null
mutant in vitro. Aortic rings were set at resting
tensions of 0.75 g, 1.0 g, and 1.25 g and depolarized
by addition of KCl (80 mM and 120 mM). Differences in the responses of the wt and mutant vessels
were quite dramatic. Aortic rings from wt mice (Fig.
3a, top three tracings) responded to KCl stimulation
whereas aortic segments from mutant mice (Fig. 3a,
lower three tracings) showed little or no response.
Similar results were obtained by stimulation with
norepinephrine (1⫻10⫺5 and 1⫻10⫺6 M) and serotonin (1⫻10⫺6 M) (data not shown). Differences in
contractility between wt and ⫺/⫺ aortas were independent of the agonist and the initial tension at the
time of stimulation (Fig. 3b), suggesting that this
phenomenon results from a fundamental dysfunction of the contractile system, presumably the absence of SM ␣-actin.
In vivo studies included measuring the systolic
blood pressure in adult conscious mice to determine
the relevance of these observations in vivo. When the
values for each pair of mice were compared, the
mean systolic blood pressure of the mutant mouse
was significantly lower that that of his wt littermate in
all five pairs examined (P⬍0.05, Table 2). Overall,
the SM ␣-actin null mice showed a significant reduction (28%) in systolic blood pressure when compared to wild-type mice. We examined the effect of
intravenous infusion of sodium nitroprusside (SNP)
on blood pressure. Typically, SNP produces rapid
vasodilatation followed by a baroreflex-mediated activation of the sympathetic nervous system, releasing
norepinephrine and resulting in increased heart rate
and vasoconstriction. In the wt mice, SNP infusion
resulted in a 50% increase in heart rate (data not
shown) and a ⬃45% decrease in systolic blood
pressure (Fig. 4a). When SNP infusion was stopped,
the systolic blood pressure rapidly returned to predrug levels after a transient overshoot, reflecting the
release of NE at the vascular level. In the two SM
␣-actin null littermates, SNP induced reduced vasoSMOOTH MUSCLE ␣-ACTIN
Figure 3. Aortic ring segments of SM ␣-actin null mice reveal
reduced contractility. a) Aortic ring segments from a wt
mouse generated the top three tracings; the bottom three
tracings were from a SM ␣-actin null mouse. 1 denotes aortic
segments most proximal to the heart; most distal sections are
indicated by 3. Aortic segments were stimulated by addition of
80 mM and 120 mM KCl, denoted by arrows. Baths were
washed out with fresh Krebs-Ringer solution when aortic
segments reached their maximal contractile response, again
denoted by an arrow, and allowed to relax back to their
resting tensions. The responses of aortic ring segments from
⫺/⫺ SM ␣-actin null mice were substantially reduced in
comparison to the response of the wt aortic segments. b) Bar
graphs indicate that regardless of the initial tension or
concentration of KCl used to stimulate the vessels, the contractility of aortic rings from mutant mice was reduced by at
least 90%. Segments of aorta from the mutant mice subjected
to the conditions of 1.25 g initial tension, 120 mM KCl had
negligible responses.
dilatation, a 26% decrease in systolic blood pressure,
and reflex tachycardia. However, when SNP infusion
was stopped, the systolic blood pressure remained
low for over an hour (Fig. 4b). Thus, resistance blood
vessels were unable to respond in response to NE in
the absence of SM ␣-actin.
Additional in vivo studies investigated the ability of
contractile vessels to relax and undergo vasodilatation. Vascular dysfunction in the mutant mice was
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TABLE 2. Reduced systolic blood pressures in SM ␣-actin null
micea
⫺/⫺ mean systolic BPs
wt mean systolic BPs
wt mouse #1
wt mouse #2
wt mouse #3
wt mouse #4
wt mouse #5
Average BP
Stnd. Dev.
118.2
128.0
103.9
107.3
115.2
114.4
9.5
⫺/⫺
⫺/⫺
⫺/⫺
⫺/⫺
⫺/⫺
mouse
mouse
mouse
mouse
mouse
#1
#2
#3
#4
#5
77.4
92.1
76.3
84.5
80.9
82.2
6.4
a
Each mean systolic BPs value represents the mean sd of 5
consecutive measurements. Statistical analysis compared the systolic
blood pressures of littermate matched pairs (wt and ⫺/⫺) using a
paired Student’s t test.
Figure 5. Impaired maintenance of blood flow in SM ␣-actin
null mice. Blood flow in anesthetized wild-type mice (filled
boxes) and SM ␣-actin null mice (filled circles) was measured
through the caudal artery with a Doppler flow probe. Increase
in body temperature (⌬2.5°C) resulted in a relative fivefold
increase in peak tail artery blood flow in normal mice whereas
in the SM ␣-actin null mice, blood flow increased only by
twofold.
indicated by a diminished peripheral blood flow in
response to thermal challenge. As shown in Fig. 5,
the relative peak tail artery blood flow velocity is
increased in normal mice in response to increasing
core body temperature whereas mutant mice showed
a significantly reduced response. Raising the body
temperature of the mice 2.5°C resulted in a fivefold
increase in flow velocity in normal mice but less than
a twofold increase in the mutant mice, demonstrating impaired vascular regulation and an inability to
modify tone in the SM ␣-actin null mice. Increased
heart rate in response to increase in temperature was
similar in both the wt and mutant mice and does not
explain this difference in blood flow. The null mice
recovered without incident, without any detection of
vasospasm, on return to normal body temperature.
DISCUSSION
Figure 4. Impaired maintenance of blood pressure and blood
flow in SM ␣-actin null mice. a) Upon tail infusion of 0.5
mg/ml sodium nitroprusside (SNP) for 15 min, illustrated by
the underlying line, the systolic blood pressure of wt mice
decreased ⬃45% and rapidly returned to predrug levels when
the infusion was stopped. b) In SM ␣-actin null mice the
systolic blood pressure did not return to basal levels after the
SNP infusion was stopped for up to 1 h, indicating that blood
vessels of the SM ␣-actin mice cannot respond to baroreflexmediated sympathetic stimulation due to the lack of SM
␣-actin.
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We asked whether SM ␣-actin gene expression relates to a specialized function during formation or
maturation of the cardiovascular system. SM ␣-actin
is the first muscle actin expressed during development of the cardiovascular system (11), but we
showed that SM ␣-actin does not seem to play an
obligatory role in the formation of the cardiovascular
system. Mutant SM ␣-actin mice appear to have no
difficulty feeding or reproducing (Table 1). Survival
in the absence of SM ␣-actin may result from other
actin isoforms partially substituting for this isoform
(19). Since SM ␥-actin is the predominant actin
isoform in tissues from the gastrointestinal and urogenital tracts (7, 13), it is not surprising that loss of
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SCHILDMEYER ET AL.
SM ␣-actin in these tissues appears to have no effect
on a gross level.
The first phenotypic difference discerned between
wild-type and SM ␣-actin null mice (Fig. 2) was
clearly revealed upon histological examination of
muscular arteries and arterioles. Cross-sectioned wt
vessels showed a highly contracted, ruffled and or
puckered like appearance whereas comparably
stained mutant aorta appeared smooth. Electron
micrographs also showed disarrayed and reduced
myofibrils in SM ␣-actin null ⫺/⫺ vessels in comparison to wt tissue sections. Large differences in vascular contractility was observed in aortic ring assays in
which aortic segments from mutant mice (Fig. 3)
showed essentially no response to KCl depolarization, revealing a fundamental deficit in the vascular
smooth muscle contractile system of the SM ␣-actin
null mouse. Blood pressure is thought to be associated with the contractile state or mass of vascular
smooth muscle by the arterial side of vasculature in
which luminal narrowing serves to increase the total
peripheral resistance (31). We observed that the
mean systolic blood pressure in adult conscious mice
SM ␣-actin null mice was significantly reduced by
28% when compared to wt littermates in all five pairs
examined (P⬍0.05, Table 2). As shown in Fig. 4,
infusion of sodium nitroprusside in SM ␣-actin null
littermates had impaired vasodilatation and vasoconstriction activities when infused with sodium nitroprusside, as demonstrated by the inability to expeditiously reduce and/or raise blood pressure.
Additional in vivo studies, shown in Fig. 5, also
indicated a diminished peripheral blood flow in
response to thermal challenge. Therefore, mice lacking the SM ␣-actin demonstrate compromised vascular responses as well as reduced basal blood pressure,
which may make them more susceptible to cardiovascular stress.
This study and others (18, 19, 32) indicate that
smooth and striated muscular tissues have the capacity to sense and respond to dramatic changes in
either the level of contractile proteins and or contractile activity. Evaluation of the expression of muscle ␣-actin genes in SM ␣-actin null mice showed that
cardiac ␣-actin gene activity was not influenced by
the SM ␣-actin-defective gene, as shown by its identical mRNA levels in wt and mutant striated muscle
tissues. Whereas cardiac and skeletal ␣-actin genes
are silent in normal vascular smooth muscle tissues,
skeletal ␣-actin RNA was up-regulated in the majority
of SM ␣-actin ⫺/⫺ aortas examined. Apparently,
up-regulation of skeletal ␣-actin gene expression in
the aorta of the mutant mice compensates to some
degree for the loss of the SM ␣-actin. In addition,
cardiac ␣-actin knockout mice have been described
(19) in which increased expression of skeletal ␣-actin
and reactivation of the SM ␣-actin were observed in
SMOOTH MUSCLE ␣-ACTIN
the hearts of newborn homozygous mutants. Increased levels of these other myogenic ␣-actins were
insufficient to maintain myofibrillar integrity in response for the loss of the ␣-cardiac actin, and most of
these cardiac ␣-actin null mice do not survive to
birth. Although mice lacking cardiac ␣-actin can be
rescued up to adulthood by the ectopic expression of
enteric smooth muscle ␥-actin using the cardiac
␣-myosin heavy chain promoter, the hearts of these
rescued cardiac ␣-actin-deficient mice are extremely
hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenic expressed enteric smooth muscle ␥-actin reduces cardiac contractility in wild-type and heterozygous mice. The
activation and substitution of alternative actin isoforms are insufficient to correct the deficit of striated
and SM actins, supporting specialized functions of
these actin isoforms. In this regard, the SM ␣-actin
null mouse may also serve as a model to further
investigate the role SM ␣-actin plays in wound repair
(33, 34), certain types of cancer (35–36), liver disease (37), and kidney disease (38, 39).
In conclusion, the complete absence of SM ␣-actin
transcripts and protein did not appear to disrupt the
formation of the cardiovascular system. However, we
found compromised vascular contractility, lowered
blood pressure, and reduced blood flow in SM
␣-actin null mice. Unexpectedly, skeletal ␣-actin
expression was activated in the vasculature of these
null mice, demonstrating greater flexibility in the
genetic reprogramming of vascular smooth muscle
tissues than previously imagined.
The authors acknowledge Dr. Joseph Miano, Dr. David
Johnson, and Dr. Charles Seidel for reagents and advice, and
Sandra Rivera, Thuy Pham, Davide Franceschini, and Evelyn
Brown for expert technical assistance. This work was supported by an American Heart Grant to M.D., The Methodist
Hospital Foundation Grant and a Chao Fellowship to A.R.B.,
and the Michael DeBakey Foundation and National Institutes
of Health grants to R.J.S.
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The FASEB Journal
Received for publication October 26, 2000.
Revised for publication May 16, 2000 .
SCHILDMEYER ET AL.