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INTRODUCTION |
Sarcomeres, the functional units of striated muscle, are composed
of >20 known proteins, precisely organized in a nearly crystalline fashion. Mechanisms by which these proteins are assembled remain largely unknown. The genetic approach of inactivating genes encoding specific sarcomeric proteins has been used to understand the role of
stoichiometry in sarcomeric assembly. One important issue is the gene
dosage effect of these proteins on muscle organization and function.
Drosophila heterozygous mutants with one functional copy of
either actin or myosin heavy chain
(MHC)1 gene have
dysfunctional flight muscle, cannot fly, and have abnormal sarcomeric
structure (1). Heterozygous mutants display a 50% reduction in either
actin or MHC protein, respectively. Interestingly, the sarcomeric
structure of muscle from double heterozygous mutants with single copies
of both actin and MHC genes is less abnormal than muscle from either of
the single heterozygous mutants. Consequently, double heterozygotes are
capable of weak flight (1). These data indicate that variations in
sarcomeric protein stoichiometry can affect myofibrillar organization
and function. Recently, similar studies have been performed in mouse
knockout models. Heterozygous
-MHC knockout mice have ~50 and 25%
reductions in
-MHC mRNA and protein, respectively, compared with
wild-type littermates. Heterozygous mice have impaired cardiac function
and abnormal sarcomeric organization (2). On the other hand,
heterozygous
-tropomyosin knockout mice display a 50% reduction in
mRNA but similar levels of
-tropomyosin protein compared with
wild-type littermates and have no detectable cardiac abnormalities (3, 4). However, to date, no studies have been performed with these mutant
mice to examine the consequences of the heterozygous state on cardiac
function under conditions that induce hypertrophy.
Myosin light chain 2 (MLC-2), the regulatory myosin light chain, plays
an essential role in vertebrate smooth muscle contraction (5-7). In
vertebrate striated muscle, MLC-2 is thought to have only a modulatory
effect on contraction (8-11). However, a recent gene targeting study
has revealed a selective requirement for the ventricular specific
isoform of mlc-2 (mlc-2v) in embryonic murine
heart function (12). Homozygous mlc-2v mutant embryos die at
approximately embryonic day 12.5. The null mutants have a significantly
reduced left ventricular ejection fraction. Ultrastructural analysis
revealed defects in sarcomeric assembly. These data indicate that
mlc-2v plays an important role in cardiac function and
myofibrillogenesis during mammalian heart development. Moreover, it has
been shown that missense mutations in the human MLC-2v gene
result in a rare form of hypertrophic cardiomyopathy (13). However,
there are no data to distinguish whether the disease results simply
from "loss" of function (gene dosage) or an alternative mechanism.
Drosophila has a single mlc-2 gene, and
homozygous mlc-2 mutants are embryonic lethal. Heterozygous
mutants can survive, but cannot fly due to dysfunctional flight muscles
that display abnormal sarcomeric structure (14). These heterozygous
mutants display a 50% reduction in mlc-2 mRNA with a
25% reduction in MLC-2 protein compared with wild-type lit4termates.
To determine whether mlc-2v exhibits a similar gene dosage
effect in mammalian hearts, we have performed in-depth analyses of
heterozygous mlc-2v mutant mice.
Furthermore, we created the original mlc-2v knockout mice
(12) by a knock-in of Cre recombinase into the endogenous
mlc-2v locus to achieve two goals: 1) to understand the
physiological importance of mlc-2v in mammalian hearts and
2) to use the heterozygous mlc-2v cre knock-in
mice for ventricular cardiomyocyte-specific gene targeting, as
described by Chen et al. (15). In this report, we
demonstrate that heterozygous mlc-2v cre knock-in
mice are indistinguishable from wild-type mice in all aspects and
display normal levels of MLC-2v protein despite a 50% reduction in
mRNA. Thus, we have validated the potential utility of the
mlc-2v cre knock-in system as a valuable approach
for ventricular restricted gene targeting.
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MATERIALS AND METHODS |
Generation of mlc-2v Knockout/cre Knock-in Mice--
The
mlc-2v targeting construct has been described elsewhere
(12). By homologous recombination, Cre recombinase cDNA was placed
into the endogenous mlc-2v locus. This study used the
heterozygous mlc-2v mutants, which have one copy of Cre
recombinase cDNA in the mlc-2v locus.
Echocardiographic Analysis--
Mice were anesthetized with 20 ml of 2.5% Avertin/kg of body weight given intraperitoneally, and
transthoracic echocardiography was performed before and 7 days after
transverse aortic constriction (TAC) as described in detail elsewhere
(16).
Induction of Pressure Overload Cardiac Hypertrophy--
Pressure
overload was produced in mice at 8 weeks of age by TAC as described
previously (17). At 7 days following surgery, the gradient of the
arterial blood pressure between the constriction was measured. Six
heterozygous mlc-2v mutants and six wild-type mice showing
an adequate pressure gradient (>25 mm Hg) were subjected to further studies.
Northern Blot Analysis--
Ten micrograms of total RNA from
adult left ventricles was used for Northern blot analyses. To
characterize the expression of mlc-2v and the atrial isoform
of MLC-2 (mlc-2a), the same probes described previously (18)
were used. Expression of atrial natriuretic factor and skeletal
-actin mRNAs was examined as a molecular marker for hypertrophy.
Western Blot Analysis--
Five micrograms of myofibrillar
protein extracts from left ventricles (19) was electrophoresed on
12.5% SDS-polyacrylamide gels and either stained with Coomassie Blue
or subjected to Western blotting analysis. Anti-MLC-2v and anti-MLC-2a
polyclonal antibodies have been described previously (18).
Myocyte Shortening Measurement (Edge Detection)--
Ventricular
myocytes were prepared from the left ventricles of heterozygous
mlc-2v and wild-type littermates at 10 weeks of age. The
mouse heart was excised, and the aorta was cannulated and mounted onto
a Langendorff perfusion apparatus. The heart was perfused for 4 min in
modified Joklik's minimal essential medium (Life Technologies, Inc.)
consisting of 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM
Na2HPO4, 1.2 mM MgSO4,
12 mM NaHCO3, 20 mM
D-glucose, 10 mM HEPES, 30 mM
taurine, 2 mM creatine, and 2 mM carnitine (pH
7.4). Perfusion was then switched to a modified Joklik's minimal
essential medium plus 0.1% (w/v) collagenase type II (Worthington).
After 15 min of enzymatic digestion, the atria and right ventricle were
removed; the left ventricle was cut into several pieces; and the cells
were dispersed by gentle agitation. Myocytes were filtered and washed,
and 1 mM calcium was reintroduced gradually to the cells.
Isolated cardiomyocytes were transferred to a temperature-controlled
perfusion chamber (HCB-101, Crescent Electronics) located on the stage
of an inverted microscope (Nikon Diaphot-TMD). Cell shortening was
measured using a video edge motion detector (Crescent Electronics)
interfaced to a standard CCD camera (Philips Technologies), which was
attached to the microscope. A 40× Nikon phase II objective was used.
Cells were stimulated at 0.5 Hz with a 5-ms pulse duration by a BMS 414 electrostimulator (Crescent Electronics). Edge detection measurements
were performed on cardiomyocytes at 22 °C in continuously circulating Tyrode's solution on cells. The concentration of calcium in Tyrode's solution was subsequently changed from 1 to 2 to 4 mM.
Myosin ATPase Assay--
Fresh myosin preparations were obtained
from each group of left ventricles by the method described previously
(20). Ca2+-ATPase, K+-EDTA-ATPase, and
actin-activated ATPase activities in myosin preparations were
determined using the method of Ikebe and Hartshorne (21) with some
modification. Ca2+-ATPase activity was measured at 23 °C
in 0.1 mg/ml myosin, 1 mM ATP, 10 mM
CaCl2, 50 mM Tris-HCl (pH 7.5), and 50 mM KCl. K+-EDTA-ATPase activity was measured at
23 °C in 0.1 mg/ml myosin, 1 mM ATP, 10 mM
EDTA, 50 mM Tris-HCl (pH 7.5), and 0.6 M KCl. Actin-activated ATPase activity was measured at 23 °C in 0.1 mg/ml myosin, 1 mM ATP, 6 mM MgCl2, 0.5 mM EGTA, 20 mM imidazole HCl (pH 7.5), and 50 mM KCl with or without 1 mg/ml F-actin.
Statistical Analysis--
Data are indicated as means ± S.E. Statistical comparisons between the wild-type mice and
mlc-2v mutants were done by unpaired Student t
test. Echocardiographic data before and after transverse aortic
constriction were compared by paired t test. A p
value of <0.05 was considered significant.
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RESULTS |
Characterization of Heterozygous mlc-2v Mutant Mice--
Our
previous study demonstrated that homozygous
mlc-2v
/
offspring die at ~12.5 days post
coitum due to cardiac dysfunction, indicating that mlc-2v is
an essential component of the myocardial contractile apparatus (12).
Heterozygous mlc-2v mutants can survive a full life span and
reproduce normally. We performed an in-depth analysis of heterozygous
mlc-2v mutants. There were no significant differences in
heart/body weight ratios between wild-type mice (4.24 ± 0.34, n = 13) and mlc-2v mutants (4.20 ± 0.33, n = 13). Histologically, heterozygous
mlc-2v mutants displayed normal sarcomeric assembly in the
ventricular wall (data not shown), although our previous study
demonstrated that homozygous mlc-2v mutants exhibit a
disrupted myofibrillar organization during cardiogenesis (12).
Post-transcriptional Regulation of MLC-2v
Expression--
Quantitative Northern blot analysis indicated that the
level of mlc-2v transcripts in ventricles of adult
heterozygous mlc-2v mutant mice decreased ~50% compared
with wild-type littermates (Fig.
1A). However, the amount of
MLC-2v protein in ventricular myofibrillar preparations of both
wild-type mice and heterozygous mlc-2v mutants was
indistinguishable when corrected for the amount of protein loaded by
using amounts of tropomyosin as a standard (Fig. 1B).

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Fig. 1.
RNA and protein analyses in heterozygous
mlc-2v mutants and wild-type littermates.
A, Northern blot analysis of adult (2-month-old) ventricular
RNA. Ten micrograms of total RNA was used for each sample.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
an internal control. B, myofilamentous proteins from adult
(2-month-old) ventricles were analyzed by SDS/glycerol-polyacrylamide
gel electrophoresis and stained with Coomassie Blue. Five micrograms of
protein was loaded in each lane. TM, tropomyosin;
TNI, troponin I.
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To examine whether the regulation of mRNA and protein expression
would be modified by cardiac hypertrophic stimuli, we compared the
levels of mlc-2v mRNA and protein following induction of
hypertrophy by TAC. Both prior to and following TAC, heterozygous
mlc-2v mutants exhibited normal levels of MLC-2v protein
(Fig. 2) and a 50% reduction in mRNA
(Fig. 3) relative to wild-type
littermates as revealed by SDS-polyacrylamide gel electrophoresis
and Northern blot analyses.

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Fig. 2.
Protein analysis before and after TAC.
Myofilamentous proteins from ventricles before and after TAC were
analyzed by SDS/glycerol-polyacrylamide gel electrophoresis and stained
with Coomassie Blue. Five micrograms of protein was loaded in each
lane. Sham, sham operation; TM, tropomyosin;
TNI, troponin I.
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Fig. 3.
RNA analysis before and after TAC.
Ventricular mRNA was analyzed by Northern blotting before and after
TAC. Ten micrograms of total RNA was used for each sample.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
an internal control. Sham, sham operation; ANF,
atrial natriuretic factor.
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In addition to MLC-2v, MLC-2a is abundantly expressed in the
ventricular chamber at the earliest stages of murine cardiogenesis (18). Our previous studies showed that MLC-2a protein content in the
ventricular chamber is dramatically increased in mlc-2v null
mutant embryos at 12 days post coitum, although there are no
significant differences in the mlc-2a mRNA levels
between wild-type and homozygous mlc-2v mutant embryos (12).
Therefore, we examined the expression of MLC-2a protein in ventricles
from adult heterozygous mlc-2v mutants by Western blot
analysis. No detectable MLC-2a protein was found in ventricles of
wild-type mice or heterozygous mlc-2v mutants before or
after TAC (data not shown).
Changes in Molecular Markers of Hypertrophy--
To elucidate
whether there are differences in expression of hypertrophic response
genes in wild-type mice and heterozygous mlc-2v mutants, we
examined the expression of atrial natriuretic factor and skeletal
-actin mRNAs following TAC. Hypertrophic stimuli resulted in
comparable increases in expression of atrial natriuretic factor and
skeletal
-actin mRNAs in both groups. Fig. 3 shows a
representative Northern blot that examined atrial natriuretic factor expression.
Examination of Global Cardiac Function by
Echocardiography--
Cardiac function was evaluated noninvasively by
echocardiography prior to and following TAC. Under basal conditions,
all echocardiographic parameters were equivalent in both groups (Table
I). Similar pressure gradients between
the aortic constriction were produced in both groups (Table
II). Left ventricular weight/body weight ratios were also similar in both groups following TAC, confirming that
the amount of hypertrophic stimulus was comparable for each group. In
both groups, left ventricular wall thickness and percent fractional
shortening increased in response to TAC. These data indicate that
global cardiac function and response to hypertrophic stimuli are
unaffected in heterozygous mlc-2v mutants.
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Table I
Echocardiographic measurements under basal conditions
All data are presented as means ± S.E. BW, body weight; HR, heart
rate; LVEDD, end-diastolic left ventricular dimension; LVESD,
end-systolic left ventricular dimension; PWth, left ventricular
posterior wall thickness; IVSth, interventricular wall thickness;
LV%FS, left ventricular percent fractional shortening; Vcf, velocity
of circumferential fiber shortening. bpm, beats/min; circ,
circumference.
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Table II
Comparative echocardiographic measurements before and after induction
of hypertrophy by TAC
See the legend to Table I for details and definitions of abbreviations.
LVW, left ventricular weight; PG, pressure gradient. Data
comparison was carried out before and after TAC in either group. bpm,
beats/min; circ, circumference.
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Mechanical Properties of Ventricular Cardiomyocytes Remain Normal
in Heterozygous mlc-2v Mutant Mice--
To characterize contractile
properties of cardiac myocytes isolated from wild-type mice or
heterozygous mlc-2v mutants, unloaded contractile motion was
monitored. Cardiac myocytes isolated from heterozygotes or wild-type
littermates were found to be morphologically indistinguishable.
Unloaded contractile motion was normalized to resting cell length.
Table III shows that percent cell
fractional shortening,
dL/dt, and
+dL/dt were comparable in wild-type and heterozygous mlc-2v mutant cells at various extracellular
calcium concentrations. Additionally, Ca2+-ATPase,
K+-EDTA-ATPase, and actin-activated ATPase activities in
myosin preparations isolated from wild-type mice and heterozygous
mlc-2v mutants displayed no statistical difference (Table
IV). These data therefore suggest that
mechanical properties of myocytes in heterozygous mlc-2v
mutants are indistinguishable from those in wild-type littermates.
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Table III
Ventricular myocyte mechanical properties with changes of calcium
concentration
All data are presented as means ± S.E. Note that measurements
were performed on nine ventricular myocytes from three wild-type mice
and on ten ventricular myocytes from three heterozygous mice.
dL/dt, the rate of shortening;
+dL/dt, the rate of relengthening.
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Table IV
ATPase activities of heterozygous mlc-2v mutants and wild-type
littermates
All data are presented as means ± S.E. Measurements were done on
six independent myosin preparations from the left ventricles in each
group.
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DISCUSSION |
There were two main goals for this study: 1) to determine whether
mlc-2v exhibits a gene dosage effect in mammalian heart and
2) to evaluate the validity of utilizing mlc-2v
cre knock-in mice for ventricular specific gene targeting.
Our previous study revealed that mlc-2v null mutants are
embryonic lethal at ~12.5 post coitum due to cardiac dysfunction and
exhibit abnormal sarcomeric assembly (12), which suggests that
mlc-2v plays an essential role in cardiac function and
myofibrillogenesis during development. However, this study has shown
that, although heterozygous mlc-2v mice display a 50%
reduction in mlc-2v mRNA levels, they exhibit no
reduction in endogenous MLC-2v protein levels. Heterozygous mice have
neither a molecular nor a physiological cardiac phenotype either at
base line or following hypertrophic stimuli. These results suggest
that, due to post-transcriptional regulation, a single mlc-2v allele is sufficient to produce normal levels of
MLC-2v protein and to maintain normal sarcomeric function and
development in mouse heart. In addition, our results provide the first
evidence that this post-transcriptional regulation can operate
following induction of cardiac hypertrophy. In a transgenic approach by other investigators, a 10-fold overexpression of mlc-2v
mRNA in murine ventricle failed to increase expression of MLC-2v
protein (22). Thus, it appears that mlc-2v is strictly
regulated at the post-transcriptional level in murine heart.
A similar post-transcriptional regulatory mechanism has been observed
for
-tropomyosin, where overexpression of
-tropomyosin mRNA
or the presence of a single
-tropomyosin allele does not affect
normal levels of
-tropomyosin protein in murine heart (3, 4). These
data may suggest that haploinsufficiency is not a causative mechanism
for human hypertrophic cardiomyopathies that result from mutations in
MLC-2v (13) or in
-tropomyosin (23). However, whether
similar post-transcriptional regulation of these myofibrillar proteins
is operative in human heart is currently unknown. In contrast to these
results with MLC-2v and
-tropomyosin, heterozygous
-MHC knockout
mice, in which cardiac function is impaired, display ~50 and 25%
reductions in
-MHC mRNA and protein, respectively. These results
suggest that two MHC alleles are necessary and that
post-transcriptional regulation cannot maintain normal protein levels
in murine hearts (2). Another compensatory mechanism has been
demonstrated in cardiac
-actin knockout mice. Cardiac
-actin
heterozygous mutants exhibit increased expression of various actin
isoforms to compensate for reduced cardiac
-actin protein expression
(24). In heterozygous mlc-2v mutants, this type of
compensatory mechanism involving other isoforms does not seem to be
activated, as our data showed that there was no detectable MLC-2a
protein in adult ventricles of heterozygous mlc-2v mutants.
These results indicate that post-transcriptional controls play a major
role in determining overall sarcomeric protein stoichiometry under
conditions of increased or decreased mRNA levels.
There is only one mlc-2 gene in Drosophila.
Interestingly, heterozygous mlc-2 mutants can survive but
cannot fly due to dysfunctional flight muscles, which display abnormal
sarcomeric structure. Heterozygous mutants display a 50%
reduction in mlc-2 mRNA with a 25% reduction in MLC-2
protein (14). Therefore, mlc-2 exhibits a gene dosage effect
in Drosophila indirect flight muscle. Similar results have also been shown in Drosophila heterozygous mutants with one
functional copy of either actin or MHC gene (1). These data suggest
that mechanisms regulating myofibrillar protein expression are specific for each gene locus in a species-dependent manner.
Gene targeting and transgenesis are strong tools for understanding the
in vivo function of a gene of interest. However, a disrupted
gene sometimes results in embryonic lethality, which makes it difficult
to undertake further studies, particularly for genes associated with
heart development (15, 25). Moreover, conventional gene targeting may
not determine whether a given phenotype is due to gene ablation in a
particular cell type, if the targeted gene is expressed in diverse cell
types. Conditional knockout and transgenesis strategies can overcome
these limitations (26). mlc-2v knockout mutants were created
by a knock-in strategy to insert the Cre recombinase coding sequence
into the endogenous mlc-2v locus (12). The mlc-2v
gene is the earliest marker of ventricular specification during murine
cardiogenesis. Thus, the mlc-2v cre knock-in mice
can be used for ventricular specific conditional gene targeting.
Recently, we achieved ventricular cardiomyocyte-specific gene targeting
of the retinoid X receptor
gene by crossing heterozygous
mlc-2v cre knock-in mice with mice homozygous for
a "floxed" retinoid X receptor
gene (15). Our laboratory and
others have been creating other ventricular specific knockout mice by
crossing our mlc-2v cre knock-in mice with
various mouse lines containing floxed alleles of interest (27).
Therefore, it is of utmost importance to fully characterize the
heterozygous mlc-2v cre knock-in mice, as all of
these conditional knockout mice will be heterozygous at the
mlc-2v locus. This study has demonstrated that
mlc-2v cre knock-in mice do not exhibit apparent
cardiac phenotypes either at base line or following a hypertrophic
stimulus. Thus, we conclude that the mlc-2v cre
knock-in system is a valid approach for further ventricular specific
knockout strategies to explore molecular mechanisms of cardiac
hypertrophy and heart development.