Selective Requirement of Myosin Light Chain 2v in Embryonic Heart
Function*
Ju
Chen
,
Steven W.
Kubalak§,
Susumu
Minamisawa
,
Robert L.
Price¶,
K. David
Becker
,
Reed
Hickey
,
John
Ross Jr.
, and
Kenneth R.
Chien
**
From the
Department of Medicine and ** Center for
Molecular Genetics, University of California at San Diego, School of
Medicine, La Jolla, California 92093-0613, and § Department
of Cell Biology & Anatomy, Medical University of South Carolina,
Charleston, South Carolina 29425-2204, and ¶ Department of
Developmental Biology and Anatomy, School of Medicine, University of
South Carolina, Columbia, South Carolina 29208
 |
ABSTRACT |
Two major myosin light chain 2 isoforms are
coexpressed in the early stages of murine cardiogenesis, a cardiac
ventricular isoform and a cardiac atrial isoform, each of which is
tightly regulated in a muscle cell-type-specific manner during
embryogenesis (Chien, K. R., Zhu, H., Knowlton, K. U.,
Miller-Hance, W., van Bilsen, M., O'Brien, T. X., and Evans,
S. M. (1993) Annu. Rev. Physiol. 55, 77-95). We have
disrupted myosin light chain 2v gene in mice and monitored in
vivo cardiac function in living myosin light chain 2v
/
embryos. The mutant embryos die at approximately embryonic day 12.5. In
mutant ventricles, the myosin light chain 2a protein level is increased
and reaches levels comparable to the myosin light chain 2v in the
ventricles of wild type littermates and is appropriately incorporated
into the thick filaments of mutant embryonic hearts. However,
despite the substitution of myosin light chain 2a,
ultrastructural analysis revealed defects in sarcomeric assembly
and an embryonic form of dilated cardiomyopathy characterized by a
significantly reduced left ventricular ejection fraction in mutant
embryos compared with wild type littermates. We conclude that myosin
light chain 2v may have a unique function in the maintenance of cardiac
contractility and ventricular chamber morphogenesis during mammalian
cardiogenesis and that a chamber-specific combinatorial code for
sarcomeric assembly may exist that ultimately requires myosin light
chain 2v in ventricular muscle cells.
 |
INTRODUCTION |
Muscle myosin, the highly conserved molecular motor, contains one
pair of heavy chains and two pairs of light chains, the essential
myosin light chains (MLC-1 or
-3)1 and the regulatory
myosin light chain-2 (MLC-2) (1). The three-dimensional structure of
myosin indicates that the light chains are arranged in tandem, with
MLC-1/-3 in the amino-terminal half of the neck and MLC-2 in the
neck/tail junction (2-4). MLC-2 plays an essential role in regulating
vertebrate smooth muscle contraction. The phosphorylation of a single
serine residue (Ser-19) of smooth muscle MLC-2 is the switch for
turning on the actin-activated myosin ATPase and hence, contraction.
However, the acto-myosin interaction in vertebrate striated muscle is
mainly regulated through the troponin-tropomyosin complex, and MLC-2 is
thought to have only a modulatory effect (5). It has been shown in an
in vitro motility assay that removal of MLC-2 from myosin
markedly reduces actin filament sliding velocity without significantly
reducing myosin ATPase activity (6, 7). Although mutation of the single
MLC-2 gene results in a flightless phenotype in
Drosophila (8), the precise in vivo physiological function of myosin light chains in vertebrate striated muscle is
unclear. Furthermore, there is no direct evidence that the highly
conserved structure of individual MLC-2 isoforms reflects a unique
functional requirement in distinct muscle cell types. Recently,
alterations in MLC-2 expression have been correlated with
the onset of cardiac morphogenetic defects during embryogenesis (9-11), point mutations in MLC-2v have been shown to be
associated with a genetic form of human cardiomyopathy (12), and
induction of the atrial MLC-2 isoform has been shown to occur in
cardiac hypertrophy and failure (13). Our efforts have been directed toward understanding the biological and physiological roles of the
MLC-2v, which is the ventricular isoform of MLC-2, by disrupting the
gene through homologous recombination in mice.
 |
MATERIALS AND METHODS |
Gene Targeting--
A 12-kilobase genomic mlc-2v
fragment was isolated from a mouse 129svj genomic DNA library
(Stratagene, La Jolla). PCR-based mutagenesis was used to convert the
1.4-kilobase pair fragment between three base pairs 5
of translational
start codon ATG and intron 2 BamHI site into an
XhoI site (see Fig. 1a). The deleted 1.4-kilobase
fragment contained part of exon 1, intron 1, exon 2, and part of intron
2. Cre recombinase cDNA (15), internal ribosomal entrance sequence
(16), green fluorescent protein cDNA (17), and pGKneo-tk cassettes were
inserted into the XhoI site. The linearized construct was
electroporated into J1 ES cells. G418-resistant ES clones were screened
for homologous recombination by Southern blotting with probe A, B, C. Chimeric males were tested for germ-line transmission of the agouti
coat phenotype of 129-derived ES cells by crossing with Black Swiss
female breeders. For PCR analysis, oligonucleotides for Cre cDNA
(Cre1, 5
-GTTCGCAAGAACCTGATGGACA-3
; Cre2,
5
-CTAGAGCCTGTTTTGCACGTTC-3
) and the mlc-2v gene
(MLC2v-P1, 5
-GGCAACTGGCCTCAGACACCAT-3
;
MLC2v-P4, 5
-TGTGGAGCCTCTGGATCAGGAC-3
) were used. Total
protein and myofibrillar protein extracts were prepared (18) from
single day 12.0 embryonic heart ventricles, of which 25% were
subjected to Western blotting analysis with MLC-2v and MLC-2a
polyclonal antibodies and tropomyosin monoclonal antibody.
Histological and Immunohistochemical Analysis--
Histological
and immunohistochemical studies were performed by a modification of
previously described methods (19, 20, 21).
Transmission Electron Microscopy--
Embryos were processed for
transmission electron microscopy as described by Price et
al. (22) and Reynolds (23).
In Vivo Videomicroscopy of Embryos--
Mice pregnant at E11.5
and 12.5 were intraperitoneally anesthetized with ketamine (100 mg/kg)
and xylazine (2.5 mg/kg), the trachea was intubated, and ventilation
was provided with a respirator. Fetuses were visualized using
intravital microscopy (11, 24), but with images recorded in color from
a charge-coupled device camera, as recently described (24). In brief,
each embryo was delivered individually from the uterus with the
umbilical circulation intact, placed on a heated stage with
superfusion, and imaged by transillumination. The left ventricular
chamber area (including the left ventricular wall, which is thin at
this age) and the chamber long axis were measured using NIH IMAGE
software. Left ventricular (LV) elliptical volumes were then calculated
using the single plane area-length method (25). The LV ejection
fraction was calculated as (LVEDV
LVESV)/LVEDV, where LVEDV is
the left ventricular end diastolic volume and LVESV is the end systolic volume.
RNase Protection Assay--
The riboprobes for RNase protection
assays were generated as described previously (26). Ventricular samples
were dissected from individual day 12 embryonic hearts, and RNase
protection assays were performed per manufacturer recommendations using
the Direct Protect Lysate Ribonuclease protection assay kit from
Ambion, Inc. (Austin, TX).
 |
RESULTS AND DISCUSSIONS |
Lack of MLC-2v Results in Embryonic Lethality at E12.5--
The
mlc-2v gene targeting construct is depicted in Fig.
1. The linearized targeting construct was
electroporated into J1 ES cells, and the cells were selected with G418.
Of a total of 47 colonies screened, 3 were identified as homologous
recombinants by Southern blot hybridization analysis of genomic ES cell
DNA. Two of the three colonies were injected into C57BL/B6 blastocysts, both resulting in the generation of chimeras, one of which displayed germ-line transmission. Heterozygous (MLC-2v+/
) offspring appeared normal in all respects and were crossed to generate MLC-2v homozygote (MLC-2v
/
) mice. Southern blot and PCR analyses were used to genotype offspring (Fig. 1, b and c). The absence
of MLC-2v proteins in MLC-2v
/
embryos was confirmed by Western
blot analysis, utilizing a polyclonal anti-MLC-2v antibody (Fig.
1d). No viable MLC-2v
/
offspring were obtained in
litters from MLC-2v +/
intercrosses (Table
I), indicating that the MLC-2v
/
null
mutation was embryonic lethal. To investigate the timing of the
lethality, mice from E9.5 to 13.5 were genotyped. All of the MLC-2v
/
mutants were viable at E11.5; however, 30% of MLC-2v
/
mutants lost viability by E12.5, and all MLC-2v
/
died by E13.5
(Table I), thereby indicating that the MLC-2v
/
embryos die at
approximately E12.5.

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Fig. 1.
Targeting the MLC-2v gene.
a, targeting strategy. A restriction map of
mlc-2v genomic region of interest is shown on top, the targeting construct is shown in the
center, and the mutated locus after homologous recombination
is shown at the bottom. ATG is the translational start site.
The arrow indicates the primers (MLC-2v-P1, MLC-2v-P4, Cre1,
and Cre2) used for PCR analysis of genomic DNA. B,
BamHI; E, EcoRI; H,
HindIII; S, SstI; X,
XbaI; X*, methylated XbaI site in
bacterial host strain. Kb, kilobases. IRES,
internal ribosomal entrance sequence, GFP, green fluorescent protein. b, detection of MLC-2v wild type (+) and mutant
( ) alleles by Southern blot analysis. DNAs from 11.5 days post-coitum
embryos derived from an intercross between heterozygote mice were
digested with XbaI and analyzed by Southern blot with probe
A. c, detection of wild type (+) and mutant ( ) alleles by
PCR analysis. DNAs used for Southern blot analysis were subjected for
PCR with oligonucleotides for the mlc-2v gene (MLC2v-P1,
5 -GGCAACTGGCCTCAGACACCAT-3 ; MLC2v-P4, 5 -TGTGGAGCCTCTGGATCAGGAC-3 ) and the Cre cDNA (Cre1,
5 -GTTCGCAAGAACCTGATGGACA -3 ; Cre2, 5 -CTAGAGCCTGTTTTGCACGTTC-3 ).
d, detection of MLC-2v protein by Western blot analysis.
Myofilament protein prepared from day 12 embryonic heart ventricles
were analyzed with anti-MLC-2v polyclonal antibody and anti-tropomyosin
C monoclonal antibody. Genotype designations are +/+, wild type; +/ ,
heterozygous; / , homozygous.
|
|
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Table I
Offspring of MLC-2v heterozygous crosses
Heterozygous mice were crossed, and offspring at different embryonic
stages and new born were genotyped. The number of dead embryos is given
in parentheses.
|
|
MLC-2v
/
Embryos Die from Heart Failure--
All MLC-2v
/
embryos that died just before dissection displayed massive cardiac
enlargement, wall thinning, chamber dilation, and pleural effusions.
Histologic examination revealed hepatic congestion and an engorged vena
cava consistent with congestive heart failure (data not shown). No
significant abnormalities in other organs were found. Analysis of
global cardiac function revealed that the left ventricular ejection
fraction of MLC-2v
/
embryos averaged 33%, which was significantly
reduced when compared with wild type and heterozygous littermates at
E11.5 (46%, 49%) and at E12.5 (50%, 53%) (Fig.
2a). Left ventricular end
diastolic volumes were similar among the three groups at E11.5, but by
E12.5, were significantly elevated in MLC-2v
/
embryos (Fig.
2b). Collectively, these results suggest that global cardiac
function is already severely impaired by E11.5 in MLC-2v
/
embryos
and that cardiac dysfunction results in progressive embryonic heart
failure around E12.5. The resulting phenotype is similar to
mid-gestational embryonic heart failure seen in a wide variety of other
gene-targeted murine embryos (14).

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Fig. 2.
left ventricular end diastolic volumes
(a) and left ventricular ejection fractions in E11.5-12.5
embryos (b). End diastolic volume and ejection
fraction were calculated as described under "Materials and
Methods."
|
|
MLC-2v
/
Embryonic Hearts Display Abnormal Myofibrillar
Organization--
Ultrastructural analysis of comparable areas from
the left ventricular free walls of wild type and mutant mice revealed
abnormalities in sarcomeric assembly in the MLC-2v
/
embryos (Fig.
3). Myocytes from E12.5 MLC-2v
/
embryonic hearts displayed prominent interruptions and myofibrillar
disorganization of the normal parallel alignment of thick and thin
filaments (Fig. 3, b and c). Additionally, total fiber width was narrower within mutant ventricular myocytes, and overall distances between Z-bands was greater than in wild type specimens. There were also several areas in mutant hearts where the
organization and alignment of Z-bands between sarcomeres was not
conserved (Fig. 3c). These observations could also be
observed in ventricular samples from younger MLC-2v
/
embryos
(E10.5 and E11.5) during earlier stages of myofibrillogenesis (data not shown).

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Fig. 3.
Ultrastructural analysis of cardiomyocyte
architecture as assessed by transmission electron microscopy.
Representative images were taken from comparable areas of the left
ventricular free walls of E12.5 wild type (a) and MLC-2v
/ (b and c) embryos. Panels b and
c represent two different areas from the same embryo. Note
narrower fiber width, lack of Z-band integrity, and deviation from the
normal parallel nature of thick and thin filaments (arrows in panels b and c) in MLC-2v /
specimens.
|
|
Taken together, these data indicate that there is a selective
requirement for MLC-2v in the normal development of ventricular cardiac
myocyte structure and function. The observed disruption of myofibrillar
organization in the absence of MLC-2v leads to reduced myocyte
contractility and cardiac function that results in death at
approximately E12.5 and suggests a specific requirement of MLC-2v in
the interaction between thick and thin filaments during sarcomere
assembly. In this regard, in vitro motility assays have
previously shown that removal of MLC-2 reduces actin filament sliding
velocity by about 63% without significantly reducing the myosin ATPase
activity (6, 7). It has also been proposed that MLC-2 can act to
stiffen the myosin neck (3, 4). If each head of the thick filament is
responsible for the arrangement of proper hexagonal packing of the
thick filament and thin filament during myogenesis, then the
disorganized and improperly functioning myosin head due to the lack of
MLC-2v would result in improper packing and organization of thick and
thin filaments during cardiac sarcomere assembly.
MLC-2a Cannot Compensate for the Deficiency of MLC-2v--
Two
different myosin light chain 2 genes, mlc-2v and
mlc-2a, are abundantly expressed in the ventricular chamber
at early stages of murine cardiogenesis, raising the issue as to
whether there is a unique role for MLC-2v versus MLC-2a
during the maintenance of heart function and morphogenesis.
mlc-2v is initially expressed at about E8 and continues to
be restricted to the ventricular chamber throughout embryonic
development and into adulthood (27, 28). The expression of
mlc-2a is uniform in the E8 linear heart tube and then
becomes down-regulated at the RNA level in the ventricular chamber by
E12.5 (29). To investigate whether MLC-2a can compensate for MLC-2v in
mutant embryos, we examined MLC-2a mRNA and protein levels in
MLC-2v
/
embryos at E12. Although there were no significant differences in the MLC-2a mRNA levels between wild type and MLC-2v
/
embryos (Fig. 4a),
Western blot analysis (Fig. 4b) demonstrated that MLC-2a
protein content in the ventricular chamber was dramatically increased
in both total cellular protein and myofibril protein extracts of these
embryos. A quantitative study of myofilament proteins isolated from E12
embryonic ventricles indicated that levels of MLC-2a protein in MLC-2v
/
ventricles were comparable to the MLC-2v protein levels in wild
type litter mates (Fig. 4c). This result is consistent with
the findings in a transgenic mouse model in which overexpression of
cardiac MLC-2v does not result in an increase in ventricular MLC-2v
protein levels despite a significantly higher level of MLC-2v mRNA
in these transgenic mice (30). In both cases, the MLC-2 protein level
appears to be regulated at the post-transcriptional level. Thus, the
current study indicates that during early stages of cardiac chamber
development, MLC-2a protein levels increase in the ventricles of mutant
animals, implying that MLC-2a may partially replace MLC-2v in the
developing ventricular myocyte. In this regard, immunohistochemical
analysis by confocal microscopy of MLC-2a expression in MLC-2v
/
embryos demonstrated that MLC-2a protein was indeed elevated in the
ventricles of mutant embryos (Fig. 4g) and incorporated into
nascent myofibrils (Fig. 4i). However, MLC-2a cannot
completely compensate for the deficiency of MLC-2v, as shown by the
lack of sarcomeric structure (Fig. 3) and depressed cardiac
contractility (Fig. 2). We conclude that there is an important
qualitative difference between MLC-2v and MLC-2a proteins, reflecting a
unique requirement for MLC-2v during functional maturation of the
ventricular chamber, thereby underscoring the potential importance of
MLC-2 in the in vivo regulation of cardiac contractility.
Furthermore, these studies provide the first direct evidence that
alterations in MLC-2v can directly lead to overt heart failure, which
will become of interest to explore in the post-natal setting of the
adult heart.

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Fig. 4.
MLC-2v and MLC-2a mRNA and protein
analysis in wild type and MLC-2v / embryos. a, RNase
protection assay on isolated ventricles of E12 embryos using riboprobes
for MLC-2v, MLC-2a, and control probe EF-1a. b, Western blot
analysis of total proteins (lanes 1-4) and purified
myofilament proteins (lanes 5-8) from E12.0 embryonic
ventricles using a polyclonal antibody for MLC-2a and a monoclonal
antibody for tropomyosin. c, myofilamental proteins from E12
embryonic ventricle were analyzed by SDS-glycerol polyacrylamide gel
electrophoresis and stained with Coomassie Blue. d-i,
immunohistochemical analysis of MLC-2v (panels d and
f) and MLC-2a (panels e, g, h, and i)
in wild type (panels d, e, and h) and MLC-2v
/ embryos (panels f, g, and i) by confocal
microscopy. Panels h and i were taken from the
left ventricular free wall rough trabecular area, similar to that shown
in Fig. 3.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Frank A. Delano, Lan Mao, Jibin
Zhao, and Mahmand Itani for their technical assistance. Dr. Jenny
Price, Jun Zhao, and Julie Sheridan were invaluable for ES cell and
transgenic mouse work. We are grateful to Dr. Jim Lin for gift of
trypomyosin monoclonal antibody, Dr. Rudolf Jaenisch for providing J1
ES cells, and Drs. Sylvia Evans and Geir Christensen for critical
reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from NHLBI, National
Institutes of Health (to K. R. C.).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.
A recipient of an endowed chair awarded by the American Heart
Association, California affiliate, San Diego division.

To whom correspondence and requests for reprints should be
addressed: University of California at San Diego, School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0613. Tel.: 619-534-6835; Fax:
619-534-8081; E-mail: kchien{at}ucsd.edu.
1
The abbreviations used are: MLC, myosin light
chains; PCR, polymerase chain reaction; LV, Left ventricular.
 |
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