© 1997 American Heart Association, Inc.
Articles |
Manipulating Myosin Light Chain 2 Isoforms In Vivo
A Transgenic Approach to Understanding Contractile Protein Diversity
Correspondence to Dr Margaret Buckingham, CNRS URA 1947, Department of Molecular Biology, Pasteur Institute, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
Key Words: myosin light chain • transgenic mice • isoform diversity • editorial
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Introduction |
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 Top Introduction References |
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The evolution of the contractile apparatus of striated muscle, from cytoskeletal systems involved in cell motility to the specialized structure of the sarcomere, has been achieved largely by the diversification of contractile protein isoforms. Despite the distinct embryological origins and transcriptional regulatory pathways of skeletal and cardiac muscle, there is a considerable overlap in the contractile protein isoforms expressed in these two striated muscle types (see Reference 11 ). Isoform switching occurs during striated muscle development and mediates plasticity in adult skeletal and cardiac myocytes. Functional diversity in different muscle types is therefore largely the result of differential expression of interchangeable sarcomeric components.
Cardiac and skeletal muscle programs are finely tuned to the functional
requirements of these striated muscles. Forced activation of the
skeletal muscle program in myocardial cells of mice expressing
transgenes coding for skeletal muscle regulatory factors in the heart
leads to varying degrees of abnormal heart morphology and
cardiomyopathy, demonstrating at a gross level the
incompatibility of these programs for correct sarcomeric
function.2 3 There are few examples to date where specific
skeletal muscle contractile protein isoforms have been targeted to the
heart. In transgenic mice expressing skeletal troponin C (TnC) in the
myocardium, it has been shown that contractile sensitivity
to acidosis was reduced, identifying functional differences between TnC
isoforms.4 In a naturally occurring model, the BALB/c line
of inbred mice, a duplication upstream from the cardiac 
-actin gene
results in reduced cardiac -actin expression and abnormally high
levels of skeletal -actin in the adult heart.5
Overexpression of this skeletal muscle isoform correlates with
nonpathological increased myocardial
contractility,6 thus allowing functional
distinction between two isoforms that differ by only 4 of 375 amino
acids.
In a series of experiments using transgenic mice to remodel the contractile apparatus of the heart, Gulick et al7 in this issue of Circulation Research have addressed the in vivo significance of myosin light chain (MLC) 2 isoform diversity. This experimental approach opens up the possibility of expressing a high level of ectopic gene product in different cardiac compartments with the potential for partial or complete replacement of one myofibrillar protein isoform by another. Assessment of physiological changes in isolated working hearts of such transgenic animals provides new insights into a functional understanding of isoform diversity in vivo.
Virtually nothing is known about the functional differences between
sarcomeric MLC2 isoforms; indeed, it is only relatively recently that
their role in the sarcomere has begun to be elucidated (see Reference
88 ). Sarcomeric myosin provides the molecular motor for force
production, and each myosin hexamer is composed of two heavy
chains and four light chains, two essential MLC1/3 molecules, and two
regulatory MLC2 molecules. MLC2 plays an important regulatory role in
smooth and nonmuscle myosin, where Ca2+-dependent MLC2
phosphorylation regulates ATPase activity. In striated
muscle, the actomyosin interaction is regulated in a
Ca2+-dependent manner by the troponin/tropomyosin complex,
although some modulation of contractile activity via increased force
production at low levels of Ca2+ activation results
from MLC2 phosphorylation (see Reference 99 ). The most
striking mechanical effect of MLC2 is on the
physiological speed of shortening of the actomyosin
complex; removal of MLC2 significantly decreases the velocity of actin
movement on skeletal myosin.10 These indications of the
physiological role of the regulatory light chain
are complemented by structural studies, which have shown that the light
chains are associated with the myosin heavy chain (MHC) in the neck
region between the globular head and the extended coiled tail of the
molecule (see Reference 1111 ). The two classes of MLCs bind to highly
hydrophobic -helical regions and, together, stabilize the -helix;
indeed, some of the MLC2 mutants discussed below have disorganized
myofilaments, indicating the importance of this structural role.
At least three MLC2 isoforms are present in mammalian striated
muscle: MLC2F in fast skeletal muscle and MLC2V and MLC2A in the
ventricular and atrial compartments of the heart,
respectively. MLC2V is also the major isoform in slow skeletal muscle
fibers. The major MHC in the adult mouse heart is -MHC, which is
present in the atria in combination with MLC2A and in the
ventricles with MLC2V. -MHC is also present in certain skeletal
muscles, including the masseter muscle, and in muscle spindle fibers,
where it is probably associated with slow isoforms, including MLC2V
(see Reference 1212 ). MLC2V is also associated with ß-MHC in the heart
and in slow skeletal fibers; reshuffling of isoform types therefore
occurs in different adult muscles and during muscle
development.1 Species differences in isoform expression
patterns are an important facet of isoform diversity; eg, in the human
heart, -MHC remains restricted to the atria and ß-MHC is the major
form associated with MLC2V in the ventricles, whereas in the mouse this
corresponds to the fetal phenotype. Although expression of fast
myosin isoforms is not a feature of the myocardium, MLC2F
transcripts have been detected in embryonic mouse
hearts13 ; therefore, MLC2F is one of a group of fast
skeletal muscle transcripts transiently expressed in the developing
mouse heart.14 15
There are a number of experimental approaches that can be adopted
to investigate the functional fine-tuning that different MLC isoforms
probably effect. In particular, biochemical studies involving
extraction and replacement of sarcomeric components in isolated fibers
have already provided basic data on the effect of different classes of
MLC on contractility,16 as have in vitro
motility assays, which allow the rate of translocation of actin
filaments on immobilized myosin to be
measured.10 Until recently, there has been very little
opportunity to examine MLC function in vivo, although elegant studies
of the differences in intracompartmental sorting between alkali MLC
isoforms have been carried out in isolated
cardiomyocytes.17 An indication of the
importance of MLC2 in vivo has come primarily from the study of MLC2
mutations. The study of null and
phosphorylation-defective MLC2 alleles in
Drosophila indirect flight muscles has demonstrated key
roles for MLC2 in myofilament structure and in modulating
contractility via
phosphorylation.18 Rare human
cardiomyopathies that are the result of point
mutations close to the phosphorylation site of MLC2V
have been documented.19 In addition, cases of dilated
cardiomyopathy in humans have been shown to be
associated with a reduction of MLC2V levels, resulting from specific
protease-mediated cleavage of this light chain.20 Changes
in MLC2 isoform content between cardiac compartments have been
documented in abnormal hearts of mutant mice that lack the regulatory
protein Nkx2.521 or RXR.22 MLC2 is
therefore of significant pathological importance, although the in vivo
role of different MLC2 isoforms has remained obscure.
In the transgenic mice generated by Robbins and
colleagues,7 23 24 different MLC2 isoforms have been
expressed in the adult mouse heart using 5' regulatory sequences (-4.5
kb from the transcription start site) of the -MHC gene. The -MHC
upstream sequence is particularly suited to target ectopic gene
expression to the mouse heart, since it gives reproducible high-level
expression in both adult atria and ventricles. Transgene expression is
approximately dependent on the number of transgene copies, suggesting
that the -MHC promoter may contain a locus control region that
buffers the transgene from context effects exerted by genomic sequences
at the integration site; a series of transgenic lines may therefore be
used to titrate gene expression in the heart. A second advantage of
using the -MHC sequence is that it is activated uniformly
throughout the myocardium. Regionalized expression has been
observed with a number of other transgenes that are presumably
detecting differences in the transcriptional potential of
subcompartments of the myocardium.14 25
Palermo et al23 studied the expression of MLC2V transgenes
in the atria as well as the ventricles. Initial
physiological studies demonstrated that with the
remodeled atrial myocardium, contractility
and ventricular relaxation were impaired. A number of
observations also apply to the work of Gulick et al7
described in this issue, which describes the remodeling of the atrial
and ventricular myocardium by the expression of
skeletal muscle MLC2F transgenes. Endogenous MLC2 mRNA
levels are not affected by high levels of transgene expression.
Strikingly, despite greatly increased quantities of MLC2 mRNA, the
overall quantity of MLC2 protein is maintained in both atria and
ventricles, suggesting that sarcomeric protein stoichiometry in the
heart is regulated at precise levels by posttranscriptional mechanisms.
This is consistent with studies of skeletal -actin
transcript levels in different inbred mouse lines, in which the
absolute level of -actin mRNA was found to vary by as much as 8-fold
while the level of actin protein was constant.26 In
isoform replacement studies, the maintenance of constant
protein levels despite variable transcript levels allows for the
separation of phenotypes resulting from protein substitution
from phenotypes resulting from protein overexpression.
The degree to which expression of ectopic MLC2F replaces the
cardiac MLC2 isoforms differs between atrial and
ventricular compartments. In the atria, saturating amounts
of MLC2F mRNA result in total replacement of the MLC2A isoform by
MLC2F, vindicating the transgenic approach to achieve complete isoform
switching. In the ventricle, however, a maximum of about 55% MLC2
replacement was obtained, even in mice with high transgene copy
numbers. Gulick et al7 have speculated that this reflects
different relative affinities of MLC2 isoforms for the contractile
apparatus in atrial and ventricular
compartments. These observations uncover an additional feature of
isoform diversity (sarcomere specificity) and raise a caveat for the
transgenic replacement approach: extremely high levels of transgene
expression may be required to totally displace isoforms when the
relative affinity of the ectopic subunit for the contractile
apparatus is less than that of the endogenous
subunit. The relative affinities of MLC2 isoforms for the MLC binding
-helical region of -MHC may thus be in the order
MLC2V>MLC2F>MLC2A, although other sarcomeric components such as the
essential light chains1 (MLC1A in the atria and MLC1V in
the ventricles) may also influence this. The upshot of differential
sarcomeric affinity appears to be preferential degradation of
unincorporated MLC2 (MLC2A in the atria and MLC2F in the ventricle),
since Gulick et al find no accumulation of nonmyofilament sarcomeric
MLC2, and polysome loading/translation of endogenous and
transgenic mRNAs appears equivalent. Control of cardiac gene regulation
at the posttranscriptional level is therefore clearly a critical,
although poorly understood, mediator of the myocardial
phenotype. This should be considered in assessing pathological
situations and in attempts to remedy them using gene therapy.
The replacement of MLC2A by MLC2V or MLC2F in the atria and the partial
replacement of MLC2V by MLC2F in the ventricle leads to reduced
contractility in transgenic hearts. Notably, in the
case of ectopic MLC2F expression, contractility and
relaxation are significantly impaired, with a decrease in maximal
ATPase activity. This conclusion is based on analysis of a
single transgenic line: it will be an important test of this transgenic
approach to assess whether the degree of contractility
reduction correlates directly with the extent of isoform replacement.
Although there is no obvious pathology, this result points to the
different functional roles of MLC2 isoforms in fine-tuning the
contractile apparatus and demonstrates the potential of
this transgenic approach. An alternative in vivo approach to studying
isoform function is to generate a null allele by gene knockout
technology using mouse embryonic stem cells, although for many cardiac
genes this may result in embryonic lethality, as in the case of
-MHC.27 Although analysis of heterozygotes may
be informative, gene knockin technology makes it possible to completely
replace one isoform with another. The transgenic approach described by
Gulick et al7 offers several advantages, including
targeting replacement to particular cardiac chambers and flexibility in
manipulating levels of replacement. However, in any approach that
involves isoform switching in vivo, compensating mechanisms may operate
(eg, see Reference 2828 ); it is important to consider the possibility
that physiological changes may be indirect and
derive from adaptive changes in sarcomeric components other than those
that have been switched.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editor or of the American Heart Association.
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References |
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TopIntroductionReferences |
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