(Received for publication, July 11, 1995; and in revised form, September 5, 1995)
From the
Tropomyosins comprise a family of actin-binding proteins that
are central to the control of calcium-regulated striated muscle
contraction. To understand the functional role of tropomyosin isoform
differences in cardiac muscle, we generated transgenic mice that
overexpress striated muscle-specific -tropomyosin in the adult
heart. Nine transgenic lines show a 150-fold increase in
-tropomyosin mRNA expression in the heart, along with a 34-fold
increase in the associated protein. This increase in
-tropomyosin
message and protein causes a concomitant decrease in the level of
-tropomyosin transcripts and their associated protein. There is a
preferential formation of the
-heterodimer in the transgenic
mouse myofibrils, and there are no detectable alterations in the
expression of other contractile protein genes, including the endogenous
-tropomyosin isoform. When expression from the
-tropomyosin
transgene is terminated,
-tropomyosin expression returns to normal
levels. No structural changes were observed in these transgenic hearts
nor in the associated sarcomeres. Interestingly, physiological analyses
of these hearts using a work-performing model reveal a significant
effect on diastolic function. As such, this study demonstrates that a
coordinate regulatory mechanism exists between
- and
-tropomyosin gene expression in the murine heart, which results in
a functional correlation between
- and
-tropomyosin isoform
content and cardiac performance.
A precise assembly of contractile proteins consisting of myosin,
actin, and associated molecules (i.e. tropomyosin
(TM)(), troponin (Tn), and
-actinin) is required for a
functional striated muscle sarcomere. Many myofibrillar proteins exist
in multiple isoforms, and major changes in their expression occur
during myogenesis. Differences in contractile and regulatory function
among isoforms have been determined for mammalian myosin, TM, and
Tn-T(1, 2, 3, 4) . For example, in
different skeletal muscle fiber types, the expression of particular TM
and Tn-T isoforms correlates with the Ca
regulation
of sarcomeric tension. Investigations on muscle assembly and function
also serve as a paradigm for understanding development and
differentiation of many biological systems. For example, genetic
mutants of Caenorhabditis elegans, Drosophila
melanogaster, and axolotl Ambystoma mexicanum illustrate
the importance of contractile protein genes for myofibrillar structure,
assembly, and function(5, 6, 7, 8) .
Insufficient maintenance of functional myofibrils can lead to cardiac
failure and mutations in ventricular myosin heavy chain (
-MHC),
-TM, and cardiac Tn-T cause familial hypertrophic
cardiomyopathy(9, 10) .
Investigations on the
functional roles of contractile protein isoforms, such as striated
muscle-specific - and
-TM are essential for understanding the
physiology of the striated muscle sarcomere. TM, which is encoded
within a multigene
family(11, 12, 13, 14, 15, 16, 17) ,
is a coiled-coil dimer that stabilizes actin filaments and is central
to the control of striated muscle contraction. In association with the
Tn complex, the function of TM in skeletal and cardiac muscle is to
regulate the calcium-sensitive interaction of actin and myosin. The
Tn
TM complex inhibits actomyosin ATPase activity under resting
intracellular calcium ion concentrations. In response to Ca
release by the sarcoplasmic reticulum, Tn-C binds additional
calcium ions and a conformational change is transmitted through the
Tn-bound TM complex. This movement coordinately releases the inhibition
of actomyosin ATPase activity and results in sarcomeric contraction.
Although there is significant amino acid similarity between the
sarcomeric - and
-TM isoforms (87.5% identity), differences
in the ratio of these isoforms have been observed between fast and slow
contracting striated muscles(18) , thereby suggesting a
functional difference between the isoforms. Fast contracting skeletal
and cardiac muscles contain more
-homodimers, and slow
contracting muscles express more
-homodimers. Nevertheless,
additional studies have demonstrated that the
-heterodimer is
preferentially formed in most
muscles(2, 4, 19) , although the biological
significance of this remains unclear. Functional differences of these
protein isoforms and their interactions with other molecules like the
Tn complex have been
studied(1, 4, 20, 21) . Preliminary
studies by Thomas and Smillie (22) indicate that strength of
binding the
- and
-TMs to Tn-T are equivalent,
whereas the strength of binding
-TM to Tn-T is less. Also,
biochemical studies suggest the
-homodimer exhibits higher
tropomyosin-actin-S1 ATPase activity and greater
Ca
-sensitive release of Tn inhibition than either the
-homodimer or
-heterodimers. Further support that
specific combinations of these isoforms have different contractile
properties is found during development of chicken and rabbit skeletal
muscle(18, 23) .
Despite the biochemical evidence,
the relationship between TM isoforms and contractile behavior of the
heart has not been delineated. Previous studies by Izumo et al.(24) have demonstrated that -TMstr transcripts and
other fetal program genes (skeletal
-actin, ANF, etc.) are
reinduced in the adult rat heart during pressure-overloaded
hypertrophy. Whether the isoform changes are directly responsible for
and sufficient to cause alterations in cardiac behavior remains
unproven. With the advent of gene-targeting and transgenic animal
technologies, it is now possible to target a specific gene/isoform for
ablation or overexpression and thus determine the function of a
specific gene/isoform product.
Recently, we conducted a quantitative
analysis on TM expression in murine cardiac tissue(25) . Both
striated - and
-TM transcripts are increased during embryonic
cardiac development (days 11-19), with the ratio of these mRNAs
changing from 5:1 to 60:1 during the embryonic to adult transition. To
understand the functional role of striated muscle-specific
- and
-TM isoforms, we have generated transgenic mice that overexpress
-TMstr specifically in the adult heart. By utilizing this
approach, we could address whether any compensatory mechanism in
cardiac musculature is activated in response to the overproduction of
-TMstr mRNA or protein and whether differences in the
:
-TM isoform ratios could morphologically and/or
physiologically change the contractile behavior of the heart. Nine
independent transgenic lines were generated, which show a 150-fold
increase in
-TMstr mRNA expression in the heart, along with a
34-fold increase in the associated protein. Expression was restricted
to the cardiac compartment. The data show that the increase in
-TMstr messages and protein in the transgenic (TG) mouse heart
also causes a concomitant decrease in the levels of
-TMstr
transcripts and their associated protein. There is also a preferential
formation of
-heterodimers that occurs in the TG mouse
hearts. No detectable alterations in the expression of any other
contractile protein genes are found in these transgenic mice.
Morphological analyses indicate that there are no structural changes in
the heart nor in the sarcomere that is associated with the increase in
-TMstr expression. Interestingly, physiological analyses of these
transgenic hearts reveal that functional parameters associated with
myocardial contractility appear normal; however, there is a significant
delay in the time of relaxation and a decrease in the maximum rate of
relaxation in the left ventricle.
Isoproterenol was added to the Krebs-Henseleit solution entering the heart using microperfusion pumps (multispeed infusion pump, model 600, Harvard Apparatus). These pumps could be operated separately or together so that drug concentration delivery could be modulated easily. Each isoproterenol concentration was infused for 3 min into the venous return line before the volume control in order not to alter venous return. In all hearts, the infusions were initiated at similar preloads, afterloads, and heart rates.
Figure 1:
Transgene construct
and expression. A, the construct used for the microinjection
is shown. The 5`-UTR of the -cardiac MHC gene is encompassed by
three exons marked as 1, 2, and 3 boxes, and the line represents introns. The
-MHC/
-TM fusion gene was
constructed by two step cloning as described under ``Experimental
Procedures.'' The unique EcoRI site is designated. The SacI-HindIII enzymes were used to release the
transgene fragment. B, single-stranded, striated
muscle-specific
- and
-TM cDNA probes were hybridized in the
same reaction to RNAs from NTG and TG mice hearts, skeletal muscle RNA (SkM), and tRNA. Full protection of the
-probe is
detected in all samples except tRNA, the negative control sample.
-TM probe is detected in all the TG RNA and skeletal muscle RNA
samples. The position of
- and
-TMstr probes are marked on
the left; marker bands are shown on the right. The darkly shaded region in the diagram represents the
3`-UTR of
-TMstr cDNA, and lightly shaded regions designate the striated muscle-specific sequences. Positions of the
corresponding amino acid (aa) numbers are marked. C,
RNA from
-5.5CAT transgenic mouse hearts (lane 1), NTG
hearts (lane 2), and TG hearts (line 10) (lane 3)
were analyzed for
- and
-TMstr transcripts as described in B. In this S1 reaction, a loading control
glyceraldehyde-3-phosphate dehydrogenase was also included along with
the
- and
-TMstr probes. The positions of
-TM,
-TM,
and glyceraldehyde-3-phosphate dehydrogenase probes are marked on the left; size markers are designated on the right.
We measured the expression of the construct using S1
nuclease and Northern slot-blot analyses following RNA isolation of
hearts obtained from TG and NTG littermates. As depicted in Fig. 1B, the -TM probe, 363 nucleotides long, was
end-labeled at codon 184 and extended into the 3`-UTR of the striated
muscle cDNA sequence. The
-TM probe, a 299-nucleotide-long PstI-BglII fragment, was end-labeled at codon 144 and
extended into codon 244, encompassing the striated muscle-specific exon
6B. To quantitate the relative levels of
- and
-TM
transcripts, S1 nuclease protection analysis was done in a single
reaction in which equivalent amounts of specific activity from both
- and
-TM probes were hybridized to an equal amount of RNA
(25 µg) from TG or NTG mouse hearts. Hybridization products were
then digested with S1 nuclease and analyzed as described(25) .
As seen in Fig. 1B, full protection of the
-TM
probe (299 bp) is readily detectable in all TG heart RNA samples. Upon
longer exposure, very low levels of the endogenous
-TMstr band are
detectable in the NTG samples. Full protection of the
-TMstr probe
(363 nucleotides) is clearly visible in all cardiac muscle samples. The
data also clearly demonstrate there is a concomitant decrease in the
-TMstr message levels in the TG cardiac RNAs as compared with
those of the NTG samples (full protection of the 363-nucleotide band).
(The NTG 28 sample spilled during loading of the gel; independent
experiments with additional NTG 28 RNA samples showed that the
-TM
transcript levels are equivalent to other NTG samples (data not
shown)). Also, additional experiments using glyceraldehyde-3-phosphate
dehydrogenase as control probes have demonstrated equivalent amounts of
total RNA were analyzed among the NTG and TG samples. A PhosphorImager
analysis was conducted to quantitate the
- and
-TMstr
transcript levels. The intensities of the bands at 363 and 299 bp were
measured, and the relative ratio was calculated. There is an increase
of
-TM mRNA amount in the different TG lines (58.0 ± 6.5
units) when compared with their NTG littermate (0.4 ± 0.01
units). These values represent a 150-fold increase in
-TMstr mRNA
expression in the heart. The
:
striated muscle TM transcript
ratio in the hearts of TG mice is 0.3:1, whereas this ratio is 58:1 in
the NTG hearts. This value for the NTG
:
ratio corroborates
our earlier published value of 60:1 in the adult mouse
heart(25) . Further, there is a 3-fold increase in the total
amount of
-TM plus
-TM transcripts in the TG verses NTG mouse
hearts. Northern slot-blot analyses confirmed these results; additional
experiments addressing the expression of the
-MHC/
-TMstr
transgene in different tissues showed that the expression of the
construct is restricted to heart tissue (data not shown). This result
is in agreement with previous studies demonstrating the
cardiac-specific expression exhibited by this
-MHC
promoter(26, 35) .
The observed decrease in the
-TMstr isoform following overexpression of the
-TMstr
strongly suggests a mechanism for coordinately regulating TM mRNA
levels. We addressed whether the endogenous
-TM expression is
altered by the transgene construct using Northern slot-blot analysis
with an oligonucleotide probe specific to the
-TMstr 5`-UTR, which
is lacking in our construct (see ``Experimental
Procedures''). Results demonstrate that the
-MHC/
-TMstr
transgene did not transactivate expression of the endogenous
-TM
gene, as similar low levels of
-TMstr mRNA were detected in TG and
NTG mouse heart samples (data not shown). We also checked if the
-MHC promoter or the SV40 fragment influenced expression of TM in
the transgenic mouse. RNA from the
-5.5 transgenic mouse (gift
from Dr. J. Robbins; (26) ), which contains the
-MHC
promoter ligated to the chloramphenical acetyltransferase gene and the
SV40 poly(A) signal, was quantitated for
- and
-TM
transcripts by S1 nuclease mapping as described previously. No changes
are found in the levels of the
- and
-TMstr messages in the
-5.5 transgenic mouse heart when compared with control mice (Fig. 1C).
We further examined whether there were
molecular compensatory reactions in the expression of other contractile
protein genes (- and
-MHC,
-cardiac and
-skeletal
actin, Tn-C, and Tn-T). Interestingly, results from Northern slot-blot
analyses demonstrate that there are no dramatic quantitative
differences in the expression of transcripts from these genes in TG
hearts as compared with their NTG littermates (data not shown). This
result is in agreement with the recent finding that overexpression of
skeletal Tn-C in cardiac muscle also does not alter the expression of
other contractile protein genes, including the endogenous cardiac
Tn-C(36) .
Differences in the levels of -TMstr
transcripts among the nine transgenic lines were observed with TG line
1, showing the least expression, and several TG lines exhibiting much
greater expression. Variability in expression of the transgene among
the TG lines may be due to differences in the copy number of the
integrated transgene construct and/or variability in the site of
integration. To determine the integration and copy number of the
microinjected DNA, we conducted a genomic Southern blot analysis.
Results demonstrate that the expected size band (6.8 kb) is present in
the genomic DNA from all TG lines when hybridized with a radiolabeled
SV40 fragment (Fig. 2). A nucleic acid slot-blot analysis
confirms that the different levels of expression of the
-TMstr
transgene are partially due to differences in the copy number among the
lines, with these values ranging from 4 copies (TG line 1) to 56 copies
(TG line 10) (data not shown).
Figure 2: Organization of the transgene. Southern blot analyses of genomic DNA from different TG lines hybridized with radiolabeled SV40 DNA were performed. The expected size band of 6.8 kb is seen in all TG lanes.
Figure 3:
Western blot analysis of TG and NTG
myofibrillar proteins. Protein gels containing equivalent amounts of TG
and NTG myofibrillar proteins were subject to Western blot analysis
using a striated muscle-specific antibody followed by S-labeled anti-mouse IgG as described under
``Experimental Procedures.'' A, autoradiogram of a
Western blot of TG and NTG heart samples. The positions of
- and
-TM proteins are indicated. B, the radioactivity
associated with
- and
-TM proteins was quantitated with a
PhosphorImager system, and the signal intensity (mean value ±
S.E.) obtained from four different gels was used to quantitate the
relative levels of
- and
-TM proteins in NTG and TG
myofibrils. Black bar,
-TM; striped bar,
-TM; stippled bar,
-TM +
-TM.
Previous investigations conducted by our laboratory (17, 25) and others (24) found that striated
muscle -TM is the primary mRNA isoform expressed in rodent cardiac
musculature. The results shown in Fig. 3demonstrate that TM
composition in wild-type cardiac musculature is essentially
-TM.
However, the dimeric species of TM protein with respect to its
and
subunits have not been examined in the heart. To ascertain
the distribution of
- and
-TM dimeric species in control and
transgenic mice, myofibrillar proteins from NTG and TG hearts were
subject to two-dimensional gel electrophoretic analysis, followed by
immunoblotting of the transfer membrane with the striated muscle TM
antibody. A minor modification of the two-dimensional electrophoretic
procedure of O'Farrell (32) was developed to resolve the
different TM dimers and determine their subunit composition (see
``Experimental Procedures''). Heart myofibrillar protein
samples from NTG mice show that a diffuse area, labeled
, is reactive with the TM antibody (Fig. 4A). As shown in the oxidized-reduced electrophoretic
conditions, the
-TM dimer is resolved into the
subunit (Fig. 4B). The protein of this spot with
respect to isoelectric focusing (pI value of 5.1) and SDS mobility
strongly suggests this protein is the
-dimer. In
concert with previous studies on TM composition in skeletal musculature
(where multiple
-TM isoform species have been described; (2) ), the results suggest multiple
isoform species are
also present in the heart. This production of several
-TM protein
species in cardiac musculature is also supported by the heterogeneity
found at the mRNA level(17, 25) .
Figure 4:
Two-dimensional electrophoretic analysis
of NTG (A and B) and TG (line 28; C and D) myofibrillar proteins. The dimeric forms of myofibrillar TM
were analyzed by oxidized-oxidized (A and C) and
oxidized-reduced (B and D) two-dimensional gel
electrophoresis as described under ``Experimental
Procedures.'' The square and star in C would indicate the expected positions of the -
and
-homodimers, respectively, if they were found. The
pI values (marked on top) were determined from first dimension
isoelectric focusing tube gels as described by
O'Farrell(32) . The
and
spots are marked. 10% SDS-polyacrylamide gel electrophoresis was used
in the second dimension. The standard molecular mass markers are on the right. The positions of
- and
-TM subunits are
indicated.
The results from Fig. 3show that in the TG mice, both - and
-TM
proteins are present in the myofibrils. When the TM subunit composition
of TG hearts is examined, a cluster of spots is detected in
polyacrylamide gels run under oxidized-oxidized conditions (Fig. 4C). The position of these spots with respect to
isoelectric focusing (pI value of 5.0) and SDS mobility is consistent
with the formation of
-heterodimers(2) . Detection of
four
-TM species at this position has been found previously
in various skeletal muscles and is partially attributable to
heterogeneity in the production of TM
isoforms(2, 17, 25) . Resolution of these
spots into
and
subunits in TG samples subject to
oxidized-reduced polyacrylamide gel electrophoresis (Fig. 4D) also strongly suggests the composition of
-heterodimers in the TG cardiac myofibrils.
To determine if hypothyroidism
would restore normal -TMstr transcript levels, relative levels of
- and
-TMstr transcripts in the ventricles of normal and
hypothyroid TG and NTG mice were determined by S1 nuclease analysis
followed by PhosphorImager quantitation. Further, to insure that equal
amounts of RNA were used in this assay, a glyceraldehyde-3-phosphate
dehydrogenase probe was also included along with the
- and
-TM probes. Results demonstrate that there is no detectable
-TMstr band (299 nucleotides) in the transgenic hearts of the
hypothyroid mice (Fig. 5A, lanes 4 and 8),
indicating that the transgenic
-TM expression is completely
down-regulated. However, in euthyroid transgenic mice (Fig. 5A, lanes 2 and 6), the
overproduction of
-TMstr message is quite clear, as we had
demonstrated earlier (Fig. 1B). Interestingly, the
levels of
-TMstr isoform expression in these hypothyroid TG hearts
are increased above euthyroid TG levels (compare the 363-nucleotide
band of lanes 4 and 8 with lanes 2 and 6). In addition to demonstrating that expression of the TM
transgene construct can be regulated by altering thyroid hormone
levels, this analysis also shows that endogenous
- and
-TMstr
isoform expression in the murine system is not affected by the
hypothyroid state; this can be seen by the relatively equal amounts of
mRNA present in euthyroid (lanes 1 and 5) and
hypothyroid (lanes 3 and 7) NTG hearts.
Figure 5:
RNA (A) and myofibrillar protein (B) analysis of normal and PTU-treated NTG and TG mouse
hearts. A, an S1 nuclease analysis of RNA from normal
(euthyroid) NTG hearts (lanes 1 and 5), TG hearts (lanes 2 and 6), PTU-treated (hypothyroid) NTG hearts (lanes 3 and 7), and PTU-treated TG hearts (lanes
4 and 8). This analysis was performed by using - and
-TMstr and glyceraldehyde-3-phosphate dehydrogenase probes as
described (see ``Results'' and Fig. 1). Lanes 2 and 4 represent TG line 10; lanes 6 and 8 represent TG line 13. B, Western blot analysis of
myofibrillar proteins using the CH1 antibody was conducted as described
in the ``Results.'' The sample order is similar to A. The migrating positions of
- and
-TMstr proteins
are marked on the left; the standard molecular mass markers
are on the right.
Since
results demonstrate there is an increase in -TMstr transcript
levels in response to hypothyroidism by the TG mice, we addressed
whether this change would be reflected at the translational level.
Using Western blot analysis, the levels of
- and
-TMstr
protein in the TG and NTG hypothyroid mouse ventricles were examined.
In results similar to those observed at the mRNA level,
-TM
protein is down-regulated in the hypothyroid TG mouse hearts (Fig. 5B, lanes 4 and 8). In
hypothyroid TG mice, the level of
-TMstr protein has been
up-regulated and is present in quantities similar to levels found in
NTG hypothyroid mice (lanes 3 and 4 and lanes 7 and 8). In addition, there are no detectable changes in
the
-TMstr protein levels between euthyroid and hypothyroid NTG
hearts (lanes 1 and 3 and lanes 5 and 7), indicating that thyroid hormone does not have any major
influences on striated muscle TM protein expression. Since there is no
effect of thyroxine on endogenous
- and
-TMstr isoform
expression in murine hearts, we can conclude from our results that
these two genes coordinately regulate their expression in the mouse
heart, as previously suggested to occur in chicken pectoralis
muscle(42) .
A detailed analysis of the
sarcomeric structure of the myocardium was conducted using electron
microscopy and immunohistochemistry. Adult cardiac tissue from TG and
NTG mice was examined by transmission electron microscopy for anomalies
of sarcomeric structure or other morphological alterations. Results
demonstrate there are no differences in cardiomyocyte structures or
organization in the TG mouse hearts (Fig. 6). Additional studies
on other mice at various magnifications confirmed this finding. In
immunological analyses, sections of TG hearts were immunostained using
a striated muscle-specific TM monoclonal antibody. There are no
apparent differences in the staining pattern between TG and NTG
sections, and there is no nonspecific binding of the TM antibody to
structures other than the myofilaments (data not shown). Thus, the
results from the histological analyses demonstrate that overexpression
of -TMstr isoform in these TG mice does not lead to morphological
or pathological alterations in cardiac muscle structure.
Figure 6:
Electron microscopy analysis of TG mouse
heart sections. Heart sections for EM were prepared as described under
``Experimental Procedures.'' A representative picture of a TG
mouse left ventricular myofilament structure is shown. Original
magnification: 15,000.
Studies on
rabbit fast skeletal muscle fibers demonstrate that different
TnTM combinations in the relaxed and active states and their
affinities for calcium appear to be a significant determinant of
myofiber contractile properties in vivo(18) . In our
investigation, we have developed an in vivo system where the
sarcomeric TM is mostly
-heterodimers instead of the usual
-homodimers. As such, one might expect changes in the
cardiac performance. To determine whether any such changes occur in the
TG mice, we implemented the isolated work-performing mouse heart
preparation. This model is a powerful technological tool used to
elucidate the quality of cardiovascular and contractile parameters in
individual mouse
hearts(33, 34, 43, 44) . The
advantage of this method is the observation of contractile and
relaxation parameters of individual mouse hearts under identical
minimal afterloads (aortic pressure of 50 mm Hg) and identical preloads
(cardiac output 5 ml/min), and similar heart rates. The contractile
parameters of seven TG mouse hearts were age (2-4 weeks old) and
sex matched with seven NTG littermate controls (2 hearts each from TG
lines 7, 10, and 28 and one heart from line 12). These results are
summarized in Table 1. It is clear that there are only a few
significant differences in cardiac performance that exist between TG
and NTG mice. Most of the physiological measurements are similar
between the two groups, including heart rate, mean aortic pressure,
cardiac output, and intraventricular pressure. When the minimal pre-
and afterload was applied, both TG and NTG hearts showed similar
systolic, diastolic, and end-diastolic intraventricular pressures.
However, there were some differences in the additional quantitative
measurements of contractile performance. Although the contractile
parameters (maximum rate of contraction and time to peak pressure) of
TG hearts showed no significant differences from the NTG values, the
relaxation parameters (maximum rate of relaxation and time to half
relaxation) of the TG hearts were significantly reduced or prolonged,
respectively, compared with the NTG hearts (Table 1).
This
effect on relaxation performance disappeared when the Starling forces
were increased: an afterload increase from a normal value of 50 mm Hg
mean aortic pressure to 62 mm Hg and/or an increase in the preload from
a normal value of 5 ml/min cardiac output to 7 ml/min. Consequently, at
maximally tolerated workload, contractile and relaxation parameters
were indistinguishable between NTG and TG hearts. Similar results could
also be accomplished by exposing the TG hearts to increasing
concentrations of the -adrenergic stimulant isoproterenol (Table 2). When 4 nM isoproterenol was administered to
the working hearts via the Krebs-Henseleit solution perfusate (a
concentration far below the ED
values of 12-40
nM), the differences in relaxation parameters were completely
removed. In summary, results from the physiological analysis show that
there is a functional alteration in cardiac muscle performance in the
TG mice. The primary function that is effected is an inability of these
hearts to relax fully coupled with a prolonged relaxation phase. As
such, these results demonstrate there is a functional correlation
between the
- and
-TMstr isoform content and cardiac
performance.
Striated muscle contraction involves the interaction of Tn
with TM on the muscle thin filament. Numerous biochemical studies have
provided insight into the importance of interactions among thin
filament proteins such as actin, Tn-T, and TM. The existence of muscle
fiber types has been correlated with various contractile protein gene
isoforms. However, understanding of the structure-function relationship
of specific isoforms remains largely unresolved. To gain new insights
into the functional role of sarcomeric TMs in murine cardiac muscle, we
generated transgenic mice overexpressing striated muscle-specific
-TM in adult hearts and addressed the response of the myocardium
at the molecular, morphological, and physiological levels. Results
demonstrate that overexpression of the striated muscle
-TM isoform
alters cardiac muscle performance by decreasing the maximum rate of
relaxation and increasing the time needed to complete the relaxation
phase of sarcomeric function. Also, this overexpression of
-TMstr
activates a compensatory mechanism to reduce the production of
-TMstr mRNA and protein, thus limiting the total amount of TM
production. As shown by the administration of PTU to shut off the
transgene promoter, this compensatory molecular mechanism is fully
reversible. The exogenous
-TMstr protein that is produced
preferentially associates to form an
-heterodimer, which data
suggests becomes integrated into the cardiac muscle sarcomere.
Interestingly, no morphological or pathological phenotype is associated
with this increased expression of
-TM in cardiac muscle.
Previous studies demonstrate that the 3`-UTR of sarcomeric actin
genes, which are highly conserved across species, may play an important
role in their regulation of expression(54, 55) . In vitro experiments coupling heterologous promoters to actin
3`-UTRs show that the expression of these chimeric constructs follows
the expression patterns of the 3`-UTR regions, not the associated
promoters. These results strongly suggest that this expression of actin
isoforms is strongly influenced by their
3`-UTRs(56, 57) . The mechanism through which the
3`-UTRs mediate actin expression is thought to be cis-regulated.
Rastinejad and Blau (58) have recently shown that regulation by
3`-UTRs of certain differentiation-specific RNAs (-TM,
-cardiac actin, and Tn-T), can operate in trans as well. Our
results demonstrate that neither the
-MHC promoter nor the SV40
poly(A) fragment used in the transgene construct is responsible for the
decreased level of
-TMstr isoform in the TG mice. Although both
the endogenous
- and
-TMstr isoforms are generated by
alternative splicing(13, 17) , it is unlikely that the
titration of splicing factors is responsible for the decrease in
-TM levels since the
-TM cDNA was used in the transgene
construct. Thus, it is possible that the 3`-UTR of the
-TMstr may
be involved in the down-regulation of
-TMstr isoform expression.
It is tempting to speculate that this could be the reason why the
relative ratio of
/
-TMstr muscle RNA in the TG mice is much
lower than the protein value; portions of the overexpressed
-TM
messages, particularly the 3`-UTR, may act as trans-acting regulators
in a feedback loop that inhibits
-TMstr mRNA isoform production.
However, we cannot rule out the possibility that translational control
mechanisms or stability of the mRNA or protein may also influence TM
production. Studies are in progress to elucidate the molecular basis of
this coordinate regulation.
It is interesting to note that small animals, such as
adult rat, rabbit, guinea pig, and mouse have virtually no cardiac
-TM component, whereas larger species (i.e. pig, sheep,
and human) have about 20% of their cardiac muscle TM in the
-form.
It has also been suggested that increases in
-TM content are
associated with a slower speed of striated muscle
contraction(60) . However, multiple contractile protein genes
undergo only partial developmental isoform transitions in larger
animals, thereby resulting in significant amounts of
-MHC,
-skeletal actin, and
-TMstr in the adult
myocardium(60, 61, 62, 63, 64, 65) .
As such, the slower contractile/relaxation properties associated with
larger animals may reflect multiple isoform differences. Our results
show that when cardiac muscle TM is present as an
-heterodimer, there are no additional changes observed in the
expression levels from other contractile protein genes. Further, it is
well known that a decisive parameter determining contraction and
relaxation of the cardiac muscle is not only the Ca
concentration per se but the calcium occupancy of Tn
molecules. Thus, the contractile parameters of the sarcomere can also
be altered by changing the calcium sensitivity of the regulatory
proteins (i.e. Tn-C) and associated proteins (i.e. TM
and Tn-T) that influence the responsiveness of the myofilaments to
calcium ions.
Measurements of contraction reflect cross-bridge
turnover, whereas relaxation is more tightly associated with myocardial
calcium handling (66) . Interestingly, the two key components
(sarcoplasmic reticulum Ca-ATPase and phospholamban)
that are involved in Ca
sequestration and
cardiomyofilament relaxation do not exhibit changes in their expression
in the
-TM TG mice (data not shown). Thus, it is reasonable to
speculate that the increased production of
-TMstr protein in the
TG mouse myocardium has a direct link to the relaxation function of the
heart muscle. Interestingly, diastolic dysfunction is a frequently
reported abnormality often associated with several cardiomyopathic
conditions, including hypertrophy, ischemia, and dilated
cardiomyopathy(67, 68, 69) . Although no
gross morphological abnormalities are detected with the overexpression
of
-TMstr in the hearts of the TG mice, this work demonstrates a
functional difference in diastole exists between the striated muscle
and
-TM dimers.
Hewett et al.(43) have demonstrated that a significant functional
correlation exists between -actin content and cardiac contractile
function; increasing levels of
-skeletal actin in the cardiac
muscle (which occurs genetically in BALB/c mice) enhances cardiac
contractility when compared with
-cardiac actin. In this study, we
have established a transgenic system that addresses functional
questions regarding specific TM isoforms. Although the
- and
-TMstr proteins possess 88% amino acid sequence homology, it is
not apparent how altering the
:
TM ratio might lead to
functional differences in the myocardium. However, it is interesting to
note that there are several amino acid differences between the
-
and
-TM molecules positioned around amino acids 150-180,
which is one of two putative Tn-T binding domains that attach TM to the
Tn complex(70, 71) . Perhaps, these differences
contribute to a weaker binding of
-TM to the Tn complex, which
subsequently affects the relaxation properties of the cardiac muscle.
Analyzing the functional significance and molecular regulatory
mechanisms controlling contractile protein isoform expression is
essential for understanding the development and differentiation of the
mammalian cardiogenic system. By establishing transgenic overexpression
and ``knockout'' animal models, we can address the functional
and molecular significance of specific isoforms that exist within
multigene and alternatively spliced gene families. By establishing a
transgenic mouse that overexpresses the striated muscle -TM mRNA
and protein specifically in the heart, the importance of
-TMstr in
muscle relaxation has been addressed in an in vivo system. In
addition, we have begun to decipher the mechanisms controlling the
molecular genetic regulation of
- and
-TM isoform production.
Through further use of transgenic animal model systems, this knowledge
can be extended to address the role of both wild-type and mutant
transcripts and proteins in the development of normal and pathological
conditions.