© 1999 American Heart Association, Inc.
Integrative Physiology |
Signal Transduction in Atria and Ventricles of Mice With Transient Cardiac Expression of Activated G Protein 
q
From the Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Ulrike Mende, MD, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail mende{at}calvin.bwh.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Abstract—We recently showed that the transient expression of a hemagglutinin (HA) epitope–tagged, constitutively active mutant of the G protein
q subunit
(HAq*) in the hearts of transgenic mice is sufficient to
induce cardiac hypertrophy and dilatation that continue to
progress after HAq* protein becomes undetectable. We
demonstrated that the activity of phospholipase Cß, the immediate
downstream target of activated Gq, is increased
at 2 weeks, when HAq* is expressed, but also at 10
weeks, when HAq* is no longer detectable. This
observation suggested that the transient HAq* expression
causes multiple, persistent changes in cellular signaling pathways. We
now demonstrate changes in the level, activity, or both of several
signaling components, including changes in the amount and hormone
responsiveness of phospholipase Cß enzymes, in the basal level of
diacylglycerol (which predominantly reflects activation of
phospholipase D), in the amount or distribution of protein kinase C
(PKC) isoforms (PKC, PKC
, and PKC
), and in the amount of
several endogenous G proteins. These changes vary depending
on the isoform of the signaling molecule, the chamber in which it is
expressed, and the presence or absence of HAq*. Our
results suggest that a network of linked signaling functions determines
the development of hypertrophy. They also suggest that
atria and ventricles represent different signaling domains. It
is likely that such changes occur in other model systems in which the
activity of a single signaling component is increased, either due to an
activating mutation or due to overexpression of the wild type.
Key Words: heart • hypertrophy • transgene • G protein • signal transduction
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The activation of signal transduction pathways mediated by heterotrimeric Gq protein can lead to cardiac hypertrophy and dilatation (for a review, see Dorn and Brown1 ). To understand better the cellular and molecular events leading to hypertrophy, several transgenic mouse models have been created with cardiac overexpression of various components of the Gq-mediated signaling pathway, such as receptors that work through Gq (eg,
-adrenoreceptors2 3 and
angiotensin receptors4 ),
Gq protein subunits (wild type5
or activated6 ), and potential downstream targets
(eg, protein kinase Cß [PKCß],7 8
calcineurin, and nuclear factor of activated T cells
[NFAT]9 ). Except for the mice expressing
angiotensin receptors, which died shortly after birth, mice
from the other lines developed cardiac hypertrophy, some
with dilatation.
One objective in the production of transgenic animals that
express single components of the transmembrane signaling machinery is
to establish systems that will eventually allow the activity of the
transgenic protein to be connected to the final phenotype. To
achieve this goal, it is first essential to distinguish changes that
arise directly in response to the activity of the transgenic protein
from changes that arise due to secondary alterations in the activity or
expression of other signaling components. So far, there has been no
systematic attempt to characterize changes in signaling components that
occur as a consequence of cardiac expression of
Gq-coupled receptors,2 3 4
wild-type Gq,5 or putative downstream
targets in the pathway.7 8 9
The transgenic mouse line that we recently developed6
makes an excellent model system in which to analyze primary
changes due to expression of the transgenic protein versus secondary
ones. This mouse line transiently expresses activated
hemagglutinin (HA) epitope–tagged G protein q
(HAq*). The protein is present at 2 weeks,
diminishes by 4 weeks, and is undetectable by 10 weeks.6
The mice develop cardiac hypertrophy and dilatation that
progress inexorably to death from heart failure between 8 to 30 weeks
of age. We previously showed that the basal activity of phospholipase C
(PLCß, the direct target of activated
q) remains elevated at 10 weeks even though
HAq* becomes undetectable.6 We
now test the hypothesis that the transient expression of
HAq* leads to multiple, persistent secondary
changes in many signaling components that continue after the
HAq* protein is downregulated and that may
perpetuate the pathology. In a comparison of younger mice expressing
HAq* with older mice with undetectable
HAq*, we found changes in the amount,
activity, or both of several signaling components, including the amount
of PLC enzyme, its hormone responsiveness, the level of diacylglycerol
(DAG), the amount and distribution of PKC, and the amounts of
several endogenous G proteins.
Our work also shows that atria and ventricles, both of which are
severely hypertrophied and dilated, are quite different in their
response to the expression of HAq*. Despite
the fact that the -myosin heavy chain promoter, which we used and
which is widely used for cardiac-specific transgene expression, is
active in all chambers of the heart,10 there is little
information so far about the biochemical consequences of overexpression
of signaling components in the atria. Our data documenting differences
between atria and ventricles and, in some cases, differences between
right and left chambers suggest that the right and left sides of the
heart, as well as the atria and ventricles, reflect not only distinct
transcriptional domains11 but also distinct signaling
domains.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Production of HA
q* Transgenic MiceTwo transgenic mouse lines (
q*52 and
q*44) were generated as previously
described.6 A homozygous line, called
q*44h, was developed from
q*44. These animals begin to reexpress atrial
natriuretic factor and ß-myosin heavy chain at 4 to 6
months and develop enlarged hearts by 14 months, confirming in a second
line that the expression of HAq* causes
cardiac hypertrophy. In this study, we used heterozygous
q*52 mice at 14 to 16 days (2 weeks) and 62 to
75 days (10 weeks) of age. The cardiac morphology of this line was
previously described.6
Western Blot Analysis
Particulate and soluble protein fractions from atrial
appendages, ventricles, and ventricular
myocytes12 from 2- and 10-week-old transgenic mice and
littermate wild-type controls were prepared, separated on 9% SDS-PAGE,
and blotted.6 (The left and right atrial appendages were
used in the present study and are referred to as left and right
atria.) The left and right atria from 8 to 10 wild-type or transgenic
2-week-old mice were pooled; only right atria were used from
10-week-old mice (8 wild-type mice and 2 or 3 transgenic mice in each
pool). Nitrocellulose membranes were cut into 5 strips to detect
proteins of different molecular weights in the same samples on the same
blot (the antibodies that were used are described in detail in the
supplemental online information). The total amount of each PKC and
PLCß isoform was calculated by multiplying the integrated signal
obtained from an equal amount of the particulate and soluble proteins
by the total protein in the particulate and soluble fractions.
Similarly, the total amount of G protein subunits was calculated by
taking into account the total amount of particulate protein.
Adenylyl Cyclase Activity
Two pools each of right atrial tissue from 10-week-old wild-type
and transgenic mice (each containing a total of 15 to 20 mg tissue from
6 wild-type or 2 or 3 transgenic animals) were homogenized
and ultracentrifuged at 100 000g. The adenylyl
cyclase (AC) activity was determined in the particulate fraction as
previously described.13
Measurement of [3H]Inositol Phosphate
Formation
Total inositol phosphate formation was measured in small pieces
of atrial and ventricular tissue6 that had
been incubated for 30 minutes with or without carbamylcholine or
endothelin-1.
DAG Levels
Lipid fractions were extracted from 15 to 25 mg atrial or 30 to
60 mg ventricular snap-frozen tissue according to the
method of Bligh and Dyer14 with minor modifications as
described in the manufacturer’s instructions for the s,n-1,2-DAG Assay
Reagent System (Amersham Life Science). At 2 weeks, the right and left
atria from 5 or 6 wild-type or transgenic mice were pooled. At 10
weeks, left atrial tissue that had been carefully freed of any blood
and thrombi was used (each pool contained left atria from 6 wild-type
mice or 2 transgenic mice). The radioactivity comigrating with a
phosphatidic acid signal from the DAG standard was quantified by
computerized densitometry. It was normalized to the signal obtained for
the internal standard included in each sample. After blank subtraction,
the data were normalized to milligrams of protein in each
homogenized sample before extraction and referred to the
age-matched wild-type control.
Statistical Analysis
Data are reported as mean±SEM (for n determinations).
Comparisons between transgenic mice and wild-type controls were made
with the use of unpaired two-tailed Student t test.
P<0.05 was considered significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Transient cardiac expression of activated HA
q* leads to hypertrophy and
dilatation.6 HAq* protein is
expressed at 2 weeks, when the hearts are morphologically barely
distinguishable from wild type, but is not detectable at 10 weeks, when
they are dilated and hypertrophied. The primary target of
activated q is activation of PLCß.
We have previously shown that PLC activity is elevated, both when
HAq* is expressed and after the protein
becomes undetectable.6 This observation suggests that
transient HAq* expression causes persistent
changes in cellular signaling pathways. We now show that the persistent
changes involve not only the activity but also the cellular levels of
several signaling proteins. The mechanism by which the proteins are
changed is not yet known but may involve differences in transcription
of the gene, stability of the protein, or both. A striking finding in
our study is that atria and ventricles respond very differently to the
initial expression of the HAq* protein. At 2
weeks, the levels of HAq* expression are
similar in atria and ventricles,6 although in atria the
protein has been expressed throughout development, whereas in
ventricles, it is turned on shortly after birth.10 Despite
the fact that activated HAq* has been
expressed throughout development, at 2 weeks, the biochemical changes
in the atrium are modest and generally similar to those in ventricle.
This finding suggests that the long-term consequences of
HAq* expression depend not only on the
immediate effects of the protein itself and the signaling pathways that
it sets in motion but also on the different hemodynamic
stresses on atria and ventricles.
Expression of G Protein and ß Subunits
To interpret any changes in downstream effectors, it is essential
to know whether the expression of HAq* causes
changes in the endogenous G protein subunits. We
demonstrated previously that at 10 weeks, when
HAq* is no longer detectable, the level of
endogenous q* is upregulated
(Reference 66 , see also Figure 1
).
We now demonstrate that the levels of other G proteins also change and
that these changes occur in a chamber- and an isoform-specific fashion
(Figure 1). At 2 weeks, the amount of the various G protein
subunits tested with Western blotting was only slightly different from
that of age-matched wild-type littermate controls in both atria and
ventricles from q*52 mice (Figure 1A and 1B, respectively). At 10 weeks, the ventricular levels
of some G protein subunits were modestly increased further. In
contrast, in the right atrium, there was a substantial increase in
q/11 (2.9-fold), i
(2.7-fold), and the long form of s (4.8-fold)
compared with wild-type controls (Figure 1A). The short form of
s was unchanged compared with the wild type,
indicating that the s isoforms are
differentially regulated. A similar observation, albeit much less
pronounced, was made in 10-week-old ventricles in which the long form
of s was slightly increased, whereas the short
form was slightly decreased (Figure 1B). In both atria and
ventricles, the relative rise in subunits exceeded the relative
rise in G protein ß
subunits. However, because the absolute levels
of the subunits in mouse heart are not known, one cannot conclude
from these data that the subunits are in excess of the ß
subunits. A large fractional increase in a very nonabundant subunit
could lead to a small change in relative ß subunit levels.
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It was not possible to obtain sufficient atrial myocytes for
biochemical studies, but we isolated ventricular myocytes
and observed qualitatively similar changes in the level of G protein
q/11 (data not shown). This suggests that the
changes in expression levels that we observed reflect changes in the
myocytes (see also later discussion of PKC expression). However,
additional changes in nonmyocytes cannot be ruled out.
To determine whether the pronounced rise in Gs
subunit levels in the right atria of 10-week-old
q*52 mice has any functional consequence, we
measured the activity of the enzyme regulated by
Gs, AC. There was no significant difference in
basal AC activity or in the response to stimulation with the
nonhydrolyzable GTP analog
guanosine-5'-O-(3-thio)triphosphate, the ß-adrenergic
agonist isoproterenol, or the diterpene forskolin, which directly
activates the catalytic units of AC synergistically with G
protein s,15 in right atrial
membranes from 10-week-old q*52 mice compared
with wild-type controls (Table 1). The
lack of enhanced AC activation despite a pronounced increase in the
long form of s may be explained by the
concomitant rise in G protein i subunits (see
earlier), which exert an inhibitory effect on AC activity.
Alternatively, because the major AC isoforms expressed in the heart, AC
V and VI, are sensitive to direct inhibition through
Ca2+ (for a review, see Taussig and
Gilman16 ), a rise in Ca2+ in
response to chronic PLC activation (see below) could counteract a
potential rise in AC activity. Other explanations are also
possible.
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Expression of PLCß Isoforms
In our previous study, we showed that although
HAq* is undetectable at 10 weeks, the basal
PLC activity continues to be elevated.6 This continued
elevation may be in part due to the secondary rise in
endogenous q (Figure 1
). We
now show that in addition, the level of PLCß enzyme itself is
relatively higher. We find that the amount of
PLCß1 and PLCß3
decreases as the animals age (Figure 2A and 2B). PLCß2 is not expressed in the
heart.17 In 10-week-old wild-type mice, the levels of both
PLCß isoforms were decreased by 30% to 65% compared with the levels
at 2 weeks. However, in the atria of q*52
mice, there was no maturational drop in PLCß1
or PLCß3 (Figure 2A). Thus, the total
amount of PLCß in atria from 10-week-old
q*52 mice was increased compared with that in
wild-type controls. In ventricles, only the decline in
PLCß1 was blunted (Figure 2B), so the
difference between wild types and q*52 was
less in ventricles than in atria. There was no significant change in
the distribution of PLCß1 or
PLCß3 between particulate and soluble fractions
(data not shown).
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) and
) mice. Tissue from both sides of
heart was always combined except for 10-week-old atria (right atria
only). Data represent mean±SEM from 2 to 4 atrial pools and 4
to 6 ventricles and are expressed in percent of 2-week-old wild
type.
Agonist Activation of PLC
Constitutively active G protein subunits activate
downstream effectors but interact poorly with receptors and with G
protein ß subunits. Therefore, the overexpression of
HAq* should not affect the
endogenous response to receptor activation. Unless the
level of effector is limiting, we expect hormonal stimulation of PLCß
in 2-week-old q*52 mice to be additive to the
elevated basal activity, with no net difference between wild-type and
q*52 mice. However, if the levels of other
signaling components are changed as a consequence of activated
HAq* expression, then the increment in
effector activity due to receptor activation may differ between
wild-type and q*52 mice. We hypothesized that
the agonist-stimulated increment in PLC activity would be additive to
the elevated basal activity at 2 weeks, but not at 10 weeks, given the
pronounced chamber- and isoform-specific secondary changes in G protein
and PLCß expression in q*52 mice at 10
weeks.
We analyzed the effect of 2 agonists, endothelin-1 and
carbachol, on total inositol phosphate formation as a measure of PLC
activity (Figure 3). Endothelin-1
activates PLC in the heart by binding to
endothelinA receptors and activating a pertussis
toxin–insensitive G protein, presumably Gq/11
(for a review, see Sugden and Bogoyevitch18 ). It is still
a matter of debate which muscarinic receptor subtypes mediate the
effect of carbachol on PLC activity. The heart contains predominantly
M2 muscarinic receptors that are coupled to
pertussis toxin–sensitive
Gi/Go proteins. It may also
contain M1 and M3
muscarinic receptors that are coupled to pertussis toxin–insensitive G
proteins.19 20 There are conflicting results regarding the
pertussis toxin sensitivity of muscarinic stimulation of cardiac PLC
activity.21 22 23 The effect of
Gi/Go proteins on PLC is
mediated by the ß subunits released on activation, whereas the
q/11 subunits are largely responsible for
mediation of the effect of pertussis toxin–insensitive G proteins (for
a review, see Rhee and Bae24 ). It is likely that in
contrast to endothelin-1, more than one mechanism contributes to the
effect of carbachol on PLC activity.
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5-fold lower in
ventricles than in atrial appendages, scales of graphs are different.
Number of mice studied in each condition is given in brackets.
In accord with our hypothesis, at 2 weeks, a maximal dose of carbachol
(100 µmol/L) caused the same increment in PLC activity in
q*52 as it did in wild-type hearts in all
chambers tested (Figure 3, left). Thus, carbachol-stimulated PLC
activity was additive with the elevated basal activity. However, a high
dose of endothelin-1 (1 µmol/L) was additive in the right
ventricle but not in the atria. Endothelin-1 causes a much more robust
activation of PLC than does carbachol. In the atria of 2-week-old
q*52 mice, the incremental response to a high
dose of endothelin-1 (1 µmol/L) was less than that in wild type,
as if PLC activation were saturated. Consistent with this
interpretation, a lower concentration of endothelin-1 (0.1
µmol/L) produced similar increments over basal activity in wild-type
and q*52 atria.
Consistent with the maturational decline in the amount of
PLCß1 and PLCß3 in
10-week-old wild-type mice, basal PLC activity and responsiveness to
both agonists tested declined with age (Figure 3, right). In
contrast to the approximately equal increment in carbachol-stimulated
PLC activity over basal levels in wild-type and
q*52 mice at 2 weeks, at 10 weeks, the
carbachol-stimulated increase in activity was greater in
q*52 than in wild-type mice in the right
atrium. In the ventricles of q*52 mice, both
carbachol and endothelin-1 responses were greater than in wild-type
mice. The increased responsiveness of PLC to stimulation with 2
different agonists is presumably due to secondary increases in other
signaling components, such as the rise in PLCß or
endogenous q/11, that may increase
the efficiency of receptor coupling to PLC. As a result,
q*52 mice may be more sensitive to hormones
that are normally present in the heart, making them more
susceptible to endogenous hypertrophic stimuli. The
increased susceptibility may help sustain the pathological process.
DAG Formation
DAG, the other second messenger generated by PLC, is also produced
by phospholipase D (PLD). The substrate for PLD, phosphatidyl choline,
is much more abundant than the substrate for PLC, phosphatidyl inositol
bisphosphate, so most of the DAG that accumulates in stimulated cells
is derived from phosphatidyl choline (for a review, see
Exton25 ) and a rise in DAG in response to PLCß
activation is not easily detectable. However, DAG activates
some forms of PKC (including the PKC isoforms expressed in the heart;
see later), and PKC, in turn, can activate PLD, thereby
providing a mechanism for cross-talk and signal amplification between
PLC and PLD in many cell types (for a review, see
Exton25 ), including the myocardium (for a
review, see Eskildsen-Helmond et al26 ). Although the
mechanisms by which PKC activates PLD are not yet clearly
defined, the positive feedback between these enzymes seems to allow a
more prolonged rise in DAG and may enhance and prolong the activation
of PKC.
In ventricles from q*52 mice, basal DAG levels
were increased at 2 weeks (147±10% of wild type; n=4,
P<0.005) and at 10 weeks (184±12% of wild type; n=4;
P<0.005). In contrast, there was a more modest rise in
basal DAG levels in atria from q*52 mice
(119±17% of wild type at 2 weeks and 157±8% of wild type at 10
weeks, 2 pools each; mean±range). In contrast to inositol phosphates,
DAG was more elevated in ventricles than in atria, suggesting that the
PLD pathway may play an important role in the perpetuation of
ventricular pathology.
PKC
The activation of PKC can change the amount of PKC protein, induce
translocation to the membrane, or both.27 Three PKC
isoforms expressed in the adult heart, PKC, PKC, and PKC (for
reviews, see Pucéat and Vassort28 and Steinberg et
al29 ), showed a differential response to the chronic
activation of PLCß in q*52 mice. We
determined the relative amount of these PKC isoforms and their
distribution between particulate and soluble fractions through Western
blotting of hearts from 2- and 10-week-old
q*52 and wild-type mice (Table 2 and Figure 4). The results obtained for
ventricular tissue were confirmed in isolated
ventricular myocytes (Table 2). Differences in
mobility on SDS-PAGE presumably reflect different
phosphorylation states (see, for example, Keranen et
al30 ). At 2 weeks, PKC and PKC increased minimally
in both atria and ventricles from q*52 mice.
At 10 weeks, the rise in both PKC isoforms was more pronounced (Table 2). Although there was no difference in the amount of
membrane-bound PKC or PKC at 2 weeks, there was a significant
increase in membrane-bound ventricular PKC and atrial
PKC at 10 weeks (Figure 4). The ratio of membrane-bound to
soluble ventricular PKC increased from 0.43±0.06 in
wild type to 0.82±0.07 in q*52 (n=12 each,
P<0.001); the ratio for atrial PKC increased from
0.32±0.06 in wild type to 0.55±0.07 in
q*52 (n=4 each,
P<0.05). The total amount of PKC was decreased in
ventricles, but not in atria, of 2-week-old
q*52 mice (Table 2) without a change in
the relative amount that was membrane bound (Figure 4). This
decline was even more pronounced at 10 weeks. PKC seems to play a
role in protection of the heart against damage from
ischemia.31 32 A decrease in PKC in the
ventricles of q*52 mice may therefore
contribute to the worsening pathology. PKC is not downregulated but
rather translocated in mice expressing unactivated
q.5 This difference may be due to
a more pronounced activation of PLC with a constitutively active
q compared with wild-type
q. Our results suggest that the different PKC
isoforms have different functions and that they are regulated by
different mechanisms.
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Conclusions
One of the advantages of transgenic mouse models is that the
effects of the targeted overexpression of one protein can be studied in
the context of the whole animal. In the present study, we
demonstrate that multiple, long-lasting changes in signaling (including
isoform- and chamber-specific changes) can be initiated by the
transient expression of the transgenic protein and that additional
secondary mechanisms continue to drive the pathology after the
transgenic protein has been downregulated. It is possible that some of
the changes observed at 10 weeks are a consequence rather than a cause
of the cardiomyopathy. However, the fact that many
of the changes begin early suggests that they do contribute to the
development of the pathology. Our findings emphasize the plasticity of
the cardiac response to the expression of an activated
signaling molecule, as well as to pronounced differences in signaling
response between atria and ventricles. Given the multitude and breadth
of changes that are observed, it is likely that many different
signaling pathways determine the hypertrophic response. Positive and
negative feedback loops may also contribute. In delineation of the
interplay of the various signaling events, their temporal sequence and
the transcription factors that are involved will allow a better
understanding of the network of these linked signaling functions and of
their role in the development of cardiac hypertrophy.
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Acknowledgments |
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This work was supported by National Institutes of Health Grants HL-52320 and GM-36359 (Dr Neer). We are very grateful to Paula McColgan for her excellent secretarial help and to Xiaofen Lou and Donna Martins for help with the myocyte experiments.
Received January 25, 1999; accepted September 17, 1999.
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