Cardiac Hypertrophic and Developmental Regulation of the
-Tubulin Multigene Family*
Takahiro
Narishige,
Kristie L.
Blade,
Yuji
Ishibashi,
Toshio
Nagai,
Masayoshi
Hamawaki,
Donald R.
Menick,
Dhandapani
Kuppuswamy, and
George
Cooper IV
From the Gazes Cardiac Research Institute, Cardiology Division of
the Department of Medicine, Medical University of South Carolina and
the Veterans Affairs Medical Center,
Charleston, South Carolina 29403
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ABSTRACT |
Increased microtubule density, through viscous
loading of active myofilaments, causes contractile dysfunction of
hypertrophied and failing pressure-overloaded myocardium, which is
normalized by microtubule depolymerization. We have found this to be
based on augmented tubulin synthesis and microtubule stability. We show here that increased tubulin synthesis is accounted for by marked transcriptional up-regulation of the
1- and
2-tubulin isoforms, that hypertrophic regulation of these genes recapitulates their developmental regulation, and that the greater proportion of
1-tubulin protein may have a causative role in the microtubule
stabilization found in cardiac hypertrophy.
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INTRODUCTION |
When under pathological circumstances the heart is forced to eject
blood against an increased impedance, the terminally differentiated cardiac muscle cell, or cardiocyte, responds by hypertrophic growth (1). The resultant increase in muscle mass constitutes the basic
compensatory cardiac response to sustained hemodynamic overloading, but
this initial compensation is frequently vitiated by a progressive decline in cardiocyte contractile function (2), so that congestive heart failure ensues.
We have found that this cardiocyte contractile defect is caused by
increased density of the cellular microtubule network (3), which
imposes a viscous load on the shortening sarcomeres during contraction
(4). Thus, microtubule depolymerization in hypertrophied cardiocytes
restores normal cellular contractile function, and induced microtubule
hyperpolymerization in normal cardiocytes causes these cells to exhibit
the same contractile abnormality found in hypertrophied cells (3).
The 
-tubulin heterodimer-microtubule system is in a dynamic
steady state. Therefore, in attempting to uncover the cause of
increased microtubule density in hypertrophied cardiocytes, we focused
on increased tubulin synthesis (5) and thus microtubule formation as
well as on increased stability of the microtubules once formed. With
respect to the latter, we have indeed found marked stabilization of the
microtubule network in hypertrophied cardiocytes associated with a
substantial increase in the predominant microtubule-associated protein
of the heart, MAP41 (6).
Although recent data (7, 8) put into question the previously accepted
role of MAP4 in microtubule assembly and stability for some cell types,
the muscle-specific variant of MAP4 appears to play a role in striated
muscle (9). Given that MAPs and the expressed proteins of the
-tubulin multigene family exhibit coordinate developmental
regulation (10) and that the latter may via their isoform-variable
carboxyl-terminal domain confer differing MAP binding affinity and
microtubule stability after assembly (11), the question of whether
there is differential regulation of the members of the
-tubulin
multigene family during cardiac hypertrophy assumed pivotal importance.
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EXPERIMENTAL PROCEDURES |
Right Ventricular Pressure Overloading--
Pressure overload
hypertrophy of the feline right ventricle (RV) was created (12) by
placement of a 2.9-mm inner diameter band around the proximal pulmonary
artery. Because the RV mass increase stabilizes by 2 weeks after a step
increase in load (5), at 2 weeks after surgery intravascular pressures
were measured in these and in control cats; values in the systemic
circulation were the same for both groups. The heart was then removed,
RV mass was determined, and Ca2+-tolerant quiescent
cardiocytes were isolated enzymatically from the RV and left ventricle
(LV) separately (13). All operative procedures were carried out under
full surgical anesthesia; all procedures and the care of the cats were
in accordance with institutional guidelines. At 2 weeks, RV systolic
pressure was doubled, and there was a 59% increase in the ratio of RV
to body weight; the mass of the normally loaded same animal control LV
was unchanged.
Western Blotting--
Peptide synthesis and coupling were
performed as described (14) with minor modifications. The peptides
underlined in Fig. 1 were synthesized, purified, keyhole limpet
hemocyanin-conjugated via glutaraldehyde cross-linking, and injected
into rabbits. Sera were monitored on slot blots using bovine serum
albumin-conjugated peptides until high antibody activity was achieved;
the IgG fractions were then purified using covalently coupled peptide
columns. BamHI fragments of the human h
1, mouse m
2,
and human h
2 (14) genes, which encode the carboxyl-terminal 100 amino acids of
1-,
2-, and
4-tubulin as noted here,
respectively, were ligated into the pET-28 histidine-tagged expression
vector (Novagen). Cultures were induced to express for 1.5 h with
1 mM isopropyl-1-thio-
-D-galactopyranoside. Cells were then spun down, resuspended in binding buffer, and lysed
with a French press. The resultant fusion proteins, after isolation on
a Ni2+-chelation column (Novagen), were used to verify the
monospecificity of each of the site-directed polyclonal antibodies.
Because of potentially differing antibody affinities, aliquots of the
purified fusion proteins for
1-,
2-, and
4- tubulin were
resolved on SDS-PAGE and silver-stained; loading was adjusted to
produce equal tubulin band intensities. For the subsequent immunoblots,
fusion protein loading normalized in this manner was then used as an internal standard for analysis of the tubulin present in the samples, where the ratio of integrated optical density for the single band for
each sample to that for each fusion protein was estimated.
Northern Blotting--
To generate isoform-specific cDNA
probes, the 3'-untranslated region of each
-tubulin isoform gene was
cloned from the cat.
1- and
4-tubulin were cloned from a cat
cDNA library (Stratagene) by PCR using oligonucleotides specific to
the conserved region of each
-tubulin isoform and a poly(T)
oligonucleotide as primers.
2-tubulin was cloned by reverse
transcriptase-PCR from total RNA isolated from cat brain using a
poly(T) primer for reverse transcription; oligonucleotides from the
conserved region of
-tubulin and a poly(T) primer were then used for
PCR amplication of the cDNA products, which were cloned into the
pT7Blue vector (Novagen) and sequenced (Sequenase, U. S. Biochemical
Corp.). Oligonucleotides that specifically amplified the
3'-untranslated region of each gene were used to generate
32P-radiolabeled PCR probes. To estimate
-tubulin
isoform mRNA levels, total RNA was extracted from frozen RV and LV
samples from the same hearts (15). The RNA samples were dissolved in 500 µl of diethylpyrocarbonate-treated water and quantified
spectrophotometrically at a 260/280 nm extinction coefficient, and to
assess RNA quality and to assure equal loading of RV and LV RNA for
Northern blots, 3-5-µg RNA samples were stained with ethidium
bromide and run on 1% agarose check gels. We then electrophoresed
5-7-µg RNA samples on denaturing 2% formaldehyde, 1% agarose gels
followed by 1.5 h of pressure-driven blotting to a nylon membrane
(Hybond-N, Amersham Pharmacia Biotech). The RNA was immobilized on the
nylon membrane by UV cross-linking (Stratalinker, Stratagene), and the
membrane was prehybridized for 4 h at 42 °C in a solution
containing 50% (v/v) deionized formamide, 0.2% (w/v) Ficoll, 0.02%
(w/v) polyvinylpyrrolidone, 5× SSC, 10 mM MOPS, pH 7.0, 2 mM EDTA, 100 µg/ml denatured salmon sperm DNA, and 0.2%
(w/v) SDS. The membrane was hybridized for 16 h at 42 °C in a
solution containing a 32P-radiolabeled probe (0.5-1.0 × 106 cpm/ml) for each
-tubulin isoform. The Northern
blots were washed 3 times in 2× SSC, 0.1% SDS for 1.5 h at
42 °C followed by a wash in 0.2× SSC, 0.1% SDS for 20 min at
42 °C and processed for autoradiography. Each blot was normalized by
stripping and reprobing with a glyceraldehyde-3-phosphate dehydrogenase
probe PCR-generated from feline glyceraldehyde-3-phosphate dehydrogenase cDNA.
mRNA Stability--
Hearts were removed from control cats
and from cats 2 weeks after RV pressure overloading; cardiocytes
isolated enzymatically (13) from the RV and LV separately were
incubated at 37 °C in 1.8 mM Ca2+
mitogen-free M-199 medium at pH 7.4. The cardiocytes were either untreated or exposed to 5 µg/ml actinomycin D for 0, 4, 8, or 18 h. To test the effect of an acute increase in the cytosolic concentration of tubulin heterodimers on tubulin mRNA stability, further control and hypertrophied cardiocytes were simultaneously exposed to both 5 µg/ml actinomycin D and 10 µM
colchicine for 0 or 4 h. Total RNA was then extracted from the
cells for Northern blot analysis using the three isoform-specific
-tubulin probes or a probe that recognizes the isoform-common region
of feline
-tubulin mRNA (5).
-Tubulin Isoform Fractionation and Localization--
For the
immunoblots, a fresh 100-mg tissue specimen from the RV and LV of each
cat was homogenized in 2 ml of hypotonic Tris buffer (20 mM
Tris, pH 7.4, 20 mM
-glycerophosphate, 1 mM
dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml pepstatin, 10 µg/ml
aprotinin, 0.5 mM phenylmethylsulfonyl fluoride) and
centrifuged at 15,000 × g at 4 °C for 15 min. The
supernatant was mixed with an equal volume of SDS-sample buffer and
saved as the cold-extractable fraction. The pellet was extracted in 2%
Triton buffer (100 mM Tris, pH 7.4, and 2% Triton X-100)
with the same protease inhibitors and centrifuged at 15,000 × g at 4 °C for 5 min; no tubulin was detected in the
supernatant. The pellet was added to SDS-sample buffer and boiled for 5 min. The SDS-solubilized pellet and the cold-extractable fraction were
resolved on 12.5% SDS-PAGE and immunoblotted with a 1:10,000 dilution
of the
1-,
2-, or
4-tubulin-specific antibodies. For double
label immunofluorescence confocal micrographs, freshly isolated RV and
LV cardiocytes in 37 °C modified M-199 medium (10 mM
Hepes, pH 7.4, 250 µM Ca2+) were sedimented
onto laminin-coated coverslips for 45 min and then, after 1 h of
exposure to either 37 or 8 °C, extracted for 1 min in 1% Triton
X-100 in microtubule stabilization buffer (16), washed three times in
the same buffer, and fixed for 30 min with 3.7% formaldehyde, all at
25 °C. After blocking with 10% donkey serum in 0.1 M
glycine, the cardiocytes were incubated overnight at 4 °C both with
a 1:200 dilution of our rabbit
1-tubulin-specific antibody and with
a 1:500 dilution of a mouse monoclonal antibody (B-5-1-2, Sigma), which
recognizes all native
-tubulin isoforms (17), followed by both
Cy3-labeled anti-rabbit IgG and Cy5-labeled anti-mouse IgG (Jackson
ImmunoResearch) secondary antibodies. Micrographs were acquired as
single 0.7-µm confocal sections taken at the level of the nuclei (LSM
GB-200, Olympus).
Cardiac Developmental Expression of
-Tubulin--
Pregnant
and 1-, 20-, and 90-day-old postpartum Sprague-Dawley rats were
anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally).
The 15-day-old embryonal and 1-day-old neonatal rats were decapitated,
the hearts were removed, and the atria and great vessels were trimmed
away. About 30 embryonal hearts and ~50 mg of ventricular myocardium
from the other stages were homogenized in 500 µl of lysis buffer (10 mM Tris, 0.5 mM dithiothreitol, 1 mM sodium vanadate, 1% sodium dodecyl sulfate, pH 7.4),
boiled for 5 min, and centrifuged at 16,000 × g at
room temperature for 10 min; the supernatants were saved. For the
subsequent 12.5% SDS-PAGE, an equal amount of protein (25 µg) as
determined by a bicinchoninic acid assay (Pierce) was processed with
SDS-sample buffer and loaded for each sample; immunoblotting was done
with the same antibodies as those specified in Fig. 1. Three samples from each stage were studied with confirmatory results.
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RESULTS AND DISCUSSION |
The five
-tubulin isoform proteins whose expression was
examined in this model, classified according to Cleveland (14, 18), are
shown in Fig. 1. Site-directed polyclonal
antibodies were generated against each of these
-tubulin isoforms
using synthetic peptides having the sequences underlined in Fig. 1. After preparing the appropriate fusion proteins, the specificity of the
peptide column-purified
1,
2, and
4 antibodies was validated, as also shown in Fig. 1. No reactivity of the
3 and
5 antibodies with homogenates from either normal or hypertrophied myocardium was
detected despite strong reactivity of each antibody with the respective
bovine serum albumin-conjugated peptide.

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Fig. 1.
Carboxyl-terminal domains of the five
vertebrate -tubulin polypeptides, the
sequences within these peptides used for antibody generation, and
validation of the specificity of three of these antibodies.
Peptides corresponding to the underlined region of each
-tubulin isoform shown in the upper panel, which
uses the one-letter amino acid code, were used to prepare
site-directed polyclonal antibodies. Fusion proteins corresponding to
the 1, 2, and 4 isoforms were then used, as shown in the
lower panel, to determine antibody specificity. These fusion
proteins, with concentrations adjusted to produce comparable staining
intensities, were loaded onto each lane of the 12.5% SDS-PAGE
before immunoblotting and visualization by enhanced chemiluminescence
(DuPont). Each isoform-specific antibody bound strongly to the
corresponding fusion protein, but there was no detectable antibody
cross-reactivity.
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Immunoblots of normal feline hearts showed that
4-tubulin expression
is greatly preponderant (see below). As shown in Fig. 2, by taking advantage of the much higher
affinity of the
1- and
2- as opposed to the
4- and common (all
isoforms)
-tubulin antibodies and by varying blot exposure times, it
was possible to visualize all four classes of
-tubulin in the same
homogenates, where the samples from the control and RV hypertrophy
hearts were treated identically. In the hypertrophied RV, despite the
marked increase in both free and polymerized isoform-common
-tubulin, there was little change in
4-tubulin. Rather, the
increased
-tubulin was accounted for by marked increases in free and
polymerized
1- and
2-tubulin. Densitometric analysis of
immunoblots from the RVs and LVs of two further control and RV
hypertrophy cats, where the total tubulin protein fraction (6) was
assayed and equal amounts of fusion proteins specific to each
-tubulin isoform were loaded along with the myocardial samples,
showed that in both ventricles from control cats and in the normally
loaded LV from RV hypertrophy cats,
1-,
2-, and
4-tubulin were
4, 1, and 95%, respectively, of total
-tubulin; in the
hypertrophied RVs,
1-,
2-, and
4-tubulin were 20, 4, and 76%
of total
-tubulin. Of singular interest, as shown by the immunoblot
summary data in Fig. 3, solely for
1-tubulin in the hypertrophied RV there was a disproportionate
increase in the microtubule-assembled pool. That is, at 2 weeks of RV
pressure overloading the increase of
1-tubulin in the
microtubule-assembled pool was about twice that in the unassembled
heterodimer pool. Although functional significance has been ascribed to
differential
-tubulin isoform expression in the male germ line of
Drosophila (19, 20) and has been inferred from heterogeneous
cellular isoform distributions (e.g. Ref. 21), the present
data represent the first such direct evidence in vertebrates, and this
is in a context of potential consequence to an important human disease
state.

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Fig. 2.
Immunoblots of
-tubulin isoforms in RV and LV myocardium from a
control cat and a RV pressure-overloaded cat 2 weeks after pulmonary
artery banding. Both free (lanes 1 and 3)
and polymerized (lanes 2 and 4) tubulin
fractions, prepared and validated as before (6), were probed with the
1-, 2-, or 4-tubulin-specific antibodies or, for total
-tubulin, with an antibody denoted as "common ," which
recognizes an epitope common to all -tubulin isoforms (DM1B,
Amersham Pharmacia Biotech). Equal proportions of the free and
polymerized samples were loaded onto the two lanes for each ventricle,
and an equal amount of protein as determined by a bicinchoninic acid
assay (Pierce) was loaded for the RV and LV samples. The LV pressure of
both cats was normal and equivalent.
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Fig. 3.
Summary of -tubulin
isoform immunoblots during development of RV hypertrophy. For this
semiquantification, fusion proteins over a concentration range, which
with the corresponding antibody produced a linear densitometric
relationship with protein concentration, were loaded onto 12.5%
SDS-PAGE gels along with cardiac samples whose protein concentration
had been adjusted to produce a densitometry signal within this same
linear range. Statistical comparisons were by two-way ANOVA (analysis
of variance) and a means comparison contrast, where n is the
number of cats at each of the four time points. *, p < 0.01 for difference from the value for free tubulin at matched time
points; , p < 0.01 for difference from the initial
control value within a group.
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When the time course of these changes was examined, where the
pressure-overloaded RV was compared with the normally loaded LV in five
cats at each time of 2 days, 1 week, and 2 weeks after an increase in
RV load, by 1 week there were increases in both the polymerized and
free
-tubulin fractions, with the greater increase being in the
polymerized fraction. This increase was accounted for in its entirety
by increases in the
1 and
2 isoforms. This pattern was found in
additional studies to be maintained for up to 6 months after the
hypertrophic response was complete (data not shown).
Expression of
-tubulin isoform transcripts was then examined by
Northern blot analysis. Fig. 4, where the
alternate polyadenylation site of
1-tubulin (22) produces two
transcripts, shows that the pattern of
-tubulin isoform expression
on the mRNA level mimics that seen on the protein level. That is,
the amount of
4-tubulin mRNA was equivalent in the RV and LV of
control cats and changed very little in either ventricle during the
development of RV hypertrophy. There was, however, a striking and
persistent up-regulation of
1- and of
2-tubulin mRNA in the
hypertrophying RV.

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Fig. 4.
Time course for expression of
-tubulin isoform transcripts during development of
cardiac hypertrophy. Northern blots of each -tubulin isoform in
myocardium from control and RV pressure-overloaded cats were prepared
from total RNA isolated from the RV and LV at the indicated times after
pulmonary artery banding. The blots were probed with -tubulin
isoform-specific cDNA probes. Equal RV versus LV loading
was confirmed by stripping and reprobing with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.
Similar results were obtained in two additional cats at each time
point.
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To gain some insight into the relationship of mRNA levels to
protein levels for these
-tubulin isoforms, we examined mRNA stability in cardiocytes from the hypertrophied RV and the control LV
of pulmonary artery-banded cats. Because it has not proved possible to
isolate transcriptionally active nuclei from these cells, we used
actinomycin D to inhibit RNA polymerase in fresh primary cultures of
these cells and then measured the rate of decline of mRNA levels
for the
-tubulin isoforms and for total
-tubulin by Northern
analysis. Lanes 1-4 of Fig. 5
show that mRNA stability estimated in this manner is quite similar
in hypertrophied RV versus control LV both for the three
-tubulin isoforms examined and for isoform-common total
-tubulin.
The same result was obtained in the RV versus LV of normal
cats (data not shown). Thus, increased
1- and
2-tubulin protein
in hypertrophied myocardium results from increased transcription of
these genes.

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Fig. 5.
Message stability for
-tubulin isoforms and isoform-common total
-tubulin in RV and LV cardiocytes from a cat RV
pressure-overloaded 2 weeks earlier. Cardiocytes were isolated
from each ventricle and treated with 5 µg/ml actinomycin D. Northern
blots show message levels for each isoform and for total -tubulin at
0 (lane 1), 4 (lane 2), 8 (lane 3),
and 18 (lane 4) h after actinomycin D treatment. Message
half-life averaged ~6 h for each mRNA species examined in this
and three further cats with 2 weeks of RV pressure overloading.
Additional RV and LV cardiocytes were exposed simultaneously to both 5 µg/ml actinomycin D and 10 µM colchicine and incubated
for 0 (lane 5) and 4 (lane 6) h. These findings
were confirmed in cardiocytes from another cat with 2 days of RV
pressure overloading.
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The concurrent up-regulation of
-tubulin on both the protein and the
message levels, which we have found to persist indefinitely in
myocardium hypertrophying in response to a pressure overload (5),
appears to contravene the co-translational negative feedback control
that tubulin exerts on its own rate of synthesis (23) via reduced
mRNA stability. That is, the stability of ribosome-bound
-tubulin mRNA is controlled by co-translational binding of
either
-tubulin itself or an intermediary factor to the
amino-terminal
-tubulin tetrapeptide as it emerges from the
ribosome. This binding then activates an RNase or causes ribosomal
stalling with the result in either case being accelerated
-tubulin
mRNA degradation, such that the net effect is an inverse relation
between
-tubulin protein concentration and
-tubulin mRNA
half-life. A breakdown of this regulatory control of tubulin synthesis,
which might selectively affect different
-tubulin gene products,
could explain increased microtubule density in hypertrophied
cardiocytes. Thus, we used colchicine to acutely increase the
concentration of tubulin heterodimers in control and hypertrophied
cells and observed the effect of this intervention on tubulin mRNA
stability. Lanes 5 and 6 of Fig. 5 show that to
an equivalent degree for both control and hypertrophied cardiocytes,
colchicine-induced microtubule depolymerization accelerates the rate of
mRNA degradation for all three
-tubulin isoforms and for total
-tubulin. Further, in intact cats given 1 mg/kg colchicine
intravenously with or without 2 or 7 days of RV hypertrophy, where
-
and
-tubulin mRNAs are markedly increased in hypertrophied RVs
(5),
-tubulin mRNA levels had decreased equivalently 4 h
later in normal and hypertrophied RVs and in the control LVs to
25.5 ± 1.7% of the respective control values for cats not given
colchicine. Thus, these data demonstrate that the co-translational
regulatory mechanism for controlling tubulin mRNA stability is
intact in normal and hypertrophied terminally differentiated
cardiocytes, such that, again, there is an authentic increase in the
transcription of the
1- and
2-tubulin genes in cardiac
hypertrophy. However, they also strongly suggest that such
co-translational control is exerted as a rate-dependent
rather than a concentration-dependent function of the
cytosolic concentration of tubulin heterodimers, a finding having
general rather than cardiocyte-restricted implications.
A central goal of this study was to determine whether if up-regulation
of specific
-tubulin isoforms during cardiac hypertrophy was
discovered, such changes in gene expression have functional significance. That is, the cardinal alterations of the extramyofilament cytoskeleton of hypertrophied cardiocytes, in terms of inducing contractile dysfunction, are interrelated increases in the quantity and
stability of the microtubule network. If there were increased expression of specific
-tubulin genes, the protein products would directly account for the persistent increases in both free and polymerized tubulin. However, if one or more of the up-regulated isoforms conferred greater stability on the microtubules once assembled, this would contribute to increased density of the
microtubule network via a mechanism independent of augmentation of the
heterodimer pool. Thus, because stable microtubules are resistant to
cold-induced depolymerization (6), we measured
1-,
2-, and
4-tubulin in the cold-stable cytoskeletal fraction of normal and
hypertrophied myocardium and cardiocytes. The left panels of
Fig. 6 show that although the proportion
of
2- or
4-tubulin in this fraction is very low for normal or
hypertrophied myocardium, a significant proportion of
1-tubulin is
found in the cold-stable cytoskeletal fraction, and this is more
pronounced for the hypertrophied RV. The right panels of
Fig. 6 show that the microtubule array of the hypertrophied cardiocyte
is more cold-stable than that of the control cardiocyte, that
microtubules of the hypertrophied cardiocyte incorporate more
1-tubulin than those of the control cardiocyte, and that this latter
finding is especially pronounced in the cold-stable microtubules of the
hypertrophied cell. These findings are consistent with densitometric
analysis of tubulin isoforms, because the data in Fig.
7 show first in normal RVs and LVs a
greater proportion of
1-tubulin in the cold-stable microtubule
fraction and second a selective further shift solely of
1-tubulin to
this fraction in the hypertrophied RV. Nonetheless, there is not
necessarily an exact correspondence between microtubule cold stability
and microtubule stability in vivo, such that these correlative data do not constitute proof that
1-tubulin-enriched microtubules are more stable in the intact cardiocyte of the heart in situ. We are therefore testing this point directly via
adenovirus-mediated overexpression of
1-tubulin in isolated
cardiocytes and via cardiac-targeted
1-tubulin overexpression in
transgenic mice.

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Fig. 6.
-Tubulin isoform localization
via immunoblotting and confocal microscopy. The left panels
show an immunoblot analysis of -tubulin isoforms in soluble
cytosolic and cold-stable cytoskeletal fractions of the RV and LV of a
cat heart 2 weeks after RV pressure overloading. The two
cytosolic lanes were loaded equally as were the two
cytoskeletal lanes; the loading ratio of the soluble
versus cold-stable fractions was constant. The same result
was obtained in the hearts from four further RV pressure-overloaded
cats. The right panels show double label immunofluorescence
confocal micrographs of cardiocytes isolated from the RV and LV of a
feline heart 2 weeks after RV pressure overloading. Prior to fixation,
the two upper cells were maintained at 37 °C and the two lower cells
at 8 °C for 1 h; the latter condition causes selective
depolymerization of labile microtubules (6). These cells were
double-stained for 1-tubulin (red) and for -tubulin
(green), where 1- and -tubulin primary antibodies were
followed by species-specific fluorochrome-conjugated secondary
antibodies; areas of coincident decoration by both primary antibodies
range in color from orange to yellow. The
inset at the lower right corner of each
micrograph is a magnified view of a segment of a single microtubule.
Cardiocyte preincubation with the 1-tubulin peptide abolished
1-tubulin labeling. When this protocol was repeated using the
2-tubulin antibody, very little microtubule decoration was apparent,
and it was not obviously selective for cold-stable microtubules (data
not shown).
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Fig. 7.
Summary of -tubulin
isoform distribution in cold-stable versus cold-labile
microtubules. These ratios were obtained by densitometric
quantification of the two fractions for each isoform when run on 12.5%
SDS-PAGE gels and immunoblotted with the corresponding antibodies.
Statistical comparisons were by two-way ANOVA and a means comparison
contrast, where n is the number of control or RV hypertrophy
cats (RVH). *, p < 0.01 for difference from
the ventricle-specific value for the other isoforms; ,
p < 0.01 for between ventricle difference for a given
isoform.
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Because many protein isoforms normally expressed in the
developing heart and then down-regulated in the adult heart are
re-expressed after hemodynamic hypertrophic stimulation and because the
specificity of these isoform switches may become important to
understanding transcriptional regulation during cardiac hypertrophy, we
examined cardiac developmental regulation of the
-tubulin multigene
family in hearts extirpated from a developmentally timed series of rats that were subjected to immunoblot analysis of
1-,
2-, and
4-tubulin as well as total isoform-common
-tubulin. Fig.
8 shows that between embryonic day 15 and
postpartum day 90 there is a rather modest decrease in total
-tubulin and a very minor increase in
4-tubulin. However, both
1- and
2-tubulin peak at postpartum day 1 and decline to very low
levels in the adult heart.

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Fig. 8.
Immunoblot analysis of the developmental
expression of -tubulin isoforms and
isoform-common total -tubulin in rat cardiac
ventricles at embryonic day 15 and postpartum days 1, 20, and 90. Equal protein loading was used for each lane and for each
isoform. The antibodies were the same as those used for Fig. 2. Three
samples from each stage were studied with confirmatory results (data
not shown).
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In common with the questionable functional significance of many other
cardiac isoform switches wherein hypertrophy recapitulates phylogeny
(1), these data do not imply a role for altered
-tubulin isoform
expression in the generative processes either of cardiac hypertrophy or of cardiac development. Rather, the functional significance of the hypertrophic cardiac
-tubulin isoform switch described here is presently of known consequence only in terms of the
resultant disordered contractile function (3). However, hypertrophic
expression of the
-tubulin multigene family clearly does
recapitulate its developmental expression, which, again, may provide
eventual insight into
-tubulin transcriptional control mechanisms
common to these two phases of cardiocyte growth.
Apart from the intrinsic interest of these observations, the major
impetus for this study was to ascertain their basis in terms of the
augmented tubulin quantity (5) and microtubule stability (6) found in
the pressure overload-hypertrophied heart. Although our finding of MAP4
up-regulation in cardiac hypertrophy (6) may well be important to the
latter phenomenon, it does not directly explain the former. Thus, the
possibility that increased expression of one or more members of the
-tubulin multigene family might explain both the greater quantity of
tubulin and the greater microtubule stability, either directly via
differing intrinsic properties or indirectly via differing MAP4
affinities (11), was quite intriguing. The data in this study indeed
show that whereas expression of the predominant cardiac
-tubulin
isoform is but little affected, there is marked up-regulation of two
ordinarily minor cardiac
-tubulin isoforms. In addition to the
possibility that this explains augmented tubulin quantity, selective
localization of the
1-tubulin isoform to stable microtubules may
also explain augmented microtubule stability. Finally, the fact that
hypertrophic regulation of
-tubulin mimics its developmental
regulation may provide the eventual insight required to understand
transcriptional regulation of the
-tubulin gene family during
cardiac hypertrophy.
 |
ACKNOWLEDGEMENTS |
We thank Sebette Hamill, Charlene Kerr, and
Mary Barnes for technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL-48788 and by research funds from the Department of Veterans Affairs.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.
To whom correspondence should be addressed: Gazes Cardiac Research
Institute, P.O. Box 250773, Medical University of South Carolina,
Charleston, SC 29403. Tel.: 843-953-6474; Fax: 843-953-6473; E-mail:
cooperge{at}musc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, microtubule-associated protein;
RV, right ventricle;
LV, left
ventricle;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase
chain reaction;
MOPS, 4-morpholinepropanesulfonic acid.
 |
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