(Received for publication, July 26, 1995; and in revised form, October 19, 1995)
From the
Cardiac hypertrophy requires protein accumulation. This results
largely from an increased capacity for protein synthesis, which in turn
is the result of an elevated rate of ribosome biogenesis. The process
of ribosome formation is regulated at the level of transcription of the
ribosomal RNA genes. In this study, we examined the amounts and
activities of various components of the ribosomal DNA transcription
apparatus in contraction-arrested neonatal cardiomyocytes and in
spontaneously contracting cardiomyocytes that hypertrophy. Nuclear
run-on assays demonstrated that spontaneously contracting
cardiomyocytes supported a 2-fold increased rate of ribosomal DNA
transcription. However, enzymatic assay of total solubilized RNA
polymerase I and Western blots demonstrated that contraction-induced
increases in ribosomal RNA synthesis were not accompanied by increased
activity or amounts of RNA polymerase I. In contrast, accelerated
ribosome biogenesis was accompanied by an increased amount of the
ribosomal DNA transcription factor, UBF. Immunoprecipitation of
[P]orthophosphate-labeled UBF from
hypertrophying, neonatal cardiomyocytes indicated that the accumulated
UBF protein was phosphorylated and, thus, in the active form. UBF mRNA
levels began to increase within 3-6 h of the initiation of
contraction and preceded the elevation in rDNA transcription. Nuclear
run-on assays demonstrated increased rates of transcription of the UBF
gene. Transfection of chimeric reporter constructs containing deletions
of the 5`-flanking region of the UBF gene revealed the presence of
contraction response elements between -1189 and -665
relative to the putative start of transcription. These results are
consistent with the hypothesis that UBF is an important factor in the
regulation of rDNA transcription during contraction-mediated neonatal
cardiomyocyte hypertrophy.
Mechanical stimulation, in its various forms, is a primary determinant of cardiomyocyte phenotype and hypertrophic growth both in vivo and in vitro (reviewed in (1) and (2) ). For instance, unattached feline cardiomyocytes maintained in cell suspension rapidly lose the organizational characteristics of differentiated cardiac muscle and resemble undifferentiated neonatal cardiomyocytes(3) . In contrast, attachment of adult cardiomyocytes to laminin-coated substrates inhibits the loss of characteristic biochemical and functional properties(3) . In recent reports, linear deformation (passive stretch) has been demonstrated to increase cellular growth as measured by rates of RNA and protein synthesis and protein accumulation in cultured neonatal cardiomyocytes(4, 5) .
Spontaneous contractile activity also appears to be an important regulator of cardiomyocyte growth(6, 7, 8) . For example, spontaneously contracting neonatal cardiomyocytes in culture accumulate RNA and protein at a faster rate than noncontracting, i.e. arrested, cells. This growth occurs in the absence of changes in DNA content or cell number (hypertrophic growth) and appears to be largely independent of humoral or neuronal factors(8) . Increased protein levels are due to an increase in the fractional rate of protein synthesis in the absence of changes in the rate constants of protein degradation. The elevated RNA content represents, for the most part, an accrual of ribosomes, i.e. ribosomal RNA (rRNA), which is necessary to support the elevated cellular capacity to produce protein(9) . However, little, if anything, is known about the signals which couple contraction to ribosome biogenesis and subsequent protein synthesis.
In other
mammalian cell types and other classes of organisms, ribosome content
is regulated largely by alterations in the rate of transcription of the
rRNA genes (rDNA) to produce 45 S preribosomal RNA, rather than by
changes in the processing or stability of ribosomal RNA
(rRNA)(10, 11) . Similarly, the accelerated levels of
ribosome biogenesis observed during contraction-induced hypertrophy of
cultured neonatal cardiomyocytes is linked to elevated rates of rRNA
synthesis(12) . Increased rDNA transcription also accounted for
the accelerated rates of ribosome biogenesis that follow exposure to
norepinephrine(13) , phorbol 12-myristate
13-acetate(14) , or endothelin I. ()Overall, these
results emphasize that rDNA transcription is a central point in the
control of cardiomyocyte protein synthesis during cardiac growth.
Efficient transcription of ribosomal DNA promoters in vitro requires at least two DNA-binding proteins, UBF and SL-1, as well
as RNA polymerase I(10, 11, 15) . In
addition, recognition by RNA polymerase I of the protein-DNA complexes
that form on the promoter requires the presence or activity of at least
one polymerase-associated factor referred to as either TF1C or
TIF-1A(16, 17) . SL-1 is absolutely required for basal
levels of transcription in vitro(10, 11) ,
and UBF increases the efficiency of template utilization raising the
level of transcription(10, 11, 18) . When
fractionated by SDS-PAGE, ()mammalian UBF consists of two
proteins, 97 and 94 kDa, that are referred to as UBF1 and UBF2,
respectively(10, 11, 20) . They are coded for
by two different mRNAs that result from alternative processing of a
single transcript(17) . UBF1 and UBF2 bind to DNA and form
homo- and heterodimers, but only UBF1 has been shown to activate
transcription in vitro(19, 21, 22) .
The activity of UBF is regulated by post-translational and/or transcriptional mechanisms(23, 24, 25, 26) . In regard to the former mechanism, UBF is a phosphoprotein whose ability to activate transcription is reduced when dephosphorylated(23, 24) . When CHO cells were serum-starved, and rDNA transcription significantly reduced, the phosphorylation state of UBF also decreased(23) . In contrast, the decreased rDNA transcription observed during serum starvation of NIH3T3 fibroblasts was characterized by decreased UBF content. In this example, the decrease in UBF protein was preceded by a reduction in UBF mRNA content(26) .
We have examined the rDNA transcription apparatus of contraction-induced hypertrophic cardiomyocytes in order to determine which components of this system are altered in response to the hypertrophic stimulus. We show here that the accelerated rate of rDNA transcription during contraction-mediated neonatal cardiomyocyte hypertrophy is not accompanied by increased amounts or activity of RNA polymerase I enzyme, but by a significant increase in the protein and mRNA levels of UBF. Nuclear run-on assays of UBF mRNA synthesis revealed that at least part of the increase in UBF content was the result of increased transcription of the UBF gene. Transient transfection of chloramphenicol acetyltransferase (CAT) reporter constructs linked to fragments 5` of the translation initiation site of the UBF gene demonstrated the presence of contraction response elements between -1189 and -665 relative to the start of transcription (+1).
Figure 6:
Analysis of UBF promoter activity by
transient expression in arrested and contracting cardiomyocytes. A, neonatal cardiomyocytes were transfected with either pCAT0 (lanes 1 and 2), pCATUB .75 (lanes 3 and 4), or pCATUB 3.2 (lanes 5 and 6), and
either arrested by the addition of 50 mM KCl to the serum-free
defined media (lanes 1, 3, and 5) or allowed
to contract (lanes 2, 4, and 6). Twenty-four
hours later, extracts were made from the cardiomyocytes and assayed for
CAT activity. B, neonatal cardiomyocytes were co-transfected
with the indicated constructs of the UBF promoter linked to the
bacterial chloramphenicol acetyltransferase gene and the pCMV-Gal
construct and treated as described in A. Twenty-four hours
after transfection, cells were harvested, and extracts were prepared
and assayed for CAT and
-galactosidase activity. The CAT activity
in each extract was normalized for the recovery of
-galactosidase
activity to correct for differences in transfection efficiency. The
normalized activity of pCATUB 3.2 in arrested cardiomyocytes is
indicated as 100%. The normalized activities of the remainder of the
constructs are shown as the percentage of pCATUB 3.2. The results
depicted are the average of 3 separate experiments ± S.D.** and
*, significant difference from control, p < 0.001 and 0.05,
respectively.
Figure 1:
Contraction regulates protein
accumulation and rDNA transcription in neonatal cardiomyocytes. A, at the times indicated, total cellular protein was
determined for cultures of contraction-arrested or spontaneously
contracting neonatal cardiomyocytes and normalized for the DNA content
per plate. Results represent 7 separate experiments and are expressed
as the average percentage increase ± S.D. in the protein to DNA
ratio of contracting cells as compared to control (arrested) cells. *,
significant difference from control cells, p < 0.001. B, neonatal cardiomyocytes, cultured as indicated, were
contraction-arrested or allowed to spontaneously contract for
6-72 h, and nuclei were isolated from 16 10
cells per time point. Ribosomal DNA transcription was measured as
described under ``Materials and Methods.'' Radiolabeled rRNA
transcripts were purified from the reaction mixture and hybridized to
45 S rDNA (clone pU5.1E/X) or control pUC19 DNA. After stringent
washes, the hybrids were visualized by autoradiography (see inset). The radioactivity of 45 S run-on transcripts obtained
from 7 separate experiments was quantified by laser densitometry and is
presented as the average percentage increase ± S.D. in rDNA
transcription in response to contraction over the transcription
observed in contraction-arrested cells. *, significance from control
cells, p < 0.001.
To initiate hypertrophy, the cardiomyocytes were allowed to resume spontaneous contraction by reduction of the concentration of KCl in the culture medium from 50 mM to 5 mM. Within 2 h of the medium change, the cardiomyocytes were contracting, and, after 3 days, the contracting cardiomyocytes had accumulated 39% more protein than control, contraction-arrested cells (Fig. 1A). The increases in protein content of the cells occurred in the absence of changes in DNA content indicating that the growth was due to hypertrophy rather than hyperplasia. These results are in good agreement with those reported previously(8, 9) .
In order to characterize the rates of ribosome biogenesis in our cultures, we used nuclear run-on analysis to measure the rate of ribosomal DNA (rDNA) transcription in contractile-arrested and spontaneously contracting neonatal cardiomyocytes. Results presented in Fig. 1B demonstrate that the rates of rDNA transcription in nuclei derived from spontaneously contracting neonatal cardiomyocytes were greater than those observed in nuclei obtained from time matched contractile-arrested cardiomyocytes. Significant increases in the rates of rRNA synthesis were observed within 12 h following initiation of contraction (159% ± 13) and reached maximal levels within 24-48 h (200% ± 21, Fig. 1B). These results confirm previous studies (8, 9, 12) indicating that contraction-induced hypertrophy of neonatal cardiomyocytes is associated with significant increases in the rate of synthesis of rRNA as the result of accelerated rates of transcription of the 45 S preribosomal DNA.
The relative enzymatic activity of total solubilized RNA polymerase
I (as determined by its ability to initiate nonspecific transcription
on calf thymus DNA) was determined in F3 extracts obtained from
arrested and spontaneously contacting neonatal cardiomyocytes as
described under ``Materials and Methods.'' The activity of
RNA polymerase I extracted from cardiomyocytes that had been
contracting for 24 h was not significantly different from that
extracted from contraction-arrested cells (106% ± 3.8, n = 3). Similarly, no significant differences were observed
in the total polymerase (106% ± 5.8) or polymerase II (103%
± 16) activities in contracting cells with respect to
contraction-arrested cells. Western analyses, using antibodies raised
to the ` subunit of RNA polymerase I (rPolI
`) (13) ,
indicated that the levels of the
` subunit of RNA polymerase I
were essentially the same in contractile-arrested neonatal
cardiomyocytes (Fig. 2A, lanes 1 and 3) and cells growing in response to spontaneous contraction (Fig. 2A, lanes 2 and 4). The results
from a number of separate experiments were quantified by laser
densitometry and are presented in Fig. 2B.
Figure 2:
Western analysis of RNA Polymerase I
` subunit (rPolI
`) protein levels in contraction-arrested and
spontaneously contracting neonatal cardiomyocytes. A, total
cellular protein was extracted at the times indicated from
contraction-arrested, neonatal cardiomyocytes (arrested, lanes 1 and 3) or from spontaneously contracting (Contract., lanes 2 and 4). After SDS-PAGE
and Western transfer, rPolI
` was detected with rabbit
anti-rPolI
` antisera and visualized by ECL as described under
``Materials and Methods.'' Equal amounts of protein (25
µg) were loaded per lane. B, the results of 3 or
more experiments similar to those presented in A were
quantified by laser densitometry and are presented as the average fold
increase ± S.D. in rPolI
` levels in spontaneously
contracting neonatal cardiomyocytes over the level found in control
cells. Note, the levels of rPolI
` are not significantly different
between arrested and contracting cardiomyocytes at any time point
examined.
In many cell lines, the cellular content of UBF is proportional to the rate of rDNA transcription(25, 26) . Accordingly, we determined whether the accelerated rates of rDNA transcription observed in spontaneously contracting neonatal cardiomyocytes might also be characterized by changes in the levels of UBF. As shown in Fig. 3A, UBF1 and UBF2 protein levels were significantly greater in rapidly contracting cardiomyocytes than in time-matched contractile-arrested cells. The UBF protein levels were maximal after 12 h of contraction (3.5-4.5-fold) and remained at similar levels for up to 72 h. The increases in UBF protein levels cannot be explained by the general increase in cellular protein observed in response to contraction, because equal amounts of protein were loaded per lane. The results from a number of separate experiments were quantified by laser densitometry and are presented in Fig. 3B.
Figure 3:
Western analysis of UBF protein levels and
phosphorylation status in contraction-arrested and spontaneously
contracting neonatal cardiomyocytes. A, total cellular protein
was extracted at the times indicated from contraction-arrested,
neonatal cardiomyocytes (arrested, lanes 1, 3, and 5) or from spontaneously contracting (Contract., lanes 2, 4, and 6). After SDS-PAGE and
Western transfer, UBF was detected with rabbit anti-UBF antisera and
visualized by ECL as described under ``Materials and
Methods.'' Equal amounts of protein (25 µg) were loaded per
lane. B, the results of 3 or more experiments similar to those
presented in A were quantified by laser densitometry and are
presented as the average fold increase ± S.D. in UBF levels in
spontaneously contracting neonatal cardiomyocytes over the level found
in control cells.***,**, and *, significance from control cells, p < 0.001, 0.01, and 0.05. respectively. C,
contraction-arrested (lanes 1 and 3) and
spontaneously contracting (lanes 2 and 4) neonatal
cardiomyocytes were metabolically labeled with
[P]orthophosphate as described under
``Materials and Methods.'' After 12 h, the cells were
harvested, lysed, and UBF-immunopurified using anti-UBF antisera bound
to protein A-agarose beads. Immunopurified UBF was removed from the
beads and resolved by SDS-PAGE. The immunoprecipitated UBF (lanes 3 and 4, Ppt.) and the resultant supernatants (lanes 1 and 2, Sup.) were subsequently
visualized directly by autoradiography (Autorad.) or
Western-blotted and probed with anti-UBF antisera (Western).
Each lane contains UBF immunoprecipitated from equivalent numbers of
cells (4
10
) per treatment. Lanes 1 and 2 and lanes 3 and 4 of the autoradiogram
were exposed for 5 h and 3 days,
respectively.
Figure 4:
Contraction induces a sustained increase
in UBF mRNA levels and UBF gene transcription in neonatal
cardiomyocytes. A, after the times indicated, total RNA was
extracted from contraction-arrested (lane 1) or spontaneously
contracting (Contract., lanes 2-5) neonatal
cardiomyocytes cultured at 4 10
cells/60-mm plate.
Following electrophoresis and transfer to Zeta-Probe membranes, the RNA
(30 µg/lane) was hybridized to
P-labeled UBF cDNA, and
the UBF mRNA transcripts were visualized by autoradiography. B, the results of 3-5 experiments similar to those shown
in A were quantified and are expressed as the average fold
increase ± S.D. in UBF mRNA levels found in contracting cells
over the level found in contraction-arrested cells. ***,**, and *
denote significant differences from control cells, p <
0.001, 0.01, and 0.05, respectively. C, neonatal
cardiomyocytes, cultured as indicated, were contraction-arrested or
allowed to spontaneously contract for 8 h, and nuclei were isolated
from 16
10
cells per time point. Nuclear run-on
assays were carried out as described under ``Materials and
Methods.'' Radiolabeled transcripts were purified from the
reaction mixture and hybridized to DNA probes for
-tubulin mRNA
and UBF mRNA or control pUC19 DNA as described under ``Materials
and Methods.'' After stringent washes, the hybrids were visualized
by autoradiography. The values are means ± S.D. of 3 separate
experiments. *, significant difference from control, p <
0.01.
Within 3 h of the
onset of contraction, the amount of UBF mRNA began to rise (Fig. 4A, lane 3). After 12 h of contraction,
the UBF mRNA levels were maximal and remained elevated for up to 3 days (Fig. 4B). This temporal pattern of UBF mRNA induction
parallels that observed for the elevation in UBF protein in response to
contraction. When similar blots were probed for mRNA encoding
glyceraldehyde-3-phosphate dehydrogenase, no significant increases were
observed (see Fig. 5A for an example). The kinetics of the
increase in UBF mRNA levels indicated that at least part of this
response might reflect increased expression of the UBF gene. Indeed,
nuclear run-on assays demonstrated that nuclei isolated from cells that
had been contracting for 12 h demonstrated a 4.5 ± 0.5-fold (n = 3) greater rate of UBF gene transcription than nuclei
isolated from time-matched control cells (Fig. 4C). The
specificity of the hybridization was demonstrated by the lack of
hybridization of the de novo synthesized transcripts to
control pUC19 DNA (Fig. 4C). Under the same conditions,
transcription of the -tubulin gene was unchanged, indicating that
the transactivation of the UBF gene was not merely the result of a
global increase in general transcription (Fig. 4C).
Figure 5:
Cardiac non-cardiomyocytes do not mediate
contraction-induced increases in UBF expression in cardiomyocytes. A, total RNA was isolated from cultures of either normally
purified (lanes 1 and 3) or Percoll gradient purified (Perc., lanes 2 and 4) cardiomyocytes that
had been either maintained in the arrested state (lanes 1 and 2) or had been allowed to spontaneously contract (Contr.,
lanes 3 and 4) for 12 h. Following electrophoresis and
blotting, the RNA (30 µg/lane) was hybridized to P-labeled UBF cDNA (upper panel) or
glyceraldehyde-3-phosphate dehydrogenase cDNA (lower panel). B, the results of 3 or more experiments similar to those
described in A were quantified and expressed as the average
fold increase ± S.D. in UBF mRNA levels found in contracting
cells over the level found in contraction-arrested cells. *,
significant difference from control cells, p < 0.001. C, after 12 h, total RNA was extracted from
contraction-arrested neonatal cardiomyocytes (lane 1),
spontaneously contracting neonatal cardiomyocytes treated with vehicle (Contract., lane 23) or spontaneously contracting neonatal
cardiomyocytes treated with DuP 753 (DuP 753, 1 µM).
Following electrophoresis and Northern blotting, the RNA (30 µg)
was hybridized to
P-labeled UBF cDNA, and the UBF mRNA
transcripts were visualized by autoradiography. The experiment was
repeated 3 times, and a representative autoradiogram is
shown.
To distinguish between these possibilities, we measured the hypertrophic response of contracting cardiomyocytes purified by centrifugation through Percoll gradients(28) . The results presented in Fig. 5demonstrate that when cardiomyocytes purified in this manner were allowed to spontaneously contract, the induction of UBF mRNA (Fig. 5A, lane 3) was not significantly different from that observed in the standard cardiomyocyte preparations (Fig. 5A, lane 4). The results from 3-5 separate experiments were quantified and are presented in Fig. 5B. Thus, the alteration of UBF expression observed following contraction occurred in the hypertrophying cardiomyocyte population and required neither additional cells types nor the permissive action of paracrine growth factors from the cardiac non-cardiomyocytes. Moreover, under the same conditions, the mRNA levels of the ``housekeeping'' gene glyceraldehyde-3-phosphate dehydrogenase were not altered in response to contractile activity (Fig. 5A, lanes 1-4, lower panel), further demonstrating that the increase in UBF mRNA following initiation of contraction was not simply part of a nonspecific transcription response.
The physiological
parameters of spontaneous contraction have not been defined, but they
are likely to include both passive stretch and increased tension. It
has been suggested that increased cardiomyocyte growth and some
alterations in gene expression induced by passive stretch may be, at
least partly, mediated by the autocrine release and action of
angiotensin II(39) . Accordingly, the possibility that
angiotensin II might mediate the contraction-induced increases in
cardiomyocyte growth and UBF expression in the present experiments was
considered. The ability of contraction to augment protein accumulation
after 48 h was the same in the presence and absence of the specific
angiotensin II receptor antagonist, DuP 753 (1 µM), 137%
and 135% of control, respectively. DuP 753 also failed to prevent
contraction-induced increases in UBF mRNA (Fig. 5C) or
protein (results not shown). Similar results were obtained with the
alternative angiotensin II antagonist
[Sar,Ile
]angiotensin II (1
µM). Both of these antagonists abolished short-term,
angiotensin II-mediated increases in MAP kinase activity in
contraction-arrested cardiomyocytes, indicating that they were active
at the concentrations used (data not shown). Therefore, it is unlikely
that the ability of contractile activity to modulate growth and UBF
expression requires the permissive action of angiotensin II in our
neonatal cardiomyocyte culture system.
Increased ribosome biogenesis is essential to the accumulation of protein during neonatal cardiomyocyte hypertrophy(9, 12, 34, 40) . This process is regulated largely as the result of alterations in the transcriptional rate of the ribosomal RNA genes (rDNA)(12, 40) . However, the molecular signals and pathways by which hypertrophic stimuli effect an increase in the rate of rDNA transcription in these cells are not known. For instance, it is possible that diverse hypertrophic stimuli augment rDNA transcription by activating common regulatory factor(s). Alternatively, they might act through divergent signaling pathways culminating in the activation of rDNA transcription through distinctly different sets of activators.
We have reported previously that the accelerated rDNA transcription
associated with norepinephrine-induced cardiomyocyte hypertrophy was
accompanied by increased cellular levels of the rDNA transcription
factor UBF(13) . We hypothesized that increased amounts and/or
post-translational modification of UBF, i.e. increased UBF
activity, might be a common mechanism by which diverse hypertrophic
stimuli effect changes in rDNA transcription in cardiomyocytes. In
order to address this question, an alternative model of cardiac
hypertrophy was examined, one in which the cardiomyocytes were
hypertrophying in response to spontaneous
contraction(6, 7, 8) . Specifically, we have
compared the levels and enzymatic activity of RNA polymerase I and the
content of the rDNA transcription factor, UBF, in contraction-arrested
and contracting cultured neonatal cardiomyocytes. We have found that
concomitant with contraction-induced increases in rDNA transcription,
the cardiomyocyte level of UBF also increased in the absence of changes
in the amounts or activity of RNA polymerase I. Immunoprecipitation of P-orthophosphorylated UBF demonstrated that the
accumulated UBF was phosphorylated and thus transcriptionally active.
Northern blots and nuclear run-on assays revealed that the accumulation
of UBF was at least partially regulated at the level of transcription
of the UBF gene. Further, transient transfection assays demonstrated
that the UBF gene contained an element that could direct increased
levels of transcription in response to contraction.
UBF can stimulate rDNA transcription in in vitro transcription reactions(10, 11, 18) . In
preliminary studies, we have found that the overexpression of UBF1 in
primary neonatal cardiomyocyte cultures increases transcription from a
co-transfected rDNA promoter in a dose-dependent manner. ()When considered together with these findings, our present
data strongly implicate changes in the amount of UBF as a common
mechanism by which diverse hypertrophic stimuli effect increases in
rDNA transcription in neonatal cardiomyocytes.
An alternative approach to this problem would start
with the analysis of the UBF promoter and the determination of the cis-acting elements and trans-acting factors
responsible for increased levels of transcription in response to
contraction. The UBF gene has been cloned, and the region immediately
5` to the transcription initiation site has been
sequenced(37) . ()In the studies described here, a
UBF promoter/CAT chimeric gene transfected into neonatal cardiomyocytes
responded to contraction in a quantitatively similar manner as the
endogenous gene. Moreover, we have identified a region between
-1189 and -665 relative to the predicted start of
transcription of the UBF gene which contains an element(s) that
mediates the response to contraction. Further studies using this
approach will allow us to define the specific cis-acting
elements within this region that regulate contractile-mediated UBF
expression and to determine the respective trans-acting
factors which bind to those sequences. These studies should put us one
step closer to elucidating the signal transduction pathways that link
the cascade initiated by contraction with accelerated rDNA
transcription during neonatal cardiomyocyte hypertrophy.