©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of rDNA Transcription Factors during Cardiomyocyte Hypertrophy Induced by Adrenergic Agents (*)

(Received for publication, October 27, 1994; and in revised form, January 4, 1995)

Ross D. Hannan (§) Joachim Luyken (¶) Lawrence I. Rothblum (**)

From the Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ribosomal DNA transcription is important to the regulation of cardiomyocyte ribosome content and, as a consequence, the rate of protein synthesis and accumulation during cardiac hypertrophy. We studied the regulation of ribosomal RNA synthesis and the levels of RNA polymerase I and the ribosomal DNA transcription factor, UBF, during norepinephrine-induced hypertrophy of contraction-arrested neonatal cardiomyocytes in culture. Nuclear run-on assays and Western blots demonstrated that, concomitant with hypertrophy, norepinephrine (1 µM) increased the rate of ribosomal DNA transcription, without causing an increase in the amount of RNA polymerase I. However, the elevated rate of rRNA synthesis was accompanied by an increased cellular content of UBF protein as determined by Western analysis. Northern blots demonstrated norepinephrine-induced increases in UBF mRNA in neonatal cardiomyocytes indicating that the response was regulated, at least in part, at the pretranslational stage. Both alpha- and beta-adrenergic agents increased the level of UBF mRNA. The beta-adrenergic response was mimicked by forskolin (1 µM) and the cyclic AMP analog dibutyryl cAMP (10 µM). However, activation of protein kinase C by phorbol 12-myristate 13-acetate (0.1 µM) did not increase expression of UBF. These results implicate UBF as a possible regulatory factor of the accelerated rDNA transcription observed during norepinephrine-mediated cardiomyocyte hypertrophy.


INTRODUCTION

The cardiomyocytes of the adult heart respond to growth stimuli by increasing in size in the absence of cellular division (hypertrophy). A variety of model systems have been employed to examine myocyte hypertrophy. Using these systems, the stimuli which either directly or indirectly modulate cardiomyocyte growth have been identified. These include mechanical stimuli such as active or passive stretch, hormones such as norepinephrine (NE), (^1)endothelin, angiotensin, or thyroid hormone, and various peptide growth factors(1, 2) . One finding common to these reports is that cardiomyocyte growth results mainly from an increase in general protein synthesis and total RNA accumulation in the absence of changes in the fractional rate constants of protein and RNA degradation(3) . Experiments in several systems indicate that the rate of general protein synthesis is limited by the protein synthetic capacity of the cardiomyocytes, i.e. the cellular ribosome content(4, 5, 6) . This suggests that ribosome biogenesis is the major rate-limiting step in the accumulation of protein during hypertrophy. In fact, McDermott et al.(6, 7) demonstrated that the increased rates of ribosomal RNA (rRNA) synthesis during beatinginduced hypertrophy of cultured, neonatal cardiomyocytes resulted from elevated transcription of the rRNA genes (rDNA) that encode the 45 S precursor of the 18 S, 5.8 S, and 28 S rRNAs. Taken together, these studies implicate transcription of the ribosomal RNA genes (rDNA transcription) as a control point in the regulation of cardiomyocyte protein synthesis and consequently cardiac growth. As ribosomal DNA transcription constitutes approximately 40-60% of the total nuclear RNA synthesis, even a relatively small increase in this activity would represent a significant increase in total cellular protein synthetic capacity. Therefore, the elucidation of the molecular mechanisms by which rDNA transcription is regulated will be of significant importance to the general understanding of cardiac hypertrophy during both normal and disease states.

Despite species-specific variations in their utilization, mammalian cells appear to utilize a set of highly homologous, if not identical, factors for transcription of the ribosomal DNA genes(8, 9) . At least two DNA-binding proteins, referred to as UBF and SL-1, as well as RNA polymerase I, are required for efficient transcription of ribosomal DNA promoters in vitro. The ability of RNA polymerase I to recognize the protein-DNA complexes which form on the promoter requires the presence or activity of at least one polymerase-associated factor referred to as either Factor C*, TF-1C, or TIF-IA. SL-1 is absolutely required for transcription, while UBF appears to be an auxiliary transcription factor. That is, UBF is not required for basal levels of transcription in vitro(10) . However, the addition of UBF to an in vitro transcription system increases the efficiency of template utilization(11) , and over-expression of UBF in NIH3T3 cells leads to increased expression of a co-transfected reporter gene under the control of the rDNA promoter(12) . UBF is a dimeric, DNA-binding protein. SDS-PAGE analysis of UBF purified from mammalian cells demonstrates two proteins of molecular sizes 97 and 94 kDa, referred to as UBF1 and UBF2, respectively(8, 9) . These two proteins are coded for by two different mRNAs, which result from alternative processing of a single transcript(13) . Both UBF1 and UBF2 can bind to DNA and form homo- and heterodimers. However, only UBF1 has been shown to activate rDNA transcription(10, 12) .

UBF is subject to regulation by at least two different mechanisms. O'Mahony et al.(14, 15) and Voit et al.(16) demonstrated that UBF is a phosphoprotein and that the activity of UBF was reduced when the protein was dephosphorylated in vitro. Further, our laboratory demonstrated that when CHO cells were serum-starved, and rDNA transcription significantly reduced, the phosphorylation state of UBF was also reduced(15) . We have also found that cells might regulate UBF and, by analogy, the rate of rDNA transcription, by altering the cellular content of UBF. When L6 myoblasts are induced to differentiate, there is a decrease in the rate of rDNA transcription of approximately 80%(17) . The amount of UBF present in these cells decreased in parallel with the decrease in the rate of rDNA transcription. Interestingly, the level of the UBF mRNA decreased with more rapid kinetics, suggesting that at least one rate-limiting step in regulating rDNA transcription during this process was transcription of the UBF gene(17) .

In the studies presented here, we have examined rDNA transcription and the levels of both the RNA polymerase I beta` subunit and an rDNA transcription factor, UBF, during norepinephrine-induced cardiomyocyte hypertrophy. Specifically, we sought to 1) characterize the changes in ribosomal transcription during norepinephrine-induced cardiac hypertrophy, 2) assess whether the levels of RNA polymerase I and/or UBF protein were increased in response to norepinephrine treatment, and, if so, 3) determine whether the quantitative and temporal patterns of expression of these proteins were consistent with the hypothesis that they might be involved in the modulation of the observed increases in rDNA transcription and consequent protein accumulation.

We show here that when neonatal cardiomyocytes undergo hypertrophy as the result of norepinephrine treatment, they exhibit increased rates of rDNA transcription as well as increased amounts of UBF protein. In contrast, the amount of RNA polymerase I (reflected by the levels of the polymerase I beta` subunit) was not altered by norepinephrine. The increased UBF protein levels were regulated at least partially at the pretranslational level since cardiomyocytes also demonstrated transient increases in UBF mRNA. Both alpha- and beta-adrenergic agents increased the level of UBF mRNA. The beta-adrenergic response could be mimicked by forskolin and cyclic AMP (cAMP) analogs. On the other hand, activation of protein kinase C by phorbol 12-myristate 12-acetate (PMA), which has been implicated in the alpha-adrenergic response(1, 2) , did not result in increased levels of UBF. These represent the first experiments linking stimulation of cardiomyocyte growth to the induction of a factor known to be intimately involved in rDNA transcription. These results are consistent with the hypothesis that UBF is an important regulatory factor in ribosome biogenesis during norepinephrine-induced neonatal cardiomyocyte growth.


MATERIALS AND METHODS

Cardiomyocyte Culture

Neonatal cardiomyocytes were isolated from the ventricles of day-old Sprague-Dawley rat pups by digestion with trypsin, chymotrypsin, and elastase as described previously(5) . After preplating for 1.5 h to remove fibroblasts, cardiomyocytes were plated on gelatin-coated plates either at 0.4 times 10^6 cells/60-mm culture plate (low density) or at 4 times 10^6 cells/60-mm culture plate (high density) and allowed to attach overnight in minimal Eagle's media containing 10% newborn calf serum and 0.1 mM 5-bromodeoxyuridine at 37 °C in an atmosphere of 5% CO(2), 95% air. The following day, the cells were washed three times in defined serum-free media containing 0.1 mM bromodeoxyuridine (18) and maintained in the same defined media for the duration of the experiments. KCl (50 mM) was added to the medium to prevent the spontaneous contraction characteristic of neonatal cardiomyocytes plated at high density(7) . Cells plated at low density (0.5 times 10^6/60-mm plate) did not spontaneously contract, in accord with previous studies (19) . Cultures prepared in this manner consisted of 85-95% cardiomyocytes. All experiments were initiated 3 days following the cardiomyocyte dispersion.

Induction of Cardiomyocyte Hypertrophy

After 2 days in defined media (day 3 of culture), hypertrophy was initiated by the addition of norepinephrine (1 µM) in phosphate-buffered saline (PBS) containing 0.1% ascorbic acid. Control cells were given vehicle alone. When the cardiomyocytes were treated for more than 24 h, the medium containing either norepinephrine or vehicle was replaced daily. After the appropriate length of treatment, cells were harvested and processed for the determination of protein and DNA or for Northern, Western, or nuclear run-on assays. Adrenergic agonists and antagonists were prepared immediately before use in 0.1% ascorbic acid. Forskolin and PMA were prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide added to the culture was less than 0.001%. When the beta-adrenergic antagonist (L-propranolol, 5 µM) was used in combination with norepinephrine, the agent was added to the culture medium 2 h prior to the addition of norepinephrine.

Protein and DNA Determination

Cardiomyocytes were released from the plates by treatment with trypsin-EDTA, washed three times in ice-cold PBS, and solubilized directly in 1 times SSC (0.15 M NaCl, 15 mM sodium citrate), 0.25% SDS, and frozen at -20 °C until analysis. DNA determinations were performed by the fluorometric method of Cesarone et al.(20) using calf thymus DNA as a standard. Protein was assayed using the Bio-Rad DC assay kit and immunoglobulin G (IgG) as the protein standard. Growth was expressed as the percentage increase in the protein-to-DNA ratio of norepinephrine-treated cells over time as compared to time-matched control cells. Experiments were repeated a minimum of five times from separate cardiomyocyte preparations.

RNA Extraction and Northern Analysis

Total RNA was extracted from cardiomyocytes as described(21) . RNA (30 µg/lane) was denatured in 50% formamide (22) by heating at 65 °C for 10 min. Following electrophoresis through 1.2% denaturing (formaldehyde) agarose gels(22) , the RNA was blotted onto Zeta-Probe (Bio-Rad) membranes according to the manufacturer's instructions. The quantity of RNA loaded and transferred was verified by staining the gels and membranes with ethidium bromide before and after transfer. cDNA probes specific for UBF were labeled to a specific activity of 10^9 cpm/µg using random priming(23) . Hybridization was carried out as described(24) . The UBF probe used was a 1.2-kilobase polymerase chain reaction fragment of the rat UBF cDNA which hybridizes to both UBF-1 and UBF-2(13) . Results were quantified using either a laser densitometer (Molecular Dynamics) or an Ambis 4000 radioanalytic imager (Ambis Systems). Experiments were repeated at least five times, and representative autoradiograms are shown.

Western Analysis

Cardiomyocytes were rinsed three times in ice-cold PBS and then scraped directly into 1 times SSC, 0.25% SDS, and stored at -20 °C until analysis. Protein and DNA determination was performed as described above. Equal amounts of protein (20 µg/lane) were denatured in Laemmli solubilizing buffer(25) , electrophoresed on 8% polyacrylamide-SDS gels (19:1, acrylamide/bisacrylamide), and electroblotted onto Immobilon-P membranes (Millipore). To verify that equal amounts of protein had been loaded per lane, the same samples were electrophoresed on a parallel gel, and the gel was stained with Coomassie Brilliant Blue. The membranes were rinsed thoroughly with PBS, and UBF and RNA polymerase I beta` proteins were detected by incubating the membranes with either polyclonal rabbit anti-UBF antisera (15) or polyclonal antisera raised to the rat RNA polymerase I beta` subunit (^2)diluted 1:10,000 in Buffer B (PBS, 5% milk powder, and 0.1% Tween 20). After 1 h, the membranes were washed 3 times 15 min in Buffer B and then exposed to a goat anti-rabbit IgG conjugated to horseradish-peroxidase (1:2000, Sigma) in Buffer B for 1 h. After 3 times 15 min rinses in PBS, UBF and RNA polymerase I beta` protein bands were visualized by the Enhanced Chemiluminescent (ECL) method (Amersham). Western blots were quantitated by laser densitometry (Molecular Dynamics). The molecular sizes of the immunodetected proteins were verified by comparison to the migration of standard protein markers (Bio-Rad) electrophoresed in parallel lanes. Experiments were repeated at least five times from separate cardiomyocyte isolations, and representative Western blots are shown.

Nuclear Run-on Transcription

Transcription from the rDNA promoter in isolated cardiomyocyte nuclei was measured by the hybridization of in vitro synthesized, P-labeled run-on transcripts to a 45 S rDNA clone (pU5.1E/X) as described previously(17) . Briefly, cells (16 times 10^6 per treatment) were released from the plates by trypsinization, washed twice in ice-cold PBS, and incubated on ice for 10 min in 10 ml of nuclear isolation buffer(25) . Nuclei were collected by centrifugation at 500 times g for 10 min, washed in ice-cold nuclear isolation buffer, and resuspended in 200 µl of nuclear storage buffer(25) . A 100-µl aliquot was removed for the determination of DNA. Run-on transcription assays, in the presence of 100 µg/ml alpha-amanitin, and the isolation of de novo synthesized RNA were carried out as described(17) . The isolated RNA was resuspended in 100 µl of TE, and unincorporated nucleotides were removed by centrifugation through Pharmacia S-400 HR columns. To control for RNA recovery, in vitro-transcribed, ^3H-labeled actin RNA was added to the run-on assays prior to extraction and purification, and the amount of [^3H]RNA recovered was quantitated by liquid scintillation spectrometry.

Specific rDNA transcription was determined by the hybridization of run-on transcripts to 45 S rDNA (clone pU5.1E/X) immobilized on Zeta-Probe nylon membranes. The hybridization conditions and the post-hybridization washes were the same as described(24) . Control pUC19 template was also immobilized on the filters to control for nonspecific hybridization. Hybridization was detected by autoradiography and quantified using a laser densitometer (Molecular Dynamics) and an Ambis 4000 radioanalytic imager (Ambis Systems).

Preparation of Figures

Autoradiograms obtained from Western, Northern, and nuclear run-on analyses were scanned and converted to 8-bit TIFF Bitmapp files using a laser densitometer and ImageQuant software (Molecular Dynamics). The TIFF Bitmapp files were subsequently imported directly and unmodified into Corel Draw! where labels and headings were attached. Completed figures were output to print film on a Montage FRL Lasergraphics.


RESULTS

Induction of Cardiac Hypertrophy and rDNA Transcription

Our experiments were designed to examine the hypothesis that the elevated rate of rDNA transcription observed during norepinephrine-induced cardiomyocyte hypertrophy would be associated with increased levels of RNA polymerase I and/or the rDNA transcription factor, UBF. As a first step in this process, we characterized both the growth and the level of rDNA transcription in control and norepinephrine-treated neonatal cardiomyocytes.

Neonatal cardiomyocytes were prepared and cultured at a relatively high density (4 times 10^6 cells/60-mm dish) in the presence of 50 mM KCl as described under ``Materials and Methods.'' Experiments were initiated after 3 days in culture, to allow the cardiomyocytes to adapt to the culture conditions(6, 7) . During the subsequent 4 days in culture, the control cardiomyocytes demonstrated no significant growth. The protein to DNA ratios of control cultures varied by 4.3% ± 2.9 over this time period. These findings are in accordance with those obtained by others using similar culture conditions(6, 7) . However, cells plated at high density and exposed to 1 µM norepinephrine for 1-4 days demonstrated a significant accumulation of total cellular protein, as reflected by increased protein to DNA ratios with respect to control cells. After 2, 3, and 4 days of treatment with norepinephrine, total cellular protein levels had increased by 27.9% ± 4.9, 25.71% ± 4.33, and 25.89% ± 5.72 respectively, as compared to time-matched control cells. Treatment with norepinephrine did not significantly increase the DNA content of the cardiomyocytes (data not shown) indicating that growth was the result of hypertrophy rather than hyperplasia. The growth of these cardiomyocytes was not due to norepinephrine-induced contractile activity since the cardiomyocytes were contraction-arrested with 50 mM KCl.

Similar increases in total protein and protein to DNA ratios (31.2% ± 5.6 increase in protein to DNA ratio after 4 days) in response to norepinephrine were observed in nonbeating cardiomyocytes plated at a relatively low density (0.5 times 10^6 cells/60-mm plate). These results are in agreement with the reports of others(18, 26) .

In order to confirm that the norepinephrine-induced increases in cellular protein levels were accompanied by increased 45 S rRNA synthesis, we measured the rate of rDNA transcription in nuclei isolated from control and norepinephrine-stimulated neonatal cardiomyocytes. The results presented in Fig. 1A demonstrate that the rate of RNA polymerase I transcription in nuclei derived from norepinephrine-treated neonatal cardiomyocytes maintained at either high density or low density was significantly greater than that derived from control cells. Further, the increased RNA polymerase I transcriptional activity in nuclei isolated from norepinephrine-treated neonatal cardiomyocytes maintained at low density was independent of contractile activity since inclusion of KCl (50 mM) in the media did not significantly reduce the norepinephrine-induced increase in the hybridization signal (Fig. 1A, slots 4 and 5). The specificity of hybridization was verified by the lack of hybridization to pUC19 DNA. The maximal increase in the rate of rDNA transcription occurred after 24-48 h of continuous norepinephrine treatment (57% ± 10.5 and 58% ± 8.8, respectively) and remained significantly above basal levels (49% ± 10.1) after 3 days of exposure to the agent (Fig. 1B).


Figure 1: rDNA transcription in cultured neonatal cardiomyocytes following norepinephrine (NE) stimulation. A, neonatal cardiomyocytes, cultured as indicated, were stimulated for 6-72 h with NE (1 µM), and nuclei were isolated from 16 times 10^6 cells per time point. When indicated, KCl was added to a final concentration of 50 mM. RNA polymerase I transcription was measured by incubating the isolated nuclei at 37 °C in the presence of [P]UTP and alpha-amanitin as described under ``Materials and Methods.'' Radiolabeled rRNA transcripts were purified from the reaction mixture and hybridized to 45 S rDNA (clone pU4.5E/X) or control pUC19 DNA, and, after stringent washes, the hybrids were visualized by autoradiography. B, the radioactivity of 45 S run-on transcripts obtained from a number of separate experiments were quantified by laser densitometry and are presented as the percent increase in rDNA transcription in response to NE over the transcription observed in time-matched control cells. For these experiments, the cells were cultured at 4 times 10^6 cells/60-mm plate, and contraction was arrested by the addition of 50 mM KCl. The vertical lines indicate the standard deviation from the mean of 5 or more separate experiments.



These results demonstrate that norepinephrine-induced hypertrophy of neonatal cardiomyocytes is accompanied by a sustained elevation in the rate rDNA transcription and that this response is independent of the density at which the cells were plated. These findings are in accordance with the model that increased rDNA transcription is a pivotal point in the regulation of ribosome accumulation and the increased rate of protein synthesis prerequisite for the growth of cardiomyocytes(3) .

Analysis of RNA Polymerase I and UBF Levels in Control and Hypertrophic Cardiomyocytes

The rate of rDNA transcription may be regulated either by alterations in the amounts and/or activities of RNA polymerase I or the various associated rDNA transcription factors. In order to determine whether the changes in the level of the RNA polymerase I enzyme might be associated with altered levels of rDNA transcription in neonatal cardiomyocytes, we determined the protein levels of the RNA polymerase I beta` subunit (rPOLIbeta`) using antisera raised to recombinant rPOLbeta` protein(8) . The specificity of the anti-rPOLIbeta` antisera is shown in the Western blot of total protein extracted from rat Novikoff hepatoma ascites cells (Fig. 2A). No immunoreaction was observed when the blot was incubated with preimmune sera (Fig. 2A, lane 1). However, a single band of the predicted size (194 kDa) was observed when the same blot was incubated with antisera obtained from a rabbit immunized with recombinant rPOLIbeta` protein (Fig. 2A, lane 2). The relative mobility of calibrated protein molecular mass markers are given for reference. We next determined the levels of rPOLIbeta` protein in control and hypertrophying neonatal cardiomyocytes.


Figure 2: RNA polymerase I beta` subunit (rPOLIbeta`) levels in neonatal cardiomyocytes following norepinephrine (NE) stimulation. A, total cellular protein extracted from exponentially growing rat Novikoff hepatoma cells (lanes 1 and 2) was fractionated by SDS-PAGE (25 µg/lane), transferred to a nylon membrane, and incubated with either preimmune antisera (lane 1) or antisera obtained from the same rabbit after immunization with recombinant rPOLIbeta` (lane 2). rPOLIbeta` was subsequently visualized by ECL as described under ``Materials and Methods.'' B, total cellular protein was extracted from contraction-arrested, neonatal cardiomyocytes cultured at 4 times 10^6 cells/60-mm plate at the times indicated after treatment with vehicle (0.1% ascorbic acid, lanes 1 and 3) or NE (1 µM, lanes 2 and 4). After SDS-PAGE and Western transfer, rPOLIbeta` protein was detected with a rabbit anti-rPOLIbeta` antibody and visualized by ECL as described under ``Materials and Methods.'' Equal amounts of protein (25 µg) were loaded per lane.



Total protein was extracted from arrested cardiomyocytes treated with vehicle or norepinephrine for 24 or 48 h, and the levels of rPOLIbeta` protein were determined by SDS-PAGE and Western analysis. As shown in Fig. 2B, after 24 and 48 h of treatment with norepinephrine, the level of rPOLIbeta` protein in neonatal cardiomyocytes was not significantly altered relative to time-matched control cells. In fact, the level of rPOLIbeta` did not change in response to norepinephrine at any time point examined (12-96 h, data not shown). These results indicate that it is unlikely that an increase in the amount of the RNA polymerase I enzyme contributes to the elevated rate of rDNA transcription observed during norepinephrine-induced hypertrophy of neonatal cardiomyocytes.

The addition of UBF to cell-free transcription assays has been shown to increase rDNA transcription in a dose-dependent manner(11) . Moreover, overexpression of UBF in cells has been shown to augment transcription from a co-transfected ribosomal DNA promoter(12) . Accordingly, we ascertained whether the increased rDNA transcription observed during norepinephrine-induced hypertrophy of neonatal cardiomyocytes, was associated with increased amounts of UBF protein.

Total protein was extracted from arrested cardiomyocytes treated with vehicle or norepinephrine for 6-72 h, and the levels of UBF1 and UBF2 protein were determined by SDS-PAGE and Western analysis. As shown in Fig. 3, A and D, cardiomyocytes treated with norepinephrine exhibited elevated levels of UBF protein as compared to time-matched control cells. The UBF levels were maximal after 12-24 h. However, even after 48 h of treatment with norepinephrine, the absolute amount of UBF in the treated cells was greater than that in the control cells. Since equal amounts of protein were loaded per lane, the changes in the level of UBF were not merely the consequence of the general increase in protein content which is observed in norepinephrine-treated cells.


Figure 3: Treatment of neonatal cardiomyocytes with norepinephrine (NE) leads to increased levels of UBF protein and mRNA. A, total cellular protein was extracted from contraction-arrested, neonatal cardiomyocytes cultured at 4 times 10^6 cells/60-mm plate at the times indicated after treatment with vehicle (0.1% ascorbic acid, lanes 1 and 3) or NE (1 µM, lanes 2 and 4). After SDS-PAGE and Western transfer, UBF1 and UBF2 protein was detected with a rabbit anti-UBF antibody and visualized by ECL as described under ``Materials and Methods.'' Equal amounts of protein (25 µg) was loaded per lane. B, total RNA was extracted from contraction-arrested, neonatal cardiomyocytes cultured at 4 times 10^6 cells/60-mm plate at the times indicated after treatment with vehicle (0.1% ascorbic acid, lanes 1, 3, and 5) or NE (1 µM, lanes 2, 4, and 6). After electrophoresis and Northern blotting, the RNA (30 µg) was hybridized to P-labeled UBF cDNA, and the UBF mRNA transcripts were visualized by autoradiography. C, total RNA was extracted from neonatal cardiomyocytes cultured at a density of 0.5 times 10^6 cells/plate at the various times indicated following exposure to vehicle (0.1% ascorbic acid, lanes 1, 2, and 5) or NE (1 µM, lanes 3, 4, and 6). After electrophoresis and Northern blotting, the RNA was hybridized to P-labeled UBF cDNA and UBF mRNA transcripts visualized by autoradiography. Where indicated, KCl was included in the media to a final concentration of 50 mM. D, the experiments depicted in A and B were repeated a number of times, and the results were quantified by laser densitometry. After normalization for load, the results were expressed as the -fold increase in signal obtained from NE-treated cells over the signal obtained from time-matched control cells. Vertical lines represent the standard deviation from the mean of 5 or more separate experiments.



The alteration in the cellular content of a protein can reflect changes in the turnover of that protein (post-translational) and/or alterations in the levels of its mRNA (pretranslational). In order to determine whether the norepinephrine-induced increase in UBF protein reflected either of these stages of regulation, UBF mRNA levels were measured in the total RNA extracted from control and norepinephrine-treated high density cardiomyocyte cultures. The results presented in Fig. 3B demonstrate that cardiomyocyte UBF mRNA levels were rapidly and significantly (3-4-fold) increased after 12 h of treatment with norepinephrine compared to the level found in time-matched control cells (Fig. 3B, lanes 1 and 2). Northern blots do not resolve the two mRNAs for UBF1 and UBF2(13) . Interestingly, and in contrast to the pattern demonstrated by the UBF protein levels, the increase in UBF mRNA levels was transient. After 24 h of treatment, the norepinephrine-treated cells exhibited UBF mRNA levels similar to those found in untreated control cells. The integrity of the RNA samples and the amounts loaded were determined by quantifying the fluorescence pattern of the 18 S ribosomal RNA observed after transfer to the Zeta Probe nylon membrane (Fig. 3B).

Recent studies have revealed that the alterations of specific transcription events observed in response to norepinephrine may vary with the density at which the cardiomyocytes are cultured(27) . In order to determine if the observed induction of UBF by norepinephrine was density-dependent, we also examined UBF mRNA levels in cardiomyocytes maintained at a relatively low density. As shown in Fig. 3C, cardiomyocytes cultured at 0.5 times 10^6 cells per 60-mm culture plate and treated with norepinephrine exhibited increases in UBF mRNA levels that were quantitatively similar to those observed in high density cultures (Fig. 3C, lanes 1-4). Once again, the observed induction cannot be explained by norepinephrine-induced increases in contractile activity of the cardiomyocytes as the inclusion of 50 mM KCl in the media did not significantly alter the increased UBF mRNA levels (Fig. 3C, lanes 5 and 6). The norepinephrine-induced increases in UBF protein and mRNA levels exhibited by 4-5 separate, high density cardiomyocyte preparations were quantitated by laser densitometry and are presented graphically in Fig. 3D. These results suggested that in high and low density cultures of cardiomyocytes, at least part of the response that leads to increased levels of UBF protein is pretranslational.

We also found that cardiomyocyte cultures purified by centrifugation through Percoll gradients (28) exhibited the same response, i.e. the induction of UBF mRNA in response to NE, as observed in less rigorously prepared cultures (results not shown). Thus, we conclude that the observed increase in UBF protein and mRNA levels reflect a direct affect of norepinephrine upon the cardiomyocytes themselves and is not mediated by other cell types.

Regulation of UBF mRNA Levels by alpha- and beta-Adrenergic Receptors

The physiological actions of adrenergic agents on cellular structure and function are mediated through the alpha- and beta-adrenergic receptors, both of which have been shown to be present in membranes of neonatal cardiomyocytes(1, 2, 3) . In order to determine if either of these adrenergic receptor subtypes could be capable of mediating the norepinephrineinduced UBF accumulation of UBF mRNA, we treated neonatal cardiomyocytes with either the selective alpha-adrenergic agonist, phenylephrine (10 µM), or the selective beta-adrenergic agonist, isoproterenol (1 µM) (Fig. 4A). Interestingly, cardiomyocyte UBF mRNA levels were elevated in response to both alpha- (Fig. 4A, lane 5) and beta- (Fig. 4A, lane 6) adrenergic stimulation, although the degree of response observed with either agent was less than that observed with norepinephrine (Fig. 4A, lane 3). The slight cross-over affinity of phenylephrine for the beta-adrenergic receptors could not account for the effect of this agent, since a similar induction was observed when phenylephrine was used in combination with the beta-adrenergic antagonist propranolol (5 µM) (Fig. 4A, lane 4). The results from a number of separate experiments were quantified by laser densitometry and are presented in Fig. 4B.


Figure 4: Accumulation of UBF mRNA in neonatal cardiomyocytes in response to alpha- and beta-adrenergic stimulation. A, total RNA was extracted from arrested neonatal cardiomyocytes at the times indicated following exposure to vehicle (CTL, 0.1% ascorbic acid, lanes 1 and 2), norepinephrine, 1 µM (NE, lane 3), a combination of the alpha(1)-adrenergic agonist phenylephrine, 10 µM, and the beta-adrenergic antagonist propranolol, 5 µM (Phe + Prop, lane 4), phenylephrine alone (lane 5), or the beta-adrenergic agonist isoproterenol, 1 µM (Iso, lane 6). All other culture conditions were as described in the legend to Fig. 1. After electrophoresis and Northern blotting, the RNA (30 µg) was hybridized to P-labeled UBF cDNA, and the UBF mRNA transcripts were visualized by autoradiography. B, the experiments in A were repeated a number of times, and the UBF hybridization signals were quantitated by laser densitometry. After normalization for loading, the results were expressed as the -fold increase in signal obtained from NE-treated cells over the signal obtained from time-matched control cells. Vertical lines represent the standard deviation from the mean of 3 or more separate experiments.



Activation of the beta-adrenergic receptors on neonatal cardiomyocytes leads to activation of adenylyl cyclase and the subsequent activation of protein kinase A in response to the increased cellular content of cAMP(1) . Since pathways distal to cAMP have been implicated in the regulation of nuclear trans-acting factors(29) , we examined the effect of forskolin, a drug known to increase cellular cAMP levels, on the expression of UBF. Treatment of cardiomyocytes with forskolin (1 µM) led to an increase in the level of cardiomyocyte UBF mRNA after 12 h of treatment (Fig. 5, lane 3), similar to that observed in response to norepinephrine (Fig. 5, lane 2). Additional studies demonstrated that the effect of beta-adrenergic activation on UBF expression could be mimicked by addition of the membrane-soluble cAMP analog, dibutyryl cAMP (10-100 µM) to the cells (results not shown). These studies demonstrate that cellular signals downstream of cAMP may regulate induction of UBF in response to beta-adrenergic agonists.


Figure 5: UBF mRNA levels in neonatal cardiomyocytes following treatment with norepinephrine, forskolin, or PMA. Total RNA was extracted from arrested neonatal cardiomyocytes after 12 h of treatment with vehicle (0.1% ascorbic acid, 0.001% dimethylsulfoxide, lane 1), norepinephrine (NE, 1 µM, lane 2), forskolin (1 µM, lane 3), or phorbol 12-myristate 13-acetate (PMA, 0.1 µM, lane 4). After electrophoresis and blotting, the RNA (30 µg) was hybridized to P-labeled UBF cDNA, and the UBF mRNA transcripts were detected by autoradiography. The experiment was repeated three times, and a representative autoradiogram is shown.



Activation of the alpha-adrenergic receptors leads to the release of inositol 1,4,5-trisphosphate and sn-(1,2)-diacylglycerol via phospholipid hydrolysis(30, 31) . Diacylglycerol is the endogenous activator of the phospholipid-dependent protein kinase C family; a group of kinases that have been variously implicated in the activation of nuclear trans-acting factors(26, 32, 33) . Accordingly, we also examined whether PMA, a synthetic activator of protein kinase C, might also increase UBF mRNA levels in neonatal cardiomyocytes. Interestingly, treatment of cells with PMA (0.1 µM) for 12 h did not increase UBF mRNA above basal levels (Fig. 5, lane 4) nor was an elevation of UBF mRNA level observed at any other time point investigated (3-72 h). In addition, treatment with PMA did not affect cardiomyocyte UBF protein levels at any time studied although, in agreement with the findings of others(26) , this treatment did lead to significant activation of protein kinase C in these cells (results not shown).


DISCUSSION

Cardiomyocyte hypertrophy requires increased ribosome synthesis in order to produce more protein since the pool of existing ribosomal subunits are already engaged in synthesizing proteins(3) . Hence, studies examining the mechanism of ribosome biogenesis in cardiomyocytes are of importance to the general understanding of cardiac growth. We have examined the hypothesis that the adrenergic mediated hypertrophy of cultured neonatal cardiomyocytes is associated with increased rates of ribosomal RNA synthesis. Secondly, we examined possible mechanisms through which the cardiomyocytes might increase the rate of rDNA transcription. Specifically, we have demonstrated that norepinephrine-induced cardiomyocyte hypertrophy is accompanied by increases in the rate of rDNA transcription. Significantly, the amount of RNA polymerase I (as reflected by the amount of the beta` subunit of the enzyme) does not change, but the amount of the rDNA transcription factor, UBF, does. Furthermore, we have demonstrated that the levels of UBF mRNA in neonatal cardiomyocytes increase in response to both alpha- and beta-adrenergic stimulation and have presented preliminary data toward defining the signal transduction pathways involved in this process.

rDNA Transcription

In previous studies it was shown that increased protein synthesis in neonatal cardiomyocytes treated with norepinephrine (18, 26, 34) was accompanied by elevated cellular content of ribosomal RNA (26) and increased polymerase I transcription (35) . Our results confirm and extend these observations since we demonstrate that the increase in rRNA content in norepinephrine-treated cardiomyocytes can be accounted for by an accelerated rate of rDNA transcription as measured by run-on transcription assays from the 45 S precursor gene. We have previously reported, in agreement with the findings reported herein, that cultured, contracting, hypertrophic neonatal cardiomyocytes synthesized rRNA at a rate approximately 70% faster than noncontracting, nonhypertrophic cells(6) . During this hypertrophy, the rate of 45 S pre-rRNA processing was not changed, rather the rate of synthesis of preribosomal RNA was accelerated(7) . We also have preliminary evidence to suggest that rDNA transcription is increased during cardiomyocyte hypertrophy induced by other stimuli such as passive stretch and endothelin. (^3)Taken together with the present results, these findings are consistent with the general hypothesis that ribosome biogenesis in cardiomyocytes is largely regulated at the level of rDNA transcription and that this process is the rate-limiting step in the synthesis of new proteins.

Regulation of RNA Polymerase I and UBF Levels

Some of the components which have been implicated in the regulation of the ribosomal DNA transcription system have been identified, purified, or cloned(8, 9) . However, their relative contribution(s) to the accelerated rate of rDNA transcription observed during norepinephrine-induced cardiomyocyte hypertrophy is unknown. In these studies, we demonstrate that norepinephrine-induced increases in rDNA transcription in neonatal cardiomyocytes are not accompanied by significant changes in the amounts of the rat RNA polymerase I beta` subunit. If the levels of the polymerase I beta` subunit accurately reflect the level of the total RNA polymerase I enzyme in the cell, then it appears unlikely that increased amounts of this enzyme can account for the altered level of rDNA transcription observed in norepinephrine-treated neonatal cardiomyocytes.

We next examined the levels of the rDNA transcription factor UBF during norepinephrine-induced cardiac hypertrophy. We have previously demonstrated that the addition of UBF to cell-free extracts depleted of UBF increases transcription from the ribosomal DNA promoter in a dose-dependent manner(11) . Further, overexpression of UBF has been shown to increase transcription from a co-transfected 45 S gene construct(12) . These two lines of evidence suggest that alterations in the amount of UBF is one mechanism by which rDNA transcription by polymerase I can be regulated. We report here that increased ribosomal RNA synthesis observed in neonatal cardiomyocytes following norepinephrine stimulation is associated with elevated levels of both UBF1 and UBF2 mRNA and protein. The maximal increase in UBF protein occurred within 12-24 h of norepinephrine treatment and was coincident with the time when the rate of rDNA transcription rate reached its maximum. Thus, the temporal profile of UBF protein induction is consistent with the hypothesis that this nucleolar transcription factor is important to both the initiation and maintenance of accelerated rDNA transcription observed during norepinephrine-induced hypertrophic growth of neonatal cardiomyocytes. During the first 12 h of norepinephrine treatment, there was a significant accumulation of UBF mRNA. We are currently using nuclear run-on experiments to determine whether alterations in the rate of transcription can account for the observed increased in UBF mRNA levels(36) .

Interestingly, and in contrast to the observed increase in UBF protein, the induction of UBF mRNA in response to norepinephrine was transient. While the UBF protein levels remained elevated for 48-72 h, the UBF mRNA levels returned to the level found in control cells within 24 h. This pattern contrasted with our previous observations of the regulation of UBF in differentiating myoblasts(17) . When L6 cells differentiated, there was a direct correlation between the reduction in UBF mRNA and UBF protein since they decreased in parallel. Comparing that study with the results presented here suggests that UBF expression during hypertrophy may also be subject to post-translational regulation, i.e. protein stabilization.

Regulation of UBF by alpha- and beta-Adrenergic Pathways

Current studies have provided evidence implicating molecular pathways linked to both the alpha- and beta-adrenergic receptors in the regulation of norepinephrine-induced cardiomyocyte hypertrophy in vivo(1, 37) . However, their relative contributions to this process in isolated cardiomyocytes in cell culture is still under discussion and appear to depend on a number of factors including the contractile status of the cells, the age of the animal from which they were prepared, the method of isolation, and the density/purity of the final cultures. In this study, the treatment of arrested neonatal cardiomyocytes with either the specific alpha-adrenergic agonist, phenylephrine, or the specific beta-adrenergic agonist, isoproterenol, resulted in elevated levels of UBF mRNA. These findings indicate that both receptor families interact in mediating the response to NE.

Stimulation of beta-receptors in cardiomyocyte membranes leads to activation of protein kinase A in response to an increased cellular content of cAMP(29) . In turn, activated protein kinase A phosphorylates a number of transcription factors including cAMP response element binding protein, cAMP response element modulator, and activating transcription factor 1 (29) which bind to their respective response elements in the nucleus and result in altered patterns of gene transcription. In the present study, the beta-adrenergic induction of UBF mRNA could be mimicked by treatment of the cells with the membrane-permeable cAMP analog, dibutyryl cAMP, or with forskolin, a drug which elevates cellular cAMP levels. Thus, it is likely that pathways distal to cAMP accumulation, such as protein kinase A activation, mediate beta-adrenergic increases in UBF mRNA levels in neonatal cardiomyocytes.

The protein kinase C family has been implicated in the hypertrophic response of neonatal cardiomyocytes to alpha(1)-adrenergic agents, although their exact role is still controversial(38) . However, in these experiments, the alpha-adrenergic induction of UBF mRNA and protein could not be reproduced by exposure of neonatal cardiomyocytes to PMA, a membrane-permeable synthetic activator of the protein kinase C family. These results suggest that activation of protein kinase C does not, or is not sufficient by itself, to increase UBF levels in neonatal cardiomyocytes. However, one cannot extrapolate the effects of synthetic activators of protein kinase C, such as PMA, to those of the endogenous activators of protein kinase C, such as norepinephrine, without caution(38) . For example, chronic treatment of cardiomyocytes with PMA down-regulates certain protein kinase C isoforms, while chronic treatment with norepinephrine up-regulates others(38, 39) . Furthermore, some members of the protein kinase C family are ``atypical'' in that they are not Ca-dependent or are phorbol ester-insensitive(39, 40) . Thus, differences in the exact subset of protein kinase C isoforms which are activated and/or their intracellular translocation may also contribute to discrepancies in the results obtained from norepinephrine- and PMA-treated cells.

It is possible that the regulation of the amount or activity of UBF is a conserved phenomenon important to the increased rate of ribosome biogenesis induced by diverse hypertrophic stimuli. In support of this hypothesis, we have preliminary data indicating that UBF mRNA and protein levels are up-regulated during neonatal cardiac hypertrophy stimulated by contraction and serum.^3 However, to definitively determine the effect of elevated levels of UBF on the rate of rDNA ribosomal transcription and protein synthesis in cardiomyocytes in vivo will require further experiments. We are currently assessing the effect of overexpressing UBF in neonatal cardiomyocytes on transcription of a co-transfected reporter gene under the control of the rDNA promoter. Further studies concerning the regulation of the UBF gene may provide clues as to the pathways by which alpha- and beta-adrenergic receptors contribute to cardiac hypertrophy.

The studies described in this report emphasize rDNA transcription as an important step in the regulation of ribosome content and, subsequently, protein synthesis during norepinephrine-induced hypertrophy of neonatal cardiomyocytes. Furthermore, this is the first report linking adrenergic stimulation of neonatal cardiomyocyte growth to the regulation of a nuclear factor, UBF, known to be intimately associated with rDNA transcription. These findings should now allow us to define the regulatory pathways which connect hypertrophic stimuli to an increase in ribosome biogenesis during more physiologically relevant settings such as the hypertrophy of adult cardiomyocytes in vivo.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL 47638 and GM46991 (to L. I. R.) and an award from the Geisinger Foundation (to L. I. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship of the American Heart Association.

Fellow of the Deutsche Forschungsgemeinschaft.

**
To whom correspondence and reprint requests should be addressed. Tel.: 717-271-6662; Fax: 717-271-6701; LROTHBLUM{at}GEISINGER.edu.

(^1)
The abbreviations used are: NE, norepinephrine; PMA, phorbol 12-myristate 13-acetate; rPOLIbeta`, beta` subunit of RNA polymerase I; IgG, immunoglobulin G; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. D. Hannan and L. I. Rothblum, manuscript in preparation.

(^3)
R. D. Hannan, J. Luyken, and L. I. Rothblum, unpublished data.


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