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Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes ¹

Molecular Physiology Laboratory, Baker Medical Research Institute, Melbourne, Victoria 8008, Australia

²Correspondence: Baker Medical Research Institute, St. Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail: ross.hannan{at}baker.edu.au

SPECIFIC AIMS

We tested the hypothesis that increased ribosome biogenesis is a prerequisite for the progression of cardiomyocyte hypertrophic growth and that this process is regulated at the level of ribosomal gene (rDNA) transcription by the nucleolar transcription factor UBF.

PRINCIPAL FINDINGS

1. Adenovirus-directed expression of UBF antisense RNA inhibits ₁-adrenergic- and contraction-induced UBF expression in cardiomyocytes
Noncontracting primary cultures of neonatal cardiac muscle cells (cardiomyocytes) were infected at a titer of 1 x 10² PFU/cell with either a control Ad-5-derived virus expressing the nonmammalian, control gene chloramphenicol transferase (Ad-CAT) or an Ad-5 virus expressing UBF cDNA (-267 to+635) in the antisense orientation under control of the CMV promoter (Ad-UBFas). At this titer, 95% of the myocytes were infected as determined by GFP fluorescence. After infection, the cardiomyocytes were stimulated for 3 days with either the ₁-adrenergic agonist phenylephrine (PE; 25 µM) or contraction to induce hypertrophy. Western blot analysis demonstrated that myocytes infected with the control virus Ad-CAT exhibited increases in UBF expression in response to 3 days of the ₁-adrenergic receptor agonist PE (2.7±0.4-fold increase) or contraction (2.4±0.4-fold increase) stimulation comparable to those we have described previously (Fig. 1A ). In contrast, infection with Ad-UBFas significantly blocked UBF accumulation in response to both PE (1.3±0.4-fold increase) and spontaneous contraction (1.5±0.3-fold increase) compared with the time-matched and stimulus-matched Ad-CAT-infected cells, but had no effect on the expression of a control protein Erk1/2 (Fig. 1A ).

View larger version (40K): [in this window] [in a new window]   Figure 1. Adenovirus-mediated expression of UBF antisense RNA inhibits increases in UBF expression, rDNA transcription, and rRNA accumulation during PE- and contraction-mediated cardiomyocyte hypertrophy. A) Cardiomyocytes were infected with either the control adenovirus expressing CAT (Ad-CAT) or the adenovirus expressing UBF antisense RNA (Ad-UBFas) at a titer of 1 x 102 PFU/cell and stimulated as indicated. Cell extracts were prepared after 48 h, and UBF1/2 and ERK1/2 levels were determined by Western blot analysis. Results were quantitated and expressed as the average (±SE, n=4) change in UBF protein content relative to arrested cells infected with Ad-CAT (control). B, C) Cardiomyocytes were infected with either Ad-CAT or Ad-UBFas and stimulated as described above. After 72 h, myocytes were pulse labeled with [3H] uridine for 10 min, then total RNA was extracted and rRNA synthesis rates were determined as described in Materials and Methods. Total RNA content was expressed as the average (±SE, n=5) change in RNA relative to the levels in unstimulated cells infected with Ad-CAT (control). *P < 0.05 vs. control cells;P < 0.05 vs. PE-stimulated cells infected with Ad-CAT; P < 0.05 vs. contracting cells infected with Ad-CAT.

2. Inhibition of UBF accumulation blocks increases in rDNA transcription and ribosomal RNA (rRNA) accumulation during PE- and contraction-mediated cardiac hypertrophic growth
Synthesis of rDNA in cardiomyocytes was measured by incorporation of [³H]UTP into pre-rRNA after hybridization to a rDNA clone containing the 5' ETS region of the rat 45S gene. [³H]UTP-labeled RNA was prepared by rapid pulse labeling of myocyte cultures with [³H] uridine. Total myocyte RNA was used as an index of ribosomal RNA content since 85–90% of total RNA is ribosomal. Infection with Ad-CAT had no effect on the ability of PE or contraction to stimulate significant increases in rRNA synthesis (1.74- and 1.92-fold, respectively) and total RNA levels (1.44±0.12-fold and 1.36±0.05-fold, respectively) relative to unstimulated cells infected with Ad-CAT (Fig. 1B , C ). In marked contrast, Ad-UBFas significantly inhibited the increase in rRNA synthesis after PE and contraction (1.10- and 1.05-fold, respectively) and RNA accumulation (1.16±0.09-fold and 1.11±0.05-fold, respectively) vs. time- and stimulus-matched cells infected with Ad-CAT (Fig. 1B , C ).

3. Synthesis of new ribosomes is a prerequisite for the progression of ₁-adrenergic- and contraction-mediated hypertrophic growth of neonatal cardiomyocytes
The ability of Ad-UBFas to inhibit rRNA synthesis allowed us to address the broader issue of whether the synthesis of new ribosomes was an absolute requirement for cardiac hypertrophic growth in response to ₁-adrenergic and contraction stimuli. Hypertrophy was defined as an increase in total cellular protein content in the absence of significant changes in DNA levels. Infection with Ad-CAT had no effect on the ability of PE or contraction to stimulate significant increases in cellular protein levels (1.34-fold±0.06-fold and 1.28±0.07-fold, respectively) relative to unstimulated cells infected with Ad-CAT (Fig. 2A ). In contrast, infection with Ad-UBFas essentially prevented any increase in protein accumulation after PE and contraction (1.0±0.1-fold and 0.98±0.06-fold, respectively) (Fig. 2A ). In agreement with this, calculation of the relative cell surface area of the myocytes after infection with control or adenovirus-infected cells confirmed that PE-stimulated cardiomyocytes expressing UBF antisense virus Ad-UBFas were significantly smaller (32%±17 increase relative to control cells infected with Ad-CAT) than PE-stimulated cells infected with equivalent amounts of the control virus Ad-CAT (180%±40 increase relative to control cells) (Fig. 2B ).

View larger version (36K): [in this window] [in a new window]   Figure 2. Increased ribosome biogenesis is required for cardiomyocyte hypertrophic growth. A) Cardiomyocytes were infected with either Ad-CAT or Ad-UBFas at 1 x 102 PFU/cell and stimulated as indicated. After 72 h, protein and DNA levels were determined and results were expressed as the average (±SE, n=4) % change in protein to DNA ratio relative to unstimulated cells infected with Ad-CAT (control). *P < 0.05 vs. control cells; P < 0.05 vs. PE-stimulated cells infected with Ad-CAT; P < 0.05 vs. contracting cells infected with Ad-CAT. B) Neonatal cardiomyocytes plated at a density of 416 cells/mm2 were infected at 1 x 102 PFU/cell with either Ad-CAT or Ad-UBFas. After 72 h of stimulation, cells were visualized under fluorescence (GFP) microscopy and the relative cell surface area was quantitated.

CONCLUSIONS AND SIGNIFICANCE

The experiments described in this study demonstrate that the cellular level of a nucleolar transcription factor termed UBF, which we have previously implicated in the regulation of cardiac rDNA transcription, is a critical determinant for ribosome biogenesis in cardiac muscle cells. Specifically, we show that inhibition of UBF protein accumulation via adenovirus-directed expression of UBF antisense RNA significantly attenuates the ability of ₁-adrenergic and contraction to stimulate rDNA transcription and rRNA accumulation in cultured neonatal cardiomyocytes. Moreover, the inhibition of rRNA synthesis by UBF antisense expression significantly repressed hypertrophic growth in response to these stimuli. This is the first direct evidence to support the hypothesis that increased translational efficiency of existing ribosomes alone is insufficient to account for sustained hypertrophic growth of cardiomyocytes and that synthesis of new functional ribosomes must also occur.

Since UBF appears to be an important regulatory step in the accelerated formation of ribosomes during cardiac hypertrophic growth, identification of the activators proximal to UBF that are responsive to hypertrophic stimuli should allow for elucidation of the bona fide signaling pathways that regulate cardiac growth (Fig. 3 ). Current data indicate that pathways that include the activation of p70 S6 kinase (p70^S6k) are likely candidates for such a role. These conclusions can be drawn from experiments demonstrating that the p70^S6k inhibitor rapamycin significantly attenuates rRNA accumulation and protein synthesis during the initiation of cardiac hypertrophy (Fig. 3) . Moreover, since p70^S6k has also been implicated in regulating translational efficiency, a model in which translational capacity (cellular ribosome content) and protein translational efficiency are coordinately regulated by p70^S6k can be envisaged. However, other than the 40S ribosomal protein S6, physiologically relevant targets for p70^s6k and its nuclear homologue p85 that might lead to increased UBF levels and rDNA transcription are yet to be defined. Moreover, we have preliminary data to suggest that components of the MAPK-dependent signaling pathway contribute, at least in part, to both UBF activation and rDNA transcription (R. D. Hannan, unpublished data) (Fig. 3) . Clearly these issues need to be resolved.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the proposed pivotal role UBF plays in regulating cardiac rRNA synthesis and growth. Hypertrophic stimuli, by yet undefined pathways, increase UBF cellular activity resulting in accelerated rates of the 45S ribosomal gene (rDNA) transcription and ribosomal RNA (rRNA) synthesis. The increased pool of rRNA together with an increased translation of ribosomal proteins (rProtein) results in more functional ribosomes (increased translational capacity). UBF antisense RNA (red text) blocks UBF expression after hypertrophic stimuli and thus prevents synthesis of new ribosomes required for the growth response. Blue lines and text indicate pathways blocked by the 70 kDa S6 ribosomal protein kinase (p70S6k) inhibitor rapamycin.

In conclusion, the studies described here implicate UBF as a major regulatory point for cardiac hypertrophy downstream of signaling pathways elicited by diverse stimuli. They also demonstrate that increased translational efficiency of existing ribosomes alone is insufficient to account for sustained enlargement of cardiomyocytes and that synthesis of new functional ribosomes must occur. Further studies assessing the molecular mechanisms regulating UBF expression/activity should provide new avenues for the identification the bona fide signaling pathways that control the protein synthetic capacity of cardiac muscle cells. Moreover, transgenic approaches targeted toward inhibition of UBF might provide a novel mechanism to dissect out the relative contribution of cardiac growth and gene reprogramming to the progression of decompensated cardiac hypertrophy and provide alternative therapeutic approaches to combat hypertrophic heart disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0853fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.00-0853fje

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