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(Hypertension. 2008;51:302.)
© 2008 American Heart Association, Inc.

Original Articles

Suppression of Cardiomyocyte Hypertrophy by Inhibition of the Ubiquitin-Proteasome System

Silke Meiners; Henryk Dreger; Mandy Fechner; Sven Bieler; Wim Rother; Christoph Günther; Gert Baumann; Verena Stangl; Karl Stangl

From the Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité - Universitaetsmedizin Berlin, Germany.

Correspondence to Karl Stangl, MD, Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité Universitaetsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. E-mail karl.stangl{at}charite.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibitors of the proteasome interfere with transcriptional regulation of growth signaling pathways and block cell cycle progression of mitotic cells. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of postmitotic cardiomyocytes. To test this hypothesis, we analyzed the effects of proteasome inhibition on hypertrophic growth of neonatal rat cardiomyocytes. Partial inhibition of the proteasome effectively suppressed cardiomyocyte hypertrophy as determined by reduced cell size, inhibition of hypertrophy-mediated induction of RNA and protein synthesis, reduced expression of several hypertrophic marker genes, and diminished transcriptional activation of the BNP promotor. Importantly, suppression of hypertrophic growth was independent of the hypertrophic agonist used. Expressional profiling and subsequent Western blot and kinase assays revealed that proteasome inhibition induced a cellular stress response with reduced expression of conserved growth signaling mediators and impaired G1/S phase transition of cardiomyocytes. In hypertensive Dahl-salt sensitive rats, inhibition of the proteasome with low doses of the FDA approved proteasome inhibitor Velcade significantly reduced hypertrophic heart growth. Our data provide important insight into the suppressive effects of proteasome inhibitors on hypertrophic growth of cardiomyocytes and establish low-dose proteasome inhibition as a new and broad-spectrum approach to interfere with cardiac hypertrophy.

Key Words: hypertrophy • stress • gene expression • myocytes • molecular biology

	Introduction

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Hypertrophic growth of postmitotic cardiomyocytes is characterized by quantitative effects on cell size, sarcomeric and constitutive proteins, and by qualitative changes in cardiac gene expression. The signal transduction pathways of hypertrophic growth involve several conserved signaling pathways of mitogenic growth and survival, such as activation of G protein–coupled receptors, receptor tyrosine kinases, phosphoinositol-3-kinase, Akt, and extracellular signal regulated kinase 1/2 (Erk1/2).¹ Activation of cardiac specific signaling mediators, eg, calcineurin, also contributes to the hypertrophic growth response of the heart.¹ Hypertrophic signals are further integrated within the cell and result in enhanced RNA and protein synthesis, and altered cell cycle regulation and hypertrophic gene expression.² Thus, cardiac hypertrophy appears to be a specialized form of cellular growth that requires mechanisms normally involved in proliferation control—such as growth-mediating signaling pathways and cell cycle regulation.²

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells.³ It tightly controls the level of many key proteins involved in cell cycle control, growth and inflammatory signaling, and transcriptional regulation.^4–6 Whereas sustained inhibition of the proteasome with high doses of specific proteasome inhibitors blocks cell cycle progression and subsequently induces apoptosis of mitotic cells, partial inhibition of the proteasome with low inhibitor doses results in cell cycle arrest and has prosurvival effects on various different cell types.⁷ Thus, partial proteasome inhibition offers an attractive tool to interfere with transcriptional regulation of growth signaling pathways and cell cycle progression of mitotic cells in the absence of apoptosis. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that partial proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of post-mitotic cardiomyocytes.

	Materials and Methods

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Reagents
MG132, MG262, and ALLM were purchased from Biomol, and Velcade was kindly provided by Millenium Pharmaceuticals. Isoproterenol, endothelin-1 (ET-1), interleukin (IL)-1, transforming growth factor (TGF)β, and triiodthyronine (T3) were purchased from Sigma. Antibodies against calcineurin and -smooth muscle actin (-SMA) were obtained from Sigma; against β-myosin heavy chain (β-MHC) from Alexis; anti-p44/42 antibodies from New England Biolabs; anti-cyclin D3 and anti-Akt antibodies from Santa Cruz; anti–cyclin-D1 antibodies from Labvision. Recombinant Rb (769) was purchased from Santa Cruz.

Primary Cardiomyocyte Cell Culture
Neonatal rat cardiomyocytes (NRCs) were prepared as described previously.⁸ For induction of hypertrophy, nonconfluent cells were set serum-free for 24 hours and cultivated in M199 medium in the presence of hypertrophic agonists for 24 or 48 hours, either with 0.1% DMSO as a solvent control or with proteasome inhibitors. Representative data from single preparations are shown. All experiments were reproduced in several other preparations of neonatal hearts.

Calculation of Cardiomyocyte Size
Cardiomyocytes were fixed in 4% paraformaldehyde and stained with Phalloidin-fluorescein isothiocyanate (FITC; Sigma, 1 µg/mL). At least 3 independent stimulations were stained and cell surface area of 10 random images was determined using SCION IMAGE Beta 4.0.2 software (Scion Corporation), and normalized to the number of cells/field and to controls.

RNA Preparation and RT-PCR Analysis
Total RNA was extracted and cDNAs were amplified by real-time PCR using a 5700 Sequence Detection System (Applied Biosystems) as described elsewhere.⁹

Microarray Analysis
Expression of RNAs was investigated by use of Atlas 1.2 rat arrays (Clontech, Takara BioCompany) according to the manufacturer’s protocol. Signals were normalized to GAPDH. Only those expression changes were included in the analysis where the mean ratio of 4 independent arrays was more than 2 or below 0.5.

Determination of RNA and Protein Synthesis
RNA and protein synthesis of NRCs were determined as described elsewhere.¹⁰

Measurement of Proteasomal Activity
The chymotrypsin-like proteasomal activity of cell extracts was determined as described previously.⁹

Western Blot Analysis
Total cell extracts were prepared and Western blot analysis was performed as published.¹¹ To ensure equal protein loading, membranes were stained with amido black solution (Sigma).

Transfections and Luciferase Assays
NRCs were transfected with DNA/lipofectin (Invitrogen), washed, cultivated in serum-free medium for 21 hours, and then stimulated with or without hypertrophic agonists in the presence or absence of MG132 (0.05 to 1 µmol/L).

For determination of transcriptional activation of the BNP promoter, we transfected pGL3-hBNP, a construct containing 1.9 kb of the human BNP promoter. Luciferase assays were performed using the Promega kit. Proteasome inhibition was assessed by transfection of a ubiquitin-conjugated variant of the green fluorescent protein (UbG76V-GFP).¹² The number of fluorescent cells was quantified by morphometric analysis using SCION IMAGE Beta 4.0.2 software.

Cyclin D1 Kinase Assay
Treated cells were lysed in lysis buffer (50 mmol/L Hepes [pH 7.5], 150 mmol/L NaCl, 0.1% Tween-20, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 10% glycerol, 1 mmol/L DTT, 10 mmol/L β-glycerophosphate, 1 mmol/L NaF, 0.1 mmol/L Na₃VO₄, 1x complete (Roche Applied Science). Extracts were immunoprecipitated by overnight incubation with anti-cyclin D1 antibodies coupled to protein A-sepharose. After washing, pellets were resuspended in kinase buffer (50 mmol/L Hepes [pH 7.5], 10 mmol/L MgCl₂, 2.5 mmol/L EGTA, 1 mmol/L DTT) containing glutathione S-transferase-retinoblastoma pocket protein. ³²P ATP (10 µCi/mL) was added to measure cyclin D1-associated kinase activities. Samples were analyzed by SDS-PAGE and autoradiography.

Animals and Study Design
Age-matched male Dahl-salt sensitive rats (DSS, SS strain) were obtained from Reinhold Kreutz (Charité). Animals were housed conforming to the institutional guidelines. From the age of 6 weeks rats were given a low- (0.2% NaCl) or high-salt (4% NaCl) diet. The high-salt group was treated with Velcade (50 µg/kg body weight) or with saline for 8 weeks (n=11 per group, twice weekly intraperitoneal injections). Systolic blood pressure was measured at the end of the study with a noninvasive tailcuff method in awake animals using a computer-assisted oscillatory detection device (TSE, Bad Homburg). Animals were euthanized (50 mg/kg body weight thiopental, Altana, Koblenz), organs were weighed and either shock-frozen or formalin-fixed.

Statistics
Data are expressed as mean±SEM, unless otherwise indicated. We calculated significance by 1-way ANOVA or t test where appropriate. P<0.05 was regarded as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of the Proteasome Suppresses Hypertrophy Irrespective of Stimulus
We evaluated the effects of proteasome inhibitors on hypertrophic growth in neonatal rat cardiomyocytes (NRCs).¹³ The more than 2-fold serum-induced increase in cardiomyocyte size was markedly suppressed by cotreatment with nontoxic doses of the proteasome inhibitors MG132 and MG262. It was unaffected by ALLM, an inhibitor of cathepsins and calpains (Figure 1A). Proteasome inhibition also effectively suppressed hypertrophic growth at doses of only 0.05 µmol/L when hypertrophy was induced by specific hypertrophic stimuli that activate distinct receptor signaling pathways, such as isoproterenol, ET-1, IL-1β, TGFβ, and T3 (Figure 1B).

View larger version (54K): [in this window] [in a new window]   Figure 1. Representative pictures of phalloidin-stained NRCs treated either with 10% FCS in the absence or presence of 1 µmol/L ALLM, 1 µmol/L MG132, or 0.1 µmol/L MG262 (A) or with 10 µmol/L Iso, 0.1 µmol/L ET-1, 10 ng/mL IL-1β, 10 ng/mL TGFβ, 0.1 µmol/L T3 in the absence or presence of 0.05 µmol/L MG132 (B) for 48 hours. Bar corresponds to 100 µm. For quantification see morphometric analysis, n=10±SD, *P<0.05 vs agonist-treated control (Tukey test for A or t test for B).

Proteasome Inhibition Suppresses Induction of Various Markers of Hypertrophy
We further characterized the hypertrophy-suppressing effects of proteasome inhibition in isoproterenol-treated NRCs. Real time RT-PCR analysis revealed that isoproterenol-induced expression of -MHC, myosin-light chain-2 (MLC-2), and cardiac -actin (CAA) was reduced dose-dependently by proteasome inhibition with 0.05 to 0.1 µmol/L MG132 (Figure 2A). On the protein level, MG132 suppressed isoproterenol-induced expression of the β-MHC isoform dose-dependently and also diminished expression of constitutively expressed -SMA (Figure 2B). Cotreatment with MG132 also significantly reduced isoproterenol-stimulated activation of the BNP promotor in reporter gene assays (Figure 2C). Already low doses of MG132 (0.05 µmol/L) completely suppressed isoproterenol-stimulated RNA and protein synthesis in NRCs (Figure 2D and 2E).

View larger version (22K): [in this window] [in a new window]   Figure 2. RNA expression of -MHC, MLC-2, and CAA was determined after treatment of NRCs with isoproterenol (Iso) in the absence or presence of MG132 for 48 hours by real-time RT-PCR analysis; n=3±SEM (A). Expression of β-MHC and -SMA was determined by Western blot analysis of protein-matched extracts (B). BNP-mediated luciferase activity was assessed in NRCs treated with Iso in the absence or presence of MG132 for 24 hours; n=12±SEM (C). Analysis of RNA (D) and protein synthesis (E), n=3±SD. *P<0.05 vs Iso-treated control (Tukey test for 2A, 2D, and 2E or t test for 2C).

Partial Inhibition of the Proteasome Suppresses Hypertrophic Growth
Low doses of MG132 (0.05 to 0.1 µmol/L) only partially inhibited the main proteolytic activity of the proteasome—the chymotrypsin-like activity—in whole cell extracts by 60% to 80% (Figure 3A). Dose- and time-dependent inhibition of the proteasome was observed in cardiomyocytes that were transiently transfected with a ubiquitinated variant of the green fluorescent protein (UbG76V-GFP, Figure 3B). This protein is rapidly degraded by the proteasome, whereas inhibition of the proteasome stabilizes UbG76V-GFP resulting in enhanced green fluorescence.¹²

View larger version (17K): [in this window] [in a new window]   Figure 3. The chymotrypsin-like activity was analyzed in NRC extracts using fluorogenic peptides (A). Values are given in percent of activity normalized to control; n=3±SD, *P<0.05 vs isoproterenol-treated control (Kruskal-Wallis test). Overall proteasomal activity was analyzed after transfection of UbG76V-GFP (B); n=10±SEM *P<0.05 vs isoproterenol-treated control (Kruskal-Wallis test).

Suppression of Hypertrophy Is Reversible
To investigate whether the observed suppressive effects of proteasome inhibition on hypertrophic cell growth are reversible, cardiomyocytes were stimulated with isoproterenol in the presence or absence of 0.05 µmol/L MG132 for 48 hours, washed, and then restimulated with 10% FCS for another 72 hours (Figure 4A). Notably, suppression of isoproterenol-induced hypertrophy by the proteasome inhibitor MG132 could be effectively reverted by serum-treatment for 72 hours (Figure 4B and 4C).

View larger version (35K): [in this window] [in a new window]   Figure 4. NRCs were stimulated with isoproterenol (Iso) in the presence or absence of 0.05 µmol/L MG132 for 48 hours and restimulated with 10% FCS (A). Representative phalloidin-stainings for the time points 0, 24, and 72 hours after serum stimulation are shown in B; morphometric analysis of single myocyte area is shown in C; n=10±SD.

Proteasome Inhibition Downregulates Multiple Hypertrophic Signaling Pathways
To gain insight into the hypertrophy-suppressive effects of proteasome inhibitors, we compared expression patterns of isoproterenol-stimulated cardiomyocytes with MG132-cotreated cells using rat Atlas cDNA arrays. Generally, genes were predominantly downregulated on proteasome inhibition (please see http://hyper.ahajournals.org, Table S1). Most strikingly, we observed uniform downregulation of genes involved in cell cycle regulation and growth signaling pathways (Table 1). Downregulation of Akt1 and erk1/2 was confirmed by Western blot analysis, and could be extended to the cardiac specific growth mediator calcineurin, which was not present on the array (Figure 5A). Reduced expression of Akt1, Erk1/2, and calcineurin was also observed in serum-treated NRCs although to a lesser extent (Figure 5A).

View this table: [in this window] [in a new window]   Table 1. Downregulation of Growth Signaling Pathways in Isoproterenol-Stimulated Versus MG132-Cotreated Cardiomocytes

View larger version (16K): [in this window] [in a new window]   Figure 5. NRCs were stimulated with 10% FCS or 10 µmol/L isoproterenol (Iso) in the presence or absence of 0.05 µmol/L MG132 for 48 hours. Expression of Akt, erk1/2, and calcineurin (A) and of cyclin D1 and D3 (B) was determined by Western blot analysis of protein matched extracts. Cyclin D1 immunocomplexes of corresponding extracts were assayed for associated kinase activities (C).

Among the genes that were most distinctly downregulated in cardiomyocytes, we found several G1/S phase specific cyclins, such as cyclin D1, D2, and D3. These data were confirmed by Western blot analysis for cyclin D1 and D3 (Figure 5B) and were also observed when cardiomyocytes were cultivated in the presence of serum. Importantly, reduced expression of G1/S phase cyclins was directly associated with diminished activation of cyclin D1 bound cyclin-dependent kinases (cdk) as shown by reduced in vitro phosporylation of retinoblastoma protein in cyclin D1 immunoprecipitates (Figure 5C).

Low-Dose Proteasome Inhibition Attenuates Cardiac Hypertrophy in Dahl Salt Sensitive Rats
To assess the effects of proteasome inhibition on development of cardiac hypertrophy in vivo, we treated DSS rats with low and nontoxic doses of the proteasome inhibitor Velcade for 8 weeks. On high-salt diet, DSS rats developed pronounced cardiac hypertrophy attributable to pressure overload (Table 2). Treatment with Velcade attenuated cardiac hypertrophy in high-salt animals as shown by significant reduction in absolute heart weights and in the heart weight/tibia length ratio. Velcade also effectively suppressed cardiomyocyte hypertrophy in vitro (please see http://hyper.ahajournals.org, Figure S1).

View this table: [in this window] [in a new window]   Table 2. Low-Dose Proteasome Inhibition Attenuates Cardiac Hypertrophy In Vivo

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Low-Dose Proteasome Inhibition Suppresses Cardiac Hypertrophy
Here, we demonstrate that low-dose inhibition of the proteasome suppresses the hypertrophic growth response of cardiomyocytes and of the heart. Our in vitro data clearly show that partial inhibition of the proteasome by low inhibitor doses does not affect cell viability of cardiomyocytes but suppresses hypertrophy by reversible changes within the cell. Our in vivo data concur with a recent publication that showed prevention of left ventricular hypertrophy by treatment with the proteasome inhibitor epoxomicin in a mouse model of short-term pressure overload.¹⁴ However, in contrast to this study by Depre et al, we observed significant suppression of cardiac hypertrophy with low and nontoxic doses of Velcade in a model of chronic pressure overload in DSS rats. The Velcade dose of only 50 µg/kg body weight is markedly lower than the maximum tolerated dose of 83 µg/kg body weight and only partially inhibited the chymotrypsin-like activity in the liver by about 25% (data not shown). Although proteasome inhibitor treatment slightly but not significantly diminished blood pressure in hypertensive rats, this did not correlate with reduced cardiac hypertrophy in single animals (data not shown).

Proteasome Inhibition Downregulates Key Signaling Pathways of Hypertrophic Growth
Suppression of hypertrophy occurred irrespective of the hypertrophic agonist used. This finding indicates that proteasome inhibition affects conserved and nodal signaling routes of hypertrophic growth. Indeed, we observed uniform transcriptional downregulation of key signaling molecules such as Akt, Erk1/2, and calcineurin as also observed by others.^15,16 As inhibition of Akt, Erk1/2, and calcineurin inhibits development of cardiomyocyte hypertrophy in vitro and in vivo,^17–19 it is well conceivable that proteasome inhibitor-mediated downregulation of these central signaling pathways contributes to suppression of hypertrophic growth of cardiomyocytes.

The hypertrophy suppressive effects of low-dose proteasome inhibition are accompanied by reduced G1/S phase transition. Induction of hypertrophic growth apparently requires the reentry of cardiomyocytes into the cell cycle with activation of G1 cdks and subsequent G1/S phase transition.^2,20–22 Preventing cell cycle reentry by overexpression of cdk inhibitors such as p16 or p21 inhibited G1 cdk activity and blocked the hypertrophic growth response of cardiomyocytes in vitro and in aortic banded mice.^23–25 Impairing hypertrophy-induced cell cycle reentry by proteasome inhibition might thus contribute to reduced hypertrophic growth similar to the proteasome inhibitior–mediated suppression of proliferative growth in mitotic cells.^5,15,16

Proteasome Inhibition Induces a Conserved Stress Response in Cardiomyocytes
In summary, the response of cardiomyocytes to low-dose proteasome inhibition strongly resembles a conserved cellular stress response with transcriptional deregulation of signal mediators, impaired G1/S phase transition, upregulation of heat shock proteins, and reduced RNA and protein synthesis. We propose that this stress response makes the cardiomyocytes "numb" for incoming growth signals thereby preventing hypertrophic cell growth. Importantly, this "numbness" does not affect cell viability but is reversible and can be overcome by strong growth stimulatory signals. In mitotic cells, proteasome inhibition induces intracellular stress that is either lethal or protective depending on the degree of proteasomal inhibition.⁷ It is tempting to speculate that the observed suppression of RNA and protein synthesis in cardiomyocytes is an integral part of the intracellular stress response to proteasome inhibition as also observed for other stressors.^26–28 Although recent data indicate that proteasome inhibition directly suppresses protein synthesis,²⁹ there are no data available concerning the inhibitory effects on RNA synthesis. As induction of RNA synthesis by hypertrophic stimuli is mainly attributable to enhanced transcription of ribosomal RNAs by RNA polymerase I,³⁰ suppression of RNA synthesis by proteasome inhibition probably involves inhibition of RNA polymerase I activity. One may speculate that proteasome inhibitor mediated stabilization of the tumor suppressor proteins p53 and p14/ARF contribute to the negative regulation of ribosomal RNA transcription.^31,32

Perspectives
In this study, we provide evidence that partial proteasome inhibition induces a protective stress response in cardiomyocytes which effectively suppresses cardiomyocyte hypertrophy irrespective of the inducing stimulus. As low-dose proteasome inhibition suppresses cardiac hypertrophy in vitro and in vivo, our data establish partial proteasome inhibition as a new and broad-spectrum approach to interfere with hypertrophic growth of cardiomyocytes.

	Acknowledgments

 
We thank Andrea Weller for excellent technical assistance and Antje Ludwig and Mario Lorenz for helpful discussions.

Sources of Funding

This study was partly supported by a grant from the Deutsche Forschungsgemeinschaft (DFG STA 567/3-1) to K.S. and S.M. S.M. was funded by a personal grant by the Charité Universitaetsmedizin Berlin.

Disclosures

None.

	Footnotes

 
S.M. and H.D. contributed equally to this study.

Received July 23, 2007; first decision August 8, 2007; accepted November 14, 2007.

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