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Cardiovascular Research 2002 56(1):64-75; doi:10.1016/S0008-6363(02)00510-2
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro

Peter K Buska,*, Jirina Bartkovab, Claes C Strøma, Linda Wulf-Andersena, Rebecca Hinrichsena, Tue E.H Christoffersena, Lucia Latellab, Jiri Bartekb, Stig Haunsøa and Søren P Sheikha

aLaboratoriet for Molekylær Kardiologi and Hjertecenteret H:S, Rigshospitalet. 20, Juliane Mariesvej., DK-2100 Copenhagen Ø, Denmark
bDepartment of Cell Cycle and Cancer, Danish Cancer Society, 49, Strandboulevarden, DK-2100 Copenhagen Ø, Denmark

* Corresponding author. Tel.: +45-3545-6737; fax: +45-3545-6500 busk{at}molheart.dk

Received 5 November 2001; accepted 5 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Present knowledge suggests that cell-cycle regulatory proteins take part in hypertrophy. We have investigated if the D-type cyclins are involved in cardiac hypertrophy. Methods: The expression and activity of the D-type cyclins and associated kinases in cardiomyocytes were studied during angiotensin II- and pressure overload-induced hypertrophy in rats (Rattus norvegicus) and in isolated, neonatal cardiomyocytes. Expression of the D-type cyclins was manipulated pharmacologically and genetically in neonatal myocytes. Results: In the left ventricle, there was a low, constitutive expression of the D-type cyclins, which may have a biological role in normal, adult myocytes. The protein level and the associated kinase activity of the D-type cyclins were up-regulated during hypertrophic growth. The increase in cyclin D expression could be mimicked in vitro in neonatal cardiac myocytes. Interestingly, the cyclin Ds were up-regulated by hypertrophic elicitors that stimulate different signalling pathways, suggesting that cyclin D expression is an inherent part of cardiac hypertrophy. Treatment of myocytes with the compound differentiation inducing factor 1 inhibited expression of the D-type cyclins and impaired hypertrophic growth induced by angiotensin II, phenylephrine and serum. The response to hypertrophic elicitors could be restored in differentiation inducing factor 1-treated myocytes by expressing cyclin D2 from a heterologous promoter. Conclusion: Our results point to the D-type cyclins as important regulators of cardiac hypertrophy. This supports the notion that cell-cycle regulatory proteins regulate hypertrophic growth.

KEYWORDS Hypertrophy; Myocytes; Signal transduction; Gene expression; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Increased cardiac workload induces cardiac hypertrophy, which leads to myocyte necrosis and apoptosis [1]. This process is an important risk factor of cardiac morbidity and mortality and can lead to clinical heart failure. Reduction of left ventricular hypertrophy by antihypertensive treatment improves the prognosis of patients [2]. However, only a partial reduction of cardiac hypertrophy is achieved with the available means.

Left ventricular wall stress, hormones, cytokines, growth factors and cardiovascular diseases increase cardiac workload and induce myocyte hypertrophy [1]. Recent evidence indicates that multiple signalling pathways are involved in the hypertrophic response. Calcium seems to be an essential second messenger in hypertrophic signalling and at least two calcium-dependent pathways mediate hypertrophy [3]. One of these pathways involves activation of the calmodulin-dependent phosphatase calcineurin, which induces transcription of hypertrophy-related genes through the transcription factor NF-AT3 [4]. The importance of the calcineurin pathway is supported by the finding that the calcineurin inhibitors cyclosporin A and FK506 impair genetic and hormone-induced hypertrophy in multiple animal models of cardiac hypertrophy [5,6]. However, in rats and mice, inhibition of calcineurin does not block pressure overload-induced hypertrophy suggesting that calcineurin-independent pathways are also important in hypertrophic signalling [5,7].

A pathway involving calcium-calmodulin-dependent protein kinases acts in parallel to the calcineurin pathway [8,9]. Other intracellular signalling molecules such as protein kinase C [10], MAP kinases [11–13] and JAK-STAT [14], mediate the hypertrophic response of cardiomyocytes. The pathways have not been elucidated in detail but it is clear that there is a significant cross-talk between the signalling molecules, e.g. between calcineurin and protein kinase C [15].

It is of great interest to find a point of convergence for the different signalling pathways. Hypertrophic growth occurs both by cell-cycle-dependent and -independent mechanisms [16,17]. Recent evidence suggests that the cell-cycle regulatory mechanism participates in cardiac hypertrophy. Many of the signalling molecules in hypertrophy are protooncogenes, which directly or indirectly stimulate cell division. Adult cardiomyocytes only divide to a limited extent [18,19] instead they develop hypertrophy in response to growth stimuli [1]. Interestingly, hypertrophic stimuli induce many cell-cycle regulatory proteins in cardiac myocytes [20–25].

Many of the decisions whether a cell should divide or not are taken in the G1-phase of the cell cycle before DNA synthesis [26]. Key regulators of G1-progression include cyclin D1, D2 and D3 that associate with and activate cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) [27]. The amount of CDK inhibitors also regulates CDK4 and CDK6 activity [28]. CDK4 and CDK6 phosphorylate the Rb family proteins thereby activating the E2F transcription factors. In late G1, E2F activates transcription of the cyclin E gene that is required for entry into the S-phase and for DNA synthesis [29]. Cyclin E binds to and activates CDK2. CDK2 activity is further modified by specific inhibitors and by phosphorylation [26]. Expression of cyclin E is also regulated by an Rb-independent pathway [30].

As hypertrophy does not involve cell-division, the events in G1-phase before DNA synthesis are especially interesting for cardiac hypertrophy. Although many different stimuli induce cell proliferation through different signalling pathways, activation of cyclin Ds is necessary for most, if not all of these pathways [31]. This places cyclin D expression at a point of convergence for mitotic signalling. The central role of the cyclin Ds in G1-phase suggests they are prime candidates for regulation of hypertrophy. Inhibition of the cyclin D-dependent kinases by overexpression of P16 impairs cardiac hypertrophy in vitro [25] and in vivo [32]. These findings indirectly show that the cyclin Ds are important for hypertrophic signalling.

In the present work, we have focused on the relation of the cyclin Ds to cardiac hypertrophy. Cyclin D expression and associated kinase activity were measured in two rat models of hypertrophy and in cardiomyocyte cultures. The effect of genetic and pharmacologic manipulation of cyclin D expression was investigated in isolated myocytes. The results point to cyclin D as important regulators of cardiac hypertrophy.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Experimental animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

Angiotensin II (200 ng/kg per min) was administered subcutaneously for 2 weeks to Wistar rats 250–300 g (Møllegård, Denmark) by implanting miniosmotic pumps (ALZET®, USA). The angiotensin II was diluted in 0.9% NaCl with 0.01 M acetic acid. Age-matched control animals were infused with vehicle starting on the same day.

Banding of the ascending aorta was carried out in Wistar rats (Møllegård, Denmark) with a body weight of 60–90 g. The rats were anesthetized, intubated and connected to a rodent ventilator. The ascending aorta was exposed through a thoracotomy, and a titanium clip (Weck Closure Systems, USA) of 0.6 mm i.d. was placed around the ascending aorta. Peroperative mortality was approximately 15%. Sham operated animals of the same age underwent the same procedure on the same day, except for placement of the clip. The animals were killed at the time indicated after the operative procedure. The left ventricle was isolated and frozen in liquid nitrogen.

2.2 Cardiomyocyte cultures
Ventricular myocytes were prepared from 1- to 5-day-old neonatal Wistar rats (University of Copenhagen, Denmark) as described [33]. Cells were plated at a density of 5x104 cells/cm2 in minimal essential medium supplemented with 1% L-glutamine; 0.1 mM bromodeoxyuridine; 0.15 mM vitamin B12, 1 µg/ml insulin, and 6250 U/ml penicillin in 35-mm wells precoated with 8% fetal calf serum for 5 h at 37 °C. Phenylephrine, angiotensin II and leucocyte inhibitory factor were from Sigma, USA. DIF-1 was purchased from Affiniti Research, UK.

2.3 Morphology
Cells were plated on chamber slides. After incubation for the time indicated, the cells were washed twice in TBS (25 mM Tris–HCl buffer (pH 7.4); 137 mM NaCl; 2.7 mM KCl) and fixed in 4% paraformaldehyde for 15 min. The cells were washed twice in TBS and prehybridized for 30 min at RT in TBS containing 5% dry milk and 0.02% Tween 20. Actin was stained with 0.4 µg/ml TRITC-labelled phalloidin (Sigma, USA) in the same buffer for 30 min at RT. The preparations were washed three times in TBS with 0.02% Tween 20 before nuclear staining with 100 nM SYTOX Green nucleic acid stain (Molecular Probes, USA) in TBS for 30 min, RT. Finally, the preparations were washed three times in TBS and mounted on glass coverslips with DAKO-fluorescent mounting medium (Dako, Denmark) and viewed by confocal laser microscopy (LSM510, Zeiss, Switzerland). Cell size was analyzed using the Metamorph software (Zeiss, Switzerland) and expressed as arbitrary units.

2.4 Recombinant adenovirus
A cDNA encoding human cyclin D2 was inserted in the XbaI site of pAdTrack-CMV [34] to create pAdD2. The cDNA for human P16 was cut out of pBSKS+/P16 [35] with NotI and HinDIII and ligated into pAdTrack-CMV digested with NotI and HinDIII resulting in pAdP16. Replication-deficient adenovirus expressing cyclin D2 or P16 from the CMV promoter were made by homologous recombination of pAdD2 or pAdP16 with pAdEasy-1 in E. coli BJ5183 [34]. The vira were multiplied and packed by transfection into HEK293 cells [34]. Cardiomyocytes were infected with adenovirus as described [32] 1 day before treatment with DIF-1, angiotensin II or serum.

2.5 Protein extraction and Western analysis
Frozen left ventricles were homogenised with a douncer in 50 mM Tris/HCl (pH 7.5); 100 mM NaCl; 5 mM EDTA; 1% (v/v) Triton X-100; 1 mM NaF; 1 mM Na3VO4; 0.2 mM phenylmethylsulfonyl fluoride; 10 µg/ml leupeptin; 10 µg/ml aprotinin. Lysates were cleared by centrifugation at 13 000xg for 10 min at 4 °C. Supernatants were frozen in liquid nitrogen. Protein from myocyte cultures was extracted by lysing the cells in 62.5 mM Tris/HCl (pH 6.8); 2% SDS; 10% glycerol. Lysates were cleared by centrifugation at 13 000xg for 10 min at 4 °C.

Twenty micrograms of protein were separated by denaturing SDS–polyacrylamide gel electrophoresis. Equal loading of protein was verified by Coomassie staining. Gels were blotted onto PVDF membranes (Biorad, USA) that were incubated with antibody diluted 1:1000. The antibodies were cyclin B: SC-752, cyclin D1: SC-450, cyclin D2: SC-593, cyclin D3: SC-6283, cyclin E: SC-481, CDK2: SC-163, CDK4: SC-260, CDK6:SC-177 (all from Santa Cruz Biotech, USA) and PCNA: 610664 (BD Biosciences, USA). Proteins were visualized by horseradish peroxidase conjugated anti-rabbit antibody (Amersham/Pharmacia, UK) followed by chemiluminescence detection (Amersham/Pharmacia, UK).

Quantification was done by scanning on a Duoscan T1200 scanner (AGFA, Belgium) and the bands were quantified with the NIH Image 1.62 software.

2.6 Cyclin D-associated kinase assay
Cyclin D (and associated kinase) was immunoprecipitated from 100 µg protein and the kinase activity was measured essentially as described [30] except that the IP buffer contained 50 mM Tris/HCl (pH 7.5); 100 mM NaCl; 5 mM EDTA; 1% (v/v) Triton X-100; 1 mM NaF; 1 mM Na3VO4; 0.2 mM phenylmethylsulfonyl fluoride; 10 µg/ml leupeptin; 10 µg/ml aprotinin and the kinase substrate was 0.1 µg truncated Rb protein (SC-4112, Santa Cruz Biotech, USA). The antibodies were diluted 1:200 for immunoprecipitation.

2.7 Immunohistochemistry
Immunohistochemistry was done as described [36,37] with the antibodies DCS-6 to cyclin D1, DCS-22 to cyclin D3 and C-17 (Santa Cruz Biotech, USA) to cyclin D2 on cryosections fixed with methanol/paraformaldehyde.

2.8 Statistics
Statistical comparison was performed by unpaired two-sided Student’s t-test. Differences were considered significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
As model for development of hypertrophy in vivo we chose pharmacologically induced hypertrophy (angiotensin II) and pressure-overload hypertrophy induced by aorta banding. After 2-week treatment with angiotensin II, the rats showed increased left ventricular weight indicating hypertrophy (Table 1). In aortic banding, the hypertrophy was more severe than in angiotensin II-treated animals (Table 1).


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  		Table 1 Animals treated with angiotensin II or by aortic banding developed left ventricle hypertrophy

 
To investigate which cell-cycle regulatory proteins might be involved in cardiac hypertrophy we analyzed the expression of the G1-related cyclins and associated kinases in both animal models. Western blotting showed that angiotensin II treatment was accompanied by an increase in the amount of cyclin D1, D2 and D3 in the left ventricle whereas aorta banding leads to an increase in cyclin D2 and cyclin D3 compared to sham operated animals and a decrease in cyclin D1 (Fig. 1). There was a higher level of cyclin D2 in operated animals (SH and AB) than in pharmacologically-treated animals (C and AII). A microarray analysis showed many other differences in gene expression between control animals (C) and sham-operated animals (SH) (CCS, unpublished). Both of the cyclin D-associated kinases, CDK4 and CDK6, were expressed in the left ventricle. CDK4 was highest in newborn animals whereas CDK6 was highest in adult animals.


Figure 1
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  		Fig. 1 Expression of G1-phase cell-cycle regulators in the left ventricle. The amount of cell-cycle regulatory proteins was measured by Western blotting for cyclins D1, D2 and D3 and cyclin E and the cyclin-dependent kinases CDK2, CDK4 and CDK6. Expression in left ventricle from newborn animals (NB); adult animals treated with vehicle (C) or angiotensin II (AII) and sham (SH) or aorta banded (AB) adult animals. Each sample was a pool of equal amounts of protein from six animals (seven for vehicle-treated). Equal loading of protein was controlled by Coomassie staining. The panel at the right is a quantification of the expression. The level of expression in sample AII is given as percent of the expression in sample C and the level of expression in sample AB is given as percent of the expression in sample SH. The horizontal line represents 100% (no change in expression compared to the respective control). The data represent the mean of three experiments.

 
Cyclin E was expressed in left ventricles of newborn animals but was undetectable in adult rat ventricles (Fig. 1). Nevertheless, the cyclin E-associated kinase, CDK2, was present in adult animals. CDK2 can also be activated by cyclin A, which was found in ventricles (not shown). CDK2 has different functions when bound to cyclin A than together with cyclin E [27]. The absence or low levels of cyclin E, which is crucial for entry into S-phase, suggests that the D-type cyclins are the most relevant candidates for regulation of hypertrophy.

Cyclin D2 expression was elevated in all animals subjected to aortic banding compared to sham operated animals 6 weeks after operation (P<0.01) whereas cyclin D1 and D3 were unaffected (P>0.05) (Fig. 2A). The high level of cyclin D2 expression was also observed at 12 (P<0.01) and 16 weeks (P<0.05).


Figure 2
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  		Fig. 2 Cyclin D expression in individual animals and expression of proliferation markers. (A) The amount of cell-cycle regulatory proteins was measured by Western blotting for cyclins D1, D2 and D3 in individual animals 6 weeks or 12 and 16 weeks after aortic banding. The bands were quantified as described in Methods and the expression level in the four sham operated animals was compared to the expression level in the five aortic banded animals by a two-sided Student’s t-test. (B) Expression of PCNA and cyclin B (cycB) in left ventricle from adult animals treated as in Fig. 1.

 
It was previously reported that hypertrophy is accompanied by increased DNA synthesis and limited cell-division [18,19,38]. To investigate if the elevated level of cyclin D expression in angiotensin II and aortic banded animals was due to increased cell division, we measured the level of PCNA and cyclin B in the animals. PCNA is a part of DNA polymerase delta that is elevated in proliferating cells and cyclin B is induced after DNA synthesis in the cell cycle and is necessary for mitosis [39]. The level of PCNA was elevated in the left ventricle of angiotensin II-treated animals but this was not accompanied by an increase in cyclin B (Fig. 2B). In aortic banded animals, both proteins were less expressed than in sham operated animals. Taken together, this suggests that the protein levels detected by Western blotting originated mainly from hypertrophic myocytes and did not come from proliferating cells.

To determine if the D cyclins were expressed in cardiomyocytes or in interstitial cells we investigated the expression by immunohistochemical analysis of left ventricles. In normal myocardium, all three D-type cyclins were detectable, and all were nuclear (Fig. 3). All three cyclin Ds were found in the myocytes. In terms of fraction of positive cells, cyclin D3 was the most expressed (detectable in the majority of cells, up to 80%), followed by cyclin D1 (around 50% cells positive), and D2 the least positive (focally, in total 10–20% positivity, in a very heterogeneous manner). In pressure-overload hypertrophy, the level of cyclin D2 is elevated whereas there is no clear induction of cyclin D1 and D3 (Fig. 3).


Figure 3
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  		Fig. 3 Immunohistochemical detection of cyclin D1, D2 and D3 in left ventricle. The three cyclin Ds show nuclear staining (arrowheads) in the ventricular cardiomyocytes of both sham operated and aortic banding animals. The nuclear staining for cyclin D2 is increased (arrows) in ventricles from animals with aortic banding. The cryosections were made from the same animals as in Fig. 1.

 
The known function of the cyclins is to activate CDKs but CDK inhibitors and CDK phosphorylation also determine the CDK activity [26]. Therefore, we measured the kinase activity associated with cyclin D1, D2 and D3 in the left ventricle (Fig. 4). The complex of cyclin D-CDK was immunoprecipitated with cyclin D-specific antibodies and the kinase activity was measured as the ability to phosphorylate a truncated Rb protein. No CDK activity was detected in samples immunoprecipitated with a control antibody (data not shown). The activity of the cyclin D-associated kinases (CDK4/6) was induced in hypertrophic ventricles from angiotensin II-treated animals. Pressure overload hypertrophy by aortic banding only induced cyclin D2-associated kinase. This result shows a correlation between hypertrophy and cyclin D-associated kinase activity although there was a differential regulation of the individual D-type cyclins in the heart.


Figure 4
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  		Fig. 4 Induction of cyclin D-associated kinase activity in the left ventricle during hypertrophy. Cyclin D1, D2 and D3 were immunoprecipitated from left ventricle protein extracts. The associated kinase activity was measured as the ability to phosphorylate a truncated Rb protein with radioactive ATP. Reactions were stopped with SDS sample buffer and the Rb protein was analysed on SDS–PAGE. The degree of phosphorylation was measured with a phosphorimager. Samples were made as in Fig. 1. The experiment was repeated three times.

 
The morphological and molecular characteristics of hypertrophy can be mimicked in cardiomyocytes isolated from newborn animals. We measured the level of cyclin D1, D2 and D3 in newborn myocytes. Cyclin D1 was expressed at a low level in myocytes and induced by hypertrophic stimulation with phenylephrine or serum (Fig. 5A). Cyclin D2 and D3 expression was slightly up-regulated by phenylephrine and more strongly by serum. The response to hypertrophic conditions resembles the regulation found in adult hearts. Both cyclin D1 and D2 were induced by different hypertrophic hormones (angiotensin II and phenylephrine) and by the cytokine leucocyte inhibitory factor (Fig. 5B). This cytokine induces hypertrophy but through a different pathway than angiotensin II and phenylephrine [1]. The expression was measured 2 days after induction of hypertrophy to determine the level of constitutive expression of the cyclins in myocytes. However, cyclin D1 induction was detectable already 4 h after addition of angiotensin II to the cultured myocytes and increased until 24 h after addition (Fig. 5C). We did not observe any transient increase in cyclin D2 and D3 during the first 24 h after hypertrophic stimulation (data not shown).


Figure 5
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  		Fig. 5 Induction of cyclin D in neonatal myocytes in response to hypertrophic stimuli. (A) The cyclin Ds are induced by hormone- and serum-induced hypertrophy in neonatal myocytes. Western blotting of cyclin D1, D2 and D3 in protein extracts from isolated, neonatal myocytes incubated in medium without serum (0), with 10 µM phenylephrine (PE) or with 5% serum (SE) for 48 h. The panel on the right is a quantification of the expression where the level of expression in serum-treated samples is normalised to 100. The data represent the mean of three experiments. (B) Induction of cyclin D expression by hypertrophic stimuli that activate different signalling pathways. Western blotting of cyclin D1, and D2 in protein extracts from isolated, neonatal myocytes incubated in medium without hormones (0), with 0.5 µM angiotensin II (AII), 5 nM leucocyte inhibitory factor (LIF) or 10 µM phenylephrine (PE) for 48 h. The panel on the right is a quantification of the expression where the level of expression in angiotensin II-treated samples is normalised to 100. The data represent the mean of three experiments. (C) Cyclin D1 is induced after 4 h of angiotensin II treatment. Western blotting of cyclin D1 in protein extracts from isolated, neonatal myocytes incubated in medium with 0.5 µM angiotensin II for the time indicated. The panel below the Western blotting is a quantification of the expression where the level of expression after 24 h is normalised to 100. The experiment was repeated three times.

 
To study the biological role of the D-type cyclins in hypertrophy, we investigated the effect of inhibiting cyclin D expression on hormone- and serum-induced hypertrophy. Others have studied the effect of inhibiting cyclin D-associated kinase activity on hypertrophy [25,32]. However, the D-type cyclins have biological roles that do not require associated kinase activity [40,41]. We therefore chose to treat the cells with Differentiation Inducing Factor 1 (DIF-1) from Dictyostelium discoideum. This compound inhibits expression of all three cyclin Ds in vascular smooth muscle cells but does not affect the expression of other G1-related cell-cycle regulatory proteins [42]. The same concentration of DIF-1 as used in vascular smooth muscle cells lowered basal and serum-induced expression of the D-type cyclins in cardiomyocytes (Fig. 6A). Cyclin D1 and D3 expression was less affected than cyclin D2 under basal conditions. This is in agreement with the observation that the basal activities of cyclin D1- and D3-associated kinases were more or less unaffected by DIF-1 (Fig. 6B). However, DIF-1 inhibited the kinase activity associated with all three D-type cyclins in the presence of serum. Interestingly, cyclin D2-kinase, which displayed the strongest induction by serum, was also the most sensitive to DIF-1.


Figure 6
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  		Fig. 6 DIF-1 inhibits cyclin D expression and associated kinase activity in neonatal myocytes. (A) Western blotting of cyclin D1, D2 and D3 in protein extracts from isolated, neonatal myocytes incubated in medium without hormone (0) or with serum (SE) for 2 days; 30 µM DIF-1 (DIF) was added as indicated at the same time as serum. The panel on the right is a quantification of the expression where the level of expression in serum-treated samples is normalised to 100. The data represent the mean of three experiments. (B) Cyclin D-associated kinase activity was measured in myocytes treated as in (A). The experiment was repeated three times.

 
To test the effect of cyclin D-inhibition on hypertrophy we incubated neonatal myocytes with DIF-1 together with hypertrophic stimuli. The hypertrophy induced by two hormones (angiotensin II and phenylephrine) and by serum could be efficiently suppressed by simultaneous treatment with DIF-1 (Fig. 7). Morphologically, cells treated with DIF-1 in combination with hormone or serum resembled cells without hypertrophic elicitor (Fig. 7A). This was confirmed by measuring the cell-size. The DIF-1-treated myocytes did not increase in cell-size in response to angiotensin II, phenylephrine or serum whereas hypertrophy was induced in controls (Fig. 7B). There was a small decrease in the basal cell size in response to DIF-1. A fraction of the myocytes are hypertrophic even under basal conditions [33] and DIF-1 may reduce the size of these cells.


Figure 7
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  		Fig. 7 Inhibition of cyclin D expression with DIF-1 impairs the development of hypertrophy in neonatal myocytes. (A) Neonatal myocytes were incubated without DIF-1 (control) or with 30 µM DIF-1 (DIF) in medium without serum (basal), with 0.5 µM angiotensin II (AII) or with 5% serum (SE) for 3 days. The cells were fixed and stained for actin (red) and nucleus (green). (B) Increase in cell size (hypertrophy) in response to hormones or serum. The average size of the cells (arbitrary unit) under each condition was determined as described in Methods. DIF-1-treated cells were statistically smaller than controls treated in the same way (P<0.01). Neither hormone, nor serum led to any statistically significant increase in the size of DIF-1-treated cells compared to Basal+DIF-1 (P>0.05). At least 50 cells were counted under each condition and two independent experiments gave the same results.

 
The absence of hypertrophy in cells where cyclin D expression is inhibited indicates that cyclin D expression is necessary for hypertrophy. However, it is possible that DIF-1 impairs hypertrophy through a mechanism that is unrelated to cyclin D. To address this possibility, we infected cardiomyocytes with an adenovirus overexpressing cyclin D2. This led to an increased expression of cyclin D2 independently of DIF-1 (Fig. 8A). The myocytes overexpressing cyclin D2 were hypertrophic under basal conditions and serum did not induce any further increase in cell size (Fig. 8B). Furthermore, DIF-1 failed to block serum-induced hypertrophy in cyclin D2 overexpressing myocytes suggesting cyclin D2 can rescue the hypertrophic response in the presence of DIF-1. The result shows that cyclin D2-overexpressing can restore the hypertrophic response in DIF-1-treated myocytes. Cyclin D2 overexpressing myocytes expressed more D2 and were larger without than with DIF-1 (P<0.05) (Fig. 8A,B). This could be caused by a stimulation of endogenous cyclin D2 expression that is impaired by DIF-1 or by destabilisation of the cyclin D2 mRNA by DIF-1. Myocytes infected with an adenovirus that expresses the cyclin D-associated kinase inhibitor P16 lowered the hypertrophic response to serum and angiotensin II (Fig. 8C and D). The hypertrophic response to serum was only partially inhibited. However, there is a high basal level of P16 expression in the myocytes showing that hypertrophy can occur in the presence of P16 (Fig. 8C). In agreement with this notion, the hypertrophic response seems to depend on the amount of P16 expressed in the cells (PKB, unpublished data). In summary, these results suggests that cyclin D-expression is necessary for hypertrophy and that the effect of cyclin D is mediated through activation of the associated kinases CDK4 and 6.


Figure 8
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  		Fig. 8 Cyclin D and cyclin D-associated kinase are necessary for hypertrophic response in neonatal myocytes. (A) Western blotting of cyclin D2 in protein extracts from neonatal myocytes with or without DIF-1 infected with a mock virus (M) or a virus overexpressing cyclin D2 (D2). (B) Increase in cell size (hypertrophy) in response to serum (Se). The average size of the cells (arbitrary unit) under each condition was determined as described in Methods. Without DIF-1, the mock-infected cells increased in size in response to serum (P<0.05) whereas serum did not affect the D2-overexpressing cells (P>0.05). In the presence of DIF-1, the mock-infected cells did not increase in size in response to serum (P>0.05) whereas the D2-overexpressing cells became larger in response to serum (P<0.05). Two independent experiments gave the same results. (C) Western blotting of P16 in protein extracts from neonatal myocytes infected with a mock virus (M) or a virus overexpressing P16 (P16). (D) Increase in cell size (hypertrophy) in response to angiotensin II (AII) or serum (Se). The average size of the cells (arbitrary unit) under each condition was determined as described in Methods. At least 50 cells were counted under each condition and two independent experiments gave the same results.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Our data indicate that cyclin D expression is necessary for cardiac myocyte hypertrophy in vitro. In addition, cyclin D and associated kinase activity was induced in two different models of left ventricle hypertrophy suggesting that these proteins are also important for hypertrophy in vivo.

The hormone DIF-1 from D. discoideum inhibited expression of the D cyclins and impaired hormone- and serum-induced hypertrophy in neonatal myocytes. This suggests that the D-type cyclins are necessary for cardiac hypertrophy at least in vitro. DIF-1 does not have any detectable effect on other G1-phase specific proteins [42]. However, it is not known how DIF-1 inhibits the expression of the D-type cyclins. It was proposed that DIF-1 acts on one or more MAP kinases but this has not been demonstrated. In any case, DIF-1 impairs the expression of the three cyclin Ds in both vascular smooth muscle cells [42] and cardiomyocytes (this study).

The cyclin Ds were necessary for hypertrophy induced by two different hormones and by serum. The primary function of the D-type cyclins is to activate CDK4 and CDK6 [27,28,31]. In agreement with previous reports [25,32] we found that inhibition of CDK4 and CDK6 by overexpression of P16 impaired hypertrophy in vitro. Thus, hypertrophy may require induced expression of the cyclin Ds and subsequent activation of CDK4/6 as in the case of proliferation. Cell division is linked to cell size and the cell-cycle regulatory mechanism seems to have an active role in determining cell size in eukaryotes [43–45]. A current view is that hypertrophy requires entry into G1-phase of the cell cycle without progression through the S-phase [46]. This mechanism seems valid for cardiac myocyte hypertrophy.

The hypertrophic response in the presence of DIF-1 could be rescued by overexpression of cyclin D2 suggesting that cyclin D2 is necessary for cardiac hypertrophy. It is possible that overexpression of cyclin D1 or D3 would have the same effect as cyclin D2 because the D-type cyclins have largely overlapping functions [27,28,47]. The best clue as to which cyclin Ds are involved in hypertrophy comes from the CDK assays. In vivo, the kinase activity associated with all three cyclin Ds was induced in pharmacological hypertrophy whereas only cyclin D2-associated kinase activity was observed in pressure overload hypertrophy. This observation shows a differential regulation of cyclin D activity in response to distinct hypertrophic stimuli. In addition, cyclin D2 expression was elevated in all animals with aortic banding suggesting that up-regulation of cyclin D2 is characteristic for pressure overload hypertrophy. In isolated myocytes the D2-associated kinase was also strongly induced during hypertrophy. The immunohistochemistry showed that cyclin D1 and D3 expression was more abundant than cyclin D2 expression in the left ventricle. It is possible that hypertrophy is mediated by the total amount of the three D cyclins through an induction of CDK 4 and 6. Despite the expression of cyclin D in the controls, we did not detect cyclin D-associated kinase activity in these animals. This suggests that the level of cyclin D is too low to overcome the inhibition by CDK inhibitors as P16 [22,28].

In isolated neonatal myocytes, cyclin D expression increased under hypertrophic conditions as found in adult left ventricle. The induction of cyclin D expression by different stimuli that activates distinct signalling pathways [11,14] suggests that cyclin D is part of a common mechanism for hypertrophic signalling and may even be a point of convergence. This is analogous to proliferating cells where cyclin D expression can be induced by different mitogens through distinct pathways and appears necessary for cell cycle progression [31]. In both adult hearts and neonatal myocytes there was a basal level of cyclin D3 expression suggesting that cyclin D3 may play a biological role in normal cardiac myocytes. The cyclin D3 expression in several non-dividing tissues suggests that this protein is involved in maintenance of a terminally differentiated phenotype [47]. This function seems to be mediated through cyclin D3 interaction with proteins other than CDK4/6 and does not involve cyclin D3-associated kinase activity [48]. In agreement with this model, we did not detect cyclin D3-associated kinase activity in left ventricle from control animals. Therefore, it is possible that blocking cyclin D-associated kinase activity might be a plausible target in treatment of cardiac hypertrophy. This could be done with specific CDK4/6 inhibitors such as P16.

Interestingly, there was a down-regulation of CDK4 and an up-regulation of CDK6 in adult ventricle compared to newborn ventricles. CDK6 may have a role in arresting proliferation [49] which fits well with the expression in terminally differentiated, adult hearts. In addition, cyclin E was low or absent in the left ventricle of adult animals. As cyclin E is necessary for DNA synthesis and cell cycle progression [26] this finding is in accordance with the limited cell division of adult myocytes [18,19]. Moreover, we did not observe any correlation of the expression of the proliferation marker PCNA and the mitotic regulator cyclin B with hypertrophy. Induced expression of cyclin E in CDK2 transgenic mice leads to S phase progression in adult myocytes but does not overcome a blockage at the G2/M phase of the cell-cycle [50]. It was previously reported that cyclin E levels are low in adult cardiac myocytes and that the protein is induced by myocardial infarction [23]. This induction is accompanied by increased cell division in the left ventricle [18,19]. The lack of cyclin E together with the increased cyclin D1-associated kinase activity indicates that cardiac hypertrophy involves an entry into the cell-cycle but that division is arrested in late G1-phase. This resembles the molecular mechanism that was outlined for renal tubule epithelial cell hypertrophy [16,51]. However, in renal tubule epithelial cells cyclin E levels are high and cyclin E-associated kinase activity is impaired by the specific inhibitor p57kip [51]. This shows that different cell types apply different mechanisms to regulate cyclin E activity during hypertrophy.

Time for primary review 24 days.


   Acknowledgements
 
We thank Katrine Kastberg and Tordis Christiansen for technical help. This work was supported by grants from the Danish Heart Association (00-2-2-17A-22836), the Danish Medical Research Council, the Birthe and John Meyer Foundation, the Villadsen Family Foundation, the European Community, the Danish Cancer Society and the Foundation of 17.12.1981.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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