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European Journal of Heart Failure 2007 9(4):352-356; doi:10.1016/j.ejheart.2006.10.002
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© 2007 European Society of Cardiology

Endogenous inhibitors of hypertrophy in concentric versus eccentric hypertrophy

Katrien Lemmensa, Vincent F.M. Segersa, Marc Demoldera, Maria Michielsa, Philip Van Cauwelaertb and Gilles W. De Keulenaera,*

a University of Antwerp, Laboratory of Physiology Groenenborgerlaan 171, 2020 Antwerp, Belgium
b Department of Cardiac Surgery, Middelheim Hospital Antwerp, Belgium

* Corresponding author. Tel.: +32 3 265 32 77; fax: +32 3 265 32 78. E-mail address: gilles.dekeulenaer{at}ua.ac.be


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Left ventricular (LV) hypertrophy (LVH) is an adaptive response to hemodynamic overload, but also contributes to the pathogenesis of heart failure. LVH can be concentric (cLVH) but subsequent dilatation and progression to eccentric hypertrophy (eLVH) may lead to global pump failure. Recently, several endogenous molecular inhibitors of hypertrophy have been identified. Using real-time PCR, we compared the myocardial mRNA expression of these inhibitors in pressure-overload induced cLVH (severe aortic stenosis) and in volume overload-induced eLVH (severe mitral regurgitation) in patients, and during the progression from cLVH to eLVH in pressure overload in rat. Each of these genes showed a unique temporal expression profile. Strikingly, except for SOCS-3, changes in gene expression of these negative regulators in rat cLVH and eLVH vs sham were recapitulated in human cLVH and eLVH. In particular, VDUP-1 and MCIP-1 were high in cLVH but expression levels were normal in eLVH, both in rat and human. These data indicate that during the progression of LVH, both in pressure and volume overload, expression levels of endogenous inhibitors of hypertrophy are modified and that these changes may have pathophysiological significance. In particular, MCIP-1 (the endogenous calcineurin inhibitor) and VDUP-1 (the endogenous inhibitor of thioredoxin) are potential molecular switches in the progression of LV hypertrophy.

Key Words: Hypertrophy • gene expression • heart failure • redox

Received April 26, 2006; Revised July 14, 2006; Accepted October 2, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Left ventricular hypertrophy (LVH) is an adaptive response of the heart to mechanical overload but also an intermediate step towards heart failure [1]. LVH can be concentric (cLVH) but subsequent dilatation and progression to eccentric hypertrophy (eLVH) indicates progression to global pump failure [2]. Little is known about the molecular mechanisms that underlie the differences in cLVH and eLVH and the transition to failure [3,4].

Numerous pro-hypertrophic molecular pathways/factors are activated during hypertrophy, many of which are controlled by biomechanical strain [5]: the mitogen-activated protein (MAP) kinases [5], Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway [6], calcineurin [1], thioredoxin [7] and NF{kappa}B [8]. For each of these pathways an endogenous inhibitory molecule has been identified [9]. These molecules are activated during mechanical overload [7,8,10-12] and thus may serve as negative feedback control signals. Their in vivo inhibitory effect has been demonstrated in transgenic mice [12,13] but the physiological relevance in human disease remains to be elucidated. In the present study we investigated the mRNA expression of 5 suppressors of hypertrophy in pressure-overload induced cLVH versus volume-overload induced eLVH in human patients and during the transition from cLVH to eLVH in pressure overload in rat.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Aortic constriction in rat
LVH was induced in adult male Sprague-Dawley rats (Harlan Netherlands BV, n=63) by a transverse aortic constriction (TAC). Rats were anaesthetized with fentanyl (0.05 mg/kg, Janssen-Cilag), diazepam (5 mg/kg, Roche) and haloperidol (3 mg/kg, Janssen-Cilag), ventilated and a left thoracotomy was performed. A 5-0 silk suture (Deknatel) ligature was tied around the transverse aorta against an 18-gauge needle. Age-matched sham operated animals served as control. Rats were randomly assigned to groups (sham or TAC) and time points (1 day, 2, 4, 8 and 16 weeks). Hypertrophy was evaluated based on heart weight (HW)/body weight(BW) ratio and echocardiographic parameters. Echocardiograms were performed on anesthetized animals using a 10MHz transducer. LV anterior (AWT) and posterior wall thickness (PWT) and end-diastolic and end-systolic internal dimensions (EDD, ESD) were measured on three consecutive cycles and averaged by a single observer in a blinded fashion. LV end diastolic volume (LVEDV) was obtained using the Teichholz method [14]: (7/(2.4+EDD))EDD3. LV mass was calculated as: 0.8(1.04)((AWT+EDD+PWT)3-EDD3)+0.6 [15]. LV systolic function was assessed by using fractional shortening: %FS=[(EDD-ESD)/EDD]x100 [16].

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1985).

2.2. Human biopsies
LV biopsies were obtained in cLVH (during surgery for severe aortic stenosis, AS, n=9), in eLVH (surgery for severe mitral regurgitation, MR, n=10) and in non-hypertrophied hearts (surgery for mitral stenosis or CABG, control, n=8). Biopsies were procured immediately after the start of the pump run at the level of the distal interventricular septum. Samples were immediately frozen in liquid nitrogen and stored at –80 °C. LV mass and LVEDV were calculated as for rat. The investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee. All patients provided informed consent.

2.3. Real-time quantitative PCR
Heart tissue was homogenized with a Polytron homogenizer in TRIzol ®Reagent (Invitrogen, Life Technologies). mRNA was isolated following instructions of the manufacturer. Reverse transcription was performed with TaqMan reverse transcription reagents (Applied Biosystems) and samples were used for real-time PCR in a 25 µl reaction containing 12.5 µl SYBR Green PCR Reaction Mix (Applied Biosystems) and 100-900 nmol/L of both primers. After incubation for 2 min at 50 °C and 10 min at 95 °C, 45 PCR cycles were carried out consisting of a denaturation step of 15 s at 95°C and a primer annealing and elongation step of 1 min at 60 °C, on an ABI Prism 7700 sequence detection system. Expressions were normalized to GAPDH. Expression levels of GAPDH were constant among different groups. PCR efficiencies were equal for the gene of interest and GAPDH.

2.4. Statistics
Data are expressed as mean±SEM. mRNA expression was quantified using the comparative threshold method. Main effects of TAC in rat were assessed using two-way ANOVA. Comparisons between TAC and sham at 8 and 16 weeks were performed with a Student's t-test with Bonferroni correction for multiple comparisons. Human data were compared with one-way ANOVA and Dunnett analysis.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Hypertrophy
Fig. 1A shows that TAC-induced pressure overload in rat resulted in cLVH at 8 weeks (HW/BW: 5.1±0.6 versus 3.4±0.1 in sham, p<0.05; LVEDV: 250±26 µl vs 220±20 µl in sham, p=1.00, n=5), but in eLVH at 16 weeks (HW/BW: 4.3±0.3 vs 3.3±0.17 in sham, p<0.05; LVEDV: 350±26 µl vs 226±13 µl in sham, p<0.05, n=11) with reduced FS (FS: 47±4% versus 58±2% in sham, p<0.05, n=11). Similarly, patients with AS suffered from cLVH (LV mass: 261±8 g vs 177±10 g in control, p<0.05; LVEDV: 104±13 µl vs 122±9 µl in control, p=1.00, n=9) and patients with MR suffered from eLVH (LV mass: 325±64 g vs 177±10 g in control, p<0.01; LVEDV: 215±20 ml versus 122±9 ml in control, p<0.01, n=11). (Fig. 1B).


Figure 01
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  		Fig. 1 Validation of Study Design, A, Pressure overload in rat induces cLVH after 8 weeks and eLVH at 16 weeks. p<0.05, B, Human LV biopsies were obtained from hearts with cLVH (aortic stenosis, AS), eLVH (mitral regurgitation, MR) and non-hypertrophied controls. p<0.05, C, Representative echocardiograms after 8 weeks (left) and 16 weeks (right) of pressure overload in rat.

 
3.2. Gene expression
As shown in Fig. 2A, each gene followed a unique temporal expression profile during pressure overload in rat. IEX-1 and SOCS-3 mRNA expression remained unchanged. MKP-1 mRNA was downregulated both in cLVH and eLVH (16 weeks: 5.8±2.3 fold versus sham, p<0.05, n=11). VDUP-1 and MCIP-1 were upregulated in cLVH (VDUP-1: 3.8±1.1 fold versus sham, p=0.07; MCIP-1: 4.6±1.5 fold versus sham, p<0.05, n=5) but expression levels returned to normal in eLVH. Strikingly, except for SOCS-3, the changes in mRNA expression in cLVH and eLVH in rat were recapitulated in human cLVH and eLVH (Fig. 2B). In particular, MKP-1 was downregulated both in cLVH and eLVH (MR: 30±10 fold versus control, p<0.05, n=11) whereas VDUP-1 and MCIP-1 were upregulated in cLVH (VDUP-1: 2.3±1.0 fold versus control, p=0.14; MCIP-1: 9.6±3.7 fold versus control, p<0.05, n=9) but unchanged in eLVH.


Figure 02
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  		Fig. 2 mRNA expression of inhibitory molecules in rat and human, A, mRNA expression in rat hearts at indicated durations of TAC expressed as fold induction versus sham. p<0.05, {varphi}=0.08, {pi}=0.07, B, mRNA expression in human LV biopsies obtained from hearts with cLVH and eLVH expressed as fold induction versus non-hypertrophied control. p<0.05, AS: aortic stenosis; MR: mitral regurgitation.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although the molecular pathways promoting hypertrophy have been extensively studied, the molecular mechanisms involved in the transition from cLVH to eLVH have remained incompletely understood. Recently, several molecules have attracted attention as in vivo endogenous inhibitors of these pathways but the physiological regulatory function of these molecules is still unclear [9,17]. In the present study we report that the gene activity of these endogenous inhibitors changes during progression from cLVH to eLVH in pressure overload in rat and that, surprisingly, most of these changes are recapitulated in pressure-overload induced cLVH and volume-overload induced eLVH in patients.

In particular, MKP-1, the MAPK inhibitor, was down regulated in LVH, with no difference between cLVH and eLVH in rat and human. In view of the previously reported activation of MAPKs in human and rat heart failure, this downregulation can be regarded as an extra activation of this pathway [18,19] In contrast, VDUP-1 (the inhibitor of thioredoxin) and MCIP-1 (the endogenous calcineurin-inhibitory protein) were upregulated in cLVH probably serving as a negative feedback regulation of induced calcineurin and thioredoxin activity [18,20], but expression levels were normal in eLVH. Importantly, the increase in VDUP-1 only occurred at the latest stages of cLVH just before dilatation and failure, resulting in a relative small time window during which VDUP-1 was upregulated, and probably responsible for the borderline significance of the alterations. This late and brief induction, however, may also indicate that thioredoxin is involved in the transition towards LV dilation.

It has been previously shown that pro-hypertrophic signaling cascades are differentially altered in cLVH versus eLVH, raising the possibility that this differential regulation plays a causal role in the transition towards LV dilatation [18,19]. Our data extend these reports by demonstrating that the inhibitory regulators of pro-hypertrophic pathways exhibit adaptations in cLVH and eLVH and that these inhibitors must be taken into account to better estimate the impact of a specific pathway.


   Acknowledgements
 
Dr Lemmens was supported by a grant from the Fund for Scientific Research-Flanders. This study was supported by a grant form the European Society of Cardiology (MO-4304).


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

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