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European Journal of Heart Failure 2007 9(6-7):568-573; doi:10.1016/j.ejheart.2007.02.009
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© 2007 European Society of Cardiology

Altered melusin expression in the hearts of aortic stenosis patients

Sebastian Brokata, Jenny Thomasa, Lars R. Herdaa, Christoph Knosallab, Reinhard Preglab, Mara Brancaccioc, Federica Accorneroc, Guido Taronec, Roland Hetzerb and Vera Regitz-Zagroseka,b,*

a Center for Gender in Medicine and Cardiovascular Research Center (CCR), Charité Berlin, Germany
b German Heart Institute (DHZB), Berlin, Germany
c Department of Genetics, Biology and Biochemistry, University of Turin, Italy

* Corresponding author. Center for Gender in Medicine and Center for Cardiovascular Research (CCR), in Cooperation with DHZB, Charité – Universitätsmedizin Berlin, Hessische Straße 3-4, D-10115 Berlin, Germany. Tel.: +49 30 450 525 172; fax: +49 30 450 525 972. E-mail address: vera.regitz-zagrosek{at}charite.de, vrz{at}dhzb.de


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusion
 References
 
Background: The role of melusin, a necessary component in pressure-induced left-ventricular hypertrophy (LVH) in mice, has not yet been determined in human cardiac hypertrophy. We analyzed for the first time the expression and regional distribution of melusin in human LVH due to aortic stenosis (AS) and determined AKT phosphorylation as a potential downstream effector of melusin signalling.

Methods: Regional distribution of melusin was evaluated in four normal hearts. Melusin staining, gene expression and protein content were assessed in biopsies from normal and diseased hearts and melusin gene expression was correlated with LV functional changes. The pAKT/AKT ratio was determined in parallel and correlated with melusin protein content.

Results: In normal hearts, melusin was found in the myocytes with a uniform regional distribution. Melusin staining, mRNA and protein were significantly decreased in human AS hearts. The reduction in melusin mRNA was significantly correlated with LVEF, LVEDD and LVESD. pAKT/AKT ratio was significantly decreased in human AS and was correlated with melusin content.

Conclusion: Reduction in melusin expression parallels the functional cardiac impairment in human AS. The simultaneous decrease of melusin and AKT phosphorylation suggests a connection between the loss of melusin and the decrease in systolic function.

Key Words: Melusin • Aortic stenosis • Hypertrophy • Gene expression • Proteinkinase B/AKT

Received July 10, 2006; Revised December 20, 2006; Accepted February 22, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusion
 References
 
In human cardiovascular pathophysiology, different forms of hypertrophy are observed. Adaptive hypertrophy such as in trained athletes is not associated with an unfavourable prognosis [1]. It is characterized by increased ventricular wall thickness and cardiomyocyte size, lack of fibrosis, maintained systolic function and is fully reversible. Pressure overload — induced concentric hypertrophy, primarily increases wall thickness, reduces wall stress and is associated with maintained cardiac function in the early stages of pressure overload. In the later stages of chronic pressure load, the hypertrophic phenotype turns into thinning of ventricular walls, ventricular dilatation, fibrosis and systolic function decreases. Molecular markers to distinguish these different stages of hypertrophy and mechanisms leading to the development of dilatation and cardiac failure are currently being actively investigated [2].

Melusin, a cytosolic protein, whose expression pattern is restricted to cardiac and skeletal muscle, is thought to play a key role in cardiac hypertrophy. Melusin physically interacts with the integrin β1 cytoplasmic domain and localizes at costameres and is thus part of the molecular machinery connecting the sarcomeric structure to the sarcolemma and to the extracellular matrix [3,4]. Gene inactivation experiments in mice suggest that melusin is not required for heart development, sarcomere organization or cardiac function in basal conditions [3]. Melusin ablation, however, strongly impairs the left ventricular hypertrophic response to pressure overload as induced by transverse aortic banding, and dramatically accelerates the transition to cardiac dilation [3,5]. At the same time, when melusin is over-expressed in the heart of transgenic mice, the left ventricle retains its compensatory concentric hypertrophy with full contractile function when hearts are subjected to long standing pressure overload [6]. These functional properties are accompanied by protection from cardiomyocyte apoptosis and lack of stromal tissue deposition, hallmarks of beneficial heart remodelling. Interestingly, endogenous melusin levels are upregulated during the initial phase of compensatory hypertrophy in mice subjected to aortic banding, but return to basal levels in hearts that have undergone the transition towards dilation. Melusin signalling includes the phosphorylation of AKT and GSK3β as well as phosphorylation of ERK1/2 [3]. These signalling kinases are known to regulate nuclear translocation of different transcription factors, including GATA 4 and NFAT, which are involved in cardiomyocyte hypertrophy [7,8].

Based on these findings, we investigated whether melusin expression is altered in human chronic aortic diseases and whether it correlates with changes in LV function. We also investigated whether regulation of melusin is accompanied by alterations in the phosphorylation of proteinkinase B/AKT in the human heart.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 5. Limitations
 6. Conclusion
 References
 
2.1. Patients
Left ventricular myocardial samples from 17 patients with aortic valve stenosis and 16 control (donor) subjects were analyzed for melusin mRNA or protein content as well as pAKT/AKT ratios, indicating AKT phosphorylation, depending on the amount of tissue available. Consecutive patients with isolated aortic stenosis, without aortic regurgitation or other valve disease exceeding grade I, after exclusion of significant coronary artery disease were included. Clinical data are summarized in Table 1. In patients with AS, biopsies were obtained from the left ventricular septum during elective aortic valve replacement surgery from an area underneath the surgical aortic valve and frozen at –80 °C. The control group was composed of donor hearts that were rejected for transplantation due to logistic reasons. All controls had normal systolic function and no history of cardiac disease. Post-mortem histology evaluated was normal in all cases, as described previously [9,10]. Four of the control donor hearts were also used to study the regional distribution of melusin within the left ventricle. All samples were obtained after written informed consent. The study was performed in accordance with the Declaration of Helsinki.


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  		Table 1 Patient characteristics

 
2.2. Quantification of mRNA by real time PCR
Total RNA preparation, deoxyribonuclease (DNase) digestion and reverse transcription were performed as described previously [9-11]. A "hot start" real time PCR procedure with SYBR Green, that was validated for reproducibility and linearity within the measuring range, was performed in duplicate with a TaqMan– 7000 (ABI). Primers are available on request. To correct for potential variance between samples in mRNA extraction or in RT-efficiency, the mRNA content of the target genes was normalized to the expression of the stably expressed reference genes GAPDH or PDH in the same sample.

2.3. Protein extraction and immunoblotting
Myocardial samples were homogenized and blotted onto nitrocellulose membranes. Rabbit polyclonal affinity purified melusin antibodies were generated as previously described [4]. Purified antibodies were used in western blotting at a final dilution of 2 µg/ml. Antibodies to phospho-Ser473-AKT and to AKT (Santa Cruz) were used as described [4]. Specific HRP-conjugated antibodies were used as secondary antibody. To normalize for different protein content, we stripped the membrane from the first antibody complex and re-hybridized for GAPDH (primary antibody: Chemicon, MAB-374, 1:50.000; secondary antibody: Donkey anti-mouse, Dianova, 1:15.000) as described [3].

2.4. Immunofluorescence microscopy
Tissue samples were fixed in frozen section medium Neg-50 (Richard Allan Scientific). After cutting the samples into 3 µm sections with the Cryostat microtom (Jung Frigocut 2800E; Leica), they were mounted on slides and air dried overnight. For immunohistochemistry the sections were fixed in acetone. After washing, samples were blocked in Peroxidase Blocking Reagent (K3467, Dako Cytomation, USA) and Avidin/Biotin Blocking Reagent (SP-2001, Vector Laboratories). The slides were incubated with the first antibody (melusin [4]) at final dilution of 2 µg/ml in Antibody Diluent Reagent (S3022, Dako Cytomation, USA). After washing slides, were incubated with the secondary antibody conjugated to Peroxidase-anti rabbit (711-035-152, Dianova, Germany; 1:50 in Antibody Diluent Reagent). Negative controls included sections incubated with secondary antibodies only, omitting primary antibody. After washing, slides were stained with Liquid DAB+ Substrate as described in the company protocol (K3467, Dako Cytomation, USA). Nuclei were stained with Haematoxylin. Micrographs were taken with a microscope (Zeiss Axiostar plus) and analyzed using Adobe Photoshop software.

2.5. Statistical methods
Values are calculated as % of controls and given as mean±SEM. For biochemical parameters, normalized gene expression or protein ratios were compared to the mean ratio of the respective control group which was set as 100%. Mean values were compared by the two-sided Student's t-test. Statistics were calculated with the Excel, Sigma Plot and SPSS software. A p-value <0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 5. Limitations
 6. Conclusion
 References
 
Seventeen consecutive patients with chronic AS, undergoing elective aortic valve replacement were included in the study. The average age of patients was 71 years; 76% were male, 76% were hypertensive and 47% diabetic; prescribed drugs were diuretics (76%), ACE-inhibitors (41%), digitalis (24%) and β-blockers (41%). Left ventricular function was impaired (LVEF 46.0±18.2%; FS 27.0±11.0%) and the left ventricle was dilated (LVEDD 54.1±8.8 mm; LVESD 40.9±11.7 mm); only 3 patients had normal LVEF and normal LVESD (Table 1).

Immunohistochemical staining of melusin was done to visualize expression changes due to cardiac hypertrophy. Melusin was found in the myocytes (Fig. 1a). Staining in the normal hearts was clearly visible (Fig. 1a; neg. control: Fig. 1c). In contrast, in the AS hearts, melusin staining was significantly weaker (Fig. 1b) and almost comparable to the negative control (Fig. 1d).


Figure 01
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  		Fig. 1 Immunohistochemical staining of biopsies from a) normal human donor heart (200 fold magnification; c) negative control) and b) human AS heart with EF 50% (200 fold magnification; d) negative control). Staining of melusin in the normal hearts was clearly visible with a location within the myocytes. In the diseased heart, melusin staining was significantly weaker.

 
To assess the regional distribution of melusin in the heart, we analyzed melusin content in 4 different regions (septum, anterior, free lateral and inferior wall) of 4 normal donor hearts. Analysis revealed that melusin expression was homogenous in these different areas of the left ventricle (Table 2).


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  		Table 2 Analysis of melusin protein content (melusin/GAPDH ratios) in 4 different regions of 4 normal donor hearts

 
In order to quantify the changes observed by immunohistochemistry, melusin expression in the control and AS hearts was evaluated both at the mRNA and protein level. Interestingly, melusin was significantly downregulated (p<0.05) in our group of patients with AS. Melusin mRNA in AS patients went down to 24.3±4.9% of control values (Fig. 2a). Concordantly, melusin protein was decreased to 32.5±5.2% of control (Fig. 2b, c). Melusin expression was positively correlated with LVEF and inversely correlated with ventricular size, i.e. LVEDD and LVESD (p<0.05; Fig. 3a-c).


Figure 02
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  		Fig. 2 Regulation of melusin a) mRNA and b) protein in human AS compared to controls. Controls were set as 100% and AS values were related to the mean value of controls. Data are presented as mean value±SEM. Controls are shown in white bars, AS in gray bars. c) Typical immunoblots showing melusin and GAPDH in patients with AS and controls.

 


Figure 03
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  		Fig. 3 Melusin was significantly correlated with parameters of LV function in human AS; positively correlated with a) LVEF (r=0.64, p=0.048) and inversely with b) LVEDD (r=–0.82; p=0.002) and c) LVESD (r=–0.77, p=0.006).

 
AKT phosphorylation is located downstream from melusin. AKT phosphorylation was significantly decreased in human AS (71.2±5.2%, p<0.001; Fig. 4a, b) whereas AKT protein content remained unchanged (103% of control; data not shown). pAKT content was positively correlated (r=0.61; p=0.022) with melusin protein; i.e. a loss of melusin was accompanied by a parallel loss of AKT phosphorylation (Fig. 4c).


Figure 04
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  		Fig. 4 a) pAKT/AKT ratios in human AS compared to controls. Controls were set as 100% and AS values were related to the mean value of controls. Data are presented as mean value±SEM. Controls are shown in white bars, AS in gray bars. b) Typical immunoblots showing pAKT and AKT in patients with AS and controls. c) Melusin protein content was significantly correlated with AKT phosphorylation (r=0.61, p=0.022).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
5. Limitations
 6. Conclusion
 References
 
Rodent models suggest a major role for melusin in the preservation of cardiac function in response to biomechanical stress [3]. We describe for the first time a decrease in melusin in patients with AS, which parallels the impairment of systolic function and the increase in left ventricular size, as well as the decrease in AKT phosphorylation in human hearts.

Melusin expression was evaluated both at transcript and protein levels in samples of human hearts from control healthy individuals and from patients with aortic stenosis showing different degrees of left ventricular remodelling. Our results clearly show that in dilated hypertrophic hearts, melusin levels are lower than in healthy hearts both at the mRNA and protein level. Moreover, immunohistochemical staining showed that melusin reduction occurred in the cardiomyocytes, thus excluding the possibility that the low melusin levels in the biochemical assays was due to the increased proportion of stromal tissue. Immunohistochemical staining also indicated that melusin expression was homogenous in different regions of the control hearts. Thus, the lower melusin expression in the area beneath the septum of AS hearts, from where the biopsies were taken, probably reflects changes in the whole heart. Interestingly, when melusin levels were compared to both structural and functional parameters of the AS hearts, an inverse correlation was found between the degree of dilation of the left ventricle (both diastolic and systolic diameters) and the melusin level, with low melusin in highly dilated hearts. A direct correlation between melusin and functional parameters such as ejection fraction was also observed. These data indicate that in human AS, decreased melusin levels are associated with a greater degree of structural and functional deterioration of the heart.

This finding is consistent with the phenotype of mice in which melusin expression has either been abrogated by gene inactivation [3] or strongly increased by transgenesis [6]. In these models, lack of melusin dramatically accelerates the evolution to LV dilation and failure in conditions of pressure overload; while sustained melusin expression maintains compensatory hypertrophy and full contractility even in conditions of pressure overload which cause dilation and failure in wild type mice [3]. Based on these findings, we speculate that decreased melusin expression in humans is predictive of a worse functional state and strategies aimed at maintaining high melusin levels could be of important therapeutic value.

In line with this hypothesis, we found that melusin expression correlated with AKT phosphorylation in human AS hearts. This is in agreement with previous data showing a link between melusin expression and AKT phosphorylation in mice [3] and shows that melusin can control downstream signalling pathways in the human heart known to be involved in positive left ventricular remodelling [12].

In a previous study, increased collagen content together with decreased MMP expression was found in a group of patients with AS [9]. These changes in collagen and MMP content occurred in the early stages of the disease. Since our patient cohort was similar to this previous study group, we compared melusin and MMP expression in a subset of 12 patients from our study. We found that melusin positively correlated with MMP1 (r=0.75, p=0.005) and MMP9 (r=0.62, p=0.043) in these 12 hearts. Parallel downregulation of melusin and MMPs in human AS may reflect a common involvement in integrin signalling and regulation by mechanical load. In addition, it may reflect a relationship between melusin regulation, impairment of systolic function and increase in fibrosis in pressure overload in the human heart.


   5. Limitations
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Limitations
6. Conclusion
 References
 
A persistent limitation in studies using human heart samples is the limited availability of tissue samples. Only small intra-operative samples can be obtained in human AS and the number of unused donor hearts is limited. Thus, we were not able to study the effects of age and sex and medical therapy in a detailed manner. However, based on the analyses which were possible, comparing patients with and without a given therapy, we did not observe an effect of medical therapy on melusin gene expression.


   6. Conclusion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusion
References
 
Reduction in melusin expression parallels the functional impairment in human hearts with AS. Reduced melusin levels are accompanied by decreased AKT phosphorylation, suggesting a connection between the loss of melusin and the impaired systolic function. These results, together with published data in genetically modified mouse models with altered melusin expression, suggest that reversing the loss of melusin may be beneficial in human AS.


   Acknowledgements
 
This work has been supported by EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart and by the DFG, Graduate course 754 on myocardial hypertrophy. This work was also supported with grants to GT from the Italian Ministry of University and Research (FIRB 2001 and PRIN 2003) as well as from Telethon.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusion
 References
 

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  3. Brancaccio M., Fratta L., Notte A., et al. Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat Med (Jan 2003) 9(1):68–75.[CrossRef][Web of Science][Medline]
  4. Brancaccio M., Guazzone S., Menini N., et al. Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain. J Biol Chem (Oct 8 1999) 274(41):29282–29288.[CrossRef]
  5. Brancaccio M., Hirsch E., Notte A., et al. Integrin signalling: the tug-of-war in heart hypertrophy. Cardiovasc Res (Jun 1 2006) 70(3):422–433.[CrossRef]
  6. De Acetis M., Notte A., Accornero F., et al. Cardiac overexpression of melusin protects from dilated cardiomyopathy due to long-standing pressure overload. Circ Res (May 27 2005) 96(10):1087–1094.[CrossRef]
  7. Antos C.L., McKinsey T.A., Frey N., et al. Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A (Jan 22 2002) 99(2):907–912.[CrossRef]
  8. Hardt S.E., Sadoshima J. Glycogen synthase kinase-3beta: a novel regulator of cardiac hypertrophy and development. Circ Res (May 31 2002) 90(10):1055–1063.[CrossRef]
  9. Fielitz J., Leuschner M., Zurbrugg H.R., et al. Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis. J Mol Med (Dec 2004) 82(12):809–820.[CrossRef][Web of Science][Medline]
  10. Nordmeyer J., Eder S., Mahmoodzadeh S., et al. Upregulation of myocardial estrogen receptors in human aortic stenosis. Circulation (Nov 16 2004) 110(20):3270–3275.[CrossRef]
  11. Martinka P., Fielitz J., Patzak A., et al. Mechanisms of blood pressure variability-induced cardiac hypertrophy and dysfunction in mice with impaired baroreflex. Am J Physiol Regul Integr Comp Physiol (Mar 2005) 288(3):R767–R776.[Abstract/Free Full Text]
  12. Matsui T., Li L., Wu J.C., et al. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem (Jun 21 2002) 277(25):22896–22901.[CrossRef]
  13. Braunwald E. Heart disease, a textbook of cardiovascular medicine. (2001) 5th ed. W B Saunders Company.

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