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Eur J Cardiothorac Surg 2006;30:737-743
© 2006 Elsevier Science NL

Reduced myocardial expression of calcium handling protein in patients with severe chronic mitral regurgitation

Przemyslaw Leszeka,*, Jerzy Korewickia, Anna Klisiewicza, Jadwiga Janasa, Andrzej Biedermana, Aldona Browareka, Danièle Charlemagneb, Pascal Trouvéb

a Institute of Cardiology, Alpejska 42 St., 04-628 Warsaw, Poland
b INSERM, Unite 127, Hopital Lariboisiere, 41 boulevard de la Chapelle, 75475 Paris, France

Received 23 January 2006; received in revised form 29 June 2006; accepted 4 July 2006.

* Corresponding author. Address: Heart Failure Department, National Institute of Cardiology, Alpejska 42 St., 04-628 Warsaw, Poland. Tel.: +48 22 34 34 483; fax: +48 22 34 34 522. (Email: leszekp{at}ikard.pl).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Objective: Left ventricle (LV) function was shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation (MR). However, the cellular mechanisms that develop in the LV remodeling secondary to volume overload in chronic severe MR is still not well defined. In single ventricular myocyte, a reduced contraction and slowed relaxation have been mainly attributed to defective intracellular Ca2+ currents. Between several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) expression and activity determines not only the extent and rate of relaxation, but also the rate and amplitude of contraction. The aim of the study was to determine whether modifications of SERCA2 gene expression occurs in LV wall remodeling process secondary to chronic severe MR. Methods: The LV samples were obtained from 12 patients presented LV wall remodeling (LV: diastolic/systolic diameter—70 ± 7 mm vs 46 ± 10 mm; diastolic/systolic volume—260 ± 65 ml vs 102 ± 68 ml) due to chronic, severe MR. Expressions of SERCA2 isoforms—SERCA2a and 2b mRNAs were estimated by semiquantitative RT-PCR and normalized to GAPDH. The protein levels of SERCA2 were determined by Western blot after normalization to actin. Results were compared with samples from non-failing human hearts (NFH). Results: On SERCA2 mRNA levels, important reduction on both SERCA isoforms SERCA2a (–40%) and SERCA2b (–49%) compared to NFH, together with significant correlation between isoforms (r = 0.89; p = 0.01) were observed. SERCA2 protein levels were decreased (–38%) in MR compared to NFH. Also significant correlations between SERCA2a/2b and SERCA2 protein expression (r = 0.83, p = 0.017; r = 0.68, p = 0.05, respectively) were observed. Moreover, a negative correlation between protein levels of SERCA2 (r = –0.64, p = 0.053) and left ventricular diastolic diameter was observed. Conclusions: In chronic volume overload the down-regulation of SERCA2a and 2b at the mRNA and SERCA2 protein levels exist. Moreover, protein levels of SERCA2 tend to correlate to the grade of left ventricular diastolic dilatation and suggest an important role LV remodeling.

Abbreviations: ALD = aldosterone • ANGII = angiotensin II • CI = cardiac index • EDD = left ventricle end-diastolic diameter • EDV = left ventricle end-diastolic volume • EF = ejection fraction • ESD = left ventricle end-systolic diameter • ESV = left ventricle end-systolic volume • HF = heart failure • LA = left atrium • LV = left ventricle • MR = mitral regurgitation • NCX = Na+/Ca2+ exchanger • NA = noradrenaline • NFH = non-failing hearts • NYHA = New York Heart Association functional class • PRA = plasma renin activity • PAP = pulmonary artery pressure • PWP = pulmonary wedge pressure • SERCA1, 2, 2a, 2b 3 = Ca2+-ATPase of the sarcoplasmic reticulum—isoforms 1, 2, 2a, 2b, 3 • SR = sarcoplasmic reticulum • SVI = stroke volume index • 2D = two-dimensional transthoracic echocardiography

Key Words: Mitral regurgitation • Left ventricle remodeling • SERCA2 expression


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
The surgical correction in mitral regurgitation (MR) performed at optimal time restores the proper cardiac function along with the improvement in functional status and survival. That is why, recently, early surgery is increasingly recommended for patients with degenerative MR in order to prevent irreversible decline in left ventricle (LV) function.

The onset of symptoms of heart failure (HF) and LV dysfunction predicts a poor prognosis after surgery. Moreover, in the patients with already diagnosed severe MR, the progression of LV muscle dysfunction is quite variable. Although both experimental and human studies suggest that LV failure due to chronic severe MR is caused, at least in part, to inherent defects in LV contractile function, the specific changes in myocardial composition and gene expression are largely unknown [1,2].

There is accumulating body of evidence that both contractility and relaxation are mainly dependent upon the rise and fall of cytosolic Ca2+, closely linked to altered expression/function of the Ca2+-regulatory proteins [3–5]. Among several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) decreased expression/function has been claimed to be a molecular early sign of decompensation, however, not confirmed by all authors. Diastolic calcium removal from the cytosol occurs by SERCA2 which transports Ca2+ from cytosol into the sarcoplasmic reticulum (SR) and also exchange of Ca2+ for Na+ outside the sarcolemma by the Na+/Ca2+ exchanger (NCX). The amount of Ca2+ released by the SR depends on its Ca2+ load and also Ca2+ gradient between the SR and cytosol, so there is a general agreement that SERCA2 determines not only the extent and rate of relaxation but also the rate and amplitude of contraction [6]. However, the role of SERCA2 expression changes in LV ventricular remodeling secondary to chronic MR is not yet established.

SERCA2 is encoded by three different genes (SERCA1, SERCA2, and SERCA3) and six distinct isoforms are expressed. In cardiac muscle cells, the SERCA2 gene encodes two alternatively spliced Ca2+-ATPase mRNA isoforms: SERCA2a and 2b [7]. Although there is an important functional difference between both SERCA2 isoforms, indeed, to our knowledge, none of studies indicated a reduced mRNA level of SERCA2 in failing heart and discriminated between both isoforms.

Accordingly, the purpose of this investigation was to determine whether and which extend myocardial SERCA2 gene on mRNA and protein expression occur in LV remodeling due to chronic severe MR. Also to determine whether a relationship exists between SERCA2 expression and parameters of LV volume overload and remodeling.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Limitations
 References
 
The investigation conforms with principles outlined in the Declaration of Helsinki. The informed consent was obtained from all patients participating in the study according to the protocol approved by the Local Ethics Committee.

The study group was composed of 12 consecutive patients who presented overt HF secondary to pure, severe, chronic nonischemic MR, excluded from mitral valve repair, during cardiac surgery due to technical reasons. Patients with primary cardiomyopathy and secondary MR, coronary artery disease, other significant valve disease apart from tricuspid regurgitation were excluded from the study group.

2.1 Patients evaluation
Each patient underwent right heart catheterization; pulmonary artery pressures (PAP) and pulmonary wedge pressures (PWP) were measured. Cardiac output was determined by the thermodilution method; cardiac index (CI) and stroke volume index (SVI) were calculated.

Two-dimensional (2D), M-mode and color Doppler transthoracic echocardiography on left lateral position on commercially available equipment (VingmedV) using standard views. 2D parasternal long/short axis and apical views were performed for mitral anterior/posterior leaflets assessment. The LV end-diastolic/end-systolic diameters (EDD/ESD) and end-diastolic/end-systolic volumes (EDV/ESV), together with ejection fraction (EF) and left atrium size (LA) were measured/calculated according to the recommendations of the American Society of Echocardiography. Color Doppler was used for assessment of the presence and severity of MR. MR severity was measured by a semiquantitative method—graded as severe MR if the percent ratio of the maximal flow disturbance produced by the MR jet to left atrial area was >40%.

Before surgery, the blood samples were obtained for the hormonal evaluation: plasma noradrenaline level (NA), plasma renin activity (PRA), angiotensin II (ANGII), aldosterone (ALD). Neurohumoral assays were performed using commercially available kits: PRA/ALD by radioimmunoassay (DiaSorin s.r.l., Italy), ANGII by RIA (Buhlmann Laboratories AG, Switzerland), after previous reverse-phase extraction of the plasma samples with phenylsilysilca columns, NA by HPLC method (BioRad, USA).

2.2 Preparation of cardiac tissue
During mitral valve replacement (with posterior leaflet and both chordals preservation), samples of LV anterior papillary muscle weighing 100–150 mg were obtained, avoiding connective tissue, endocardium, and vessels. The biopsies were rinsed immediately, blotted dry, frozen in liquid nitrogen, and kept at –80 °C until use.

Non-failing hearts (NFH), from four adults aged 22–58 years, without previous medical history or any abnormalities in ECG and echocardiography (LV dimensions/contractility within normal ranges) who died from head trauma and received dopamine (2–5 µg/(kg min)) before death were used as controls. They were rejected for transplantation for other reasons than depressed cardiac function. The samples were obtained from LV papillary muscle.

2.3 RNA isolation and RT-PCR analysis
Total cellular RNA was isolated from samples using Chomczynski and Sacchi's modified procedure [8]. Total RNA concentrations were assessed spectrophotometrically, RNA yield were similar in NFH and MR group (0.52 ± 0.04 µg RNA/mg tissue and 0.49 ± 0.08 µg RNA/mg tissue, respectively).

Reverse transcription (RT) was performed using RNAsine (40 U), dNTP mix 25 mM (0.75 mM), Oligo (dT) (1750 ng), MgCl2, (2.5 mM), TPCR 4.0 µl, H20 DEPC, MMLV (400 U), and 3 µg of total RNA all in a total volume of 50 µl. Reverse transcriptase was inactivated at 99 °C for 5 min and used for cDNAs synthesis by the PCR method.

For PCR experiments, MgCl2 (0.5 mM), TPCR 10X, sense primers (150 ng), antisense primers (150 ng) and Taq polymerase (2.5 U) were added to 10 µl of the RT reaction mixture, for a total reaction volume of 50 µl. For SERCA2a, upper primer (5'-sense) TTT CCT ACA GTG TAA AGA GGA CAA CC3' (26 mer) and lower primer (3'-antisense) TTC CAG GTA GTT GCG GGC CAC AAA3' (24 mer, predicted length of the PCR product 364 bp) were used. For SERCA2b, upper primer (5'-sense) TTT CCT ACA GTG TAA AGA GGA CAA CC3' (26 mer, same as for SERCA2a) and lower primer (3'-antisense) AGA CCA GAA CAT ATC GCT AAA GTT3' (24 mer, predicted length of the PCR product 513 bp) were used [9,10]. For GAPDH, [11] upper primer (5'-sense) CTT CAT TGA CCT CAA CTA CAT GGT and lower primer (3'-antisense) CTC AGT GTA GCC CAG GAT GCC CTT3' (predicted length of the PCR product 726 bp) were used. Thirty-five PCR amplification cycles were run using a thermal cycler (Perkin-Elmer, San Diego, USA) as following: denaturation at 95 °C, 0.5 min; annealing at 58 °C, 1 min; extension at 72 °C, 1 min, and heating at 72 °C, 10 min.

Semi quantitative analysis of PCR products was estimated after electrophoresis on 1% agarose gels using ethidium bromide as a dye. Quantifications of SERCA2a/2b and GAPDH PCR products were performed by densitometric analysis after gel scanning (GelDoc 1000, Biorad) using Molecular Analyst (BioRad) software and normalized to GAPDH (Fig. 1 ).


Figure 1
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  		Fig. 1. RT-PCR amplification of mRNA SERCA2a, SERCA2b. Lines 1–3: SERCA2a (364 bp); line 4: molecular weight marker; and lines 5–7: SERCA2b (513 bp).

 
2.4 Protein studies
The protein studies were performed on crude homogenates (CSR) according to Rannou et al. [12]. Protein concentration was determined in triplicates by the method of Lowry et al. [13] using bovine serum albumin as a standard. The yield of protein per gram of wet weight was 75 ± 9.5 mg/g and 66.5 ± 12.5 mg/g from NFH and MR group, respectively.

Equal amounts of protein from all samples were subjected to SDS–PAGE according to Laemmli method [14] and blotted to PVDF membrane. Membranes were saturated in 5% fat-free milk, incubated for 2 h with a specific monoclonal antibody anti-SERCA2 (gift from Professor D.O. Levitsky) diluted in 1:1500 in TBS containing 0.1% Tween 20. Lack of specific antibodies did not allow differentiating between the two isoforms. The specific bands were revealed by ECL technique on Kodak X-ray films. Quantifications were performed by densitometric analysis after ECL film scanning. Signals from proteins stained with Coomassie Blue R 250 and actin were used to normalize the specific signal in each lane (Fig. 2 ).


Figure 2
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  		Fig. 2. Western blot analysis of proteins form MR and NFH groups. Lines 1–4: NFH (non-failing heart)—control group; lines 5–10: MR (mitral regurgitation (MR) group)—study group; SERCA2: Ca2+-ATPase 2 bands; Actin: actin bands; Total protein levels: proteins stained with Coomassie Blue R 250; and MWM: molecular weight marker (kD).

 
2.5 Statistical analysis
Data were expressed as means ± standard deviations. The two-sided significance level was p < 0.05. The analysis comparing study and control group was performed with t-test. In the assessment between mRNA of both SERCA2 isoforms, proteins and clinical variables Pearson's correlations were calculated.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Limitations
 References
 
3.1 Study group population
The MR group composed of 12 patients (4 women, 8 men) with mean age 58 ± 11 years and echocardiographically documented severe, chronic nonischemic MR (myxomatous mitral valve degeneration: anterior leaflet—4 patients; posterior leaflet—6 patients; both leaflets—2 patients). No further histological analysis of the excised leaflets was performed.

All patients presented symptoms of HF and met the criteria for NYHA class III (nine patients) or IV (three patients). The mean period from the initial MR diagnosis to study inclusion was 24 years (±2). Atrial fibrillation was present in all patients.

All patients had echocardiographic evidence of secondary LV dilatation: diastolic (EDD—70 ± 7 mm; EDV—260 ± 65 ml), systolic (ESD—46 ± 10 mm; ESV—102 ± 68 ml) with LA enlargement (66 ± 13 mm). They presented with elevated PAP/PWP and depressed CI/SVI and also elevated NA, PRA, and ALD plasma levels (supine position) (Table 1 ).


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  		Table 1. Clinical characteristics and hemodynamic data of the MR patients
 
3.2 Comparison of mRNA levels between MR patients and non-failing myocardium
The mRNA levels of SERCA2a and SERCA2b (Fig. 3a) were significantly lower in MR group than in NFH. A significant correlation between SERCA2a and 2b mRNA expressions was observed (Table 2 ).


Figure 3
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  		Fig. 3. (a) mRNA levels of SR Ca2+-ATPase 2a (panel A) and SR Ca2+-ATPase 2b (panel B) normalized to internal standard (GAPDH). (b) Protein levels of SR Ca2+-ATPase 2 normalized to actin protein levels (panel A) and SR Ca2+-ATPase 2b (panel B) normalized to total protein levels (panel B). Mitral regurgitation patients—MR; control group—non-failing heart (NFH), results presented in arbitrary densitometric units.

 

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  		Table 2. Relation between mRNA SERCA2a/2b levels normalized to GAPDH; and protein SERCA2 normalized to actin and total protein levels
 
3.3 Comparison of protein accumulation between MR group and non-failing myocardium
Protein levels of SERCA2 were normalized to actin or total protein levels. MR group did not differ from NFH group neither in actin protein levels (1.8 ± 0.15 vs 1.9 ± 0.1, ns, arbitrary densitometric units per milligram of protein, respectively) nor after normalization actin to total protein levels (0.35 ± 0.03 vs 0.38 ± 0.04, ns, arbitrary densitometric units per milligram of protein).

A significant decrease of the protein accumulation of the SERCA2 (after normalization to both actin and all protein levels) in whole MR group compared to NFH was observed. However, within the MR group the lack of homogeneity in the changes in SERCA2 protein expression was discovered. The changes in SERCA2 protein levels range from normal levels to decrease by 60% or 58% after normalization to actin protein levels or all proteins (Fig. 3b), respectively.

Moreover, a significant correlation between SERCA2a/2b mRNA and SERCA2 protein expressions was observed (Table 2).

3.4 Relation between clinical parameters and SERCA2 expression
SERCA2a and 2b mRNA did not correlate with clinical and plasma hormonal parameters, a negative correlation between the protein levels of SERCA2 normalized to actin (r = –0.64; p = 0.053) and trend towards negative correlation between the protein levels of SERCA2 normalized to all protein levels (r = –0.67; p = 0.07) and LV diastolic diameter were observed (Table 3 ).


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  		Table 3. Relation between clinical parameters and SERCA2 expression
 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Limitations
 References
 
LV function has been shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation. Even paradoxically mitral valve surgery abruptly increases afterload by elimination of the unloading effect of the regurgitant flow to the low-pressure pulmonary circuit and may precipitate development/progression of overt, left ventricle dysfunction. Moreover, also the cellular mechanisms that develop in the myocardium during transition from the compensated to the decompensated phase are largely unknown [2].

Over the past decade, both human (on LV papillary muscle—which in fact is believed to represent the changes in LV free wall) and experimental studies of mitral regurgitation show reduced myosin content within the myocardium [15]. However, Mulieri et al. [16] did not confirm the previous finding indicating that a deficiency in cyclic adenosine monophosphate production might be the cause of reduced contractility. More recently, Oral et al. [17] reported the relationship between cytokines and level of LV remodeling in volume overload.

The present work, to our knowledge, for the first time assessed the changes on SERCA2 expression in LV dilatation due to chronic severe MR.

All of the studies on the failing human hearts on SERCA2 published by now indicated a reduced mRNA level [18]. However, in spite of the obvious functional difference between both SERCA2 isoforms indeed, none of the study discriminate the two isoforms SERCA2a and 2b. Whereas SERCA2a is expressed at high levels in cardiac muscle, the ubiquitous SERCA2b is found in almost all cell types. While SERCA2b, in comparison to SERCA2a, has nearly twofold higher affinity for calcium ion, it also presents lower maximal catalytic turnover rate than SERCA2a.

Moreover, the recent transgenic mouse experiments have shown that even the full suppression of the SERCA2a could be partially compensated by the expression of SERCA2b isoform alone, and the knockout animals presented only moderate functional LV impairment [19].

In the present study we showed that both SERCA2a and 2b isoforms (at the mRNA levels) presented significant and parallel down-regulation. Our findings did not reveal any compensational shifts between both isoforms at the mRNA levels.

However, we found the lack of correlation between SERCA2a/2b at the mRNA levels with the LV parameters, confirming the previous findings that mRNA content of SERCA2 is not necessarily transferable to the grade of LV dilatation/dysfunction [20].

Moreover, the steady state mRNA levels cannot always be assumed to be a certain predictor of protein content and function. The differences in mRNA processing, translation, posttranslational modification, protein synthesis, degradation ratio and function may be attributed to differences in mRNA/protein levels. However, our results confirmed that the decrease in SERCA2 mRNA content parallels a decrease in protein concentration.

The results obtained at the protein levels of SERCA2 in human are more conflicting than at the mRNA levels. The recent results have confirmed that decrease in SERCA2 protein levels in human HF is not an universal finding. Moreover, the changes in SERCA2 protein content can be attributed to the grade of myocardial dysfunction, which was assessed by the force–frequency relation [21]. Also, recently, Hasenfuss et al. [22] reported that discrimination within an heterogeneous group of failing human hearts based on diastolic function lead to two different phenotypes: first with disturbed diastolic function related to unchanged protein levels of the NCX and decreased SERCA2 level and second with preserved diastolic function related to increased protein levels of the NCX and unchanged SERCA2 level. Our results are in concordance with previous findings. Although significantly lower SERCA2 protein levels in MR group to NFH were observed, also the lack of homogeneity within the study group was documented. Moreover, the important negative correlation between SERCA2 protein levels and the LV diastolic size may suggest the association with the preservation diastolic dilatation. Although all correlations have to be interpreted cautiously because in severe MR, due to abnormal loading conditions the assessment of LV function is difficult.

However, more recently works presenting gene transfer techniques, confirmed the importance of reduced SERCA2 protein levels to myocardial dysfunction. Several studies in animal models have reported that SERCA2 gene transfer restored SERCA2 protein expression and activity to non-failing level which was reflected in restoration of systolic as well as diastolic functions [23,24].

In summary, to our knowledge this is the first study showing the decreased mRNA and protein expression of the SERCA2 in LV remodeling due to chronic severe MR. Moreover, correlation of the decreased protein levels which tends to relate to the grade of left ventricular diastolic dilatation suggests that changes in the SERCA2 expression may play an important role in LV volume overload remodeling. We can assume that reduction of SERCA2 protein levels could be attributed to the maladaptation to chronic overload in patients with chronic mitral regurgitation.


   5. Limitations
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
References
 
The results from our study, as from all studies that use tissues obtained from MR and NFH human hearts should be interpreted cautiously. Although the homogeneity in the etiology and standardization of applied treatment in MR group was achieved, the substantial heterogeneity in age, gender, and genes expressions were observed. In addition, non-failing hearts are not necessarily representative of the normal human population. These hearts also did not receive the same treatment as hearts from MR group, moreover, they additionally received low dose of dopamine.

As the LV performance was based on echocardiography that utilized load-dependent parameters, the relation to molecular assessment should be interpreted cautiously. The use of the load-independent methods, e.g., tissue Doppler velocities or pressure/volume loops could enrich LV assessment.

Additionally, between different proteins involved in Ca2+ movement, we focused only on SERCA2 expression but without differentiation between SERCA2a and 2b on protein level. In spite of all the limitations listed above, we added the new insights into the bases of human LV remodeling secondary to the pure MR in volume overload model, which is still not well defined.


   Acknowledgments
 
We are very grateful to Drs B. Swynghedauw and J.-M. Moalic from INSERM for the support and suggestions during the work. We also would like to thank Dr T. Rywik for his assistance during manuscript preparation. This study was supported by ESC Research Grant, and State Committee for Scientific Research Grant No. 1241/P05/98/14.


   Footnotes
 
{star} The paper has been presented at Heart Failure Update 2000—Venice, June 2000: Leszek P, Trouve P, Korewicki J, Browarek A, Biederman A, Klisiewicz A, Janas J, Charlemagne D. Contribution of abnormal sarcoplasmic reticulum ATPase expression in heart failure due to volume overload in mitral regurgitation model.

{star}{star} The paper has been presented at 4th International Congress of Polish Cardiac Society—Wroclaw 2000: Leszek P, Trouve P, Korewicki J, Browarek A, Biederman A, Klisiewicz A, Janas J, Charlemagne D. Sarcoplasmic reticulum ATPase expression in heart failure due to volume overload in mitral regurgitation model. Kard Pol 2000;53(Suppl. 2):26.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 

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