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European Journal of Clinical Investigation (2004) 34, 723– 730
Down-regulation of sarcolipin mRNA expression in chronic
atrial fibrillation
N. Uemura*†, T. Ohkusa‡, K. Hamano‡, M. Nakagome*, H. Hori*, M. Shimizu§, M. Matsuzaki‡,
S. Mochizuki†, S. Minamisawa* and Y. Ishikawa*¶
*Yokohama City University Graduate School of Medicine, Yokohama, †Jikei University School of Medicine, Tokyo,
‡Yamaguchi University Graduate School of Medicine, Yamaguchi, §Jikei University Kashiwa Hospital, Kashiwa, Japan,
¶New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, USA
Abstract
Background Abnormal intracellular Ca2+ homeostasis is an important modulator of chronic
atrial fibrillation. Sarcolipin, a homologue of phospholamban, is specifically expressed in
the atria, and may play an important role in modulating intracellular Ca2+ homeostasis in
the atria. The aim of this study was to investigate the expression of sarcolipin mRNA in the
atrial myocardium of patients with chronic atrial fibrillation.
Methods We analyzed the expression of sarcolipin, phospholamban, cardiac calsequestrin
and sodium calcium exchanger mRNAs in the right atrial myocardium from nine patients
with mitral valvular disease with atrial fibrillation (MVD/AF), nine patients with MVD who
had normal sinus rhythm (MVD/ NSR), and 10 control patients with normal sinus rhythm
who received open heart surgery (controls). The expression of mRNA was measured using
the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA).
Results Relative expression levels of sarcolipin mRNA were significantly lower in MVD/AF
(0·60 ± 0·11) than in either MVD/NSR (1·28 ± 0·17, P < 0·01) or controls (1·10 ± 0·10,
P < 0·05). The expression levels of sarcolipin mRNA were significantly lower in the group
with high values for right atrial pressure. The expression levels of phospholamban, cardiac
calsequestrin and sodium calcium exchanger mRNAs were comparable among all three
groups.
Conclusions Chronic electrical and mechanical overload decreased the expression of
sarcolipin mRNA in the right atrial myocardium in patients with chronic atrial fibrillation.
Down-regulation of sarcolipin mRNA may be part of atrial fibrillation-induced atrial
remodelling.
Keywords Arrhythmia, calcium, electrical remodelling, gene expression.
Eur J Clin Invest 2004; 34 (11): 723–730
N.U. and T.O. contributed equally to this work.
Department of Physiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan (N. Uemura, M. Nakagome, H. Hori, S. Minamisawa, Y. Ishikawa); Division of Cardiology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan (N. Uemura, S. Mochizuki); Division of Cardiovascular Medicine (T. Ohkusa, M. Matsuzaki) and Division of Cardiovascular Surgery (K. Hamano), Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Yamaguchi; Division of Cardiology, Department of Internal Medicine, Jikei University Kashiwa Hospital, Kashiwa, Japan (M. Shimizu); Departments of Medicine (Cardiology) and Cell Biology and Molecular Medicine, Cardiovascular Research Institute, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA ( Y. Ishikawa).
Correspondence to: Susumu Minamisawa, Department of Physiology, Yokohama City University Graduate School of Medicine, 3 – 9 Fukuura, Kanazawa-ku, Yokohama 236 – 0004, Japan. Tel.: +81–[0]45–787–2575; fax: +81–[0]45–788–1470; e-mail: [email protected] Received 7 July 2004; accepted 26 September 2004 Introduction
Atrial fibrillation (AF) is the most prevalent arrhythmia Selection of patients
in clinical practice in adults. Atrial fibrillation is a progres-sive disorder; an increasing body of evidence indicates that We examined right atrial appendages from: (1) nine patients AF itself produces electrical and structural remodelling with mitral valvular disease (MVD) in whom AF had been of the atria which exacerbates the disorder [1–5]. Atrial sustained for more than 6 months (MVD/AF), (2) nine fibrillation-induced electrical remodelling is characterized MVD patients with normal sinus rhythm (NSR, MVD/ by rapid shortening of the atrial effective refractory period NSR), and (3) 10 non-MVD patients with NSR (control).
[2,3,6,7]. -type Ca2+ channel and several potassium chan- None of the MVD/NSR patients or controls had a docu- nels are thought to be involved in the rapid shortening of mented history of AF. Haemodynamic and echocardiographic the atrial effective refractory period [6,8,9]. In addition, data were obtained by reviewing data from preoperative recent studies have suggested that abnormal intracellular cardiac catheterizations and echocardiograms. Patients were Ca2+ homeostasis and perturbations in Ca2+ handling starved and took no medication at least for 8 h before play an important role in AF-induced atrial remodelling cardiac the catheterizations. Patients with MVD/AF exhibited AF during the cardiac catheterizations. These tissues were The -type Ca2+ channel and cardiac sarcoplasmic reticu- obtained during cardiac surgery for MVD or coronary lum proteins co-ordinately regulate the intracellular Ca2+ artery bypass and were frozen immediately and stored at concentration in the heart. Ca2+ entry via the -type Ca2+ −80 °C until needed. Informed consent was obtained from channel triggers the release of a large amount of Ca2+ from each patient. The protocols were in accordance with the the sarcoplasmic reticulum through the ryanodine receptor.
guidelines laid down by the Institutional Review Board, Intracellular Ca2+ is restored from the cytosol to the sarco- Yamaguchi University Hospital and Yokohama City Univer- plasmic reticulum lumen via SERCA2a. The activity of the sity School of Medicine. The investigation conforms with ryanodine receptor and SERCA2a is modulated by their the principles outlined in the Declaration of Helsinki.
intrinsic regulatory domains and associated sarcoplasmicreticulum proteins in integrated manners. In this regard,phospholamban has been known to be the strongest endo- RNA and cDNA preparation
genous inhibitor of SERCA2a to date. Ablation of thephospholamban gene resulted in maximal inotropic and Total cellular RNA was isolated from each frozen tissue lusitrophic effects on myocardium without any catecho- sample (∼50 mg) by the method of acid guanidinium lamine stimulation [17] and improved cardiac function in thiocyanate/phenol /chloroform extraction, and then stored the failing heart in mice [18]. Masaki et al. reported that at −80 °C. The cDNA was prepared from 2 µg of total RNA ablation of the phospholamban gene decreased the duration in a 50-µL reaction using the SuperScript First-Strand of the -type Ca2+ channel activity, which may shorten the Synthesis System for reverse transcription-polymerase action potential duration [19]. Therefore, phospholamban chain reaction (RT-PCR) (Invitrogen, La Jolla, CA) as may also contribute to perturbations in Ca2+ handling in patients with AF. However, many reports have demon-strated that the expression levels of phospholamban mRNAor protein are not changed in patients with AF [14 –16], TaqMan Quantitative RT-PCR
suggesting no involvement of phospholamban in thepathogenesis of AF-induced electrical remodelling or the Using 5 µL of the reverse reaction as a template, quantita- presence of an alternative molecule mimicking phospho- tive RT-PCR was performed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster Sarcolipin is a 31-amino acid proteolipid in the SR City, CA) using the 5′ nuclease activity of TaqDNA and interacts with SERCA1 and SERCA2 to modulate polymerase to generate real-time DNA analysis [22]. The their activity [20]. As sarcolipin’s structure and protein sarcolipin gene was quantified using the Assays-on-Demand sequence are similar to those of phospholamban, they Gene Expression probe (Applied Biosystems). PCR primers may belong to the same family. We recently reported that and TaqMan probes for phospholamban, cardiac sodium sarcolipin is specifically expressed in the atria, but not calcium exchanger, cardiac calsequestrin, brain natriuretic in the ventricles [21]. As sarcolipin also interacts with peptide and glyceraldehyde-3-phosphate dehydrogenase SERCA2a, like phospholamban [20], it may play an were designed using PrimerExpress (Applied Biosystems) important role in modulating intracellular Ca2+ homeostasis [Table 1]. Amplifications were generated by 10 min at in the atria. However, the transcriptional regulation of 95 °C and then 40 cycles of denaturation at 95 °C for 15 s, sarcolipin gene has not been investigated in the atrial annealing, and extension at 60 °C for 1 min using a TaqMan myocardium of patients with chronic AF. In the present universal PCR Master Mix kit (Applied Biosystems). The study, we compared the expression level of sarcolipin TaqMan assay was repeated either twice (cardiac calseques- mRNA in the right atrial myocardium between patients trin and brain natriuretic peptide) or three times (sarcolipin, with mitral valvular disease with AF and those without phospholamban and cardiac sodium calcium exchanger).
Glyceraldehyde-3-phosphate dehydrogenase was used as 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 723–730
Sarcolipin expression in atrial fibrillation Table 1 Oligonucleotide primers and fluorescent probes used to quantify cDNA
*Covalently linked at the 5′ end to 5-carboxyfluorescein and at the 3′ end to N,N,N-tetramethyl-6-carboxyrhodamine.
SLN, sarcolipin; PLN, phospholamban; NCX, sodium calcium exchanger; CSQ2, cardiac calsequestrin; BNP, brain natriuretic peptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
an internal control to standardize the expression of six controls. Left atrial dimension and pulmonary capillary genes. The comparative CT method was used to determine wedge pressure were significantly larger in the MVD/AF or the ratio of targets and the internal control according to MVD/NSR patients than in the controls.
Expression levels of sarcolipin mRNA and other
Northern-blot analysis
transcripts
To verify the results of quantitative RT-PCR, Northern-blot As shown in Fig. 1, the relative expression levels of sarcoli- analysis was performed for the selected patients as previously pin mRNA were significantly lower in MVD/AF (0·60 ± 0·11) described with modification [18]. Total RNA was electro-phorezed on a 1% formaldehyde-agarose gel and transferredonto Nytran membranes (Schleicher & Schuell Inc., Keene,NH). Blots were hybridized at 68 °C with radiolabelledprobes in PerfectHyb solution (TOYOBO, Tokyo, Japan) asrecommended by the manufacturer. Human sarcolipin,brain natriuretic peptide and glyceraldehyde-3-phosphatedehydrogenase cDNA probes were used for hybridization.
Statistics
All data are presented as means ± SEM. Comparisonsbetween data from multiple groups were performed byunpaired analysis of variance followed by the Student-Newmann-Keuls test. A value P < 0·05 was considered tobe statistically significant.
Figure 1 Expression of sarcolipin in patients with atrial fibrillation
(AF). (a) Representative Northern-blot analysis is shown. The
expression levels of sarcolipin mRNA were markedly decreased in patients with mitral valvular disease/atrial fibrillation (MVD/AF). Clinical characteristics and haemodynamic data
The expression levels of BNP were increased in MVD/AF. (b) Relative abundance of sarcolipin mRNA was measured by TaqMan quantitative RT-PCR. The expression levels of sarcolipin The preoperative haemodynamic and echocardiographic mRNA were significantly lower in the MVD/AF than in the MVD/ data for the three groups are shown in Table 2. The age and NSR patients or controls (Control). AF: atrial fibrillation; BNP: the left ventricular ejection fraction were similar in all three brain natriuretic peptide; GAPDH: glyceraldehyde-3-phosphate groups. Right atrial pressure was significantly higher in dehydrogenase; MVD: mitral valvular disease; NS: not significant; the MVD/AF patients than in the MVD/ NSR patients or NSR: normal sinus rhythm; SLN: sarcolipin.
2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 723–730
Table 2 Clinical characteristics and haemodynamic data
digoxin, furosemide, spironolactone, enalapril maleate, warfarin potassium digoxin, furosemide, enalapril maleate, warfarin potassium spironolactone, enalapril maleate, warfarin potassium digoxin, furosemide, spironolactone, enalapril maleate, warfarin potassium digoxin, furosemide, spironolactone, warfarin potassium digoxin, furosemide, spironolactone, warfarin potassium digoxin, ISDN, furosemide, captopril, warfarin potassium 7·4 ± 0·9* 14·7 ± 1·4† 52·9 ± 2·7† MVD/NSR
1
digoxin, furosemide, spironolactone, temocapril hydrochloride furosemide, enalapril maleate, ubidecarenone Controls
1
metroprolol tartrate, ISDN, amlodipine besilate 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 723–730
Sarcolipin expression in atrial fibrillation Table 2 Continued
diltiazem hydrochloride, ISDN, nicorandil, nitroglycerin atenolol, diltiazem hydrochloride, ISDN, nicorandil, enalapril *P < 0·05, MVD + AF vs. MVD + NSR or controls; †P < 0·01, MVD + AF or MVD + NSR vs. controls.
LVEF, left ventricular ejection fraction; RAP, right atrial pressure; PCWP, pulmonary capillary wedge pressure; LAD, left atrial diameter; AF, atrial fibrillation; MVD, mitral valve disease; MR, mitral valve regurgitation; ASD, atrial septal defect; MS, mitral valve stenosis; AS, aortic valve stenosis; VSD, ventricular septal defect; IHD, ischaemic heart disease; ISDN, isosorbide dinitrate; ISMN, isosorbide mononitrate; AD, aortic dissection; NE, not examined.
than in either MVD/ NSR (1·28 ± 0·17, P < 0·01) or control are decreased in the right atrium of MVD/AF patients [11].
patients (1·10 ± 0·10, P < 0·05). The relative expression There were no significant differences in the expression levels levels of brain natriuretic peptide mRNA were significantly of phospholamban, cardiac sodium calcium exchanger and higher in the patients with MVD/AF (3·20 ± 1·53) than in cardiac calsequestrin among all three groups [Fig. 3].
the controls (0·59 ± 0·19, P < 0·05). There was no statisticaldifference in the relative expression levels of brain natriureticpeptide mRNA in the patients with MVD/ NSR (0·91 ± 0·30)compared with controls.
Discussion
We then analyzed the expression levels of sarcolipin mRNA according to the patient’s right atrial pressure and Main findings
pulmonary capillary wedge pressure. The expression levelsof sarcolipin mRNA were significantly lower in the group Sarcolipin is a unique SR protein which is preferentially with high values for right atrial pressure, but not for expressed in the atria [21]. The present study demonstrated that pulmonary capillary wedge pressure (Fig. 2a,b). Figure 2(c) the expression levels of sarcolipin mRNA in the right atrium shows the weak inverse relation between right atrial pressure were significantly decreased in MVD/AF. According to haemo- and the expression of SLN mRNA, although it is not dynamic data and the levels of brain natriuretic peptide statistically significant [correlation coefficient (r) = −0·35, mRNA, not only electrical overload (tachyarrhythmia itself ) P-value = 0·074]. When this analysis was applied for only but also mechanical overload was present in the right atrium subjects with NSR (n = 18), no significant inverse relation of MVD/AF. In addition, we found that the expression levels was observed between right atrial pressure and the expres- of sarcolipin mRNA were lower at higher levels of right atrial pressure, but not significantly affected by the levels of pul- We also investigated other intracellular Ca2+ regulatory monary capillary wedge pressure. Therefore, it is possible that proteins (phospholamban, cardiac sodium calcium exchanger both mechanical and electrical overloads down-regulated and cardiac calsequestrin) in atrial myocardial tissues from the sarcolipin mRNA expression in the MVD/AF patients.
these patients, as we have already reported that the expres- In contrast to phospholamban, the role of sarcolipin in sion levels of the cardiac ryanodine receptor and SERCA2a the heart remains unknown [23]. According to a recent 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 723–730
publication, sarcolipin inhibited polymerization of phos-pholamban, resulting in super-inhibition of SERCA1 andSERCA2a in skeletal muscles [24]. Thus, it is highlypossible that sarcolipin inhibits Ca2+ uptake into the sarco-plasmic reticulum lumen in the atria and down-regulationof sarcolipin mRNA increases SERCA2a activity and Ca2+restore in the sarcoplasmic reticulum. Our previous studydemonstrated that SERCA2 mRNA was also down-regulated in MVD/AF [11]. Therefore, down-regulation ofsarcolipin mRNA may compensate the down-regulation ofSERCA2 mRNA or vice versa. In this regard, such changesmay reflect electrical remodelling caused by chronic elec-trical and /or mechanical overload. In this study, we did notmeasure sarcoplasmic reticulum Ca2+ uptake in the atrialtissue because of the limited amount of tissue samples avail-able. To our knowledge, however, SERCA2 activity or Ca2+uptake has not been examined in patients with AF to date.
Only an animal study has demonstrated impaired Ca2+handling in the electrically remodelled atria [13]. Down-regulation of sarcolipin mRNA may also actively contributeto decreased duration of -type Ca2+ channel activity, asphospholamban inactivation has been known to decreaseit [19]. This may play an important role in progressivelyshortening the plateau phase of the action potential andmaintaining and /or perpetuating electrical remodellingof AF.
No changes in the expression of phospholamban,
cardiac sodium calcium exchanger and cardiac
Figure 2 Comparison among the expression levels of sarcolipin
calsequestrin mRNAs in patients with AF
mRNA and (a) right atrial pressure (RAP) and (b) pulmonary capillary wedge pressure (PCWP). The expression levels of In addition to sarcolipin, we also investigated other intra- sarcolipin mRNA were significantly lower in the group with high cellular Ca2+ regulatory proteins (phospholamban, cardiac values for RAP, but not for PCWP. As RAP was not measured in sodium calcium exchanger and cardiac calsequestrin).
any control patient, the total number of patients was 27 in (a). (c) Weak inverse relation between right atrial pressure and the There were no significant alternations in the expression of expression of sarcolipin (SLN) mRNA, although it was not phospholamban, cardiac sodium calcium exchanger and statistically significant [correlation coefficient (r) = −0·35, cardiac calsequestrin in MVD/AF, which is consistent with P-value = 0·074]. Atrial fibrillation: atrial fibrillation; MVD: mitral other reports [15,16]. These data suggest that phospho- valvular disease; NS: not significant; NSR: normal sinus rhythm; lamban, cardiac sodium calcium exchanger and cardiac PCWP: pulmonary capillary wedge pressure; RAP: right atrial calsequestrin may not play a major role in the electrical Figure 3 Relative abundance of
phospholamban (PLN), sodium calcium
exchanger (NCX) and cardiac
calsequestrin (CSQ2) mRNAs were
measured by TaqMan quantitative RT-
PCR. Expression levels of PLN, NCX and
CSQ2 mRNAs are comparable among all
three groups. Atrial fibrillation: atrial
fibrillation; CSQ2: cardiac calsequestrin;
MVD: mitral valvular disease; NCX:
sodium calcium exchanger; NS: not
significant; NSR: normal sinus rhythm;
PLN: phospholamban.
2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 723–730
Sarcolipin expression in atrial fibrillation Limitations of the study
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digitalis and Ca2+-channel blockers can mediate the expres- 8 Bosch RF, Zeng X, Grammer JB, Popovic K, Mewis C, sion of Ca2+-cycling genes [26,27]. As shown in Table 2, Kuhlkamp V. Ionic mechanisms of electrical remodeling in medications excluding warfarin potassium were basically human atrial fibrillation. Cardiovasc Res 1999;44:121–31.
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We have speculated that the decrease in the expression 11 Ohkusa T, Ueyama T, Yamada J, Yano M, Fujumura Y, of sarcolipin mRNA may contribute to abnormal intracel- Esato K et al. Alterations in cardiac sarcoplasmic reticulum lular Ca2+ homeostasis in initiating or sustaining AF; Ca2+ regulatory proteins in the atrial tissue of patients with however, we did not directly measure intracellular Ca2+ chronic atrial fibrillation. J Am Coll Cardiol 1999;34:255–63.
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patients with chronic AF would be of great interest.
13 Kneller J, Sun H, Leblanc N, Nattel S. Remodeling of Ca(2+)- In conclusion, chronic electrical and mechanical overload handling by atrial tachycardia: evidence for a role in loss of decreased the expression of sarcolipin mRNA in the right rate-adaptation. Cardiovasc Res 2002;54:416–26.
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15 Brundel BJ, van Gelder IC, Henning RH, Tuinenburg AE, Deelman LE, Tieleman RG et al. Gene expression of proteins Acknowledgements
influencing the calcium homeostasis in patients with persistent
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We thank Dr Yoshio Yasumura for generously providing the 16 Lai LP, Su MJ, Lin JL, Lin FY, Tsai CH, Chen YS et al. PCR primers and TaqMan probes for NCX, phospholam- Down-regulation of 1-type calcium channel and sarcoplasmic ban, cardiac calsequestrin, BNP and GAPDH, and Remi reticular Ca(2+)-ATPase mRNA in human atrial fibrillation Haruno for excellent technical assistance. Yokohama Foun- without significant change in the mRNA of ryanodine receptor, dation for Advancement of Medical Science supported this calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling. J Am Coll Cardiol
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