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European Journal of Heart Failure 2007 9(1):37-43; doi:10.1016/j.ejheart.2006.04.007
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© 2007 European Society of Cardiology

Mutational screening of phospholamban gene in hypertrophic and idiopathic dilated cardiomyopathy and functional study of the PLN –42 C>G mutation

Maria Medin, Manuel Hermida-Prieto, Lorenzo Monserrat*, Rafael Laredo, Jose Carlos Rodriguez-Rey, Xustox Fernandez and Alfonso Castro-Beiras

Hospital Universitario Juan Canalejo Spain

* Corresponding author. Servicio de Cardiologia, Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, A Coruna 15006, Spain. E-mail address: lorenzo_monserrat{at}canalejo.org


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: Phospholamban is an endogenous sarcoplasmic reticulum calcium ATPase inhibitor with a regulatory effect on cardiac contraction/relaxation coupling. Mutations in the phospholamban gene (PLN) have been associated with primary cardiomyopathies.

Aims: To screen for PLN mutations in our population of patients with primary cardiomyopathies and to perform functional analysis of the mutations identified.

Methods: We performed SSCP mutational screening and DNA sequencing of the PLN gene in 186 patients with either hypertrophic or dilated cardiomyopathy. To study promoter strength we constructed reporter plasmids containing the luciferase gene and performed transient transfection analysis in C6 and C2C12 cell lines.

Results: The PLN –42 C>G mutation was found in one patient with late onset familial apical hypertrophic cardiomyopathy. This mutation decreased phospholamban promoter activity by 43% and 47%, in C6 and C2C12 cell lines respectively. One son had mild apical hypertrophic cardiomyopathy and carried the mutation, another son with normal ECG and echocardiogram also had the mutation.

Conclusion: The PLN –42 C>G mutation is associated with a benign form of apical hypertrophic cardiomyopathy in this family, though the presence of a healthy adult carrier suggests that other genetic and environmental factors could be involved. Otherwise, mutations in the PLN gene are not a frequent cause of cardiomyopathies in our population.

Key Words: Phospholamban • Mutation • Promoter region • Hypertrophic cardiomyopathy

Received July 28, 2005; Revised February 14, 2006; Accepted April 11, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Hypertrophic cardiomyopathy (HCM) has been defined as a primary disease characterised by unexplained left ventricular hypertrophy usually caused by mutations on sarcomeric genes [1-3]. More than 400 sarcomeric gene mutations associated with HCM have been described [3-8]. However, the mutations identified in these genes only account for the disease in about 60% of HCM patients [1,4,5]. Similarly, although different mutations in cytoskeletal, sarcomeric, intermediate filaments and other genes have been implicated in dilated cardiomyopathy (DCM) aetiology; they can explain only a very limited number of cases [9,10].

There is evidence for the implication of calcium regulatory proteins in the pathogenesis of heart failure [11-13] and therefore, changes in genes encoding for proteins involved in the re-uptake of calcium might harbour mutations leading to cardiomyopathies. Phospholamban is an endogenous sarcoplasmic reticulum calcium ATPase (SERCA 2a) inhibitor with a regulatory function on cardiac contraction/relaxation coupling. Phospholamban reversibly inhibits SERCA 2a by direct protein-protein interactions and therefore plays an important role in sarcoplasmic reticulum calcium reuptake.

Very recently, two different mutations in the PLN gene have been associated with DCM and one mutation has been reported in one patient with HCM [14-16]. The objective of this study was to screen for PLN mutations in our patients with DCM and HCM, and to perform functional studies of the mutations that could be identified in this population.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Study population
We screened 186 patients with either HCM (n=101) or DCM (n=85). HCM and DCM were defined according to the criteria of the WHO and of the ESC working group on myocardial and pericardial diseases [17]. All patients gave written informed consent to the clinical and the genetic study. The local ethics committee approved the study. All patients were followed in a cardiomyopathy clinic at our institution. The patients were evaluated by a detailed history, physical examination, 12-lead electrocardiography (ECG), general biochemistry and M-mode, two-dimensional and Doppler echocardiography. A pedigree was drawn for each patient and all first-degree relatives were invited to participate in familial screening (history, physical examination, biochemistry, ECG and echocardiography).

2.2. Mutation screening
Mutation screening was performed on two genomic PLN sequences, one corresponding to proximal promoter and exon 1 (GenBank accession no.: AF177763) and the other corresponding to exon 2 (GenBank accession no.: AF177764). DNA samples were extracted from peripheral blood lymphocytes using the GFX Genomic Blood DNA Purification Kit (Amersham Biosciences). The PCR amplification generated three fragments: the first fragment corresponding to the promoter region and exon 1 and two fragments corresponding to fragmented exon 2.

The oligonucleotides used for PCR amplification are available as supplementary information. PCR products were run in SSCP polyacrylamide gels in order to determine electrophoretic mobility changes. For this, GeneGel SSCP Starter Kit (Amersham Biosciences) was used according to the manufacturer's instructions. Electrophoresis conditions were pH 8.9 and pH 9.0 at 12 °C and 20 °C respectively. Bands were developed by silver nitrate staining. The nucleotide sequence of all the samples showing an abnormal electrophoretic mobility in SSCP gels, was determined. PCR products were purified with EXOSAP-it purification kit (USB Corporation) and direct sequence was done with the CEQ 2000 Dye Terminator Cycle Sequencing Kit (Beckman Coulter) using CEQ 8000 Genetic Analysis System model (Beckman Coulter). Sequencing primer sequences were taken from the literature [14,16].

2.3. ARMS (Amplified Refractory Mutation System)
The heterozygous single nucleotide transition PLN –42 C>G found by sequencing was subsequently confirmed by ARMS. Specific primers for nucleotide change PLN –42 C>G and PCR control primers are available as supplementary information. We tested the presence of the mutant allele in 120 control subjects (240 chromosomes). Controls were randomly selected from a population of anonymous adult blood donors from our region.

2.4. Promoter activity assays
2.4.1. Reporter plasmids
A 219 bp fragment extended from –162 to +56 of PLN gene was amplified by PCR using primers modified to contain KpnI-HindIII sites and cloned in the PCR vector PGEMT-easy (Promega, Madison, WI). After excision with KpnI and HindIII, the fragments were subcloned in the promoterless PGL3-basic vector (Promega, Madison, WI) and used in transient transfection assays.

2.5. Cell culture, transfection and luciferase assays
Transient transfection assays were done in C6 and C2C12 cell lines. Both cell lines were obtained from the ECACC (European Collection of Cell Cultures). These cell lines had been previously used in the study of the PLN promoter. C6 allows a high basal luciferase expression under the control of PLN promoter and C2C12 had been chosen for the study of PLN promoter activity in a muscular tissue without endogenous phospholamban expression [18,19]. Both C6 and C2C12 cell lines were cultured in Dulbecco's-modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotic/antimicotic mix (Gibco. BRL). All the cells were grown at 37 °C and 5% CO2.

Twenty hours before transfection the cells were seeded in T6 (C6) and T12 (C2C12) plates at a cell density of 200.000 and 100.000 cells per well, respectively, with serum free medium Optimem (Gibco. BRL). This cell density allows the necessary confluence (90-100%) at the moment of transfection.

For transfecting both C6 and C2C12 we used 1 µg of pGL3-derived plasmids and 20 ng of phRL vector (Promega) were included as a control of transfection efficiency. Transfection reagents FuGene (Roche, Postfach, Switzerland) and Lipofectamine (Invitrogen) were used for the transfection of C6 and C2C12 cells respectively, always according to manufacturer's instructions. After transfection DNA-cell mixes were incubated for 48 h in the indicated medium before analysing luciferase expression with the Dual Luciferase Assay System (Promega) according to manufacturer's instructions. Luciferase activity measures in C6 and C2C12 cell lines were made with the luminometer TD-20/20 (Turner Designs).

Results were expressed as Relative Luciferase Activity, defined by the ratio Firefly luciferase (PGL3 vector)/Renilla Luciferase (phRL vector). Duplicated cells were assayed for each transfection condition and at least three independent transfection assays were performed. Data were expressed as mean±S.E.M. We compared luciferase activity between PGL3 vectors containing wild type and mutant PLN promoter sequences (P<0.05 in a non-paired Student T test was considered statistically significant).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
The analysis of the DNA samples from the 186 patients (101 HCM and 85 DCM) included in this study revealed the existence of a single-point mutation, a nucleotide C to G transition at position –42 in the phospholamban promoter region of one patient with HCM (Fig. 1A and B). No changes in the coding region of phospholamban gene were found in any of the 186 patients affected with either HCM or DCM included in this study. The index case with the PLN –42 C>G mutation was an 85- year-old woman with apical HCM (Fig. 2). Maximum wall thickness was 22 mm at the apex with normal thickness in basal segments. She originally presented with palpitations and chest pain with normal coronary angiography aged 59 years (in 1977) and developed atrial fibrillation 2 years later. She was diagnosed with apical HCM at 67 years (in 1985). At present she is in NYHA functional class II and continues to have occasional episodes of angina. Her brother was diagnosed with apical HCM at 72 years of age (19 mm at apex with normal basal segments wall thickness) after presenting with palpitations. He was in atrial fibrillation since diagnosis, NYHA functional class II, and with atypical chest pain. He died suddenly at 81 years of age (DNA sample not available). Three sons of the index case were screened and one 59-year-old male had apical hypertrophic cardiomyopathy with mild hypertrophy (maximal wall thickness 15 mm) and carried the mutation. The other two sons were asymptomatic with normal ECG and echocardiogram. Following DNA testing, we found that one son also had the mutation. In the index patient, additional mutations in the MYH7 and MyBPC3, encoding beta myosin heavy chain and myosin binding protein C, were excluded by SSPC and sequencing of all the coding sequence of both genes. We also performed a screening of previously described mutations in TNNT2, ACTC, TNNI3 and TPM1 genes with negative results.


Figure 01
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  		Fig. 1 (A) PCR-SSCP gel of the HCM patient and a healthy control. The sample shows different electrophoresis mobility in polyacrylamide gels compared to the control, indicating a possible nucleotide change in PLN promoter sequence. (B) Genomic DNA sequence analysis of the PLN promoter region of a HCM patient. The arrow indicates a heterozygous C/G 42 bp upstream the transcription start site in PLN promoter. (C) ARMS. Amplified Refractory Mutation System (ARMS) that confirms the mutation PLN –42 C>G. The figure shows PCRs from wild-type (N) and mutant (M) alleles in the HCM index case, and in the control subject. The 360 bp band shows the PCR product from the housekeeping ATT gene, used as amplification control. The 232 bp bands are specific for the PLN –42 C>G mutation. M: Mutant allele. N: Normal allele (Wild Type).

 


Figure 02
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  		Fig. 2 Pedigree of the family and echocardiogram of the index patient. (A) Pedigree of the family. Squares represent males and circles females. Patients with apical hypertrophic cardiomyopathy are in black. + indicates carriers of the mutation, – indicates subjects without the mutation and ? indicates subjects with unknown genetic status. The numbers indicate the age at the last evaluation. (B) Two-dimensional end diastolic apical 4 chamber (right) and 2 chamber (left) views of the heart of the index patient. There is clear apical hypertrophy (22 mm at the apex) with normal left ventricular wall thickness in the basal segments. The left atrium is dilated.

 
The nucleotide change PLN –42 C/G was confirmed by ARMS (Fig. 1C). The –42 C to G transition was not found in more than 100 control subjects. Thus PLN –42 C>G is a heterozygous point mutation present in a HCM patient and two first-degree relatives: one affected and one healthy carrier.

The mutation is located at the 217-bp fragment, which has been defined as the promoter fragment with maximal transcriptional activity. The nucleotide mutation point is phylogenetically conserved: nucleotide at –42 upstream transcription start site is identical in the promoter sequences of four mammalian species (Fig. 3). All these data suggest that the mutation could result in changes in promoter activity.


Figure 03
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  		Fig. 3 Alignment of different promoter PLN sequences in four different species. The alignment shows the 238 bp fragment upstream the PLN transcription start site (+1). High homology is present at consensus boxes (E-box, MCAT-box, CP/NFY-box and GATA-box), at the AT-rich region (70% conservation), and at –169/–134 region, which shows 85% homology between species. Nucleotide at –42 position (mutation point) is identical in all species. (GenBank accession numbers; AF177763, AF037348, LO3381 and M63600).

 
In order to test this possibility we performed a series of experiments in which we transiently transfected pGL3-constructs containing either the wild type or the mutated promoter. The first series of experiments were done in the C6 glyoma cell line. As shown in Fig. 4A the –42 C>G mutation resulted in a 43% decrease of transcriptional activity compared to the wild-type promoter (P=0.024). As C6 is a cell line derived from non-muscle tissue we performed another series of transfection assays in C2C12; a cell line derived from muscle. The results of these second series of experiments indicated that the –42 C<G mutation resulted in a 47% decrease of transcriptional activity also compared to the wild-type promoter (P=0.022) (Fig. 4B).


Figure 04
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  		Fig. 4 Effect of the PLN –42 C>G H mutation on promoter activity. The figure shows transcriptional activity of wild type and mutant PLN promoter in C6 (A) and C2C12 (B) cell lines. Transcriptional activity was defined as a ratio between Firefly luciferase activity to Renilla luciferase activity and was normalized with the mean transcriptional activity of the wild-type PLN promoter. The values are expressed as means±S.E.M. of four independent experiments (*P<0.05 in a non-paired T-Student Test). S.E.M.: standard error mean.

 
A second variation in the promoter sequence, a nucleotide A to C transition at position –36 was found in one patient with DCM (PLN –36A>C) but it was also present in 5% of the healthy controls and it did not change transcriptional activity in functional studies.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We have found a novel mutation in the PLN gene promoter in a family with a benign form of apical hypertrophic cardiomyopathy, and we have demonstrated that this mutation decreases promoter activity in two different cellular models. We have also confirmed that PLN mutations are a rare cause of hypertrophic or dilated cardiomyopathies, accounting for less than 1% of the cases.

Cardiac muscle contraction is initiated by calcium extrusion from cardiac sarcoplasmic reticulum by different regulated channels. SERCA 2a (sarcoplasmic reticulum calcium ATPase) transports cytosolic calcium inside sarcoplasmic reticulum once sarcomeric contraction has happened, being fundamental for sarcomeric relaxation. Phospholamban reversibly inhibits SERCA 2a by direct protein-protein interaction and therefore plays an important role in sarcoplasmic reticulum calcium reuptake. Different studies in animal models and in human heart failure suggest that alterations in cardiac contraction/relaxation coupling could underlie myocardial dysfunction [20-22] and three different mutations in the PLN gene have been associated with both DCM and HCM. Initially Schmitt et al. [14] described the PLN R9C mutation, which encodes a mutant phospholamban protein that constitutively inhibits SERCA 2a and results in DCM and heart failure. Subsequently, Haghighi et al. [15] described the mutation PLN L39STOP, which results in the absence of phospholamban expression (loss of function mutation), and is associated with two different phenotypes, DCM in homozygous individuals and HCM in heterozygous individuals. Finally, Minamisawa et al. [16] described the mutation PLN –77 A<G, which results in an 80% increase in phospholamban transcriptional activity, and is associated with HCM.

The organisation of the human PLN gene resembles that reported for avian and other mammalian species. It comprises two exons separated by a large 10.5 kb intron. The whole coding sequence is located within the second intron, where multiple potential polyadenylation sites have been reported [23,24]. The expression of the gene is driven by a promoter located within the 238 bp upstream of the transcription start site (GenBank accession no.: AF177763) [24]. Within this region, motifs for E-box (–11/–6), GATA (–108/–103), M-CAT-like (–68/–62) and CP1/NF (–84/–80) boxes are highly conserved among different mammalian species. Two additional regions also show a marked conservation: an AT-rich region (–217/–170) is approximately 70% conserved and the region between –169 and –134 shows 85% conservation. PLN promoter is a strong promoter, although all the transcriptional proteic factors still remain unknown [18]. Our experiments showed that the PLN –42 C<G mutation affects PLN promoter strength: PLN –42 C<G results in a 43% and 47% decrease of promoter activity compared to wild-type PLN promoter in C6 and C2C12 cell lines, respectively.

Both inhibition and overexpression of phospholamban have been associated with the development of primary cardiomyopathies in humans [14-16] and with heart failure in animal models [20,22]. Previous reports in primary cardiomyopathies suggest a possible association between the decrease in phospholamban activity with DCM [14] and the increase in activity with HCM [16]. Our findings apparently contradict these previous observations, as we found an association of HCM with a mutation that produces a decrease in promoter activity. The discrepancy of our findings with those previously reported by Minamisawa et al. could be related to the use of different promoter constructs, and we have to remember that we are talking about different point mutations [16]. Moreover, promoter activity in vivo may be different to that found in "in-vitro" studies. Finally, even though HCM and DCM are apparently opposed phenotypes, we know that mutations in the same genes may cause both HCM and DCM and the same patient may even progress from HCM to DCM [1,9,10]. These diverse phenotypic manifestations are a consequence of the interaction of the primary effect of the mutations with multiple other genetic and environmental factors. How either the increase or the decrease in phospholamban activity can lead to cardiomyopathy is an issue that deserves further research.

In our study, the change in promoter activity resulting from a mutation affecting a highly conserved residue, together with the fact that the mutation appears in two members of a family affected with apical HCM and is not found in the healthy population makes its association with the disease possible. Despite this, we cannot forget that another family member that also shows the PLN –42 C>G mutation has no clinical manifestations of the disease. This could be explained in the context of incomplete penetrance of the mutation or in expression variability of HCM, reported in so many cases [1,3,5]. Incomplete penetrance is particularly described in mutations that cause late onset disease [1,25]. In our family, although we cannot be sure that the disease was not phenotypically expressed at younger ages, the late onset of the clinical manifestations, with clinical diagnosis around the sixth decade of life, suggest that we are dealing with late onset HCM. In this elderly population, the phenotypic expression of the disease is probably more dependent on the presence of additional genetic and environmental factors. Although laboratory DNA analysis for mutant genes is the most definitive method for establishing the diagnostic of HCM there still remain important obstacles for translating the genetic studies to the clinical strategies, and one of these obstacles is the recognition of age-related disease onset and incomplete penetrance [26]. Interestingly, the mutation reported by Minamisawa et al. [16] was also associated with late onset disease. It is possible that our clinically unaffected patient who carries the PLN mutation could still develop the disease.

Apical HCM is present in about 3-10% of western patients and it is usually associated with a benign course with long-term survival [27]. Several reports have described familial aggregation of apical HCM, suggesting that specific genetic causes could account for this pattern of hypertrophy [28,29]. To elucidate whether apical HCM is a characteristic presentation of the –42 C<G PLN mutation, or is a particular expression of the mutation in our patients, we need to identify the mutation in other families.

Our study confirms the fundamental role of familial screening to define genotype-phenotype correlation. The mutation described by Minamisawa et al. was only identified in the index patient [16] and our work shows that in vitro studies of gene function are useful to evaluate the potential pathogenic role of a mutation, but familial study is necessary to get a full perspective of the clinical implications of each genetic variant. Genetic screening in other populations and long term follow-up of mutation carriers are warranted.

In conclusion, the mutation PLN –42 C>G, which decreases the transcriptional activity of PLN promoter, is associated with apical hypertrophic cardiomyopathy with a benign course in the reported family. The presence of a healthy adult carrier suggests that other genetic and environmental factors could be implicated. Otherwise, mutations in the PLN gene are not a frequent cause of hypertrophic or dilated cardiomyopathy in our population.


   Acknowledgements
 
Spanish cardiovascular research network: RECAVA; Xunta de Galicia PGIDT03-san 91603PR, and FIS PI020880 supported this work. Lorenzo Monserrat is supported by a research grant from Aventis Foundation.

We specially thank Raquel Duran for her excellent technical assistance and Sandra Pampín and JP Vaqué for their help with cell cultures and luciferase experiments.


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

  1. Maron B.J., McKenna W.J., Danielson G.K., et al. ACC/ESC clinical expert consensus document on hypertrophic cardiomyopathy: a report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology Committee for Practice Guidelines (Committee to Develop an Expert Consensus Document on Hypertrophic Cardiomyopathy). J Am Coll Cardiol (2003) 42:1687–1713.[Free Full Text]
  2. McKenna W.J., Monserrat L. Identification and treatment of patients with hypertrophic cardiomyopathy at risk of sudden death. Rev Esp Cardiol (2000) 53:123–130.[Web of Science][Medline]
  3. Maron B.J. Hypertrophic cardiomyopathy: a systematic review. Review. JAMA (2002) 287:1308–1320.[Abstract/Free Full Text]
  4. Erdmann J., Daehmlow S., Wischke S., et al. Mutation spectrum in a large cohort of unrelated consecutive patients with hypertrophic cardiomyopathy. Clin Genet (2003) 64:339–349.[CrossRef][Web of Science][Medline]
  5. Richard P., Charron P., Carrier L., et al. EUROGENE Heart Failure Project. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation (2003) 107:2227–2232.[Abstract/Free Full Text]
  6. Van Driest S.L., Ellsworth E.G., Ommen S.R., Tajik A.J., Gersh B.J., Ackerman M.J. Prevalence and spectrum of thin filament mutations in an outpatient referral population with hypertrophic cardiomyopathy. Circulation (2003) 108:445–451.[Abstract/Free Full Text]
  7. Van Driest S.L., Jaeger M.A., Ommen S.R., et al. Comprehensive analysis of the beta-myosin heavy chain gene in 389 unrelated patients with hypertrophic cardiomyopathy. J Am Coll Cardiol (2004) 44:602–610.[Abstract/Free Full Text]
  8. http://cardiogenomics.med.harvard.edu/project-list.
  9. Chien K.R. Genotype, phenotype: upstairs, downstairs in the family of cardiomyopathies. J Clin Invest (2003) 111:175–178.[CrossRef][Web of Science][Medline]
  10. Castro-Beiras A., Monserrat L., Hermida M. Familial dilated cardiomyopathy. Current status and clinical benefits of basic research. Rev Esp Cardiol (2003) 56:7–12.[Medline]
  11. Arai M., Alpert N.R., MacLennan D.H., Barton P., Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res (1993) 72:463–469.[Abstract/Free Full Text]
  12. Meyer M., Schillinger W., Pieske B., et al. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation (1995) 92:778–784.[Abstract/Free Full Text]
  13. Hasenfuss G. Alterations of calcium-regulatory proteins in heart failure. Review. Cardiovasc Res (1998) 37:279–289.[Free Full Text]
  14. Schmitt J.P., Kamisago M., Asahi M., et al. Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science (2003) 299:1410–1413.[Abstract/Free Full Text]
  15. Haghighi K., Kolokathis F., Pater L., et al. Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest (2003) 111:869–876.[CrossRef][Web of Science][Medline]
  16. Minamisawa S., Sato Y., Tatsuguchi Y., et al. Mutation of the phospholamban promoter associated with hypertrophic cardiomyopathy. Biochem Biophys Res Commun (2003) 304:1–4.[CrossRef][Web of Science][Medline]
  17. Richardson P., McKenna W.J., Bristow M., et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies. Circulation (1996) 93:841–842.[Free Full Text]
  18. Yabuki M., Toyofuku T., Otsu K., et al. Involvement of NF-Y in transcriptional regulation of the phospholamban gene. Eur J Biochem (1998) 258:744–751.[Web of Science][Medline]
  19. Harrer J.M., Ponniah S., Ferguson D.G., Kranias E.G. Expression of phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity. Mol Cell Biochem (1995) 146:13–21.[CrossRef][Web of Science][Medline]
  20. Minamisawa S., Hoshijima M., Chu G., et al. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell (1999) 99:313–322.[CrossRef][Web of Science][Medline]
  21. Iwanaga Y., Hoshijima M., Gu Y., et al. Chronic phospholamban inhibition prevents progressive cardiac dysfunction and pathological remodeling after infarction in rats. J Clin Invest (2004) 113:727–736.[CrossRef][Web of Science][Medline]
  22. Song Q., Schmidt A.G., Hahn H.S., Carr A.N., Frank B., Pater L. Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J Clin Invest (2003) 111:859–867.[CrossRef][Web of Science][Medline]
  23. Johns D.C., Feldman A.M. Identification of a highly conserved region at the 5' flank of the phospholamban gene. Biochem Biophys Res Commun (1992) 188:927–933.[CrossRef][Web of Science][Medline]
  24. McTiernan C.F., Frye C.S., Lemster B.H., et al. The human phospholamban gene: structure and expression. J Mol Cell Cardiol (1999) 31:679–692.[CrossRef][Web of Science][Medline]
  25. Niimura H., Bachinski L.L., Sangwatanarog S., et al. Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy. N Engl J Med (1998) 338:1248–1257.[Abstract/Free Full Text]
  26. Maron B.J., Seidman J.G., Seidman C.E. Proposal for contemporary screening strategies in families with hypertrophic cardiomyopathy. Review. J Am Coll Cardiol (2004) 44:2125–2132.[Abstract/Free Full Text]
  27. Klues H.G., Schiffers A., Maron B.J. Phenotypic spectrum and patterns of left ventricular hypertrophy in hypertrophic cardiomyopathy: morphologic observations and significance as assessed by two-dimensional echocardiography in 600 patients. J Am Coll Cardiol (1995) 26:1699–1708.[Abstract]
  28. Maron B.J., Bonow R.O., Seshagiri T.N., Roberts W.C., Epstein S.E. Hypertrophic cardiomyopathy with ventricular septal hypertrophy localized to the apical region of the left ventricle (apical hypertrophic cardiomyopathy). Am J Cardiol (1982) 49:1838–1848.[CrossRef][Web of Science][Medline]
  29. Penas M., Fuster M., Fabregas R., Llorente C., Cosio F.G. Familial apical hypertrophic cardiomyopathy. Am J Cardiol (1988) 62:821–822.[CrossRef][Web of Science][Medline]

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