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Cardiovascular Research 2006 71(3):496-505; doi:10.1016/j.cardiores.2006.04.004
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Copyright © 2006, European Society of Cardiology

Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy

Hendrik Miltinga,*, Nina Lukasb, Bärbel Klaukea, Reiner Körfera, Andreas Perrotc, Karl-Josef Osterzielc, Jürgen Vogta, Stefan Petersd, Rolf Thieleczekb,1 and Magdolna Varsányib

aHerz- und Diabeteszentrum NRW, Klinik der Ruhr-Universität Bochum, Erich und Hanna Klessmann-Institut für Kardiovaskuläre Forschung und Entwicklung, Georgstr. 11, 32545 Bad Oeynhausen, Germany
bInstitut für Physiologische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany
cCharité-Universitätsmedizin Berlin/Kardiologie am Campus Buch and Virchow-Klinikum and Max-Delbrück-Centrum für Molekulare Medizin, 13125 Berlin, Germany
dKlinikum Quedlinburg, Innere Medizin, Abteilung Kardiologie, Ditfurter Weg 24, 06484 Quedlinburg, Germany

* Corresponding author. Tel.: +49 5731 973510; fax: +49 5731 972476. Email address: hmilting{at}hdz-nrw.de

Received 10 November 2005; revised 29 March 2006; accepted 6 April 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective: Mutations in the cardiac ryanodine receptor (RYR2) gene have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). The molecular mechanisms by which genetic modifications lead to ARVC are still not well understood.

Methods ARVC patients were screened for mutations in the RYR2 gene by denaturing HPLC and DNA sequencing. Single channel measurements were carried out with RyR2 channels purified from explanted hearts of ARVC patients.

Results: None of the published RYR2 mutations were found in our ARVC-cohort. However, we identified two single nucleotide polymorphisms (SNPs) in exon 37 of the human RYR2 gene which lead to the amino acid exchanges G1885E and G1886S, respectively. Both SNPs together were found exclusively in 3 out of 85 ARVC patients in a composite heterozygous fashion (genotype T4). This genotype was associated with ARVC (p<0.05) but not with dilated cardiomyopathy (DCM, 79 patients) or none-failing controls (463 blood donors). However, either one of the two SNPs were identified in further 7 ARVC patients, in 11 DCM patients, and in 64 blood donors. The SNP leading to G1886S may create a protein kinase C phosphorylation site in the human RyR2. Single channel recordings at pCa4.3 revealed four conductance states for the RyR2 of genotype T4 and a single open state for the wild type RyR2. At pCa7.7, the lowest subconductance state of the RyR2 channel of genotype T4 persisted with a greatly enhanced open probability indicating a leaky channel.

Conclusion The RyR2 channel leak under diastolic conditions could cause SR-Ca2+ depletion, concomitantly arrhythmogenesis and heart failure in a subgroup of ARVC patients of genotype T4. A change in the RyR2 subunit composition due to the combined expression of both SNPs alters the behaviour of the tetrameric channel complex.

KEYWORDS Arrhythmia; Ca-channel; Cardiomyopathy; Gene polymorphisms; Single channel currents

This article is referred to in the Editorial by I. Jóna and P.P. Nánási (pages 416–418) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
The cardiac sarcoplasmic reticulum (SR) calcium release channel, ryanodine receptor (RyR2), is central to myocardial excitation contraction coupling. During systolic calcium induced calcium release, Ca2+-ions are passing through this channel on their route from the SR to the cytosol. Tetrameric RyR2 forms the core of a macromolecular complex to which numerous endogenous modulatory ligands can associate and control the gating of the calcium release channel [1,2].

RYR2 is one of the largest human genes (105 exons) encoding an mRNA of about 15 kb. Mutations in this gene have been associated with catecholaminergic polymorphic ventricular tachycardia, CPVT [3,4], and arrhythmogenic right ventricular cardiomyopathy, ARVC [5,6]. Mutations in the gene of calsequestrin, another protein of the RyR2 complex that interacts with the Ca2+ release-channel from the inside of the SR, are associated with CPVT [7,8]. Functionally impaired cardiac calsequestrin can cause spontaneous Ca2+ transients and arrhythmogenic delayed afterdepolarisations in cardiac myocytes [9].

Mutations in RYR2 linked to CPVT have been shown to reduce the affinity of FK506-binding-protein (FKBP12.6) to RyR2. The release of FKBP12.6 from RyR2 leads to a destabilized channel with an increased single channel open probability at diastolic conditions. These leaky SR-Ca2+ release-channels can trigger arrhythmia and sudden cardiac death [10]. However, the proposed molecular mechanism for the development of CPVT has been challenged recently [11,12]. RyR2 can be phosphorylated at serine 2809 (Ser2808 in the human sequence) by protein kinase A (PKA) and Ca2+-calmodulin dependent kinase II [13] and probably contains further phosphorylation sites [14]. Phosphorylation of Ser2809 by PKA, causing dissociation of FKBP12.6 from RyR2 and thereby an activation of the Ca2+-channel, has been considered important for the regulation of the RyR2 channel activity and for cardiac dysfunction in heart failure [10]. However, studies from different laboratories could not find a correlation between Ser2808-phosphorylation and FKBP12.6-dissociation [14–16]. Recently, a novel PKA phosphorylation site, Ser2030, has been identified in RyR2 which appeared to be the only phosphorylated residue after acute β-adrenergic stimulation [12]. This phosphorylation occurred stoichiometrically and did not dissociate FKBP12.6 from RyR2. Thus, the molecular mechanisms by which the RyR2 channel activity is modified in the failing heart are still not well understood and need further elucidation.

In the present study we identified two common single nucleotide polymorphisms (SNPs) in the human RYR2 gene which cause the non-conservative amino acid exchanges G1885E and G1886S, respectively, and are associated with ARVC in a composite heterozygous fashion. The single channel properties of RyR2 from two ARVC patients of different RYR2 genotype indicate that only the combined expression of the two polymorphic alleles of RyR2 is associated with increased diastolic channel activity and may contribute to the development of ARVC in a subgroup of patients.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1 Study population and clinical evaluation of the patients
Mutations screening of the human RYR2 gene was carried out with 79 heart transplantation (HTx) candidates with DCM and 85 patients with ARVC. DNA from 463 anonymous blood donors was analysed for allele frequencies as a control. The ARVC patients were from the clinical programs of the Heart and Diabetes Center NRW (HDZ), Bad Oeynhausen, from the Charité Berlin, and from the Klinikum Quedlinburg, Germany. The diagnosis of ARVC was according to the criteria of the European Society of Cardiology [17]. DCM patients were from the HTx-program of the HDZ and listed for HTx due to endstage heart failure. All patients gave informed consent and the study was approved by the local ethics committee in accordance with the Declaration of Helsinki.

2.2 Mutation screening of the human RYR2 gene
DNA samples from patients and control persons were screened by denaturing high-performance liquid chromatography (dHPLC) at two different temperatures (see below). Genomic DNA was extracted from blood samples by QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). PCR products with divergent dHPLC elution profiles were analysed by automated DNA sequencing. Genotypes of individuals with two heterozygous SNPs in exon 37 of RYR2 were analysed by TOPO-TA-cloning of the PCR-fragments in Escherichia coli (Invitrogen, USA). Cloned fragments were identified by sequencing of plasmid-DNA in 17 different bacterial clones. All RYR2-exons of those patients whose hearts were used for RyR2 purification and single channel measurements were sequenced completely in both directions.

2.3 dHPLC analysis
dHPLC analysis was performed on a Wave DNA Fragment Analysis System MD with a DNASep column (Transgenomic, USA). PCR fragments were denatured for 2 min at 94 °C and then re-annealed in a thermoblock using a ramp of – 2 °C/min to a final temperature of 8 °C. Separation was performed at 59.5 °C with a gradient of 53.8–62.8% buffer B and at 63 °C with a gradient of 50.8–59.8% buffer B. The gradient was obtained by mixing buffer A (0.1 M triethylamine acetate, pH7.0) and buffer B (buffer A containing 250 ml/l acetonitril). The increase in buffer B was 2% per min at a flow rate of 0.9 ml/min with a total gradient time of 4.5 min. Column temperatures were calculated with the NAVIGATOR software. Temperature standards (Transgenomic) were used to confirm system performance and the accuracy of the oven temperature. DNA samples from three patients with a RYR2-SNP previously identified by DNA sequencing were used as positive controls to verify the dHPLC-sensitivity. All samples were analysed for heteroduplexes using wild type DNA as a control. DNA-samples of wildtype-RyR2 were mixed with patients DNA for the detection of homozygous sequence changes. Divergent conformers were analysed by automated DNA sequencing.

2.4 PCR amplification of the RYR2 exons
PCR amplification was performed on a GeneAmp PCR System 9600 (Applied Biosystems) in a final volume of 50 µl containing 25 ng of genomic DNA, PCR buffer with 1.5 mM MgCl2 (Qiagen, Hilden, Germany), 40 pM of each primer (TIB MolBiol, Berlin, Germany), 12.5 µM deoxynucleotide triphosphates (Invitrogen, USA), and 1.5 units of HotStarTaq (Qiagen). Cycling conditions were: 95 °C, 10 min; 95 °C, 30 s; 57 °C, 40 s; 72 °C, 50 s; 35 cycles. Primer sequences are available from the authors upon request.

2.5 DNA sequencing
PCR amplicons were purified (JETquick; Genomed, Löhne, Germany) and sequenced on a ABI310 Genetic Analyzer with BIG DYE dideoxy-terminator chemistry in both directions (Applied Biosystems, USA). Amplicons showing changes in base composition were re-sequenced using the product of an independent PCR reaction as a template.

2.6 Enrichment of heavy SR (HSR) vesicles from human heart muscle
HSR was isolated from explanted hearts of transplantation candidates at the HDZ, Bad Oeynhausen, according to a modified method of Meissner and Henderson [18]. 60 g ventricle muscle was homogenized in a Warring Blendor for 4 x 25'' in 9 volumes of microsome buffer (100 mM NaCl, 0.5 mM EGTA, and 10 mM Na-HEPES, pH7.5, containing a protease inhibitor cocktail consisting of 0.2 mM Pefabloc, 100 nM Aprotinin, 1 µM Leupeptin, 1 µM Pepstatin, 1 µM Calpain I, 1 µM Calpain II, and 1 mM Benzamidine). Cell organelles were removed by centrifugation at 3700 x g for 30 min. From the resulting supernatant, crude microsomes were collected by centrifugation at 35,000 x g for 30 min. Actomyosin was extracted with a buffer containing 600 mM KCl, 10 mM K-PIPES, pH7.0, 250 mM sucrose, 0.1 mM EGTA, 90 µM CaCl2, and the above protease inhibitor cocktail. After 1 h of incubation at 4 °C, the microsome fraction was collected by centrifugation at 70,000 x g for 30 min and the pellet was resuspended in ice cold microsome buffer. This suspension was loaded on top of a linear sucrose gradient (15–45%, w/w) containing 100 mM NaCl, 0.5 mM EGTA, 10 mM Na-HEPES, pH7.4 and the protease inhibitor cocktail and centrifuged for 16 h at 100,000 x g. Fractions containing RyR2 were collected, diluted in three volumes of microsome buffer, and centrifuged at 100,000 x g for 1 h. The pellet was resuspended in 0.3 M sucrose and 10 mM K-PIPES, pH7.0, immediately frozen in liquid nitrogen, and stored at – 80 °C.

2.7 Solubilization of RyR2 from HSR
Human cardiac HSR vesicles were solubilized in twice the volume of buffer containing 1 M NaCl, 40 mM Na-HEPES, pH7.5, 0.3 mM CaCl2, 0.2 mM EGTA, 1.6% (w/v) CHAPS, 5 mg/ml phosphatidylcholine, 1 mM dithiotreitol, and the protease inhibitor cocktail described above, for 1 h on ice followed by 30 min at room temperature. This solubilisate was separated by sucrose density centrifugation on a linear sucrose gradient (10% to 30%, w/w) at 120,000 x g for 16 h. After fractionation of the gradient, RyR2 was localized by [3H]-ryanodine binding [18]. Aliquots of the peak RyR2 fractions were quickly frozen in liquid nitrogen and stored at – 80 °C.

2.8 Single-channel measurements and analyses
RyR2 purified from explanted hearts of patients whose genotype was determined previously by complete sequencing of the RYR2-DNA were used for single channel measurements. CHAPS-solubilized RyR2 was incorporated into a Müller-Rudin type planar lipid bilayer [19] containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in n-decane at a weight ratio of 5:4:1 (total phospholipid 20 mg/ml n-decane). Small aliquots of the solubilized RyR2 were added to one side of the bilayer designated as the cis (cytoplasmic) side. The trans side was defined as ground. Single-channel currents were recorded in symmetric KCl buffer solutions (250 mM KCl, 100 µM EGTA, 150 µM CaCl2, 20 mM K-PIPES, pH7.2) with additions as indicated in the text. The current signals were filtered at 2 kHz employing a 4-pole low-pass Bessel filter and digitized at 10 kHz with a 16-bit analog/digital–digital/analog converter Digidata 1322A in concert with an Axopatch 200B amplifier with a CV 203BU head stage (all from Axon Instruments, Union City, CA, USA) and a conventional PC operated under Windows 2000 professional (Microsoft, USA). Data acquisition and analysis were performed using pClamp 9.2 (Axon Instruments). Channel opening events were detected by time-course fitting using a minimal event duration of 0.2 ms and tolerating a maximum deviation in event amplitude of 10% of the corresponding full amplitude (both applied to all levels if applicable). For analysis of the data a resolution of 0.4 ms was imposed on the experimental record (the rise time of the recording system was 166 µs). After changing the experimental conditions, an equilibration time period of at least 5 min was allowed during which both, the cis and the trans solution were gently stirred. Po values were calculated from representative data segments with stable open probability (estimated from plots of a moving average of Po against time, carried out for all open levels if applicable) using the pClamp software. Free Ca2+-concentrations were calculated using winmaxc32 [20].


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
Two SNPs were identified in exon 37 of the human RYR2 gene by means of dHPLC and subsequent DNA sequencing (Fig. 1). Deviation from the single-peak wild type elution profile (Fig. 1A, trace 1) indicates heteroduplex formation of an RYR2 amplicon with the corresponding wild type DNA template (Fig. 1A; traces 2–4). The nucleotide exchanges responsible for these elution profiles are shown in Fig. 1B. The identified SNPs 5654G>A and 5656G>A in the codons 1885 and 1886 (nucleobases 5653–5658 of the open reading frame, Genbank Accession No. X98330 [GenBank] ) of the RYR2 gene lead to the non-conservative amino acid exchange G1885E and G1886S, respectively. Both polymorphisms were common in blood donors and in patients with DCM or ARVC. Three of the 85 ARVC patients but none of the DCM patients or blood donors contained both SNPs (Fig. 1B, profile 3). Cloning of the corresponding PCR fragments revealed a composite heterozygous genotype. Thus, both SNPs are located on different chromosomes.


Figure 1
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  		Fig. 1 Single nucleotide polymorphisms identified in exon 37 of the human RYR2 gene. A: Heterozygous sequence alterations detected by dHPLC in the RYR2 gene of ARVC patients (profiles: 1 to 4) and DCM patients (profiles: 1, 2, and 4). See Table 1 for the corresponding genotypes WT, T1, T2, and T4. Heteroduplexes were eluted at 63 °C. Absorbance of the cDNA fragments is given as output voltage. B: Nucleotide sequencing of the amplicons analysed in A. The wild type sequence (profile 1) of the two adjacent codons 1885 and 1886, 5653GGGGGC5658 (bold), is either changed to 5653GAGGGC5658 (profile 3, genotype T4; profile 4, genotype T2) or 5653GGGAGC5658 (profile 2, genotype T1; profile 3, genotype T4) resulting in the non-conservative amino acid exchanges G1885E and G1886S, respectively. Note that the composite heterozygous nucleotide alteration 5654G>A-5656G>A (profile 3, genotype T4) was identified in ARVC patients only.

 
Results of the RYR2 gene screening of 85 ARVC patients, 79 DCM patients, and 463 blood donors are summarized in Table 1. The wild type RyR2 G1885/G1886 was observed to about the same extend in all three groups investigated (88.2%, 86.1%, and 85.7% for ARVC patients, DCM patients, and blood donors, respectively). Two of the four SNP-affected RYR2 genotypes identified leading to the expression of the wild type isoform and either RyR2 G1885/G1886S (genotype T1) or RyR2 G1885E/G1886 (genotype T2), were found in all three groups to a similar degree (genotype T1: 5.9%, 6.3%, and 7.8%, genotype T2: 2.4%, 7.6%, and 6.0% for ARVC patients, DCM patients, and blood donors, respectively). Only altered non-wild type RyR2 are expressed in carriers of genotype T3 and T4. RyR2 G1885E/G1886 was found in 2 out of 463 blood donors (genotype T3) but not in ARVC and DCM patients. Three of the ARVC patients (3.5%) but none of the DCM patients and blood donors, respectively, were composite heterozygous carries of both SNPs (genotype T4), i. e. they are expressing RyR2 G1885E/G1886 and G1885/G1886S but not a RyR2 monomer with both amino acid exchanges. Testing the data in Table 1 for statistical independence by means of a contingency table reveals a significant (p<0.05) association between disease and SNP only for ARVC and patients of genotype T4 (Type 1 error of 0.004 according to the two-sided Fisher's exact test).


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  		Table 1 SNPs identified in the exon 37 of the RYR2 gene of patients with terminal heart insufficiency resulting from ARVC or DCM compared to a control group of blood donors

 
A comparison of the human RyR2 amino acid sequence with available RyR2 sequences of other species is compiled in Table 2. The amino acid exchanges due to the identified SNPs are located in a stretch of the primary sequence which is highly variable among the three known RyR isoforms [21]. Nevertheless, when focussing on the cardiac isoform, the affected residues 1885 and 1886 are part of a cluster of about 27 amino acids which seems relatively highly conserved among mammals (Table 2). The SNP leading to the amino acid substitution G1886S may create a phosphorylation site for protein kinase C (PKC) considering a minimum consensus sequence of S–X–K/R or K/R–X–S (X stands for any amino acid).


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  		Table 2 Comparison of the amino acid changes identified in the present study with known respectively predicted RyR2 sequences

 
The hearts of two ARVC patients, genotype T4 (female, age 54) and wild type RYR2 (male, age 36), were available for purification of RyR2 during orthotopic HTx. The genotype of both patients was verified by sequencing all exons of the RYR2 gene. There were no other non-synonymous changes in the RyR2 open reading frame. Thus, the measured effects described below are entirely dependent on the SNPs identified in the RyR2 of genotype T4. Both hearts showed typical severe damage of the right ventricle with fibrolipomatosis and isolated islets of remaining myocardium.

The single channel characteristics of the wild type RyR2 of an ARVC patient are summarized in Fig. 2. At systolic free Ca2+ (pCa4.3) on the cytoplasmic side of the RyR2, the open probability of the channel was 0.135 (Fig. 2A). Upon reduction of the free Ca2+ concentration to diastolic levels (pCa7.7), the open probability dropped to 0.015 (Fig. 2C). Increased time resolution of the trace in A indicates a single open state of the channel (Fig. 2B) which is confirmed by histograms of the events detected before (Fig. 2E) and after (Fig. 2F) imposing a resolution of 0.4 ms (see Methods). The latter, reflecting the true channel opening events, reveals a conductivity of about 473 pS. The mean open channel conductance derived at various holding potentials was 433±11 pS (Fig. 2G). The channel was blocked irreversibly in a subconductance state of about 85 pS upon the addition of 3 µM ryanodine to the cytoplasmic side (Fig. 2D).


Figure 2
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  		Fig. 2 Single-channel characteristics of a native RyR2 purified from the heart of an ARVC-patient homozygous for the wild type RYR2 gene. A–D: Single-channel currents recorded in symmetrical 250 mM KCl shown as upward inflections from a closed state indicated by the arrow-marked horizontal line across each trace. The time domain indicated in A is shown expanded in B. The free [Ca2+] of the cis chamber and the channel open probability (Po) are given on top of each trace. In D, 3 µM ryanodine was added to the cis chamber. The holding potential was 71 mV and the free [Ca2+] of the trans chamber was 50 µM. E, F: crude (E) and true (F) event amplitude histogram obtained from the recording in A before (E) and after (F) imposing a time resolution of 0.4 ms. Single Gaussians fitted to the peaks in F suggest a mean channel current amplitude of about 33.6 pA. The obtained mean open dwell times are 0.63 ms for A and 0.44 ms for C. G: current–voltage relationship measured at pCa7.7 and 4.3. Linear regression reveals a single channel conductivity of 433±11 pS (mean value±S.E.M.).

 
The single channel characteristics of the RyR2 of an ARVC patient of genotype T4 (Fig. 3A) suggests several subconductance states. A crude event histogram derived from this recording by assuming one closed and one open state shows a blurred event amplitude distribution with a densely occupied transition region (Fig. 3G). The corresponding open dwell time distribution is fit by a sum of three exponentials which also suggests a more complex gating model. In accordance with previous reports on RyR2 [10,15] we have used four open levels (Fig. 3B) for event detection leading to the crude event histogram shown in (Fig. 3H). The corresponding amplitude histogram of true opening events at 59 mV holding potential (Fig. 3I) reveals subconductivities of about 76, 176, and 290 pS between the closed and the fully open state (398 pS). An apparent unitary conductivity of 90±24 pS was estimated as mean of the increments in slope of the current–voltage relationship of the event amplitudes (Fig. 3J). A decrease of the free Ca2+ concentration on the cytoplasmic side of the channel from pCa4.3 to 7.7 causes reversible suppression of the open probability of the three highest conductance states, Po2, Po3, and Po4, whereas the one of lowest conductivity, Po1, is rather increased at diastolic free Ca2+ (compare Fig. 3A, C, and D). In this particular experiment, addition of 1.6 mM ATP to the cytoplasmic side suppressed Po2 and Po3 while stabilizing Po1 and enhancing Po4 (Fig. 3E). Further addition of 3 µM ryanodine to the cytoplasmic side blocks the channel in a subconductance state of about 83 pS similar to the former open state of lowest conductivity (Fig. 3F). This amounts to about 20% of the fully open conductivity which is less than the commonly reported 30–50%.


Figure 3
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  		Fig. 3 Single-channel characteristics of a native RyR2 purified from the heart of an ARVC-patient composite heterozygous for both SNPs in the RYR2 gene (genotype T4). A–F: Single-channel currents recorded in symmetrical 250 mM KCl. The closed state is indicated by the arrow-marked horizontal line across each trace. The holding potential was 93 mV for A, B, and 59 mV for C–F, respectively. The time domain boxed in A is shown expanded in B with dotted lines indicating the open levels used for event detection. In C–F, sequential additions of EGTA (C), CaCl2 (D), ATP (1.6 mM, E), and ryanodine (3 µM, F) were made to the cis chamber. The free [Ca2+] of the cis chamber and the open probabilities of the four open states of the channel (Po1,..., Po4) are given on top of the traces. The free [Ca2+] of the trans chamber was 50 µM. G, H: crude event amplitude histograms obtained from the recording in A by assuming one (G) and four (H) open states. I: true event amplitude histogram obtained from H after imposing a time resolution of 0.4 ms. Single Gaussians fitted to the peaks in I yield subconductance current amplitudes of about 4.5, 10.4, 17.1, and 23.5 pA. The mean dwell times for the obtained open states {tau}o1,...,{tau}o4 are (ms): 0.46, 0.45, 0.45, 0.50 for A, 1.68, 0.47, 0.33, 1.04 for C, 0.46, 0.64, 0.55, 0.46 for D, and 24.1, 2.7, 13.2, 14.4 for E. J: current–voltage relationship measured at pCa7.7 and 4.3. Linear regression reveals single-channel substate conductivities (mean value±S.E.M.) of about 90±13 (substate 1, {square}), 190±24 (substate 2, {bigtriangleup}), 254±24 (substate 3, {bigtriangledown}), and 376±53 pS (fully open state, O).

 
Fig. 4 summarizes the effects of the experimental conditions described before on the open probability of the RyR2 channel obtained from the ARVC patient of genotype T4 (Fig. 4A, 6 experiments) and from the one of wild type RYR2-genotype (Fig. 4B, 2 experiments). In short, the combined results are in agreement with the results of the individual experiment shown in Figs. 2 and 3Go except that ATP is stabilizing the open probability of all conductance states on high levels (Fig. 4A) rather than affecting them differently (Fig. 3E). The most striking observation is the high mean open probability at pCa7.7 (Po=0.57±0.17) of the lowest conductance state (~90 pS) of the RyR2 derived from the heart of the ARVC-patient of genotype T4 (Fig. 4A). It is almost 50-times higher than the value obtained at diastolic free Ca2+ (Po=0.012±0.003) with the RyR2 from the ARVC patient with the wild type RYR2-gene (Fig. 4B). These results indicate a strongly enhanced conductivity of the functional tetrameric RyR2 complex at pCa7.7, i. e. a leaky SR Ca2+ release channel under diastolic conditions in ARVC patients with both SNPs in the RYR2-gene.


Figure 4
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  		Fig. 4 Single-channel open probabilities Po of native RyR2 from ARVC-patients of genotype T4 (A) and those with the wild type RYR2 gene (B). The experimental conditions are indicated in the boxes above the bars. The open conductance states 1 to 4 refer to the RyR2 of the composite heterozygous carrier of both SNPs and o refers to the single open state of the wild type RyR2. Note the high open probability (mean Po=0.57) of the open conductance state 1 (~90 pS) at diastolic free Ca2+ (pCa7.7) and the markedly different character of Po of the two channel types at systolic free Ca2+ (pCa4.3). The bars represent mean values±S.E.M. of the number of experiments given beneath each column. The same bar pattern refers to the same open conductance state under different experimental conditions.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
We have identified two SNPs in the human RYR2-gene by means of dHPLC and DNA sequencing, which lead to the expression of the RyR2 isoforms G1885E and G1886S. Both SNPs were previously denoted as common polymorphisms ([22] and NCBI rs3766871). Here we present evidence that a combination of these common polymorphisms (genotype T4) is associated with ARVC (stochastic dependence at p<0.05) in a subgroup of ARVC patients. The composite heterozygous carriers of both SNPs are not expressing the wild type RyR2 in contrast to heterozygous carriers of just one of the two SNPs (genotype T1 or T2). There is no statistical association between the other non-WT genotypes (T1, T2, and T3) and ARVC or DCM. In addition, an association between ARVC and the genotype T4 is also supported by an analysis of the allele frequencies. From the data in Table 1, frequencies of the alleles G1885/G1886, G1885E/G1886, and G1885/G1886S of 0.924, 0.029, and 0.047, respectively, are obtained for ARVC patients. The corresponding values for blood donors are 0.927, 0.035, and 0.039, respectively (Supplementary Information). Based on these data for blood donors, a frequency of the heterozygous allele combination G1885E/G1886–G1885/G1886S of 0.0027 would be expected. The observed frequency of 0.0353 for this allele combination in ARVC patients of genotype T4 is, however, 13-times higher than expected. The homozygous combination of allele G1885E (genotype T3) was identified in blood donors only (0.0043 compared to 0.0012 expected), whereas a homozygous combination of allele G1886S was never found in 627 individuals screened.

In this context it is interesting that the substitution G1886S creates a putative PKC phosphorylation site which in homozygous carriers would be present in every RyR2-subunit. In heart failure, neurohumoral stimulation of several signalling pathways which are merging downstream at PKC is activated chronically. The predominant myocardial PKC isoform, PKC-{alpha}, plays a key role in regulating contractility and Ca2+ turnover [23]. Further experimental evidence will be needed to clarify (i) whether serine 1886 in RyR2 can be phosphorylated by PKC and (ii) whether this posttranslational modification is of physiological relevance.

An increase in the free cytoplasmic Ca2+ concentration from pCa7.7 to 4.3 activates RyR2-channel of both genotypes investigated by increasing their open probabilities. This applies to the single open state of the wild type channel and all conductance states of the RyR2-channel of genotype T4. ATP further enhances the open probabilities of these latter multiple open states. However, at pCa7.7 the open probability of the lowest subconductance state (~90 pS) of the RyR2 channel of genotype T4 (Po=0.57±0.17) is almost 50-times higher than the one of the wild type RyR2 channel (Po=0.012±0.003). Thus, the expression of the RYR2 genotype T4 likely leads to a SR Ca2+-release channel which is leaky under diastolic conditions.

The clamp-shaped structure of the cytoplasmic part of RyR2 represents the three-dimensional location at which several single amino acid changes linked to the development of CPVT and ARVC are clustered [24]. This region, which includes domains 5, 6, 9, and 10, undergoes conformational changes when the channel is switched from the closed to the open state [25,26]. The glycine residues 1885 and 1886 affected by the SNPs are located in a region known as divergent region 3 (DR3) which comprises residues 1852–1890 of RyR2 [21]. It has been mapped to domain 9 in the clamp structure of RyR2 adjacent to the FKBP12.6 binding site and may be involved in channel regulation by Ca2+, Mg2+ or FKBP12.6 [21,26]. The appearance of subconductance states of the channel has been linked to the dissociation of FKBP12.6 from RyR2 (for review see [10]) but was not confirmed in other studies [27,28]. We regularly have observed subconductance states only with RyR2 purified from the myocardium of the ARVC patient of genotype T4. Since both ARVC patients were catecholamine dependent and received phosphodiesterase inhibitors immediately before HTx, FKBP12.6 removal from RyR2 due to chronic ß-adrenergic stimulation cannot be the cause for the different channel properties observed here. The molecular mechanism by which the DR3 region of RyR2 participates in channel regulation is not known. Our results suggest that the combination of two amino acid exchanges in the DR3 region, which is unique to RyR2 channels of genotype T4, causes a modified channel gating with a highly increased channel activity at diastolic free Ca2+ concentrations. An intracellular myocardial Ca2+ leak can cause SR Ca2+ store depletion leading to a reduced amplitude of the intracellular Ca2+ transient and diminished force production [10]. Ca2+ released during diastole can provoke delayed afterdepolarizations which can initiate fatal cardiac arrhythmias [9,29]. In heart failure, increased Ca2+ extrusion via the Na+–Ca2+ exchanger has been reported [30] which would contribute to SR Ca2+ store depletion. Thus, contractile dysfunction in heart failure can, at least in part, result from RyR2-dependent Ca2+ leak (for review see [10]).

The actual allele combination of the genotype expressed will influence the composition (see Table 1) and possibly the properties of the functional tetrameric RyR2 complex. If we designate the expression products of the RyR2 alleles G1885/G1886, G1885/S1886, and E1885/G1886 with {alpha}, β and {gamma}, respectively, there are different possible combinations of these subunits in the RyR2 tetramer. As discussed above, an association between disease and SNP exists only for ARVC and the composite heterozygous occurrence of both SNPs in genotype T4. These patients could express one or both of the RyR2-subunits β and {gamma} in the following combinations: β4, β3{gamma}, β{gamma}β{gamma}, ββ{gamma}{gamma}, β{gamma}3, and {gamma}4. Formation of β4 would be possible also for carriers of genotype T1 which is neither associated with ARVC nor DCM. The homo-tetramer {gamma}4 may be formed also in carriers of genotype T2 and T3 (here it represents the only RyR2 complex) which both are also not associated with ARVC or DCM. This suggests that one or more of the hetero-tetrameric RyR2 complexes β3{gamma}, ββ{gamma}{gamma}, β{gamma}β{gamma}, and β{gamma}3, which are unique to ARVC patients of genotype T4, could be associated with the observed leakiness of the RyR2 channel at diastolic free Ca2+. Thus, in a subgroup of ARVC patients, the combination of differently altered RyR2-subunits to the functional tetrameric complex and not the amino acid exchange per se seems to be relevant for the development of the disease.

There are ARVC patients who may have a different RyR2 tetramer composition (the {alpha}–β and {alpha}–{gamma} combinations resulting for genotype T1 and T2, respectively) and a majority of them (88.2%) is sharing the wild type RyR2 complex {alpha}4. Surprisingly, we could not detect any of the published RYR2 mutations [5,6] linked to the development of ARVC in our patients by means of complete screening of the RYR2-gene (data not shown). Thus, while polymorphisms in the RYR2-gene are associated with ARVC in a subgroup of patients, there seem to be other molecular routes towards the development of ARVC like those involving desmosomal proteins [31].


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.04.004.


   Acknowledgements
 
This work was supported by grants from the Medical Faculty of the Ruhr-Universität Bochum to M.V. and H.M. (FoRUM F314/02 and F 399A6-2003) and from the Erich and Hanna Klessmann-Stiftung, Gütersloh, Germany.


   Notes
 
1 Present address: Herz- und Diabeteszentrum NRW, Klinik der Ruhr-Universität Bochum, Erich und Hanna Klessmann-Institut, Georgstr. 11, 32545 Bad Oeynhausen, Germany. Back

Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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A. Koop, P. Goldmann, S. R. W. Chen, R. Thieleczek, and M. Varsanyi
ARVC-Related Mutations in Divergent Region 3 Alter Functional Properties of the Cardiac Ryanodine Receptor
Biophys. J., June 15, 2008; 94(12): 4668 - 4677.
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