Download Composite polymorphisms in the ryanodine receptor 2 gene

Cardiovascular Research 71 (2006) 496 – 505
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Composite polymorphisms in the ryanodine receptor 2 gene associated
with arrhythmogenic right ventricular cardiomyopathy
Hendrik Milting a,*, Nina Lukas b, Bärbel Klauke a, Reiner Körfer a, Andreas Perrot c,
Karl-Josef Osterziel c, Jürgen Vogt a, Stefan Peters d, Rolf Thieleczek b,1, Magdolna Varsányi b
a
c
Herz- und Diabeteszentrum NRW, Klinik der Ruhr-Universität Bochum, Erich und Hanna Klessmann-Institut
für Kardiovaskuläre Forschung und Entwicklung, Georgstr. 11, 32545 Bad Oeynhausen, Germany
b
Institut für Physiologische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany
Charité-Universitätsmedizin Berlin/Kardiologie am Campus Buch and Virchow-Klinikum and Max-Delbrück-Centrum für Molekulare Medizin, 13125
Berlin, Germany
d
Klinikum Quedlinburg, Innere Medizin, Abteilung Kardiologie, Ditfurter Weg 24, 06484 Quedlinburg, Germany
Received 10 November 2005; received in revised form 29 March 2006; accepted 6 April 2006
Available online 18 April 2006
Time for primary review 25 days
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.
D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
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.
* Corresponding author. Tel.: +49 5731 973510; fax: +49 5731 972476.
E-mail address: [email protected] (H. Milting).
1
Present address: Herz- und Diabeteszentrum NRW, Klinik der RuhrUniversität Bochum, Erich und Hanna Klessmann-Institut, Georgstr. 11,
32545 Bad Oeynhausen, Germany.
1. Introduction
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].
0008-6363/$ - see front matter D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2006.04.004
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Abstract
H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
2. Methods
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 PCRfragments 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. DNAsamples of wildtype-RyR2 were mixed with patients DNA
for the detection of homozygous sequence changes.
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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 h-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.
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H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
Divergent conformers were analysed by automated DNA
sequencing.
pH7.0, immediately frozen in liquid nitrogen, and stored
at 80 -C.
2.4. PCR amplification of the RYR2 exons
2.7. Solubilization of RyR2 from HSR
PCR amplification was performed on a GeneAmp PCR
System 9600 (Applied Biosystems) in a final volume of
50 Al 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 AM
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.
Human cardiac HSR vesicles were solubilized in twice
the volume of buffer containing 1 M NaCl, 40 mM NaHEPES, 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
120,000g 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.
2.5. DNA sequencing
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 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 AM Leupeptin,
1 AM Pepstatin, 1 AM Calpain I, 1 AM Calpain II, and 1
mM Benzamidine). Cell organelles were removed by
centrifugation at 3700g for 30 min. From the resulting
supernatant, crude microsomes were collected by centrifugation at 35,000g 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 AM CaCl2, and the above protease inhibitor cocktail.
After 1 h of incubation at 4 -C, the microsome fraction
was collected by centrifugation at 70,000g 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
100,000g. Fractions containing RyR2 were collected,
diluted in three volumes of microsome buffer, and
centrifuged at 100,000g for 1 h. The pellet was
resuspended in 0.3 M sucrose and 10 mM K-PIPES,
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 AM EGTA, 150 AM 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 timecourse 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 As).
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. P o values were calculated from representative data
segments with stable open probability (estimated from
plots of a moving average of P o against time, carried out
for all open levels if applicable) using the pClamp
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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.
H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
499
software. Free Ca2+-concentrations were calculated using
winmaxc32 [20].
3. Results
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) of the RYR2 gene
lead to the non-conservative amino acid exchange G1885E
and G1886S, respectively. Both polymorphisms were
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
N
Genotype abbreviation
Allele combinationb
RyR2 expressedc
ARVC
%
DCM
%
Blood donors
%
85
100
79
100
463
100
Wild type
WT
5654G/5656G
5654G/5656G
G1885/G1886
G1885/G1886
75
88.2
68
86.1
397
85.7
SNP(s)a
5656G > A
5654G > A
5654G > A
5654G > A
Homozygous
5656G > A
T1
5654G/5656G
5654G/5656A
G1885/G1886
G1885/G1886S
5
5.9
5
6.3
36
7.8
T2
5654G/5656G
5654A/5656G
G1885/G1886
G1885E/G1886
2
2.4
6
7.6
28
6.0
T3
5654A/5656G
5654A/5656G
G1885E/G1886
G1885E/G1886
0
0
0
0
2
0.4
T4
5654A/5656G
5654G/5656A
G1885E/G1886
G1885E/G1886S
3
3.5
0
0
0
0
The remaining homozygous allele combination 5654G/5656A, which would lead to the expression of solely RyR2 G1885/G1886S, was not detected in either
of these groups (see also Supplement). N is the total number of individuals in each group.
a
The nucleotide numbering given starts at the ATG start codon of the cDNA (Genbank accession no. X98330).
b
The nucleotides at both positions of interest on both alleles are shown. A changed nucleotide is indicated boldly.
c
Shown are the amino acids of the gene products of both alleles at both positions. An amino acid exchange is indicated boldly.
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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.
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H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
Table 2
Comparison of the amino acid changes identified in the present study with known respectively predicted RyR2 sequences
The last two sequences indicate the amino acid exchanges found in the present study. Identical amino acids are indicated by a horizontal dash.
*Predicted.
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.
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).
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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: Singlechannel 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 ( P o) are given on top
of each trace. In D, 3 AM ryanodine was added to the cis chamber. The holding potential was 71 mV and the free [Ca2+] of the trans chamber was 50 AM. 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 T 11 pS (mean
value T S.E.M.).
H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
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
501
of genotype T4 (Type 1 error of 0.004 according to the
two-sided Fisher’s exact test).
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).
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
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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 AM, 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 ( P o1 , . . . , P o4) are given on top of
the traces. The free [Ca2+] of the trans chamber was 50 AM. 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 s o1,. . .,s 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 T S.E.M.) of about 90 T 13 (substate 1, g), 190 T 24 (substate 2, ‚),
254 T 24 (substate 3, 3), and 376 T 53 pS (fully open state, >).
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H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
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 90T 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, P o2, P o3,
and P o4, whereas the one of lowest conductivity, P o1, 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 P o2 and P o3 while stabilizing P o1
and enhancing P o4 (Fig. 3E). Further addition of 3 AM
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%.
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 3 except that
ATP is stabilizing the open probability of all conductance
Fig. 4. Single-channel open probabilities P o 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 P o = 0.57) of the open conductance state 1 (¨90 pS) at
diastolic free Ca2+ (pCa7.7) and the markedly different character of P o of the two channel types at systolic free Ca2+ (pCa4.3). The bars represent mean
values T 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.
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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 T 11 pS (Fig. 2G). The channel was blocked irreversibly
in a subconductance state of about 85 pS upon the addition of
3 AM ryanodine to the cytoplasmic side (Fig. 2D).
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
H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
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 ( P o = 0.57 T 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+ ( P o = 0.012 T 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.
4. Discussion
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 ( P o =
0.57 T 0.17) is almost 50-times higher than the one of the wild
type RyR2 channel ( P o = 0.012 T 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
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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 RyR2subunit. In heart failure, neurohumoral stimulation of
several signalling pathways which are merging downstream
at PKC is activated chronically. The predominant myocardial PKC isoform, PKC-a, 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.
503
504
H. Milting et al. / Cardiovascular Research 71 (2006) 496 – 505
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.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.cardiores.2006.04.004.
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