Circulation: Arrhythmia and Electrophysiology

ORIGINAL ARTICLE

Human RyR2 (Ryanodine Receptor 2) Loss-of-

Function Mutations
Clinical Phenotypes and In Vitro Characterization
Yanhui Li , MD, PhD; Jinhong Wei , PhD; Wenting Guo , PhD; Bo Sun , PhD; John Paul Estillore, MD;
Ruiwu Wang, PhD; Ayhan Yoruk, MD, MPH; Thomas M. Roston , MD, PhD; Shubhayan Sanatani , MD*;
Arthur A.M. Wilde , MD, PhD*; Michael H. Gollob , MD*; Jason D. Roberts , MD, MAS*; Zian H. Tseng, MD*;
Henrik K. Jensen , MD, DMSc*; S.R. Wayne Chen , PhD

BACKGROUND: The overall objective of the present study is to extend our understanding of the clinical phenotype and underlying
mechanism of a newly discovered cardiac arrhythmia syndrome through a multicenter study. Gain-of-function mutations in
the cardiac Ca2+ release channel (RyR2 [ryanodine receptor 2]) cause catecholaminergic polymorphic ventricular tachycardia,
whereas loss-of-function RyR2 mutations are linked to a new cardiac arrhythmia disorder termed Ca2+-release deficiency
syndrome (CRDS). Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmia disorder characterized
by stress-induced bidirectional and polymorphic ventricular tachyarrhythmias and is routinely diagnosed by using exercise
stress testing. Conversely, RyR2-CRDS is characterized by ventricular arrhythmias and sudden cardiac death but a negative
exercise stress testing for catecholaminergic polymorphic ventricular tachycardia. There are currently no clinical diagnostic
tests for CRDS and affected patients may manifest with sudden cardiac death as their first symptom. In the absence of
effective clinical diagnostic tools, in vitro functional characterization of associated RyR2 mutations provides an alternative
means to identify potential cases of CRDS.
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METHODS: We searched for patients presenting with phenotypes compatible with CRDS that have RyR2 mutations and
performed in vitro functional characterization.

RESULTS: We found that 3 novel (G570D, R4147K, and A4203V) and 2 previously reported (M4109R and A4204V) RyR2
mutations associated with CRDS phenotypes markedly reduced caffeine-induced Ca2+ release and store overload-induced
Ca2+ release. We also characterized 2 additional loss-of-function RyR2 mutations previously reported (Q3925E and L4769S)
that are located in the central and channel pore-forming domains critical for Ca2+ activation and channel gating. Q3925E
was identified through postmortem genetic testing in an individual who died suddenly, while L4769S is a variant of uncertain
significance reported in ClinVar, suggesting that RyR2 CRDS may be under detected.
CONCLUSIONS: These findings provide further support for the existence of an emerging RyR2 loss-of-function associated
arrhythmia syndrome (CRDS) and shed new insights into the disease mechanism.

GRAPHIC ABSTRACT: An online graphic abstract is available for this article.

Key Words: caffeine ◼ death ◼ mutation ◼ phenotype ◼ ryanodine

T
he cardiac Ca2+ release channel (RyR2 [ryanodine in cardiac muscle.1,2 Mutations in the RYR2 gene are linked
receptor type 2]) mediates sarcoplasmic reticulum to catecholaminergic polymorphic ventricular tachycardia
Ca2+ release that is essential for excitation-contraction (CPVT), an inherited life-threatening arrhythmia disorder

Correspondence to: S.R. Wayne Chen, PhD, Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, 3330 Hospital Drive
N.W., Calgary, Alberta, T2N 4N1, Canada. Email swchen@ucalgary.ca *Additional corresponding authors are listed in an Appendix at the end of this article.
The Data Supplement is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCEP.121.010013.
For Sources of Funding and Disclosures, see page 884.
© 2021 American Heart Association, Inc.
Circulation: Arrhythmia and Electrophysiology is available at www.ahajournals.org/journal/circep

Circ Arrhythm Electrophysiol. 2021;14:e010013. DOI: 10.1161/CIRCEP.121.010013 September 2021 874

 Li et al
WHAT IS KNOWN?
• Cardiac RyR2 (ryanodine receptor 2) gain-of-func-
tion mutations cause catecholaminergic polymor-
phic ventricular tachycardia, which is diagnosed
based on exercise stress testing showing bidirec-
tional or polymorphic ventricular tachyarrhythmias.
• Loss-of-function RyR2 mutations lead to a recently
described cardiac arrhythmia syndrome, termed
RyR2 calcium release deficiency syndrome, char-
acterized by ventricular arrhythmias and sudden
cardiac death, but an exercise stress testing not
diagnostic for typical catecholaminergic polymor-
phic ventricular tachycardia.
WHAT THE STUDY ADDS
• An international multicenter collaboration identi-
fied and characterized 5 RyR2 mutations associ-
ated with calcium release deficiency syndrome
phenotypes and 2 RyR2 mutations of uncertain
significance, all of which suppress store overload-
induced calcium release.
• In the absence of effective clinical diagnostic tests
for calcium release deficiency syndrome, in vitro
functional characterization may provide an alterna-
tive means to identify potential cases of calcium
release deficiency syndrome.
• These clinical and in vitro functional analyses fur-
ther support the existence of an emerging RyR2
calcium release deficiency syndrome and shed new
insights into the causal mechanism of the disease.
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Nonstandard Abbreviations and Acronyms
CPVT catecholaminergic polymorphic ventricu-
lar tachycardia
CRDS Ca2+ release deficiency syndrome
EST exercise stress testing
GOF gain-of-function
ICD implantable cardioverter-defibrillator
LOF loss-of-function
RyR2 ryanodine receptor 2
SCD sudden cardiac death
VF ventricular fibrillation
characterized by syncope, malignant ventricular arrhyth-
mias, and sudden cardiac death (SCD).3–7 The hallmark of
CPVT is emotional or physical stress-induced bidirectional
and polymorphic ventricular tachycardias in the absence
of structural heart disease. This hallmark of CPVT can be
reproduced on exercise stress testing (EST). As a result,
EST is commonly used as a diagnostic tool for CPVT.3–10
In vitro functional characterization of pathogenic RyR2
mutations have established that CPVT is associated with
aberrant RyR2 gain-of-function (GOF).6 It is generally
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RyR2 LOF and Sudden Cardiac Death
believed that RyR2 GOF mutations increase the propen-
sity for spontaneous sarcoplasmic reticulum Ca2+ release
and delayed afterdepolarizations that can lead to trig-
gered activity and malignant ventricular arrhythmias.6
In addition to the typical CPVT phenotype, naturally
occurring mutations in RyR2 are also associated with phe-
notypes distinct from CPVT. For instance, patients harboring
the RyR2-A4860G mutation presented with idiopathic ven-
tricular fibrillation (VF) and SCD but with normal EST.11 Fur-
thermore, patients harboring the RyR2 mutations K4594R/
I2075T,12 S4938F,13 and I4855M14 also displayed pheno-
types distinct from those of CPVT. Interestingly, functional
studies demonstrated that each of these atypical CPVT-
associated RyR2 mutations (A4860G, K4594R/I2075T,
S4938F, and I4855M) are loss-of-function (LOF),13–16
contrasting to the GOF RyR2 mutations that are associ-
ated with typical CPVT. These findings raise an intriguing
and important possibility that RyR2 LOF mutations may
be linked to a unique malignant cardiac arrhythmia disor-
der distinct from CPVT. Indeed, a recent systematic clinical,
genetic, and functional study provided compelling evidence
for a link between RyR2 LOF mutations and a novel disease
entity that we have termed RyR2 Ca2+ release deficiency
syndrome (CRDS).16 Unlike typical CPVT, RyR2-CRDS
shows negative EST for CPVT. Furthermore, the underlying
pathophysiology of RyR2-CRDS also differs from that of
CPVT. Notably, RyR2 LOF mutations protect against spon-
taneous sarcoplasmic reticulum Ca2+ release and delayed
afterdepolarizations but promote Ca2+ alternans, early after-
depolarizations and reentrant arrhythmias.16 There is cur-
rently no clinical diagnostic test to distinguish patients with
RyR2-CRDS from normal individuals.
At present, the only method to ascertain the potential
presence of CRDS is through in vitro functional evaluation
of the RyR2 variant. To this end, we searched for patients
presenting with phenotypes compatible with CRDS that
possess RyR2 mutations and performed in vitro func-
tional characterization. We identified and demonstrated 3
novel and 2 previously reported RyR2 mutations associ-
ated with CRDS phenotypes to be LOF mutations. We
also characterized 2 additional LOF RyR2 mutations pre-
viously reported that are located in structurally important
domains. Thus, our data add to the growing evidence that
RyR2 LOF is associated with a new malignant cardiac
arrhythmia disorder distinct from CPVT.
MATERIALS AND METHODS
The data that support the findings of this study and the meth-
ods and materials used in this study are available from the cor-
responding authors upon reasonable request.
Construction of RyR2 Mutations
All RyR2 point mutations were generated by using the over-
lap extension polymerase chain reaction method and the full-
length mouse RyR2 cDNA as described previously.17
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Caffeine-Induced Ca2+ Release Measurements
in HEK293 Cells
Free cytosolic Ca2+ concentration in transfected HEK293 cells
was measured using the fluorescence Ca2+ indicator dye fluo-
3-AM as described previously.17
Single-Cell Cytosolic Ca2+ Imaging of HEK293
Cells
Cytosolic Ca2+ levels in stable, inducible HEK293 cells express-
ing RyR2 WT or mutant channels were monitored using single-
cell Ca2+ imaging and the fluorescent Ca2+ indicator dye Fura-2
acetoxymethyl ester as described previously.18
Single-Cell Luminal Ca2+ Imaging of HEK293
Cells
Luminal Ca2+ concentrations in HEK293 cells expressing RyR2
WT or mutants were measured using single-cell Ca2+ imaging
and the fluorescence resonance energy transfer-based ER
luminal Ca2+-sensitive chameleon protein D1ER as described
previously.19,20
Statistical Analysis
All values shown are mean±SEM unless indicated otherwise.
To test for differences between groups, we used 1-way ANOVA
followed by Dunnett multiple comparisons test or Student t test
(2 tailed) using GraphPad Prism version 8. A P<0.05 was con-
sidered to be statistically significant.
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Human Studies
The human aspects of the study were performed as part of
protocols approved by the research ethics boards of the partici-
pating institutions. All subjects gave informed consent.
RESULTS
Identification of RyR2 Mutations Associated
With CRDS Phenotypes
To further uncover potential RyR2 LOF mutations, we
searched for RyR2 mutant carriers who displayed CRDS
phenotypes, including ventricular arrhythmias, SCD, and
aborted sudden cardiac arrest with negative EST for
CPVT. We identified 3 novel RyR2 mutations G570D,
R4147K, and A4203V. These mutations are located in
the N-terminal disease-mutation cluster 1 and near the
C-terminal disease-mutation cluster 3 in the 3-dimen-
sional structure of RyR2 (Figure 1). The clinical manifes-
tations of patients harboring these RyR2 mutations are
summarized as follows.
G570D
The proband harboring the RyR2-G570D mutation had a
cardiac arrest at the age of 34 years old while standing in
the kitchen and was successfully resuscitated with defi-
brillation shocks by emergency responders. Clinical eval-
uation postarrest was entirely normal, including cardiac
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RyR2 LOF and Sudden Cardiac Death
imaging and ECG, and the event remained unexplained.
The patient has an implantable cardioverter-defibrillator
(ICD) and had some short runs of polymorphic VT and
2 appropriate ICD shocks. Two exercise stress tests
showed no PVCs during exercise, but PVCs during
recovery, which may be seen but is not typical in CPVT
patients. The patient’s father (age 72 years) and mother
(age 66 years) are both alive, and his paternal grandfa-
ther apparently died suddenly at the age of 40 years old.
The patient has 2 healthy sisters and 2 healthy children.
Whole-exome sequencing revealed the RyR2-G570D
mutation in the proband and no other suspicious vari-
ants in genes known to cause genetic-based inherited
arrhythmias or cardiomyopathies.
R4147K
The proband harboring the RyR2-R4147K variant is a
19-year-old girl who had an aborted cardiac arrest when
she was 14 years old (Figure 2). The parents found her
with cardiac arrest and had not observed her for the last
10 to 15 minutes. Paramedics identified VF and after
2 attempts at defibrillation, sinus rhythm was restored.
Subsequently, 12-lead ECG, echocardiography, and cor-
onary computed tomography scan were found to be nor-
mal. Due to anoxic brain damage, it was not possible to
perform EST and medical treatment with β-blocker was
omitted initially. An ICD was implanted. Four years later
in relation to very light rehabilitation exercise she had a
very short syncope with documented VF and a subse-
quent shock by the ICD converted her to sinus rhythm.
The electrogram recorded by the ICD shows a period of
sinus tachycardia, followed by a short run of ventricular
tachycardia, a long-pause and a short-coupled ventricu-
lar beat before the onset of VF. This pattern of initiation
of VF is similar to the programed electrical stimulation
protocol consisting of a long-burst, a long-pause, and
a short-coupled ventricular extra-stimuli (LBLPS) used
to induce ventricular tachycardia/VF in a RyR2-CRDS
mouse model and patients as described previously.16
Medical treatment with metoprolol 50 mg once a day
was initiated. Other carriers of the RyR2-R4147K variant
in the family (mother 47 years, sister 23 years, mater-
nal uncle 50 years, and maternal grandmother 69 years
old) are still asymptomatic. These relatives have normal
ECGs, echocardiography, and exercise stress tests.
A4203V
A 25-year-old woman without cardiac history had a
witnessed seizure and paramedics discovered VF on
arrival. She had a past medical history of seizures and
depression treated with levetiracetam and citalopram,
respectively, but no concerning family history. Follow-
ing defibrillation to sinus rhythm, her 12-lead ECG was
normal. Echocardiography revealed a structurally nor-
mal heart and treadmill testing performed to screen for
coronary disease was normal without ventricular ectopy;
QT length and hysteresis were also normal. She was
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Figure 1. Identification and location of RyR2 (ryanodine receptor 2) loss-of-function (LOF) mutations in the 3-dimensional
structure of RyR2.
A, A schematic diagram of the linear sequence of RyR2. Major structural domains of RyR2 are depicted as solid blue boxes. The orange boxes
indicate 4 disease-associated mutation clusters (mutation hotspots) in RyR2. B, Locations of RyR2 LOF mutations in the 3-dimensional (3D)
structure of RyR2 (PDB: 6JI0).

diagnosed with idiopathic VF and a single-chamber ICD
was implanted; no cardiac medications were initiated.
Genetic testing (113 gene panel) identified an RyR2-p.
Ala4203Val variant of unknown significance. Over the
subsequent 17 years, she had 2 episodes of VF. ICD
interrogation for the second episode revealed sinus
tachycardia (133 beats per minute) and a PVC resulting
in a long-short initiation of VF (Figure 2). She has since
been treated with flecainide without further arrhythmia or
seizure recurrences. We suspect her seizures were due
to cerebral hypoperfusion resulting from VF.
We also identified 2 previously reported RyR2 muta-
tions M4109R and A4204V that are associated with
CRDS phenotypes. These mutations are also located
in disease-mutation cluster 3 (Figure 1). The RyR2-
M4109R mutation was reported by Nof et al21 in a
31-year-old female who had aborted cardiac arrest
due to VF while she was at work early in the morning.
The proband’s father and brother both died suddenly.
Exercise-induced ventricular arrhythmias were not doc-
umented in all tested patients with the RyR2-M4109R
mutation. The RyR2-A4204V mutation was reported by
Kron et al22 in a 16-year-old girl who had aborted cardiac
arrest while running to the school bus. The patient had no
history of syncope, dizziness, palpitations, or chest pain.
ECG showed normal left ventricular function, and there
was no evidence of hypertrophic cardiomyopathy. EST
was normal.
Effect of RyR2 Mutations on Caffeine-Induced
Ca2+ Release in HEK293 Cells
We monitored intracellular Ca2+ release triggered by
sequential additions of increasing concentrations of caf-
feine. As shown in Figure 3, Ca2+ release in HEK293
cells expressing RyR2 WT was activated by caffeine with
an EC50 of 0.19 mmol/L. Increasing caffeine concentra-
tion from 0.05 to 1.0 mmol/L progressively increased the
level of Ca2+ release for each consecutive addition of caf-
feine. Further additions of caffeine from 2.5 to 5 mmol/L
decreased the level of Ca2+ release. This decreased caf-
feine-induced Ca2+ release is likely due to the depletion

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Figure 2. Family pedigrees and ECG/electrogram (EGM) recordings.

A, Pedigree of the family carrying the RyR2 (ryanodine receptor 2)-R4147K+/− mutation. Squares and circles indicate males and females,
respectively; blue symbols represent mutation carriers/obligate carriers; open symbols represent mutation negative/unaffected persons;
symbols with a slash represent deceased persons. Probands are indicated by an arrow. B, Surface ECG lead and implantable cardioverter-
defibrillator (ICD) near- and far-field ventricular electrogram tracings showing a long-short initiation of ventricular arrhythmia in a RyR2-
R4147K+/− mutant carrier. C, ICD near-field and far-field ventricular electrograms showing a long-short initiation of polymorphic ventricular
tachycardia/ventricular fibrillation in a RyR2-A4203V+/− mutant carrier. The numbers shown at the bottom indicate intervals in ms. aSCA
indicates aborted SCA; PVC, premature ventricular contraction; SCA, sudden cardiac arrest; and VT, ventricular tachycardia.

of intracellular Ca2+ stores by previously added caffeine
(0.025–1.0 mmol/L; Figure 3A). The caffeine response
of HEK293 cells expressing the novel G570D, R4147K,
or A4203V mutations was shifted to the right compared
with that of WT cells with an EC50 of caffeine activa-
tion of 0.36, 0.39, and 0.41 mmol/L, respectively (Fig-
ure 3B). Thus, the G570D, R4147K, or A4203V mutation
significantly reduced the caffeine activation of RyR2.
Similarly, we found that the previously reported M4109R
and A4204V mutations also suppressed caffeine activa-
tion of RyR2 with an EC50 of 0.76 and 0.59 mmol/L,
respectively, all of which are significantly greater than
that of the WT (Figure 3A through 3C).
The Q3925 residue, known to be critical for Ca2+
activation of RyR2,23–25 was found through postmortem
genetic testing to be mutated to glutamate (Q3925E) in

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Figure 3. Effect of RyR2 (ryanodine receptor 2) mutations on caffeine-induced Ca2+ release in HEK293 cells.
HEK293 cells were transfected with RyR2 wild type (WT; 1) and mutants G570D (2), Q3925E (3), M4109R (4), R4147K (5), A4203V (6),
A4204V (7), and L4769S (8; A). Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after
each caffeine addition. The numbers (under the traces) indicate caffeine concentrations (mmol/L). B, Cumulative caffeine concentration–Ca2+
release relationships in HEK293 cells transfected with RyR2 WT and mutants. C, The apparent EC50 (mmol/L) values of caffeine-induced
Ca2+ releases in HEK293 cells transfected with RyR2 WT or mutants. Data shown are mean±SEM (n=5; 1-way ANOVA with Dunnett multiple
comparisons post hoc test, * P<0.05 vs WT).

an individual who died suddenly.5 This mutation would be
predicted to impair channel activation. Indeed, we found
that Q3925E dramatically reduced caffeine activation of
RyR2 with an EC50 of 3.95 mmol/L (Figure 3). Thus,
all 5 RyR2 mutations associated with CRDS phenotypes
and one sudden unexplained death-associated RyR2
mutation located in the Ca2+ activation site impaired
channel activation (LOF).
Effects of RyR2 Mutations on Store Overload-
Induced Ca2+ Release in HEK293 Cells
A common defect of CPVT RyR2 mutations is enhanced
susceptibility to store overload-induced Ca2+ release
(SOICR).6,18 To assess the effect of RyR2 mutations
associated with CRDS phenotypes on SOICR, we
measured spontaneous Ca2+ oscillations in HEK293
cells expressing RyR2 WT and the mutations G570D,
Q3925E, M4109R, R4147K, A4203V, and A4204V. We
perfused the cells with elevating extracellular Ca2+ (0–2.0
mmol/L) to induce SOICR and monitored SOICR activ-
ity using a fluorescence Ca2+ indicator, Fura-2 acetoxy-
methyl ester, and single-cell Ca2+ imaging as described
previously.18 As shown in Figure 4, the G570D, R4147K,
A4203V, and A4204V mutations markedly suppressed
SOICR, while the M4109R mutation nearly abolished
SOICR in HEK293 cells. The Q3925E mutation com-
pletely abolished SOICR (Figure 4). Thus, opposite to the
effect of CPVT RyR2 GOF mutations, these RyR2 muta-
tions suppressed or abolished SOICR.

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Figure 4. Effects of RyR2 (ryanodine receptor 2) mutations on store overload-induced Ca2+ release (SOICR) in HEK293 cells.
Stable, inducible HEK293 cells expressing RyR2 wild type (WT; A) or mutants G570D (B), Q3925E (C), M4109R (D), R4147K (E), A4203V
(F), A4204V (G), and L4769S (H) were loaded with 5 μmol/L Fura-2 acetoxymethyl ester (Fura-2 AM) in KRH buffer. The cells were then
perfused continuously with KRH buffer containing increasing levels of extracellular Ca2+ (0–2 mmol/L) to induce SOICR. Fura-2 ratios of
representative RyR2 WT and mutant cells were recorded using single-cell Ca2+ imaging. I, The percentages of RyR2 WT and mutant cells
that display Ca2+ oscillations at various extracellular Ca2+ concentrations. Note that no SOICR was detected in HEK293 cells expressing the
Q3925E mutant. Data shown are mean±SEM (n=5).

Effects of RyR2 Mutations on the Activation and
Termination of Spontaneous Ca2+ Release in
HEK293 Cells
We next determined the impact of these mutations on the
activation and termination thresholds for SOICR. To be
able to measure the thresholds for SOICR activation and
termination, we promoted spontaneous ER luminal Ca2+
oscillations in HEK293 cells expressing RyR2 WT and
mutants by perfusing the cells with 1 mmol/L caffeine
and increasing concentrations (from 0 to 2 mmol/L) of
extracellular Ca2+. Note that elevating extracellular Ca2+
alone (without 1 mmol/L caffeine) was insufficient to
induce Ca2+ oscillations in these RyR2 mutant-expressing

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 Li et al RyR2 LOF and Sudden Cardiac Death

cells. We used single-cell Ca2+ imaging to monitor the
ER luminal Ca2+ dynamics in HEK293 cells using the
D1ER-based fluorescence resonance energy trans-
fer imaging.19,20 As shown in Figure 5, HEK293 cells
expressing RyR2 WT displayed spontaneous ER Ca2+
oscillations (shown as downward deflections of the fluo-
rescence resonance energy transfer signal) upon eleva-
tion of extracellular Ca2+ from 0 to 1 and 2 mmol/L. The
profile of these ER Ca2+ oscillations could be described
by a threshold level (FSOICR) at which SOICR occurs when
the ER luminal Ca2+ content increases, and another
threshold level (Ftermi) at which SOICR terminates when
the ER luminal Ca2+ content decreases. Under these
conditions, RyR2-WT expressing HEK293 cells exhib-
ited a SOICR activation threshold of 70%, a termination
threshold of 37%, and a fractional Ca2+ release (activa-
tion threshold−termination threshold) of 33% (Figure 6).
Under the same conditions, HEK293 cells expressing
the G570D, R4147K, A4203V, and A4204V mutations
significantly increased the SOICR activation threshold
with unchanged termination threshold. As a result, the
fractional Ca2+ release (activation threshold−termination
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threshold) during SOICR was also significantly increased
in these mutant-expressing cells (Figures 5 and 6). How-
ever, these mutations had no significant impact on the
store capacity (Fmax−Fmin; Figures 5 and 6). Note that no
luminal Ca2+ oscillations were detected in the HEK293
cells expressing mutations Q3925E and M4109R even
in the presence of 1 mmol/L caffeine. Importantly,
SOICR did not occur in control HEK293 cells express-
ing no RyR2 and that SOICR was not affected by the
IP3R inhibitor, xestospongin C,26 indicating that SOICR
is mediated by RyR2. Collectively, these data indicate
that RyR2 mutations associated with CRDS phenotypes
suppressed SOCIR by increasing the SOICR activation
threshold.
The RyR2-L4769S Variant Located in the S5
Outer Helix Is a LOF Mutation
The channel pore-forming domain is critical for chan-
nel gating (Figure 1). Mutations in this domain are likely
to impair channel function. Indeed, the S6 inner helix is
a hotspot for RyR2 LOF mutations, including I4855M,

Figure 5. Effect of RyR2 (ryanodine receptor 2) mutations on store overload-induced Ca2+ release (SOICR) activation and
termination.
Stable, inducible HEK293 cell lines expressing RyR2 wild type (WT; A), G570D (B), R4147K (C), A4203V (D), A4204V (E), and L4769S
(F) were transfected with the fluorescence resonance energy transfer (FRET)-based ER luminal Ca2+-sensing protein D1ER for 48 h. The
cells were perfused with KRH buffer containing 1 mmol/L caffeine and increasing levels of extracellular Ca2+ (0–2 mmol/L) to induce SOICR.
This was followed by the addition of 1 mmol/L tetracaine to inhibit SOICR and then 20 mmol/L caffeine to deplete the ER Ca2+ store. FRET
recordings from representative RyR2 WT and mutant cells (a total of 36–76 cells each) are shown.

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Figure 6. RyR2 (Ryanodine receptor 2) loss-of-function (LOF) mutations increase the thresholds for store overload-induced Ca2+
release (SOICR) activation and the fractional Ca2+ release.
To minimize the influence by CFP/YFP cross-talk, we used relative fluorescence resonance energy transfer (FRET) measurements for
calculating the activation threshold (A) and termination threshold (B). The activation threshold was determined by the equation ([FSOICR−Fmin]/
[Fmax−Fmin])×100%, and the termination threshold was determined by the equation ([Ftermi−Fmin]/[Fmax−Fmin])×100%. The fractional Ca2+ release
(C) was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal Fmax is defined as the
FRET level after tetracaine treatment. The minimum FRET signal Fmin is defined as the FRET level after caffeine treatment. The store capacity
(D) was calculated by subtracting Fmin from Fmax. Data shown are mean±SEM (n=8–10; 1-way ANOVA with Dunnett multiple comparisons
post hoc test, *P<0.05 vs wild type [WT]).

A4860G, and Q4879H.16 However, it is unclear whether
mutations in the S5 outer helix could also lead to LOF
phenotypes. To address this question, we identified and
characterized an RyR2 mutation L4769S in ClinVar
(National Center for Biotechnology Information Clin-
Var; [VCV000392074.2], https://www.ncbi.nlm.nih.gov/
clinvar/variation/VCV000392074.2). We found that the
L4769S mutation markedly suppressed caffeine activa-
tion (Figure 3), inhibited SOICR activity (Figure 4), and
increased the SOICR activation threshold (Figures 5 and
6). This finding suggests that more RyR2 LOF mutations
most likely exist, but their disease association has yet to
be determined.
Expression of RyR2 LOF Mutations in HEK293
Cells
To assess whether RyR2 LOF mutations alter the expres-
sion of the RyR2 protein, we performed immunoblotting
analysis of cell lysate from HEK293 cells transfected

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 Li et al
with RyR2-WT and the G570D, Q3925E, M4109R,
R4147K, A4203V, A4204V, and L4769S mutants. As
shown in Figure 7, the levels of protein expression of
these RyR2 mutants in HEK293 cells were compatible
to that of the WT.
DISCUSSION
We have recently shown that LOF RyR2 mutations are
linked to a new inherited arrhythmia disorder, termed
RyR2-Ca2+ release deficiency syndrome (CRDS).16
Unlike CPVT, which is associated with GOF RyR2
mutations and is characterized by emotional or physi-
cal stress-induced bidirectional and polymorphic ven-
tricular tachycardias,3–7 RyR2-CRDS is characterized
by ventricular arrhythmias and SCD but a negative
CPVT. Notably, EST is routinely used to diagnose
CPVT, but it is ineffective for CRDS.16 CRDS patients
typically present with normal clinical testing. There
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Circ Arrhythm Electrophysiol. 2021;14:e010013. DOI: 10.1161/CIRCEP.121.010013
RyR2 LOF and Sudden Cardiac Death
is currently a lack of an effective clinical diagnostic
tool for RyR2-CRDS. This is particularly problematic
when an RYR2 variant of unknown significance is
identified in survivors of unexplained cardiac arrest
because it can be challenging to clarify its relevance
on clinical grounds alone. Therefore, it is important
to perform in vitro functional characterization of the
associated RyR2 variants to facilitate the diagnosis
of RyR2-CRDS. In the present study, we sought to
further investigate the link between RyR2 LOF and
cardiac arrhythmias through identification of patients
with CRDS phenotypes that were found to possess
a RyR2 mutation. We identified 3 novel and 2 previ-
ously reported RyR2 mutations associated with CRDS
phenotypes and demonstrated these to be LOF on in
vitro functional analysis. The clinical features of these
patients provide additional insight into the phenotypic
spectrum associated with RyR2 LOF and further high-
light the distinction between CPVT and CRDS.
Figure 7. Effect of RyR2 (ryanodine
receptor 2) loss-of-function (LOF)
mutations on protein expression in
HEK293 cells.
HEK293 cells were transfected
with RyR2 wild type (WT) and LOF
mutants. Cell lysates were prepared
from these transfected cells and used
for immunoblotting analysis (A). The
same amount of cell lysate was used
for immunoblotting using the anti-
RyR2 antibody. B, Quantification of the
expression of RyR2 WT and mutants.
Dashed lines indicate where 2 parts of
the same immunoblot were joined. The
expression level of each RyR2 mutant
was normalized to that of the WT in
each experiment and data shown are
mean±SEM from 5 separate experiments
(1-way ANOVA with Dunnett multiple
comparisons post hoc test).
September 2021 883
 Li et al
We have also characterized 2 additional RyR2 LOF
mutations previously reported (Q3925E and L4769S)
given their important structural localizations in the
3-dimensional structure of RyR2, although detailed clini-
cal phenotypes associated with these mutations are not
available. The RyR2 Q3925E mutation was uncovered
through postmortem genetic testing in an individual who
had unexplained sudden death.5 This Q3925 is one of
the residues involved in coordinating Ca2+ binding for the
activation of RyR2 by cytosolic Ca2+. As expected, the
Q3925E mutation dramatically reduced RyR2 channel
activation. Thus, although the sudden death of the RyR2
Q3925E mutant carrier is unexplained, it is conceivable
that the Q3925E LOF mutant carrier may have suffered
from RyR2-CRDS.
The RyR2-L4769S variant of uncertain significance
was reported in ClinVar (VCV000392074.2; https://www.
ncbi.nlm.nih.gov/clinvar/ variation/VCV000392074.2).
The clinical phenotype of the RyR2 L4769S mutant car-
rier is not available. This variant was not detected in the
Genome Aggregation Database (gnomAD),27 indicat-
ing that it is not a common benign variant. The L4769S
mutation is located in the S5 outer helix important for
channel gating. Indeed, we found that the L4769S muta-
tion substantially inhibited RyR2 channel activation. Our
data suggest that the RyR2-L4769S mutation could
potentially contribute to a RyR2-CRDS phenotype.
Downloaded from http://ahajournals.org by on August 6, 2023
Study Limitations
We have recently shown that a CRDS associated RyR2
LOF mutation D4646A abolishes SOICR but increases
the propensity for early afterdepolarizations and reen-
trant activities in a mouse model due, in part, to the com-
plex cardiac remodeling, including increased L-type Ca2+
current, prolonged action potential duration, and elevated
sarcoplasmic reticulum Ca2+ content. The present study
focused on the identification of patients with phenotypes
compatible with CRDS and in vitro characterization of
the impact of associated RyR2 mutations on the intrinsic
properties of the RyR2 channel. However, it remains to
be determined whether the RyR2 LOF mutations identi-
fied here also increase the propensity for early afterdepo-
larizations and reentrant activities. Generation of mouse
models expressing each of these RyR2 LOF mutations
would be needed to address this question.
In summary, here we identified and characterized 6
RyR2 LOF mutations that are associated with ventricu-
lar arrhythmias, SCD, and sudden unexplained death.
These data further support the link between RyR2 LOF
and CRDS. Our findings also suggest that RyR2 LOF
mutations may be under detected, as there are currently
no effective clinical diagnostic tests for RyR2-CRDS,
and as RyR2 LOF mutant carriers could remain asymp-
tomatic until later into adulthood due likely to incom-
plete penetrance. Effective diagnosis tools are urgently
Circ Arrhythm Electrophysiol. 2021;14:e010013. DOI: 10.1161/CIRCEP.121.010013
RyR2 LOF and Sudden Cardiac Death
needed to detect and manage patients with CRDS and
their potentially vulnerable family members before they
suffer lethal events.
ARTICLE INFORMATION
Received March 23, 2021; accepted August 17, 2021.
Affiliations
Department of Physiology and Pharmacology, Libin Cardiovascular Institute, Uni-
versity of Calgary, AB, Canada (Y.L., J.W., W.G., B.S., J.P.E., R.W., S.R.W.C.). Depart-
ment of Internal Medicine, Institute of Hypertension, Tongji Hospital, Tongji Medi-
cal College, Huazhong University of Science and Technology, Wuhan, China (Y.L.).
Medical School, Kunming University of Science and Technology, China (B.S.). Car-
diac Electrophysiology Section, Division of Cardiology, Department of Medicine,
University of California, San Francisco (A.Y., Z.H.T.). Division of Cardiology, Depart-
ment of Medicine (T.M.R.) and Child and Family Research Institute, Department of
Pediatrics (S.S.), University of British Columbia, Vancouver, Canada. Heart Center,
Department of Clinical and Experimental Cardiology, Amsterdam University Medi-
cal Centre, location AMC, the Netherlands (A.A.M.W.). Member of the European
Reference Network ‘ERN GUARD-Heart’ (A.A.M.W.). Inherited Arrhythmia and
Cardiomyopathy Program, Arrhythmia Service, Division of Cardiology, Toronto
General Hospital (M.H.G.) and Department of Physiology (M.H.G.), University of
Toronto, ON, Canada. Population Health Research Institute, McMaster Univer-
sity and Hamilton Health Sciences, Hamilton, ON, Canada (J.D.R.). Department
of Cardiology, Aarhus University Hospital and Department of Clinical Medicine,
Health, Aarhus University, Denmark (H.K.J.).
Acknowledgments
Dr Wei is the recipient of the Libin Cardiovascular Institute and Cumming School
of Medicine Postdoctoral Fellowship Award. Dr Guo is the recipient of the Alberta
Innovates-Health Solutions (AIHS) Studentship Award. Dr Sun is the recipient of
the AIHS Fellowship Award. Dr Roston is supported by the University of British
Columbia Clinician Investigator Program, and a UBC Friedman Scholar in Health
and CHRS George Mines Travelling Fellow in Cardiac Electrophysiology. Dr Chen
is the Heart and Stroke Foundation Chair in Cardiovascular Research.
Appendix
Additional corresponding authors contributed to this article: Shubhayan Sanatani,
ssanatani@cw.bc.ca; Arthur A.M. Wilde, a.a.wilde@amsterdamumc.nl; Michael
Gollob, michael.gollob@uhn.ca; Jason D. Roberts, jason.roberts@phri.ca; Zian H.
Tseng, Zian.Tseng@ucsf.edu; and Henrik K. Jensen, hkjensen@clin.au.dk.
Sources of Funding
This work was supported by research grants from the Canadian Institutes of
Health Research (PJT-155940), the Heart and Stroke Foundation of Canada
(G-19-0026444), and the Heart and Stroke Foundation Chair in Cardiovascular
Research (END611955) to Dr Chen; the Novo Nordisk Foundation, Denmark
(NNF18OC0031258) to Dr Jensen; the Canadian Institutes of Health Research
(408226) to Dr Gollob, the Royal Netherlands Academy of Sciences (PRE-
DICT2) to Dr Wilde; the E-Rare Joint Transnational Call for Proposals 2015 Im-
proving Diagnosis and Treatment of Catecholaminergic Polymorphic Ventricular
Tachycardia: Integrating Clinical and Basic Science to Drs Chen, Sanatani, and
Wilde; and the National Institutes of Health (NIH/NHLBI HL R01 102090, HL
R01 126555, and HL R01 147035) and Centers for Disease Control and Pre-
vention (CDC DP14-1403) to ZHT.
Disclosures
None.
Supplemental Materials
Supplemental Methods
References 28–32
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