Circulation Journal

Official Journal of the Japanese Circulation Society

ORIGINAL  ARTICLE
http://www. j-circ.or.jp Arrhythmia/Electrophysiology

Dantrolene, a Therapeutic Agent for Malignant

Hyperthermia, Inhibits Catecholaminergic
Polymorphic Ventricular Tachycardia
in a RyR2R2474S/+ Knock-In Mouse Model
Shigeki Kobayashi, MD, PhD; Masafumi Yano, MD, PhD; Hitoshi Uchinoumi, MD, PhD;
Takeshi Suetomi, MD; Takehisa Susa, MD; Makoto Ono, MD, PhD; Xiaojuan Xu, MD, PhD;
Hiroki Tateishi, MD, PhD; Tetsuro Oda, MD, PhD; Shinichi Okuda, MD, PhD;
Masahiro Doi, MD, PhD; Takeshi Yamamoto, MD, PhD; Masunori Matsuzaki, MD, PhD

Background:  Dantrolene, a specific agent for the treatment of malignant hyperthermia, was found to inhibit
Ca2+ leak through not only the skeletal ryanodine receptor (RyR1), but also the cardiac ryanodine receptor (RyR2)
by correcting the defective inter-domain interaction between N-terminal (1–619 amino acid) and central (2,000–
2,500 amino acid) domains of RyRs. Here, the in vivo anti-arrhythmic effect of dantrolene in a human catechol-
aminergic polymorphic ventricular tachycardia (CPVT)-associated RyR2R2474S/+ knock-in (KI) mouse model was
investigated.

Methods and Results:  ECG was monitored in KI mice (n=6) and wild-type (WT) mice (n=6), before and after
an injection of epinephrine (1.0 mg/kg) or on exercise using a treadmill. In all KI (but not WT) mice, bi-directional
ventricular tachycardia (VT) was induced after an injection of epinephrine or on exercise. Pre-treatment with
dantrolene (for 7–10 days) significantly inhibited the inducible VT (P<0.01). In KI cardiomyocytes, Ca2+ spark fre-
quency (SpF; s–1 ∙ 100 μm–1: 5.8±0.3, P<0.01) was much more increased after the addition of isoproterenol than in
WT cardiomyocytes (SpF: 3.6±0.2). The increase in SpF seen in KI cardiomyocytes was attenuated by 1.0 μmol/L
dantrolene (SpF: 3.6±0.5, P<0.01).

Conclusions:  Dantrolene prevents CPVT, presumably by inhibiting Ca2+ leak through the RyR2.   (Circ J  2010;
74: 2579 – 2584)

Key Words: Calcium; Excitation-contraction (E-C) coupling; Ventricular tachycardia

T
he N-terminal (1–619 amino acid) and central (2,000–
2,500 amino acid) domains of the ryanodine recep-
tors (skeletal: RyR1, cardiac: RyR2) harbor many
mutations associated with malignant hyperthermia (MH),
catecholaminergic polymorphic ventricular tachycardia
(CPVT), and arrhythmogenic right ventricular cardiomyopa-
thy type 2.1 There is strong evidence to suggest that the inter-
domain interaction between these regions plays an important
role in the mechanism of channel regulation.2–16 We reported
that dantrolene, a specific agent for the treatment of MH,
prevented abnormal Ca2+ leak by correction of the defective
inter-domain interaction between the N-terminal and central
domains within MH RyR1 (i.e., aberrant formation of a chan-
nel-activating unzipped configuration of the N-terminal/cen-
tral domain pair in an otherwise resting state).9 We further
showed that, in failing hearts, dantrolene corrected the defec-
tive inter-domain interaction within the RyR2, thereby inhib-
iting Ca2+ leak through RyR2.11 More recently, by using the
knock-in (KI) mouse model with a human CPVT-associated
RyR2 mutation (R2474S), we clarified that a single amino
acid mutation within the RyR2 sensitizes the RyR2 channel
to activation by luminal [Ca2+] (i.e., a decreased threshold
of luminal [Ca2+] for channel activation), and in turn induces
spontaneous Ca2+ sparks and DAD, leading to CPVT, and
that danrrolene stabilized the leaky RyR2 by correcting the
defective inter-domain interaction.13 Here, we investigated
the in vivo anti-arrhythmic effect of dantrolene in the KI
mice model.

Received July 13, 2010; revised manuscript received August 3, 2010; accepted August 9, 2010; released online October 7, 2010   Time
for primary review: 9 days
Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Japan
Mailing address:  Masafumi Yano, MD, PhD, Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi Uni-
versity Graduate School of Medicine, 1-1-1 Minamikogushi, Ube 755-8505, Japan.   E-mail: yanoma@yamaguchi-u.ac.jp
ISSN-1346-9843   doi: 10.1253/circj.CJ-10-0680
All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cj@j-circ.or.jp

Circulation Journal  Vol.74,  December  2010

2580
Table.  ECG Characteristics
n
HR (beats/min)
WT 6 540±86　　 24±2
RyR2R2474S/+ 6 576±102
P value (WT vs KI) NS
Data are expressed as mean ± SD.
SVT, sustained ventricular tachycardia; WT, wild-type mice; RyR2R2474S/+, heterozygous mice carrying R2474S mutation;
KI, knock-in.
Methods
ECG Telemetry
ECG was monitored in R2474S/+ KI mice (n=6) and wild-
type (WT) mice (n=6) in a conscious state by using ECG
telemetry, as previously described,13 with slight modification.
Briefly, transmitters (Data Sciences International, St. Paul,
MN, USA) were implanted in the back space with s.c. elec-
trodes in a lead II configuration. Telemetry was recorded after
96 h of recovery from surgery in a conscious state at baseline
and after the injection of epinephrine (1 mg/kg of body
weight, i.p.) for measurement of the number of VT episodes
for 30 min. A subset of telemetered KI (n=6) and WT mice
(n=6) were injected with epinephrine (1 mg/kg of body
weight, i.p.) and monitored for 30 min. Another set of KI
(n=6) and WT mice (n=6) were exercised on a treadmill
(Panlab, Barcelona, Spain). After recording the ECG before
treatment with dantrolene, these KI mice were treated with
dantrolene i.p. (20 mg/kg) for 7–10 days before being exposed
to an epinephrine injection or exercise on a treadmill.
The QT interval was measured from the beginning of the
QRS complex to the end of the T wave based on the 5-min
stable ECG segments of sinus rhythm, as previously
described.17 Then, the QT interval was rate-corrected using
Bazett’s formula (QT interval divided by the square root of
the heart rate) and expressed as QTc in the present study.
Arrhythmias were defined as follows: non-sustained ventric-
ular tachycardia (VT) was defined as a series of 4–10 con-
secutive repetitive ventricular ectopic beats (VEBs), and
sustained VT (SVT) was defined as a run of >10 consecutive
VEBs.18
Isolation of Cardiac Cardiomyocytes
The enzymatic isolation of mice cardiomyocytes was per-
formed as described previously.13 In brief, R2474S/+ KI and
WT mice (2–3 months old) were anesthetized with pentobar-
bital sodium (70 mg/kg of body weight, i.p.), intubated and
ventilated with ambient air. An incision in the chest was
made, and the heart was quickly removed and retrogradely
perfused with a collagenase-free buffer via the aorta under
constant flow. The LV myocardium was minced with scis-
sors in a fresh collagenase-containing buffer and the rod-
shaped adult mice cardiomyocytes were prepared by retro-
grade perfusion of the hearts with 95%O2/5%CO2-bubbled
Minimal Essential Medium (Sigma, St Louis, MO, USA)
supplemented with 50 μmol/L [Ca2+], 0.5 mg/ml collagenase
B, 0.5 mg/ml, collagenase D, and 0.02 mg/ml protease type
XIV. The Ca2+ concentration was then gradually increased to
a final concentration of 1 mmol/L by changing the incubation
medium (50 μmol/L, 100 μmol/L, 300 μmol/L, 600 μmol/L
and then 1 mmol/L).
The isolated mice cardiomyocytes were transferred to
laminin-coated glass culture dishes, and incubated for 12 h at
Circulation Journal  Vol.74,  December  2010
KOBAYASHI S et al.
Baseline Exercise stress
QT (ms) QTc (ms) SVT (n)
74±7 0
24±2 73±5 6
NS NS 0.0047
37°C in a 5%CO2/95%O2 atmosphere. Experiments were
carried out at room temperature.
Analysis of Local Ca2+ Release Events With Laser  
Scanning Confocal Microscopy
Ca2+ sparks in intact isolated cardiomyocytes were measured
by confocal microscopy to examine the effect of dantrolene
on the local Ca2+ release, as previously described.13,19 The
Ca2+ sparks were measured using a laser scanning confocal
microscope system (LSM-510, Carl Zeiss) equipped with an
argon ion laser coupled to an inverted microscope (Axiovert
100, Carl Zeiss) with a Zeiss ×40 oil-immersion Plan-Neofluor
objective (numerical aperture, 1.3; excitation at 488 nm; emis-
sion >505 nm). Briefly, intact cardiomyocytes were loaded
with fluo-4 AM (20 μmol/L; Molecular Probes) for 30 min at
room temperature. Line-scan mode was used, where a single
cardiomyocyte was scanned repeatedly (520.8 Hz) along a
line parallel to the longitudinal axis, avoiding nuclei. To
monitor Ca2+ sparks, cardiomyocytes were stimulated until
the Ca2+ transient reached a steady state, then stimulation was
stopped, and Ca2+ sparks were then recorded during the sub-
sequent ~10 s rest.
Statistical Analysis
A paired or unpaired t-test was used for statistical compari-
son of the data between 2 different situations. The chi-square
test was used to estimate the effectiveness of dantrolene to
prevent occurrence of CPVT. Data are expressed as
mean ± SD except for the data of Ca2+ spark frequency. The
data of Ca2+ spark frequency are expressed as mean ± SE. We
accepted a P value less than 0.05 as statistically significant.
Results
Table shows the ECG characteristics obtained from WT and
KI mice by telemetry. There was no significant difference in
baseline parameters of HR, QT interval, and QTc between
WT and KI mice. In all KI mice, bi-directional VT was
induced after injection of epinephrine or on exercise, but not
in WT mice (Table). In KI mice, pre-treatment with a daily
intraperitoneal injection of 20 mg/kg dantrolene (7–10 days)
decreased the frequency of premature ventricular contrac-
tions at rest, and prevented the inducible VT (Figures 1–3).
In KI (but not WT) mice, pre-treatment with dantrolene sig-
nificantly increased the total running distance during exer-
cise on a treadmill (Treated; 188.5±77.1 m, vs Untreated;
22.3±9.0 m, P<0.01). In KI cardiomyocytes, Ca2+ spark fre-
quency (SpF; s–1 ∙ 100 μm–1: 5.8±0.3, P<0.01) was much more
increased after the addition of isoproterenol than in WT car-
diomyocytes (SpF: 3.6±0.2) (Figures 4,5). In KI cardiomyo-
cytes, the SpF was inhibited by 1.0 μmol/L dantrolene (SpF:
3.6±0.5, P<0.01) (Figures 4,5).
Anti-Arrhythmic Effect of Dantrolene 2581

Figure 1.    Representative telemetry ECGs in RyR2R2474S/+ KI mice on exercise using a treadmill. RyR2, ryanodine receptor; KI,
knock-in.

Figure 2.    Representative telemetry ECGs in RyR2R2474S/+ KI mice before and after an epinephrine injection. RyR2, ryanodine
receptor; KI, knock-in.

Circulation Journal  Vol.74,  December  2010

2582 KOBAYASHI S et al.

Figure 3.    Summarized data for the effect of dantrolene on the inducible VT in RyR2R2474S/+ mice. RyR2, ryanodine receptor; VT,
ventricular tachycardia.

Figure 4.    Effect of dantrolene on the aberrant Ca2+ sparks in intact RyR2R2474S/+ KI cardiomyocytes. Ca2+ sparks were mea-
sured at 2 mmol/L extracellular [Ca2+]. RyR2, ryanodine receptor.

Circulation Journal  Vol.74,  December  2010

Anti-Arrhythmic Effect of Dantrolene
Figure 5.    Summarized data for the frequency of Ca2+ sparks in intact RyR2R2474S/+ KI cardiomyocytes. RyR2, ryanodine recep-
tor; KI, knock-in.
Discussion
Catecholaminergic polymorphic VT is regarded as a highly
lethal disease and β-blockers can be used as first line treat-
ment of CPVT. Unfortunately, however, the efficacy of β-
blockers in CPVT is known to be low. Most of the patients
with CPVT require an implantable cardioverter defibrillator,
with 50% of implanted patients receiving appropriate shocks
during a 2-year follow up.20
The most important new aspect of the present study is the
finding that pre-treatment with dantrolene, a specific agent
for MH, prevented CPVT induced by either epinephrine or
exercise, and significantly improved exercise tolerance in KI
mice. In addition, in confirmation with our previous report
using saponin-permeabilized cardiomyocytes that dantrolene
inhibited the PKA-phosphorylation-induced aberrant Ca2+
release,13 it markedly suppressed the isoproterenol-induced
spontaneous Ca2+ sparks in intact cardiomyocytes as well.
The mechanism by which dantrolene prevented CPVT is
likely to be attributable to stabilization of the CPVT-associ-
ated, mutated RyR2. This notion is based on the following
findings, which have been previously noted: (1) in either
RyR1 or RyR2, dantrolene specifically binds to the domain
with the same amino-acid sequence; Leu590-Cys609 in RyR1
or Leu600-Cys619 in RyR2),9,11,21,22 and (2) dantrolene corrects
the defective inter-domain interaction between N-terminal:
1–619 and central: 2,000–2,500 domains (i.e., domain unzip-
ping to zipping), thereby inhibiting Ca2+ leak through
RyRs.9,11,13
More recently, we further demonstrated that in the
RyR2R2474S/+ KI mice, the affinity of calmodulin (CaM) bind-
ing to the RyR2 is reduced upon PKA-mediated phosphory-
lation, which seems to be a critical cause of spontaneous
Circulation Journal  Vol.74,  December  2010
2583
local Ca2+ release events.14 As dantrolene restored a normal
level of CaM-binding affinity in the PKA-phosphorylated KI
hearts,14 it is suggested that the defective inter-domain inter-
action between the N-terminal domain and the central
domain of the RyR2 is involved in the reduction of the CaM
binding affinity.
It is quite advantageous for the clinical use of dantrolene
that it showed no appreciable effect on cardiac function in
normal hearts, but it substantially improved the contractile
function in pacing-induced failing hearts.11 This beneficial
effect is clearly different from other anti-arrhythmic drugs.
The clinical dose of dantrolene for the treatment for MH
is less than 7 mg/kg. In the present study, however, we
treated KI mice with a relatively high dose of dantrolene
(20 mg ∙ kg–1 ∙ day–1). This dosage was determined by a titra-
tion study that was aimed to find out the minimum dose to
achieve complete prevention of sustained VT on exercise.
Before we further move on to clinical studies, we should
pay great attention to the effective and safety dosage of dan-
trolene for the treatment of patients with CPVT or heart
failure.
Acknowledgments
This work was supported by grants-in-aid for scientific research from
The Ministry of Education in Japan (grant nos. 20390226 to M.Y.,
20590868 to T.Y., 20591805 to S.K., 19209030 to M.M.), and grants
from Takeda Science Foundation (to M.Y. and S.K.). The authors declare
no competing financial interests.
Disclosures
None.
2584
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