Cardiovascular Research (2016) 111, 295–306
doi:10.1093/cvr/cvw095
Exercise training prevents ventricular tachycardia
in CPVT1 due to reduced CaMKII-dependent
arrhythmogenic Ca21 release
Ravinea Manotheepan 1,2*, Tore K. Danielsen 1,2, Mani Sadredini 1,2, Mark E. Anderson 3,
Cathrine R. Carlson 1,2, Stephan E. Lehnart 4, Ivar Sjaastad1,2, and Mathis K. Stokke 1,2,5
1
Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway; 2Center for Heart Failure Research, University of Oslo, Oslo, Norway;
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Clinic of Cardiology and Pulmonology, Heart Research Center Göttingen, Göttingen, Germany;
and 5Clinic for Internal Medicine, Lovisenberg Diakonale Hospital, Oslo, Norway
3
Received 10 November 2015; revised 12 April 2016; accepted 1 May 2016; online publish-ahead-of-print 8 May 2016
Time for primary review: 34 days
Aims
Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine
receptor (RyR2) that lead to disrupted Ca2+ handling in cardiomyocytes and ventricular tachycardia. The aim of this
study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2-R2474S by restoring normal Ca2+ handling.
.....................................................................................................................................................................................
Methods
Ryr2-R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session
and results
comprised five 8 min intervals at 80– 90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active
rest periods at 60%. VO2max increased by 10 + 2% in exercise trained RyR-RS (ET), while no changes were found in
sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED,
coinciding with fewer Ca2+ sparks and waves, less diastolic Ca2+ leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca2+ –calmodulin-dependent kinase type II (CaMKII) compared with
RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-L-cysteine reduced Ca2+ wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data
indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining
reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET.
.....................................................................................................................................................................................
Conclusion
Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -2+- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Keywords
Arrhythmias † CPVT1 † Exercise training † Ca
1. Introduction
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an
inherited cardiac disease predisposing to ventricular tachycardia (VT)
and sudden cardiac death triggered by physical or psychological stress.1
The prevalence of CPVT is 1:10 000, and the mortality rate in untreated
patients is 30– 33% at the age of 35.2 Even with b-blocker treatment,
breakthrough VT occurs in almost 50% of patients.3 Distinct genetic
syndromes are known: CPVT1 is caused by mutations in the cardiac ryanodine receptor 2 (RyR2) gene with autosomal dominant inheritance.3
homeostasis † CaMKII
The very rare autosomal recessive variant CPVT2 results from mutations in the cardiac calsequestrin 2 gene,4 while rare mutations responsible for CPVT3 were found in KCNJ2 leading to inward-rectifier
potassium channel dysfunction.5 RyR2 gain-of-function in CPVT1 disrupts normal control of excitation – contraction coupling, resulting in
abnormally increased Ca2+ leak from the sarcoplasmic reticulum
(SR) in diastole.6 Diastolic Ca2+ leak originating from only one RyR2
cluster may lead to abnormal activation of neighbouring RyR2 clusters
and thereby trigger regenerative, cell-wide Ca2+ release, i.e. Ca2+
waves. Regenerative Ca2+ release activates excess depolarizing Na+/
* Corresponding author. Institute for Experimental Medical Research, Oslo University Hospital, Ullevål, Kirkeveien 166 (Ullevål sykehus), Building 7, 4th Floor, N-0450 Oslo, Norway.
Tel: +47 23016800; fax: +47 23016799, E-mail: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: [email protected].
296
Ca2+-exchanger (NCX) current in diastole, resulting in delayed afterdepolarizations that can trigger action potentials and ultimately
arrhythmias.7
Current guidelines strongly recommend that patients with CPVT abstain from high-intensity, strenuous, and competitive exercise. However, there is a lack of systematic clinical studies on the effects of
monitored exercise at controlled levels in patients with CPVT.8,9 Based
on pathophysiological considerations about differences between acute
effects of exercise and long-term effects of regular exercise training,
our group conducted a pilot study where CPVT1 patients were allowed to exercise at an individualized level during careful monitoring.10
Monitored exercise at a safe intensity level for 12 weeks resulted in increased aerobic capacity and an increased threshold for arrhythmias in
the exercise trained patients compared with control patients.10 Regular
exercise training has been shown to reduce the propensity for VT in a
CASQ2 mutant CPVT2 mouse model.11 Furthermore, results from
mice with RyR2 dysfunction due to diabetic cardiomyopathy indicated
that exercise training might improve RyR2 function through a CaMKIIdependent mechanism.12 However, the therapeutic strategy that exercise directly reduces RyR2-dependent Ca2+ leak in CPVT1 has never
been shown before. Therefore, we hypothesized that interval treadmill
running exercise training can reduce the propensity for VT in mice with
a proven CPVT1-causative RyR2 leak mechanism.13
2. Methods
A full description of Methods is provided in the Supplementary material
online.
2.1 Animals
The experimental protocol was approved by the Norwegian National Animal Research Authority (project license no. FOTS 3649, 5669, and 7169).
The animal experiments were performed in accordance with the European
Directive 2010/63/EU. The Ryr2-R2474S (RyR-RS) mice used in this study
have been described previously.13 Heterozygote RyR-RS based on a
C57BL/6Ncr background were used for this study. All mice used in the
study were bred locally at the Institute for Experimental Medical Research
(IEMR), Oslo University Hospital. We used both male and female mice, and
in total, 130 RyR-RS mice and 12 wild type (WT) mice were included in the
study.
2.2 Exercise training protocol
A 2 week interval treadmill exercise training protocol was developed, consisting of treadmill running 6 days/week (Figure 1A). Each exercise session
lasted 1 h, and comprised a 10 min warm-up period, followed by five
8 min intervals at 90% of the running speed at which maximum oxygen uptake (VO2max) was achieved during a weekly test. The intervals were interspersed by 2 min periods of moderate intensity with 60% of the speed at
VO2max. The protocol was validated in WT mice, i.e. C57BL6/J (WT) (Supplementary material online, Figure S1A), before experiments with RyR-RS
mice were initiated. The mice were adapted to exercise training on the
treadmill for 3 days before the start of the exercise programme, and tested
for VO2max in a separate metabolic chamber (Columbus Instruments, Columbus, OH, USA). The running speed for each week was set after a weekly
VO2max test. The running speed during VO2max testing was increased every
second minute by 1.8 m/s, starting with 5.4 m/s, until exhaustion. For each
speed, the software calculated seven VO2 values. An average of the last
three VO2 values for each speed was calculated manually after each test.
The highest VO2 level was defined as VO2max for each mouse. The speed,
which the mouse was running with at the time of maximum VO2, was
defined as the maximum speed.
R. Manotheepan et al.
Mice were sacrificed after 2 weeks from the start of the training period,
or a similar time period for sedentary controls (SED), before the hearts
were excised and used for cellular experiments or western blot analysis.
A subgroup of mice were trained with the same protocol, followed by detraining, i.e. sedentary caging conditions, for 2 weeks before retesting of
VO2max and cardiac cellular experiments at Week 5.
2.3 Telemetric ECG surveillance
ECG recordings by telemetric ECG surveillance were performed in a subset of mice. Telemetric ECG transmitters (Data Sciences International,
St. Paul, MN, USA) were implanted as previously described.14 ECG recordings were made during VO2max tests at Weeks 0, 1, 2, and 4. At the end of
the VO2max test in Week 2, mice were run to exhaustion, followed by an
intraperitoneal injection of isoprenaline sulfate (ISO) (NAF, Norway)
20 mg/kg, and further ECG recordings for 20 min. VT episodes were
defined as three or more consecutive ventricular beats.
2.4 Echocardiography
Vevo 2100 (VisualSonics, Ontario, Canada) was used to perform echocardiography on anaesthetized mice before the first VO2max test and after 2
weeks of training. Doppler signals were collected as previously described.15
Fractional shortening was calculated with the following formula: (LVDd 2
LVDs)/LVDd (LVDd, left ventricular diameter in diastole; LVDs, left ventricular diameter in systole). Furthermore, cardiac output was calculated
2
with the following formula: heart rate × VTILVOT × praorta
(VTI, velocity
time integral; LVOT, left ventricular outflow tract).
2.5 Cell isolation
Mice were anaesthetized in 2% isoflurane inhalation prior to sacrifice by
cervical dislocation. The thoracic cavity was opened before rapid excisement and transfer of the heart to the cold buffer containing (in mM) 130
NaCl, 25 HEPES, 22 D-glucose, 5.4 KCl, 0.5 MgCl2, 0.4 NaH2PO4 (pH
7.4). Ventricular cardiomyocytes were isolated by enzymatic digestion after
perfusion of the excised heart with collagenase type 2 (Worthington Biochemical Corporation, Lakewood, NJ, USA), as previously described.16
2.6 Field-stimulation experiments
Ca2+ transients, SR Ca2+ content, and Ca2+ waves were recorded by
whole-cell Ca2+ imaging with a PTI Microscope Photometer D-104G
(PhotoMed, Køge, Denmark). Ca2+ wave measurements were repeated
during exposure to the CaMKII inhibitor autocamtide-2-related inhibitory
peptide (AIP) (5 mM) and antioxidant N-acetyl-L-cysteine (NAC) (5 mM),
respectively. Cardiomyocytes were stimulated at 1 and 4 Hz for 30 s, followed by a 20 s pause. SR Ca2+ content was measured as peak Ca2+ fluorescence after rapid exposure to 10 mM caffeine. The protocol was
repeated in the presence of 100 nM ISO (NAF, Norway). Fluorescence signals were normalized to diastolic levels during stimulation (F0).
SR Ca2+ leak was measured with a protocol described by Shannon
et al. 17 Briefly, cytosolic Ca2+-dependent fluorescence was measured in
resting cells after a period of 1 Hz stimulation with steady-state Ca2+ transients. Recordings were performed in the absence and presence of 1 mmol/
L tetracaine, which blocks RyR-dependent SR Ca2+ leak. By comparing
cytosolic Ca2+-dependent fluorescence in these two situations, RyRdependent leak can be calculated. The cytosolic Ca2+-dependent fluorescence was measured as an average of a 10 s period 30 – 40 s after the last
Ca2+ transient or Ca2+ wave. The protocol was performed in the presence
and absence of ISO.
Ca2+ sparks were recorded by confocal line-scan imaging with an inverted
water-immersion objective on a Zeiss LSM 7 live microscope (Zeiss Observer Z1, Micro imaging, GmbH, Germany). Pinhole size was set to 1 Airy unit.
The scan line was 512 pixels in length along the longitudinal axis of the cell,
avoiding cell nuclei. Consecutive line scans were made every 1.5 ms. The recordings lasted for 6 s, and Ca2+ sparks were counted from 600 ms after the
Exercise training in mice with CPVT1
297
Figure 1 Increased VO2max and reduced arrhythmias in RyR-RS after exercise training. (A) Illustration of the exercise training protocol. (B) Average
VO2max from RyR-RS ET and RyR-RS SED at the end of Weeks 0 and 2 of the exercise protocol. *P , 0.05 RyR-RS SED vs. RyR-RS ET at Week 2,
calculated with nested ANOVA. **P , 0.05 RyR-RS ET Week 0 vs. Week 2, calculated with Student’s t-test. Number of RyR-RS mice in each group
(SED/ET): 59/71. (C) In vivo ECG recording from a RyR-RS SED mouse towards the end of a VO2max test close to exhaustion. Left box: ventricular premature beat. Centre box: bidirectional VT. Right box: sinus beats. (D) Average number of VT episodes in a 20 min period immediately after the end of
VO2max test and intraperitoneal ISO injection. Number of RyR-RS mice (SED/ET): 6/5.
last transient triggered by 1 and 4 Hz stimulation, respectively. Measurements
were repeated in the presence of ISO. Ca2+ sparks were analysed using CaSparks (Daniel Ursu, &2003, University of Ulm, Germany).5
The field-stimulation experiments were performed at 378C and in parallell with cells from the same animals, with a few exceptions to allow for
added number of experiments after interim statistical analysis.
298
2.7 Protein analysis
For protein analysis, cardiectomy was performed, and the left ventricles
were isolated and rapidly frozen in liquid nitrogen. Left ventricular tissue
was stored at 2708C before the tissue was prepared for western blotting.
2.8 Statistics
Results are reported as the mean + standard error of mean. Student’s
t-test for paired data was used for comparison of the effect of exercise
training on VO2max from Week 0 to Week 2 within each group. Nested
ANOVA analysis was used for comparison of VO2max in ET and SED in
WT and RyR-RS mice, respectively. The nested ANOVA approach was
also used to calculate the statistics for all the cellular experiments. This statistical method use the average data from all cells from each animal to calculate the average in each group. With the nested ANOVA approach the
statistical calculations take into account that the data are from different cells
but from the same animal. The nested ANOVA was performed using the
lme function in the nlme R library.18 Specifically, a linear mixed-effect model
was fitted with treatment (ET/SED) as fixed effect and animal as random
effect. A shiny application for such analyses was developed and made available at www.staaln.shinyapps.io/nested-anova. Student’s t-test for unpaired
data was used for comparison of the protein abundance (Figure 5 and Supplementary material online, Figure S1) between SED and ET mice, except
for MDA; for this, a one-sided t-test for unpaired data was used to specifically test if protein abundance of MDA was reduced in ET mice. Ca2+ spark
frequency was analysed using Poisson test to adjust for a skewed distribution. P , 0.05 was considered statistically significant for all analyses.
3. Results
3.1 Increased aerobic capacity, running
speed, and reduced frequency of VT in
RyR-RS ET compared with RyR-RS SED
RyR-RS mice were randomized into RyR-RS SED or RyR-RS ET that
performed the interval exercise training protocol previously validated
in WT (Figure 1A and Supplementary material online, Figure S1). RyR-RS
ET increased VO2max by 10 + 2% compared with baseline levels
(P , 0.05), while no changes in VO 2max were observed in RyR-RS
SED (Figure 1B). RyR-RS ET also increased their maximum running
speed by 16 + 3% compared with baseline levels (P , 0.05).
ECGs were recorded from RyR-RS mice during the last VO2max test
of the interval treadmill exercise training protocol and in a 20 min period following exhaustion (Figure 1C). No mice died during or after exercise. To provoke arrhythmias, all mice were given an intraperitoneal
injection of 20 mg/kg ISO immediately upon exhaustion on the treadmill. QRS complexes during VT were typically bidirectional and sometimes also polymorphic. Compared with RyR-RS SED, RyR-RS ET
exhibited remarkably fewer episodes of VT in the 20 min period after
ISO injection (P , 0.05) (Figure 1D). Echocardiography at Week 0 and
after 2 weeks of interval treadmill exercise training did not show any
differences (Supplementary material online, Table S2).
3.2 Lower Ca21 wave frequency in RyR-RS
ET compared with RyR-RS SED
To study the effect of exercise training on the cellular events underlying
arrhythmias in CPVT1, Ca2+ transients and Ca2+ waves were recorded
by whole-cell Ca2+ imaging. No differences were found in basic parameters of Ca2+ cycling between RyR-RS SED and RyR-RS ET (Supplementary material online, Table S1). The frequency of arrhythmogenic
Ca2+ waves was measured in a 10 s pause after 1 and 4 Hz field
R. Manotheepan et al.
stimulation (Figure 2A and B). As expected, few Ca2+ waves occurred
in the absence of ISO at low stimulation frequency (1 Hz) (Figure 2C,
left panel). However, during increased stimulation frequency (4 Hz)
and in the presence of ISO, the frequency of Ca2+ waves was clearly
reduced in RyR-RS ET compared with RyR-RS SED (P , 0.05)
(Figure 2C and D).
3.3 Lower SR Ca21 leak in RyR-RS ET
compared with RyR-RS SED
SR Ca2+ leak, reflecting RyR dysfunction, was measured with wholecell Ca2+ imaging and quantification of cytosolic Ca2+ before and after
the addition of tetracaine to block RyR2. The protocol was performed
in the absence and presence of ISO (Figure 3A). In line with the results
from in vivo arrhythmias and Ca2+ wave measurements, SR Ca2+ leak
normalized to SR Ca2+ content was significantly decreased in RyR-RS
ET compared with SED mice both in the absence and presence of ISO
(P , 0.05) (Figure 3B).
3.4 Lower SR Ca21 spark frequency in
RyR-RS ET compared with RyR-RS SED
To investigate SR Ca2+ release from individual release units, Ca2+
sparks were measured during a pause after 1 and 4 Hz field stimulation
(Figure 4A and B). As expected, few Ca2+ sparks and no differences between RyR-RS SED and RyR-RS ET were observed in the absence
of ISO (Figure 4C). However, in the presence of ISO, Ca2+ spark
frequency was clearly lower in myocytes from RyR-RS ET animals
(P , 0.05) (Figure 4D).
3.5 Lower CaMKII-dependent Ser2814
phosphorylation of RyR2 and Ox-CaMKII
levels in RyR-RS ET compared with RyR-RS
SED
Western blot analysis was used to quantify the abundance of proteins
and phosphoproteins in tissue homogenates from the left ventricles of
RyR-RS SED and RyR-RS ET. RyR-RS ET hearts showed significantly decreased levels of Ser2814 phosphorylation of RyR2 compared with
RyR-RS SED (P , 0.05) (Figure 5A). Contrary to this observation,
RyR-RS ET exhibited increased levels of PKA-dependent phosphorylation of RyR2 at Ser2808 compared with RyR-RS SED (P , 0.05)
(Figure 5B). Since exercise appeared to reduce CaMKII-dependent
phosphorylation, we designed experiments to further elucidate the
mechanism behind this effect. First, total CaMKII abundance was equal
in the two groups (Figure 5C). Second, the effect was not due to altered
autophosphorylation of CaMKII, as Thr286-phosphorylated CaMKII levels were also unchanged in RyR-RS ET compared with RyR-RS SED
(Figure 5D). However, intriguingly, left ventricular tissue from RyR-RS
ET exhibited a small but significant reduction in Ox-CaMKII abundance
in RyR-RS ET compared with RyR-RS SED (P , 0.05) (Figure 5E). Considering that the quantitative difference in Ox-CaMKII abundance
seemed small, these results were confirmed in a second Western
blotting performed by a different person (data only shown for the
last series). Having confirmed the small, yet consistent reduction, we
examined if the results coincided with signs of a general reduction in
cellular oxidation products, a known effect of exercise training. Indeed,
a small reduction in MDA protein abundance was also observed in
RyR-RS ET (P , 0.05) (Figure 5F). Still, the level of Ox-CaMKII reduction needed to have functional effects is unknown. Therefore, we
decided to pursue this in separate cellular experiments. First, Ca2+
Exercise training in mice with CPVT1
299
Figure 2 Reduced Ca2+ wave frequency in RyR-RS mice after exercise training. Example tracing of whole-cell calcium imaging in field-stimulated cardiomyocytes from (A) RyR-RS SED and (B) RyR-RS ET. Average Ca2+ wave frequency after (C) 1 and 4 Hz stimulation in the absence of ISO. Average
Ca2+ wave frequency in the presence of ISO after (D) 1 and 4 Hz stimulation. *P , 0.05. Number of RyR-RS mice (SED/ET): 15/13, number of cells (SED/
ET): 40/38, ISO: 46/50.
wave frequency was measured in cardiomyocytes in the presence of
the CaMKII inhibitor AIP, which prevents substrate-binding of CaMKII
regardless of the mechanism for CaMKII-activation (Figure 6A). Pharmacological CaMKII inhibition reduced the frequency of Ca2+ waves in
RyR-RS SED (Figure 6B), supporting a causal relationship between
reduced CaMKII-dependent Ser2814 phosphorylation of RyR2 and a
reduced Ca2+ wave frequency in RyR-RS ET. After these encouraging
results, we tested the effect of reduced oxidation levels in RyR-RS SED
cardiomyocytes by exposure to the antioxidant NAC (Figure 6C). In line
with a mechanism depending on reduced Ox-CaMKII after exercise
300
R. Manotheepan et al.
Figure 3 Reduced SR Ca2+ leak in RyR-RS mice after exercise training. SR Ca2+ leak in resting cardiomyocytes after 1 Hz stimulation. (A) Illustration of
protocol in the presence of ISO. SR Ca2+ leak in (B) the absence and presence of ISO. The Y axis shows the SR Ca2+ leak levels normalized to SR Ca2+
content levels (F/F0). *P , 0.05, number of RyR-RS mice in protocol in the absence and presence of ISO (SED/ET): 12/10, 5/7, number of cells (SED/ET):
12/9, ISO: 13/19. The cells used in the experiments in the presence and absence of ISO were from different animals.
training, this reduced the frequency of Ca2+ waves equal to the effects
of AIP and exercise training (Figure 6B and C).
3.6 Reversed effects of interval treadmill
exercise training in RyR-RS ET after
detraining
To further test a causal relationship between exercise training and reduced propensity for arrhythmias, a subset of RyR-RS ET animals was
exposed to 2 weeks of detraining, i.e. spontaneous sedentary behaviour, directly following completion of the 2 week interval treadmill exercise training protocol. Indeed, after detraining, VO2max levels were
reduced by 18 + 5% in RyR-RS ET compared with peak VO2max at 2
weeks (P , 0.05) (Figure 7A and B). Remarkably, after the detraining
period, the number of VT episodes in RyR-RS ET during VO2max testing
was reversed back to the initial levels prior to the exercise program
(P , 0.05) (Figure 7C). Furthermore, Ca2+ wave frequency in detrained
mice was not significantly different from SED mice at 4 weeks
(Figure 7D– G), confirming complete reversibility of the effects of exercise training.
4. Discussion
To our knowledge, this is the first study to explore the therapeutic potential of exercise training in a physiological model of CPVT1.
Importantly, 2 weeks of interval treadmill exercise training of mice
with CPVT1 significantly reduced the number of VT episodes, arrhythmogenic SR Ca2+ release, and CaMKII-dependent Ser2814 phosphorylation of RyR2. Causality was further supported by complete reversal
of the beneficial effects by detraining.
4.1 Exercise training as therapy for
exercise-triggered arrhythmias
VT in CPVT is usually triggered in situations associated with increased
sympathetic activity and high heart rates, such as exercise or mental
stress.1 The relationship between heart rate and initiation of arrhythmias is individual and reproducible.19 In a recent pilot study, we used
this heart rate threshold for ventricular arrhythmias to allow patients
with CPVT1 to participate in an individualized exercise training
program.10 The pilot study indicated that exercise training at a safe submaximal intensity, under appropriate medical therapy and in a controlled environment, can be performed safely by patients with
CPVT1, and could have a beneficial effect on ventricular arrhythmias.
The CPVT mouse model in the current study reproduced rather well
the arrhythmic phenotype observed in humans, as well as the beneficial
effects of exercise training. Furthermore, this experimental study suggests a mechanism that is supported by current knowledge about the
mechanism for arrhythmias in CPVT and exercise physiology. Thus,
the long-term effects of regular exercise training should probably be
Exercise training in mice with CPVT1
301
Figure 4 Reduced Ca2+ spark frequency in RyR-RS mice after exercise training. Confocal imaging of cardiomyocytes from (A) RyR-RS SED and (B)
RyR-RS ET after 1 Hz stimulation in the presence of ISO. Average Ca2+ spark frequency in a 6 s pause after (C) 1 and 4 Hz stimulation in the absence of
ISO. Average Ca2+ spark frequency in a 6 s pause in the presence of ISO after (D) 1 and 4 Hz stimulation. Density blots illustrate Poisson analysis used for
comparison of Ca2+ spark frequency in RyR-RS ET and RyR-RS SED groups. *P , 0.05. Number of RyR-RS mice (SED/ET): 6/6, number of cells (SED/
ET): 30/31, ISO: 21/25.
separated from the acute effects during individual bouts of physical activity even in patients with CPVT1. An interesting question to come
from this is what level and duration of exercise is necessary to induce
this effect and for this to outweigh the risk of individual sessions. Our
study was not designed to answer such questions that would demand
comparison of different exercise protocols. We chose a protocol
302
R. Manotheepan et al.
Figure 5 Reduced CaMKII-dependent phosphorylation of RyR in RyR-RS mice after exercise training. Left ventricle lysates from RyR-RS SED and
RyR-RS ET were immunoblotted with antibodies against (A) pSer2814-RyR2, (B) pSer2808-RyR2, (C ) total CaMKII, (D) pThr286-CaMKII, (E) OxCaMKII, and (F) MDA. pSer2814-RyR2 and pSer2808-RyR2 were normalized to total RyR2 levels; pThr286-CaMKII and Ox-CaMKII levels were normalized to total CaMKII levels. The values are presented in percentage and are normalized to RyR-RS SED. *P , 0.05, **P , 0.05 with one-sided Student’s
t-test. Number of hearts used from RyR-RS mice (SED/ET): 9/9.
based on previous studies that were likely to induce adaptive
changes.20,21 The protocol used in the present study was somewhat
shorter than the ones used in comparable studies by others. 11,12
However, a relevant exercise effect was proved by increased VO2max
and maximum running speed, and the protocol was therefore sufficient
for the aim of the study, i.e. to test if effects of exercise beyond the
Exercise training in mice with CPVT1
303
Figure 6 Reduced Ca2+ wave frequency in RyR-RS after CaMKII inhibition and antioxidant treatment. (A) Example tracing from whole-cell calcium
imaging in field-stimulated cardiomyocytes isolated from RyR-RS SED with AIP added to the experimental solution in the presence and absence of ISO.
Separate experiments were performed with the same protocol with NAC replacing AIP. Average Ca2+ wave frequency in pause in the absence of ISO
after (B) 1 and 4 Hz stimulation. Average Ca2+ wave frequency in pause in the presence of ISO after (C ) 1 and 4 Hz stimulation. *P , 0.05. Number of
RyR-RS mice experiments with AIP and NAC (SED): 6, number of cells (SED): 22, ISO: 32, SED cells with AIP: 10, ISO: 28, SED cells with NAC: 6, ISO: 35.
acute effects during a single bout, i.e. exercise training, could have effects on the pathological SR Ca2+ leak in CPVT1.
Nevertheless, caution is required before extrapolating the data
from these studies into the clinical setting. First, the present study
shows a beneficial effect on the occurrence of the pathognomonic bidirectional VT in RyR-RS. However, we have not investigated the effect on VF, which have also been observed in this mouse model.13 VT
degenerating into VF is probably the cause of SCD in CPVT patients,
but not lethal in mice. Thus, even if repeated interval exercise training
could be confirmed to attenuate the effect of acute exercise on the
occurrence of bidirectional VT, we cannot be sure that the risk of
SCD is equally reduced in patients. Second, a more nuanced view
of CPVT is emerging, possibly identifying different subpopulations
of patients with a clinical CPVT diagnosis or carriers of mutations
with CPVT-associated genes that vary even within the identified types
of CPVT.22 These subgroups seem to have very different risk profiles.
The patients included in our previous clinical pilot study were probably from a low-risk group, since none of them had any episodes of
syncope and only two had diagnosed VT. Indeed, the majority were
diagnosed after discovery of disease-causing mutations in symptomatic family members. Thus, it is likely that the risk associated with exercise may vary even between patients with CPVT. The effects of
exercise in different subgroups should be pursued in future studies.
Of note, beneficial effects of exercise training have also been shown
304
R. Manotheepan et al.
Figure 7 Detraining reverses VO2max, VT episodes, and Ca2+ waves in RyR-RS. (A) VO2max levels for RyR-RS SED. (B) VO2max levels for RyR-RS ET.
(C) Average number of VT episodes during VO2max test after detraining in Week 4. Data from Week 2 for RyR-RS ET in (C) is equal to the RyR-RS ET
data in Figure 1D. Number of RyR-RS mice (SED/ET): 6/5. (D and E) Ca2+ wave frequency after (D) 1 and (E) 4 Hz stimulation in the absence of ISO. (F and
G) Ca2+ wave frequency in the presence of ISO after (F ) 1 and (G) 4 Hz stimulation. Data from Week 2 for RyR-RS SED and RyR-RS ET in D– G are equal
to data in Figure 2C– D. *P , 0.05. Number of RyR-RS mice exposed to detraining: 6 SED/11 ET, number of cells: absence of ISO 11 SED/38 ET, presence
of ISO 25 SED/42 ET.
in a CASQ2 CPVT mouse model.11 However, as CPVT1 is typically
caused by missense mutations in RyR2 with a very different role in
cardiomyocyte Ca2+ handling, the mechanisms for the effect of exercise training may differ between the different mouse models. This
might also be true for different Ryr2 mutations in CPVT1, as these
can differ in their biophysical properties.22
4.2 Possible mechanisms for beneficial
effects of exercise training in CPVT1
Our data indicate that the beneficial long-term effects of exercise training in CPVT1 are due to reduced CaMKII-dependent Ser2814 phosphorylation of RyR2. Pharmacological inhibition of CaMKII has
305
Exercise training in mice with CPVT1
previously been shown to prevent arrhythmias in a mouse model of
CPVT1,23 and our findings add further support to the concept that
CaMKII inhibition may prevent arrhythmias in this disease.24 In a very
different model comprising increased SR Ca2+ leak, i.e. a mouse model
of diabetic cardiomyopathy, the effects of exercise training were very
much similar to our findings, with reduced SR Ca2+ leak equal to the
effects of pharmacological CaMKII inhibition.12 This potential mechanism for the effect of exercise training works upstream of the target protein for the mutations. We speculate that the beneficial effect on RyR2
is due to a reduction in the total sum of destabilizing influences. In this
perspective, our results indicating an increased PKA-dependent phosphorylation of Ser2808 of RyR2 are interesting. PKA-dependent phosphorylation probably has an essential role in the normal response to
sudden sympathetic stimulation.25 The findings in the present study
could indicate that exercise training reduces the detrimental effects
of CaMKII-activation while preserving or even improving the physiological PKA-dependent Ser2808-phosphorylation of RyR2.
As for the mechanism for reduced CaMKII-dependent phosphorylation, our data show that reduced Ox-CaMKII levels could be an explanation. Even though the quantitative effect on protein abundance
was small, the finding was supported by the generally decreased ROS
levels in RyR-RS ET measured by MDA abundance, and even further by
the functional effects of the antioxidant NAC on Ca2+ wave frequency
in RyR-RS ET. A reduced level of ROS-production after exercise training is well documented in the literature, although the link to CaMKII has
yet to be explored.26 Our results indicate that future studies should investigate whether the indicated effect on the ROS-CaMKII system
could be a general effect of exercise training. However, the role of
CaMKII in the physiological adaptation to exercise training is likely complex as others have shown that inhibition of CaMKII blunted the response to exercise in WT mice.27 Furthermore, the effect of
exercise training on Ca2+ homeostasis is likely to be different in WT
mice than in mice with CPVT-related mutations due to the differences
in key aspects of Ca2+ homeostasis before onset of training. Thus, comparison of subtle aspects of training effects in WT and CPVT should
only be made with caution.
In conclusion, we show that interval treadmill exercise training prevents VT in mice with a CPVT1-causative RyR2 mutation and decreases
arrhythmogenic SR Ca2+ release due to lower CaMKII-dependent
phosphorylation of RyR2. Sub-maximal, individualized, and safely monitored exercise training has beneficial long-term effects on RyR2 and
might attenuate the acute effects of exercise in CPVT1.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Acknowledgements
We thank the following persons for technical assistance: Ulla Enger and
Marianne Lunde (western blotting); Marita Martinsen and Hilde Dishington (mouse breeding and genotyping); and Ståle Nygård (statistics);
all from IEMR, OUH; and Daniel Ursu who programmed the CaSparks
software (University of Ulm, Germany). We also want to thank the KG
Jebsen Cardiac Center and The Olav Raagholt and Gerd Meidel Raagholt Research Foundation for the support.
Conflict of interest: none declared.
Funding
This work was supported by Norwegian Health Association, Anders Jahres
Foundation for the Promotion of Science, The Research Council of
Norway, and Rakel and Otto Bruuns Foundation; and by a grant of the
Deutsche Forschungsgemeinschaft (SFB 1002, subproject B05A09 to
S.E.L.). M.E.A. was supported, in part, by grants from the National Institutes
of Health (R01 HL079031, R01 HL070250, R01 HL096652, and R01
HL113001).
References
1. Leenhardt A, Lucet V, Denjoy I, Grau F, Ngoc DD, Coumel P. Catecholaminergic polymorphic ventricular tachycardia in children. A 7-year follow-up of 21 patients. Circulation 1995;91:1512 –1519.
2. Lehnart SE, Wehrens XH, Laitinen PJ, Reiken SR, Deng SX, Cheng Z, Landry DW,
Kontula K, Swan H, Marks AR. Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak. Circulation
2004;109:3208 –3214.
3. Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, DeSimone L,
Coltorti F, Bloise R, Keegan R, Cruz Filho FE, Vignati G, Benatar A, DeLogu A. Clinical
and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. Circulation 2002;106:69–74.
4. Lahat H, Pras E, Eldar M. A missense mutation in CASQ2 is associated with autosomal
recessive catecholamine-induced polymorphic ventricular tachycardia in Bedouin
families from Israel. Ann Med 2004;36(Suppl. 1):87 –91.
5. Kalscheur MM, Vaidyanathan R, Orland KM, Abozeid S, Fabry N, Maginot KR,
January CT, Makielski JC, Eckhardt LL. KCNJ2 mutation causes an adrenergicdependent rectification abnormality with calcium sensitivity and ventricular arrhythmia. Heart Rhythm 2014;11:885 –894.
6. Marks AR. Ryanodine receptors, FKBP12, and heart failure. Front Biosci 2002;7:
d970 –d977.
7. Pogwizd SM, Bers DM. Cellular basis of triggered arrhythmias in heart failure. Trends
Cardiovasc Med 2004;14:61 –66.
8. Vanhees L, Rauch B, Piepoli M, van Buuren F, Takken T, Borjesson M,
Bjarnason-Wehrens B, Doherty P, Dugmore D, Halle M. Importance of characteristics
and modalities of physical activity and exercise in the management of cardiovascular health
in individuals with cardiovascular disease (Part III). Eur J Prev Cardiol 2012;19:1333–1356.
9. Zipes DP, Camm AJ, Borggrefe M, Buxton AE, Chaitman B, Fromer M, Gregoratos G,
Klein G, Moss AJ, Myerburg RJ, Priori SG, Quinones MA, Roden DM, Silka MJ, Tracy C,
Smith SC Jr, Jacobs AK, Adams CD, Antman EM, Anderson JL, Hunt SA, Halperin JL,
Nishimura R, Ornato JP, Page RL, Riegel B, Priori SG, Blanc JJ, Budaj A, Camm AJ,
Dean V, Deckers JW, Despres C, Dickstein K, Lekakis J, McGregor K, Metra M,
Morais J, Osterspey A, Tamargo JL, Zamorano JL. ACC/AHA/ESC 2006 guidelines
for management of patients with ventricular arrhythmias and the prevention of sudden
cardiac death: a report of the American College of Cardiology/American Heart Association Task Force and the European Society of Cardiology Committee for Practice
Guidelines (Writing Committee to Develop Guidelines for Management of Patients
With Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death). J Am
Coll Cardiol 2006;48:e247– e346.
10. Manotheepan R, Saberniak J, Danielsen TK, Edvardsen T, Sjaastad I, Haugaa KH,
Stokke MK. Effects of individualized exercise training in patients with catecholaminergic
polymorphic ventricular tachycardia type 1. Am J Cardiol 2014;113:1829 –1833.
11. Kurtzwald-Josefson E, Hochhauser E, Katz G, Porat E, Seidman JG, Seidman CE,
Chepurko Y, Shainberg A, Eldar M, Arad M. Exercise training improves cardiac function
and attenuates arrhythmia in CPVT mice. J Appl Physiol 2012;113:1677 –1683.
12. Stolen TO, Hoydal MA, Kemi OJ, Catalucci D, Ceci M, Aasum E, Larsen T, Rolim N,
Condorelli G, Smith GL, Wisloff U. Interval training normalizes cardiomyocyte function, diastolic Ca2+ control, and SR Ca2+ release synchronicity in a mouse model of
diabetic cardiomyopathy. Circ Res 2009;105:527 –536.
13. Lehnart SE, Mongillo M, Bellinger A, Lindegger N, Chen BX, Hsueh W, Reiken S,
Wronska A, Drew LJ, Ward CW, Lederer WJ, Kass RS, Morley G, Marks AR. Leaky
Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death
in mice. J Clin Invest 2008;118:2230 –2245.
14. Stokke MK, Hougen K, Sjaastad I, Louch WE, Briston SJ, Enger UH, Andersson KB,
Christensen G, Eisner DA, Sejersted OM, Trafford AW. Reduced SERCA2 abundance
decreases the propensity for Ca2+ wave development in ventricular myocytes. Cardiovasc Res 2010;86:63–71.
15. Sjaastad I, Sejersted OM, Ilebekk A, Bjornerheim R. Echocardiographic criteria for detection of postinfarction congestive heart failure in rats. J Appl Physiol (1985) 2000;89:
1445– 1454.
16. Louch WE, Sheehan KA, Wolska BM. Methods in cardiomyocyte isolation, culture, and
gene transfer. J Mol Cell Cardiol 2011;51:288–298.
17. Shannon TR, Ginsburg KS, Bers DM. Quantitative assessment of the SR Ca2+ leak-load
relationship. Circ Res 2002;91:594 –600.
18. Pinheiro J, Bates D, DebRoy S, Sarkar D. and R Core Team. nlme: Linear and Nonlinear
Mixed Effects Models, 2016. R package version 3.1-126, http://CRAN.R-project.org/
package=nlme.
306
19. Haugaa KH, Leren IS, Berge KE, Bathen J, Loennechen JP, Anfinsen OG, Fruh A,
Edvardsen T, Kongsgard E, Leren TP, Amlie JP. High prevalence of exercise-induced
arrhythmias in catecholaminergic polymorphic ventricular tachycardia mutationpositive family members diagnosed by cascade genetic screening. Europace 2010;
12:417 – 423.
20. Kemi OJ, Loennechen JP, Wisloff U, Ellingsen O. Intensity-controlled treadmill running
in mice: cardiac and skeletal muscle hypertrophy. J Appl Physiol (1985) 2002;93:
1301 –1309.
21. Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training
improves skeletal muscle function and Ca2+ handling-related protein expression in
sympathetic hyperactivity-induced heart failure. J Appl Physiol (1985) 2010;109:
702 –709.
22. van der Werf C, Wilde AA. Catecholaminergic polymorphic ventricular tachycardia: disease with different faces. Circ Arrhythm Electrophysiol 2015;8:523 – 525.
R. Manotheepan et al.
23. Liu N, Ruan Y, Denegri M, Bachetti T, Li Y, Colombi B, Napolitano C, Coetzee WA,
Priori SG. Calmodulin kinase II inhibition prevents arrhythmias in RyR2 (R4496C+/2 )
mice with catecholaminergic polymorphic ventricular tachycardia. J Mol Cell Cardiol
2011;50:214–222.
24. Rokita AG, Anderson ME. New therapeutic targets in cardiology: arrhythmias and
Ca2+/calmodulin-dependent kinase II (CaMKII). Circulation 2012;126:2125 –2139.
25. Shan J, Kushnir A, Betzenhauser MJ, Reiken S, Li J, Lehnart SE, Lindegger N, Mongillo M,
Mohler PJ, Marks AR. Phosphorylation of the ryanodine receptor mediates the cardiac
fight or flight response in mice. J Clin Invest 2010;120:4388 –4398.
26. Campos JC, Gomes KMS, Ferreira JCB. Impact of exercise training on redox signaling in
cardiovascular diseases. Food Chem Toxicol 2013;62:107 –119.
27. Kaurstad G, Alves MN, Kemi OJ, Rolim N, Hoydal MA, Wisloff H, Stolen TO, Wisloff U.
Chronic CaMKII inhibition blunts the cardiac contractile response to exercise training.
Eur J Appl Physiol 2012;112:579 – 588.