Arrhythmia-preventive effects of exercise training in
catecholaminergic polymorphic ventricular tachycardia type 1.
From experimental models to patients
Dissertation for the degree of Philosophiae Doctor (PhD) University of Oslo, Oslo, Norway
2017
Ravinea Manotheepan, M.D
Institute for Experimental Medical Research Institute for Clinical Medicine
Faculty of Medicine University of Oslo
© Ravinea Manotheepan, 2018
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-8377-232-6
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
Supported by:
Norwegian Health Association
Oslo University Hospital
University of Oslo
Institute for Experimental Medical Research
Center for Heart Failure Research
Norwegian PhD School of Heart Research
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Dedicated to Pancharatnam Vishvalingam and Sivampikai Thiyagarajah, my grandparents, strong, inspiring individuals, who lived their lives showing that one can overcome anything with hard work and dedication.
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Acknowledgements
I am sincerely grateful to have worked at such an amazing place as Institute for Experimental Medical Research (IEMR). The inspiring research environment with high competence, positive people and fantastic social atmosphere have been incredible. It made the time here better than what I have ever imagined for a work place. I have to thank Professor Ole M. Sejersted leading the institute in such a great way, and also Lisbeth H. Winer and for contributing to this fine leadership.
I have been very lucky to have two excellent supervisors Mathis Korseberg Stokke and Professor Ivar Sjaastad. Thank you Mathis for being my main supervisor and mentor! You have from day one been enthusiastic, inspiring and supporting. I am forever grateful for your generosity, advice and positive energy, the last being very contagious giving me boost of energy when things are tough. You and Ivar have both been a great inspiration when it comes to plan and structure research work but also how to combine this with clinical work and family life. Thank you Ivar for introducing me to the institute as a medical student which triggered my interest for research even more. Your guidance and wise advice along the way have been highly appreciated. You and Mathis have always had open doors (and phones lines) and been available to consult with at all times. I will forever be grateful for all the help and knowledge you both have given me and hope I will get the opportunity to contribute to more research with you in the future.
As for most research, the articles in this thesis would not have been possible to complete without collaborations. Tore Kristian Danielsen and Mani Sadredini, thank you for the solid team work, I could not have made this without both of you. You are amazing colleagues and great friends. Thank you so much for the contributions, discussions and for being there to share frustrations and successes.
You both have been invaluable and I am lucky to be part of such a great team of good friends.
I also have to thank Professor Thor Edvardsen and Assosiate Professor Kristina H. Haugaa for the great collaborations which have resulted in two of the articles in this thesis. Thank you very much Thor for allowing us to use the fantastic research facilities available at Rikshospitalet for collection of the clinical data to Article 1 and 3. I also have to thank Jørg Saberniak, Ida Skrinde Leren and
Margareth Ribe. It has been a great pleasure working with you all. Thank you to Cathrine Carlson for engaging in interesting discussions and contributions to Article 2. Andrew G. Edwards and Kevin Vincent, your knowledge related to computer modeling was invaluable, thank you so much for the collaborations to Article 3.
The mouse model used in two of the three articles in this thesis was extremely important for us. I highly appreciate the collaboration with Professor Stephen Lehnart who has provided us the mice. My gratitude also goes to Professor Mark E. Anderson for the collaboration in article 2 and for continuing to work further on with our group. A big thanks also goes to Askim Family Sports club and Domus
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Atletica who gave us the facilities for exercise training which was necessary for Article 1. The excellent spinning instructors and nurses also have to be thanked.
I have to thank Professor Geir Christensen for giving me the opportunity to participate in
administrative work planning courses and seminars for Norheart and CHFR. The experience gained with this work is invaluable. Professor William (Bill) Louch, thank you for always being available.
You have been a lifesaver when problems occurred with the microscopy set-ups during experiments.
Ståle Nygård, thank you very much for your advice in statistical analysis, it is much appreciated. Per Andreas Norseng and Vidar Magne Skulberg, without both of you, there would be no life to my PCUS273, your IT assistance is invaluable. Roy Trondsen, you have an amazing way to find practical solutions for us when it comes to anything we need for the microscopy equipment, parts for the treadmill or the cell isolation setup, thank you. Jo-Ann (Joselan) Fabe Larsen thank you for always being there and for the relaxing chats. The technical staff particularly Ulla Enger and Marianne Lunde, thank you for your expertise on Western blotting. Also great thanks to Hilde Dishington and Marita Martinsen helping out with the genotyping. Marita, you have taught me a lot about how to handle and breed mice, and together with Siv Leng Tran, Siv Rong Tran, Ann Christin Josefsen, Lindy Eliassen Høyum, Oda Landfald, Marie Rousset, Hanne Nathalie Bækkelund, Iván Eiriz Delgado and Gro Furset Flatekval, thank you all for providing first class expertise on animal care and handling.
Professor emeritus Arnfinn Ilebekk, Trude Aspelin and Morten Eriksen thank you for letting me participate in your experiments when I was a medical student. That inspired me even more to continue with scientific work after medical studies. Pim, Terje, Emil, Tandi, Kine, Olav E, Ingunn, Christoffer and Karina, some of you have finished your time at the institute and some still connected to the institute, thank you for making the office feel like a second home and for sharing frustrations and happiness. All you other fantastic friends, colleauges and last but not least the “lunch club” people at IEMR, thank you for making IEMR the amazing place that it is.
I also need to thank all my family and friends. A special thanks to my in-laws and for always being understanding and motivating regarding my work. To all other extended family members and friends, thank you so very much for your support and interest for my work. Prathepa, your help with the graphic touch did wonders!
Finally, I would specially thank the people very close to me, starting with my parents, amma Buvanadevi and appa Kirupamurthy, you have thought and shown me that one can achieve anything with hard work and dedication. You have always been motivating and helpful in everything I do, and prioritizing us, before yourselves. I am incredibly grateful and lucky to have you as my parents. You are my biggest inspiration, thank you! Darshan, it have been invaluable to have a brother who can relate to different aspects of my work within research. Thank you for being my discussion partner and truly understanding the different aspects of the research field, I am forever grateful. You are an extremely kind and understanding brother for me and uncle for Tansi, thanks for always being there!
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Manotheepan you are my best friend, husband and the person who handles all my frustration and joy to the fullest. I am incredibly lucky to have you in my life. You balance me out, I would never have made this without your patience, care, and love. Thank you for putting out with me during the final run of this thesis. You are a great father and life partner, more than what I ever could wish for.
Thanks for everything.
Tansi, you make the whole world complete and make me understand the real importance of life. You were born during the first year of my PhD and have constantly been reminding me how grateful and lucky I am no matter what happens. You blow away all problems with your wonderful smile and happy mood. Thank you for being you!
Oslo, December 2017, Ravinea Manotheepan
8 Table of contents
1 Abbreviations ... 9
2 List of articles in thesis ... 10
3 Introduction ... 11
3.1 Sudden cardiac death ... 11
3.2 Catecholaminergic polymorphic ventricular tachycardia ... 12
3.3 Potential antiarrhythmic effects of exercise training ... 23
4 Aims of the thesis ... 25
5 Methods ... 26
5.1 Patient studies ... 26
5.2 Basic research methods ... 28
6 Brief summary of the main results in the thesis ... 34
6.1 Article 1 ... 34
6.2 Article 2 ... 34
6.3 Article 3 ... 35
7 Discussion ... 36
7.1 Feasibility of exercise training in CPVT1 and effects on aerobic capacity ... 36
7.2 Mechanisms for antiarrhythmic effects of exercise training in CPVT1 ... 39
7.3 Potential therapeutic interventions and targets in CPVT1 ... 42
8 Conclusions ... 44
9 Reference list ... 45
10 Appendix: Article 1-3 ... 55
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1 Abbreviations
AIP - autocamtide-2-related inhibitory peptide CaM - Calmodulin
CASQ2 – calsequestrin 2
CaMKII - Ca2+/calmodulin-dependent protein kinase type II CICR - Ca2+ induced Ca2+ release
CPVT - catecholaminergic polymorphic ventricular tachycardia DAD - delayed afterdepolarization
ET - exercise training
FKBP12.6 - FK-binding protein 12.6, calstabin2 HEK293 - human embryonic kidney cells 293 HF - heart failure
HR - heart rate
ICD - implantable cardioverter defibrillator MDA - methyl-dealdehyde
NAC - N-acetyl-L-cysteine
NCX1 - Na+/Ca2+ exchanger, cardiac isoform Nnt - nicotinamide nucleotide transhydrogenase
Ox-CaMKII - oxidized Ca2+/calmodulin-dependent protein kinase type II PKA - protein kinase A
PLB - phospholamban PP1 - protein phosphatase 1 PP2A - protein phosphatase 2A ROS - reactive oxygen species
RyR2 - ryanodine receptor, SR Ca2+ release channel, cardiac isoform RyR-RS - mice with RyR R2474S mutation
SCD - sudden cardiac death SED - sedentary
SERCA2 - sarcoplasmic reticulum Ca2+ ATPase, cardiac isoform SR - sarcoplasmic reticulum
VA - ventricular arrhythmia VES - ventricular extrasystole VF - ventricular fibrillation
VO2max - maximum oxygen uptake VT - ventricular tachycardia
WT - wild type mice
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2 List of articles in thesis
Article 1
Effects of individualized exercise training in patients with catecholaminergic polymorphic ventricular tachycardia type 1
Ravinea Manotheepan, Jørg Saberniak, Tore K. Danielsen, Thor Edvardsen, Ivar Sjaastad, Kristina H.
Haugaa, Mathis K. Stokke
Am J Cardiol 2014;113:1829-1833
Article 2
Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII- dependent arrhythmogenic Ca2+ release
Ravinea Manotheepan, Tore K. Danielsen, Mani Sadredini, Mark E. Anderson, Cathrine R. Carlson, Stephan E. Lehnart, Ivar Sjaastad, and Mathis K. Stokke
Cardiovasc Res 2016;111:295-306
Article 3
Arrhythmia initiation in catecholaminergic polymorphic ventricular tachycardia type 1 depends on both heart rate and sympathetic stimulation
Tore K. Danielsen, Ravinea Manotheepan, Mani Sadredini, Ida S. Leren, Andrew G. Edwards, Kevin Vincent, Stephan E. Lehnart, Ole M. Sejersted,Ivar Sjaastad, Kristina H. Haugaa, Mathis K. Stokke Submitted
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3 Introduction
3.1 Sudden cardiac death
Sudden cardiac death (SCD) is defined as unexpected death from a cardiovascular cause that occurs within one hour of symptom onset, in persons without prior conditions that would appear fatal.1 If the episode was not witnessed, the individual should have been observed alive within 24 hours before their death.2 SCD occurs in 50-100 per 100 000 persons every year, and is a major public health problem.3 The most common cause of SCD is coronary heart disease (app. 80 % of cases), followed by cardiomyopathies (10-15 %), and congenital heart disease (5-10 %).2 The last group includes
conditions associated with cardiac structural abnormalities, as well as conditions where such abnormalities are not present, including ion channelopathies. There is an age-dependent variation in the causes of SCD: SCD that occurs above the age of 35 is commonly caused by ischemic and valvular heart diseases, while below the age of 35 inherited cardiomyopathies and channelopathies are more prevalent.4 Reports estimate that 27-43 % of persons that die from SCD before the age of 35 years show no structural heart disease on autopsy,5, 6 and thus are presumed to be arrhythmia related SCDs.
Approximately 50 % of the autopsy negative SCD cases are caused by inherited arrhythmia syndromes, such as long QT-syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT) and early repolarization syndrome.4 The present thesis focuses on CPVT type 1. Although CPVT1 is responsible for only a small percentage of all cases of SCD, further understanding of this disease is important because of its potentially fatal outcome in young persons, and its role as a model disease for Ca2+-dependent arrhythmias.7
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3.2 Catecholaminergic polymorphic ventricular tachycardia
3.2.1 General aspects
The phenotype of the disease later termed CPVT was described in 1975 by Reid et al.8 They
characterized a disease with bidirectional ventricular tachycardia (VT) which occurred during physical or emotional stress in a six-year-old girl with a structurally normal heart. Later, Leenhardt and co- workers reported a series of cases with syncope, and introduced the term CPVT.9 They performed a study where 21 children with stress-induced syncope were followed for seven years. The children were described as having normal ECGs and no evidence of structural heart disease. The disease was
characterized by cathecholamine-induced bidirectional and/or polymorphic VT. The VT episodes often followed the same pattern of ventricular extrasystoles (VES) in quadrigeminy, trigeminy or bigeminy, followed by bidirectional tachycardia or polymorphic VT. The arrhythmia tendency increased with increasing heart rate (HR). Other groups later described the same phenomenon.10 The risk for VT and SCD makes this a malignant disease, with a mortality rate of 30-33 % by the age of 35 in untreated patients, and 10-24 % within seven years of follow-up, in some reports.9, 11 The estimated prevalence of CPVT is 1:10 000.12
3.2.2 Diagnosis of CPVT
The following diagnostic criteria for CPVT were formulated in a consensus statement in 201313:
“1. CPVT is diagnosed in the presence of a structurally normal heart, normal ECG, and unexplained exercise or cathecholamine-induced bidirectional VT or polymorphic ventricular premature beats or VT in an individual younger than 40 years.
2. CPVT is diagnosed in patients (index case or family member) who have a pathogenic mutation.
3. CPVT is diagnosed in family members of a CPVT index case with a normal heart who manifest exercise-induced premature ventricular contractions or bidirectional/polymorphic VT.
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4. CPVT can be diagnosed in the presence of a structurally normal heart and coronary arteries, normal ECG, and unexplained exercise or cathecholamine-induced bidirectional VT or polymorphic
ventricular premature beats or VT in an individual older than 40 years.”13 One of these criteria has to be met to give the diagnosis CPVT.
3.2.3 Genetics of CPVT
From analyses of families with several cases of CPVT, an autosomal dominant inheritance was suggested.9 Later, Swan et al. was one of the groups to describe that CPVT could be caused by a mutation in the gene encoding the cardiac specific ryanodine receptor (RyR2),14 a key protein in cardiac cellular Ca2+ homeostasis.15 They performed a linkage study and found that the 1q42-q43 region was the locus for dominant CPVT.14 Priori et al. performed a candidate gene analysis and identified mutations on the RyR2 gene in four families.16 These were four different single nucleotide substitutions leading to missense mutations in the RyR2 gene. One of them was an arginine to serine substitution at position 2474 (exon 49),16 which is the mutation carried by the mouse model of CPVT1 used in the present thesis. A study by Laitinen et al. confirmed the findings of Priori et al. and also identified three mutations in the RyR2 gene.16, 17 The mutations were located in functionally important regions of the gene, and RyR2 dysfunction was considered to explain bidirectional VT and delayed afterdepolarizations (DADs) in the patients.18
CPVT1 caused by RyR2 mutations is the most common type of CPVT, and accounts for more than 50 % of the cases.10 Other types are rare: CPVT2 is a recessively inherited disease caused by an abnormality in the gene encoding calsequestrin 2 (CASQ2), and the second most common type of CPVT.19, 20 CPVT3 is caused by mutations in the KCNJ2 gene leading to an inward-rectifier potassium channel dysfunction.21 A CPVT phenotype associated with mutations in calmodulin (CaM) is termed CPVT4.22 Lastly, CPVT caused by abnormalities in triadin is termed CPVT5.23 The majority of CPVT cases are diagnosed early in life, but some individuals do not get any symptoms before later in life and are diagnosed with CPVT in adulthood.24, 25 This late-onset subtype of CPVT seems to be more prevalent in females with symptom debut around 30-40 years of age.10 Thus, one has speculated that
14
female sex-hormones might be involved in triggering this type of CPVT.25 It is also discussed if this is just a mild form of the other known CPVT subtypes, and not a separate subtype.25
Approximately 150 mutations have been described in RyR2, predominantly in three different regions.26 The regions where the majority of the mutations were detected are responsible for
physiological roles of the protein, and many of the mutations are found in parts of the gene that encodes the region where FK-binding protein 12.6 (FKBP12.6, also called calstabin2) binds to RyR2.
Genetic testing is most (cost-) effective in patients who have the typical bi-directional or polymorphic VT, of whom 62 % of tests are positive, while in patients who do not have the typical symptoms or signs, less than 15 % of tests are positive.27 This may partly be due to the fact that in most cases, the entire RyR2 gene is not screened for mutations. Thus, the prevalence of mutations outside the three most common domains, and the importance of polymorphisms, is still unknown.26 Genetic screening is not recommended as the first diagnostic option for CPVT in index cases, and only analysis of the RyR2 gene is recommended on a routine basis when a decision to perform genetic has been made.28
However, genetic testing is suggested for first-degree relatives of patients with confirmed CPVT, even with a negative clinical phenotype.28
3.2.4 Cellular electrophysiology and RyR2 biology
In order to understand details in the pathophysiology of CPVT1, a brief introduction to normal cellular electrophysiology is necessary: The process from electrical excitation to contraction of
cardiomyocytes is termed the cardiac excitation-contraction coupling.29, 30 The contraction of each cardiomyocyte is initiated by a depolarization of the cell membrane. This depolarization starts from specialized cells in the sinoatrial node. The impulse spreads from cell to cell through gap junctions that ensure an efficient conduction of the depolarizing currents. Electrical impulses conducted through the atria depolarize cells in the atrioventricular node. In the atrioventricular node the impulse
conduction is slowed down. This gives the ventricles time to fill before activation. The electrical impulse will then spread through the Bundle of His and Purkinje fibres to reach cardiomyocytes in the
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ventricles. Any disturbance in the conduction of electrical impulses, or spontaneous impulses arising in this system, can lead to disrupted cardiac function, arrhythmias and SCD.
The normal and regular cardiomyocyte contraction is a result of ion channel activity, protein interactions and electrical activity described by the cardiac action potential.30 The action potential of ventricular cardiomyocytes can be described with five phases (Figure 1).31 In phase 0, depolarization is initiated by opening of voltage-gated Na+ channels. The early repolarization in phase 1 is determined by K+ channels allowing an outward flow of K+. In phase 2, L-type Ca2+ channels are active with an inward current of Ca2+. Ca2+ entering through the L-type Ca2+ channels binds to RyR2 and triggers release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+-induced Ca2+ release (CICR).15 Ca2+
released from the SR then binds to troponin C in the myofilaments and initiates cardiomyocyte contraction. In phase 3, the Ca2+ current is gradually inactivated. This repolarization phase is
dominated by outflow of K+. The cytosolic Ca2+ concentration is still high in this phase. For relaxation to occur, the SR Ca2+ ATPase (SERCA2) removes most of the Ca2+ from the cytosol. The remaining Ca2+ will be removed by the Na+/Ca2+ exchanger (NCX1), and the plasma membrane Ca2+-ATPase.32,
33 By the end of phase 3, the membrane potential is reduced back to the resting level. In phase 4, the resting membrane potential is approximately -80 mV and dominated by an outward leak of K+.
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Figure 1: Illustration of a cardiac action potential (left) and (right) key ion channels and transporters involved in excitation-contraction coupling in ventricular cardiomyocytes.
Left panel is modified with permission from Ikonnikov G, Wong E, and Chaudhry S, McMaster Pathophysiology Review, www.pathophys.org. Right panel is modified with permission from IEMR, www.iemr.no
RyR2 is the dominant isoform of RyR in the heart and the main SR Ca2+ release channel in cardiomyocytes during excitation-contraction coupling.34 RyR2 is one of the largest ion channel proteins, with 105 exons in the gene, and consists of 4976 amino acids.16 The RyR2 forms tetramers consisting of four 565 kd RyR2 proteins, four 12 kd FK-506 binding proteins and FKBP12.6 (Figure 2). The RyR2 protein consists of three major domains, termed the N-terminal domain, central domain and C-terminal domain. The first two domains are located in the cytoplasmic region of the protein, while the C-terminal domain consists of a transmembrane part.12 The C-terminal is a pore-forming domain, and the N-terminal forms a foot structure in the space between the T-tubule and the SR. The
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N-terminal domain acts as a regulatory part which is available for interactions with other proteins, regulating the conductivity of the pore.33, 35, 36
Each RyR2 subunit binds a single FKBP12.6 molecule, which is one of the major interaction partners of the channel.37 FKBP12.6 interacts with an N-terminal site, and stabilizes the closed state of RyR2.38 This function is important to prevent Ca2+ leak from the SR.38 Other important modulators of RyR2 are CaM,39 sorcin,40 protein kinase A (PKA),41 protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A),42 and Ca2+/calmodulin-dependent protein kinase type II (CaMKII).43 CaM is known to stabilize the closed state of RyR2.39 Sorcin is thought to play a role in stabilizing RyR2 both in resting conditions (preventing Ca2+ leak), but also when RyR2 is open (decreasing amplitude of Ca2+
transients).44 PKA and CaMKII phosphorylate RyR2 on serine 2808 (Ser2808) and serine 2814 (Ser2814), respectively,41, 43 while PP1 and PP2A dephosphorylate these sites.42 We will later discuss how phosphorylation and dephosphorylation of Ser2808 and Ser2814 affects RyR2 and SR Ca2+
release. Other proteins interacting with the RyR2 macromolecular complex are junctin and triadin, which anchor RyR2 to the SR membrane,45 and junctophilin-2, which ensures the connection to L-type Ca2+ channels in the plasma membrane.46
RyR2 activity is mainly regulated by phosphorylation of serine and threonine residues47 by PKA41 and CaMKII43, but also by protein kinase C.48 PKA phosphorylates RyR2 at Ser2808 in small rodents and humans, and Ser2809 in rabbits.49 There has been much focus on the hypothesis stating that hyper-phosphorylation of Ser2808 results in increased RyR2-dependent SR Ca2+ leak in HF.41 Some studies indicate that PKA-dependent phosphorylation increases the open probability of RyR by increasing the sensitivity of Ca2+ dependent activation.38, 41 It has also been suggested that PKA phosphorylation of Ser2808 leads to dissociation of FKBP12.6, destabilizing the RyR2 channel and increasing diastolic SR Ca2+ leak.41 However, these effects of PKA-dependent phosphorylation have been opposed in later studies.50, 51 Genetic ablation of Ser2808 was not protective against HF,52 and Ser2808 seems to be less involved in the pathogenesis and remodeling process in heart failure than Ser2814.53 In non-failing hypertrophy, however, PKA-dependent phosphorylation of RyR2 has a functional effect on the channel gating.53 This was found in a study where cardiomyocytes from mice with non-failing cardiac hypertrophy showed less Ca2+ sparks when adding a PKA inhibitor.53 The
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same study showed no changes in Ca2+ spark frequency in cardiomyocytes from mice with HF using the same PKA inhibitor.
Figure 2: Illustration of the RyR2 protein with some of the interaction sites. Modified from Mohler et al.54
RyR2 can be phosphorylated by CaMKII at Ser2814, or serine 2815 in some species.55 In normal physiological situations, without beta adrenoceptor stimulation or pacing, 16 % of Ser2814 residues and 54 % of Ser2808 residues are phosphorylated.56,55 The CaMKII pathway, unlike the PKA pathway, is thought to play an important role in pathological SR Ca2+ leak.57 Knock-in mice with an inactivated Ser2814 CaMKII phosphorylation site in RyR were more resistant than WT mice to developing cardiac fibrosis, pulmonary congestion and cardiac dysfunction following chronic beta adrenoceptor stimulation.58 CaMKII activation and phosphorylation of Ser2814 is also involved in the development of atrial fibrillation in angiotensin-exposed mice,59 while genetic ablation of Ser2814 protected against pacing-induced arrhythmias.60 Interestingly, mice with a constitutive activation of
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Ser2814 did not develop arrhythmias in basal conditions, without beta adrenoceptor stimulation, but developed VT and SCD in response to beta adrenoceptor stimulation.60 Thus, it seems that CaMKII- dependent phosphorylation of RyR2 can play an important role in the development of arrhythmias.
CaMKII can be activated by Ca2+, reactive oxygen species (ROS),61 glycosylation,62 and
nitrosylation.63 Furthermore, and important for Article 3 in this thesis, high HR also increases CaMKII activity, possibly due to increased intracellular Ca2+ cycling.64
Another mechanism for modulation of RyR2 activity is by oxidation. Production of ROS is increased by stimulation of beta adrenoceptors and angiotensin II receptors.65, 66 Oxidizing conditions increase the open probability of RyR2,67 but increased oxidative stress can also cause irreversible loss of channel activity.68 The FKBP12.6 binding domain on RyR2, which is responsible for keeping the channel closed, can become unstable by increased ROS levels, leading to SR Ca2+ leak.69 Increased oxidation of RyR2 also reduces the binding affinity between CaM and RyR2.69 Interpretation of ROS effects on RyR2 in cellular data is complicated since oxidation also affects the activity of RyR2 modulators and other Ca2+ handling proteins: Oxidation of CaM reduces its binding to RyR2, and oxidation of RyR2 also reduces the binding affinity between RyR2 and CaM.69 Important for Article 2 in this thesis, CaMKII can also be oxidized and activated by ROS, even without increased cytosolic Ca2+.70
3.2.5 Mechanisms for arrhythmias in CPVT1
Under physiological conditions, RyR2 is activated through Ca2+ entering the cell via the L-type Ca2+
channel, which initiates CICR. In CPVT1, where there is a mutation in the RyR2 gene, arrhythmias are initiated by increased leak of Ca2+ through the dysfunctional channel, causing DADs and triggered activity.38, 71, 72
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Figure 3: Illustration of a normally functioning RyR2 and a normal action potential (upper panel) with synchronized SR Ca2+ release. A dysfunctional channel due to a mutation in the RyR2 gene, leads to unsynchronized SR Ca2+ release, which can trigger delayed after depolarizations (DADs) and action potentials (lower panel). Both panels are modified and used with permission from IEMR, www.iemr.no
The molecular mechanism behind increased leak of Ca2+ caused by CPVT-associated mutations is still not determined, but different theories have been proposed.73 One theory is that FKBP12.6 dissociates from RyR2. Normal RyR2 function requires stabilization by FKBP12.6 to keep the channel closed during diastole. In some RyR2 mutations, the binding affinity between FKBP12.6 and RyR2 is thought to be weakened. In these mutations, PKA-dependent phosphorylation of RyR2 can lead to dissociation of FKBP12.6 from RyR2, which increases channel opening probability and might cause Ca2+ leak in diastole.38, 74 Another theory for SR Ca2+ leak CPVT1 is “store overload-induced Ca2+ release”, which suggests that a mutation in RyR2 leads to hypersensitivity to Ca2+ in the SR, reduced threshold for SR
21
Ca2+ release, and Ca2+ to spill-over in diastole.75 The third theory suggests that RyR2 mutations can lead to defective protein-protein interactions. Normally, “zipping” of the intramolecular structure stabilizes RyR2. A mutation causing unstable “zipping” between the N- and central domains, is suggested to cause SR Ca2+ leak.76 Regardless of the exact mechanism, the result of RyR2 dysfunction caused by CPVT-associated mutations is increased SR Ca2+ leak that activates the NCX1.38 The resulting net inward current causes depolarisation of the cell membrane, i.e. a DAD.77
In the bidirectional VT characteristic for CPVT1, the QRS axis rotates 180° from beat to beat.10 This can be hemodynamically tolerated, but might degenerate into rapid polymorphic VT and ventricular fibrillation (VF). It has been speculated that the bidirectional VT seen in CPVT1 might originate from the Purkinje fibers, and then develop into polymorphic VT.78 Purkinje fibers are more susceptible to Ca2+ overload than cardiomyocytes due to higher cytosolic Na+ concentration and prolonged action potential duration,79and were shown to be important for the development of focal arrhythmias in a CPVT1 model with the RyR2 R4496C mutation.80 When the right ventricular Purkinje network was chemically ablated, the bidirectional VT turned into monomorphic VT with wide QRS.80 The authors suggest that alternating firing of triggered activity from Purkinje fibers in the right and left ventricles might be responsible for the bidirectional morphology of VT in the RyR2 R4496C model.80
Paavola et al. confirmed that DADs and triggered activity was the mechanism for arrhythmogenesis in CPVT1 patients.81 They recorded endocardial monophasic action potentials during invasive electrophysiological procedures in patients with CPVT1, and found that DADs occurred even in the absence of beta adrenoceptor stimulation, but with an increased frequency in presence of beta adrenoceptor stimulation.81 None of the control patients developed DADs.
Experiments with the RyR2 R4496C knock-in mice supported these findings: Fifty percent of these mice developed polymorphic VT and/or bidirectional VT after an exercise test following beta adrenoceptor stimulation, similar to the arrhythmias seen in patients with CPVT1.71, 72
Electrophysiological recordings showed that beta adrenoceptor stimulation elicited DADs in ventricular cardiomyocytes from RyR2 R4496C mice.72 Similar findings have been made in other RyR2 mutation models (R176Q and R2474S).77, 82 Noteworthy, however, in all of these models, increased frequency of DADs in cardiomyocytes have also been found under normal physiological
22
conditions, without beta adrenoceptor stimulation.72, 77, 82 Similar results were obtained in HEK293 cells expressing RyRs with a CPVT1 mutation.83 The findings suggest that diastolic SR Ca2+ leak is present to some extent even in the absence of beta adrenoceptor stimulation in CPVT1.83
3.2.6 Clinical management of CPVT1
Beta adrenoceptor antagonists are the recommended first line medical treatment for patients with CPVT1.84 Genetically positive family members should receive beta adrenoceptor antagonists despite a negative exercise test.85 Leren et al. found that both the complexity and number of ventricular
arrhythmias (VA) triggered by exercise testing was lower during treatment with nadolol compared to metoprolol.12, 86 In clinical practice, the effect of medical treatment is evaluated by the arrhythmia tendency during exercise stress testing. However, VA even occur in some patients treated according to guidelines and with no arrhythmias during exercise stress testing.9-11 In one study, 46 % of patients on beta adrenoceptor antagonists still experienced episodes of VT during a mean follow-up period of 40 months.10 Important for the interpretation of these results, is the fact that individual variation in pharmacokinetics and pharmacodynamics complicate treatment, and that noncompliance-related SCD in CPVT1 patients is reported to be high.12
Flecainide can be added to a beta adrenoceptor antagonist for patients who continue to have symptoms.84 Flecainide inhibits fast Na+ channels,87 and might have a direct RyR2 blocking effect.87, 88 In one study, flecainide also reduced maximum HR, even if patients reached higher workload during exercise testing, compared to before flecainide treatment was started.89 Importantly, in several studies, flecainide reduced the occurrence of VA in CPVT1 patients with pronounced symptoms.87-89
Left cardiac sympathetic denervation might be considered in patients continuing to have symptoms despite treatment with beta adrenoceptor antagonists. 90 This treatment is now recommended for patients experiencing recurrent syncopes, polymorphic/bidirectional VT or inappropriate shocks from implantable cardioverter defibrillators (ICDs) despite recommended medical treatment.84 In one study, left cardiac sympathetic denervation reduced the percentage of
23
patients with major cardiac events from 100 % to 32 %.90 However, left cardiac sympathetic denervation is only available in a few centres worldwide.
The guidelines recommend ICD implantation to CPVT patients who are survivors of cardiac arrest or continue to have symptoms despite adequate treatment.84 However, the decision to implant an ICD in patients with CPVT is difficult, as the stress associated with discharges can lead to a vicious cycle and electrical storm.91
3.3 Potential antiarrhythmic effects of exercise training
Psychological and/or physiological stress such as exercise immediately reduces vagal tone and
increases sympathetic activity, resulting in an increase in HR, atrioventricular conduction velocity and cardiac contractility.92 Increased sympathetic activity increases cAMP production with subsequent PKA activation.93 ROS production can also increase during exercise, but the role of ROS in the normal exercise response is unknown.94 In CPVT, an increase in beta adrenoceptor activation during exercise increases SR Ca2+ leak through the already dysfunctional RyR2, potentially leading to arrhythmias.
Chronic exercise training (ET) reduces risk factors for cardiac disease and VA, including type 2 diabetes,95 obesity and hypercholesterolemia.96 Long-term ET reduces basal sympathetic activity and increases basal parasympathetic activity.97 Myocardial perfusion is also improved after aerobic ET.98,
99 ET can improve aerobic capacity, and aerobic interval training leads to a higher increase in maximal oxygen levels (VO2max) than moderate continuous training.100 Endurance capacity is limited by three major factors, which are: VO2max, lactate threshold and work economy.101, 102 To improve endurance performance, at least one of these factors have to increase. Experimental studies with rodents show an increase in VO2max after ET, but the effect of exercise seems to be dependent on the ET protocol.103 Long term ET is also known to improve Ca2+ handling in cardiomyocytes,104 and reduce SR Ca2+ leak and arrhythmias.105 This has been attributed to the effect of ET on the RyR receptor. There are also suggestions that other proteins involved in Ca2+ handling can be altered after ET. In particular, studies have detected both up-106, 107 and down- regulation106, 108 of NCX1 activity after ET in healthy rats. In
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disease models, however, ET was associated with decreased NCX1 activity.109 In a myocardial infarction model with dogs, the animals were first divided into “VT susceptible” and “VT resistant groups”. The animals in these two groups were then divided into subgroups of SED and ET. The NCX1 protein abundance was found to be increased in SED animals susceptible to VF compared to SED animals who were resistant to developing VF.110 After a 10 week ET program, NCX1 protein abundance was down-regulated in the animals with a high risk of VF, and no difference to the group which was resistant to VF. This suggests that NCX1 expression was increased following myocardial infarction, and that ET gave a beneficial reduction in NCX1.110 In another study, NCX1 protein abundance was also reduced following ET, and was comparable to the abundance in healthy animals.111 Others have found that high intensity ET improved left ventricular systolic contraction, diastolic filling and Ca2+ sensitivity, more than low intensity ET.104 The contraction-relaxation rates increased, and the rise and decay of Ca2+ transients were faster in cardiomyocytes from ET animals.
One suggested mechanism for the increased rate of rise of Ca2+ transients is a more effective coupling between the L-type Ca2+ channel and RyR2,112 while the faster diastolic Ca2+ decay can be explained by increased SERCA2 activity due to higher SERCA2 protein abundance and increased SERCA2/PLB ratio.113 However, increased SERCA2 abundance after ET is not found in all studies.114 The different effects on SERCA2 activity or protein abundance after ET are considered to be due to different ET programs, age of the animals and type of ET.114-118 In healthy Wistar rats, RyR2 expression increased after ET.118 More importantly, however, reduced Ca2+ leak through dysfunctional RyR2s has also been demonstrated after long term ET in diabetic cardiomyopathy.105 The mechanism behind this decrease in SR Ca2+ leak is thought to be CaMKII-dependent. However, chronic ET can also reduce ROS production, which might contribute to this effect.119 Based on these general considerations, ET has many potential antiarrhythmic effects that might be especially beneficial in CPVT.
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4 Aims of the thesis
The main aim of this thesis was to investigate whether exercise training stabilizes RyR2 function in CPVT1, and thereby reduces the probability of ventricular arrhythmias.
Specific aims:
Article 1
To test if exercise training is feasible and increases the arrhythmia threshold in patients with CPVT1.
Article 2
To test the effect of exercise training on arrhythmia tendency, SR Ca2+ leak and arrhythmogenic Ca2+
leak in mice with a CPVT1-causative RyR2 mutation.
Article 3
To separate the effects of heart rate and beta adrenoceptor stimulation on the occurrence of VA in CPVT1.
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5 Methods
This section discusses the methods used in Article 1-3 and highlights key aspects. Further details regarding the methods are found in the individual articles and in their respective supplementary materials.
5.1 Patient studies
5.1.1 Patient cohort
In Article 1 and 3, patients diagnosed with CPVT1 were enrolled from the Department of Cardiology, Oslo University Hospital Rikshospitalet, Oslo, Norway. All patients had confirmed CPVT1-associated RyR2 mutations and typical symptoms or VA during exercise testing, and did not have any other cardiac diseases, disabilities, comorbidity, or recent or pending surgery.
5.1.2 Exercise training and testing
We used ergometer bicycling for exercise testing and training. The most relevant alternative was treadmill running. Ergometer bicycle testing is the standard at the hospital from which the patients were recruited. Therefore, ergometer bicycling allowed the same form of exercise during training and testing. Also, the quality of ECG recordings is generally better during bicycling than treadmill running, and gradual regulation of HR is easier to control. During the ergometer bicycle test, the threshold for non-sustained VT was set. Exercise testing is usually performed according to the standard “Bruce protocol”.120, 121 We used a protocol where the patients started the test at 25 W and then increased by 25 W every 2 min. For patients who were not accustomed to exercise, the work load needed to be increased even more slowly, and was kept constant until an increase in HR was detected, then slowly increased while communicating with the patient. This was continued until the patients could not maintain 60 revolutions/min. Optimally, the exercise test protocol should follow the same progression of intensity for all patients. However, our aim for the exercise tests was that each patient
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should reach their maximum exercise capacity and VO2max. To achieve this, some of the patients were allowed to keep the workload at the same level for longer than 2 min, before it was further increased, so that they could reach VO2max with an increase in HR before development of fatigue.
Peripheral muscle fatigue in the lower limbs during ergometer bicycling (exercise testing and training) was common, reflecting a low baseline aerobic capacity in the participants compared to the normal population. This was expected as these patients had been advised to avoid exercise since the time of diagnosis.
We analysed ECGs for single VES, VES in bigeminy, couplets, triplets, non-sustained VT or sustained VT. The HR before VES in bigeminy was set as the threshold HR for each patient. If the patient did not develop any VAs during the bicycle test, the threshold was set at maximal HR or at the HR where single VESs occurred. As shown in previous studies, this threshold is reproducible, which made it possible to use this threshold to plan an individualized exercise program for each patient.122 The threshold in each individual patient was used as a marker to set the intensity for the exercise sessions and to measure the effect of ET on arrhythmia threshold in this group of patients. A safety margin of 5 beats per minute (bpm) was subtracted from the threshold HR after each exercise test, giving a “modified threshold HR for arrhythmias.” The target HR during the exercise sessions was calculated as 80-90 % and 50-60 % of the modified threshold HR in the high intensity and active rest periods. The patients were continuously motivated to regulate their effort to keep their pulse at the target frequency.
To ensure safety during exercise, at least two medically trained personnel were present during all sessions. All personnel participating went through a course in cardio-pulmonary resuscitation especially designed for the patient group and training sessions in Article 1. Local ambulance services responsible for the areas where training sessions occurred were informed about the project, as well as the time and place for each session. Standard semi-automatic defibrillators were available during all training sessions.
28 5.1.3 Pacing protocol
Article 3 includes the results of pacing through atrial electrodes. These electrodes were part of combined devices with ICD as the main function. Pacing was performed for 30 s at 10 bpm above the individual threshold for VES in each patient. The short and limited pacing protocol was chosen out of safety concerns, as these tests were performed during regular visits to the outpatient clinic as opposed to more extensive testing that would have required anesthesiological and invasive electrophysiological personnel and equipment.
5.2 Basic research methods
5.2.1 Mouse models and human arrhythmia syndromes
In this thesis, a transgenic mouse model was used to investigate mechanisms underlying clinical observations. Mouse models share some electrophysiological features with humans that make them suitable for studies of basic mechanisms, but important differences between mice and humans should be noted. For example, resting HR in an unstressed mouse is around 500 bpm.123 In mice, HR
increases 10 to 20 % during exercise, 124, 125 while in humans it can increase by 100 % or even 200 %.
The ECG in mice is also different from humans, as a prominent T wave clearly separated from the QRS-complex is absent.126 The small size of the mouse heart is suggested to make the heart less susceptible to sustained arrhythmias.127 Nevertheless, mouse models of CPVT share important ECG features with the human diseases.128
The duration of the mouse action potential is approximately 10 times shorter than the human action potential.129 Importantly, the plateau phase (phase 2) is much shorter or even absent in mice.
This is due to the lack of Ca2+ mediated depolarization phase in mice, which is responsible for the plateau phase in the human cardiomyocytes.129 SR Ca2+ content in humans increases with increasing
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HR or stimulation frequency, while in mice there is little or no increase in SR Ca2+ content with increasing stimulation frequency.130
5.2.2 The RyR2 R2474S CPVT mouse model
A transgenic mouse model of CPVT1 was used in Article 2 and 3. These mice are heterozygous for a missense mutation in the RyR2 gene (R2474S) identified in patients with CPVT1.77 The mutation leads to a gain of RyR2 function resulting in increased diastolic SR Ca2+ leak. The mechanism is suggested to be decreased binding of FKBP12.6,77 causing increased channel activation and sensitivity for phosphorylation. The same mechanism is suggested for other mutations causing CPVT1: The RyR R4496C mutation is suggested to cause decreased affinity between RyR2 and FKBP12.6,71 while some mutations on the C-terminal and central terminal affect RyR2 function independently of the
FKBP12.6. However, all CPVT-causative mutations result in disrupted cellular Ca2+ homeostasis and arrhythmogenic SR Ca2+ leak.131
5.2.3 Exercise training and testing
The ET protocol used in Article 2 was developed in pilot experiments with WT mice prior to initiation of experiments with mice with the human mutation of CPVT1 with the RyR2 R2474S mutation (RyR- RS mice). Although other methods than treadmill running could have been used, exercise intensity is not as easily controlled during swimming or voluntary wheel running. Injuries to nails, paws or the tail can occur during treadmill running. With proper adaptation to the protocol and continuous
surveillance, these types of injuries can be avoided. The electrical stimuli to encourage running might also be a stress factor to the mice, but in our experience mice learn to avoid this stimulus during the adaptation phase, and keep running even if the electrical stimulation is turned off. Otherwise, slight physical contact is enough to encourage running.
To detect the effect of ET, VO2max was measured once per week. The results were also used to set the speed for training sessions the following week. During the test we also collected data on how far each mouse ran. Many mice continued to run at higher speed, even after reaching their VO2max
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levels. Others have found similar results and suggested that the speed measured at VO2max during an exercise test should be used to assess exercise intensity.132 Any speed that the mice manage to run which was higher than the speed at VO2max was termed anaerobic distance capacity.132 This is thought to be determined by individual factors such as anaerobic metabolism and running motivation.
The anaerobic distance capacity in mice even of the same strain is known to vary between 30-40 %.133 In Article 2 the VO2max was measured in a closed chamber with each mouse running during an incremental speed increase. Six values of VO2 levels were measured at each speed. An average VO2
level was calculated from the last three VO2 measurements taken during each running speed. The speed at highest average VO2 was set as the maximal speed for each mouse. When a maximal speed was calculated for all mice, the average value for max speed for all ET mice was calculated and set as the speed for the following exercise sessions. If there were large differences within the group, the mice with lowest speed ran at a higher speed than the speed corresponding to their VO2max. If these mice struggled to run with the high speed group, these low speed mice had to exercise separately with lower speed corresponding to the speed at their VO2max level.
The threshold for exhaustion in the mice was defined as five electrical stimuli within 15 s.
When this occurred, the exercise test was terminated as soon as the 6 measurements for the current speed were obtained. In some cases, the mice did not seem to perform with maximal capacity during an exercise test. This was assumed to be the case when the mice either did not want to run at all, or did not run at the same speed as used during the ET protocol the previous week. Such mice were retested the following day. To reduce variation in test conditions, we preferred to use the same investigator to test each cohort of mice.
5.2.4 Telemetric ECG surveillance and echocardiography
The mice used in this thesis were anesthetized with isoflurane 2% inhalation combined with a 0.1 mg/kg subcutaneous injection of buprenorphine (Temgesic, RB Pharmaceuticals Ltd.) for analgesia.
Isoflurance has a cardio-depressive effect, however, this is less pronounced than for other anesthetics such as pentobarbital or ketamine.134, 135 Immediately after surgery the mice show an increased
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frequency of arrhythmias even with isoflurane. We have found that a recovery period of five days is necessary to avoid the effects of anesthesia and surgery. Therefore, implantation of telemetry devices was performed one week before ET was initiated. Echocardiography was performed at two time-points in Article 2. Light isoflurane anesthesia was also used during echocardiography. For this reason, VO2max testing was not performed immediately after echocardiography. Mice used for telemetry did not undergo echocardiography.
5.2.5 Cardiomyocyte isolation
All cellular experiments in the articles included in this thesis were performed with isolated
cardiomyocytes from the left ventricle. Ca2+ cycling in isolated cardiomyocytes is very similar to Ca2+
cycling in cardiomyocytes from intact tissue.136, 137 Collagenase was used to isolate cardiomyocytes.
Collagenase batches do not have standardized activity or mixture of associated proteases. Therefore, each batch of collagenase was tested on hearts from mice with the RyR-RS mutation. For cellular experiments, cardiomyocytes from SED and ET mice hearts were isolated at the same time, and the investigator performing these isolations were blinded to experimental group.
5.2.6 Field-stimulation of isolated cardiomyocytes
We triggered Ca2+ transients in the cardiomyocytes by field-stimulation. In vivo action potentials are initiated by a Na+ current into the cell. The depolarization of cell surface membrane and voltage change activates the L-type Ca2+ channel leading to Ca2+ induced Ca2+ release from the SR, triggering contraction of the cardiomyocytes. During field stimulation, the biphasic (or monophasic) electrical pulses are thought to create a brief electric field that triggers the activation of the L-type Ca2+ channel since the channel activity is voltage dependent. Potentially, this makes the activation of L-type Ca2+
channels in field stimulated cardiomyocytes independent of the Na+ current.138, 139
32 5.2.7 Western blotting
Tissues from mice hearts were used in western blot analysis in Article 2 and 3. To investigate the effects of phosphoproteins after ET, the hearts were exposed to both frequency and beta adrenoceptor stimulation. A modified Langendorff setup was used to achieve standardized stimulation. Hearts were then frozen in liquid nitrogen. Fast freezing is particularly important to avoid the effect of
phosphatases, as it is shown that increased time to freezing affects the degree of protein
phosphorylation. 140 Some of the results after western blotting turned out to detect small differences between the groups. These experiments were repeated several times to confirm reproducibility.
5.2.8 Mathematical modelling
Results from cellular experiments in Article 2 and 3 were used to make a computational model suitable to mimic Ca2+ handling and RyR2 function in CPVT1. This model was used in Article 3 and was developed based on an existing model described by Morotti et al.141 A detailed explanation is given in Article 3. The computational model was used to simulate different combinations of frequency and beta adrenoceptor stimulation.141 It was also possible to simulate CaMKII knock out.
5.2.9 Statistics and general aspects
In Article 1 and 3, unpaired t-tests were used to analyse the differences between groups of patients with regards to threshold HR for arrhythmias. In Article 2, a paired t-test was performed to compare the VO2max results between week 0 and week 2 for the ET group and SED group. When the ET group at week 2 was compared to the SED group at week 2, unpaired t-test was performed, but also a nested ANOVA method was used, which is a repeated measures approach to detect the differences between the groups. The same method was also used to analyse data from the cellular experiments in Articles 2 and 3. The only exception was Ca2+ spark analyses, where sparks per area within a set time after the last Ca2+ transient or wave were counted. This could possibly give variations in the absolute area in
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each cell. Also, many cells had zero sparks, i.e. the data did not have a normal distribution. Therefore, the Poisson test was used for these data, adjusting for a skewed distribution.
Both in Articles 2 and 3, cells from a single isolation were used for parallel experiments on different setups to improve project efficiency and data comparability, and to reduce the number of mice used. Hearts used for western blotting were harvested by the same investigator for all analyses.
Data analysis was performed by investigators blinded for group (SED vs ET).
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6 Brief summary of the main results in the thesis
6.1 Article 1
Effects of individualized exercise training in patients with catecholaminergic polymorphic ventricular tachycardia type 1
In Article 1 we investigated the effects of ET in patients with CPVT1. The individual threshold HR for VA in each patient was used to limit the intensity of the exercise, and to measure the effect of exercise training on the propensity for VA. On average, the ET patients completed 28±3 (n±SEM) exercise sessions (78±8 % program completion) with 13±3 % increase in VO2max vs. baseline (20.2±1.6 ml/kg/min vs. 17.9±1.3 ml/kg/min, p<0.05). Average VO2max was unaltered throughout the study period for SED patients (31.1±4.1 ml/kg/min vs 29.5±4.2 ml/kg/min, n.s.). No adverse events occurred. Baseline threshold for VA was 100±6 bpm in ET patients and 135±4 bpm in SED patients.
After the training period, threshold HR for VA was 111±10 bpm in ET patients and 123±6 bpm in SED patients. The threshold for VA increased in ET compared to SED patients (+11 bpm vs. -12 bpm, p<0.05). Six months after completion of the exercise program, the ET patients no longer exhibited differences in VO2max or threshold HR for arrhythmias compared to baseline.
6.2 Article 2
Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII- dependent arrhythmogenic Ca2+ release
In Article 2 we investigated the mechanisms behind the results from Article 1. Mice with CPVT1 (RyR-RS mice) were used, and randomized into ET and SED. We found that two weeks of ET increased VO2max by 10±2 % (133.3±1.7 vs 144.7±1.8, p<0.05), while no changes were found in SED mice 1±1 % (135.2±1.7 vs 135.8±2.0, n.s.). The ET group showed fewer episodes of VT
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compared to SED, coinciding with fewer Ca2+ sparks and waves, less diastolic Ca2+ leak from the SR, and lower phosphorylation levels at RyR2 sites associated with CaMKII-dependent phosphorylation compared with SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-L-cysteine reduced Ca2+ wave frequency in cardiomyocytes from SED equally to cells from ET. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after ET. Two weeks of detraining reversed the beneficial effects of the interval treadmill ET protocol in RyR-RS ET.
6.3 Article 3
Arrhythmia initiation in catecholaminergic polymorphic ventricular tachycardia type 1 depends on both heart rate and sympathetic stimulation
In Article 3 we investigated the relative roles of HR associated factors and beta adrenoceptor
stimulation for arrhythmia initiation in CPVT1. ECGs from 17 patients with CPVT1 recorded during a standard bicycle stress-test showed that VAs were HR associated. Atrial pacemaker stimulation in four patients did not induce VES even at higher frequencies than the threshold HR for VES during the bicycle test. Whole-cell Ca2+imaging was performed in isolated ventricular cardiomyocytes from mice with the RyR2-R2474S missense mutation (RyR2-RS). These cardiomyocytes were stimulated in presence and absence of ISO at 0.5, 4 and 8 Hz. In absence of ISO, no differences in Ca2+ wave frequency were found between wild-type (WT) and RyR-RS. However, in the presence of ISO, Ca2+
wave frequency was higher in RyR2-RS (p<0.05). RyR-RS developed Ca2+ waves at lower SR Ca2+
content compared to WT (p<0.05). A computational model of CPVT1 that recapitulated differences in Ca2+ wave frequency and latency between RyR RS and WT was employed. This model suggested that the effect of frequency and beta adrenoceptor stimulation in CPVT1 were due to interactions between kinetics of SR Ca2+ reloading and CaMKII-dependent RyR2 phosphorylation.
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7 Discussion
7.1 Feasibility of exercise training in CPVT1 and effects on aerobic capacity
According to current guidelines, it is not recommended for patients with CPVT1 to be involved in competitive sports or perform endurance ET.142 However, there is a lack of data on the effect of monitored ET in CPVT1. The low prevalence of the disease and the high risk of syncope and VT during ET might be the reasons for why this type of study has not previously been performed in this patient group. Admittedly, to use ET as therapy in a condition characterized by exercise-induced arrhythmias might seem counterintuitive at first. However, we are not the first to test this idea in CPVT: One group investigated the effect of ET in a mouse model of CPVT2, showing a beneficial effect of ET.143 The rare autosomal recessive variant of CPVT caused by mutations in the
calsequestrin 2 gene stands for only 2 % of all CPVT cases, and the potential for extrapolation to other
diseases is much lower than for CPVT1. CPVT1, with a mutation in the RyR2 gene, gives an isolated dysfunction in RyR2 and thereby shares a key pathophysiological aspect with common diseases such as heart failure and ischemic heart disease. We are the first group to investigate the effects of ET in patients (Article 1) and mice (Article 2) with CPVT1.
The patients with CPVT1 included in Article 1 of this thesis had notably lower absolute VO2max than the general population of comparable age.144, 145 The most obvious reason for this is exercise restrictions. Ongoing treatment with beta adrenoceptor antagonists might also explain the low VO2max.146 However, little is known about the hemodynamics and aerob capacity of patients with CPVT1, especially in the absence of treatment. Leenhardt et al. showed that there was a marked bradycardia at rest in the CPVT patients included in their study.9 This was before the initiation of treatment with beta adrenoceptor antagonists, and might indicate that the cardiac function in CPVT patients is affected in more ways than only the increased propensity for arrhythmias. Nevertheless, we found that ET increased VO2max by 13 % in patients with CPVT1, which is comparable to studies of
