Phospholamban p.Arg14del cardiomyopathy

University of Groningen

Phospholamban p.Arg14del cardiomyopathy te Rijdt, Wouter

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te Rijdt, W. (2019). Phospholamban p.Arg14del cardiomyopathy: Clinical and morphological aspects supporting the concept of arrhythmogenic cardiomyopathy. Rijksuniversiteit Groningen.

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Phospholamban p.Arg14del cardiomyopathy

Wouter P. te Rijdt

Phospholamban p.Arg14del cardiomyopathy

Clinical and morphological aspects supporting the

concept of arrhythmogenic cardiomyopathy

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Layout Bianca Pijl, www.pijlldesign.nl

Groningen, the Netherlands

Cover design Bianca Pijl Printed by Ipskamp Printing

Enschede, the Netherlands

ISBN 978-94-034-1492-8 (print)

978-94-034-1491-1 (digital)

© Copyright: 2019 W.P. te Rijdt, Groningen, the Netherlands

All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author, or when appropriate, of the publishers of the publications included in this thesis.

Financial support by the University of Groningen, the Graduate School of Medical Sciences, Stichting PLN, Stichting C & W de Boer and the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged.

The research described in this thesis was supported by a grant of the Dutch Heart Foundation (DHF CVON2012-10 PREDICT), Fondation Leducq (CurePLaN), Young Talent Program grant (CVON PREDICT; 2017T001), and ZonMW Goed Gebruik Geneesmiddelen grant (836011002).

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Phospholamban p.Arg14del cardiomyopathy

Clinical and morphological aspects supporting the concept of arrhythmogenic cardiomyopathy

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties

De openbare verdediging zal plaatsvinden op woensdag 27 maart 2019 om 12.45 uur

door

Wouter P. te Rijdt geboren op 10 september 1985

te Groningen

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Promotores

Prof. dr. M.P. van den Berg Prof. dr. R.A. de Boer Prof. dr. A.J.H. Suurmeijer Copromotor

Dr. J.P. van Tintelen Beoordelingscommissie Prof. dr. V.V.A.M. Knoers Prof. dr. P.G.A. Volders Prof. dr. A.C. van der Wal

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5 Paranimfen

Thomas Vernooij David Vanneste

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Chapter 1 Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Table of contents

PART I - GENERAL INTRODUCTION

Introduction and outline of the thesis

Arrhythmogenic cardiomyopathy: pathology, genetics and concepts in pathogenesis

Cardiovascular Research. 2017; 113: 1521-1531.

Clinical utility of genetic testing for arrhythmogenic right ventricular cardiomyopathy

European Journal of Human Genetics. 2014; 22: e1-4.

PART II - MORPHOLOGICAL ASPECTS

Phospholamban p.Arg14del cardiomyopathy is characterized by phospholamban aggregates, aggresomes, and autophagic degradation Histopathology. 2016; 69: 542-550.

Phospholamban immunostaining is a highly sensitive and specific method for diagnosing phospholamban p.Arg14del cardiomyopathy Cardiovasc Pathol. 2017; 30: 23-26.

High resolution systematic digital histological quantification of cardiac fibrosis and adipose tissue in phospholamban p.Arg14del cardiomyopathy PLoS One. 2014; 9: e94820.

Distinct molecular signature of phospholamban p.Arg14del arrhythmogenic cardiomyopathy

Cardiovasc Pathol. 2018; 40: 2-6. [Epub ahead of print]

PART III - PHENOTYPICAL INSIGHTS USING MULTIPLE IMAGING MODALITIES

Myocardial fibrosis as an early feature in phospholamban p.Arg14del mutation carriers: phenotypic insights using cardiovascular magnetic resonance imaging

Eur Heart J Cardiovasc Imaging. 2019; 20: 92-100.

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7 Chapter 9

Chapter 10

Chapter 11

Chapter 12 Chapter 13

Early biventricular abnormalities in presymptomatic phospholamban p.Arg14del mutation carriers detected by echocardiography.

Manuscript in preparation.

Incremental prognostic value of late gadolinium enhancement in early-stage phospholamban p.Arg14del cardiomyopathy

Manuscript in preparation.

PART IV - PREVENTIVE TREATMENT

Rationale and design of the intervention in PHOspholamban RElated CArdiomyopathy STudy (i-PHORECAST).

Manuscript in preparation.

PART V - SUMMARY AND GENERAL DISCUSSION

Summary

General discussion PART VI - APPENDIX Nederlandse samenvatting List of abbreviations List of publications About the author Dankwoord

145

159

171

187 193

203 209 215 221 225

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Introduction and outline of the thesis

CHAPTER 1

PART I - GENERAL INTRODUCTION

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11 1.1 General introduction

Cardiomyopathies are a major cause of heart disease worldwide, aff ecting around 7.9 million people in all age groups.^1,2 In the Netherlands, the prevalence of cardiomyopathy is approximately 40,000 (0.25%).³ Cardiomyopathies comprise a group of heart disorders where a primary defect in the cardiac myocytes leads to abnormal structure and/or function of the myocardium. Therefore, other possible secondary causes (e.g. coronary artery disease, hypertension and valvular heart disease) must fi rst be excluded in order to make the diagnosis. The most common forms, classifi ed according to their phenotypic expression, are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy (ACM), but overlapping phenotypes do exist and are well recognized nowadays.⁴ They can be further classifi ed into inherited forms and non-inherited forms (e.g. DCM due to alcohol abuse or HCM due to amyloidosis etc.). In inherited forms the inheritance pattern is most often autosomal dominant, but autosomal recessive, X-linked and mitochondrial inheritance patterns have been observed.

Inherited cardiomyopathies are typically late-onset diseases: usual start of symptoms is in adult life although (severe) phenotypical expression in childhood has been described. In general, symptomatology in individuals carrying a cardiomyopathy-related mutation steadily increases until advanced age but some carriers will remain unaff ected, i.e. incomplete and age-related penetrance.

Since the discovery of the fi rst causative mutations for cardiomyopathies in the early 1990s, the importance of identifying the underlying genetic cause has been increasingly recognized.^5,6 Moreover, in the past decade the development and implementation of next- generation sequencing (NGS) techniques have allowed identifi cation of a still increasing number of pathogenic gene mutations related to human cardiomyopathies. These discoveries have led to a better understanding of disease pathogenesis and introduced genetic evaluation into clinical practice for aff ected individuals and their relatives via genetic cascade screening.^6,7 This allows early identifi cation of asymptomatic mutation carriers who may be at increased risk for sudden cardiac death even before the onset of any symptoms.

This thesis focuses on ACM caused by the non-desmosomal c.40_42delAGA (p.Arg14del) mutation in the phospholamban (PLN) gene.

1.2 Arrhythmogenic cardiomyopathy

ACM is a heritable myocardial disorder characterized by fi bro-fatty replacement of the myocardium that predisposes patients to ventricular arrhythmias and to slowly progressive ventricular dysfunction.⁸ Sudden cardiac death may be the presenting symptom in up to 50% of index cases.⁹ ACM encompasses a broad spectrum of disease that includes the classical right-dominant forms (ARVC, arrhythmogenic right ventricular cardiomyopathy), predominant left-sided involvement (also referred to as left-dominant arrhythmogenic cardiomyopathy (LDAC) and biventricular subtypes. The diagnosis is based on International Task Force Criteria (table1), which were modifi ed in 2010 but are in its current form still focused on the right-dominant form of ACM.¹⁰

Introduction and outline of the thesis

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12 Chapter 1

The pathology, genetics, pathogenesis, translational aspects and the clinical utility of genetic testing in ACM will be illustrated and discussed extensively in chapter 2 and 3. Mutations in genes encoding proteins of the cardiac desmosome are found in up to 60% of ACM-cases.^11,12 However, more recently, non-desmosomal genes have also been identified. One of these is the PLN gene.

Table 1. 2010 revised Task Force criteria for the diagnosis of ARVCTable 1. 2010 revised Task Force criteria for the diagnosis of ARVC

Revised Task Force criteria

I. Global or regional dysfunction and structural alterations*

Major By 2D echo:

 Regional RV akinesia, dyskinesia, or aneurysm

 and 1 of the following (end diastole):

- PLAX RVOT ≥32 mm (corrected for body size [PLAX/BSA] ≥19 mm/m²) - PSAX RVOT ≥36 mm (corrected for body size [PSAX/BSA] ≥21 mm/m²) - or fractional area change ≤33 percent

By MRI:

 Regional RV akinesia or dyskinesia or dyssynchronous RV contraction

 and 1 of the following:

- Ratio of RV end-diastolic volume to BSA ≥110 mL/m² (male) or ≥100 mL/m² (female) - or RV ejection fraction ≤40 percent

By RV angiography:

 Regional RV akinesia, dyskinesia, or aneurysm Minor By 2D echo:

 Regional RV akinesia or dyskinesia

 and 1 of the following (end diastole):

- PLAX RVOT ≥29 to <32 mm (corrected for body size [PLAX/BSA] ≥16 to <19 mm/m²) - PSAX RVOT ≥32 to <36 mm (corrected for body size [PSAX/BSA] ≥18 to <21 mm/m²) - or fractional area change >33 percent to ≤40 percent

By MRI:

 Regional RV akinesia or dyskinesia or dyssynchronous RV contraction

 and 1 of the following:

- Ratio of RV end-diastolic volume to BSA ≥100 to <110 mL/m² (male) or ≥90 to <100 mL/m² (female)

- or RV ejection fraction >40 percent to ≤45 percent II. Tissue characterization of wall

Major  Residual myocytes <60 percent by morphometric analysis (or <50 percent if estimated), with fibrous replacement of the RV free wall myocardium in ≥1 sample, with or without fatty replacement of tissue on endomyocardial biopsy

Minor  Residual myocytes 60 percent to 75 percent by morphometric analysis (or 50 percent to 65 percent if estimated), with fibrous replacement of the RV free wall myocardium in ≥1 sample, with or without fatty replacement of tissue on endomyocardial biopsy

III. Repolarization abnormalities

Major  Inverted T waves in right precordial leads (V1, V2, and V3) or beyond in individuals >14 years of age (in the absence of complete right bundle-branch block QRS ≥120 ms)

Minor  Inverted T waves in leads V1 and V2 in individuals >14 years of age (in the absence of complete right bundle-branch block) or in V4, V5, or V6

 Inverted T waves in leads V1, V2, V3, and V4 in individuals >14 years of age in the presence of complete right bundle-branch block

IV. Depolarization/conduction abnormalities

Major  Epsilon wave (reproducible low-amplitude signals between end of QRS complex to onset of the T wave) in the right precordial leads (V1 to V3)

Minor  Late potentials by SAECG in ≥1 of the following 3 parameters in the absence of a QRS duration of ≥110 ms on the standard ECG

 Filtered QRS duration (fQRS) ≥114 ms

 Duration of terminal QRS <40 µV (low-amplitude signal duration) ≥38 ms

 Root-mean-square voltage of terminal 40 ms ≤20 µV

 Terminal activation duration of QRS ≥55 ms measured from the nadir of the S wave to the end of the QRS, including R', in V1, V2, or V3, in the absence of complete right bundle-branch block

V. Arrhythmias

Major  Nonsustained or sustained ventricular tachycardia of left bundle-branch morphology with superior axis (negative or indeterminate QRS in leads II, III, and aVF and positive in lead aVL)

Minor  Nonsustained or sustained ventricular tachycardia of RV outflow configuration, left bundle-branch block morphology with inferior axis (positive QRS in leads II, III, and aVF and negative in lead aVL) or of unknown axis

 >500 ventricular extrasystoles per 24 hours (Holter) VI. Family history

Major  ARVC/D confirmed in a first-degree relative who meets current Task Force criteria

 ARVC/D confirmed pathologically at autopsy or surgery in a first-degree relative

 Identification of a pathogenic mutation^¶ categorized as associated or probably associated with ARVC/D in the patient under evaluation

Minor  History of ARVC/D in a first-degree relative in whom it is not possible or practical to determine whether the family member meets current Task Force criteria

 Premature sudden death (<35 years of age) due to suspected ARVC/D in a first-degree relative

 ARVC/D confirmed pathologically or by current Task Force Criteria in second-degree relative

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13 III. Repolarization abnormalities

Major  Inverted T waves in right precordial leads (V1, V2, and V3) or beyond in individuals >14 years of age (in the absence of complete right bundle-branch block QRS ≥120 ms)

Minor  Inverted T waves in leads V1 and V2 in individuals >14 years of age (in the absence of complete right bundle-branch block) or in V4, V5, or V6

 Inverted T waves in leads V1, V2, V3, and V4 in individuals >14 years of age in the presence of complete right bundle-branch block

IV. Depolarization/conduction abnormalities

Major  Epsilon wave (reproducible low-amplitude signals between end of QRS complex to onset of the T wave) in the right precordial leads (V1 to V3)

Minor  Late potentials by SAECG in ≥1 of the following 3 parameters in the absence of a QRS duration of ≥110 ms on the standard ECG

 Filtered QRS duration (fQRS) ≥114 ms

 Duration of terminal QRS <40 µV (low-amplitude signal duration) ≥38 ms

 Root-mean-square voltage of terminal 40 ms ≤20 µV

 Terminal activation duration of QRS ≥55 ms measured from the nadir of the S wave to the end of the QRS, including R', in V1, V2, or V3, in the absence of complete right bundle-branch block

V. Arrhythmias

Major  Nonsustained or sustained ventricular tachycardia of left bundle-branch morphology with superior axis (negative or indeterminate QRS in leads II, III, and aVF and positive in lead aVL)

Minor  Nonsustained or sustained ventricular tachycardia of RV outflow configuration, left bundle-branch block morphology with inferior axis (positive QRS in leads II, III, and aVF and negative in lead aVL) or of unknown axis

 >500 ventricular extrasystoles per 24 hours (Holter) VI. Family history

Major  ARVC/D confirmed in a first-degree relative who meets current Task Force criteria

 ARVC/D confirmed pathologically at autopsy or surgery in a first-degree relative

 Identification of a pathogenic mutation^¶ categorized as associated or probably associated with ARVC/D in the patient under evaluation

Minor  History of ARVC/D in a first-degree relative in whom it is not possible or practical to determine whether the family member meets current Task Force criteria

 Premature sudden death (<35 years of age) due to suspected ARVC/D in a first-degree relative

 ARVC/D confirmed pathologically or by current Task Force Criteria in second-degree relative

Diagnostic terminology for revised criteria:

Defi nite diagnosis: 2 Major, OR 1 Major and 2 Minor criteria, OR 4 Minor from diff erent categories Borderline diagnosis: 1 Major and 1 Minor, OR 3 Minor criteria from diff erent categories Possible diagnosis: 1 Major, OR 2 Minor criteria from diff erent categories

PLAX indicates parasternal long-axis view; RVOT: RV outfl ow tract; BSA: body surface area; PSAX: parasternal short- axis view; aVF: augmented voltage unipolar left foot lead; aVL: augmented voltage unipolar left arm lead.

* Hypokinesis is not included in this or subsequent defi nitions of RV regional wall motion abnormalities for the proposed modifi ed criteria.

¶ A pathogenic variant is a DNA alteration associated with ARVC/D that alters or is expected to alter the encoded protein, is unobserved or rare in a large non-ARVC/D control population, and either alters or is predicted to alter the structure or function of the protein or has demonstrated linkage to the disease phenotype in a conclusive pedigree.

# Adapted from Marcus et al. (2010)¹⁰

Introduction and outline of the thesis

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1.3 Phospholamban

Phospholamban, encoded by the PLN gene (locus 6q22.31; OMIM #172405), is expressed in the sarcoplasmic reticulum (SR) membrane as a 52 aminoacids 30-kD homopentameric phosphoprotein. PLN is a reversible regulator of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) pump.^13,14 It alters the Ca²⁺ affinity of SERCA in cardiac muscle (SERCA2a isoform) by a mechanism that depends on PLN’s phosphorylation. PLN inhibits the calcium uptake by SERCA2a in the non- phosphorylated state. When phosphorylated, by cAMP-dependent protein kinase (at Ser16; i.e. via the beta-adrenergic pathway, figure 1) or Ca²⁺/calmodulin-dependent protein kinase II (at Thr17;

predominantly during pathophysiological conditions), PLN dissociates from SERCA2a resulting in a higher activation with increased SR Ca²⁺ uptake, accelerated relaxation, enhanced SR Ca²⁺

load and increased Ca²⁺ release during systole. In this way, dynamic PLN/SERCA interaction plays an important role in regulating intracellular calcium homeostasis and subsequent cardiac contractility and relaxation.

Figure 1. Function of phospholamban (PLN). PLN is a reversibly phosphorylated transmembrane protein, which binds to and regulates the activity of SERCA2a, the sarcoplasmic reticulum Ca²⁺-ATPase pump. (From: MacLennan et al. Nat Rev Mol Cell Biol. 2003)¹³

Chapter 1

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15 1.4 Mutations in the PLN gene

As to be expected because of the key role of PLN in cardiac calcium cycling and pathophysiology, genetic variants in the PLN gene may cause human inherited cardiomyopathies. However, the exact underlying mechanism(s) have not been fully elucidated yet. So far, fi ve naturally occurring mutations have been identifi ed in humans in the coding region of PLN (table 2).

The fi rst mutation described in the PLN gene was c.146T>G (p.(Val49Gly)) which results in potent inhibition of the Ca²⁺ affi nity of SERCA2a. Cardiac overexpression of the p.(Val49Gly) mutant PLN in mice led to super-inhibition of cardiac contractility and remodeling, with progression to DCM, heart failure and early death.¹⁵

The second PLN mutation, c.25C>T (p.(Arg9Cys)), was shown to have no eff ects on SERCA2a activity under basal conditions but appeared to prevent phosphorylation of endogenous PLN, resulting in chronic inhibition of SERCA2a activity by PLN. Such chronic inhibition, never being able to draw on the full cardiac reserve, resulted in DCM and heart failure.¹⁶

The third mutation, c.116T>G (p.(Leu39*)), results in a truncated form of PLN without inhibitory eff ect on the calcium affi nity of SERCA2a. Heterozygous carriers had hypertrophy but no signs of cardiac contractile dysfunction. However, in homozygous carriers onset of DCM and heart failure during the teenage years was observed.¹⁷

The fourth PLN mutation described, c.40_42delAGA, resulting in a deletion of Arg-14 (p.Arg14del). The current knowledge and new insights regarding this mutation will be discussed in this thesis (starting from chapter 1.5).

The fi fth, most recently identifi ed, mutation c.73C>T p.Arg14del was found in a pedigree with DCM and malignant ventricular arrhythmia. Both SR Ca²⁺- uptake (super-inhibition of SERCA2a due to enhanced interaction between the p.(Arg25Cys) mutant PLN and SERCA2a) and SR Ca²⁺- leak (increased due to increased Cam kinase II activity associated with hyper-phosphorylation of Serine 2814 in the ryanodine receptor) seem to be impacted in this mutation, leading to depressed myocyte contractile and Ca²⁺-kinetic parameters with increased arrhythmias.¹⁸ Table 2. Overview of human PLN mutations and phenotypic characteristics

PLN, phospholamban; DCM, dilated cardiomyopathy; HF, heart failure; ACM, arrhythmogenic cardiomyopathy;

VA, ventricular arrhythmia.

Introduction and outline of the thesis

1

PLN mutation Phenotype Reference

c.146T>G, p.(Val49Gly) DCM, HF, premature death ¹⁵ c.25C>T, p.(Arg9Cys) DCM, HF, premature death ¹⁶

c.116T>G, p.(Leu39*) DCM, HF, hypertrophy ¹⁷

c.40_42delAGA,

p.(Arg14del) DCM, ACM, early myocardial fibrosis ^19-25

c.73C>T, p.(Arg25Cys) DCM, VA ¹⁸

.

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1.5 The PLN p,Arg14del mutation

This mutation, a deletion of three nucleotides (c.40_42delAGA) in the coding region of the PLN gene leading to deletion of amino acid arginine (p.Arg14del) in the PLN protein, was first described in a large Greek family with hereditary DCM,¹⁹ soon followed by others.^20,21 Heterozygous carriers were shown to exhibit inherited DCM, both mild and severe forms were observed, showing episodic malignant ventricular arrhythmias and left-ventricular dilation with contractile dysfunction leading to overt heart failure in some cases.

Van der Zwaag et al. screened a large cohort of Dutch DCM and ARVC patients for PLN- mutations and found the pathogenic PLN p.Arg14del variant to be present in ≈15% of patients clinically diagnosed with idiopathic DCM and ≈12% of patients with ARVC.²² Interestingly, a significant overlap between phenotypes was observed, in which left- or right-sided forms may predominate, compatible with the concept of ACM. Using haplotype analysis and postal code mapping, the pathogenic PLN p.Arg14del variant was characterized as a common Dutch founder mutation²³ (figure 2), but carriers have meanwhile also been identified in many other European countries (Germany, Belgium, Spain, the United Kingdom and Norway), Canada, and the USA.

By now over 1000 carriers of the PLN p.Arg14del mutation have been identified (http://www.

phorecast.nl), making this mutation the most prevalent single cardiomyopathy-related mutation identified in the Netherlands.

Figure 2. Postal code map illustrating the likely origin of the founder haplotype containing the PLN p.Arg14del mutation. The number of points based on the grandparents’ birthplaces is shown (in parenthesis: the number of postal code regions, 90 in total). On average, each region contains 180,000 inhabitants. The province of Friesland is enclosed by the bold border. (From: van der Zwaag et al. Neth Heart J. 2013; 21: 286-93.)

Chapter 1

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17 Analogous to other inherited cardiomyopathies (see 1.1), the natural course of the disease is age- related (“age-related penetrance”); after a presymptomatic phase of variable length many carriers progress to overt disease, and are diagnosed with ACM. Symptomatic carriers were found to often exhibit a malignant arrhythmogenic phenotype, refl ected by high rates of VT/VF as a presenting symptom, appropriate ICD interventions, and a positive family history for premature sudden cardiac death (SCD).²² Besides the high risk for malignant ventricular arrhythmias, a high risk of end-stage heart failure with subsequent high mortality was observed with a poor prognosis from late adolescence.²⁴ Left ventricular ejection fraction of <45% and sustained or nonsustained ventricular tachycardia were identifi ed as independent risk factors for malignant ventricular arrhythmias. Electrocardiographically, the p.Arg14del pathogenic variant is characterized by low amplitudes of the QRS complexes on the surface ECG and repolarization abnormalities (present in 41-46 % of carriers).^{21, 23, 24} These ECG changes, which often occur early, are likely a refl ection of myocardial fi brosis.

Haghighi et al. extensively studied the eff ects of the PLN p.Arg14del pathogenic variant in an overexpression mouse model.¹⁹ Transgenic mice overexpressing this variant exhibited depressed cardiac function, histopathological abnormalities (i.e. extensive myocardial fi brosis), and premature death, recapitulating the phenotype of the human mutation carriers. It remains, however, unclear how exactly the PLN p.Arg14del mutation leads to such severe cardiomyopathy and arrhythmia.

Experimental evidence from the mouse model has suggested a link between the PLN p.Arg14del mutation and impairment of cardiac Ca²⁺ cycling: SERCA2a super inhibition due to a disturbance in the structure of PLN (partial destabilization of PLN’s pentameric structure, leading to the production of highly inhibitory monomers and a persistent PLN-SERCA association).^{19, 25} As a result, phosphorylation is still possible but the inhibitory eff ect is no longer relieved. Coexpression of wild-type PLN and mutant PLN-R14Del in human embryonic kidney (HEK) cells, confi rmed the super inhibition of the SERCA channel. The dominant inhibitory eff ect of this mutant was not alleviated by phosphorylation, leading to myocellular calcium dysregulation, calcium overload, cardiomyocyte damage, and eventually to myocardial fi brosis.

Introduction and outline of the thesis

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Aims and outline of this thesis

This thesis presents multiple clinical and morphological studies in PLN p.Arg14del mutation carriers. Considering the ever-growing number of mutation carriers identified and the importance of more detailed phenotyping PLN p.Arg14del cardiomyopathy to improve early diagnostic-, risk stratification- and treatment options, the aims of this thesis were:

To study the morphological characteristics and molecular signature of PLN p.Arg14del cardiomyopathy; and

To further elucidate the clinical phenotype using different imaging modalities; and

To evaluate the clinical implications of these findings on prognostication, i.e. follow-up study, and treatment, i.e. intervention trial.

Part I provides a general introduction. Chapter 2 and 3 describe the genetics, pathology, pathogenesis, translational aspects and the clinical utility of genetic testing in ACM.

Part II of this thesis focuses on the morphological features of phospholamban p.Arg14del cardiomyopathy. In Chapter 4 and 5 the characteristics and localization of PLN protein aggregates in complete heart, LV myocardial apex samples and right ventricular endomyocardial biopsy samples are shown, studied using both light- and electron microscopy. In Chapter 6 and 7 distinct pathological characteristics of PLN p.Arg14del cardiomyopathy are presented. In complete heart specimens we studied differential protein distribution patterns, using multiple immunohistochemical markers, and the fibrosis pattern in PLN p.Arg14del cardiomyopathy. In a selected group the presence of fibrosis was quantified using a high resolution systematic digital quantification technique.

Part III presents phenotypical insights into phospholamban p.Arg14del cardiomyopathy using different imaging modalities. In Chapter 8 the results of a large multicenter cardiac magnetic resonance (CMR) study, consisting of mainly presymptomatic mutation carriers, are described. Our main focus for this study was the presence of myocardial fibrosis in this group, and the association between myocardial fibrosis, electrocardiographical findings and the occurrence of ventricular arrhythmia. Chapter 9 presents a echocardiographic study in presymptomatic PLN p.Arg14del mutation carriers where we investigated whether subtle abnormalities in cardiac structure and function can already be observed in this group. In Chapter 10, the follow-up results of the CMR cohort (chapter 8) are shown. Our aim was to investigate whether the presence of late gadolinium enhancement on CMR is of Incremental prognostic value in early-stage phospholamban p.Arg14del cardiomyopathy.

Part IV, Chapter 11, part IV, describes the design and rationale of iPHORECAST (intervention in PHOspholamban RElated CArdiomyopathy Study). Cardiac fibrosis appears to be an early feature of PLN p.Arg14del cardiomyopathy, occurring in many presymptomatic mutation carriers before onset of overt disease. No proven treatment is available for this group. We designed and initiated iPHORECAST to demonstrate that pre-emptive treatment of presymptomatic PLN p.Arg14del mutation-carriers with eplerenone reduces disease progression and postpones onset of overt disease. The study has a multicenter, prospective, randomized, open-label, blinded endpoint (PROBE) design with a follow-up time of three years.

Chapter 1

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19 References

McKenna WJ, Maron BJ, Thiene G. Classifi cation, Epidemiology, and Global Burden of Cardiomyopathies.

Circ Res. 2017 Sep 15;121(7):722-30.

Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013.”. Lancet 2013; 386 (9995): 743–800. doi:10.1016/s0140- 6736(15)60692-4 Brochure Hartfalen & Cardiomyopathie. Versie 4.7. Den Haag: Nederlandse Hartstichting; februari 2018.

Watkins H, Ashrafi an H, Redwood C. Inherited cardiomyopathies. N Engl J Med. 2011 Apr 28;364(17):1643-56.

Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, et al. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation.

Cell. 1990 Sep 7;62(5):999-1006.

Jacoby D, McKenna WJ. Genetics of inherited cardiomyopathy. Eur Heart J. 2012 Feb;33(3):296- 304.

Ackerman MJ, Priori SG, Willems S, Berul C, Brugada R, Calkins H, et al. HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies: this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). Europace. 2011 Aug;13(8):1077-109.

Corrado D, Link MS, Calkins H. Arrhythmogenic Right Ventricular Cardiomyopathy. N Engl J Med. 2017 Jan 5;376(1):61-72.

Dalal D, Nasir K, Bomma C, Prakasa K, Tandri H, Piccini J, et al. Arrhythmogenic right ventricular dysplasia:

a United States experience. Circulation. 2005 Dec 20;112(25):3823-32.

Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA, et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modifi cation of the Task Force Criteria. Eur Heart J. 2010 Apr;31(7):806-14.

Groeneweg JA, van der Heijden JF, Dooijes D, van Veen TA, van Tintelen JP, Hauer RN. Arrhythmogenic cardiomyopathy: diagnosis, genetic background, and risk management. Neth Heart J. 2014 Aug;22(7- 8):316-25.

Lazzarini E, Jongbloed JD, Pilichou K, Thiene G, Basso C, Bikker H, et al. The ARVD/C genetic variants database: 2014 update. Hum Mutat. 2015 Apr;36(4):403-10.

MacLennan DH, Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003 Jul;4(7):566-77.

Young HS, Ceholski DK, Trieber CA. Deception in simplicity: hereditary phospholamban mutations in dilated cardiomyopathy. Biochem Cell Biol. 2015 Feb;93(1):1-7.

Haghighi K, Schmidt AG, Hoit BD, Brittsan AG, Yatani A, Lester JW, et al. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure. J Biol Chem. 2001 Jun 29;276(26):24145-52.

Schmitt JP, Kamisago M, Asahi M, Li GH, Ahmad F, Mende U, et al. Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science. 2003 Feb 28;299(5611):1410-3.

Haghighi K, Kolokathis F, Pater L, Lynch RA, Asahi M, Gramolini AO, et al. Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical diff erence between mouse and human. J Clin Invest. 2003 Mar;111(6):869-76.

Liu GS, Morales A, Vafi adaki E, Lam CK, Cai WF, Haghighi K, et al. A novel human R25C- phospholamban mutation is associated with super-inhibition of calcium cycling and ventricular arrhythmia. Cardiovasc Res. 2015 Jul 1;107(1):164-74.

Haghighi K, Kolokathis F, Gramolini AO, Waggoner JR, Pater L, Lynch RA, et al. A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy. Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1388-93.

Introduction and outline of the thesis

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DeWitt MM, MacLeod HM, Soliven B, McNally EM. Phospholamban R14 deletion results in late- onset, mild, hereditary dilated cardiomyopathy. J Am Coll Cardiol. 2006 Oct 3;48(7):1396-8.

Posch MG, Perrot A, Geier C, Boldt LH, Schmidt G, Lehmkuhl HB, et al. Genetic deletion of arginine 14 in phospholamban causes dilated cardiomyopathy with attenuated electrocardiographic R amplitudes.

Heart Rhythm. 2009 Apr;6(4):480-6.

van der Zwaag PA, van Rijsingen IA, Asimaki A, Jongbloed JD, van Veldhuisen DJ, Wiesfeld AC, et al.

Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy.

Eur J Heart Fail. 2012 Nov;14(11):1199-207.

van der Zwaag PA, van Rijsingen IA, de Ruiter R, Nannenberg EA, Groeneweg JA, Post JG, et al.

Recurrent and founder mutations in the Netherlands-Phospholamban p.Arg14del mutation causes arrhythmogenic cardiomyopathy. Neth Heart J. 2013 Jun;21(6):286-93.

van Rijsingen IA, van der Zwaag PA, Groeneweg JA, Nannenberg EA, Jongbloed JD, Zwinderman AH, et al. Outcome in Phospholamban R14del Carriers: Results of a Large Multicentre Cohort Study. Circ Cardiovasc Genet. 2014 7(4):455-65.

Haghighi K, Pritchard T, Bossuyt J, Waggoner JR, Yuan Q, Fan GC, et al. The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K- ATPase. J Mol Cell Cardiol. 2012 Mar;52(3):773-82.

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25 Chapter 1

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Arrhythmogenic cardiomyopathy: pathology, genetics, and concepts in pathogenesis

CHAPTER 2

Edgar T. Hoorntje^1,2, Wouter P. te Rijdt¹, Cynthia A. James³, Kalliopi Pilichou⁴, Cristina Basso⁴, Daniel P. Judge³, Connie R. Bezzina⁵, and J. Peter van Tintelen^2,6

1Department of Genetics, University of Groningen, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands;

2Netherlands Heart Institute, Moreelsepark 1, 3511 EP, Utrecht, The Netherlands;

3Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, 1800 Orleans Street, Baltimore, MD, USA;

4Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua 35121, Italy;

5Department of Clinical and Experimental Cardiology, Heart Centre, Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands;

6Department of Clinical Genetics, Academic Medical Centre Amsterdam, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

Cardiovascular Research. 2017; 113: 1521-1531.

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Abstract

Arrhythmogenic cardiomyopathy (ACM) is a rare, heritable heart disease characterized by fibro- fatty replacement of the myocardium and a high degree of electric instability. It was first thought to be a congenital disorder, but is now regarded as a dystrophic heart muscle disease that develops over time. There is no curative treatment and current treatment strategies focus on attenuating the symptoms, slowing disease progression, and preventing life-threatening arrhythmias and sudden cardiac death. Identification of mutations in genes encoding desmosomal proteins and in other genes has led to insights into the disease pathogenesis and greatly facilitated identification of family members at risk. The disease phenotype is, however, highly variable and characterized by incomplete penetrance. Although the reasons are still poorly understood, sex, endurance exercise and a gene- dosage effect seem to play a role in these phenomena. The discovery of the genes and mutations implicated in ACM has allowed animal and cellular models to be generated, enabling researchers to start unravelling it’s underlying molecular mechanisms. Observations in humans and in animal models suggest that reduced cell–cell adhesion affects gap junction and ion channel remodelling at the intercalated disc, and along with impaired desmosomal function, these can lead to perturbations in signalling cascades like the Wnt/b-catenin and Hippo/YAP pathways. Perturbations of these pathways are also thought to lead to fibro-fatty replacement. A better understanding of the molecular processes may lead to new therapies that target specific pathways involved in ACM.

Chapter 2

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25 1. Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC), which is now considered a subform of arrhythmogenic cardiomyopathy (ACM) with right ventricular (RV) pre-dominance, is a heritable condition characterized by fi bro-fatty replacement of the myocardium that predisposes patients to ventricular arrhythmias (VA), which are frequently life-threatening, and to slowly progressive ventricular dysfunction.^1–4 Structural involvement of the RV predominates,⁵ although left- dominant forms of ACM are also well-recognized.⁶ Patients typically present in their second to fi fth decade with symptoms associated with VA.⁷ Sudden cardiac death may be the presenting symptom in up to 50% of index cases.⁸ The diagnosis is based on International Task Force Criteria⁹ and mutations in genes encoding proteins of the cardiac desmosome are found in up to 60%

cases.^1,10 Cardiac desmosomes are composed of a symmetrical group of proteins (cadherins, armadillo proteins, and plakins) that provide mechanical connections between myocytes.

However, non-desmosomal genes have also been identifi ed.¹¹ The current management strategies focus on lifestyle advice (restriction of physical exercise), attenuating symptoms, and slowing disease progression with anti-arrhythmic and heart failure medications, catheter ablation, and implantable cardioverter defi brillator (ICD) implantation. In cases of end-stage heart failure or refractory VA, a heart transplantation may be required.¹² Unravelling the genetic basis of ACM has led to the generation of animal and cellular models, enabling researchers to uncover the molecular mechanisms underlying ACM and even to discover new therapies.¹³ This review will discuss the pathological fi ndings, the genetic basis and the proposed mechanisms underlying ACM.

2. Pathological fi ndings in ACM 2.1 Morphological features

In ACM, part of the myocardium is replaced by fi brous and fatty tissue with either localized or diff use myocardial atrophy due to cumulative myocyte loss.¹⁴ The pathological hallmarks of the disease, the fi bro-fatty replacement and myocyte atrophy, are usually distinctly present in the RV but may also occur in the left ventricle (LV), and can be segmental or patchy. Traditionally, the typical localization in the RV was described as the ‘triangle of dysplasia’,^14,15 consisting of the RV infl ow tract, RV outfl ow tract, and RV apex. However, recent cardiac magnetic resonance data¹⁶ have revealed that limited ACM preferentially aff ects the basal inferior RV, with involvement of the RV apex only in advanced cases as part of global RV involvement. LV involvement has been observed in 76–84% of ACM cases,^6,14 with a predilection for the thin posterolateral and posteroseptal areas.

Typically, the LV is aff ected to a lesser extent than the RV; however, there are disease variants characterized by pre-dominant LV involvement, these are also referred to as arrhythmogenic left ventricular cardiomyopathy (ALVC).¹⁷

Involvement of the ventricular septum is rare, probably because it is not a subepicardial structure. The fi bro-fatty scar tissue progresses from the subepicardial muscle layer towards the endocardium, ultimately resulting in transmural lesions with focal or diff use wall thinning (Figure 1). This implies the ventricular wall is weakened, especially the relatively thin, free RV wall, which may lead to typical aneurysmal dilatation.

ACM pathology, genetics & pathogenesis

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26

From: Arrhythmogenic cardiomyopathy: pathology, genetics, and concepts in pathogenesis Cardiovasc Res. 2017;113(12):1521-1531. doi:10.1093/cvr/cvx150

Cardiovasc Res | Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Figure 1 Development of (ACM) over time.

Development of ACM over time. Evidence from the Dsg2^N271Smouse model for ACM. (A) At birth a structurally normal heart is presence. (B) Early myocardial injury start on the epicardial side, extends transmurally (C) and is followed by wall thinning with fi brous repair and aneurysm development (D). Figure adapted from Basso et al.¹⁰⁴

Microscopic examination typically shows islands of surviving myocytes, with fi bro-fatty tissue in between.^14,17 These changes may account for intraventricular conduction delay and re-entry circuits triggering VA. Aff ected cardiomyocytes show non-specifi c degenerative features of myofi brillar loss and hyperchromatic changes in nuclear morphology.^14,17 Cardiomyocyte death (acquired injury), by either apoptosis¹⁸ and/or necrosis,¹⁹ accounts for the progressive loss of the ventricular myocardium. These changes may be accompanied by infl ammatory infi ltrates, seen in up to 67% of hearts at autopsy.¹⁴ Importantly, active infl ammation might account for worsening of electrical instability and the onset of life-threatening arrhythmias. Whether the infl ammatory cells are reactive to cell death or a primary event due to infection²⁰ or non-infective immune factors needs to be investigated.²¹

2.2 Clinical utility of RV endomyocardial biopsy (EMB)

RV EMB may be useful for the diagnosis of ACM, through an in vivo histological demonstration of fi bro-fatty replacement. Moreover, EMB may provide additional information to rule out phenocopies, such as myocarditis or sarcoidosis, particularly in sporadic cases in which non- invasive evaluation remains inconclusive. The optimal EMB site is the RV free wall, which may, however, be severely thinned due to ACM.

In a normal heart, with increasing age and body weight, intramyocardial fat is, to a limited extent, present in the RV. Therefore, adipose tissue should be accompanied by replacement fi brosis and myocyte degeneration to be a suffi cient morphologic diagnostic feature of ACM.²²

Chapter 2

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27 In addition to conventional histology, immunohistochemical analysis can be a valuable tool, because plakoglobin (PG) signal levels at intercalated disk (ID) can be diff usely diminished in most ACM patients, also in samples from the LV or interventricular septum, irrespective of the underlying mutation.²³ However, the reliability and validity of this test for routine clinical practice still has to be confi rmed.²⁴

3. Genetic basis of ACM

Clustering of ACM within families was appreciated early.²⁵ Recognition that the cardiac phenotype of Naxos disease, a rare, familial, cardio-cutaneous condition, overlapped with familial ACM²⁶ was a key insight. Following the discovery that mutations in JUP, encoding PG, was the cause of Naxos disease,²⁷ the ACM-associated mutations in the desmosomal genes were rapidly unveiled, including DSP encoding desmoplakin,²⁸ PKP2 encoding plakophilin-2,²⁹ DSG2 encoding desmoglein-2,³⁰ and DSC2 encoding desmocollin-2.³¹

Up to two-thirds of ACM patients harbour mutations in these desmosomal genes.^1,7 Heterozygous mutations resulting in pre-mature termination of the protein product and/or abnormal splicing in PKP2 are the most prevalent.^10,32 Inheritance of desmosomal mutations follows an autosomal dominant pattern with age-related, incomplete penetrance and variable expressivity.

However, ACM patients with multiple mutations (compound heterozygosity and digenic) are not uncommon and their occurrence ranges widely (4–21% reported in various cohorts).^7,32–34 This range is likely related to how stringently missense variants are adjudicated and how many genes are sequenced.³⁵ Cases with homozygous mutations are also seen.^36,37 In addition, there are pedigrees in which siblings of the index case are more likely to be aff ected than their parents or their parents’ siblings. These phenomena raise the suspicion that other genetic and/or environmental factors may play a modifying role.³⁸

Although most reported ACM-associated pathogenic variants are in desmosomal genes (as in 95.5% of the variants reported in the ARVC Genetic Variant Database¹⁰), extra-desmosomal mutations have been identifi ed in a few patients. The fi rst of these was the p.S358L founder mutation in TMEM43, encoding transmembrane protein 43, which was identifi ed in patients in Newfoundland and Europe.^39,40 Pathogenic mutations have also been reported in genes associated with other cardiomyopathies and arrhythmia syndromes including desmin (DES),⁴¹ titin (TTN),⁴² lamin A/C (LMNA),43 phospholamban (PLN),⁴⁴ Na_V1.5 (SCN5A),45 and Filamin C (FLNC).⁴⁶ Together, these discoveries refl ect the clinical and genetic overlap of ACM with dilated cardiomyopathy at one phenotypic extreme⁴⁷ and with arrhythmia syndromes at the other. Supporting this concept, pathogenic ACM-associated PKP2 missense mutations also have been identifi ed in Brugada syndrome patients.⁴⁸

Genes encoding proteins in the ‘area composita’ (composed of desmosomes, adherens junctions (AJ), ion channels, and gap junctions) have also emerged as potentially important in the pathogenesis of ACM. Mutations in CTNNA3, encoding αT-catenin, have been identifi ed in families with classic ACM.⁴⁹ Recently, two families, with right-pre-dominant ACM, were found to have likely pathogenic mutations in CDH2, encoding cadherin-2, a calcium-dependent cell surface adhesion molecule.⁵⁰

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Mutations in transforming growth factor-beta3 (TGFB3)⁵¹ and the cardiac ryanodine receptor-2 (RYR2)⁵² genes have been described in ACM, although this association needs to be confirmed.

Finally, there are some ACM cases with no identifiable mutation. In the largest study of ACM, amongst 439 index cases, 37% had no identifiable mutation in the desmosomal genes, PLN, or TMEM43.⁷ Amongst these gene-elusive cases, only one-fifth had evidence of familial disease. A recent meta-analysis confirmed a lower prevalence of family history amongst ACM patients without desmosomal mutations.⁵³ This raises the question whether these gene-elusive cases have a primarily monogenic disease or whether they represent an oligogenic form of ACM with unknown, low-penetrant genetic variants and/or with external factors playing a role in their disease pathogenesis. Recent research showed that gene-elusive ACM cases without a positive family history weredisproportionately observed in high-level endurance athletes,^54,55 which points to exercise as a key lifestyle risk factor in these cases.

3.1 Genotype-phenotype association in ACM

Several clinically useful genotype–phenotype associations have been identified. Broadly, neither the cardiac phenotype nor clinical course differ substantially between ACM patients with and without a mutation.⁷ A recent meta-analysis identified inverted anterior pre-cordial T-waves (V_1–3) but not structural abnormalities, epsilon waves, or arrhythmias with a left-bundle branch block morphology, as being more common amongst ACM patients with desmosomal mutations.⁵³ Patients with mutations do have earlier onset of ACM.^7,53,56

In addition to an increased penetrance, carrying multiple mutations seems to be an important risk factor for malignant VA and sudden death.⁵⁷ Similarly, in 577 desmosomal, PLN, and TMEM43 mutation carriers, the 4% of patients with multiple mutations had significantly earlier occurrence of malignant VA and more frequent LV dysfunction, class C heart failure, and transplantation.³² Together these data suggest there is a gene-dosage effect in ACM.

Other associations between genotype and ACM phenotype include a higher prevalence of LV involvement and heart failure amongst ACM patients with FLNC, DSP, and PLN mutations.^32,46,58 The TMEM43 p.S358L founder mutation is associated with high disease penetrance and arrhythmic risk amongst male carriers.³⁹

Table 1 provides an overview of the genes implicated in ACM and the yield of genetic testing. Caution is warranted as variants in ACM-related genes are also often found in the general population.⁵⁹

Chapter 2