Phospholamban p.Arg14del cardiomyopathy te Rijdt, Wouter

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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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Myocardial ﬁ brosis as an early feature in phospholamban p.Arg14del mutation carriers:

phenotypic insights from Cardiovascular Magnetic Resonance imaging

CHAPTER 8 Wouter P. te Rijdt MD

^a-c

, Judith N. ten Sande MD

^b,d

, Thomas M. Gorter MD

^a

, Paul A. van der Zwaag MD, PhD

^c

, Ingrid A. van Rijsingen MD, PhD

^d

, S. Matthijs Boekholdt MD, PhD

^d

, J. Peter van Tintelen MD, PhD

^e

, Paul L. van Haelst MD, PhD

^f,g

, R. Nils Planken MD, PhD

^h

, Rudolf A. de Boer MD, PhD

^a

, Albert J.H. Suurmeijer MD, PhD

ⁱ

, Dirk J. van Veldhuisen MD, PhD

^a

, Arthur A.W. Wilde MD, PhD

^d

, Tineke P. Willems MD, PhD

^j

, Pascal F.H.M. van Dessel MD, PhD

^d,k

, Maarten P. van den Berg MD, PhD

^a

aDepartment of Clinical and Experimental Cardiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.

bNetherlands Heart Institute (Nl-HI), Utrecht, the Netherland

cDepartment of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

dHeart Center, Department of Clinical and Experimental Cardiology, University of Amsterdam, Academic Medical Center, Amsterdam, the Netherlands

eDepartment of Clinical Genetics, University of Amsterdam, Academic Medical Center, Amsterdam, the Netherlands

fDepartment of Cardiology, Antonius Hospital, Sneek, the Netherlands

gRoche Diagnostics, Basel, Switzerland

hDepartment of Radiology, University of Amsterdam, Academic Medical Center, Amsterdam, the Netherlands

iDepartment of Pathology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

jDepartment of Radiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

kDepartment of Cardiology, Medisch Spectrum Twente, Enschede, the Netherlands

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

MULTIPLE IMAGING MODALITIES

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128 Abstract Aims

The p.Arg14del founder mutation in the gene encoding phospholamban (PLN) is associated with an increased risk of malignant ventricular arrhythmia (VA) and heart failure. It has been shown to lead to calcium overload, cardiomyocyte damage, and eventually to myocardial fibrosis. This study sought to investigate ventricular function, the extent and localization of myocardial fibrosis and the associations with ECG features and VA in PLN p.Arg14del mutation carriers.

Methods and Results

CMR data of 150 mutation carriers were analyzed retrospectively. Left ventricular (LV) and right ventricular (RV) volumes, mass, and ejection fraction (EF) were measured. The extent of late gadolinium enhancement (LGE) was expressed as a percentage of myocardial mass. All standard ECG parameters were measured. Occurrence of VA was analyzed on ambulatory 24-hour and/or exercise electrocardiography, if available. Mean age was 40±15 years, 42% males, and 7% were index patients while 93% were presymptomatic carriers identified after family cascade screening.

Mean LVEF and RVEF were 58±9% and 55±9%, respectively. LV-LGE was present in 91% of mutation carriers with reduced LVEF (<45%) and in 30% of carriers with preserved LVEF. In carriers with positive LV-LGE, its median extent was 5.9% (IQR 3.2-12.7). LGE was mainly observed in the infero lateral wall. Carriers with inverted T-waves in the lateral ECG leads showed LV-LGE more often on CMR (p < 0.01) than carriers without. Finally, the presence of LV-LGE, but not attenuated R-waves and inverted lateral T-waves, was independently associated with VA.

Conclusion

LV myocardial fibrosis is present in many PLN p.Arg14del mutation carriers who still have a

preserved LVEF. It is seen predominantly in the LV inferolateral wall and corresponds with electro-

cardiographic repolarization abnormalities. Although preliminary, myocardial fibrosis was found

to be independently associated with VA. Our findings support the use of CMR with LGE early in

the diagnostic work-up.

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129

8

Introduction

The pathogenic c.40_42delAGA (p.Arg14del) founder mutation in the phospholamban (PLN;

locus 6q22.31; OMIM gene description number 172405) has been identifi ed in 10-15% of patients diagnosed with dilated cardiomyopathy (DCM) and/or arrhythmogenic cardiomyopathy (ACM) in the Netherlands.1-3 It has also been found in several other European countries, Canada, and the USA. PLN is a transmembrane sarcoplasmic reticulum (SR) phosphoprotein that regulates sarcoplasmic reticulum Ca

²⁺

-ATPase (SERCA) activity and the p.Arg14del mutation has been shown to lead to calcium overload and consequent cardiomyocyte damage, and eventually to myocardial fi brosis.4, 5 Indeed, examination of 20 whole heart specimens (autopsies and explants) of PLN p.Arg14del mutation carriers revealed extensive myocardial fi brosis in all cases.

^6,7

A striking clinical manifestation of PLN p.Arg14del mutation-related cardiomyopathy is the development of low- amplitude QRS complexes on the surface ECG

^1,8

and it is likely that this is a refl ection of underlying fi brosis, although this has not been proven. In addition, repolarization changes on the ECG, particularly of negative T-waves in the lateral leads, are an early manifestation in mutation carriers.

Late gadolinium-enhanced (LGE) cardiovascular magnetic resonance imaging (CMR) has become the gold standard for non-invasive in vivo assessment of ventricular myocardial fi brosis;

it allows the early identifi cation and evaluation of both the extent and localization of myocardial fi brosis in diff erent forms of cardiomyopathy.

^9-11

Importantly, LGE has consistently been shown to be a strong risk factor for sudden cardiac death (SCD) and overall mortality in a wide range of cardiomyopathies, e.g. DCM.

^12-16

For the present study, we formulated three hypotheses based on the previous electrocardio- graphic and histopathologic fi ndings: (1) that LGE is present in a distinct subgroup of PLN p.Arg14del mutation carriers (2) that the ECG changes refl ect fi brosis and (3) that, assuming that fi brosis is a substrate for ventricular arrhythmia (VA) in mutation carriers, the presence of LGE is associated with VA. We investigated CMR- and ECG parameters, and VA occurrence, in a large cohort of 150 mutation carriers to test these hypotheses. In particular, we analyzed the extent and localization of CMR LGE together with ECG parameters to investigate whether the development of low-voltage QRS amplitude and/or repolarization changes are associated with left ventricular (LV) LGE. We also investigated whether these fi ndings are associated with VA.

Methods

Source population

Adult (>18 years old) PLN p.Arg14del mutation carriers who had undergone CMR imaging were selected from the PHORECAST registry (PHOspholamban RElated CArdiomyopathy STudy; http://

www.phorecast.nl). Demographic and clinical parameters at the time of CMR were collected

retrospectively in three Dutch hospitals (University Medical Center Groningen, Academic Medical

Center Amsterdam, and Antonius Hospital Sneek). Our group included both index patients and

their relatives referred to a cardiogenetics outpatient clinic for family cascade screening. Index

patients in the cohort were not known to be related to each other.

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130 Cardiovascular magnetic resonance imaging protocol

CMR studies in all three centers were performed on a 1.5 Tesla whole-body CMR scanner (Magnetom Avanto, Siemens Healthcare GmbH, Erlangen, Germany) using a phased-array cardiac receiver coil. Then ECG-gated cine, steady-state free-precession (True FISP) sequences were acquired during repeated breath holds in contiguous short-axis slices (6 or 8 mm per slice) covering the entire LV and RV. The following scan parameters were used: TE 1.1 ms, TR 42 ms, flip angle 55°; matrix 192 x 192, voxel size 1.82 x 1.82 x (6 or 8) mm.

Using identical slice locations, LGE images were acquired 10 minutes after intravenous administration of 0.2 mmol/kg gadolinium-based contrast agent (Dotarem, Gorinchem, the Netherlands; 0.2 mmol/kg) with a single-shot 2D phase-sensitive inversion recovery sequence (TE 3.2 ms, TR 700 ms; flip angle 25°; matrix 360 x 360 mm, voxel size 1.4 x 1.4 x (6 or 8) mm).

The inversion time was set individually to null the signal of normal myocardium. All procedures were performed according to the standardized protocols recommended by the Society for Cardiovascular Magnetic Resonance.

¹⁷

Cardiovascular magnetic resonance imaging analysis

All CMR analyses were performed using QMass 7.6 (Medis medical imaging systems BV, Leiden, The Netherlands). The endo- and epicardial contours of the LV and RV were manually traced on the short- axis slices in the end-diastolic and end-systolic phases by a single experienced observer (T.M.G.), who was blinded for the patients’ clinical data. Papillary muscle and trabeculae were included in the blood volume. End-diastolic and end-systolic volumes were calculated using the summation of slice multiplied by slice thickness method, indexed to body surface area (BSA), and compared to reference values.

¹⁸

LV volume was dichotomized based on reference values

¹⁸

into

“non-dilated” or “dilated” (>112 ml/m

²

for males and >99 ml/m

²

for females).

For LGE imaging, first the presence of delayed-enhanced signal intensity was visually determined by two experienced independent observers (by agreement), who were blinded for patients’ clinical data (T.M.G.: >5 years experience in CMR; and T.P.W.: >15 years experience in CMR, level 3 certified cardiovascular radiologist). Subsequently, the extent of LV-LGE was quantified by one observer (T.G.) using the full-width at half-maximum method, by defining the enhanced area using 50% of the maximum signal found within the enhanced area as previously described.

¹⁹

LV- LGE size was expressed as a percentage of total LV mass. LV-LGE location was determined using the 17-segment model.20 The amount of right ventricular (RV) LGE was quantified using the 12-segment model of the RV and classified as small (≤4 segments involved) or large (>4 segments involved).

²¹

ECG analysis

Standard 12-lead resting ECGs, recorded around the time of CMR (median time span between

CMR and ECG: 35 days (interquartile range (IQR) 1-100), were analyzed after digitalization

using ImageJ (http://rsb.info.nih.gov/ij/). Each ECG was time-calibrated and conduction and

repolarization parameters during sinus rhythm were determined. Measurements of time-related

parameters (heart rate, PQ-interval, QRS-duration and QT-interval) were performed manually on-

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131 screen, in lead II whenever possible. Parameters were averaged from up to 3 consecutive beats with similar preceding RR-intervals. For the QT- and heart-rate corrected QT- interval (QTc), we used the tangent method with Bazett’s correction.

²²

R-waves of all 12 leads were summed and dichotomized based on the median value (5.3 mm) into “normal voltage” or “low voltage”. Inverted T-waves were determined and considered present if inverted in the right precordial leads (V1 as well as V2) and/or in at least in two adjacent lateral leads (V4, V5 or V6). ECGs were not analyzed if there was a left- or right bundle branch block, or bifascicular block.

Ventricular arrhythmia

To determine the association between ECG parameters, fi brosis and VA, the occurrence of VA (non- sustained or sustained ventricular tachycardia) was analyzed on ambulatory 24-hour (Holter) and/or exercise ECG. The indication was at the discretion of the attending physician, and ambulatory 24-hour and/or exercise electrocardiography were therefore not available for every mutation carrier. Non- sustained ventricular tachycardia (VT) was defi ned as at least three consecutive ventricular complexes at a heart rate >100 beats/min with a duration of less than 30 seconds. Sustained VT was defi ned as an arrhythmia at a heart rate >100 beats/min that lasted

≥30 seconds and/or required termination because of hemodynamic compromise in <30 seconds.

Statistical analysis

Statistical analyses, including bootstrapping, were performed using SPSS software, version 24.0 (SPSS for Windows, 2016 release 24.0.0.0, Chicago, Ill, USA). Continuous variables are presented as a mean with standard deviation (SD) and compared with the unpaired t-test for a normal distribution, or presented as the median with an IQR for a skewed distribution, as determined by the Kolmogorov- Smirnov Goodness-of-Fit test. Categorical variables are presented as frequencies with percentages and analyzed using the Fisher exact test. The Pearson’s correlation test was used for correlation analysis.

Associations between demographic-, ECG- and CMR variables were initially analyzed using univariable regression. All variables that were statistically signifi cantly associated with LV-LGE presence in the univariable analysis were then included in a multivariable regression model. Bootstrapping, using 1000 bootstrap samples, was used to evaluate the performance of the model. Bootstrapping is the most effi cient validation procedure as all aspects of the model development, including variable selection, are validated.

²³

The association with VA was also analyzed, For this analysis only the mutation carriers where ambulatory and/or exercise ECG data were available were included in the analysis. Due to the low prevalence of VA we had to limit our selection of variables for the corresponding multivariable analysis. The selection was based on clinical relevance and prevalence and included: low voltage, inverted lateral T-waves, LVEF, and LV-LGE.

Odds ratios and 95% confi dence intervals were calculated. A p-value of less than 0.05 was considered to be statistically signifi cant.

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132

Table 1. Clinical characteristics (n=150)

Patient characteristics All patients Index (n=10) Relative (n=140)

Age (years) 40±15 44±10 40±15

Sex Male

Female 63 (42%)

87 (58%)

5 (50%) 5 (50%)

58 (41%) 82 (59%)

BSA (m²) 1.92±0.16 1.99±0.26 1.91±0.17

NYHA functional class I II

III/IV

139 (93%) 11 (7%) 0 (0%)

8 (80%) 2 (20%) 0 (0%)

131 (94%) 9 (6%) 0 (0%)

Ventricular Arrhythmia 23 (15%) 6 (60%) 17 (12%) *

ECG parameters

PR interval (ms) 150±21 151±10 150±21

QRS width (ms) 85±11 91±15 85±11

QTc (ms) 408±23 411±29 409±23

R-wave amplitude (mV; median) 5.3 (1.4-17.0) 3.4 (1.6-6.1) 5.5 (1.4-17.0) * Inverted right precordial T-waves (V1 and

V2) 17 (11%) 0 (0%) 17 (12%)

Inverted lateral T-waves (V4, V5 or V6) 43 (23%) 8 (80%) 35 (25%) * Medication

Anti-arrhythmic 6 (4%) 2 (20%) 4 (3%)

Beta-blocker 22 (15%) 7 (70%) 15 (11%) *

RAAS inhibitors 15 (10%) 6 (60%) 9 (7%) *

Spironolactone/Eplerenone 2 (1%) 2 (20%) 0 (0%) *

Diuretics 9 (6%) 4 (10%) 5 (4%) *

Anti-coagulant 7 (5%) 2 (20%) 5 (4%)

Anti-platelet therapy 4 (3%) 1 (10%) 3 (2%)

Abbreviations: BSA = Body surface area; NYHA = New York Heart Association; ECG = electrocardiogram; RAAS

= renin-angiotensin system.

*p<0.05

Results

Patient characteristics

We identified 194 mutation carriers who had undergone CMR imaging in the three centers. For 28 patients, there were no clinical, ECG data and/or CMR LGE available and LGE could not be evaluated in six patients due to insufficient image quality. Ten patients were excluded based on their ECG (3 bifascicular block, 4 right bundle branch block, 1 left bundle branch block, 1 atrial fibrillation, and 1 Wolff-Parkinson-White syndrome). Our final study group consisted of 150 mutation carriers (table 1). Their mean age was 40±15 years and 42% were male. Ten carriers (7%) were index patients (mean age 44±10 years), while the other 140 participants were relatives identified by family cascade screening (mean age 40±15 years). The vast majority (93%) of participants were in New York Heart Association functional class I and did not take heart failure medication. The number of prescriptions for beta-blockers, renin-angiotensin system (RAAS) inhibitors, aldosterone-blocking agents (spironolactone or eplerenone), and diuretics were significantly higher (p<0.05) in the index patients.

Table 1. Clinical characteristics (n = 150)

Abbreviations: BSA = Body surface area; NYHA = New York Heart Association; ECG = electrocardiogram; RAAS = renin-angiotensin system. *p<0.05

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133 CMR fi ndings (table 2)

Mean end-diastolic LV and RV volumes, LVEF and RVEF were normal, but we observed signifi cant diff erences between index patients and their relatives for LVEDV (240±105 vs. 174±35 ml, p<0.05), LVEDVi (119±43 vs. 91±15 ml/m

²

, p<0.05), LVEDM (125±47 vs. 92±22 g, p<0.05), LVEDMi (62±17 vs. 48±9 ml/m

²

, p<0.05), LVEF (40±14 vs. 59±7 %, p<0.05), and RVEF (45±11 vs. 56±9 %, p<0.05).

Eleven (7%) mutation carriers had reduced LVEF (i.e. <45%), fi ve of these were index patients (index patients 5/10 vs. relatives 6/140, p<0.05). There was a signifi cant correlation between LVEF and RVEF (r=0.78, p<0.001) (fi gure 1).

Table 2. Cardiac magnetic resonance imaging parameters (n = 150)

Abbreviations: LGE, late gadolinium enhancement; LVEDV, left ventricular end-diastolic volume; LVEDVi, left ventricular end-diastolic volume index; LVEDM, left ventricular end-diastolic

mass; LVEDMi, left ventricular end-diastolic mass index; LVEF, left ventricular ejection fraction; RVEDV, right ventricular end-diastolic volume; RVEDVi, right ventricular enddiastolic volume index; RVEF, right ventricular ejection fraction. *p<0.05.

Figure 1. Scatter plot depicting the relationship between LVEF and RVEF in phospholamban p.Arg14del mutation carriers (n = 150; p <0.01).

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Table 2. Cardiac magnetic resonance imaging parameters (n=150)

Left ventricle All patients Index (n=10) Relative (n=140)

LVEDV (ml) 179±46 240±105 174±35 *

LVEDVi (ml/m²) 93±19 119±43 91±15 *

LVEDM (g) 95±25 125±47 92±22 *

LVEDMi (g/m²) 49±10 62±17 48±9 *

LVEF (%) 58±9 40±14 59±7 *

LVEF <45% 11 (7%) 5 (50%) 6 (4%) *

LV-LGE present (%) 50 (33%) 9 (90%) 41 (29%) *

LGE % LV mass (median), if present 5.9 (3.2-12.7) 18 (8.1-30.2) 4.6 (3.0-8.3) * Right ventricle

RVEDV (ml) 186±42 203±78 185±38

RVEDVi (ml/m²) 97±17 100±28 97±16

RVEF (%) 55±8 45±11 56±9 *

RV-LGE present (%) 8 (5%) 2 (20%) 6 (4%) *

Abbreviations: LVEDV = LV end-diastolic volume; LVEDVi = LV end-diastolic volume index; LVEDM = LV end- diastolic mass; LVEDMi = LV end-diastolic mass index; LVEF = LV ejection fraction; LGE = late gadolinium enhancement; RVEDV = Right ventricular end diastolic volume; RVEDDi = Right ventricular end diastolic volume index; RVEF = RV ejection fraction.

*p<0.05

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134 LV-LGE was seen in 50 mutation carriers (index patients 9/10 vs. relatives 41/140, p<0.05).

Mutation carriers with LV-LGE were significantly older (47±15 vs. 36±14 years, p<0.01) than those without LV-LGE. Almost all mutation-carriers with reduced LVEF (10/11; 91%) also had LV-LGE, while it was also present in 29% (40/139) of mutation carriers with preserved LVEF (figure 2). In carriers with LV-LGE, the median volume of enhanced LV myocardium was 5.9% (3.2-12.7), with index patients showing higher volumes than relatives (18.0% (8.1-30.2) vs. 4.6% (3.0-8.3), p<0.05).

Delayed enhancement was mainly present in the basal inferolateral wall of the LV (most abundant in segments 5 and 11), whereas segments 1-3, 7-9 and 14 were least affected (figure 3). In one case, we were able to examine the explanted heart to compare it with LV-LGE CMR findings, showing extensive interstitial fibrosis in the area of late gadolinium enhancement (figure 4).

Figure 2. Scatter plot depicting the relationship between the amount of LV myocardial fibrosis (%) and LVEF (%) in phospholamban p.Arg14del mutation carriers (n = 150; p <0.01). The dotted line represents an LVEF of 45%.

Figure 3. Bull’s eye plot (17 left ventricular segments model) depicting the presence and localization of myocardial fibrosis in PLN p.Arg14del mutation carriers (n = 150; % per segment represents the percentage of mutation carriers with CMR LGE in that segment).

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135

Figure 4. Clinico-pathological correlation between late gadolinium enhanced CMR- and histo pathologic fi ndings in a phospholamban p,Arg14del mutation carrier. Coronary angiography revealed no coronary artery disease. A) Short-axis delayed enhancement image of a mutation carrier showing inferolateral wall thinning and extensive LGE (arrow) of the inferolateral wall of the LV. The observed subendocardial LGE pattern is probably due to wall thinning in this mutation carriers, as also observed in some other cases B) Mid-ventricular cross-section of the explanted heart (gross examination) of the same mutation carrier showing macroscopically visible fi bro-fatty replacement of the RV wall (arrow) and limited fi bro-fatty alteration in the LV wall. C) Microscopic analysis of Masson trichrome-stained LV free wall sample from the same explanted heart showing extensive interstitial fi brosis.

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136 In the right ventricle, we observed LGE in only 8 (5%) mutation carriers (index patients 2/10 vs. relatives 6/140, p<0.05). RVEF was significantly lower in mutation carriers with RV-LGE than in those without RV-LGE (43±8 vs. 56±7 %, p<0.05).

ECG findings and ventricular arrhythmia occurrence

ECG conduction and repolarization parameters were, on average, within the normal range (table 1). The median R-wave amplitude was 5.3mV (index 3.4mV vs. relatives 5.5mV, p<0.05). Carriers with low voltage (mean R-value below median) were significantly older than carriers with normal voltage (44±15 vs. 36±14 years, p<0.01). Inverted T-waves in the right precordial leads were seen in 17 (11%) carriers and inverted T-waves in the lateral leads were seen in 43 (29%) carriers (index 8/10 vs. relatives 36/140, p<0.05). A representative example of electrocardiographic and CMR findings in a PLN mutation-carrier is shown in figure 5.

Figure 5. A) Typical Cine CMR images in a PLN p.Arg14del mutation carrier showing left lateral delayed contrast enhancement (arrow). B) Twelve-lead ECG of the same mutation carrier showing normal sinus rhythm with low voltages in all leads (<0.5 mV) and flattened or inverted T waves in all precordial leads and inferolateral limb leads.

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137 In 23/150 carriers (15%), either sustained or non-sustained ventricular tachycardia was documented (index patients 6/10 vs. relatives 17/140, p<0.05).

Association between ECG fi ndings and CMR LV-LGE (table 3)

In univariable analysis, the presence of low voltage on the surface ECG was associated with the presence of LV-LGE on CMR (OR=3.06, p<0.01). If the surface ECG showed inverted T-waves in the lateral leads (V4-6), then LV-LGE was also seen more often on CMR (OR=8.48, p<0.01).

In a multivariable regression model including age, low-voltage, inverted lateral T-waves, LVEF, LV dilatation and RVEF (all p<0.05 in univariable analysis), only two factors − age (OR=1.05, p<0.01) and inverted lateral T-waves (OR=5.70, p<0.01) − were independently associated with the presence of LV-LGE. Bootstrapping yielded comparable results in comparison with conventional computation.

Table 3. Univariable and multivariable analysis of the association between demographic-, ECG- and CMR variables and the presence of LV-LGE.

Abbreviations: LGE = late gadolinium enhancement; NYHA = New York Heart Association; LVEF = left ventricular ejection fraction; RVEF = RV ejection fraction; B = regression coeffi cient.; CI, confi dence interval.

Association between ECG fi ndings, CMR LV-LGE and ventricular arrhythmia occurrence (table 4) In the univariable analysis, inverted lateral T-waves, low voltage, and LVEF<45% were associated with the occurrence of VA. We also found that the presence of CMR LV-LGE was signifi cantly associated with the occurrence of VA (OR=10.7, p<0.01; fi gure 6).

The presence of LV-LGE (OR 5.52, p<0.01) remained independently associated with the occurrence of VA, but low voltage, inverted T-waves and LVEF<45% were not independently associated with VA occurrence.

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LV-LGE

(n=50) No LV-LGE

(n=100) Univariable

(OR 95%CI) Multivariable (OR 95%CI)

Age (years) 47±15 36±14 1.04 (1.02-1.07)

p<0.05 1.05 (1.01-1.08) p<0.01; B=0.45 Sex Male

Female 25 (50%)

25 (50%) 38 (38%)

62 (62%) 1.63 (0.82-3.24) p=0.16 NYHA functional class (≥2) 6 (12%) 5 (5%) 2.59 (0.75-8.95)

p=0.13 Low voltage

(present) 34 (68%) 41(41%) 3.06 (1.50-6.25)

p<0.05 1.09 (0.45-2.62) p=0.85; B=0.85 Inverted lateral T-wave

(present) 29 (58%) 14 (14%) 8.48 (3.83-18.8)

p<0.05 5.70 (2.28-14.26) p<0.01; B=1.74 LV dilatation (present) 16 (32%) 13 (13%) 3.15 (1.37-7.24)

p<0.05 2.51 (0.83-7.61) p=0.10; B=0.92 LVEF <45% (present) 10 (20%) 1(1%) 24.8 (3.07-199)

p<0.05 5.34 (0.52-54.8) p=0.16; B=1.67 RVEF <45% (present) 6 (12%) 1 (1%) 13.5 (1.58-116)

p<0.05 2.11 (0.12-37.59) p=0.61; B=0.75

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138

Table 4. Univariable and multivariable analysis of the association between ECG- and CMR variables and VA occurrence.

Abbreviations: VA = ventricular arrhythmia; LVEF = left ventricular ejection fraction; LGE = late gadolinium enhancement; p = p Value; B = regression coefficient; CI = confidence interval.

*Only the mutation carriers where ambulatory and/or exercise ECG data were available were included in this analysis

Figure 6. Bull’s eye plot (17 left ventricular segments model) showing localization of LGE (% per segment represents the mutation carriers with CMR LGE in that segment) in PLN p.Arg14del mutation carriers who experienced VA (n = 23; right panel) versus no VA (n = 127; left panel). VA = ventricular arrhythmia.

Table 4. Univariable and multivariable analysis of the association between ECG- and CMR variables and VA occurrence.

VA (n=23) No VA

(n=110*) Univariable

OR 95%CI Multivariable OR 95%CI Low voltage (present) 16 (70%) 50 (45%) 2.55 (0.97-6.68)

p<0.05 0.73 (0.20-2.59) p=0.62; B=-0.32 Inverted lateral T wave

(present) 16 (70%) 26 (24%) 7.38 (2.74-19.89)

p<0.05 3.05 (0.85-10.91) p=0.09; B=1.11 LVEF<45% (present) 7 (30%) 3 (3%) 15.6 (3.66-66.58)

p<0.05 4.91 (0.95-25.47) p=0.06; B=1.59 LV-LGE (present) 18 (78%) 28 (25%) 10.5 (3.58-31.04)

p<0.05 5.52 (1.62-18.77) p<0.01; B=1.71 Abbreviations: VA = ventricular arrhythmia; LVEF = left ventricular ejection fraction; LGE = late gadolinium enhancement; p = p Value; B = regression coefficient.

*Only the mutation carriers where ambulatory and/or exercise ECG data were available were included in this analysis

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139 Discussion

PLN p.Arg14del cardiomyopathy is characterized by early low voltage and repolarization changes on the ECG

^{1, 8}

and a high risk for VA,

³

both likely a refl ection of fi brosis although previously unproven.

The present study provides new insights into the association between these observations.

As hypothesized, fi rstly, we found that LGE is present in a distinct subgroup of PLN p.Arg14del mutation carriers in this cohort. Index patients showed more extensive structural and functional evidence of disease progress but LV-LGE on CMR was also seen in many subjects with a preserved LV systolic function (LVEF >45%). This important fi nding suggests that fi brosis is an early feature in PLN p.Arg14del cardiomyopathy. Secondly, both the presence of low voltage and inverted lateral T- waves were associated with LV-LGE on CMR in PLN p.Arg14del mutation carriers. LV-LGE was most abundant in the LV inferolateral wall, where we observed a high prevalence of negative T-waves. Our fi ndings strongly suggest that these ECG changes are a refl ection of myocardial fi brosis. Thirdly, in line with these fi ndings, we could demonstrate that LV- LGE is independently associated with the occurrence of VA, attesting to the clinical importance of fi brosis in this disease. Finally, in addition to previous clinical and histopathological fi ndings,

^1,6,7

our current data support the notion of biventricular involvement in PLN p.Arg14del cardiomyopathy, given the strong correlation between LV and RV systolic function on CMR.

The pathophysiological mechanisms responsible for these fi ndings in PLN p.Arg14del cardiomyopathy are not fully understood, but it is likely that disturbed calcium homeostasis plays an important role. The mutation in the PLN gene leads to reduced SERCA activity, which causes calcium overload leading to cardiomyocyte damage and eventually myocardial fi brosis.7 We observed a specifi c predominance of this fi brosis in the inferolateral wall of the LV, comparable to desmosomal ACM24 and specifi c other forms of cardiomyopathy, for example in Duchenne muscular dystrophy.

^25,26

It is unclear how a pathogenic mutation that presumably aff ects the heart in a diff use manner may result in a segmental distribution of disease. Whether the inferolateral wall is more vulnerable due to regional molecular changes caused by the mutation, or whether this regional susceptibility results from exposure to higher mechanical stress has not yet been elucidated.

Current guidelines for the primary prevention of sudden cardiac death in patients with DCM generally recommend that a defi brillator be implanted in patients who have NYHA functional class II/III and an LVEF of less than 35%.

^27-29

However, previous studies suggest that LV-LGE on CMR imaging is an extra independent risk factor in these patients.12-16 In our previous study on PLN p.Arg14del mutation carriers, we showed that an LVEF of less than 45% (rather than 35%) is an independent risk factor for VA.3 In the present study, we refi ne this fi nding by showing that LV-LGE on CMR is an even stronger risk factor than LVEF. In fact, even in the setting of preserved LVEF, the mere presence of LV-LGE is associated with a higher risk of VA in PLN p.Arg14del mutation carriers.

Taken together, these data strongly support the use of CMR with LGE in this patient group, and should include the presymptomatic carriers.

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140 Study Considerations

CMR imaging was only performed in PLN p.Arg14del mutation carriers without a pacemaker or implantable cardioverter defibrillator, leading to a preferential inclusion of patients with early- stage disease. Moreover, the majority were not index patients, but carriers identified after family cascade screening. However, these circumstances provided us with a unique opportunity to study early-stage disease.

The main limitation of the LGE technique is the inability to evaluate diffuse myocardial fibrosis. The enhanced area is defined on the basis of the difference in signal intensity relative to that of the normal myocardium. If the myocardial fibrosis is diffuse instead of focal, no differences in signal intensity will be observed. T1 mapping, a CMR sequence to visualize and quantify diffuse myocardial interstitial fibrosis in the whole heart, better reflects the total myocardial fibrosis burden

³⁰

but has only recently become available at our centers.

We speculate that the presence of RV myocardial fibrosis is underestimated in the present cohort. We believe the observed low prevalence of RV-LGE is mainly due to the thin wall of the RV, which makes the RV much harder to visualize.

The occurrence of VA was determined on ambulatory 24-hour (Holter) and/or (exercise) electrocardiography which were not available for every patient Therefore, we have only included mutation carriers where ambulatory and/or exercise ECG data were available for the VA-analysis. . This may have led to selection bias.

Finally, this was a retrospective study with inherent limitations, in particular regarding the collection and analysis of our data. Although this does not negate our observed associations between ECG and CMR findings, caution is definitely warranted regarding the findings on prognostication.

Conclusions

This multicenter CMR study is the largest carried out so far in a genetically homogeneous cardio-

myopathy cohort worldwide. We observed LV myocardial fibrosis even in the presence of preserved

LVEF, which suggests that the development of fibrosis occurs as an early phenomenon in PLN

p.Arg14del mutation carriers. Myocardial fibrosis is mainly present in the LV inferolateral wall and

corresponds with ECG repolarization abnormalities. Although preliminary, the presence of LV-

LGE was found to be independently associated with VA. Based on our findings, we recommend

integrating CMR findings into the diagnostic work-up for both symptomatic and presymptomatic

mutation carriers.

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141 Funding

This work was supported by the Netherlands Cardiovascular Research Initiative (CVON), an initiative supported by the Dutch Heart Foundation (the Hague, the Netherlands): CVON [grant number 2012-10] PREDICT, CVON [grant number 2014-40] DOSIS and CVON [grant number 2015- 12] eDETECT projects.

Confl ict of interest

AWWW is a Member of the Scientifi c Advisory Board of LilaNova. PLH recently became an employee of F. Hoff mann-La Roche Ltd. The authors report no further confl icts of interest or any relationships with industry.

Acknowledgements

We thank Jackie Senior for carefully editing the manuscript.

Declaration of Helsinki

The study conformed to the principles of the Helsinki Declaration and the institutional medical ethics committees. The data were collected retrospectively and the clinical investigations were performed as part of routine patient care.

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