University of Groningen 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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Phospholamban p.Arg14del cardiomyopathy is

characterized by phospholamban aggregates,

aggresomes, and autophagic degradation

CHAPTER 4

Wouter P te Rijdt,1,2,3_{J Peter van Tintelen,}4_{Aryan Vink,}5_{Allard C van der Wal,}6

Rudolf A de Boer,1_{Maarten P van den Berg}1_{& Albert J H Suurmeijer}2

1_{Department of Cardiology,University of Groningen, University Medical Centre Groningen, Groningen,} 2_{Department of Pathology, University of Groningen, University Medical Centre Groningen, Groningen,} 3_{Interuniversity Cardiology Institute of The Netherlands (ICIN), Utrecht} 4_{Department of Clinical Genetics, University of Amsterdam, Academic Medical Centre, Amsterdam,} 5_{Department of Pathology, University Medical Centre Utrecht, Utrecht, and} 6_{Department of Pathology, University of Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands}

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Abstract

Aims

The non-desmosomal phospholamban (PLN) p.Arg14del mutation was identified in patients diagnosed with dilated cardiomyopathy (DCM) and/or arrhythmogenic cardiomyopathy (ACM). We aimed to investigate whether this mutation leads to aggregation, aggresome formation and autophagy of mutant PLN protein.

Methods and results

We studied 20 complete heart specimens of PLN p.Arg14del mutation carriers [age 48 ± 15 years (mean ± standard deviation); 55% males], either from autopsies or from explants. Gross and microscopic examination showed biventricular cardiomyopathy with histopathological features of both ACM and DCM, i.e. a combination of fibrofatty replacement and interstitial fibrosis. Immunohistochemistry for PLN showed large perinuclear PLN protein aggregates in cardiomyocytes in both ventricles in all examined hearts. The median numbers of PLN-containing aggregates were 12 per 5 mm2_(range

3–48 mm2_{) in right ventricular myocardium and 13 per 5 mm}2_{(range 5–89 mm}2_{) in left}

ventricular myocardium. Double immunohistochemical staining showed colocalization of autophagy markers p62 (sequestosome-1) and microtubule-associated protein light chain 3 with PLN in all aggregates, suggestive of degradation by selective autophagy. On electron microscopy, the ultrastructural appearance of these PLN-containing aggregates was typical of aggresomes; they were not surrounded by a membrane, and were located adjacent to the microtubular organizing centre. PLN-containing aggregates were not found in 10 PLN-negative cases of idiopathic and genetic DCM or in seven cases of desmosomal ACM.

Conclusions

PLN p.Arg14del cardiomyopathy is a biventricular cardiomyopathy characterized by large perinuclear PLN protein aggregates with a typical ultrastructural appearance of aggresomes. PLN detected by immunohistochemistry appears to be a sensitive and specific marker for this disease.

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4

Introduction

Inherited cardiomyopathies are generally caused by mutations in genes coding for sarcomeric, cytoskeletal, nucleoskeletal, mitochondrial, desmosomal, ion channel and calcium-handling proteins. These mutations are most commonly associated with hypertrophic, dilated or arrhythmogenic phenotypes, but overlapping phenotypes do exist and are well recognized.1

An overlapping arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy (DCM) phenotype has been described in Dutch patients carrying a mutation in the phospholamban (PLN) gene.2

PLN acts as a reversible regulator of calcium uptake by inhibiting the sarcoplasmic reticulum

Ca2+_{-ATPase 2a (SERCA2a) pump in the non-phosphorylated state. Upon phosphorylation (e.g. via}

the β-adrenergic pathway), PLN dissociates from SERCA2a, resulting in greater activation of this Ca2+ pump and, subsequently, an increased rate of calcium uptake in the sarcoplasmic reticulum. In this way, the dynamic PLN–SERCA2a interaction plays an important role in cardiac contractility and relaxation.3

Several pathogenic PLN mutations causing inherited cardiomyopathy have been described.4–6_{In a Dutch cohort, an Arg14 deletion (p.Arg14del; c.40_42delAGA) in the coding}

region of PLN was identifi ed in 15% of patients clinically diagnosed with idiopathic DCM and in 12% of patients diagnosed with arrhythmogenic right ventricular (RV) cardiomyopathy (ARVC), making this mutation the most prevalent single cardiomyopathy-related mutation identifi ed in The Netherlands.2,7

However, it remains unclear how exactly the PLN p.Arg14del mutation leads to such severe cardiomyopathy and arrhythmia. The underlying mechanisms involve super-inhibition of SERCA2a activity6_{and hindering of PLN phosphorylation by the protein kinase A catalytic}

subunit,8_{but other mechanisms remain to be elucidated.}

It has been established that, in terminally diff erentiated cardiomyocytes, cellular function depends on protein homeostasis, and this protein balance is particularly important during remodelling of the heart. Autophagy is of paramount importance in the clearance of stress-induced protein aggregates and aggresomes in cardiomyopathies.9_{Recently, it has been}

shown, in primary cultured neonatal mouse cardiomyocytes, that PLN is degraded by selective p62-mediated autophagy after polyubiquitinylation.10_{p62 acts as an adaptor protein during}

aggregation, sequestration [hence its synonym, sequestosome-1 (SQSTM-1)] and degradation of misfolded proteins by the ubiquitin– proteasome pathway and selective autophagy.11

Microtubule-associated protein light chain 3 (LC3) is a more specifi c diagnostic marker of autophagy. LC3 plays a key role in the selective recruitment of autophagic cargoes into autophagosomes, and serves as docking site for adaptor proteins such as p62.

In the present study, by using immunohistochemistry (IHC) for PLN, p62, and LC3, as well as conventional electron microscopy, we show that PLN p.Arg14del mutation cardiomyopathy is characterized by the formation of large perinuclear inclusion bodies (so-called aggresomes) in ventricular cardiomyocytes. The ultrastructural morphology of these aggregates and the immunohistochemical colocalization of PLN with p62 and LC3 in these aggresomes provides further evidence for their degradation via autophagosomes and lysosomes. When other DCM and ACM specimens were studied, it appeared that immunohistochemical analysis of PLN-containing

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aggregates is sensitive and specific. The presence of these aggregates could be of help in the postmortem diagnosis of PLN p.Arg14del mutation cardiomyopathy.

Materials and methods

Clinical and pathological examination

We evaluated 20 complete hearts from Dutch patients who were heterozygous carriers of the identical PLN p.Arg14del mutation. These hearts were from autopsies (including cases of sudden cardiac death) or from patients who underwent heart transplantation.

Information on gross examination of these hearts was available in all cases. Formalin-fixed and paraffin-embedded samples from the left ventricular (LV) wall and the right ventricular (RV) wall were available in all cases. Tissue sections (4 μm) were stained with Masson’s trichrome.

All clinical cardiological data available from these 20 mutation carriers were collected and evaluated retrospectively. Clinical data were missing for six autopsy patients, because they died suddenly without any prior medical documentation. The widely accepted clinical criteria proposed by Mestroni et al.12_{were used to diagnose DCM, and the revised task force criteria were}

used to diagnose ARVC.13

RV endomyocardial biopsies obtained from a second cohort of 25 patients with the PLN p.Arg14del mutation were also studied (immunohistochemical analysis). These biopsies were taken for diagnostic purposes.

Ethics statement

All specimens were retrieved from the archive of the Department of Pathology of the University Medical Centre Groningen and the consultation cases of A.J.H.S. The study met the criteria of the code of conduct for responsible use of human tissue that is used in The Netherlands (Dutch Federation of Biomedical Scientific Societies; http://www.federa.org). Specific approval from the hospital ethics committee was not necessary for this study, because all material was originally obtained for diagnostic procedures, and the tissue samples were completely de-identified before inclusion in the study.

IHC

IHC for PLN was performed to visualize PLN-containing protein aggregates in cardiac muscle cells. We used mouse monoclonal antibody 2D12 (Abcam, Cambridge, MA, USA) at a dilution of 1:10 000. With this dilution, strong immunostaining of PLN-containing aggregates was still easy to discern in the weaker background staining of PLN in the cytoplasm of the cardiomyocytes. IHC for SQSTM1 (p62), a scaffold protein that links ubiquinated proteins to autophagosomes, was performed with mouse monoclonal antibody D3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:100. IHC for LC3 was performed with monoclonal antibody 5F10 (Nanotools, Teningen, Germany) at a dilution of 1:150. A punctate distribution of LC3 is considered to represent autophagic activity. Immunohistochemical staining was performed on an automated immunostaining platform (Ventana Benchmark Ultra; Ventana Medical Systems, Tucson, AZ, USA) with the CC1 standard antigen retrieval protocol (Tris-HCl buffer, pH 9, for 1 h at 95°C). IHC sequential double staining for PLN and both p62 and, separately, LC3 was performed on

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69 10 specimens with the ultraView alkaline phosphatase and 3,3’-diaminobenzidine detection kits from Ventana Medical Systems, respectively. Appropriate positive and negative controls were used throughout. Specifi cally, for the PLN antibody, the myocardium served as an internal positive control. For the SQSTM1 and LC3 antibodies, cirrhotic liver tissue from a case of α1-antitrypsin defi ciency served as a positive control.

The numbers of PLN-containing aggregates in cardiomyocytes in the posterolateral LV wall and posterolateral RV wall were scored in PLN-immunostained sections in an area of 5 mm2_,

corresponding to 20 high-power fi elds (HPFs) (the fi eld of the ×40 lens), with an Olympus BX40 microscope. At low-power magnifi cation, the area with the highest number of aggregates was selected.

In order to establish the specifi city of the PLN-containing aggregates, PLN IHC was also performed on LV samples from 10 hearts of patients with idiopathic and genetic DCM (four cases represented genetic DCM, owing to mutations in the desmin, RNA-binding motif protein 20, lamin A/C and dystrophin genes), and on LV and RV samples of seven hearts with ACM with known mutations in desmosomal genes (four in the plakophilin-2 gene, one in the desmocollin-2 gene, one in the junctional plakoglobin gene, and one in the desmoplakin gene).

Sample preparation for electron microscopy

Two heart specimens were processed for electron microscopy. Four 2-mm myocardial fragments were fi xed in 2% glutaraldehyde in 0.1 M sodium cacodylate buff er (pH 7.4), washed in 0.1 M sodium cacodylate, and postfi xed in 1% osmium tetroxide and 1.5% potassium ferrocyanide. Samples were dehydrated, embedded in Epon, and sectioned. Ultrathin sections (70 nm) were positioned on A600 Nickel single slot holders (Fisher Scientifi c, Schwerte, Germany) supported by Formvar, and contrasted with 2% uranyl acetate (methanol) followed by Reynolds lead citrate. Acquisition was performed by use of a Zeiss supra 55 electron microscope with ATLAS software developed by Fibics (Ottawa, Canada). Samples were recorded at a pixel size of 2.5 nm. Scans were stitched and rendered as HTML fi les with ATLAS VE VIEWER software. Images were exported with ATLAS VE VIEWER as TIFF fi les.

Results

Patient characteristics

The complete heart cohort consisted of 11 males and nine females. The mean (± standard deviation) age at the time of autopsy or heart transplantation was 48 ± 15 years (range 25–72 years).

The initial presentation was variable; 12 of 20 patients presented with an arrhythmic event (of which six were sudden cardiac death events), and eight of 20 patients presented with signs and/or symptoms related to heart failure. Fourteen patients (the six sudden cardiac death cases had no prior medical documentation) were clinically evaluated by applying the Mestroni criteria for DCM12_{and the revised task force criteria for ARVC.}13_{All 14 patients had LV dysfunction (LV}

ejection fraction of <45%), 12 patients had LV dilatation (LV end-diastolic diameter of >117%), nine patients fulfi lled the revised task force criteria for a defi nitive diagnosis of ARVC, and eight patients were diagnosed with both ARVC and DCM.

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Pathology

In all 20 complete hearts, we observed areas of fibrofatty replacement of RV myocardium that were indistinguishable from fibrofatty replacement found in ARVC caused by desmosomal gene mutations (Figures 1 and 2). In trichrome-stained tissue sections, distinct features of fibrofatty change were areas with atrophic and hypertrophic cardiomyocytes embedded in collagen and surrounded by fat cells. Both fatty and fibrofatty changes were most prominent in the epicardial half of the compact myocardium, which effaced the normally sharp border between myocardium and epicardial fat (Figures 1 and 2). Moreover, areas of fibrofatty replacement were often seen in the LV wall, in particular in non-sudden cardiac death cases (Figure 1). In addition, in all 20 hearts, the LV myocardium showed moderate to severe interstitial replacement fibrosis (Figures 1

and 2), which was most prominent in the posterolateral wall, as found earlier by our group with

a high-resolution digital histological quantification technique.14_{Thus, PLN p.Arg14del-related}

cardiomyopathy is a biventricular cardiomyopathy with overlapping histopathological features of ACM and DCM in most cases. Notably, our previous study showed no difference in the distribution pattern of myocardial replacement fibrosis or fatty change between patients presenting with a clinically predominant DCM phentotype and those presenting with an ACM phenotype.14

Figure 1. The explanted heart of a 57-year-old female phospholamban p.Arg14del mutation carrier who was clinically diagnosed with dilated and arrhythmogenic cardiomyopathy. A, Midventricular transverse slice of

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the heart showing extensive fatty infi ltration of the outer compact layer of both right ventricular (RV) and left ventricular (LV) myocardium (arrows). B,C, Masson trichrome stain of a microscopic section of the lateral LV wall showing extensive fatty and fi brofatty infi ltration of the outer compact myocardium and replacement fi brosis of the inner compact myocardium. D,E, Masson trichrome stain of a microscopic section of the anterior RV wall (of the outfl ow tract) showing a wavefront of extensive fatty and fi brofatty infi ltration of the compact myocardium with areas of replacement fi brosis with collagen surrounding hypertrophic and atrophic cardiomyocytes.

Figure 2. Microscopy of the heart of a 25-year-old male who presented with sudden unexpected cardiac death resulting from arrhythmogenic phospholamban p.Arg14del mutation-related cardiomyopathy. A,B, Masson trichrome stain of a microscopic section of the lateral LV wall showing moderate replacement fi brosis of the compact LV myocardium. C,D, Masson trichrome stain of a microscopic section of the posterior right ventricular wall, showing a wavefront of extensive fatty and fi brofatty infi ltration of the compact myocardium including replacement fi brosis with collagen surrounding hypertrophic and atrophic cardiomyocytes.

IHC

In all examined hearts from patients with a heterozygous PLN p.Arg14del mutation, immunohistochemical examination showed large PLN-staining aggregates in single isolated cardiomyocytes of the RV and LV myocardium, which were detectable at low-power magnifi cation. Notably, these aggregates were not visible in routine haematoxylin and eosin-stained heart tissue. Cardiomyocytes with PLN-containing aggregates were transmurally and randomly distributed in the LV wall. In the RV wall and septum, we observed a patchy distribution. Typically, PLN-containing inclusion bodies were large globular aggregates oriented perinuclearly (sometimes circumnuclear). In longitudinal cross-sections, tubular cardiomyocytes contained a single large aggregate that had an elongated globular structure (Figure 3A). In transverse cross-sections, aggregates were also easily detectable (Figure 3B).

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The median numbers of PLN-containing aggregates were 13 per 5 mm2_{(range 5–89 mm}2_{) in}

LV myocardium and 12 per 5 mm2_{(range 3–48 mm}2_{) in RV myocardium, corresponding to an}

average of one aggregate in two HPFs in LV samples, and one aggregate in three HPFs in RV samples. Strikingly, cardiomyocytes with PLN-containing aggregates detected by IHC showed significantly weaker cytoplasmic staining (of PLN in the sarcoplasmic reticulum) than surrounding cardiomyocytes without PLN-containing aggregates. This was apparent in both longitudinal and transverse cross- sections of myocardium (Figure 3A,B).

By IHC, the perinuclear PLN-containing aggregates were also positive for p62/SQSTM1 and LC3. p62 showed a globular staining pattern of large aggregates, in addition to fine punctate cytoplasmic staining (Figure 4A), whereas LC3 showed a punctate staining pattern adjacent to and in the periphery of the large aggregates (Figure 4B). Immunohistochemical double staining showed colocalization of both p62 and LC3 in PLN-containing aggregates in all PLN-positive cardiomyocytes (Figure 4C,D).

In the 17 hearts used as negative control specimens (10 hearts with idiopathic or genetic DCM, and seven hearts with genetic ACM resulting from desmosomal gene mutations), PLN-positive aggregates were completely absent, indicating that PLN-containing aggregates are specific for PLN p.Arg14 cardiomyopathy.

After we had established that PLN-containing aggregates can be detected in slices of whole heart specimens, we tested whether they could also be detected in endomyocardial biopsies from the right ventricle. A minimum of two RV endomyocardial biopsy samples were available for histology in all cases. In two of 25 (8%) cases, PLN-containing aggresomes were observed in RV endomyocardial biopsies of patients with PLN p.Arg14del cardiomyopathy.

Figure 3. A. Large elongated perinuclear and circumnuclear globular phospholamban (PLN)-stained aggregates in a single cardiomyocyte sectioned longitudinally. B, PLN-stained aggregates (aggresomes) in cross-sections of cardiomyocytes. Note the weaker PLN staining in cardiomyocytes with aggresomes (circled) than in other cardiomyocytes.

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Figure 4. A. Granular cytoplasmic and globular staining of p62, suggestive of autophagy. B, Punctate staining of microtubule-associated protein light chain 3 (LC3) around and in aggresomes, indicative of autophagy. C, Colocalization of p62 (brown) and phospholamban (PLN) (red) in a perinuclear aggresome shown by immunohistochemical double staining. D, Colocalization of LC3 (brown) and PLN (red) in a perinuclear aggresome shown by immunohistochemical double staining.

Electron microscopy

In the two samples processed for electron microscopy, single cardiomyocytes were seen to contain large perinuclear aggregates. These giant aggregates were round, electron-dense globules that diff ered in size (Figure 5A). At high magnifi cation, in one cardiomyocyte the aggregate was amorphous, but had a structured granular appearance. This aggregate was not surrounded by a membrane, but was located adjacent to a network of microtubules (Figure 5B). These features are consistent with the aggresome, a large paranuclear protein aggregate formed by the transport of aggregated proteins on microtubules to the perinuclear area, where the Golgi complex is also located. Moreover, in the core of this aggregate, membranous structures resembling lysosomes were seen (Figure 5C).

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Figure 5. Ultrastructural visualization of a cardiomyocyte (A) with globular perinuclear aggresome formation (arrow). The aggresome is located adjacent to an array of microtubules (arrow) (B), with a membrane-free perinuclear aggresome containing lysosomes (arrow) (C).

Discussion

The p.Arg14del (c.40_42delAGA) mutation in PLN is the most prevalent single cardiomyopathy-related mutation identified in The Netherlands, and the mutation has also been found in several other European countries, Canada, and the USA. This PLN mutation has been associated with DCM,6,15,16_{and recently also with ACM.}2_{Carriers of this mutation are at high risk for malignant}

ventricular arrhythmias and end-stage heart failure, with subsequent high mortality and a poor prognosis from late adolescence.17_{However, it remains unclear how exactly the PLN p.Arg14del}

mutation leads to such severe cardiomyopathy and arrhythmia. The present study has three significant and novel results. First, we show that the PLN p.Arg14del mutation leads to the formation of large aggregates of PLN protein in ventricular cardiomyocytes that are degrated via autophagosomes and lysosomes. These aggregates have a typical ultrastructural appearance

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75 of aggresomes. Second, these aggresomes can easily be detected by IHC with a highly diluted PLN monoclonal antibody. Finally, immunohistochemical detection of these PLN-containing aggresomes appeared to have high sensitivity and specifi city for PLN cardiomyopathy, as we observed them in all PLN cardiomyopathy hearts and in none of the hearts with cardiomyopathy caused by other genetic mutations.

The perinuclear and circumnuclear location of the PLN-containing aggregates that we observed with IHC and light microscopy, and the observation made with electron microscopy that these large perinuclear aggregates are membrane-free and are found adjacent to microtubule arrays, are consistent with aggresomes.18,19_{Aggresomes are single intracytoplasmic juxtanuclear}

inclusion bodies consisting of insoluble protein aggregates (e.g. mutant PLN) that are formed by the retrograde transport of aggregated protein via the microtubular network.19_Aggresome

formation along microtubules most likely explains why PLN-containing aggregates have an elongated shape in longitudinal sections of tubular cardiomyocytes, as was observed in this study.

Another human cardiomyopathy with aggresome formation is desmin-related cardiomyopathy.20,2_{1 In this cardiomyopathy, mutations in the coding region of the DES gene}

may cause conformational changes, and these mutant proteins have a strong tendency to form insoluble aggregates and aggresomes, as seen in PLN cardiomyopathy, which are hypothesized to be proteotoxic. One of our controls was desmin-related cardiomyopathy, and in this case no PLN- containing aggregates were observed. Our observations confi rm the hypothesis that the PLN p.Arg14del mutation also induces protein changes that have a tendency to result in the formation of aggresomes, and are in line with an in-vitro study in which PLN was found to be polarized at one site of the cytoplasm in PLN p.Arg14del cardiomyocytes, whereas in cells where the mutation was corrected a homogeneous reticular distribution of PLN was observed.22

In addition to aggregated mutant proteins, aggresomes contain several adaptor proteins, such as heat shock proteins, ubiquitin, and p62.19_{p62 (SQSTM-1) is a multifunctional protein}

involved in the formation of homotypic protein aggregates that may be degraded by the ubiquitin–proteasome pathway or by selective autophagy. In this IHC study, we observed that PLN-containing aggresomes were, indeed, positive for p62. By IHC, p62 was seen both in aggresomes and as fi ne granular intracytoplasmic dust, possibly representing smaller ubiquitinylated aggregates. Besides its roles in tumour necrosis factor-α signalling, control of reactive oxygen species, and protein degradation in the ubiquitin–proteasome pathway, another function of p62 is to act as a cargo receptor for degradation by autophagy of organelles (e.g. mitophagy) or macromolecules (e.g. mutant proteins) by interacting with LC3.23_{The interaction of LC3 with}

p62 aggregates facilitates fusion of autophagosomes with lysosomes for protein aggregate degradation, as was recently shown by Teng et al.10_{in an experimental model of mutant PLN R9C}

in transgenic mice. With double immunohistochemical staining, we showed that PLN-containing aggresomes contained not only p62 but also LC3. Punctate LC3 staining in autophagosomes was located around and in PLN-containing aggresomes. Moreover, with electron microscopy, we demonstrated the presence of lysosomes within perinuclear aggresomes. These light and electron microscopic fi ndings support the concept that p62-mediated selective autophagy of aggregates and aggresomes occurs in PLN p.Arg14del cardiomyopathy.

The fact that we observed large aggresomes of PLN might indicate at least partially

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inadequate degradation of mutated proteins via the autophagy pathway. In previous studies of human cardiomyopathy, it has been suggested that inadequate degradation of protein conjugates may eventually contribute to so-called autophagic cell death.24_{We observed that}

single cardiomyocytes with PLN-containing aggresomes showed weaker cytoplasmic PLN staining in the sarcoplasmic reticulum than in surrounding cardiomyocytes. This may indicate that the cardiomyocytes with PLN- containing aggresomes are indeed dysfunctional. Given the role of PLN in calcium homeostasis in cardiomyocytes, it is possible that these isolated cardiomyocytes are foci of electric instability and a source of arrhythmic events, one of the hallmarks of this cardiomyopathy, but this warrants further investigation.

PLN-containing perinuclear aggresomes appeared to be specific for PLN p.Arg14del cardiomyopathy, as they were not encountered in other examined hearts of patients with idiopathic or genetic DCM and genetic ACM. However, it is conceivable that PLN-containing aggresome formation can also occur in other genetic PLN cardiomyopathies. To date, five different PLN mutations have been reported in familial DCM, including PLN p.Arg14del. In The Netherlands, the PLN p.Arg14del founder mutation has a high prevalence, and was identified in 15% of Dutch patients clinically diagnosed with DCM and in 12% of patients diagnosed with ARVC. Unfortunately, no cardiomyopathic hearts other than PLN p.Arg14del cardiomyopathy hearts were available for inclusion in this study, owing to their low prevalence and consequent availability.

The number of randomly distributed PLN-containing aggresomes in LV and RV myocardium was relatively low. On average, we found one in two HPFs in LV myocardium, and one in three HPFs in RV myocardium. This relatively low number of aggresomes per mm2 in RV myocardium may explain why we detected PLN-containing aggresomes in only two of 25 (8%) cases of RV endomyocardial biopsies of patients with PLN p.Arg14del cardiomyopathy. The distribution of aggregates in the RV wall and septum is patchy, so sampling error may occur. Moreover, visualization of PLN-containing aggresomes in RV endomyocardial biopsies was severely hampered by contraction band artefacts in cardiomyocytes. LV assist device implantation is becoming increasingly common in cardiomyopathy patients with heart failure. During implantation of the device, a larger part of the apex of the left ventricle becomes available for diagnostic purposes. In these larger myocardial tissue parts, PLN- containing aggresomes can probably be detected at a higher rate than in small RV biopsies.

Gross and microscopic examination of PLN p.Arg14del mutation cardiomyopathy revealed a biventricular cardiomyopathy with features of both ACM, often with LV involvement, and DCM. When sudden death coronial autopsies are performed, this mixed pattern should prompt retention of appropriate samples for genetic analysis, and referral of family members for further investigation. When PLN p.Arg14del cardiomyopathy is suspected clinically and/or pathologically, additional immunohistochemical analysis for PLN may be very helpful in the diagnostic workup, as shown in this study.

In conclusion, our novel finding is that PLN p.Arg14del cardiomyopathy is a protein aggregate- associated biventricular disease characterized by large cytoplasmic perinuclear PLN-containing aggregates representing aggresomes that are degraded by p62-selective autophagy. IHC for PLN is a sensitive and specific method for detecting PLN-containing aggregates in whole heart specimens of this cardiomyopathy.

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Confl icts of interest

The authors state that they have no confl icts of interest.

Author contributions

A. J. H. Suurmeijer and W. P. te Rijdt designed the study and examined the cases. W. P. te Rijdt and A. J. H. Suurmeijer wrote the manuscript. J .P. van Tintelen, A. Vink, A. C. van der Wal, R. A. de Boer and M. P. van den Berg revised the manuscript critically for important intellectual content. All authors approved the fi nal version of the manuscript.

Acknowledgements

This study was supported by the Interuniversity Cardiology Institute of The Netherlands (ICIN), project number 12301 (W. P. te Rijdt, R. A. de Boer, and J .P. van Tintelen). We also acknowledge support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, the Dutch Federation of University Medical Centres, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON-PREDICT).

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