DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters

Abstract

BACKGROUND:

The p.Arg14del variant of the phospholamban (PLN) gene causes cardiomyopathy, leading to severe heart failure (HF). Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease. Dwarf open reading frame (DWORF) has been shown to counteract PLN regulatory calcium handling function in the sarco/endoplasmic reticulum (S/ER). Here we investigated the potential disease modulating action of DWORF in this cardiomyopathy and its effects on calcium handling and PLN aggregation.

METHODS:

We studied a PLN-R14del mouse model, which develops cardiomyopathy with similar characteristics as human patients, and explored whether cardiac DWORF overexpression could delay cardiac deterioration. To this end, homozygous PLN-R14del (R14^Δ/Δ) mice carrying the DWORF transgene (R14^Δ/ΔDWORF^Tg) were used.

RESULTS:

DWORF expression was suppressed in hearts of R14^Δ/Δ mice with severe HF. Restoration of DWORF expression in R14^Δ/Δ mice delayed cardiac fibrosis and HF, and increased life span more than 2-fold (from 8 to 18 weeks). DWORF accelerated SR calcium re-uptake and relaxation in isolated cardiomyocytes with wildtype PLN, but in R14^Δ/Δ cardiomyocytes SR calcium re-uptake and relaxation were already enhanced and no differences were detected between R14^Δ/Δ and R14^Δ/ΔDWORF^Tg. Rather, DWORF overexpression delayed the appearance and formation of large pathogenic perinuclear PLN clusters. Careful examination revealed co-localization of SR markers with these PLN clusters in both R14^Δ/Δ mice and human p.Arg14del PLN heart tissue and hence these previously termed aggregates are comprised of abnormal organized S/ER. This abnormal S/ER organization in PLN-R14del cardiomyopathy contributes to cardiomyocyte cell loss and replacement fibrosis, consequently resulting in cardiac dysfunction.

CONCLUSIONS:

Disorganized S/ER is a major characteristic of PLN-R14del cardiomyopathy in humans and mice, and results in cardiomyocyte death. DWORF overexpression delayed PLN-R14del cardiomyopathy progression and extended life span in R14^Δ/Δ mice, by reducing abnormal S/ER clusters.

INTRODUCTION

A number of pathogenic mutations in the gene coding for phospholamban (PLN) have been identified that cause cardiomyopathy. The p.Arg14del pathogenic variant is the best described and most prevalent variant, and may cause dilated cardiomyopathy (DCM) or arrhythmogenic cardiomyopathy (ACM), often resulting in severe heart failure (HF) with characteristic perinuclear PLN aggregates ^1,2. So far, no specific treatment for this cardiomyopathy is available and heart transplantation often is needed as a last resort.

PLN is a small cardiac-specific protein that localizes to the sarcoplasmic reticulum (SR) membrane, where it negatively regulates the re-uptake of cytosolic calcium ions (Ca²⁺) into the SR via its interaction with the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) ³. Calcium handling studies have shown different effects of PLN-R14del on SERCA regulation and the precise contribution of dysfunctional SERCA regulation in the pathology of this disease is still under debate ^4–8. Additionally, functions of PLN have been attributed to nuclear and mitochondrial mechanisms ^9,10, suggesting more complex PLN interactions and function. A recent study using human induced pluripotent stem cell (hiPSC)-derived cardiac tissues suggested impairment of the endoplasmic reticulum (ER)/mitochondria compartment as a novel disease mechanism underlying PLN-R14del cardiomyopathy ¹⁰. Interestingly, human PLN-R14del cardiomyopathy is characterized by the presence of dense perinuclear PLN protein aggregates in cardiomyocytes ¹¹. Furthermore, in a mouse model of PLN-R14del cardiomyopathy, clustered perinuclear PLN protein was identified and was consistently found to be the first pathological sign, preceding the onset of cardiac dysfunction, suggesting a causal role ^12,13. So far, the exact nature of the perinuclear PLN aggregates in PLN-R14del cardiomyopathy is unknown, but the strong association with disease development warrants further investigations.

A recently discovered SR localized micropeptide, termed dwarf open reading frame (DWORF), with a function opposite to PLN, was strongly down-regulated in humans and mice with HF ^14–16. Using a transgenic mouse line, DWORF overexpression was shown to improve SERCA mediated calcium re-uptake and prevent HF in the muscle LIM protein knock-out (MLP-KO) DCM mouse model ¹⁷. In addition, DWORF overexpression improved ventricular function and reduced cardiac dilation in a mouse model of myocardial infarction (MI) ¹⁸. These studies have thus shown that DWORF overexpression is cardioprotective in HF from genetic but also common etiology, most likely via enhanced calcium re-uptake. Whether DWORF affords cardioprotection in PLN-R14del cardiomyopathy, with its characteristic dense perinuclear PLN clusters, is unknown.

A mouse model that recapitulates the human PLN-R14del pathological phenotype has been described ¹². Mice harboring the heterozygous mutation develop disease characteristics at around 18 months of age, which is similar to what clinically is observed in humans where age of onset often is around 40–50 years of age. Mice harboring both pathogenic PLN-R14del alleles (homozygous, R14^Δ/Δ) showed an enhanced and accelerated phenotype developing in 4–8 weeks, while standard HF treatments could not attenuate disease progress, again in accordance with clinical observations ¹².

Here, we investigated the effect of DWORF overexpression in PLN-R14del cardiomyopathy. In particular, DWORF^Tg mice were crossbred with PLN-R14^Δ/Δ mice to generate R14^Δ/ΔDWORF^Tg mice. We found that overexpression of DWORF in PLN-R14^Δ/Δ mice reduced PLN-R14del-induced S/ER cluster formation, improved cardiac function and extended life span.

METHODS

Data Availability.

The data, methods, and study materials used to conduct the research are available from the corresponding authors on reasonable request. Expanded Methods are available in the Supplemental Material, also please see the Major Resources Table in the Supplemental Materials.

Animals

Animal studies were approved by the Central Committee of Animal experiments (CCD, license number AVD1050020199105) and the animal ethical committee of the University of Groningen (permit numbers IVD199105-01-003, IVD199105-01-010 and IVD199105-01-022 for the current study, IVD1583-02-001 for DWORF gene expression in 3- to 7-week-old R14^Δ/Δ mice and DEC6827A, DEC6920A and IVD16487-03-01 for the samples we used from the study from Du et al.¹⁹) and conformed with the guidelines from the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and reported following the ARRIVE guidelines ²⁰.

Study design

To determine the effect of DWORF overexpression on the development of PLN-R14del cardiomyopathy, the DWORF^Tg mice were crossed with the PLN-R14^Δ/Δ mouse model (R14^Δ/ΔDWORF^Tg). Mice were assigned to the experimental groups by their genotype. The life span of R14^Δ/Δ mice with and without DWORF overexpression (male and female, in total n=7–9 per group) was determined. Furthermore, cardiac function of these mice and their wildtype (WT, transgene-negative) littermates was evaluated using echocardiography at 4, 6, 10, 14 and 18 weeks of age. In addition, electrocardiography (ECG) and several histological and molecular analyses were performed at 3, 5, 7 and 18 weeks of age (both male and female, mice of each genotype were randomly assigned to the different experimental time points, per group n=3, 8, 10 and 8 respectively). R14^Δ/ΔDWORF^Tg mice that reached the humane endpoint (which occurred between 17 and 20 weeks of age) and their 22-week-old WT controls, will be referred to as 18 weeks of age.

Echocardiography

Two-dimensional transthoracic echocardiography measurements were performed using the Vevo 3100 system with a 40-MHz MXX550D linear array transducer (FUJIFILM VisualSonics, Canada) as described previously ²¹. During imaging, mice were anaesthetized with a mixture of oxygen and isoflurane (2.5%). Cardiac dimensional and functional parameters were determined using Vevo LAB software (version 3.2.6; FUJIFILM VisualSonics). Data acquisition and analysis were conducted in accordance with the recommendations of the European Society of Cardiology (ESC) Working Group on Myocardial Function ²² and were performed in a blinded fashion.

Surface electrocardiography

Lead II configuration ECG recordings were acquired on anesthetized mice (2.5% isoflurane mixed with oxygen), by a previously reported procedure ¹². Subdermal needle electrodes were connected to a PowerLab 8/30 data acquisition device (model ML870; ADInstruments, New-Zealand) and an animal Bio Amp biological potential amplifier (model ML136; ADInstruments), which transferred recordings to LabChart Pro software (version 8; ADInstruments). The ECG Analysis module was used for data analysis.

Histological analysis

Heart tissue was fixed overnight in 4% buffered formaldehyde solution (4% formalin; Klinipath, the Netherlands). Afterwards, tissues were dehydrated using an automated system (Leica TP1020, Leica Microsystems, Germany), embedded in paraffin (Klinipath) and cut into 4-μm thick sections. Slides were deparaffinized before proceeding with the subsequent staining protocol. Staining protocols are available in the Supplemental Material and the antibodies used are presented in Table S1.

Western blot analysis

Radioimmunoprecipitation assay (RIPA) lysis buffer was used to isolate proteins from snap-frozen LV tissue as described previously ¹². Quantification was performed using ImageJ software (National Institutes of Health) ²³ and the measurements of the protein of interest were corrected for the measured total amount of loaded protein that was determined using the Revert 700 Total Protein Stain (LI-COR Biosciences, NE, USA). The calculated values were presented as a fold change compared to the age-matched control group. The primary and secondary antibodies that were used are presented in Table S3.

Calcium transient and contraction measurements in isolated adult mouse cardiomyocytes

Cardiomyocytes were isolated from WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice at 3 weeks or 6 weeks of age (n=4 per genotype) using the protocol of Ackers-Johnson et al. ²⁴. An IonOptix Calcium and Contractility System (IonOptix corporation, MA, USA) was used to measure calcium transients and video-based sarcomere length (SL) in Fura-2 loaded cardiomyocytes under pacing conditions (IonOptix MyoPacer; 2 Hz, 4 ms pulse duration, 20 V) at 37°C. Calcium and contractility data were analyzed using CytoSolver, a cloud-based analysis program of IonOptix.

Electron microscopy

Electron microscopy (EM) with PLN-quantum dot immunolabeling was performed as reported previously ^25,26.

Statistical analyses

All data are presented as means ± standard errors of the mean (SEM), with the exception of calcium transient and contraction data that are presented as truncated violin plots (median and quartiles are indicated by lines). Nonparametric tests were performed because of small group sizes. Therefore, the Mann-Whitney U test was performed for comparisons between two groups and the Kruskal-Wallis test followed by Dunn’s post hoc test for multi-group comparisons. For statistical analysis of survival curves, a log-rank test was performed. Since individual cardiomyocytes instead of individual animals were plotted for calcium transient and contraction data, a hierarchical cluster analysis using customized R software (version 4.3.1) was applied according to the method described by Sikkel et al. ²⁷, after log-transformation of the data because of non-normal distribution. All other statistical analyses were performed using GraphPad Prism (version 8.4.2, GraphPad Software, MA, USA). P-values < 0.05 were considered statistically significant. Additional details on all statistical tests performed can be found in a supplemental statistical report.

RESULTS

DWORF overexpression extended life span and improved cardiac function in R14^Δ/Δ mice

DWORF has been shown to be downregulated in HF in both mice and humans ^14,17. We confirmed downregulation of cardiac DWORF gene expression in our PLN-R14del cardiomyopathy mouse model (Figure S1A). At end-stage disease, DWORF gene expression levels in PLN-R14del mice were significantly lower than in mice with myocardial ischemia reperfusion (I/R) injury and in mice that underwent myocardial infarction (MI) (Figure S1B). Since DWORF and PLN have been reported to have opposite functions, we investigated whether elevated DWORF levels could have beneficial effects against PLN-R14del cardiomyopathy development. To this aim, DWORF^Tg and PLN-R14^Δ/Δ mice were crossed (R14^Δ/ΔDWORF^Tg) and an elevated DWORF protein level in the R14^Δ/ΔDWORF^Tg strain was confirmed by Western blot analysis (Figure S1C,D). To determine the effect of DWORF overexpression on the development of PLN-R14del cardiomyopathy, cardiac function and cardiac histological and molecular changes were evaluated over time and life span was determined, as outlined in Figure 1A.

Figure 1. DWORF overexpression extended life span and inhibited cardiac impairment in R14^Δ/Δ mice.

A, Overview of the experimental design. B, Survival curve of WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice, expressed as a percentage over time (n=7–9 per group). Significance was examined by Log-rank (Mantel-Cox) test. Representative (average-based) serial M-mode echocardiographic images of the LV (C) with quantification of the ejection fraction (D), end-systolic diameter (LVESD) (E) and end-diastolic diameter (LVEDD) (F). Group sizes for echo data are n=8–10, except for age 18 weeks for which n=5–7. G, Schematic representation of ECG tracings taken at different time points. The average from 1-min ECG recordings of multiple mice was used. The x-axis per ECG complex has a duration of 90 milliseconds. H, Quantification of the ECG peak-to-peak amplitude (n=3–4 at 3 weeks, n=9–11 at 5 weeks, n=5–10 at 7 weeks, n=4 at 18 weeks). WT in green, R14^Δ/Δ in red and R14^Δ/ΔDWORF^Tg in purple. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Significance was examined by Kruskal-Wallis with Dunn’s multiple comparisons test, or the Mann-Whitney U test when two groups were compared.

A striking extension of life span was achieved by DWORF overexpression in PLN-R14^Δ/Δ mice. While R14^Δ/Δ mice have a limited life span of ~8 weeks, this was substantially prolonged to 17–20 weeks by DWORF overexpression (R14^Δ/ΔDWORF^Tg) (Figure 1B). Moreover, at 6 weeks of age, cardiac function (left ventricular ejection fraction [LVEF]) of R14^Δ/Δ mice was already severely impaired (LVEF<35%), whereas cardiac function of R14^Δ/ΔDWORF^Tg mice was still preserved (LVEF>55%) and comparable to WT (Figure 1C,D). At about 10 weeks of age, cardiac dysfunction was detected in the R14^Δ/ΔDWORF^Tg mice and at 14 weeks the LVEF was as severely impaired as in the 6 weeks R14^Δ/Δ group. Other parameters of PLN cardiomyopathy were also attenuated by DWORF overexpression, including the end-systolic diameter (Figure 1E), a measure of cardiac dilatation during contraction. The end-diastolic diameter did not significantly change in R14^Δ/Δ mice and in R14^Δ/ΔDWORF^Tg mice at 6 weeks, but it increased in R14^Δ/ΔDWORF^Tg mice starting at 14 weeks of age (Figure 1F). Since low-voltage ECGs are a hallmark of PLN-R14del cardiomyopathy in patients, the ECG potential was quantified by the peak-to-peak amplitude at different time points in this study. At 7 weeks of age, ECG peak-to-peak amplitude was significantly reduced in the R14^Δ/Δ group (0.48±0.04 mV, p<0.05 vs. WT 1.41±0.27 mV), whereas ECG potentials were preserved in R14^Δ/ΔDWORF^Tg mice (1.06±0.07 mV) (Figure 1G,H). At 18 weeks of age, the amplitude of the R14^Δ/ΔDWORF^Tg group was decreased to 0.42±0.12 mV as well. The quantification of P, R and S amplitudes is displayed in the Supplemental Material (Figure S2). Together these results indicate that DWORF overexpression delayed cardiac impairment in R14^Δ/Δ mice, thereby extending the life span of these mice.

DWORF overexpression delayed cardiac remodeling in R14^Δ/Δ mice

The positive effects of DWORF on the development of HF were also validated at the molecular level. The well-documented increases in gene expression of the HF marker Nppa (atrial natriuretic peptide [ANP]) and the ratio Myh7/Myh6 (Myosin Heavy Chain 7, encoding β-MHC; Myosin Heavy Chain 6, encoding α-MHC) in R14^Δ/Δ mice, was significantly reduced by DWORF overexpression at both 5 and 7 weeks of age (Figure 2A,B). For both Nppa and the ratio Myh7/Myh6, an expression level comparable to 7-week-old R14^Δ/Δ mice was reached in R14^Δ/ΔDWORF^Tg mice at 18 weeks of age. Moreover, elevation of gene expression of the fibrotic markers Col1a1 (collagen type I alpha 1 chain) and Lgals3 (Galectin-3) was delayed by overexpression of DWORF in PLN-R14^Δ/Δ mice (Figure 2C,D).

Figure 2. DWORF overexpression inhibited pathological gene expression and cardiac fibrosis in R14^Δ/Δ mice.

Relative LV gene expression of the HF marker Nppa (A), the Myh7/Myh6 ratio (B) and fibrotic markers Col1a1 (C) and Lgals3 (D), measured by qPCR and displayed as a fold change (FC) difference to age-matched WT controls (n=3 at 3 weeks, n=9 at 5 weeks, n=10 at 7 weeks, n=8–9 at 18 weeks). E, Representative (average-based) Masson’s trichrome-stained cardiac tissue sections (scale bar: 100 μm). F, Quantification of cardiac fibrosis (n=3 at 3 weeks, n=5 at 5 weeks, n=10 at 7 weeks, n=8–9 at 18 weeks). WT in green, R14^Δ/Δ in red and R14^Δ/ΔDWORF^Tg in purple. * P<0.05, *** P<0.001, **** P<0.0001. Significance was examined by Kruskal-Wallis with Dunn’s multiple comparisons test, or the Mann-Whitney U test when two groups were compared.

Consistent with these findings, histological analyses revealed that fibrosis occurred significantly slower in R14^Δ/ΔDWORF^Tg mice as compared to R14^Δ/Δ mice (2.6±0.4% vs. 12.5±1.0% at age 7 weeks, p<0.05; WT 1.2±0.2%) (Figure 2E,F). At 18 weeks of age, cardiac fibrosis was elevated in the R14^Δ/ΔDWORF^Tg group as well (12.1±0.4%, p<0.05 vs. WT).

DWORF overexpression did not alter calcium re-uptake in R14^Δ/Δ mice

To investigate calcium handling and sarcomere contraction and relaxation, mouse cardiomyocytes were isolated at 3 weeks of age, before disease onset. Calcium transient investigations (Tau, Time to 80% decay of calcium peak, T₈₀, and relaxation velocity) showed accelerated relaxation in both R14^Δ/Δ and R14^Δ/ΔDWORF^Tg compared to WT, and no difference between R14^Δ/Δ and R14^Δ/ΔDWORF^Tg (Figure 3A,B). In agreement with previous data¹⁴, DWORF^Tg in a wildtype PLN background significantly decreased Tau (Figure S3A,B), but DWORF overexpression had no additional effect in R14^Δ/Δ cardiomyocytes, because of the already low Tau value in R14^Δ/Δ. No significant differences in calcium amplitudes were observed between cardiomyocytes from the different genotypes (Figure 3A and Table S4). Consistent with calcium observations, sarcomere relaxation velocity was significantly enhanced in both 3-week-old R14^Δ/Δ and R14^Δ/ΔDWORF^Tg derived cardiomyocytes compared to WT, and there was no difference between R14^Δ/Δ and R14^Δ/ΔDWORF^Tg in sarcomere relaxation time and relaxation velocity (Figure 3C,D). Beneficial effects by DWORF overexpression could also not be explained by an increase in sarcomere fractional shortening (FS) (Figure S3C,D) since there was no significant difference in FS between R14^Δ/ΔDWORF^Tg and R14^Δ/Δ cardiomyocytes (Figure 3C,D). β-Adrenergic stimulation with isoproterenol (ISO) enhanced contraction and relaxation in all genotypes, but had a reduced effect on relaxation parameters in R14^Δ/ΔDWORF^Tg and R14^Δ/Δ cardiomyocytes, which is in accordance with the improved baseline values in these genotypes (Figure 3A–D). Cardiomyocytes isolated at 6 weeks of age, when disease is already present, were also investigated. Cardiomyocyte isolation was more challenging for the 6 week R14^Δ/Δ hearts, and yields of viable cardiomyocytes were consistently lower (about 8 fold lower as compared to WT). Data from these apparent more vulnerable cardiomyocytes, which at this age also have become hypertrophic (Figure S4A), should therefore be interpreted with caution. Nevertheless, even though sarcomere data depict reduced quality of the 6-week-old R14^Δ/Δ isolated cardiomyocytes, calcium transients at this age are similar to the findings at 3-weeks-of age (Figure S4B–E). Together, the above results demonstrate that calcium re-uptake is accelerated in R14^Δ/Δ mice and indicate that the mechanism by which DWORF inhibits disease progression R14^Δ/Δ mice is unrelated to a change in calcium handling.

Figure 3. DWORF overexpression did not alter calcium handling in R14^Δ/Δ mice.

A, Normalized calcium transients under pacing-conditions with and without ISO stimulation. B, decay time constant (Tau), time to reduce systolic calcium level by 80% (T₈₀) and relaxation velocity. C, Normalized pacing-induced sarcomere contraction-relaxation curves with and without ISO stimulation. D, Fractional shortening, time to reach 80% of the diastolic sarcomere length after systole and relaxation velocity. Calcium and contraction measurements were simultaneously performed in isolated adult mouse cardiomyocytes from 3-week-old WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice (−ISO: n = 60–100 cells from 4 mice per group, +ISO: n = 30–40 cells from 4 mice per group). ISO= isoproterenol, ISO condition was compared with the untreated matched genotype. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Significance was examined by Hierarchical cluster analysis using ANOVA with Bonferroni correction.

DWORF overexpression inhibited the formation of characteristic PLN aggregates in R14^Δ/Δ mice

Since hearts of PLN-R14del patients and R14^Δ/Δ mice display characteristic dense perinuclear PLN localization, the effect of DWORF on this phenomenon was evaluated. While perinuclear PLN accumulation was present in almost all cardiomyocytes of R14^Δ/Δ mice at 7 weeks of age, R14^Δ/ΔDWORF^Tg mice had only a few PLN aggregate positive cardiomyocytes (Figure 4A,B). Strikingly, even at 18 weeks of age only a limited number of cardiomyocytes showed this characteristic PLN mislocalization. This demonstrates that DWORF overexpression is associated with less perinuclear PLN mislocalization. In correspondence with previous data ^12,28, there was a trend towards decreased PLN protein levels in 7-week-old R14^Δ/Δ mice compared to WT (p=0.07), and the slower electrophoretic PLN phosphorylated forms were absent in R14^Δ/Δ mice (Figure 4C,D). PLN protein levels were comparable between R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice at both 7 and 18 weeks of age (Figure 4C,D), even though fewer perinuclear PLN clusters were present in R14^Δ/ΔDWORF^Tg mice (Figure 4A,B). Thus, the difference in PLN accumulation was not a result of altered PLN protein expression. Together this indicates that perinuclear PLN accumulation is a key pathological process in PLN-R14del cardiomyopathy and its reduction can delay disease development.

Figure 4. Inhibition of PLN cluster formation accounts for the beneficial effects of DWORF in R14^Δ/Δ mice.

A, Representative (average-based) IF staining of PLN (red), together with WGA staining the extracellular matrix (green) and DAPI staining the nuclei (blue) in LV tissue sections of WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice of 7 or 18 weeks of age (scale bar: 100 μm). B, PLN cluster positive cardiomyocyte count per 0.1mm² (n=6–9 per group) C, Representative (average-based) Western blot images of PLN protein levels in LVs of WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice of 7 or 18 weeks of age. D, Total PLN protein level quantified and shown as fold change compared to age-matched WT mice (n=4 per group). WT in green, R14^Δ/Δ in red and R14^Δ/ΔDWORF^Tg in purple. * P<0.05, **** P<0.0001. Significance was examined by Kruskal-Wallis with Dunn’s multiple comparisons test, or the Mann-Whitney U test when two groups were compared.

Dense PLN perinuclear structures are comprised of mislocalized S/ER

Despite the pathological relevance of PLN accumulation in PLN-R14del cardiomyopathy, little is known about the composition of these perinuclear PLN clusters. To obtain more information about the ultrastructure of this clustered PLN, electron microscopy (EM) was performed on LV tissue from 7-week-old R14^Δ/Δ mice, 18-week-old R14^Δ/ΔDWORF^Tg mice and WT controls. Using quantum dot labeling of an anti-PLN antibody, PLN clusters were visualized in the EM samples.

In R14^Δ/Δ mice, highly dense clusters of labeled PLN were mainly present in the perinuclear region (Figure 5A, PLN clusters outlined in red, nucleus outlined in blue), which corresponds with the initial description of perinuclear localization of PLN aggregates in PLN-R14del patient material ^11,29. Importantly, the clustered PLN labeling was present in large membrane-like structures (Figure 5B). These structures were also present in 18-week-old R14^Δ/ΔDWORF^Tg mice (Figure 5C,D), but were not as abundant as compared to R14^Δ/Δ mice, in agreement with fluorescent imaging (Figure 4A,B). In WT hearts, no PLN clusters were detected, but labeled PLN was distributed throughout the cell and localization to organized sarco/endoplasmic reticulum (S/ER) structures was observed (Figure 5E,F). The clustered membrane like structures in R14^Δ/Δ cardiac tissue did not correspond with vesicles like autophagosomes, which have double layer membranes, or lysosomes, which are spherical vesicles. Since PLN is a transmembrane protein anchored in the SR membrane, these large membrane structures could be disorganized clusters of the S/ER membrane.

Figure 5. Peri-nuclear clustering of PLN-positive membranes in R14^Δ/Δ cardiomyocytes.

Representative (best illustrative) electron microscopic images from cardiac tissue labeled for PLN of (A) 7-week-old R14^Δ/Δ mice showing peri-nuclear PLN clustered signal, with (B) a close-up EM image showing PLN quantum dot positive aberrant membrane structures; (C) 18-week-old R14^Δ/ΔDWORF^Tg mice showing peri-nuclear PLN clustered signal, with (D) a close-up EM image showing PLN quantum dot positive aberrant membrane structures; (E) 7-week-old WT mice showing peri-nuclear PLN signal in the S/ER with (F) a close-up of PLN quantum dot positive S/ER. PLN = PLN signal cluster; M = mitochondrion; Cyt = cytosol, S/ER or PLN-positive membrane clusters outlined in red, nucleus outlined in blue. Scale bar 2 μm (A, C, E) or 500 nm (B, D, F) (n=4 for 7-week-old WT and R14^Δ/Δ mice, n=2 for 18-week-old R14^Δ/ΔDWORF^Tg mice).

To investigate the organization of the S/ER in cardiomyocytes from R14^Δ/Δ mice, immunofluorescence (IF) microscopy was performed on LV tissue from R14^Δ/Δ and WT mice to visualize PLN together with other S/ER resident proteins. In contrast to the equal distribution throughout the cardiomyocytes observed in WT mice (Figure S5), clustering of the longitudinal tubular SR resident proteins SERCA2, histidine-rich calcium-binding protein (HRC) and calnexin was observed in cardiac tissue from R14^Δ/Δ mice (Figure 6). Moreover, these SR resident proteins co-localized with PLN at the perinuclear clusters (Figure 6). The structural ER protein atlastin-3 (ATL3) clustered and co-localized with the PLN clusters as well (Figure 6). In contrast, the sarcomere protein troponin and mitochondrial protein Tom20 were distributed throughout the cell, as in WT mice (Figure S5), and did not co-localize with the PLN clusters in R14^Δ/Δ mice (Figure 6). Although fewer clusters were present at 3 weeks of age, when present, SERCA2, HRC, calnexin and ATL3 clustered and co-localized with PLN in R14^Δ/Δ mice, while troponin and Tom20 did not and comparable results with more clusters were observed at 5 weeks of age (Figure S6, S7). Similarly, in 18-week-old R14^Δ/ΔDWORF^Tg mice, cardiomyocytes that contained large PLN clusters showed co-localization of SERCA2, HRC, calnexin and ATL3 and with PLN, whereas troponin and Tom20 did not co-localize (Figure S8). These data indicate that more cardiomyocytes develop an abnormal S/ER structure in R14^Δ/Δ mice with age and this process is delayed in R14^Δ/ΔDWORF^Tg mice.

Figure 6. Clustering of S/ER proteins in R14^Δ/Δ cardiomyocytes.

Representative (average-based) IF double staining for PLN together with SERCA2, calnexin, HRC, ATL3, troponin or Tom20 in LV tissue sections from 7-week-old R14^Δ/Δ mice (n=6; scale bar: 50 μm).

To corroborate this finding, we investigated the localization of these SR proteins in cardiac tissue from PLN-R14del human patients and non-PLN-R14del DCM patients. Perinuclear PLN localization could be observed in some cardiomyocytes in PLN-R14del patient-derived heart tissue, as described previously ¹¹. Importantly, SERCA2, HRC, calnexin and ATL3 all colocalized with these PLN clusters in these patient samples (Figure S9), confirming our observations in mice. In non-PLN-R14del DCM patient-derived cardiac tissue, perinuclear PLN clusters were not observed, and S/ER markers showed a normal distribution in all cardiomyocytes (Figure S10). Taken together, these data confirm perinuclear PLN clustering as a key characteristic of PLN cardiomyopathy and show that these previously identified PLN aggregates are comprised of disorganized S/ER and DWORF overexpression can delay this disorganization.

DWORF overexpression delays S/ER disorganization and cell death by restraining PLN cluster size

Closer investigation of abnormal PLN localization revealed that adjacent to cells with large PLN clusters, about 80% of all cardiomyocytes of 18-week-old R14^Δ/ΔDWORF^Tg mice showed the presence of small sized PLN speckles (Figure S11, some PLN speckles are indicated by white arrowheads and Figure S12 for quantification). These PLN speckles could also be observed in about 30% of the cardiomyocytes in 3-week-old R14^Δ/Δ mice, often located next to some cells with larger clusters, suggesting that these speckles represent an initial stage that progresses into larger clusters (Figure S11,S12). In 7-week-old R14^Δ/ΔDWORF^Tg mice, these speckles were much less prevalent compared to 18-week-old R14^Δ/ΔDWORF^Tg mice (only in about 30% of all cardiomyocytes), indicating that this phenomenon occurs at a slower rate in the presence of DWORF and/or develop less frequently into larger clusters (Figure S11,S12).

Although we currently do not understand how DWORF interferes with these processes, we believe that PLN clustering and SR disorganization have major consequences for cardiomyocyte cell viability. Severe accumulation of the stress inducible protein, p62, was observed within cardiomyocytes that contain large PLN clusters (Figure 7A). When quantified, R14^Δ/Δ mice had a significantly higher count of p62-positive cardiomyocytes compared to R14^Δ/ΔDWORF^Tg mice (Figure S13). About 10% of the p62-positive cardiomyocytes had p62 accumulation throughout the entire cell together with a strongly reduced signal for the troponin protein, which suggest cardiomyocyte death, most likely by necrosis (Figure 7B, Figure S13B). In vivo Evans Blue staining confirmed the presence of necrotic cardiomyocytes in R14^Δ/Δ mice (Figure 7C,D). Consistent with these findings, plasma cardiac troponin levels were strongly elevated in 6-week-old R14^Δ/Δ mice while skeletal troponin levels remained low, indicative of troponin leakage from the heart (Figure 7E,F). Moreover, plasma cardiac troponin levels were lower in R14^Δ/ΔDWORF^Tg mice compared to R14^Δ/Δ mice (Figure 7E,F). The extensive increase in extracellular matrix in 7 weeks R14^Δ/Δ mice and 18 weeks R14^Δ/ΔDWORF^Tg (Figure 2) therefore most likely reflects replacement fibrosis, which is in agreement with observations in human PLN-R14del cardiomyopathy hearts ³⁰. Since large PLN-R14del cluster formation is less prominent in R14^Δ/ΔDWORF^Tg mice, the delayed cardiac dysfunction and decreased fibrosis can be explained by a reduced rate of cardiomyocyte cell loss in the hearts of R14^Δ/ΔDWORF^Tg mice.

Figure 7. R14^Δ/Δ cardiomyocyte cell death.

A, Representative (best illustrative) IF double staining for PLN (red) together with together with p62 (green) and DAPI (blue) in LV tissue sections of 7-week-old R14^Δ/Δ mice. B, Representative (best illustrative) IF double staining for troponin (red) together with together with p62 (green) and DAPI (blue) in LV tissue sections of 7-week-old R14^Δ/Δ mice. (n=4; scale bar: 50 μm). C, Representative (average-based) images of Evans Blue (pink), WGA (green) and DAPI (blue) stained hearts from 6-week-old WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg hearts (35x magnification, scale bar: 100 μm). D, Evans Blue positive cardiomyocyte count per 1mm² at 6 weeks of age, indicative of cardiomyocyte necrosis occurrence (n=3–4 per group). E, Skeletal troponin I and (F) cardiac troponin I levels in plasma of 6-week-old WT, R14^Δ/Δ and R14^Δ/ΔDWORF^Tg mice (n=6–7 per group). * P<0.05, *** P<0.001, **** P<0.0001. Significance was examined by Kruskal-Wallis with Dunn’s multiple comparisons test.

DISCUSSION

In this study, we demonstrate that DWORF expression can substantially extend life span of mice with PLN-R14del cardiomyopathy. DWORF is substantially reduced in heart failure and restoration of DWORF levels by crossbreeding DWORF^Tg mice with R14^Δ/Δ mice, delayed cardiac dysfunction and fibrosis in these mice. This cardioprotective effect could not be attributed to the role of DWORF in calcium handling. Instead, we provide evidence that characteristic PLN aggregates present in PLN-R14del disease, are comprised of harmful perinuclear clustered S/ER and DWORF has an inhibitory effect on this S/ER disorganization, suggesting a novel cardioprotective role of DWORF in PLN-R14del cardiomyopathy.

We show for the first time that the previously termed perinuclear PLN aggregates, observed in both patients and mice with PLN-R14del cardiomyopathy, are not only composed of PLN protein, but are comprised of aberrantly clustered cardiac S/ER. Using electron microscopy, we revealed perinuclear localized PLN-dense membrane clusters in hearts of R14^Δ/Δ mice. Further investigations showed that other S/ER markers, including SERCA2, calnexin, HRC and ATL3, all colocalized with PLN in these membrane clusters in both R14^Δ/Δ mouse and PLN-R14del human patient samples. These findings provide clear evidence that in both in mouse and men, PLN-R14del-induced S/ER disorganization is a major characteristic of PLN-R14del cardiomyopathy. So far, the mechanistic aspects of this abnormal S/ER clustering remain elusive. Further investigations will be needed to investigate whether this process involves abnormal PLN-R14del membrane interactions and/or PLN aggregation and complex formation with other proteins in the SR membrane.

In addition to the occurrence of abnormal perinuclear PLN clusters in cardiomyocytes before the onset of cardiac dysfunction ¹³, our data indicates that cluster formation involves a sequence of events. At an early stage, when the majority of both PLN and S/ER distribution is still normal, the first PLN speckles can be observed in cardiomyocytes. At a later stage, these speckles formed larger PLN clusters and caused severe disorganization of the S/ER. Concurrently, p62 accumulation was observed in about half of the cardiomyocytes with large PLN clusters. Importantly, in about 10% of the cardiomyocytes with p62 cluster formation troponin was strongly reduced, which was accompanied by an increase in plasma troponin levels, which collectively indicates necrotic death of these cardiomyocytes. These results were further confirmed by injection of Evans Blue, a previously described necrotic cell death marker ³¹, and are again in line with the high vulnerability of these cardiomyocytes during isolation. This also explains the excessive replacement fibrosis in these hearts and concomitant development of hypertrophy in the remaining cardiomyocytes and is in congruence with the cell death-fibrosis phenomenon in HF ³². Importantly, in PLN-R14del mutation carriers fibrosis is an early feature which can already be observed in carriers that still have a preserved LVEF ³³. Thus, although the exact molecular processes require further investigations, our observations indicate that abnormal perinuclear S/ER cluster formation is an early phenomenon that drives a sequence of pathological processes resulting in PLN-R14del cardiomyopathy.

Interestingly, DWORF overexpression inhibited the presence of large perinuclear PLN clusters with severe S/ER disorganization in R14^Δ/Δ mice. At 3 weeks of age the majority of the R14^Δ/ΔDWORF^Tg cardiomyocytes showed normal PLN distribution and at 18 weeks of age the majority of the R14^Δ/ΔDWORF^Tg cardiomyocytes have PLN speckles. By restraining PLN cluster size and inhibiting the formation of large perinuclear PLN clusters, that are associated with cardiomyocyte death, DWORF overexpression could delay disease development in R14^Δ/Δ mice. Currently, we do not know how DWORF mediates PLN cluster size, but since SERCA is present in these clusters as well it may still involve some form of competitive SERCA interactions. The knowledge about the regulation and associations of PLN and other micropeptides, like DWORF, in biological membranes is still limited, but sophisticated technologies like electron paramagnetic resonance spectroscopy might provide answers to this underlying complexity ³⁴. Other mechanisms, like control of dynamic localization via Neprilysin mediated changes in oligomerization states, as shown for Drosophila Sarcolamban should not be excluded, although our results currently do not point into this direction ³⁵. Unfortunately, the absence of a DWORF antibody for histological investigations does currently not allow us to investigate the presence of DWORF in these S/ER clusters. Despite strong reduction in PLN S/ER cluster formations, cells with clusters still develop and were also observed in 7-week-old and 18-week-old R14^Δ/ΔDWORF^Tg mice. This reduced rate of detrimental cluster formation can explain the strongly delayed deterioration of cardiac function of R14^Δ/ΔDWORF^Tg mice overtime (Figure S14).

Studies with the PLN-R14del pathogenic variant have repeatedly shown altered calcium handling properties, but the exact effects and mechanisms remain unclear ^{4–7,10,13,36}. Here we described enhanced calcium re-uptake in cardiomyocytes isolated from R14^Δ/Δ mice, which is in line with several other publications on altered calcium re-uptake by mutant PLN-R14del ^6–8,36. Likewise, calcium S/ER re-uptake is accelerated in PLN^−/− (knockout) mice, but in contrast to R14^Δ/Δ mice, PLN knockout mice do not develop HF ^37,38. Thus, the p.Arg14del pathogenic variant may behave as a loss-of-function mutation in calcium handling, but this cannot explain disease development in R14^Δ/Δ mice. Since calcium re-uptake was already enhanced in R14^Δ/Δ cardiomyocytes, DWORF overexpression did not result in an additional increase in calcium re-uptake in R14^Δ/ΔDWORF^Tg cardiomyocytes. Hence, improving calcium S/ER re-uptake appears to be ineffective in PLN-R14del cardiomyopathy. Therefore, in contrast to the protective effects of DWORF in other heart failure models ^14,17,39, its protective effect in PLN-R14del cardiomyopathy does not appear to be related to calcium handling. Based on the data presented above we therefore consider it more likely that the protective effect of DWORF involves the prevention of abnormal S/ER cluster formation in PLN-R14del cardiomyopathy.

A limitation of this study is that we did not use a heterozygous PLN-R14del mouse model (R14^+/Δ), that would resemble the same genetic situation as human carriers with one normal and one pathogenic allele. Instead, we used a homozygous PLN-R14del mouse model (R14^Δ/Δ) that shows similar characteristics as the R14^+/Δ mice and human patients but has the advantage of accelerated disease development. In addition to previously published data, we confirmed that the R14^+/Δ mouse model is a mild disease variant with regard to calcium handling and sarcomere function. Relaxation is also accelerated in adult cardiomyocytes from R14^+/Δ mice, with relaxation time and velocity values intermediate of R14^Δ/Δ and WT mice (Figure S15). Moreover, our finding that perinuclear PLN structures are composed of disorganized S/ER in homozygous mice was confirmed in PLN-R14del patient tissue, which further validates the translatability of this mouse model. The strategy of increasing the DWORF level to delay disease progression could be even more effective in human patients compared to the effects observed in the current study in homozygous mice, considering that disease progression is milder due to the presence of WT PLN in patients.

In summary, PLN-R14del induced perinuclear clustering of PLN and S/ER structural deformations trigger cardiomyocyte dysfunction. This causes cardiomyocyte cell loss as indicated by elevated troponin levels, explaining the excessive fibrosis, which finally culminates in severe cardiac dysfunction and death (Figure 8). Whether other PLN pathogenic variants also cause structural S/ER alterations is not known but deserves future attention. Importantly, DWORF can diminish PLN-R14del-induced S/ER cluster formation and significantly extend life span in an established mouse model for PLN-R14del cardiomyopathy (Figure 8).

Figure 8. The proposed new view on PLN-R14del cardiomyopathy with respect to S/ER cluster formation and the interference of DWORF.

ER = endoplasmic reticulum, SR = sarcoplasmic reticulum, EF = ejection fraction.

To conclude, we reveal that the characteristic PLN clusters present in the PLN-R14del mouse model and human patients are comprised of disorganized perinuclear S/ER and argue that aberrant S/ER formation is the key mechanism in the development of PLN-R14del cardiomyopathy. Preventing structural S/ER alterations by DWORF overexpression or by other interventions could be a promising therapeutic strategy in this disease.

CIRCULATION RESEARCH.

Novelty and Significance

What is known?

• The p.Arg14del variant of the phospholamban (PLN) gene causes cardiomyopathy, leading to severe heart failure (HF), which cannot be properly treated using current HF therapies.

• Mechanistic insights in disease development are required for a specific treatment and a causal role in disease development has been suggested for clustered perinuclear PLN protein.

• DWORF (dwarf open reading frame) is a SERCA (sarco/endoplasmic reticulum Ca²⁺ ATPase) activator, with a function opposite to PLN and has shown to be cardioprotective in several murine HF models.

What New Information Does This Article Contribute?

• The current work shows that the characteristic dense perinuclear PLN structures in the PLN-R14del mouse model and human patients are composed of strongly clustered and disorganized cardiac sarco/endoplasmic reticulum (S/ER).

• DWORF overexpression delays cardiac dysfunction and extends life span in a PLN-R14del cardiomyopathy mouse model.

• DWORF exerts its positive effects in PLN-R14del cardiomyopathy via the reduction of perinuclear PLN clustering and associated toxic S/ER disorganization, not via a change in SR calcium re-uptake.

These data support S/ER disorganization as a detrimental mechanism in the development of PLN-R14del cardiomyopathy. Preventing structural S/ER alterations by DWORF overexpression or by other interventions could be a promising therapeutic strategy in this disease.

Non-standard Abbreviations and Acronyms:

ATL3

atlastin-3

DWORF

dwarf open reading frame

EM

electron microscopy

HRC

histidine-rich calcium binding protein

IF

immunofluorescent

PLN

phospholamban

R14^Δ/Δ

homozygous PLN-R14del mice

R14^Δ/ΔDWORF^Tg

R14^Δ/Δ mice carrying the DWORF transgene

S/ER

sarco/endoplasmic reticulum

SERCA

sarco/endoplasmic reticulum Ca²⁺ ATPase

WT

Wildtype; transgene-negative