In PLN-R14del mice, SR structure restoration, rather than calcium cycling, is the dominant effector of PLN-ASO treatment

The generation and phenotypic characterization of the PLN-R14del mouse model, and housing of mice have been described before.⁵ Male and female WT and homozygous PLN-R14del (R14^Δ/Δ) mice were enrolled in this study. Before treatment initiation at 5 weeks of age, baseline echocardiography was performed. Based on sex, weight, and baseline cardiac function, R14^Δ/Δ mice were subsequently randomized to six different groups receiving PLN-ASO (5′-GCATATCAATTTCCTG-3′) injections, at a dose of 0 (vehicle only; Dulbecco's PBS), 3, 7, 15, or 25 mg/kg/injection or a scrambled ASO (5′-GGCCAATACGCCGTCA-3′) of 25 mg/kg/injection (n = 8 per group). Doses were selected based on dose–response-modelling from previous in vivo studies^7,8 and were chosen to cover PLN inhibition between 20% and 70%. The time to initiate treatment with ASO was informed by previous studies to give optimal opportunity to show a treatment effect. ASOs were designed and produced by Ionis Pharmaceuticals (CA, USA). Six WT mice were included as healthy controls, receiving vehicle injections. Injections started at 5 weeks of age, with four injections in the first week, followed by a weekly maintenance injection of the same dose in the subsequent weeks. R14^Δ/Δ mice were monitored until they reached the humane endpoint (ejection fraction (EF) < 10%, >20% weight loss or lethargy and dyspnoea), which generally occurs at 7–8 weeks of age.⁵ In addition, when the average EF of an ASO dosing group was <20% or when >30% of the mice of an ASO dosing group reached the humane endpoint, the entire experimental group was terminated. Surviving R14^Δ/Δ groups, together with the WT mice, were kept until 21 weeks of age, with follow-up echocardiograms at 7, 15, and 21 weeks of age. At sacrifice, hearts were collected for histological and molecular analyses. Echocardiography and termination were conducted under anaesthesia with 2.5% isoflurane mixed with oxygen. In a separate experiment for calcium handling and sarcomere measurements in isolated cardiomyocytes, an additional 10 WT and 10 R14^Δ/Δ mice were treated with vehicle or 25 mg/kg PLN-ASO between 3 and 5 weeks of age, following the abovementioned regimen. This study was approved by the Dutch Central Authority for Scientific Procedures on Animals (license numbers AVD1050020199105 and AVD10500202115446) and the Animal Welfare Body of the University of Groningen (permit numbers IVD199105-01-011 and IVD2115446-02-003), followed the guidelines set out in the Directive 2010/63/EU of the European Parliament for using animals in research and were reported in line with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.¹⁴

Left ventricular (LV) morphology and function were monitored using a Vevo 3100 preclinical imaging system equipped with a 40 MHz MX550D linear array transducer (FUJIFILM VisualSonics, Canada), as previously described.⁹ Recordings were analysed using Vevo LAB software version 5.7.1 (FUJIFILM VisualSonics). Data acquisition and analysis adhered to the guidelines provided by the European Society of Cardiology Working Group on Myocardial Function.¹⁵

Euthanasia was performed under anaesthesia using a mixture of oxygen and 2.5% isoflurane. After ensuring deep anaesthesia, the abdominal and thoracic cavities were opened, blood was collected via the abdominal aorta, and saline was used to flush the circulatory system via the heart. The heart was carefully excised, rinsed in an ice-cold 1 M potassium chloride (KCl) solution (Merck Millipore, Germany). A transverse mid-section of the heart was preserved for histological examination, and the remaining tissue samples were promptly snap-frozen in liquid nitrogen for further molecular analyses.

Transverse cardiac slices of both ventricles were preserved in a 4% formaldehyde solution (10% formalin; Klinipath, The Netherlands) for at least 24 h. After fixation, heart tissues were dehydrated using an automated system (Leica TP1020; Leica Microsystems, Germany), embedded in paraffin, and sectioned into 4 µm thick slices for histological analysis. Tissue sections were deparaffinized before proceeding with staining procedures.

Masson's trichrome staining was performed to identify collagen deposition, adhering to standard protocols. Subsequently, the entire stained sections were scanned using a Nanozoomer 2.0-HT digital slide scanner (Hamamatsu Photonics, Japan). Quantification of fibrosis in each section was accomplished by analysing the percentage of the total section area occupied by fibrotic tissue using the positive Pixel Count v9 algorithm of Aperio's ImageScope software version 12.4.0 (Leica Microsystems).

To evaluate PLN clustering, ventricular tissue sections were stained with a rabbit anti-PLN primary antibody (Cell Signaling Technologies, MA, USA), followed by labelling with an Alexa 555-conjugated secondary antibody. Cell boundaries were visualized using fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (WGA), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). PLN clusters were quantified in 30–50 cardiomyocytes per mouse (n = 6 per group). The cluster-positive cells were presented as per 0.1 mm², as indicated by the scale bar.

To show the colocalization of PLN and other SR proteins in mouse cardiac tissue samples, SERCA2a or HRC (histidine-rich calcium-binding protein) were co-stained with PLN using fluorescently-labelled secondary antibodies. Specific antibodies used in immunohistochemistry are listed in Supplementary material online, Table S1.

Fluorescent staining images were acquired using a Leica TCS SP8X confocal microscope (Leica Microsystems). PLN/WGA/DAPI localization and colocalization of SERCA2a or HRC with PLN were captured using a 63× objective (numerical aperture 1.4, pinhole size 95.5 µm).

Total RNA was extracted from snap-frozen ventricular tissue using TRI Reagent (Sigma-Aldrich, MO, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) synthesis was performed using the QuantiTect reverse transcription kit (Qiagen, Germany). Quantitative polymerase chain reaction (qPCR) was conducted with iQ SYBR Green Supermix (Bio-Rad, CA, USA) on a C1000 Thermal Cycler CFX 384 Real-Time PCR Detection System (Bio-Rad). Gene expression levels were analysed using Bio-Rad CFX Manager 3.0 software. All gene expression levels were normalized to the reference gene Rplp0 (36B4) and reported as fold changes relative to the WT control group. The primer sequences used for qPCR are listed in Supplementary material online, Table S2. The qPCR primers used for PLN mRNA quantification were designed downstream of the R14del mutation site. Primer binding and amplification efficiency are therefore similar for the WT and PLN-R14del transcript.

Total protein from snap-frozen ventricular tissue was extracted using Radioimmunoprecipitation Assay (RIPA) buffer as described previously.⁵ Protein concentrations were measured using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, MA, USA). Samples were heated to 95°C for 5 min for PLN or 50°C for 15 min for SERCA2a, followed by loading onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system. Equal amounts of protein were loaded, with 5 µg per well for PLN and 10 µg per well for SERCA2a. Proteins separated by SDS-PAGE were then transferred to an immunoblot polyvinylidene fluoride (PVDF) membrane (Bio-Rad) using a semi-dry transfer system (Amersham Biosciences, UK). The total protein level in each sample was determined using the Revert 700 Total Protein Stain Kit (LI-COR Biosciences, NE, USA), which was subsequently used for protein normalization. Membranes were blocked with blocking buffer and incubated overnight at 4°C with a primary antibody, followed by a one-hour incubation at room temperature with a secondary antibody. Detection was carried out using enhanced chemiluminescence with the Western Lightning Ultra Chemiluminescent Substrate (PerkinElmer, MA, USA) and visualized with the Amersham ImageQuant 800 Western blot imaging system (Cytiva, MA, USA). Quantification of target proteins was performed using Image Studio Lite Software version 5.2 (LI-COR Biosciences). Protein expression values were reported as fold changes relative to the WT control group. Primary and secondary antibodies that were used are presented in Supplementary material online, Table S3.

Total protein from snap-frozen isolated cardiomyocytes was extracted using RIPA buffer. To quantify PLN protein levels, a 12–230 kDa Separation Module (ProteinSimple, CA, USA) was used on an automated western blot system (Jess Simple Western; ProteinSimple) following the manufacturer's instructions. Protein lysates were used at 1 mg/mL together with an anti-PLN antibody (1:50, Cell Signaling Technologies) and an Anti-Rabbit Detection Module (ProteinSimple). Protein expression was normalized by protein normalization module (ProteinSimple) following the manufacturer's protocol.

Electron microscopy (EM) was performed as reported previously.^9,16 Briefly, LV myocardial tissue was cut into 1 mm³ pieces and fixed in EM fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate (PH 7.4)) overnight at 4°C. The fixative was subsequently replaced with fresh buffer (0.5% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4) to allow for prolonged storage and improved preservation of cellular ultrastructure. Post-fixation was carried out using 1% osmium tetroxide and 1.5% potassium ferrocyanide. The samples were then sectioned, mounted on single-slot cupper grids, and contrasted with 4% neodymium acetate. Sections were imaged using scanning transmission electron microscopy (STEM) (Zeiss Supra55, Germany).

Cardiomyocytes were isolated using a Langendorff-free method.¹⁷ Cardiomyocytes were loaded with the calcium fluorophore Fura-2, AM and single cell calcium transients and sarcomere shortening were recorded using a MultiCell High Throughput system (IonOptix, MA, USA) under pacing conditions (1.5 Hz, 20 V) at 37°C. At least 40 cells were included per mouse (n = 5 per group). Measurements were done at baseline and following beta-adrenergic stimulation using 100 nM isoproterenol. Calcium and contractility data were analysed using CytoSolver with default settings, while excluding cells with <5 transients and outliers outside mean ± 1.5 ∗ interquartile range (IQR).

Data are presented as means ± standard error of the mean (S.E.M.). Sarcomere length and ratiometric calcium transients are shown as line graphs. Quantification of SR-EM parameters and contractile and calcium parameters are displayed in SuperPlots presenting both individual cell values (transparent dots) and mean values per mouse (solid dots) with the matching colours.¹⁸ Data of isolated cardiomyocytes were log-transformed and a hierarchical test with Bonferroni correction was applied in R version 4.4.0.¹⁸ All other statistical analyses were performed using GraphPad Prism version 9.1.0. For statistical analysis of survival curves, a log-rank test was performed. For all other comparisons, nonparametric tests were performed due to small group sizes (n = 5–8). Echocardiographic parameters at each timepoint were compared separately to the WT group of the same age using a Kruskal–Wallis test with post hoc Dunn's multiple comparisons test. At the 7-week time point, comparisons were made among all PLN-R14^Δ/Δ groups. For other assays, a Mann–Whitney test was used to compare vehicle-treated R14^Δ/Δ mice to WT mice (to evaluate differences between genotypes), followed by a Kruskal–Wallis test with post hoc Dunn's multiple comparisons test for comparisons among the vehicle-treated and ASO-treated R14^Δ/Δ. P-values of <0.05 were considered statistically significant. All data acquisition and analyses were performed in a blinded manner.

PLN-ASO or vehicle treatment started at 5 weeks of age, in accordance to a previous study,⁸ with four injections during the first week of treatment, followed by weekly injections of the indicated doses (Figure 1A). As expected, vehicle-treated R14^Δ/Δ mice reached the humane endpoint at 7 weeks of age, which is in agreement with previous studies.⁵ PLN-ASO treatment showed a dose dependent increase in survival of R14^Δ/Δ mice, with the 3 mg/kg group surviving until 9 weeks of age, while those receiving 7 mg/kg reached the humane endpoint and were terminated at 11 weeks of age (Figure 1B). Importantly, the higher ASO dose groups of 15 and 25 mg/kg did not reach the humane endpoint and remained viable until the end of the experiment, except for one mouse in the 15 mg/kg group. All WT mice remained viable until the end of the experiment (Figure 1B).

Investigation of cardiac function, using echocardiography, also revealed a clear dose–response effect in the R14^Δ/Δ mice (Figure 1C–F). From 5 weeks (start of treatment) to 7 weeks of age, a rapid decline in LVEF was observed in the vehicle-treated R14^Δ/Δ group (R14^Δ/Δ Ctrl), whereas the EF of WT mice remained >60% (Figure 1C). ASO treatment prevented heart function decline and with increasing doses, a more preserved EF was observed. Since it takes some time to reach sufficient depletion of PLN by ASO-mediated silencing, an immediate stabilizing effect on cardiac function was not expected. No additional echocardiograms were made of the R14^Δ/Δ mice receiving vehicle and 3 mg/kg of ASO, because these animals reached the humane endpoint within 2 weeks after the first follow-up measurement. As R14^Δ/Δ mice receiving 7 mg/kg of ASO became lethargic around 11 weeks of age, we performed an additional echo and this revealed a further decline in cardiac function compared to the previous analysis at 7 weeks of age (Figure 1C). Subsequently, these mice were terminated at this time point, because this group reached the predefined humane endpoint (group average EF < 20%). Importantly, the EF of the 15 and 25 mg/kg groups did not decline further after the first follow-up echocardiogram (7 weeks of age), indicating stabilization of cardiac function (Figure 1C). This was even more profound in the highest dose group, suggesting that at this dose even some improvement may occur. LV end-systolic and end-diastolic internal diameters were significantly increased in vehicle-treated R14^Δ/Δ mice as compared to WT mice, indicating ventricular dilatation, which was dose-dependently rescued by PLN-ASO administration (Figure 1D and E). All observed effects were specific to PLN-ASO administration, as R14^Δ/Δ mice injected with a scrambled ASO at the highest dose (25 mg/kg) showed the same rapid decline in cardiac function as untreated mice with an average EF of 13.0 ± 1.1%, and reached the humane endpoint by 7 weeks of age (see Supplementary material online, Figure S1). Together, these data demonstrate that PLN-ASO therapy exerts a dose-dependent effect on cardiac function and lifespan in PLN-R14^Δ/Δ mice.

Next, to confirm effective depletion of PLN following PLN-ASO treatment, PLN expression was determined at both mRNA and protein level. Since R14^Δ/Δ mice receiving vehicle or 3 or 7 mg/kg PLN-ASO did not survive until the 21-week time point, it is important to note that these cardiac samples are from time points corresponding to their maximal lifespan (see Figure 1B). As shown, both gene (see Supplementary material online, Figure S2A) and protein (Figure 2A) expression levels were dose-dependently decreased in hearts of ASO-treated R14^Δ/Δ mice. We like to note that at 7 weeks of age, PLN levels are also lower in the R14^Δ/Δ vehicle group as compared to the WT group (Figure 2A and Supplementary material online, Figure S2A). This has been observed before and is at least partially related to downregulation of PLN in this animal model, as present in this group at 7 weeks.¹⁹ At the protein level this difference may also partly be explained by differences in antibody binding to wild-type PLN and PLN-R14del and therefore we have to be cautious with direct protein comparisons between WT and R14^Δ/Δ groups. Moreover, cardiomyocyte density was approximately 25% lower in the R14^Δ/Δ vehicle group compared to WT, consistent with previously reported cell death, which may contribute to the observed reduction in PLN levels (see Supplementary material online, Figure S3). However, a direct comparison of immunohistochemical staining for PLN and cardiac troponin revealed a pronounced decrease in PLN expression within cardiomyocytes (see Supplementary material online, Figure S3). While some bias in this quantification cannot be excluded, due to certain PLN clusters being out of focus, western blot analyses corrected for cardiac troponin I and α-actinin levels also confirmed reduced PLN protein levels in the R14^Δ/Δ vehicle group (see Supplementary material online, Figure S4). These findings corroborate earlier reports of cellular PLN downregulation in the context of heart failure.⁸ The dose-dependent efficacy of ASO-mediated PLN protein depletion, calculated as per cent reduction compared to vehicle-treated R14^Δ/Δ mice, is shown in Supplementary material online, Table S4. Besides PLN, also SERCA2a protein and gene (Atp2a2) expression levels were investigated (Figure 2B, Supplementary material online, Figure S2B). SERCA2a was reduced in the R14^Δ/Δ vehicle group and in the low dose ASO3 and ASO7 treatment groups, but was restored by the high dose ASO treatment. The observed reduction in SERCA2a is consistent with findings in both mice and human, where decreased SERCA2a expression is associated with heart failure.^20,21 The restoration of SERCA2a expression in the high dose groups therefore aligns with improved cardiac function. Since a low SERCA2a/PLN ratio may contribute to heart failure,²⁰ we also measured the ratio in this study. Although no significant decrease was observed in vehicle-treated R14^Δ/Δ mice, at high ASO doses SERCA2a/PLN ratios were increased (Figure 2C). Similar changes were also observed for SERCA2a gene expression and the ratio between gene expression levels of SERCA2a and PLN (see Supplementary material online, Figure S2C). Overall, these results confirm the expected dose-dependent reduction of cardiac PLN-R14del levels following PLN-ASO administration.

Molecular changes related to cardiac remodelling were also investigated. Gene expression of the cardiac wall stress and hypertrophy biomarker atrial natriuretic peptide (encoded by the Nppa gene) was significantly elevated in R14^Δ/Δ vehicle group compared to WT (Figure 3A). Consistent with improved cardiac function in the 25 mg/kg PLN-ASO-treated R14^Δ/Δ groups, Nppa was significantly reduced as compared to the vehicle-treated R14^Δ/Δ group (Figure 3A). A similar dose–response effect was observed for fibrotic gene expression (Timp1 and Col1a1), indicative of diminished fibrosis formation (Figure 3B and C). To corroborate this finding, histochemistry was performed, using Masson's trichrome staining to visualize myocardial fibrosis in ventricular sections (Figure 3D). As shown before,⁵ in vehicle-treated R14^Δ/Δ mice, cardiac fibrosis was significantly elevated as compared to WT mice (Figure 3D and E). Hearts from R14^Δ/Δ mice treated with 3 and 7 mg/kg of ASO showed more collagen deposition as compared to the vehicle-treated R14^Δ/Δ group. We believe that the increased lifespan of these low dose groups, as compared to the vehicle group, provides an increased time window for fibrosis development, ultimately resulting in higher levels of extracellular matrix. Most importantly, however, is that in fibrosis was not further elevated in the high dose groups. Thus, PLN-ASO therapy dose-dependently reduces natriuretic peptide expression and halts fibrosis formation in hearts of R14^Δ/Δ mice, corroborating our in vivo findings.

A key hallmark of PLN-R14del cardiomyopathy in humans and mice is the occurrence of detrimental cardiac SR clusters, as evidenced by strong perinuclear PLN staining in R14^Δ/Δ cardiomyocytes.^5,9 In vehicle-treated R14^Δ/Δ mice, PLN clusters were highly prevalent with 17.7 ± 1.3 cluster-positive cells per 0.1mm,² whereas such clusters were not observed in WT mice (Figure 4A and B). Importantly, these SR clusters were dose-dependently reduced after ASO treatment and were nearly absent at the highest doses. Absence of PLN-positive clusters suggests that the organization of the SR is restored. We previously demonstrated that HRC and SERCA2a colocalize with PLN in aberrant SR clusters.⁹ To confirm restructuring of reticulum membranes, we performed immunohistochemical double staining for SERCA2a/PLN and HRC/PLN in cardiomyocytes from both WT and R14^Δ/Δ mice treated with either vehicle or 25 mg/kg ASO, assessing protein colocalization. (Figure 5A and B). Confocal imaging validated colocalization of these proteins and showed enrichment in the SR clusters of R14^Δ/Δ mice. Importantly, ASO treatment normalized SERCA2a and HRC localization in the majority of R14^Δ/Δ cardiomyocytes, confirming that ASO treatment restored SR organization. As PLN is not completely silenced upon ASO administration, low amounts of PLN and small SR clusters were observed in some cardiomyocytes of ASO-treated R14^Δ/Δ mice. Together, these findings demonstrate that PLN-ASO therapy resulted in dose-dependent restoration of SR structure in hearts of PLN-R14^Δ/Δ mice.

To visualize the sarcoplasmic reticulum (SR) structure in greater detail, electron microscopy (EM) was performed. Ventricular tissue from vehicle-treated wild-type (WT) mice and from both vehicle- and ASO25-treated R14^Δ/Δ mice (all terminated at 7 weeks of age) was analysed to assess SR restoration following ASO treatment (Figure 6A–C). In vehicle-treated R14^Δ/Δ mice, cardiomyocytes exhibited a markedly aberrant and SR distribution, in contrast to the well-organized SR network seen in WT mice. Notably, SR density between sarcomeres was significantly reduced (Figure 6B), while SR accumulation around mitochondria was increased and appeared disorganized (Figure 6C). Strikingly, irregular membrane-like structures were frequently observed near mitochondria, reflecting severe SR disarray. PLN-ASO25 treatment largely restored the normal SR architecture. Minimal distances between the SR and mitochondria, as well as the relative SR–T-tubule distances, were unchanged across groups (see Supplementary material online, Figure S5). Collectively, these findings indicate a pronounced remodelling of the longitudinal SR in R14^Δ/Δ mice, which is substantially reversed by ASO treatment.

Since PLN regulates SERCA2a activity and the effect of PLN-R14del on SERCA2a has been debated, we investigated calcium cycling and sarcomere contraction in isolated cardiomyocytes of vehicle- and ASO-treated WT and R14^Δ/Δ mice. Due to the challenges of isolating cardiomyocytes in advanced disease, including the presence of extensive cardiac fibrosis and the frailty of diseased cardiomyocytes, cardiomyocyte isolation was performed at 5 weeks of age (an early disease stage). To reach sufficient PLN depletion, ASO treatment (with the same frequency as in the abovementioned experiment) was initiated at 3 weeks of age with the highest ASO dose (25 mg/kg) (Figure 7A). This 2-week treatment period resulted in strong reduction of PLN protein expression in cardiomyocytes isolated from WT and R14^Δ/Δ mice, as compared to the vehicle-treated mice of the same genotype (see Supplementary material online, Figure S6). For calcium measurements, cardiomyocytes were loaded with the ratiometric calcium fluorophore Fura-2, after which calcium transients and sarcomere shortening were measured simultaneously in single cells. As expected, silencing of PLN and subsequent relief of SERCA2a inhibition resulted in faster SR calcium uptake in ASO-treated WT cardiomyocytes as evidenced by significantly smaller tau (time constant of calcium transient decay) values and significantly greater calcium peak amplitudes (Figure 7B–D). In R14^Δ/Δ cardiomyocytes, tau was significantly lower and calcium transients were significantly higher than in WT cardiomyocytes, indicating a loss of SERCA2a inhibitory function in mice carrying the PLN-R14del pathogenic variant. Consistently, ASO-mediated depletion of PLN had a much smaller effect on calcium transient decay and did not alter calcium transient amplitudes in R14^Δ/Δ mice. The ASO effects on calcium cycling were also reflected in cardiomyocyte relaxation, resulting in significantly accelerated sarcomere relaxation (tau) and significantly increased shortening amplitude in WT cardiomyocytes (Figure 7E–G). R14^Δ/Δ cardiomyocytes demonstrated significantly smaller tau for sarcomere relaxation and sarcomeric shortening amplitude was significantly stronger as compared to WT cardiomyocytes. Consequently, the PLN silencing effects on contraction and relaxation were significant, though less pronounced in R14^Δ/Δ compared to WT cardiomyocytes. We also performed beta-adrenergic stimulation using isoproterenol, which had a profound effect on all indicated parameters in vehicle-treated WT cardiomyocytes (see Supplementary material online, Figure S7). The effect of isoproterenol is partially dependent on protein kinase A (PKA)-mediated phosphorylation of PLN, resulting in reduced SERCA2a inhibition and enhanced calcium cycling, contraction and relaxation.²² Not surprisingly, isoproterenol had only limited additive effect on these parameters in ASO-treated WT cardiomyocytes (see Supplementary material online, Figure S7). Similarly, isoproterenol had limited additive effects in R14^Δ/Δ cardiomyocytes with or without ASO treatment (see Supplementary material online, Figure S7), indicating that the R14del pathogenic variant of PLN is a partial loss-of-function mutation. Nevertheless, isoproterenol stimulation of ASO-treated WT and R14^Δ/Δ cardiomyocytes further increase calcium transient amplitude, showing that PLN depletion did not deplete cardiac reserve. Altogether, the current study shows that PLN-ASO therapy has limited effects on calcium handling in PLN-R14del cardiomyopathy.

All data that support the findings of this study are available from the corresponding author upon reasonable request.