Fig 2: Monomeric PLN expression is decreased (A) and phosphorylated PLN is increased (B) in both young (3-5 month) and old (10-12 month) TazKD mice compared with WT. A two-way ANOVA was used with age and genotype as main effects. For both (A) and (B), a significant main effect of genotype was detected and is depicted with an asterisk across the bar legends. *P < 0.05 (n = 4-6 per group)

Fig 3: Repeated subcutaneous injection of PLN ASO in mice with PLN R14?/? prevents cardiac dysfunction and improves survival.a Experimental design of the PLN R14?/? intervention study, consisting of ASO#27 treatment with a 4-week loading phase with weekly dosing of 100 mg/kg and a 20-week maintenance phase with dosing of 50 mg/kg once every 4 weeks. Data points for functional and biochemical assessments were obtained at treatment week 4 (T4) which was the primary endpoint, 15 (T15), 19 (T19), and 23 (T23) at the end of the study. b SYBR green qPCR results of cardiac Pln mRNA expression (n = 3 for wild-type [WT] and n = 4 for PBS and PLN-ASO). c Semi-quantified western blot analysis of PLN protein expression using LV protein lysates. Intensities were normalized to total protein loading control and the PBS treated mice (n = 3 per group). d Immunofluorescence of WT, PBS treated and PLN-ASO treated PLN R14?/? mice after 4 weeks treatment. Sections were co-stained for cardiac Troponin T (green), PLN (red), and DAPI (blue). Note the lower presence of visible PLN aggregations (white arrows) and the higher structural integrity of the PLN-ASO treated hearts vs. PBS control. Stainings were performed in two animals per group. e Representative MRI images after 4 weeks of treatment. f Quantification of LVEF (left ventricular ejection fraction), LVESV (left ventricular end-systolic volume), and LVEDV (left ventricular end-diastolic volume) (n = 12 for PBS T4, n = 13 for PLN-ASO T4). Wild-type data were previously published, and the average is presented here for reference. g Principle component analysis plot of the first 2 principle components derived from the RNA-sequencing of mice left ventricles. h Representative ECG tracing of mice after 4 weeks of treatment. i Quantification of R, S, and R + S amplitude (n = 14 for T4) Wild-type data were previously published and the average is presented here for reference. j Representative Masson’s trichrome staining after 4 weeks of treatment of PBS and PLN-ASO treated PLN R14?/? cardiac sections. Stainings were performed in four animals per group. k Quantification of myocardial fibrosis based on Masson’s trichrome staining whereby the blue, fibrotic area is presented as the fold change compared to wild-type in percentage of the total tissue slice surface (n = 4 for all groups). l Kaplan–Meier survival plot of PLN R14?/? animals treated with PLN-ASO (n = 13) and PBS (n = 14). Three PBS treated animals died during MRI imaging, the other PBS treated animals died at around 8 weeks of age. The “x” marks the 3-time points at which animals were sacrificed for tissue analyses. One-way analysis of variance was used for statistical analyses, with PBS treated animals as the reference group in multiple comparison analyses. Asterix denotes significance level based on two-sided P values compared to PBS with: *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001. Single values are depicted, and error bars represent the standard error of the mean (SEM). ASO antisense oligonucleotide, MRI magnetic resonance imaging, and ECG electrocardiogram. Source data are provided as a Source Data file.

Fig 4: A graphical study summary showing increased beneficial PLN ASO treatment effects with increased contribution of PLN to disease.Summary figure of study results. Three murine models of a dilated cardiomyopathy were studied, including the genetically engineered mouse models of PLN R14?/? and Csrp3/Mlp-/-, and the rat myocardial infarction model. PLN functional defects are causative to the pathology observed in PLN R14?/?, early contributing to the pathology in Csrp3/Mlp-/-, and consequential and aggravating in myocardial infarction. In line with this, the beneficial effects of the PLN-ASO were most abundant in the PLN R14?/? and Csrp3/Mlp-/- models, showing almost complete prevention or reversal of disease phenotype. In our myocardial infarction model, the PLN-ASO restored cardiac dimensions and contractility, but not relaxation or left ventricular ejection fraction. ASO antisense oligonucleotide, ANP atrial natriuretic peptide.

Fig 5: The effects of losartan and mirabegron on the protein expression at week 13 assessed by Western blot. Left ventricular expression and cropped representative images of (a) beta-3 adrenergic receptor (ß3-AR), (b) endothelial nitric oxide synthase (eNOS), (c) phospho-eNOS, (d) phospho-eNOS/ eNOS ratio, (e) sarcoendoplasmic reticulum calcium ATPase 2a (SERCA2a), (f) phospholamban (PLN), (g) phospho-PLN (pPLN), and (h) phospho-PLN/PLN ratio. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham-operated group, #p < 0.05 vs. CKD group (n = 6–7, One-Way ANOVA, Holm-Sidak post hoc test). Sham sham-operated group, CKD chronic kidney disease group, CKD + L losartan-treated chronic kidney disease group, CKD + M mirabegron-treated chronic kidney disease group. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Western blots are presented in Supplementary Figures S1-12.