Protein Aggregation Is an Early Manifestation of Phospholamban p.(Arg14del)–Related Cardiomyopathy: Development of PLN-R14del–Related Cardiomyopathy

Figure 1.. PLN-R14Δ/Δ mice develop progressive dilated cardiomyopathy with heart failure between 4 and 7 wk of age. A through E, Representative 3-dimensional echocardiographic reconstructions of left ventricular (LV) end-diastolic (top) and end-systolic (bottom) volumes of male WT (wild-type; 5 wk old) and PLN-R14Δ/Δ mice (A) with quantification of end-diastolic volume (B), end-systolic volume (C), stroke volume (D), and ejection fraction (E) in WT and PLN-R14Δ/Δ mice at 4, 5, 6, and 7 wk of age (n=4 per group). F and G, Representative long-axis B-mode echocardiographic images of male WT (5 wk old) and PLN-R14Δ/Δ mice with delineation of LV epicardial and endocardial borders showing vectors that indicate direction and magnitude of wall motion (scale bars, 1 mm; F) with quantification of global longitudinal strain in WT and PLN-R14Δ/Δ mice at 4, 5, 6, and 7 wk of age (n=4 per group; G). H and I, Representative average tracings of 1-min ECG recordings of male WT (5 wk old) and PLN-R14Δ/Δ mice (H) with quantification of peak-to-peak amplitudes in WT and PLN-R14Δ/Δ mice at 4, 5, 6, and 7 wk of age (n=4 per group, except n=3 for 5-wk-old PLN-R14Δ/Δ mice; I). Data are presented as mean±SD.…

Figure 2.. Hearts of PLN-R14Δ/Δ mice undergo extensive remodeling between 3 and 8 wk of age. A and B, Ratios of biventricular (A) and atrial (B) weights to tibia length to the power 3 of WT (wild-type) and PLN-R14Δ/Δ mice at 3, 4, 5, 6, 7, and 8 wk of age (n=8 per group, mixed males and females). C and D, Quantification of myocardial fibrosis in WT and PLN-R14Δ/Δ hearts at 3, 4, 5, 6, 7, and 8 wk of age, shown as fold change compared with age-matched WT mice (n=8 per group, mixed males and females; C) with representative images of Masson trichrome–stained cardiac tissue sections (scale bars, 70 μm; D). E, Representative fluorescent staining images of WT (5 wk old) and PLN-R14Δ/Δ hearts at 3, 4, 5, 6, 7, and 8 wk of age stained for nuclei (4′,6-diamidino-2-phenylindole [DAPI]) in blue (top), extracellular matrix (WGA [wheat germ agglutinin]) in green (second), PLN (phospholamban) in red (third), and an overlay of all channels (bottom; scale bars, 70 μm). F, Left ventricular gene expression levels of Nppa normalized to housekeeping gene Rplp0 (36B4) of WT and PLN-R14Δ/Δ mice at 3, 4, 5, 6, 7, and 8 wk of age as measured by quantitative polymerase chain reaction (qPCR), shown as fold change…

Figure 3.. RNA sequencing identified differential regulation of protein homeostasis systems in PLN-R14Δ/Δ hearts but not in models of myocardial ischemia. A, Schematic overview of the left ventricular tissues that were included for RNA sequencing and the corresponding stage of heart failure development. B, Principal component (PC) analysis plot of RNA sequencing results of 3-, 5-, and 8-wk-old WT (wild-type; circles) and PLN-R14Δ/Δ (squares) mouse hearts, 8-wk-old PLN-R14Δ/+ (diamonds) mice, mice 3 d post-ischemia/reperfusion (I/R; triangles, up) or 8 wk post–myocardial infarction (MI; triangles, down), and corresponding sham mice (open circles) with 95% confidence ellipses of clusters marked in gray (n=4 per group, except n=3 for 8-wk-old WT mice, all male). Information on genes contributing to variance in PC1 (x axis) and PC2 (y axis) is shown in Figure VII in the Data Supplement. C and D, Venn diagrams of significantly upregulated (C) and downregulated (D) genes in 3-, 5-, and 8-wk-old PLN-R14Δ/Δ mice and mice 3 d post-I/R or 8 wk post-MI as compared with corresponding controls. E, Top 10 most significantly upregulated and downregulated gene ontology biological processes in 3-, 5-, and…

Figure 4.. Mass spectrometry revealed differential regulation of proteins involved in protein quality control in hearts of PLN-R14Δ/Δ mice. A, Principal component (PC) analysis plot of mass spectrometry results of 3-, 5-, and 8-wk-old WT (wild-type; circles), PLN-R14Δ/+ (diamonds), and PLN-R14Δ/Δ (squares) mice (n=6 per group, except n=5 for 8-wk-old WT mice, all male). B and C, Venn diagrams of significantly upregulated (B) and downregulated (C) proteins in 3-, 5-, and 8-wk-old PLN-R14Δ/Δ mice as compared with age-matched WT mice. D, Top 10 most significantly upregulated and downregulated gene ontology biological processes in 3-, 5-, and 8-wk-old PLN-R14Δ/Δ mice as compared with age-matched WT mice. An overview of the enrichment data can be found in Tables VII through IX in the Data Supplement. MHC indicates myosin heavy chain; n.s., not significant; Padj, adjusted P; and TAP, transporter associated with antigen processing.

Figure 5.. Graphic abstract summarizing the main findings of the current study. In PLN-R14Δ/Δ mice, the PLN-R14del pathogenic variant resulted in intracardiomyocyte PLN (phospholamban) protein aggregation together with alterations in protein homeostasis systems at 3 wk of age, before onset of other abnormalities or functional deficits. At 4 wk of age, slightly increased amounts of myocardial fibrosis were observed, and left ventricular tissue deformation was impaired. At the age of 5 wk, ventricular dilatation, impaired contractility, and reduced ECG voltages became apparent, which progressively worsened in the weeks thereafter. Ultimately, volume overload resulted in atrial hypertrophy after 7 wk and premature death around the age of 2 mo. While myocardial remodeling and contractile dysfunction were also observed in models of ischemic heart failure (HF), aberrations in protein quality control were exclusively found in PLN-R14Δ/Δ mice. DCM indicates dilated cardiomyopathy.