Phosphoproteomics of PLN R14del heart tissue reveals major myocyte degenerate phenotype, and is attenuated by PLN antisense treatment in vitro

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Abstract Background/Purpose: Phospholamban (PLN) p.Arg14del (R14del, R14Δ/+) is a pathogenic variant that can cause cardiomyopathy, characterized by PLN protein aggregation, ultimately leading to heart failure (HF). The exact pathophysiology is unknown, we aim to uncover R14Δ/+ disease mechanisms and study potential treatment using PLN antisense oligonucleotides (PLN-ASOs). Methods Phosphoproteomics was performed on human heart tissue from end-stage R14Δ/+ patients (N=6) versus other etiologies of HF (N=10) as a control. CRISPR-Cas9 engineered R14Δ/+ and isogenic control (WT) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were extensively characterized, including the phosphoproteome, calcium transients (FLUO 4-AM), mitochondrial respiration (Seahorse Mito Stress Test) and contractility in dynamic engineered heart tissues (dyn-EHTs) before and after PLN-ASO treatment, and the results were validated in R14Δ/+ iPSC-CMs derived from R14Δ/+ patients. Results Phosphoproteomics of heart tissue from R14Δ/+ vs. other etiologies of HF identified 138 differentially expressed proteins (DEPs) and 317 differentially expressed phosphoproteins (DEPPs), therewith establishing a disease-specific signature. Phosphoproteomics of WT vs. R14Δ/+ iPSC-CMs identified 451 DEPs and 1046 DEPPs. Gene ontology analysis of end-stage R14Δ/+ heart tissue and iPSC-CMs revealed a large overlap. Here, R14Δ/+ showed to have great impact on contraction (e.g. SYNPO2L, MYH10, TTN) and calcium (e.g. AHNAK, HRC, RYR2). In line with this, functional characterization revealed that R14Δ/+ iPSC-CMs have an increased Ca2+ systolic velocity and accelerated calcium transient (decreased T50 and T90), that associated with altered calcium-related DEPPs (e.g. RYR2). In dyn-EHTs, R14Δ/+ dyn-EHTs have a decreased contraction duration, time-to-peak and ability to pace, associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently altered PLN mRNA expression and protein levels in iPSC-CMs, and 62 DEPs and 372 DEPPs were identified. PLN-ASOs increased Ca2+ diastolic velocity, which associated with altered calcium-related DEPPs (e.g. PLN, AHNAK, HRC). PLN-ASOs increased the ability of dyn-EHTs to pace and therewith improved contractility, which associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently increased basal respiration and ATP production, which associated with altered mitophagy-related DEPs (GABARAPL2 and MAP1LC3). These functional characteristics were validated in R14Δ/+ patient iPSC-CMs. Discussion: R14Δ/+ cardiomyocytes have a degenerative phenotype characterized by altered calcium transients and reduced contractility and PLN-ASOs attenuate these characteristics and improve metabolism.