Phosphoproteomics distinguishes disease-specific mechanisms for human phospholamban cardiomyopathy reversible by RNA therapy

Phospholamban (PLN) p.Arg14del (R14^Δ/+, also known as R14del) is a pathogenic variant that causes inherited cardiomyopathy. RNA therapy improves cardiac function and survival in murine PLN R14^Δ/+. However, the molecular disease mechanisms and potential therapeutic effects of RNA therapy in the human setting remain poorly defined. Proteomic and phosphoproteomic profiling was performed on cardiac tissue from R14^Δ/+ patients (N = 6) and compared to other causes of dilated cardiomyopathy (DCM; N = 10). Findings were validated in CRISPR-Cas9-engineered R14^Δ/+ induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and isogenic controls. To assess reversibility, PLN-targeted RNA therapy using antisense oligonucleotides was applied to iPSC-CMs. Proteomics revealed enrichment of fibrotic pathways, while phosphoproteomics highlighted altered actomyosin structural organization uniquely distinguishing R14^Δ/+ from other DCM. This phosphoproteomic profile was recapitulated in R14^Δ/+ iPSC-CMs. RNA therapy concentration-dependently reduced PLN expression and modified the disease-specific phosphorylation profile. Twenty-eight phosphorylation sites were consistently altered across patient tissue and iPSC-CMs; twenty-two were reversed by RNA therapy and were enriched for cadherin- and actin-binding functions, implicating cytoskeletal remodeling. PLN/LC3 protein aggregates, a hallmark of PLN cardiomyopathy, were reduced after RNA therapy. Functionally, R14^Δ/+ cardiomyocytes exhibited accelerated calcium handling and contractile kinetics, which increased further upon RNA therapy. Human PLN R14^Δ/+ cardiomyopathy is characterized by a distinct phosphoproteomic signature involving cytoskeletal and contractile machinery. PLN-targeted RNA therapy reduced PLN expression, partially normalized these alterations, diminished protein aggregation, and enhanced calcium handling and contractile performance. These findings clarify the molecular mechanisms underlying R14^Δ/+ pathogenesis and support RNA therapy as a promising therapeutic strategy for PLN cardiomyopathy.