Phosphoproteomics distinguishes disease-specific mechanisms for human phospholamban cardiomyopathy reversible by RNA therapy
F E Deiman, N Bomer, P Davidsson, D Spater, A N Linders, I B Dias, K F Arevalo Gomez, A Esquivel Gaytan, A Walentinsson, C Ahlstrom, A E Mullick, K M Hansson, H H W Sillje, N Grote Beverborg, P Van Der MeerAbstract
Background
Phospholamban (PLN) p.Arg14del (R14del; R14Δ/+) is a pathogenic variant which causes an inherited cardiomyopathy. PLN-targeted RNA therapy improved cardiac function and survival in murine PLN R14del. However, the molecular disease mechanisms and potential therapeutic effects of RNA therapy in the human setting remain poorly defined.
Methods
Proteomic and phosphoproteomic profiling was performed on cardiac tissue from PLN R14del patients (N=6) and compared to other genetic causes of dilated cardiomyopathy (DCM; N=10). Findings were validated in CRISPR-Cas9 engineered PLN R14del induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and isogenic control. To assess reversibility of disease signatures, RNA therapy with PLN antisense oligonucleotides (ASOs) was applied to iPSC-CMs.
Results
Proteomics revealed a disease signature mainly enriched for fibrotic pathways, while phosphoproteomics highlighted altered actomyosin structural organization uniquely distinguishing PLN R14del from other DCM. This phosphoproteomic profile was recapitulated in PLN R14del iPSC-CMs versus controls. RNA therapy treatment with ASOs dose-dependently lowered PLN expression and modified the disease-specific phosphorylation profile. Twenty-six phosphorylation sites were consistently altered across patient tissue and iPSC-CMs; twenty 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, PLN R14del cardiomyocytes exhibited accelerated calcium handling and contractile kinetics, both of which increased further with RNA therapy.
Conclusion
Human PLN R14del cardiomyopathy is characterized by a distinct phosphoproteomic signature involving cytoskeletal and contractile machinery. PLN-targeted RNA therapy reduces PLN expression, partially normalizes the disease-specific phosphorylation profile, diminishes pathogenic protein aggregation and enhances calcium handling and contractile performance. These findings clarify molecular mechanisms underlying PLN R14del pathogenesis and support RNA therapy as a promising therapeutic strategy for PLN cardiomyopathy.For image description, please refer to the figure legend and surrounding text.For image description, please refer to the figure legend and surrounding text.