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<p>Phospholamban (PLN) p.Arg14del (R14<sup>Δ/+</sup>, also known as R14del) is a pathogenic variant that causes inherited cardiomyopathy. RNA therapy improves cardiac function and survival in murine PLN R14<sup>Δ/+</sup>. 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<sup>Δ/+</sup> patients (<i>N</i> = 6) and compared to other causes of dilated cardiomyopathy (DCM; <i>N</i> = 10). Findings were validated in CRISPR-Cas9-engineered R14<sup>Δ/+</sup> 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<sup>Δ/+</sup> from other DCM. This phosphoproteomic profile was recapitulated in R14<sup>Δ/+</sup> 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<sup>Δ/+</sup> cardiomyocytes exhibited accelerated calcium handling and contractile kinetics, which increased further upon RNA therapy. Human PLN R14<sup>Δ/+</sup> 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<sup>Δ/+</sup> pathogenesis and support RNA therapy as a promising therapeutic strategy for PLN cardiomyopathy.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月28日

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  • Frederik E. Deiman,
  • Nils Bömer,
  • Pia Davidsson,
  • Daniela Später,
  • Annet N. Linders,
  • Itamar B. Dias,
  • Karla F. Arevalo Gomez,
  • Antonio Esquivel Gaytan,
  • Jumo Zhu,
  • Anna Walentinsson,
  • Susanna Engberg,
  • Neil Hattersley,
  • Damla Etal,
  • Christine Ahlstrom,
  • Adam E. Mullick,
  • Kenny M. Hansson,
  • Herman H. W. Silljé,
  • Niels Grote Beverborg,
  • Peter van der Meer

摘要

Phospholamban (PLN) p.Arg14del (R14Δ/+, also known as R14del) is a pathogenic variant that causes inherited cardiomyopathy. RNA therapy improves cardiac function and survival in murine PLN R14Δ/+. 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 R14Δ/+ patients (N = 6) and compared to other causes of dilated cardiomyopathy (DCM; N = 10). Findings were validated in CRISPR-Cas9-engineered R14Δ/+ 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 R14Δ/+ from other DCM. This phosphoproteomic profile was recapitulated in R14Δ/+ 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, R14Δ/+ cardiomyocytes exhibited accelerated calcium handling and contractile kinetics, which increased further upon RNA therapy. Human PLN R14Δ/+ 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 R14Δ/+ pathogenesis and support RNA therapy as a promising therapeutic strategy for PLN cardiomyopathy.