PXD020175

PXD020175 is an original dataset announced via ProteomeXchange.

Title	Impairment of the rough endoplasmic reticulum/mitochondria compartment in human cardiomyocytes with phospholamban p.Arg14del mutation
Description	Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
HostingRepository	PRIDE
AnnounceDate	2021-04-12
AnnouncementXML	Submission_2021-05-19_07:33:32.275.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD020175
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Xiaoke Yin
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2020-07-03 01:22:40	ID requested
1	2021-04-12 08:09:59	announced
⏵ 2	2021-05-19 07:33:33	announced	2021-05-19: Updated publication reference for PubMed record(s): 33998164.

Cuello F, Knaust AE, Saleem U, Loos M, Raabe J, Mosqueira D, Laufer S, Schweizer M, van der Kraak P, Flenner F, Ulmer BM, Braren I, Yin X, Theofilatos K, Ruiz-Orera J, Patone G, Klampe B, Schulze T, Piasecki A, Pinto Y, Vink A, H, ü, bner N, Harding S, Mayr M, Denning C, Eschenhagen T, Hansen A, Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation. EMBO Mol Med, 13(6):e13074(2021) [pubmed]

submitter keyword: cardiomyopathy, phospholamban, hiPSC, CRISPR/Cas9, engineered heart tissue, proteomics

Manuel Mayr
contact affiliation	King’s British Heart Foundation Centre of Research Excellence, King's College London, United Kingdom
contact email	manuel.mayr@kcl.ac.uk
lab head
Xiaoke Yin
contact affiliation	Cardiovascular Division, King's College London
contact email	xiaoke.yin@kcl.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD020175
     1. Label: PRIDE project
     2. Name: Impairment of the rough endoplasmic reticulum/mitochondria compartment in human cardiomyocytes with phospholamban p.Arg14del mutation