Dataset Information

Impairment of the rough endoplasmic reticulum/mitochondria compartment in human cardiomyocytes with phospholamban p.Arg14del mutation

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Skin Fibroblast

DISEASE(S): Dilated Cardiomyopathy

SUBMITTER: Xiaoke Yin

LAB HEAD: Manuel Mayr

PROVIDER: PXD020175 | Pride | 2021-04-12

REPOSITORIES: Pride

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Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.

Cuello Friederike F Knaust Anika E AE Saleem Umber U Loos Malte M Raabe Janice J Mosqueira Diogo D Laufer Sandra S Schweizer Michaela M van der Kraak Petra P Flenner Frederik F Ulmer Bärbel M BM Braren Ingke I Yin Xiaoke X Theofilatos Konstantinos K Ruiz-Orera Jorge J Patone Giannino G Klampe Birgit B Schulze Thomas T Piasecki Angelika A Pinto Yigal Y Vink Aryan A Hübner Norbert N Harding Sian S Mayr Manuel M Denning Chris C Eschenhagen Thomas T Hansen Arne A

EMBO molecular medicine 20210516 6

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca<sup>2+</sup> transient decay time, Ca<sup>2+</sup> -load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic re ...[more]

PMID: 33998164

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Project description:BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies, but can result in cardiac failure if left untreated. We hypothesized that the tail-anchored protein Dysferlin with multiple Ca2+-binding C2-domains is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes, and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. OBJECTIVE: To reveal the impact of the membrane fusion and repair protein Dysferlin on TAT network stabilization and proliferation necessary for the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: Super-resolution light and electron microscopy of mouse cardiomyocytes identified a specific localization of Dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum (SR), a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Mass spectrometry was used to characterize the cardiac Dysferlin interactome, thereby identifying a novel protein interaction with the membrane-tethering SR protein Juncophilin-2, a known interactor of L-type Ca2+ channels and Ryanodine Receptor Ca2+ release channels in junctional complexes. While the Dysferlin knockout caused a mild progressive phenotype of dilated cardiomyopathy in the mouse heart, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction (TAC), Dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while Dysferlin knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging demonstrated a profound reorganization of the TAT network in wild-type left-ventricular myocytes post-TAC with robust proliferation of axial tubules, which critically depended on the increased expression of Dysferlin within newly-emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-SR junctional complexes for regulated excitation-contraction coupling, and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure-overload.

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