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:Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable improvement in managing the progression of heart disease, developing effective therapies for heart failure (HF) remains a challenge. A better understanding of molecular mechanisms that maintain proper Ca2+ levels and contractility in the injured heart could be of therapeutic value. Here, we report that the transcription factor Zinc finger E-box-binding homeobox2 (ZEB2) is induced by Hypoxia-inducible factor 1-alpha (HIF1a) in hypoxic cardiomyocytes and regulates a network of genes involved in Ca2+-handling and contractility during ischemic heart disease. Gain- and loss-of-function studies in genetic mouse models revealed ZEB2 in cardiomyocytes to be necessary and sufficient to protect the heart against ischemia-induced diastolic dysfunction and structural remodeling. Moreover, RNA sequencing (RNA-seq) of ZEB2-overexpressing (Zeb2 cTg) hearts post-injury implicated ZEB2 in regulating numerous Ca2+-handling and contractility-related genes. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), thereby augmenting SR Ca2+ uptake and contractility. Furthermore, we observed a decrease in the activity of Ca2+-dependent calcineurin/NFAT signaling in Zeb2 cTg hearts, which is the main driver of pathological cardiac remodeling. On a post-transcriptional level, we showed that ZEB2 expression can be regulated by the cardiomyocyte-specific microRNA-208a (miR-208a). Blocking the function of miR-208a with antimiR-208a increased ZEB2 expression in the heart and effectively protected from the development of pathological cardiac hypertrophy. Together, we present ZEB2 as a central regulator of contractility and Ca2+-handling components in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in regulating Ca2+ homeostasis in cardiomyocytes is an essential step towards the development of improved therapies for HF.

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