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Abstract
Background
In most cell types, replicative senescence (RS) is supposed to be a principle causative factor for aging. Atrial fibrosis, pathologically characterized by proliferation of atrial fibroblasts (AFs) and excessive accumulation of extracellular matrix proteins, is the most common substrate of atrial fibrillation (Afib) in the elderly. However, whether AFs’ RS develops in the aged and fibrotic left atrium (LA) and, if yes, what is the key regulator for the pathogenesis of AFs’ RS remain largely unknown.
Methods
We obtained the left atrial tissues from young (6–8 weeks old) and aged (24 months old) C57BL/6 male mice. Screening and validation of differential genes were performed using comparative analysis of RNA-seq results. Replicative senescence was examined in primary AFs after cell passage. Further gain-of-function and loss-of-function experiments were performed to explore the regulation of the AFs’ RS progression.
Results
In the present study, we demonstrated that there was a considerable extent of AFs’ RS in the aged and fibrotic LA. Transcriptome screening showed that Ezh2 (Enhancer of zeste homolog 2) was significantly downregulated in the LA tissue of aged mice. Ezh2 is a histone methyltransferase that catalyzes H3K27me3 and mediates transcriptional silencing. We confirmed that Ezh2 was downregulated in the isolated pure senescent AFs. Knockdown of Ezh2 by siRNA or inhibition of Ezh2ʹs methyltransferase activities by GSK-126 and GSK-343 accelerated RS in the early passage of AFs, while its overexpression deaccelerated RS in the late passage of AFs. Mechanistically, Ezh2 suppressed CDKN2a (p16, p19) and Timp4 gene transcription by forming canonical H3K27me3 modifications in their promoter regions. Furthermore, the functional balance between Timp4 and MMP8 in AFs could be collapsed by changes in Ezh2 expression.
Conclusion
These results thus indicate that Ezh2 is a key regulator of AFs’ RS and this work may provide a basis for future treatments for atrial fibrosis in the elderly.
Graphical Abstract
Abbreviations
Afib, atrial fibrillation; AFs, atrial fibroblasts; CDKI, Cyclin-dependent kinase inhibitor; CUT&Tag, Cleavage Under Targets and Tagmentation Assay; DEGs, differentially expressed genes; ECM, Extracellular matrix; EF, Ejection fraction; Ezh2, Enhancer of zeste homolog 2; FS, Fractional shortening; H3K27me3, trimethylated versions of lysine residue 27 within histone H3; IL-1α, Interleukin-1α; IL-1β, Interleukin-1β; IL-6, Interleukin-6; LA, Left atrium; LAD, Left atrial dimension; MCP-1, Monocyte chemotactic protein-1; MMP, Matrix metalloproteinase; qRT-PCR, Quantitative Real-Time PCR; RNA-seq, RNA Sequencing; ROS, Reactive oxygen species; RS, Replicative senescence; SASP, Senescence-associated secretory phenotype; SA-β-gal, Senescence-associated beta-galactosidase; siRNA, Small-interfering RNA; TGF-β, transforming growth factor-β; Timp, tissue inhibitors of metalloproteinase; WB, Western Blotting.
Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that there is no conflict of interests.