Transcriptome and methylome analyses of stromal cells from ACM patients reveal the additional role of alterations in mitochondrial homeostasis and chromatin organization in disease pathogenesis
Authors: Lippi M , Maione A S , Chiesa M , Perrucci G L , Iengo L , Sattin T , Cencioni C , Zeiher A M , Tondo C , Pompilio G , Sommariva E 
Published: November 2023

Abstract

Background
Arrhythmogenic cardiomyopathy (ACM) is a rare genetic disorder of the heart, characterized by ventricular arrhythmias and contractile dysfunctions, which can cause sudden cardiac death and heart failure. Typically, the hallmarks of ACM ventricular myocardium are cardiomyocyte death, inflammation and fibro-adipose replacement, resulting in electrical and mechanical abnormalities of the tissue. Cardiac mesenchymal stromal cells (CMSC) participate to disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many pathogenetic molecular mechanisms are yet to be discovered.

Purpose
Our work aims to contribute to a comprehensive understanding of the pathogenic mechanism occurring in ACM, focusing on the molecular pathways underlying stromal cell involvement, through the functional validation of gene expression and epigenetic profile of ACM-CMSC.

Methods
We conducted total RNA transcriptome and DNA methylome analyses in ACM- and healthy control (HC)- CMSC to identify pathways differentially regulated in the two groups. We performed category enrichment and gene network analyses and we built transcript functional networks. To validate our findings, we measured the protein levels coded by selected genes differentially expressed by Western Blot analysis and we evaluated the relative pathways, such as mitochondria homeostasis, proliferation rate and epithelial-to-mesenchymal transition (EMT) through cytofluorimetric analyses.

Results
By transcriptomic analysis we found 529 genes differentially expressed; in particular, 327 genes were more expressed and 202 less expressed in ACM- vs HC-CMSC. Among those with the highest logFC and the lowest p value, we identified COX7C (implicated in mitochondrial respiration), S100A11 (involved in EMT) and CCND2 (one of the promoters of the cell cycle), whose protein expression was validated. Through the enrichment and gene network analysis we identified some pathways differentially regulated in ACM vs. HC. Some of these are known to be altered in ACM hearts, as matrix homeostasis and cell junction arrangement. Other pathways have never been associated to ACM, such as mitochondrial functioning and chromatin organization. By the DNA-methylation investigation, we identified 74 differentially methylated nucleotides and most of them were located on the mitochondrial genome. We also found that ACM-CMSC exhibited a greater number of mitochondria and a greater amount of mitochondrial reactive oxygen species than HC. Furthermore, ACM-CMSC showed a higher proliferation rate and a more pronounced EMT, compared to controls.

Conclusions
ACM-CMSC -omics revealed some new altered molecular pathways, not yet explored, which are relevant in the pathogenesis of the disease and may constitute novel targets for specific therapeutic approaches.