Omics analyses of stromal cells from ACM patients reveal alterations in chromatin organization and mitochondrial homeostasis [RNA-Seq]

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder, characterized by ventricular ar-rhythmias, contractile dysfunctions and fibro-adipose replacement of the myocardium. Cardiac mesenchymal stromal cells (CMSC) participate to disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many are yet to be discovered. In order to define changes in the gene expression profiles between ACM- and HC-CMSC, we performed a high throughput RNA-seq on 6 ACM and 6 HC samples. Out of 15031 expressed genes, 529 resulted differentially expressed. Through enrichment and gene network analyses, we identified differentially regulated pathways, some of which never associated with ACM, including mitochondrial functioning and chromatin organization. Functional validations confirmed that ACM-CMSC exhibited a higher amount of active mitochondria and ROS production, a lower proliferation rate and a more pronounced epicardial-to-mesenchymal transition, compared to controls. In conclusion, ACM-CMSC -omics revealed some additional altered molecular pathways, relevant in the disease pathogenesis, which may constitute novel targets for specific therapies. Cells were obtained through the digestion of endomyocardial biopsies and they were characterized. The culture medium for CMSC maintenance was Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific), supplemented with 20% Fetal Bovine Serum (EuroClone), 10 ng/mL basic fibroblast growth factor (Peprotech), 10,000 U/mL Penicillin (EuroClone), 10,000 µg/mL Streptomycin (EuroClone), and 0.02 M L-Glutamine (EuroClone). All the assays were per-formed on CMSC cultured in these conditions. For sequencing via Illumina HiSeq (50-100 Million reads/sample), we extracted total RNA of CMSC from the 6 ACM and 6 control subjects, using the total RNA purification kit (Norgen Biotek Corp.) and following the recommended protocol. High-quality libraries were prepared using the PrepXTM RNA-Seq Library Kit for Illumina platforms. Sequential aligning of raw reads was performed against the GRCh38 Human Genome reference (version 99) with ‘STAR’, and ‘Bowtie 2’ to locally align any reads not mapped by STAR. Gene expression quantification and annotation was computed by “featureCounts”. Then, when less than 10 reads aligned in at least 40% of the gene, that gene was deemed as not expressed. The expressed genes were further normalized (variance stabilizing normalization) by the ‘DaMiRseq R package.