Cardiomyocytes and stromal cells cross-talk influences the pathogenesis of arrhythmogenic cardiomyopathy: a multi-level analysis uncovers DLK1-NOTCH pathway role in fibro-adipose remodeling

Different cell types individually contribute to set the Arrhythmogenic Cardiomyopathy (ACM) phenotype with either functional abnormalities (cardiomyocytes; CM) or fibro-fatty substitution (cardiac mesenchymal stromal cells; cMSC). The relative contribution of the CM and cMSC derangements to determine disease evolution and electrical instability is still poorly understood. Here we showed that a patient-specific multicellular cardiac system, which models the functional interaction between CM and cMSC, provides insight into the relative contribution of different cell types to ACM phenotypes. Cell cultures (CM and cMSC) were lysed in RL lysis buffer (Norgen Biotek corp.). RNA was isolated from cells by using a Total RNA Purification kit (Norgen Biotek corp.). The quantification of the isolated RNA was determined by Qubit™ 4 Fluorometer (Invitrogen), and a quality check was performed. The RNA samples that passed the quality check were then sent to Eurofins Ltd Genomic Service for sequencing.RNA libraries were prepared for transcriptome sequencing using standard Illumina protocols in frame of the INVIEW Transcriptome Discover protocol. For library preparation, mRNA was fragmented, and random hexamer-primed cDNA synthesis was performed. Paired-end read sequencing (2 x 150 bp) was performed on the Illumina HiSeq 4000. Reads were aligned on human genome reference (GRCh38/hg38 version 104 ) by BWA software (v0.7.1) and mapped reads were used to quantify the gene expression, exploiting FeatureCounts (v2.0.0) .