Molecular Insights into the Role of Desmin Intermediate Filament Network in Chromatin Landscape, Early Cardiogenic Commitment and Cardiomyocyte Differentiation and Maturation (RNA-Seq Kap)

The cardiac cytoskeleton is essential for the proper intracellular and intercellular integration of structure and function, failure of which leads to heart disease. However, its role during development and cardiac differentiation is often overlooked. ­­Here, we sought to understand the role of the desmin intermediate filament cytoskeletal network in cardiac cell differentiation and homeostasis during embryonic development, post birth, adulthood and upon different stimuli. We show that desmin is highly expressed in cardiac progenitor cell populations during embryogenesis. In addition, using direct cellular reprogramming of fibroblasts to induced cardiomyocytes, we demonstrated that ectopic desmin expression directly influences cardiomyogenesis through a timely transcriptional regulation of the Notch1 signaling pathway. On the contrary, the absence of desmin in induced cardiomyocytes leads to substantial perturbations in cardiac maturation by diminishing the expression and proper localization of cardiac specific proteins, impairing calcium homeostasis and delaying myofibril formation. RNA and ChIP sequencing, along with chromosome conformation capture (HiC) in desmin-depleted cardiomyocytes revealed that the observed gene expression changes reflect the identified corresponding changes in chromatin architecture that cause loss of genome organization and thus contributing to pathophysiological phenotype. total 2 samples: induced cardiomycytes from WT (Des+/+) fibrobllasts transduced with a viral cocktail containing: one sample Gata4,Mef2C, Tbx5 plus Desmin and second Gata4,Mef2C, Tbx5 plus DsRED (control). RNAseq experiments were carried out in the Greek Genome Center (GGC) of the Biomedical Research Foundation of the Academy of Athens (BRFAA). RNAseq libraries were prepared with the illumina TruSeq RNA v2 kit using 1 μg of total RNA. Libraries were checked with the Agilent bioanalyzer DNA1000 chip, quantitated with the qubit HS spectrophotometric method and pooled in equimolar amounts for Sequencing. 100 bp Single-End reads were generated with the illumina NovaSeq sequencer. RNA-seq raw sequencing data were aligned to human genome version GCCh37/hg19 with the use of tophat (version 2.0.9) [39] with the use of «--b2-very-sensitive» parameter. Same tools (version 0.1.19) [40] were used for data filtering and file format conversion while HT-seq count (version 0.6.1p1) algorithm was performed for assigning aligned reads into exons using the following command line «htseq-count –s no –m intersection -nonempty». Finally, differentially expressed genes were identified with the use of DESeq R package and genes with fold change cut off 1.5 and p-value<=0.05 were considered to be differentially expressed (DEGs). Gene ontology and pathway analysis was performed in the DEGs with DAVID knowledgebase and Ingenuity Pathway Analysis software (IPA). Only pathways and biological processes with p-value <=0.05 were considered to be significantly enriched.