Molecular Insights into the Role of Desmin Intermediate Filament Network  in Chromatin Landscape, Early Cardiogenic Commitment and  Cardiomyocyte Differentiation and Maturation (ChIP-Seq)
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. Overall design: total 4 samples: adult Cardiomycytes (ACM) from WT (Des+/+) and Null (Des-/-) mouse hearts (2 samples), adult cardiomyocytes (ACM) from WT (Des+/+) and Null (Des-/-) hearts (2 samples) of mice that have undergone swimming stress exercise. ChIP was performed per protocol described in Katsouda, A., D. Valakos, G. Vatsellas, D. Thanos, and A. Papapetropoulos, An optimized protocol for chromatin immunoprecipitation from murine inguinal white adipose tissue. STAR Protoc, 2023. 4(4): p. 102594. H3K27Ac and LaminA/C chromatin immunoprecipitation was performed on isolated chromatin using 2ugs of antibody (H3K27Ac : Abcam ab4729 and LaminA/C:Millipore).Input and IP DNAs were quantified with qubit. 5-10 ng DNA from each sample were used as input for ChIPSeq library prep with the NEBNext Ultra II DNA library prep kit for Illumina, according to manufacturer's instructions. QC of the final purified libraries was performed with qubit and Agilent bioanalyzer DNA1000 and sequencing was carried out in the Illumina Novaseq 6000 sequencer. Approximately 25 Million, 100 bp long Single End Reads were generated per sample. Quality control of the raw sequencing reads was performed with the FASTQC and FastxToolkit software. Raw reads from ChIP-Seq experiments were aligned to the GRCm38 (mm10) reference genome using the Bowtie2 aligner with default options .  SAM files were converted to sorted and indexed BAM files using Samtools [39]. Peak calling was performed using in-house scripts (https://github.com/supatt-lab/rChIPSeqTools/blob/master/sPeakDetection.R). Annotation of the peak lists was done using the ChIPseeker R package. Common and unique peak regions between ChIP-Seq experiments were computed using ChIPpeakAnno. Gene ontology analysis was performed using clusterProfiler.