Pluto Bioinformatics

GSE89885: Transcriptional profiling reveals gene programs that are differentially regulated in cardiac fibroblasts during exercise and disease

Bulk RNA sequencing

Purpose: Cardiac fibroblasts (CFs) contribute to pathological remodeling by proliferating and secreting extracellular matrix. In contrast, physiological remodeling is an adaptive response to excessive demands of exercise that results in increased heart size without accompany fibrosis or deterioration of cardiac function. We hypothesize that these divergent outcomes are partially a function of cell-autonomous and paracrine cardioprotection afforded by CFs specifically during physiological remodeling. Our objective is to define the gene expression program (GEP) of CFs isolated from mouse models of physiological (swim) and pathological (thoracic aortic constriction (TAC) or myocardial infarction (MI)) remodeling to identify novel mechanisms underlying the development of pathological cardiac fibrosis and the cardioprotective benefits of exercise.; Methods: Cardiomyocytes (CMs) were isolated by gravity sedimentation and CFs were isolated by differential plating for 2 hours from single-cell suspensions of 10-14 week old male C57BL/6 hearts subjected to the following 7 conditions: sedentary CFs (n=3), sham surgery CFs (n=2), 3 day TAC CFs (n=3), 10 day TAC CFs (n=3), 4 day swim CFs (n=3), 10 day swim CFs (n=3), and 5 day MI CFs (n=2). CMs were isolated from sedentary 10-14 week old male C57BL/6 hearts (n=3). Total RNA was isolated using the Qiagen RNeasy Plus Kit and genomic DNA removed using Qiagen gDNA columns. RNA quality was assessed with the Agilent Bioanalyzer and only samples with RIN >8.5 were used for sequencing. Library construction was performed using the TruSeq Stranded mRNA Sample Preparation Kit from 100ng poly-A mRNA. An Illumina HiSeq 2500 was used to generate 20-25 million single end reads of 100nt for each sample. The sequence reads that passed quality filters (Trimmomatic-0.32) were analyzed at the transcript isoform level using STAR_2.4.2a for SHRiMP_2_2_3 mapping to mouse genome assembly GRCm38.p4 version mm10 and processed with Cufflinks-2.0.2.; Results: We determined that CFs display distinct GEPs that are inversely regulated in pathological and physiological remodeling. Spearman correlation and Principal Component Analysis (PCA) of genes with FPKM >1 and q >0.05 revealed consistency between biological replicates and highlighted active changes in GEPs during physiological and pathological remodeling both of which diverged from control samples in principal component (PC) 1 and PC2. CFs isolated from mice 5 days after initiation of MI also diverged from controls, TAC, and swim samples, demonstrating a different fibroblast GEP in ischemic and pressure overload induced remodeling. This analysis also revealed that our control models (sham surgery and sedentary) were indistinguishable from each other, therefore we typically treated sham and sedentary mice as a single control group in further studies. These initial results reveal that CFs differentially respond to pathological and physiological cues in as few as 3 days by enacting dynamic transcriptional changes in vivo. Consistent with previous reports of myofibroblast activation, Ingenuity Pathway Analysis (IPA) revealed that the most significantly enriched GO terms in pathological fibroblasts included stimulation of Rho-dependent contractile pathways and integrin-linked kinase (ILK) signaling, the latter of which was downregulated in a comparison between 10-day swim vs. control CFs. IPA transcription factor analysis comparing 10-day swim and 10-day TAC CFs further identified upregulation of detoxification and inflammatory transcription factors and downregulation of the serum response factor (SRF) after exercise. Furthermore, independent motif analysis using HOMER 2.0 identified the CArG box (SRF binding site) as the most enriched transcription factor binding site within 5kb of genes that are upregulated in 10-day TAC vs sham CFs.; Conclusions: We have defined the GEPs of CFs that underlie physiological and pathological cardiac remodeling. Not only is the GEP of pathological CFs enriched in Rho-dependent pathways and SRF target genes, but the GEP of physiological CFs is enriched in detoxification and inflammatory target genes. We also demonstrate that the CFs change their transcriptional signature consistently and rapidly. Within 3-4 days of initiating extracellular stimuli, distinct profiles are observed in CFs isolated from swim-trained, pressure overload, or ischemic models of cardiac remodeling. SOURCE: Eric Small ( - University of Rochester

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