A crucial function for c-Kit(+) in cardiac regeneration: role of Wnt inhibition. Mus musculus

Purpose: To explore the effects of Wnt inhibitor Sfrp2 and to clarify the changes that in situ c-Kit(+) cells undergo in the infarcted heart we performed transcript profiling (RNA-seq) Overall design: Methods: Eight week C57BL6 c-KitCreERT2/mTeG mice were injected for 14 consecutive days with tamoxifen (0.5 mg/day by i.p) to induce Cre expression and to label c-Kit(+) cells with eGFP. Mice underwent MI and two days later were treated with vehicle or Sfrp2. C-Kit(+) cells were isolated one day prior to MI, one day post-MI and 12 days post-MI (vehicle and Sfrp2). Cardiac biopsies were isolated for c-Kit(+) cells. Total RNA from the purified c-Kit(+) population was isolated using a PicoPure Arcturus kit (Invitrogen). Complimentary cDNA was generated with an Ovation Pico WTA System V2 kit (NuGEN). RNA-seq was performed with 2 lanes on a HiSeq 2000 instrument yielding a total of >400,000,000 PF clusters. Gene Ontology analysis was performed by the Duke Genomic Core. RNA-seq data was processed using the TrimGalore toolkit which employs Cutadapt to trim low quality bases and Illumina sequencing adaptors from the 3’ end of the reads. Only reads that were 20nt or longer after trimming were kept for further analysis. Reads were mapped to the GRCm38v68 version of the mouse genome and transcriptome using the STAR RNA-seq alignment tool. Reads were kept for further analysis if they mapped to a single genomic location. Gene counts were compiled using the HTseq tool. Only genes that had at least 10 reads in any given library were used in subsequent analysis. Normalization and differential expression was carried out using the DESeq2 Bioconductor package with the R statistical programming environment. The false discovery rate was calculated to control for multiple hypothesis testing. Gene set enrichment analysis was performed to identify differentially regulated pathways and gene ontology terms for each of the comparisons performed. Results: Sfrp2 promotes the differentiation of c-Kit(+) cells into cardiomyocyte-like cells