Anne-Marie Koop

268 banded animals. Red and blue points mark the genes with significantly increased or decreased expression, respectively, in Hand2F/F sham to Hand2F/F PAB (FDR<0.01). The x-axis shows log2-fold expression changes and the y-axis the log10-fold likelihood of a gene being differentially expressed; b) Heatmap of the 100 top differentially expressed genes in the RV tissue of Hand2F/F sham compared to Hand2F/F PAB showing log2 FPKM (color scale) values of dysregulated genes, with red and blue colors representing increased and decreased expression, respectively; c) Number of differentially expressed genes enriched in GO terms with p value and rich factor shown in a scatterplot. The summarized GO terms are related to the biological processes upon pulmonary artery banding in the heart. Rich factor=number of differentially expressed genes in GO term/total number of genes in GO term. The larger the rich factor, the higher enrichment is. Circle size is proportional to the frequency of the GO term, whereas color indicates the log10 p value (red higher, blue lower); d) KEGG pathway analysis of differentially expressed genes with p value and rich factor shown in a scatterplot. Differentially enriched pathways in the RV tissue of control animals subjected to PAB, in comparison to sham. Mechanisms underlying the role of Hand2 in pressure overload- induced RV pathological remodelling Next, and to better understand how RV remodelling induced by pressure overload is affected by Hand2 silencing, we performed RNA-sequencing to assess the transcriptomic changes in total RNA from RV tissue derived from control or knockout animals subjected to PAB. Transcriptome analysis identified 1783 transcripts potentially regulated by Hand2 ( figure 5a, supplemental table 3 ). Generation of a heatmap representing the 100 most differentially expressed transcripts between the 2 experimental groups revealed 33 downregulated and 67 upregulated genes in the RV of Hand2 knockout mice ( figure 5b, table 5 ). Upon silencing of Hand2, RV stress resulted in downregulation of genes that are mostly associated with cellular components related to nucleic acid binding, regulation of transcription and transcription factor activity (U3B small nuclear RNA 2, Rnu3b2 ; GINS complex subunit 2, Gins2 ; Telomerase RNA component, Terc ; Transcription factor 7, TCF7 ) and cell adhesiongenes (a desintegrin andmetallopeptidasedomain 8, Adam8 ; Angiopoietin- like protein 4, Angptl4 ; Rho GDP dissociation inhibitor alpha, Arhgdia ; Ankyrin repeat domain 63, Ankrd63 ; Ephrin B3, Efnb3 ) ( figure 5b,c ). In turn, the upregulated genes are associated with hypertrophy ( Nppb ; Myotrophin, Mtpn ; Distrophin, Dmd ) and cell cycle inhibitors (Interferon activated gene 205, Ifi205 ; cell division cycle 73, CDC73 ) ( figure 5b, 5c ). Interestingly, genes such as Bone morphogenetic protein receptor, type II ( Bmpr2 ), Rho-associated coiled-coil containing protein kinase 2 ( Rock2 ) and Tissue inhibitor of metalloproteinase 3 ( Timp3 ), previously associatedwith pulmonary hypertension (PH), showed an even higher expression in the absence of Hand2. Furthermore, ontology (GO) and pathway (KEGG) enrichment analysis associated the observed gene expression patterns with regulation of transcription, muscle cell differentiation, cellular response to hypoxia, as well as with development of different types of cancer and different signaling pathways in cancer, platelet activation, p53 signaling pathway and cell communication through gap junctions ( figure 5c, 5d ). These results reveal the role of Hand2 in specific molecular and cellular processes that key in the response of the RV to pressure overload.