Pathological heart remodelling is characterized by cardiomyocytes (CM) transcriptional changes, dedifferentiation and substantial loss with consequent phenotypic changes of non-CMs leading to imbalanced intercellular communication and failure. We have previously demonstrated that Wnt/b-catenin/TCF7L2 signaling activation in CMs contribute to disease progression. We further aimed to resolve the dynamic of Wnt/b-catenin/TCF7L2 induced transcriptional profile in CMs and their cellular crosstalk at the single cell resolution by investigating an inducible CM-specific β-catenin stabilization (gain-of-function (GOF)) model that mimics hypertrophic remodeling.

Transcriptional regulation triggered by β-catenin/TCF7L2 activation was explored in the β-catenin CM-GOF hearts, by a recently established single cell (SC) transcriptome profiling platform able to capture cells of a variety of size range including CMs. SC suspensions from heart tissue were prepared with a modified Langendorff method, dispensed and subjected to sequencing on a HiSeq4000 (Illumina). Cells of poor quality (<5%) are identified by the documentation system and artifacts perturbing analysis are eradicated. An average of 5,000-6,000 genes/ cells are uniquely mapped. Unbiased clusters were identified and differentially regulated genes between CM-GOF and control hearts were extracted. Enrichment of processes including stress response, unfolded protein binding, proteasome, chaperones, extracellular vesicle, cell cycle, alternative splicing, protein transport and vascular smooth muscle (VSM) response was observed in the CM cluster in GOF hearts. Analysis of the vascular clusters showed enrichment of cell adhesion, actin cytoskeleton organization, cell migration, circulatory system development, angiogenesis, regulation of response to stimulus in GOF hearts (p<0.05, total of 1500 cells). Similar observations were done in wild-type hearts subjected to transaortic constriction (TAC) induced-pressure overload. In line with previous unpublished data in the lab, Insulin growth factor binding protein (Igfbp) 5 was the top three highest upregulated candidate in the CM-population followed by an upregulation in VSCMs-like (top 45) and an EC-like (top 60) in comparison to the corresponding cluster in the control heart. Applying RNA-scope and immunofluorescence, IGFBP5 transcript and protein expression was confirmed in CMs and vascular cells in mouse heart upon six weeks TAC and in CMs of human heart with cardiomyopathies (biopsies kindly provided by H. Milting, Bad Oeynhausen, Germany). In order to test the hypothesis that CM producing IGFBP5 affect non-CM landscape in vivo, we injected adult mice with AAV9 carrying the Igfbp5 ORF. Whole transcriptome analysis showed enrichment of metabolic, secretion, cell fate determination, cell differentiation and intracellular signal transduction processes in AAV9-Igfbp5 injected mice in comparison to controls (n=3 per group, DEGs with log2FC≥1.0 and p≤0.05).