Updated project metadata.
After reporting that mice with inducible CM-specific Wnt activation (β-catΔex3) mimic pathological remodelling condition and heart failure, we further explored the cardiac-derived extracellular vesicles (EVs). EVs analysis in vivo are challenging, therefore we used a genetic model to analyze the extracellular compartment as well as the cell composition in vivo, which provided advantageous by focusing only on the relative changes between control and transgenic cells. With an improved protocol we isolated EVs from CT and β-catΔex3 hearts. The fraction containing small EVs was confirmed by electron microscopy and nanoparticle-tracking-analysis and further characterized with EV, endoplasmic reticulum and Golgi markers as well as mass spectrometry analysis. This analysis confirmed the cardiac origin of EVs and showed 47% proteins included in Exocarta Top100 candidate with a major exosome (Exo) representation. By proteomic analysis, 391 significantly differentially enriched proteins in β-catΔex3 hearts were identified, which were significantly loaded with all seven α and β chains of the constitute 20S proteasome, molecular chaperones and co-chaperones as well as proteasome associated proteins previously described in Exo (HSPA70, HSP90AB1, CRYAB, PKACA and BAG2). CRYAB, a cardiac chaperone protein that triages misfolded proteins for proteasomal degradation or repair, was confirmed to co-localize with labelled Exo in vitro. We performed single whole cell transcriptome, followed by pairwise comparison of differential expressed gene analysis (DEG) and their functional enrichment between β-catΔex3 and CT cell clusters. This revealed that different cardiomyocytes and endothelial cell subpopulations of β-catΔex3 CM were significantly active transcriptional activity of EVs secretion and protein quality control pathways. This finding was confirmed in cardiomyocytes populations of old mice subjected to experimental pressure overload to induce hypertrophy at early and late stages of remodeling, including an acute early upregulation of exosome biogenesis processes and chaperons transcripts including CRYAB at early stages. In order to model this molecular mechanism in human cells, human induced pluripotent stem cells (IPSC)-derived cardiomyocytes were subjected to pharmacological Wnt activation, which recapitulated the increased cell cycle activation as well as exosomal markers in vitro. Our results indicate a contribution of Exo-mediated maintenance of cardiomyocytes proteostasis upon pathological remodeling. Upon stress, CRYAB and co-chaperones increase binding to damaged-sarcomeric-proteins heading towards degradation, which overload the intracellular proteasomal capacity. This may activate EV-mediated cellular extrusion of misfolded proteins as a mechanism of cellular protection. Cardiac extracellular proteosome may serve as read-out of disease progression and for monitoring cellular remodeling in vivo.