A Stress Inducible Protein 1 (STI1) Em Fibroblastos Cardíacos: um estudo in vitro da expressão, da secreção e da localização

Abstract

Studies developed by our group have contributed to the elucidation of the protective role of the co-chaperone stress inducible protein 1 (STI1) in the heart. STI1 is a protein induced in stress situations, which is widely studied and understood in the context of the central nervous system, where it participates in the regulation of the protein maturation process, with a relevant contribution to proteostasis. In this work, we investigated both the presence and location of STI1 in cultured cardiac fibroblasts, as well as the conditions that modulate its expression and distribution along the cell surface, since such cells are relevant to the cardiac microenvironment, actively participating in the crosstalk process with cardiomyocytes (modulating their function). Initially, we evaluated the expression of STI1 in cardiac fibroblasts, using the immunofluorescence technique and gene expression analysis via real-time quantitative PCR (qPCR), in a context of normality and stress induction when treated with cobalt chloride (CoCl2 - chemical hypoxia inducer). Our findings confirmed the presence of STI1 in fibroblasts and indicated an increase in its expression after treatment with CoCl2 for 3h and 6h. In order to elucidate the location of this co- chaperone in fibroblasts, we transfected these cells with a plasmid expressing STI1 conjugated to a GFP reporter protein and subjected them to treatment with CoCl2. In control conditions, STI1 was distributed throughout the cell, including the cytosol, nucleus, and perinuclear region. Interestingly, in cells treated with CoCl2 for 3h, GFP fluorescence showed a typical vesicular pattern. In order to further investigate this STI1 labeling pattern, we performed a double labeling protocol for STI1 and exocytosis recycling vesicles, the latter using the FM 1-43 probe. Intriguingly, we found co- localization points between the vesicles and STI1, which corroborates and strengthens the evidence of the presence of this co-chaperone in vesicles. In the immunofluorescence experiments for STI1, a reduction in the fluorescence ratio of the nucleus to the cytoplasm was observed in the group treated with CoCl2, which is a strong indication of its mobilization and possible secretion by the cell. We also used the TIRF (Total Internal Reflection Fluorescence Microscopy) technique to visualize the trafficking of STI1-GFP in the cell in response to CoCl2 treatment. This increase in fluorescence is an important indication of the approach of STI1 to the cell surface, and is another important indicator of secretion.