Abstract 620: Hsf-1 Enhances Cardioprotective Potential Of Stem Cells Via Exosome Biogenesis And Their Mirna Cargo Enrichment.

Abstract

Introduction: Stem cells work via different mechanisms of which paracrine mechanism is able to justify most of the effects. Therefore, identifying the control arms for paracrine cocktail production is necessary to tailor stem cell functions. Objective: This study shows how HSF1 determines stem cell potential to repair ischemic heart via affecting production of paracrine factors including exosome and their miRNA cargo. Methods and Results: CRISPR-Cas9 gene editing tool was used to knock out HSF1 gene in nMSCs while lentiviral hsRNA constructs for HSF1 were used to overexpress it in aMSCs. We identified that HSF1 knockout resulted in reduced cardiac regeneration abilities in nMSCs while its overexpression in aMSCs had opposite effects as depicted by functional characteristics including LVEF, LVEDV, LVESV and LVFS in our rat ischemic model. Further, we found that exosome number in nMSCs decreased significantly after HSF1 knockout while it went up in aMSCs after HSF1’s overexpression as determined by NanoSite particle tracking analysis. The exosomes from nMSCs (nEXOs), aMSCs (aEXOs) and their genetically modified versions (nKO-EXOs) and aOE-EXOs) respectively) were analysed for miRNA cargo using RT-PCR. nKO-EXOs showed significantly reduced candidate miRNA expression (miR-145, miR-146, 199-3p, 199b and miR-590) than nEXOs while aOE-EXOs had increased candidate miRNA expression compared to aEXOs. Since HSF1 does not bind to RNA molecules, we hypothesised that YBX1, another protein highly expressed by nMSCs than aMSCs and has RNA binding nature, under the influence of HSF1 sorts miRNAs into exosomes. YBX1 expression was found to be controlled by HSF1 as shown by RT-PCR and western results. HSF1 controls YBX1 expression, at transcriptional level, is strongly supported by our finding that YBX1 promoter possesses heat shock elements (HSEs) as HSF1 binding sites. We further went on to see whether YBX1 alteration affects miRNA loading of exosomes and it was demonstrated that absence of YBX1 resulted in accumulation of candidate miRNAs into the cells. Conclusion: Together, our data shows that HSF1 plays a critical role in determining the regenerative potential of stem cells. HSF1 does that by affecting exosome biogenesis and miRNA cargo sorting via YBX1 protein.