Ageing is the greatest risk factor for many life-threatening disorders, including cardiovascular disease. As we age our cells age and they become dysfunctional or abnormal. Ageing leads to an increase in a phenomenon known as cellular senescence in several tissues, including the heart. These senescent cells have a limited ability to replicate, but they build up in your body and refuse to die. The main problem is that senescent cells release chemicals that can be harmful to nearby cells, eventually making them become senescent and dysfunctional cells too. The build-up of senescent cells in our bodies promotes organ degeneration, tissue and physical dysfunction and contribute to age-related diseases, like dementia and cardiovascular disease.
We and others have shown that if you eliminate or modulate senescent cells in aged mice using a new class of agents called senotherapeutics you can improve the function and healthiness of the heart. Preliminary data from our lab show a role for miRNAs (miRNA-34a and miRNA-22) in cardiomyocyte cell senescence.
Overall Aim: To investigate the role of miRNAs and anti-miRNAs in inhibiting or preventing senescence of human cardiomyocytes and cardiac endothelial cells. The overall purpose is to identify miRNAs that regulate senescence and can prevent this process, thus informing us on new targets for the development of senotherapeutics specific to cell types of the heart.
Specific objectives:
1) To perform functional high-throughput screening (HTS) of a whole genome miRNA library and antimiR library to identify miRNAs preventing/blocking senescence of human iPSC-CMs and endothelial cells. Readouts will be SA-beta gal (CellEvent™ Senescence Green Detection Kit, ThermoFisher), p21/p16, EdU and 3 SASP factors (i.e. IL-6 and IL8, CXCL1) (Year 1).
2) To investigate the effects of mimics or inhibitors of miRNAs identified according to Objective 1, on preventing senescence induction in in vitro on cardiomyocytes and endothelial cells (Year 2) and cardiac organoids (Year 3).
Techniques: in vitro assays – working with iPSCs and differentiation into cardiomyocytes and endothelial cells, image-based high-throughput screening, automated image analysis, generation of cardiac organoids, bioinformatic analysis, qRT-PCR, immunostaining and confocal microscopy.
Potential 3 month rotation project: Induction of senescence to cardiomyocytes and endothelial cells. Determine the expression profile of select miRNAs in senescent cardiomyocytes and endothelial cells. Bioinformatic analysis of small RNAseq data of senescent cardiomyocytes.