Epigenetic regulation of donor heart preservation mediated by Brd4 - Project Summary/Abstract While heart transplantation is the gold standard to treat patients with end-stage heart failure, there is a shortage of donor hearts for recipients. Unfortunately, a minority of donor hearts on offer are accepted for transplant. This can be for multiple reasons, but the risk of primary graft dysfunction (PGD) is a major factor in turning down donor hearts. The risk of PGD is increased when cold static preservation time is >4 hours although machine perfusion technique can prolong this time by several hours. Even with these advances, a finite and significant geographical limitation is placed on organ matching. Expanding our molecular understanding of cardiac preservation is needed to position the organ preservation field for leaps in therapeutic advancements. Brd4 is a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers. It plays an important role in many cardiac diseases such as cardiac hypertrophy and coronary atherosclerosis. Brd4 interacts with numerous factors that regulate transcription, histone modification, chromatin accessibility and architecture. We show that human failing hearts and donor hearts with prolonged preservation times have preferential short Brd4 isoform (Brd4-S) expression. We show that in-vivo knockdown of Brd4-S in cardiomyocytes greatly improves ex-vivo donor heart function with prolonged preservation. We also show that cold cardiac perfusion with histidine-tryptophan-ketoglutarate improves ex-vivo donor heart function and is associated with a significantly reduced Brd4-S expression. We hypothesize that cold preservation of donor hearts induces a switch towards Brd4-S expression leading to genome architectural restructuring with epigenetic changes that promote cardiac injury. In Aim1, we will determine if increased osmolality, cold perfusion and expression of splicing mediators contribute to Brd4-S expression during cardiac preservation. We used ex-vivo perfusion and heterotopic transplant models to test these hypotheses. In Aim 2, we will define the chromatin configuration induced by Brd4 isoforms during cold static cardiac preservation. We will use mass spectrometry to identify Brd4-S binding partners in donor heart preservation. To characterize the genomic landscape, we will examine the genome occupancy of Brd4-S and its cofactor, as well as histone modification, chromatin accessibility and looping in preserved donor hearts with Brd4 isoform knockdown. To determine the specific effects of cold perfusion on genomic organization, we will perform similar studies in cold HTK-perfused hearts. In Aim 3, we will determine if inhibition of Brd4 improves the preservation quality of pig and human hearts in a cold cardiac perfusion model. The proposed work will define Brd4-S's role in modulating chromatin configuration during cardiac preservation and represents a preclinical organ preservation study of combined Brd4 inhibition and cold HTK perfusion. This is expected to increase donor heart utilization and expand the donor pool with broad implications for other solid organ transplants and ischemic pathologies.
