Thursday, September 19, 2024
9/19/2024
Project Summary/Abstract Heart transplantation is recognized as the gold standard therapy for end-stage heart failure. However, demand for donor hearts currently far outstrips supply due to multiple factors. An important limitation is primary graft dysfunction (PDG) in 10-20% of transplants and is an important contributor to adverse clinical outcomes and increased resource utilization. PGD occurs when donor heart function is inadequate for end organ perfusion and the risk increases once cold preservation time is >4 hours. Mineralocorticoid receptor (MR) signaling has a key role in many cardiovascular diseases including heart failure, and cardiac hypertrophy. It mediates harmful processes such as oxidative stress, inflammation, and fibrosis. Using murine models with MR deletions in cardiomyocytes as well as clinically utilized mineralocorticoid receptor antagonists (e.g. canrenone), we show that MR antagonism can greatly improve donor heart function following preservation. We also demonstrate that cardiac preservation is associated with increased MR protein expression coupled with organization of MR into molecular condensates with organizational structures that are known to augment protein transcription and translation. Importantly we find this occurs both in murine models as well as human hearts suggesting conserved events during evolution. We also confirm that MR contains intrinsic disordered regions which mediate phase separation and condensate formation. Our plan is to: (1) Identify the MR domains that can mediate condensate formation at differing temperatures. We will also determine if MR ligands, MR response elements and histone deacetylases (HDAC) can facilitate MR condensate formation. This will be performed by in-vitro evaluation of condensate formation by cloning truncated MR constructs missing specific domains and then adding different mediators of interest (e.g. HDAC) in the presence of a crowding agent. (2) We will examine the effects of MR condensate formation by first deleting MR in cardiomyocytes and then use adeno- associated virus to reconstitute cardiomyocytes with a MR mutant that has an impaired ability to form condensates because it is missing the intrinsic disordered region. We will then compare the inflammatory, oxidative stress and cell death responses associated with full length MR and the MR mutant. This will be evaluated in-vivo in a transplant model which incorporates recipient responses, ex-vivo in a perfusion model consisting only of the native cardiac cells and in an in-vitro neonatal cardiomyocyte preservation-reperfusion culture model. (3) We will determine the efficacy of canrenone (a MR antagonist) with or without valproic acid (a HDAC inhibitor) for improving donor heart function in pigs and humans. Single-cell RNA sequencing will be performed in human hearts to determine the cell population in which MR signaling is important for preservation. Our findings will contribute towards decreasing PGD occurrence, increasing donor heart utilization, and improving transplant outcomes. This is expected to have broad clinical implications such as transplantation of other solid organs (e.g. liver, kidneys) and for warm cardiac ischemia settings (e.g. myocardial infarction).
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