Date of Award

Fall 9-1-2023

Embargo Period

9-7-2028

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Regenerative Medicine and Cell Biology

College

College of Graduate Studies

First Advisor

Ge Tao

Abstract

Every 40 seconds, someone in the United States has a myocardial infarction (MI), or heart attack. Following MI, mature mammalian cardiomyocytes (CMs) are unable to repopulate the injured myocardium due to a low turnover rate of 0.5-2% per year. The lack of CM renewal and cardiac regeneration requires infiltration of cardiac fibroblasts (CFs) to deposit ECM preventing cardiac rupture and maintaining tissue integrity. The infiltration and increased ECM deposition result in a fibrotic scar that lacks contractility and leaves the heart with decreased function. However, the neonatal mammalian heart retains a regenerative capacity during the first week of life. Understanding the mechanisms that underlie cardiac regeneration is imperative to developing therapeutic techniques and treatments for patients who experience MI.

Recent work by our lab has identified developmental transcription factor Pitx2 as a regulator of Gpx4, a selenoprotein essential to ferroptosis resistance. In this study, we attempted to determine the extent of ferroptosis after MI. Regenerative postnatal day 1 (P1) and non-regenerative postnatal day 7 (P7) mice were subjected to permanent left anterior descending artery occlusion surgery (LAD-O) and hearts were collected at multiple timepoints to profile scar progression and cell death. Three forms of cell death including ferroptosis were profiled using confocal microscopy. To determine if Pitx2 expression affected ferroptosis relevant genes, RNA sequencing was done on human induced pluripotent stem cells differentiated into cardiomyocytes (iCMs) as well as in Pitx2 CM conditional knockout (Pitx2-cKO) and overexpression (Pitx2-OE) mice.

Our work also identified Pitx2 as a regulator of Tsp1, an activator of latent TGF-β, a potent fibrotic factor. To examine if Pitx2 expression could alter fibroblast activation and fibrotic scar formation, western blot analysis of Pitx2cKO and Pitx2OE was performed. We found that Pitx2 expression was negatively correlated with Tsp1 expression. Further, we investigated if ferroptosis affected CFs and found CFs exhibit greater resistance to ferroptosis than CMs in a density-dependent manner. We confirmed that at mid to high density, CFs express greater amounts of anti-ferroptotic iron storage proteins, the ferritins. We then explored the relationship between CFs and CMs during ferroptosis. Using a Rosa26-eGFP-DTA reporter under a periostin (Postn) promoter, PostnMCM/+;Rosa26eGFP-DTA, we ablated activated fibroblasts from the mouse heart and saw increased lipid peroxidation, a hallmark of ferroptosis. To elucidate the mechanism of protection by CFs, we co-cultured human cardiac fibroblasts (HCFs) with iCMs and observed that the cell types share iron burden after iron overload.

This project has led to new understanding of the mechanisms of CM loss after MI as well as to the role of CFs in protecting CMs from cellular death and in scar formation in the regenerative and non-regenerative heart.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Thursday, September 07, 2028

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