Author ORCID Identifier

https://orcid.org/0009-0004-8281-0783

Semester

Spring

Date of Graduation

2026

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Exercise Physiology

Committee Chair

Dharendra Thapa

Committee Member

John M. Hollander

Committee Member

Emidio Pistilli

Committee Member

Eric Kelley

Committee Member

Jianhai Du

Abstract

For over a century, cardiovascular disease (CVD) has been and remains the leading cause of death globally. CVD risk and severity increase significantly with advanced age, and mitochondrial dysfunction has been implicated in the pathogenesis of aging. Furthermore, mitochondrial health is crucial for cardiac function, as one-third of total cardiomyocyte volume is occupied by mitochondria in order to meet the high energy demand of contractile and relaxational function. It is thus crucial that we understand the pathological mechanisms driving mitochondrial dysfunction in aging and how this contributes to age-associated cardiovascular disease. The current manuscript aims to fill this knowledge gap by exploring the role of lysine acetylation, a post-translational modification of proteins, in regulating the deterioration of mitochondrial function in the aged heart. We demonstrate that the aged cardiac mitochondrial proteome exhibits a pro-acetylation phenotype mediated by increased content of mitochondrial acetyltransferase general control of amino acid synthesis 5-like 1 (GCN5L1). This increase in GCN5L1 is accompanied by a decrease in cardioprotective mitochondrial deacetylase SIRT3, promoting acetylation of proteins involved in fuel substrate metabolism, bioenergetic function, and redox homeostasis. The primary goal of this dissertation is to elucidate the mechanisms by which GCN5L1-mediated hyperacetylation promotes dysregulation of mitochondrial function in the aged heart. The central hypothesis of this dissertation is that mitochondrial protein acetylation mediated by GCN5L1 negatively regulates fuel substrate utilization and redox homeostasis in the aged heart via increased acetylation of proteins facilitating fatty acid oxidation (FAO) and mitochondrial bioenergetics. We further hypothesize that age-associated hyperacetylation of these proteins drives dysregulation of fuel substrate metabolism and increased oxidative damage to cardiomyocytes, driving pathological remodeling and cardiac dysfunction in the aged heart. Using a novel tamoxifen-inducible GCN5L1 cardiac knockout (KO) mouse model, we show that GCN5L1 regulates the acetylation state of key FAO proteins in the aged heart, suppressing their activity and promoting cardiac diastolic dysfunction. Alterations in cardiac fuel substrate metabolism have been linked to several other cardiac pathologies including heart failure, ischemia/reperfusion injury, and diabetic cardiomyopathy. Further, we show that GCN5L1 cardiac KO was able to reduce the acetylation state of FAO proteins and increase their activity, rescuing cardiac diastolic function in aged mice. To further explore the role of GCN5L1-mediated lysine acetylation in regulating mitochondrial function, we next investigated mitochondrial redox milieu in the aged heart. Changes in fuel substrate metabolism have been linked to suppressed function of the oxidative phosphorylation (OXPHOS) system and increased generation of oxidative stress. The mitochondrion is the largest source of cellular ROS production, therefore the observed suppression of fatty acid metabolism in the aged heart could have downstream impact on the OXPHOS system. We report that the aged heart exhibits increased markers of oxidative damage, suppression of electron transport chain (ETC) complex activity, increased electron leak from the OXPHOS system, and suppressed quantity and activity of antioxidant enzymes, altogether promoting oxidative damage and mitochondrial bioenergetic dysfunction in the aged myocardium. We further report that GCN5L1 KO attenuated markers of oxidative damage, improved mitochondrial respiratory function, and increased antioxidant protein activity. These findings are in agreement with the increased fatty acid utilization and improved diastolic function observed in the aged GCN5L1 KO heart. Lastly, we sought to explore a direct mechanism to connect mitochondrial protein hyperacetylation in the aged heart to myocardial remodeling promoting the observed diastolic dysfunction. We next assessed fibrotic collagen deposition in the aged heart using histochemical staining with Masson’s trichrome and found significantly increased myocardial fibrosis compared to the young heart. Furthermore, this fibrosis was significantly reduced in the aged GCN5L1 KO mouse heart compared to WT. We discovered that GCN5L1 promotes acetylation of glycogen synthase kinase 3 (GSK3), inactivating it and stabilizing content of pro-fibrotic transcription factor Smad3. GCN5L1 KO reduced GSK3 acetylation, activating it and promoting the degradation of Smad3. Taken together, the age-associated increase in mitochondrial acetyltransferase GCN5L1 promotes suppression of fatty acid metabolism, suppressed OXPHOS function, and increased oxidative damage altogether promoting the induction of cardiac fibrosis and diastolic dysfunction in the aged heart. This research identifies GCN5L1 as a novel regulatory protein in age-associated mitochondrial dysfunction in the heart. Targeting GCN5L1-mediated acetylation in the aging heart offers a promising therapeutic avenue to promote healthy cardiac aging, improve healthspan, and reduce the rate of cardiovascular disease in the elderly population.

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