Defining the role of decreased estradiol in modulating crotonylation of myofilament proteins in aged female hearts

The incidence of diastolic dysfunction increases dramatically in women after menopause, which has long been attributed to loss of protection from female sex hormones, specifically estradiol. The mechanisms underlying estradiol-mediated cardioprotection are still unknown. A better understanding of biochemical pathways that are modulated in the female heart in response to estradiol has the potential to improve how we treat post-menopausal women with diastolic dysfunction. We have found that sarcomeric relaxation is significantly prolonged in healthy women after menopause compared to women who are pre-menopausal. We hypothesize that changes in sarcomeric PTMs due to decreased estradiol may contribute to this prolongation in relaxation. In addition to prolonged relaxation, we have also found that crotonylation of proteins is upregulated in old female hearts. In an unbiased screen of aged human hearts using mass spectrometry, we found a specific enzyme, short-chain enoyl-CoA hydratase (ECHS1), is significantly down regulated in the hearts of females over 60 years of age. Lower levels of ECHS1 increase crotonyl-CoA. This increase in crotonyl-CoA, in turn, leads to higher protein crotonylation, which is a novel PTM that occurs on lysine residues. The enzymes that facilitate this PTM include p300 and the enzymes that remove crotonylation from proteins are class I histone deacetylases (HDACs). While it has been shown that sarcomeric proteins are crotonylated, the role that estradiol plays in crotonylation and the functional effects of crotonylation are completely unknown. The objective of this proposal is to determine the role of estradiol in regulating cardiomyocyte crotonyl-CoA thereby modifying sarcomeric crotonylation. In this proposal, we will establish if lower ECHS1 in aged female cardiomyocytes is due to decreased estradiol (Aim 1) and determine the functional role of sarcomeric crotonylation (Aim 2). We hypothesize that reduced estradiol levels in aged female hearts lead to decreased ECHS1 in cardiomyocytes, increasing cellular crotonyl- CoA, sarcomeric protein crotonylation, thereby contributing to prolongation of sarcomeric relaxation. This R21 exploratory proposal will identify a completely novel mechanism underlying changes in cardiac diastolic function in response to estradiol. There are no data examining the role estradiol plays in regulating crotonylation nor data about how crotonylation of sarcomeric proteins modulate function. Completion of the proposed experiments will provide critical insights into specific modifications that occur in female hearts after menopause as well as contributing critical data necessary to elucidate our understanding of how an unexplored post-translational modification impacts cardiac function. Project Number: 1R21AG093397-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: KATHLEEN WOULFE | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $429,000 | Activity Code: R21 | Study Section: Integrative Myocardial Physiology/Pathophysiology A Study Section [MPPA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11303063