PROJECT SUMMARY Cardiovascular disease is the leading cause of death among men and women in the United States. Recent epidemiological findings support an intrinsic mechanism that protects pre-menopausal women from many cardiovascular pathologies and this protection is lost with menopause. Similar protective effects have been observed in preclinical models, suggesting that an understanding of the normal biology of the female heart could yield insight for effective cardiovascular therapeutics in humans. Recent work from our group demonstrates that free formaldehyde levels are two-fold higher at baseline in female hearts vs. males. Formaldehyde is a highly reactive electrophile produced as a byproduct of many cellular metabolic reactions and is known to induce cytotoxicity by forming adducts with DNA and protein. To counteract this cytotoxicity, formaldehyde dehydrogenase (FDH or GSNOR) and mitochondrial aldehyde dehydrogenase 2 (ALDH2) have evolved to oxidize formaldehyde to formate, which can act as a one-carbon source. One-carbon metabolism is essential for generating the building blocks required for amino acid, purine and thymine biosynthesis, but also for generating critical antioxidants and maintaining tetrahydrobiopterin (BH4), which is essential for nitric oxide synthase (NOS) function. Interestingly, female hearts exhibit increased FDH and ALDH2 activity vs. males, as well as higher endothelial NOS (eNOS) activity. Our preliminary findings show that FDH activity and formaldehyde levels decrease with ovariectomy (OVX) in female hearts, suggesting that endogenous formaldehyde is sex hormone- dependent. As such, the formaldehyde-formate arm of the one-carbon cycle may represent a resilient mechanism that benefits and protects female hearts by turning cytotoxic formaldehyde into formate to maintain BH4 levels and NOS activity, but this mechanism has not been examined in female hearts. Furthermore, we were the first to discover that the loss of FDH exacerbates ischemic injury in female hearts, resulting from a two-fold increase in post-ischemic formaldehyde, but ALDH2 activation rescued this injurious phenotype in female FDH-/- hearts. These results suggest that alterations to FDH activity and/or formaldehyde can drive myocardial pathology in females, but additional studies are needed to define a novel role for the formaldehyde-formate one- carbon arm in the biology of the female heart. This proposal will address this important knowledge gap in the following specific aims: 1) define the source and localization of endogenous formaldehyde generation in female hearts and 2) define a potential link between the formaldehyde-formate one-carbon arm and eNOS function in female hearts. If completed successfully, this study will advance our mechanistic understanding of the formaldehyde-formate one-carbon arm in the female heart and provide valuable insight into how this pathway may be targeted for the therapeutic treatment of cardiovascular disease.