Hexokinase 1 mitochondrial binding and its role in cardiac endothelial cell function and the development of HFpEF

Heart failure (HF) with preserved ejection fraction (HFpEF) is a prevalent disorder; however, our treatment options are limited. Studies show that endothelial cell (EC) dysfunction precedes the development of HFpEF and is a key player in the pathogenesis of this disorder. Hexokinases (HK) catalyze glucose phosphorylation, and one HK isoform (HK1) binds to the outer mitochondrial membrane via its N-terminal hydrophobic domain, encoded in its exon 1. We have generated a mouse model with deletion of the mitochondrial binding domain (MBD) of HK1 (designated as ΔE1HK1 herein). These mice display HFpEF and reduced cardiac microvascular density as they age, and isolated ECs show lower angiogenesis. Accordingly, ECs from mice with HFpEF display increased HK1 mitochondrial dislocation, supporting that HK1 cellular localization plays an important role in the development of HFpEF. Metabolomic studies on ECs from ΔE1HK1 mice showed that hexosamine biosynthetic pathway (HBP) intermediates are decreased compared to WT mice. In addition, protein samples in ECs from ΔE1HK1 mice showed less N-glycosylation of proteins, while increased post-translational O-GlcNAcylation was observed compared to WT mice. These results suggest that HK1 dislocation causes a shift from protein N- glycosylation to O-GlcNAcylation in ECs, and that this switch contributes to EC dysfunction and the development of HFpEF. In this proposal, we will address the fundamental gap in knowledge of the mechanism of HFpEF and whether increased protein O-GlcNAcylation in ECs contribute to the pathogenesis of this disease. Our hypothesis is that HK1 mitochondrial dislocation leads to increased protein O- GlcNAcylation and that HFpEF is associated with increased HK1 dislocation and higher protein O- GlcNAcylation. We also hypothesize that a reduction in protein O-GlcNAcylation can reverse the pathogenesis of HFpEF. In Aim 1, we will determine whether HK1 mitochondrial dislocation and increased protein O-GlcNAcylation in ECs cause EC dysfunction and angiogenic defects. We will inhibit protein O- GlcNAcylation with O-GlcNAc transferase (OGT) inhibitors or “force” HK1 to the mitochondria in ECs from WT, ΔE1HK1 mice and mouse models of HFpEF and will assess their angiogenic potential. In Aim 2, we hypothesize that O-GlcNAcylation of histones are increased in ECs from HFpEF hearts. To test this hypothesis, we will perform mass spectrometry (MS) on the cellular, cytosolic and nuclear fractions of ECs from HFpEF and ΔE1HK1 mice. We will also perform ChIP-Seq and RNA-seq in ECs from WT, HFpEF and ΔE1HK1 mice to determine the differential gene expression in HFpEF. In Aim 3, we will determine whether inhibition of protein O-GlcNAcylation reduces the progression of HFpEF. We have crossed the ΔE1HK1 mice with mice that overexpress O- GlcNAcase (OGA, an enzyme that opposes OGT and reduces protein O-GlcNAcylation) in ECs and will assess cardiac function in these mice. Additionally, we will assess whether treatment of mice with a novel OGT inhibitor reverses the progression of HFpEF, providing a clinical relevance for our studies.