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Introduction: It is reported that endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF). EC are known to highly rely on glycolysis to keep their function, but the metabolism of EC under the situation with HFpEF is poorly understood. Objective: We sought to elucidate the role of hexokinase 1 (HK1) in ECs for the development of HFpEF. Results: Isolated ECs from mouse hearts showed higher protein expression of HK1 than isolated cardiomyocytes and fibroblasts, suggesting HK1 has an important role in EC function. Immunogold-staining of HK1 in hearts from C57BL6 mice treated with high fat diet and LNAME, a mouse model of HFpEF, showed increased dislocation of HK1 from the mitochondria in ECs. To study the role of HK1 dislocation, we generated ΔE1HK1 mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag. Subcellular fractionation confirmed that HK1 was dislocated from mitochondria in these mice. Echocardiography showed that the mice developed impaired cardiac relaxation at 20 weeks of age and HFpEF at 40 weeks of age. Significant increased fibrosis and microvascular rarefaction (MR) were observed at 40 weeks of age, but only MR was observed at 20 weeks of age, suggesting EC dysfunction likely precedes and promotes the development of HFpEF in these mice. To study the angiogenic ability of ECs with HK1 dislocation, we isolated ECs from ΔE1HK1 hearts and performed a tubing assay, and observed significantly less tubing in ΔE1HK1 ECs. To elucidate the mechanism by which angiogenesis is reduced in ΔE1HK1 ECs, we next performed metabolomics analysis in these cells. Our data indicated that the levels of metabolites in hexosamine-biosynthetic pathway (HBP) were altered between wild-type (WT) and ΔE1HK1 EC. We next analyzed the levels of O-GlcNAcylation, and showed that ECs from ΔE1HK1 hearts have higher O-GlcNAcylation than those from WT. Finally, treatment with ST045849, an inhibitor of O-GlcNAc transferase (OGT), rescued the less angiogenic ability in ECs from ΔE1HK1 hearts. Conclusion: Our studies demonstrate that dislocation of HK1 plays an important role in the development of HFpEF through hyper-O-GlcNAcylation. Drugs that inhibit OGT may provide a therapeutic option for HFpEF.

Rationale : Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Ca v β subunit of the L-type Ca 2+ channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation–contraction (EC) coupling is unknown. Objective : This study aimed to investigate possible role of Rad in cardiac EC coupling and β-adrenergic receptor (βAR) inotropic mechanism. Methods and Results : Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current ( I Ca ), [Ca 2+ ] i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the “gain” function of EC coupling was significantly increased, due perhaps to reduced “redundancy” of LCC in triggering sarcoplasmic reticulum release. Conversely, ≈70% Rad knockdown by RNA interference increased I Ca (50%), [Ca 2+ ] i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on I Ca . Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na + /Ca 2+ exchange activity, sarcoplasmic reticulum Ca 2+ content, properties of Ca 2+ sparks and propensity for Ca 2+ waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated βAR effects on I Ca and Ca 2+ transients. Conclusion : These results establish Rad as a novel endogenous regulator of cardiac EC coupling and βAR signaling and support a parsimonious model in which Rad buffers Ca v β to modulate LCC activity, EC coupling, and βAR responsiveness.