Heart failure with preserved ejection fraction (HFpEF) is a common cause of morbidity and mortality worldwide, but its pathophysiology remains unclear. Here, we report a mouse model of HFpEF and show that hexokinase (HK)-1 mitochondrial binding in endothelial cells (ECs) is critical for protein O-GlcNAcylation and the development of HFpEF. We demonstrate increased mitochondrial dislocation of HK1 within ECs in HFpEF mice. Mice with deletion of the mitochondrial-binding domain of HK1 spontaneously develop HFpEF and display impaired angiogenesis. Spatial proximity of dislocated HK1 and O-linked N-acetylglucosamine transferase (OGT) causes increased OGT activity, shifting the balance of the hexosamine biosynthetic pathway intermediates into the O-GlcNAcylation machinery. EC-specific overexpression of O-GlcNAcase and an OGT inhibitor reverse angiogenic defects and the HFpEF phenotype, highlighting the importance of protein O-GlcNAcylation in the development of HFpEF. Our study demonstrates a new mechanism for HFpEF through HK1 cellular localization and resultant protein O-GlcNAcylation, and provides a potential therapy for HFpEF.

The cellular source of positive signals that reinvigorate T cells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8+ T cell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3+ DCs and CD8+ T cells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3+ DCs within the TME is associated with CD8+ T cell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3+ DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process.

Toxic megacolon and pulmonary nodules are not seen frequently on diagnosis in pediatric ulcerative colitis patients. This report emphasizes the importance of carefully evaluating and managing complications in pediatric ulcerative colitis cases, especially in the presence of pulmonary nodules.

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this article, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in PLWH and HFpEF and assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-γ or TNF-α) impairs their Ca 2+ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca 2+ -decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca 2+ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions.

BACKGROUND/AIMS:To describe the role of ustekinumab in inducing remission and endoscopic healing in anti-tumor necrosis factor α nonresponsive pediatric ulcerative colitis patients at a tertiary care inflammatory bowel disease center. METHODS:A retrospective chart review was performed on patients with ulcerative colitis receiving ustekinumab. Primary outcome was steroidfree clinical remission at follow-up. Secondary outcomes were biochemical remission and endoscopic healing. RESULTS:Ten children were analyzed; 7 (70%) had ulcerative colitis, and 3 (30%) had inflammatory bowel disease unspecified with colitis. Median follow-up period was 56 weeks. Nine patients (90%) achieved steroid-free clinical remission and biochemical remission. Seven patients had follow-up colonoscopies, out of which 6 (86%) achieved endoscopic remission, while 1 (14%) underwent colectomy. Out of the 3 patients without a follow-up colonoscopy, fecal calprotectin levels downtrended to < 150 mg/kg in 2 patients and < 400 mg/kg in 1 patient from baseline level of > 2,000 mg/kg. CONCLUSIONS:Ustekinumab appears efficacious in achieving not only clinical and biochemical remission but also has promising role in inducing endoscopic healing end point in patients who fail other biologics.

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of heart failure (HF) and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 ( Sirt2 -/- ) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2 -/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitors reduces cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in the progression of HF and cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of this disorder.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me 2 at enhancers of a high-affinity leucine transporter, LAT3 , and RPTOR . By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

INTRODUCTION: The American Gastroenterological Association (AGA) has compiled risk factors that may be predictive of disease complications in Crohn's disease (CD) and ulcerative colitis (UC). The aim of this study was to evaluate the performance of the AGA risk factors for risk stratification in UC and CD. METHODS: We included participants of 2 cohorts: the Ocean State Crohn's and Colitis Area Registry cohort and the Mayo Clinic cohort. Baseline clinical risk factors were extracted according to the AGA pathway. Our primary end point was defined as follows: (i) any inflammatory bowel disease related–hospitalization, (ii) any inflammatory bowel disease–related bowel surgery, or (iii) any progression of disease. We analyzed the association of the number of AGA risk factors with our end point. Statistical multivariable modeling was performed with Cox proportional hazards model. RESULTS: A total of 412 patients with CD were included. Comparing ≥3 risk factors with 0–1 risk factor, we found a significantly increased risk of complications in both the Ocean State Crohn's and Colitis Area Registry cohort (hazard ratio [HR] 2.75, 95% confidence interval 1.71–4.41) and Mayo Clinic cohort (HR 2.07, 95% confidence interval 1.11–3.84). Diagnosis at younger age (HR 2.07), perianal disease (HR 1.99), and B2/B3 behavior (HR 1.92) were significantly associated with disease complications. We did not observe a consistent association between number of risk factors nor any specific individual risk factors and risk of disease complications in the 265 patients with UC included. DISCUSSION: We found a significant association between the number of AGA risk factors and the risk of disease complication in CD; this association was not significant in UC. The presence of ≥ 3 risk factors in CD leads to the highest risk of complications. The AGA care pathway is a useful tool to stratify patients who are at higher risk of disease complications in patients with CD.

The authors have requested that this preprint be removed from Research Square.

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria, and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of ischemic injury and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2-/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitor reduced cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in cardiac response to injury and the progression of cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy and injury by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of these disorders.

BACKGROUND: Proper nuclear organization is critical for cardiomyocyte function, because global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy; however, the mechanism for this process is not well delineated. AMPK (AMP-activated protein kinase) family of proteins regulates metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNRK (SNF1-related kinase), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. METHODS: We subjected cardiac-specific Snrk –/– mice to transaortic banding to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phosphoproteomics to identify novel proteins that are phosphorylated by SNRK. Last, coimmunoprecipitation was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. RESULTS: Cardiac-specific Snrk –/– mice display worse cardiac function and cardiac hypertrophy in response to transaortic banding, and an increase in DDR marker pH2AX (phospho-histone 2AX) in their hearts. In addition, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3-dimensional volume. Phosphoproteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor proteins that directly bind to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of G-actin to F-actin. Last, jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK-downregulated cells. CONCLUSIONS: These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper cardiomyocyte nuclear shape and morphology.

BACKGROUND & AIMS: We aimed to model infliximab (IFX) pharmacokinetics (PK) in pediatric acute severe ulcerative colitis (ASUC) and assess the association between PK parameters, including drug exposure, and clinical response.METHODS: We studied a multicenter prospective cohort of hospitalized children initiating IFX for ASUC or IBD-unclassified. Serial IFX serum concentrations over 26 weeks were used to develop a PK model. We tested the association of PK parameter estimates with day 7 clinical response, week 8 clinical remission, week 26 corticosteroid-free clinical remission (CSF-CR) (using the Pedi-atric Ulcerative Colitis Activity Index), and colectomy-free survival. RESULTS: Thirty-eight participants received IFX (median initial dose, 9.9 mg/kg). Day 7 clinical response, week 8 clinical remission, and week 26 CSF-CR occurred in 71%, 55%, and 43%, respectively. Albumin, C-reactive protein, white blood cell count, platelets, weight, and antibodies to IFX were significant covariates incorporated into a PK model. Week 26 non-remitters exhibited faster IFX clearance than remitters (P [ .013). However, cumulative IFX exposure did not differ between clinical response groups. One (2.7%) and 4 (10.8%) participants underwent colectomy by week 26 and 2 years, respectively. Day 3 IFX clearance >0.02 L/h was associated with colectomy (hazard ratio, 58.2; 95% confidence interval, 6.0-568.6; P < .001).CONCLUSIONS: At median higher-than-label IFX dosing for pediatric ASUC, baseline faster IFX CL was associ-ated with colectomy and at week 26 with lack of CSF-CR. IFX exposure was not predictive of clinical outcomes. Higher IFX dosing may sufficiently optimize early outcomes in pediatric ASUC. Larger studies are warranted to determine whether sustained intensification can over-come rapid clearance and improve later outcomes. ClinicalTrials.gov identifier: NCT02799615.

Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon. Citation Format: Robin Reschke, Jason W Shapiro, Jovian Yu, Sherin J Rouhani, Daniel J Olson, Yuanyuan Zha, Thomas F Gajewski. Checkpoint blockade–induced dermatitis and colitis are dominated by tissue-resident memory T cells and Th1/Tc1 cytokines [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr IA20.

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.

Abstract Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.

Introduction: Endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF), however, the mechanism is unclear. ECs are highly dependent on glycolysis; however, it is not known how their metabolic profile is altered in the setting of HFpEF. Hexokinase 1 (HK1) carries the first step in glycolysis and is mostly bound to the mitochondria in ECs. Objective: We sought to elucidate the role of EC glucose metabolism, and specifically HK1, in the development of HFpEF. Results: We first showed that among glycolytic enzymes, HK1 is highly specific to ECs, suggesting the significance of HK1 in EC physiology. Although HK1 mRNA and protein levels are not changed, we noted increased dislocation of HK1 from the mitochondria in ECs from HFpEF mice. To study the role of HK1 dislocation in the development of HFpEF, we generated mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag (ΔE1HK1). ΔE1HK1mice develop impaired cardiac relaxation at 20 weeks of age and HFpEF by 40 weeks, and ECs from these mice displayed impaired angiogenic potential. Since HK1 is involved in glucose metabolism, we then performed metabolomic studies and demonstrated that intermediates in hexosamine biosynthetic pathway (HBP) are significantly decreased in ECs from ΔE1HK1 mice. To assess the mechanism for the decrease in HBP, we assessed protein modification, and noted reduced protein N-glycosylation and increased O-GlcNAcylation in ECs from ΔE1HK1. We also demonstrated that inhibition of the key enzyme in O-GlcNAcylation (OGT) reverses the EC dysfunction noted in ΔE1HK1 mice, indicating that O-GlcNAcylation is responsible for the angiogenic defect in ECs from ΔE1HK1. Finally, to provide a mechanism for altered protein glycosylation with HK1 mitochondrial dislocation, we showed that HK1 associates with N-glycosylation machinery when it is attached to mitochondria, while it binds to OGT when it is dislocated from mitochondria. Conclusion: Our studies demonstrate a role for HK1 mitochondrial binding and protein O-GlcNAcylation in the pathogenesis of HFpEF, and that HK1 cellular localization plays a major role in the fate of HBP intermediates into either N-glycosylation or O-GlcNAcylation machinery in ECs.

Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocyte hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved postpartum cardiac function. Collectively, these data provide what we believe to be a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.

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.

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.

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, ferroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.