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Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

ABSTRACT: The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-N-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

SUBMITTER: Prisco SZ

PROVIDER: S-EPMC7582480 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

Prisco Sasha Z SZ Rose Lauren L Potus Francois F Tian Lian L Wu Danchen D Hartweck Lynn L Al-Qazazi Ruaa R Neuber-Hess Monica M Eklund Megan M Hsu Steven S Thenappan Thenappan T Archer Stephen L SL Prins Kurt W KW

International journal of molecular sciences 20201001 19

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate-<i>N</i>-acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the rel ...[more]

PMID: 33019763

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Project description:BackgroundRight ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca(2+) sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH.Methods and resultsRV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to protein-kinase-A (PKA), Cα (PKCα) and Ca(2+)/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca(2+) sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca(2+)-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca(2+) clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05).ConclusionsIncreased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca(2+) handling proteins contribute to RV diastolic dysfunction in PAH.

Project description:BackgroundThe cardioprotective properties of sevoflurane have been reported in studies of the left ventricle. However, whether this volatile anesthetic would also be beneficial for pulmonary vascular remodeling and associated right ventricular hypertrophy (RVH) remained to be explored. Here, we investigated the potential benefit of sevoflurane to right heart function in experimental pulmonary arterial hypertension (PAH).MethodsAdult Wistar rats received one dose peritoneal injection of monocrotaline (MCT, 60 mg/kg) or the equal volume of normal saline. Two weeks later, rats were treated with sevoflurane or sham exposure. PAH status and cardiac function were assessed by echocardiography weekly, and the body weight (BW) was monitored every week. After 6 weeks of exercise, Fulton's index calculation, histological observation, IL-6 and TNF-α immunohistochemical analyses, evaluation of MDA, SOD and GSH-Px levels and NF-κB and MAPK active determination were performed in lung and RV tissue samples.ResultsMCT induced pulmonary vascular remodeling, RVH, increased Fulton's index (P<0.01), and right ventricular failure (RVF) in rats. Animals inhaled sevoflurane had an increased cardiac output (P<0.05) and lower incidence of RVF (P<0.05). Also, these animals had a reduced RVEDD, RVWTd and PAID (P<0.05), increased PV (P<0.05), reduced wall thickness and vascular wall area of pulmonary small vascular (vascular external diameter 50-150 um) (P<0.01), reduced RV fibrosis, and increased RV cardiomyocyte area (P<0.01). Furthermore, sevoflurane reduced IL-6 and TNF-α expression in lungs and heart (P<0.01), decreased level of MDA (P<0.01) and increased activity of SOD and GSH-Px (P<0.01). In addition, it decreased the activities of NF-κB and MAPK pathways (P<0.01).ConclusionSevoflurane reduces pulmonary vascular remodeling and RVH in PAH induced by MCT in rats. This effect is likely due to down-regulation of inflammatory factors IL-6 and TNF-α, reduced level of oxidative stress and the inhibition of NF-κB and MAPK pathways.

Project description:BackgroundHeightened glycolytic flux is associated with right ventricular (RV) dysfunction in pulmonary arterial hypertension (PAH). Methylglyoxal, a glycolysis byproduct, is a highly reactive dicarbonyl that has toxic effects via non-enzymatic post-translational modifications (protein glycation). Methylglyoxal is degraded by the glyoxylase system, which includes the rate-limiting enzyme glyoxylase-1 (GLO1), to combat dicarbonyl stress. However, the potential consequences of excess protein glycation on RV function are unknown.MethodsBioinformatics analysis of previously identified glycated proteins predicted how protein glycation regulated cardiac biology. Methylglyoxal treatment of H9c2 cardiomyocytes evaluated the consequences of excess protein glycation on mitochondrial respiration. The effects of adeno-associated virus serotype 9-mediated (AAV9) GLO1 expression on RV function in monocrotaline rats were quantified with echocardiography and hemodynamic studies. Immunoblots and immunofluorescence were implemented to probe the effects of AAV-Glo1 on total protein glycation and fatty acid oxidation (FAO) and fatty acid binding protein levels.ResultsIn silico analyses highlighted multiple mitochondrial metabolic pathways may be affected by protein glycation. Exogenous methylglyoxal minimally altered mitochondrial respiration when cells metabolized glucose, however methylglyoxal depressed FAO. AAV9-Glo1 increased RV cardiomyocyte GLO1 expression, reduced total protein glycation, partially restored mitochondrial density, and decreased lipid accumulation. In addition, AAV9-Glo1 increased RV levels of FABP4, a fatty acid binding protein, and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunits alpha and beta (HADHA and HADHB), the two subunits of the mitochondrial trifunctional protein for FAO. Finally, AAV9-Glo1 blunted RV fibrosis and improved RV systolic and diastolic function.ConclusionExcess protein glycation promotes RV dysfunction in preclinical PAH, potentially through suppression of FAO.

Dataset Information

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

Publications

Excess Protein O-GlcNAcylation Links Metabolic Derangements to Right Ventricular Dysfunction in Pulmonary Arterial Hypertension.

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