Glyoxylase-1 combats dicarbonyl stress and right ventricular dysfunction in rodent pulmonary arterial hypertension.

Background: Heightened 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. Methods: Bioinformatics 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. Results: In 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. Conclusion: Excess protein glycation promotes RV dysfunction in preclinical PAH, potentially through suppression of FAO.

With No Lysine Kinase 1 Promotes Metabolic Derangements and RV Dysfunction in Pulmonary Arterial Hypertension.

Small molecule inhibition of with no lysine kinase 1 (WNK1) (WNK463) signaling activates adenosine monophosphate-activated protein kinase signaling and mitigates membrane enrichment of glucose transporters 1 and 4, which decreases protein O-GlcNAcylation and glycation. Quantitative proteomics of right ventricular (RV) mitochondrial enrichments shows WNK463 prevents down-regulation of several mitochondrial metabolic enzymes. and metabolomics analysis suggests multiple metabolic processes are corrected. Physiologically, WNK463 augments RV systolic and diastolic function independent of pulmonary arterial hypertension severity. Hypochloremia, a condition of predicted WNK1 activation in patients with pulmonary arterial hypertension, is associated with more severe RV dysfunction. These results suggest WNK1 may be a druggable target to combat metabolic dysregulation and may improve RV function and survival in pulmonary arterial hypertension.

Intermittent Fasting Enhances Right Ventricular Function in Preclinical Pulmonary Arterial Hypertension.

Background Intermittent fasting (IF) confers pleiotropic cardiovascular benefits including restructuring of the gut microbiome and augmentation of cellular metabolism. Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by right ventricular (RV) mitochondrial dysfunction and resultant lipotoxicity and microbiome dysbiosis. However, the effects of IF on RV function in PAH are unexplored. Therefore, we investigated how IF altered gut microbiota composition, RV function, and survival in the monocrotaline model of PAH. Methods and Results Male Sprague Dawley rats were randomly allocated into 3 groups: control, monocrotaline-ad libitum feeding, and monocrotaline-IF (every other day feeding). Echocardiography and invasive hemodynamics showed IF improved RV systolic and diastolic function despite no significant change in PAH severity. IF prevented premature mortality (30% mortality rate in monocrotaline-ad libitum versus 0% in monocrotaline-IF rats, P=0.04). IF decreased RV cardiomyocyte hypertrophy and reduced RV fibrosis. IF prevented RV lipid accrual on Oil Red O staining and ceramide accumulation as determined by metabolomics. IF mitigated the reduction in jejunum villi length and goblet cell abundance when compared with monocrotaline-ad libitum. The 16S ribosomal RNA gene sequencing demonstrated IF changed the gut microbiome. In particular, there was increased abundance of Lactobacillus in monocrotaline-IF rats. Metabolomics profiling revealed IF decreased RV levels of microbiome metabolites including bile acids, aromatic amino acid metabolites, and gamma-glutamylated amino acids. Conclusions IF directly enhanced RV function and restructured the gut microbiome. These results suggest IF may be a non-pharmacological approach to combat RV dysfunction, a currently untreatable and lethal consequence of PAH.

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

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.