PT - JOURNAL ARTICLE AU - Sasha Z. Prisco AU - Megan Eklund AU - Thenappan Thenappan AU - Kurt W. Prins TI - With No Lysine Kinase 1 Promotes Right Ventricular Dysfunction Via Glucotoxicity AID - 10.1101/2021.06.22.449476 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.06.22.449476 4099 - http://biorxiv.org/content/early/2021/06/22/2021.06.22.449476.short 4100 - http://biorxiv.org/content/early/2021/06/22/2021.06.22.449476.full AB - Objectives Investigate how WNK1 inhibition modulates glucotoxicity, mitochondrial/peroxisomal protein regulation and metabolism, and right ventricular (RV) function in pulmonary arterial hypertension (PAH). Determine how hypochloremia impacts RV function in PAH patients.Background In PAH-induced RV failure, GLUT1/GLUT4 expression is elevated, which increases glucose uptake and glycolytic flux to compensate for mitochondrial dysfunction. However, the resultant consequences of the glucose-mediated post-translational modifications (PTM), protein O-GlcNAcylation/glycation in RV failure are understudied. WNK1, a chloride-sensitive kinase, increases GLUT1/GLUT4 expression in skeletal muscle, but its regulation in RV dysfunction is unexplored.Methods Rats were treated with WNK463 (small molecule WNK inhibitor) or vehicle starting two weeks after monocrotaline injection. Immunoblots quantified protein abundance/PTMs. Mitochondrial/peroxisomal proteomics and global metabolomics evaluated glucose metabolism and mitochondrial/peroxisomal function. Pulmonary vascular and cardiac histology, echocardiography, and pressure-volume loop analysis quantified RV function and PAH severity. Finally, the relationship between hypochloremia, a WNK1-activating state, and RV function was evaluated in 217 PAH patients.Results WNK463 decreased WNK1/GLUT1/GLUT4 expression, normalized glucose metabolite levels, which dampened excess protein O-GlcNAcylation/glycation. Integration of RV mitochondrial/peroxisomal proteomics and metabolomics identified fatty acid oxidation (FAO) as the most dysregulated metabolic pathway. WNK463 enhanced FAO as demonstrated by increased expression of mitochondrial FAO proteins and normalization of RV acylcarnitines. WNK463 reduced glutaminolysis induction and lipotoxicity, two secondary consequences of diminished FAO. WNK463 augmented RV systolic and diastolic function independent of pulmonary vascular disease severity. In PAH patients, hypochloremia resulted in more severe RV dysfunction.Conclusions WNK463 combated RV glucotoxicity and impaired FAO, which directly improved RV function.HighlightsSmall molecule inhibition of WNK1 (WNK463) signaling mitigates upregulation of the membrane glucose channels GLUT1 and GLUT4, restores levels of several glucose metabolites, and normalizes protein O-GlcNAcylation and glycation in the RV.Quantitative proteomics of RV mitochondrial enrichments shows WNK463 treatment prevents downregulation of mitochondrial enzymes in the tricarboxylic acid cycle, fatty acid oxidation pathway, and the electron transport chain complexes.Integration of proteomics and metabolomics analysis reveals WNK463 reduces glutaminolysis induction and lipotoxicity due to impaired fatty acid oxidationWNK463 augments RV systolic and diastolic function independent of PAH severity.Hypochloremia, a condition of predicted WNK1 activation, in PAH patients results in more severe RV dysfunction.Competing Interest StatementKWP served on an advisory board for Actelion and Edwards and receives grant funding from United Therapeutics. TT served on an advisory board for Actelion, United Therapeutics, Altavant Sciences, and Aria CV. TT receives research funding for clinical trials from United Therapeutics, Aria CV, Gossimer Bio, and Acceleron. The other authors have declared that no conflict of interest exists.Aco2Aconitase 2AS160160 kDa substrate of the Akt serine/threonine kinaseATPAdenosine triphosphateDCADicarboxylic fatty acidDJ-1Protein deglycaseEaEffective arterial elastanceEesEnd-systolic elastanceETCElectron transport chainFAOFatty acid oxidationGFAT1Glutamine-fructose-6-phosphate transaminase 1GLO1Glyoxalase 1GLO2Glyoxalase 2GLSGlutaminaseGLUT1Glucose transporter 1GLUT4Glucose transporter 4LVLeft ventricle/ventricularMCTMonocrotalineMCT VMonocrotaline-vehicleME2Malic enzyme 2OGAO-GlcNAcaseOGTO-linked β-N-acetylglucosamine transferasePAATPulmonary artery acceleration timePAHPulmonary arterial hypertensionPPARγPeroxisome proliferator-activated receptor gammaPTMPost-translationally modify/modificationsPVPressure-volumePVRPulmonary vascular resistanceRARight atrialRVRight ventricle/ventricularRVEDPRight ventricular end-diastolic pressureRV-PARight ventricular-pulmonary arteryRVDRight ventricular dysfunctionRVSPRight ventricular systolic pressureSdhaSuccinate dehydrogenase complex flavoprotein subunit ASdhbSuccinate dehydrogenase complex iron sulfur subunit BTAPSETricuspid annular plane systolic excursionTau/τRight ventricular relaxation timeTCATricarboxylic acidTNFαTumor necrosis factor-αUDP-GlcNACUridine diphosphate N-acetylglucosamineWNKWith No Lysine kinase