RESUMO
Background: In PAH metabolic abnormalities in multiple pathways are well-recognized features of right ventricular dysfunction, however, prior work has focused mainly on the use of a single "omic" modality to describe a single deranged pathway. We integrated metabolomic and epigenomic data using transcriptomics in failing and non-failing RVs from a rodent model to provide novel mechanistic insight and translated these findings to accessible human specimens by correlation with plasma from PAH patients. Methods: Study was conducted in a doxycycline-inducible BMPR2 mutant mouse model of RV failure. Plasma was collected from controls and PAH patients. Transcriptomic and metabolomic analyses were done on mouse RV tissue and human plasma. For mouse RV, we layered metabolomic and transcriptomic data for multiple metabolic pathways and compared our findings with metabolomic and transcriptomic data obtained for human plasma. We confirmed our key findings in cultured cardiomyocyte cells with BMPR2 mutation. Results: In failing mouse RVs, (1) in the glycolysis pathway, glucose is converted to lactate via aerobic glycolysis, but may also be utilized for glycogen, fatty acid, and nucleic acid synthesis, (2) in the fatty acid pathway, FAs are accumulated in the cytoplasm because the transfer of FAs to mitochondria is reduced, however, the ß-oxidation pathway is likely to be functional. (3) the TCA cycle is altered at multiple checkpoints and accumulates citrate, and the glutaminolysis pathway is not activated. In PAH patients, plasma metabolic and transcriptomic data indicated that unlike in the failing BMPR2 mutant RV, expression of genes and metabolites measured for the glycolysis pathway, FA pathway, TCA cycle, and glutaminolysis pathway were increased. Lactate was the only metabolite that was increased both in RV and circulation. We confirmed using a stable isotope of lactate that cultured cardiomyocytes with mutant BMPR2 show a modest increase in endogenous lactate, suggesting a possibility of an increase in lactate production by cardiomyocytes in failing BMPR2 mutant RV. Conclusion: In the failing RV with mutant BMPR2, lactate is produced by RV cardiomyocytes and may be secreted out, thereby increasing lactate in circulation. Lactate can potentially serve as a marker of RV dysfunction in PAH, which warrants investigation.
RESUMO
Insulin resistance and right ventricular (RV) dysfunction are associated with lipotoxicity in heritable forms of pulmonary arterial hypertension (PAH), commonly due to mutations in bone morphogenetic protein receptor type 2 (BMPR2). How BMPR2 dysfunction in cardiomyocytes alters glucose metabolism and the response of these cells to insulin are unknown. We hypothesized that BMPR2 mutation in cardiomyocytes alters glucose-supported mitochondrial respiration and impairs cellular responses to insulin, including glucose and lipid uptake. We performed metabolic assays, immunofluorescence and Western analysis, RNA profiling, and radioactive isotope uptake studies in H9c2 cardiomyocyte cell lines with and without patient-derived BMPR2 mutations (mutant cells), with and without insulin. Unlike control cells, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity as indicated by reduced mitochondrial respiration with increased mitochondrial superoxide production. These mutant cells show enhanced baseline phosphorylation of insulin-signaling protein as indicated by increased Akt, AMPK, and acetyl-CoA carboxylase phosphorylation that may negatively influence fatty acid oxidation and enhance lipid uptake, and are insulin insensitive. Furthermore, mutant cells demonstrate an increase in milk fat globule-EGF factor-8 protein (MFGE8), which influences the insulin-signaling pathway by phosphorylating AktSer473 via phosphatidylinositol 3-kinase and mammalian target of rapamycin. In conclusion, BMPR2 mutant cardiomyocytes have reduced metabolic plasticity and fail to respond to glucose. These cells have enhanced baseline insulin-signaling pattern favoring insulin resistance with failure to augment this pattern in response to insulin. BMPR2 mutation possibly blunts glucose uptake and enhances lipid uptake in these cardiomyocytes. The MFGE8-driven signaling pathway may suggest a new mechanism underlying RV lipotoxicity in PAH.