Metabolic regulation of mitochondrial reactive oxygen species (mtROS) production and transient receptor potential vanilloid‐4 (TRPV4) activation in microvascular endothelial cells in PAH

Abstract

Rationale In pulmonary arterial hypertension (PAH), highly proliferative microvascular endothelial cells (MVECs) are thought to contribute to vaso-occlusive lesion formation and increased pulmonary artery pressures. Using MVECs isolated from the Sugen/Hypoxia (SuHx) model of PAH, we recently showed that increased mtROS production activates the TRPV4 calcium channel and contributes to increased MVEC proliferation in vitro. However, the mechanistic basis for sustained mtROS production in MVECs isolated from SuHx rats (SuHx-MVECs) was unclear. Along with increased mtROS production, we observed evidence of significant mitochondrial dysfunction, including increased fragmentation and glycolytic shift. Since mtROS production can be increased by changes in the type of fuel (e.g. fatty acids)used to generate TCA metabolites, we hypothesized that use of non-glucose sources (specifically, increased fatty acid oxidation - FAO) could be the source of mitochondrial dysfunction and mtROS production in SuHx-MVECs. Methods Intracellular Ca2+ concentration ([Ca2+]i) was measured using Fura-2AM loaded MVEC in a flow chamber perfused with modified Krebs solution. Mitochondrial morphology was measured computationally using validated algorithms applied to confocal images of mitochondria in N- and SuHx-MVEC stained with MitoTracker. Lipid droplets were measured using BODIPY staining. Targeted metabolomics were performed on MVEC cell lysates using a standardized LC/MS approach as described previously (Suresh et al., AJP-Lung 2019). Human PAH metabolomics and genomics data were obtained from NIH Metabolomics Workbench or GEO Omnibus, respectively, and analyzed using R. Results Despite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites, measured using targeted metabolomics, were not significantly lower in SuHx-MVECs compared to normoxic (N-MVEC) controls, suggesting presence of anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks, while intracellular lipid droplet number was increased in SuHx-MVECs. Analysis of human PAH metabolomics and genomics data revealed increased serum FFA levels and increased expression of enzymes involved in breaking down long chain fatty acids for use in the TCA cycle (i.e. FAO). In SuHx-MVECs, untargeted metabolomics and metabolite-specific assays revealed increased levels of beta-hydroxybutyrate (BOHB), a ketone body produced by FAO. In N-MVECs, BOHB supplementation was sufficient to increase TRPV4 sensitivity to pharmacologic agonists. In contrast, inhibition of FAO (with Etoximir) in SuHx-MVECs attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as TRPV4 inhibition or mtROS quenching. Conclusion We conclude that intermediary signaling molecules such as BOHB and mtROS, produced as a consequence of increased FAO, act in concert to sensitize and activate the TRPV4 channel in lung MVECs. These data suggest an exciting role for metabolism as a regulator of TRPV4 channel activity in PAH.