Increased fatty acid oxidation promotes mitochondrial ROS (mtROS) production and Ca2+ entry via TRPV4 in microvascular endothelial cells in PAH

Abstract

RationaleIn 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 transient receptor potential vanilloid‐4 (TRPV4) 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 regulated by shifts in TCA fuel substrates, we hypothesized that generation of TCA metabolites using non‐glucose sources (i.e. anaplerosis) could be the source of mitochondrial dysfunction and mtROS production in SuHx‐MVECs.MethodsCa2+ was measured using Fura‐2AM loaded MVEC in a flow chamber perfused with Krebs buffer. Mitochondrial morphology was measured computationally using previously validated algorithms applied to confocal images of mitochondria in N‐ and SuHx‐MVEC treated with MitoTracker. ROS measurements were made using the ratiometric ROS sensor roGFP. Lipid droplets were assessed using BODIPY staining in live MVECs, and quantified using CellProfiler. Targeted metabolomics were conducted using a standardized LC/MS approach and MS‐grade reagent standards.ResultsDespite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites (measuring using targeted metabolomics) were not significantly lower in SuHx‐MVECs compared to normoxic (N‐MVEC) controls, suggesting presence of significant anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks. Intracellular lipid droplet number was increased in SuHx‐MVECs, while droplet size was decreased, suggesting increased FA utilization. Untargeted metabolomics performed in N‐MVEC and SuHx‐MVEC cell extracts revealed evidence of metabolite shunting towards nucleotide and amino acid synthesis as well as increased levels of fatty acid oxidation byproducts. In cultured SuHx‐MVECs, inhibition of mitochondrial fatty acid import (with Etoximir) attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as mtROS quenching and TRPV4 inhibition.ConclusionIncreased utilization of fatty acid oxidation for anaplerotic carbons is associated with mitochondrial fragmentation and mtROS production, which in turn increases [Ca2+]I via a TRPV4‐dependent mechanism. These data suggest a novel link between EC metabolism, mitochondrial dysfunction and TRPV4 activation in the SuHx model of PAH.Support or Funding InformationThis work was supported by by the National Heart, Lung and Blood Institute Grants F32HL124930 and K08HL132055 (KS), R01HL073859, R25HL084762 and R01HL126514 (LAS), F32HL124727, and K08HL133475 (JH) and T32HL007534 (KS and JH).