Mitochondrial ROS (mtROS) production and TRPV4 translocation/activation in lung microvascular endothelial cells
Authors: Suresh Karthik , Servinsky Laura , Philip Nicolas , Hill Zack , Jiang Haiyang , Bigham Zahna , Zaldumbide Joel , Huetsch John , Damarla Mahendra , Shimoda Larissa 
Published: June 2021

RationaleIn pulmonary arterial hypertension (PAH), endothelial cells (ECs) exhibiting increased migration/proliferation are thought to contribute to formation of vaso‐occlusive lesions. Using microvascular endothelial cells (MVECs) isolated from rats after experimental (i.e. Sugen/Hypoxia) PAH (SuHx‐MVEC), we recently observed evidence of increased mitochondrial ROS (mtROS) leading to activation of the membrane calcium channel TRPV4, increased intracellular Ca2+ ([Ca2+]i) levels, and increased migration/proliferation. Interestingly, both quenching mtROS (with the targeted antioxidant MitoQ) or inhibiting TRPV4 similarly attenuated basal [Ca2+]i, migration and proliferation in SuHx‐MVECs, suggesting a link between mtROS and TRPV4 in SuHx‐MVECs. To better understand this relationship, we observed the effect of a) quenching mtROS in SuHx‐MVECs and b) stimulating mtROS production (using Antimycin A; AA) in MVECs isolated from normoxic rats (N‐MVECs) on TRPV4 membrane localization and activity.MethodsCa2+ was measured using Fura‐2AM loaded MVEC in a flow chamber perfused with Krebs buffer. Mitochondrial morphology was measured computationally using 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. Biotinylation of membrane proteins was performed using biotin‐coated magnetic beads.ResultsTreatment of N‐MVECs with AA increased mtROS levels, induced fragmentation and increased basal [Ca2+]‐i levels similar to untreated SuHx‐MVECs. Treatment with the TRPV4 inhibitor HC‐067047 (HC) attenuated the effect of AA on mitochondrial fragmentation and [Ca2+]i in N‐MVECs. In SuHx‐MVECs, membrane‐bound TRPV4 and TRPV4 activity (as measured by biotinylation and change in [Ca2+]i following treatment with the TRPV4 agonist GSK, respectively) were increased at baseline. TRPV4 response to GSK was attenuated in MitoQ‐treated SuHx‐MVECs. Conversely, GSK‐induced Ca2+ entry and biotinylated TRPV4 levels were increased in AA treated N‐MVECs compared to untreated N‐MVEC controls.ConclusionThese data suggest a link between mtROS production and TRPV4 localization/activity in MVECs. Increasing mtROS production in N‐MVECs increased TRPV4 activity while quenching basal mtROS elevations in SuHx‐MVECs attenuated TRPV4 activity.Support or Funding InformationSupport for this study was provided 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). Additional support includes a Gilead PAH Research Scholar Award and Parker B. Francis Research Opportunity Award (KS), as well as NIH S10OD016374 (S. Kuo, JHUSOM Microscope facility)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.