Mitochondrial ROS production in lung microvascular endothelial cells sensitizes the transient receptor potential vanilloid‐4 (TRPV4) channel to physical and chemical stimuli.

Rationale Increased proliferation of lung microvascular endothelial cells (MVECs) is a known feature of pulmonary arterial hypertension (PAH). We have previously shown that increased mitochondrial reactive oxygen species (mtROS) and basal intracellular calcium ([Ca 2+ ] i ) are associated with increased proliferation and migration in MVECs isolated from animals undergoing experimental PAH (Sugen Hypoxia model; SuHx‐MVECs) compared to MVECs isolated from normoxic controls (N‐MVECs). In these studies, we observed that quenching mtROS in SuHx‐MVECs attenuated TRPV4 activity, basal [Ca 2+ ] i , migration and proliferation in SuHx‐MVECs. However, whether induction of mtROS production alone, in N‐MVECs, was sufficient to activate TRPV4 was unclear. Methods To measure [Ca 2+ ] i in MVECs, cells were incubated on glass coverslips with the calcium‐sensing dye FURA‐2 AM and transferred to a temperature‐controlled flow chamber set at baseline to 37 degrees and flow rate of 0.1 dynes/cm 2 . Cells were perfused with freshly‐prepared Krebs buffer. TRPV4 inhibition was achieved with HC‐067047 (HC) treatment. mtROS production was induced using treatment with antimycin A (AA) or Mito‐Paraquat (MPQ). TRPV4 was activated using either the chemical agonist GSK1016790A (GSK) or by increasing chamber and perfusate temperature to 40 degrees (since heat is a known activator of this channel). Results Treatment of N‐MVECs with antimycin A (AA) increased basal [Ca 2+ ] i . GSK‐induced Ca 2+ influx was also significantly higher in AA‐treated N‐MVECs. Both increased basal Ca 2+ and increased GSK‐induced Ca 2+ influx were attenuated with HC pre‐treatment (i.e. TRPV4 inhibition). Using temperature as a TRPV4 stimulus, we observed greater heat‐induced Ca 2+ in AA‐treated N‐MVECs. Since AA may have off‐target effects, we repeated our GSK‐induced Ca 2+ experiments following treatment with MitoPQ, a redox cycler also known to increase mtROS. Similarly to AA, Mito‐PQ treatment increased basal [Ca 2+ ] i and GSK‐induced calcium influx. Conclusion These data suggest that, in N‐MVECs, mitochondrial ROS elevate basal intracellular calcium levels and both activate TRPV4 and sensitize the channel to subsequent activation by either chemical or physical stimuli. Support or Funding Information This 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).