Effect of Distinct Induced Calcium Entry Pathways on Mitochondrial Respiration in Pulmonary Microvascular Endothelial Cells

Abstract

Pulmonary microvascular endothelial cells (PMVECs) are distributed in a unique vascular bed exposed to the entire cardiac output and thus are influenced by circulating molecules that regulate calcium influx. Despite recognition of mitochondria's ability to buffer calcium, disparate results assessing the role of mitochondrial calcium and oxidative respiration are reported. The impact of distinct calcium entry pathways on mitochondrial respiration in PMVEC is unknown. We hypothesize that calcium entry pathways originating in spatially distinct cellular regions lead to unique changes in PMVEC mitochondrial respiration. Calcium entry dynamic patterns were assessed using an Andor spinning disk confocal microscope in confluent PMVEC monolayers loaded with the cytosolic calcium sensor CAL520. Thapsigargin (1 μM), an alkaloid that inhibits the sarcoendoplasmic reticulum calcium ATPase and elicits store‐depletion‐induced calcium entry, resulted in a rapid, global increase in cytosolic calcium. In contrast, 4α‐phorbol 12,13‐didecanoate (4αPDD, 5 μM), an agonist of the transient receptor potential vanilloid 4 channel (TRPV4), resulted in a slower increase in cytosolic calcium when compared to thapsigargin and elicited repetitive, short‐lived dynamic calcium events that spread into local cellular microdomains. In parallel studies, mitochondrial respiration was assessed by measuring oxygen consumption rate (OCR) using the Seahorse XF24 analyzer. 40,000 PMVECs were seeded per well 24 hours prior to running the assay. Experiments followed a standardized sequential protocol to measure different mitochondrial states. At t=21 min, after obtaining three basal measurements, cells were exposed to substrates for mitochondrial complex I (pyruvate 1 mM) and complex II (succinate 1 mM), then either thapsigargin (1 μM, n=20) or 4αPDD (5 μM, n=20) were added to activate distinct calcium entry pathways. At t=38 min, the complex V inhibitor oligomycin (1 μM) was added. At t=55 min, the mitochondrial respiration uncoupler FCCP (1 μM) was added. At t=73 min, the complex I inhibitor rotenone (1 μM) and the complex III inhibitor antimycin A (1 μM) were added. Control groups (n=20) were exposed to a similar protocol with addition of vehicle instead of thapsigargin or 4αPDD. In the control group, under basal conditions OCR measured 195 pmol/min. Addition of oligomycin decreased OCR to 68 pmol/min, while addition of uncoupler FCCP induced maximal respiration of 389 pmol/min. Addition of rotenone and antimycin A ablated mitochondrial respiration as expected. In controls, the respiratory ratio, defined as the ratio of basal to oligomycin OCR, was 3.05 ± 0.08 (mean ± SEM). Addition of thapsigargin significantly decreased the respiratory ratio (2.27 ± 0.01), while in contrast, the ratio increased after exposure to 4αPDD (8.02 ± 0.89) (p<0.001, one‐way ANOVA). We conclude that the cytosolic calcium dynamics elicited by activation of store‐operated channels or TRPV4 in PMVECs differentially impact mitochondrial oxidative respiration.Support or Funding InformationNIH PO1 HL066299/Project 3 to M.I.T., NIH R01 HL118334 to D.F.A., and NIH S10OD02149 to USA microscopy core 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.