Mitochondrial control of calcium signaling in native endothelium
Authors: Wilson Calum , Lee Matthew , McCarron John G 
Published: November 2023

Endothelial cells have a dense mitochondrial complement. However, ATP production in endothelial cells is largely independent of mitochondrial oxidative pathways. As a result, the regulation of endothelial function by mitochondria has, in contrast to smooth muscle cells, been neglected and the precise role of the organelle is unresolved. We recently provided the first direct evidence that polarized mitochondria are required for flow‐mediated calcium waves in the endothelium of intact arteries [1]. Flow‐evoked endothelial calcium waves originate in distinct subcellular locations, propagate within and among endothelial cells, and ultimately promote vascular smooth muscle cell relaxation via the generation of nitric oxide. Thus, in intact arteries, calcium regulation enables endothelial mitochondria to critically regulate vascular tone. Here, we extended these initial studies in endothelial cells by examining: (1) the structure and distribution of mitochondria; (2) the contribution of the organelle both to local and stimulus‐evoked calcium signaling; and (3) the relationship between calcium signals, mitochondria and myoendothelial gap junctions.Mitochondria and IEL holes were visualized in large fields of endothelia (~40 cells) of intact rat second‐order mesenteric arteries using the membrane potential sensitive fluorescent probe, tetramethylrhodamine ethyl ester (TMRE), and IEL autofluorescence, respectively. In these same fields of native endothelium, basal and stimulus‐evoked calcium signaling was examined using the fluorescent calcium indicator, Cal‐520. Endothelial mitochondria were morphologically heterogeneous throughout the cytosol of individual endothelial cells and were observed as small spheres, globules, rods, as well as looped and branched rods. Although mitochondrial distribution was denser in the perinuclear region, mitochondrial networking was rarely observed. Fenestrations in the IEL were widespread (~150 per field‐of‐view; 0.7 – 35 um2). Because of their extensive distribution, mitochondria and IEL holes were found to overlap. However, there was neither an apparent increased nor decreased density of the organelles at IELs. Low‐amplitude, spontaneous (local) calcium signals occurred preferentially at IEL holes. Larger abortive and propagating calcium waves also occurred under basal conditions. However, there was no apparent correlation between IEL hole location and the initiation sites of abortive/propagating calcium waves. Interestingly, mitochondrial uncoupling using the proton uncoupler, CCCP, applied together with the H+‐ATP‐synthase blocker, oligomycin, abolished endothelial calcium signals occurring under basal conditions, those evoked by fluid flow, and those evoked by the application of exogenous ACh. Together, these results suggest that mitochondria are punctate organelles that are widely distributed throughout the cytoplasm of native endothelial cells. Mitochondrial activity maintains calcium release from internal stores and exerts significant control over various endothelial calcium signaling modalities.Support or Funding InformationThis work was supported by the Wellcome Trust and the British Heart Foundation. Their support is gratefully acknowledged.