Introduction and aims Hypoxia triggers pulmonary artery contraction by mobilising calcium from two distinct compartments of the sarcoplasmic reticulum (SR), one sensitive to block by the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) and the other insensitive [1]. Furthermore, the AMPK-activated protein kinase (AMPK) mediates acute hypoxic pulmonary vasoconstriction in the mouse lung in-vivo [2]. Therefore, we examined the nature of AMPK‑induced initiates calcium signalling across the cell-wide web of pulmonary arterial myocytes, a recently discovered network of cytoplasmic nanocourses demarcated by SR nanojunctions [3].

Methods Isolation of pulmonary arterial myocytes and confocal imaging were 18‑22 °C; excitation 494 nm; emission 506 nm), ER-tracker (Thermo Fisher) and Draq5 (Thermo Fisher). Confocal images were acquired at 22 °C using a Nikon A1R+ confocal, Galvano scanner and 1.25 n.a. water immersion objective (Nikon).

Results and conclusions ER and Fluo-4 positive nnuclear envelope invaginations (NEIs) were thus identified in live cells. Blind and transnuclear NEIs projected deep into the nucleus and were tubular, ~200nm in diameter, and branched. Asynchronous calcium signals were evident at rest within cytoplasmic nanocourses demarcated by NEI and across the cell-wide web beyond the nucleus. Strikingly, when AMPK activators (MK8722 (0.1-1μM; Compound 13, 1-30μM; 991 10μM) [1] were applied extracellularly marked increases in Fluo-4 fluorescence were evoked in NEI subsequent to signal initiation proximal to the plasma membrane by a multi-stage process. For example, extracellular application of 1μM MK8722 reduced the fluorescence intensity of the cytoplasmic calcium indicator Fluo-4 (Phase 1) across all cytoplasmic nanocourses of pulmonary arterial myocytes (fluorescence change in F/F0 = -0.065±0.014; n=6 from n=5 mice). A transient rise (~60s) in fluorescence within peripheral aspects extraperinuclear nanocourses followed (peak change in F/F0 = 0.923±0.126; n=6 from n=5 mice) that propagated inward across the cell‑wide web and induced concomitant contraction (Phase 2). A secondary increase in fluorescence within perinuclear but not extraperinuclear nanocourses followed (peak change in F/F0 = 1.315±0.369; n=6 from n=5 mice) which maintained myocyte contraction (Phase 3). In 3 of 6 cells, a further, prolonged increase in Fluo-4 fluorescence occurred in perinuclear nanocourses only (Phase 4; peak change in F/F0 = 0.854±0.18; n=3 from n=3 mice). Intriguingly, MK98722 induced calcium flux into cytoplasmic nanocourses demarcated by NEI, and these signals were maintained between marked oscillations in calcium flux at proximal perinuclear nanocourses. Consistent with previous proposals [1-3], activation of AMPK, likely triggers contraction of pulmonary arterial myocytes by pre-loading of SR stores and then sequential mobilisation of peripheral SR, central SR and latterly NEI calcium stores, where calcium flux into cytoplasmic nanocourses demarcated by NEI remained elevated between phases of calcium release and contraction across the wider cell.