Research ArticlePhysiology

The LKB1–AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria

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Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaau0296
DOI: 10.1126/scisignal.aau0296
  • Fig. 1 The LKB1-AMPK but not the CaMKK2-AMPK signaling pathway is required for HPV.

    (A to C) Top panels show spectral Doppler analyses of peak velocity versus time for the systolic waveform within the main pulmonary artery of (A) AMPK-α1 and AMPK-α2 floxed controls (AMPK-α1/α2 FLX), (B) AMPK-α1 knockout, and (C) AMPK-α2 knockout mice during normoxia, hypoxia (12, 8, or 5% O2), and recovery. Bottom panels show example records of peak velocity during normoxia, hypoxia, and recovery. (D) Bar chart shows the means ± SEM for the maximum change in peak velocity observed during 5% O2 for AMPK-α1/α2 FLX, C57Bl6, CaMKK2 KO, hypomorphic LKB1 floxed (LKB1 KD), AMPK-α1 KO, and AMPK-α2 KO mice; n = 3 to 7 mice per group, **P < 0.01, ***P < 0.001. (E to G) Top panels show spectral Doppler analyses of peak velocity versus time for the systolic waveform within the main pulmonary artery of (E) AMPK-α1/α2 FLX controls, (F) AMPK-α1 knockout, and (G) AMPK-α2 knockout mice during intravenous injection of 5-HT (serotonin). (H) Bar chart shows the means ± SEM for the maximum change in peak velocity observed for AMPK-α1/α2 FLX, AMPK-α1 KO, and AMPK-α2 KO mice; n = 3 to 5 mice per group.

  • Fig. 2 Deletion of AMPK-α1 or AMPK-α2 does not affect mitochondrial membrane potential.

    (A) Images show a bright-field image of a pulmonary arterial myocyte, deconvolved three-dimensional reconstructions of a Z-stack of images of MitoTracker fluorescence, tetramethylrhodamine ethyl ester (TMRE) fluorescence, and a merged image from AMPK-α1/α2 FLX, AMPK-α1, and AMPK-α2 knockout mice. (B) Bar chart shows the means ± SEM for TMRE fluorescence (n = 23 to 34 cells from 4 mice per group). (C to E) Records of rhodamine 123 (Rhod123) fluorescence ratio (F/F0) plotted against time, recorded from pulmonary arterial myocytes of (C) AMPK-α1/α2 FLX, (D) AMPK-α1, and (E) AMPK-α2 knockout mice during normoxia and hypoxia. The nonquenching mode was confirmed by adding the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 10 μM). (F) Bar charts show the means ± SEM for the maximum change in Rhod123 fluorescence during application of hypoxia (~6% O2) and oligomycin (3 μM). n = 3 to 6 cells from 3 mice per group. AU, arbitrary units.

  • Fig. 3 Deletion of AMPK-α1 but not AMPK-α2 prevents the inhibition of KV1.5 currents by hypoxia and mitochondrial poisons.

    (A to C) Top panels show the voltage steps. Bottom panels show raw records of KV currents activated at each voltage step during normoxia and hypoxia. (D to F) Plots (means ± SEM) for the current-voltage (I/Imax) relationship of steady-state KV currents recorded (average from the last 20 ms of voltage step) during normoxia and hypoxia (~6% O2) in acutely isolated pulmonary arterial myocytes from (D) AMPK-α1/-α2 floxed (control), (E) AMPK-α1 smooth muscle knockout (AMPK-α1 KO), or (F) AMPK-α2 smooth muscle knockout (AMPK-α2 KO) mice. (G) Bar charts show the means ± SEM for the peak inhibition in KV current at 0 mV (I/Imax) for pulmonary arterial myocytes from each genotype during hypoxia and after extracellular application of the mitochondrial poison oligomycin (3 μM), the AMPK-α1–selective agonist C13 (30 μM), and the selective KV1.5 blocker DPO-1 (1 μM); n = 4 to 9 cells from 3 mice per group; *P < 0.05, **P < 0.01.

  • Fig. 4 AMPK inhibits recombinant KV1.5 by direct phosphorylation of two AMPK recognition motifs incorporating Ser559 and Ser592.

    (A) Alignment of recognition motif for AMPK with sequences of KV1.5. Phosphorylation sites are marked in pink; β, basic residue; ϕ, hydrophobic residue. Schematic indicates position of residues within the KV1.5 α subunit. Coomassie blue stain (B) and autoradiogram (C) show the effect of S559A (n = 3 independent experiments), S592A (n = 3 independent experiments), and S559A/S592A (n = 2 independent experiments) mutations on KV1.5 phosphorylation in cell-free assays by AMPK in the presence of AMP. (D) Fold change in phosphorylation (mutant/wild type) of WT KV1.5 and KV1.5 mutants incorporating S559A, S592A, and S559A/S592A. **P < 0.01, ***P < 0.001. (E) Example records show the effects of C13 on whole-cell K+ currents measured by voltage ramp and voltage step protocols, during extracellular application of 30 μM C13 in HEK293 cells expressing WT KV1.5 and the KV1.5 S559A/S592A mutant. (F) Time course for the reductions in whole-cell K+ currents carried by WT and S559A/S592A mutant KV1.5 channels during extracellular application of 30 μM C13. (G) Bar chart shows the means ± SEM for reductions in steady-state K+ currents (average from last 20 ms of voltage step) carried by WT and mutant KV1.5 channels 5 min after extracellular application of 30 μM C13; n = 5 to 7 cells per group; *P < 0.05, **P < 0.01, ***P < 0.001. DMSO, dimethyl sulfoxide.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/550/eaau0296/DC1

    Fig. S1. End point reverse transcription PCR confirms AMPK deletion.

    Fig. S2. AMPK-α1 deletion blocks HPV during moderate and severe hypoxia.

    Fig. S3. Effect of deletion of LKB1, AMPK-α1, or AMPK-α2 on peak velocity and VTI.

    Fig. S4. HPV requires LKB1 but not CaMKK2.

    Fig. S5. Deletion of AMPK-α1 but not AMPK-α2 blocks KV1.5 current inhibition by hypoxia.

    Fig. S6. Deletion of AMPK-α1 blocks KV1.5 current inhibition by hypoxia.

    Fig. S7. Deletion of AMPK-α1 but not AMPK-α2 blocks KV1.5 current inhibition by hypoxia.

    Table S1. Basal cardiopulmonary hemodynamics.

    Table S2. Electrophysiological characteristics of KV currents.

  • This PDF file includes:

    • Fig. S1. End point reverse transcription PCR confirms AMPK deletion.
    • Fig. S2. AMPK-α1 deletion blocks HPV during moderate and severe hypoxia.
    • Fig. S3. Effect of deletion of LKB1, AMPK-α1, or AMPK-α2 on peak velocity and VTI.
    • Fig. S4. HPV requires LKB1 but not CaMKK2.
    • Fig. S5. Deletion of AMPK-α1 but not AMPK-α2 blocks KV1.5 current inhibition by hypoxia.
    • Fig. S6. Deletion of AMPK-α1 blocks KV1.5 current inhibition by hypoxia.
    • Fig. S7. Deletion of AMPK-α1 but not AMPK-α2 blocks KV1.5 current inhibition by hypoxia.
    • Table S1. Basal cardiopulmonary hemodynamics.
    • Table S2. Electrophysiological characteristics of KV currents.

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