Research ArticlePulmonary Arterial Hypertension

Inhibition of pyruvate dehydrogenase kinase improves pulmonary arterial hypertension in genetically susceptible patients

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Science Translational Medicine  25 Oct 2017:
Vol. 9, Issue 413, eaao4583
DOI: 10.1126/scitranslmed.aao4583
  • Fig. 1. PDK is important in the metabolic remodeling of PAH and is up-regulated in human PAH lungs.

    (A) Schematic demonstrating how PDK and SIRT3 and UCP2 suppress mitochondrial function by independent mechanisms (inhibition of PDH and glucose oxidation, global increase in mitochondrial acetylation, and global decrease in mitochondrial calcium, respectively). This mitochondrial suppression inhibits apoptosis and promotes proliferation, leading to the proliferative vascular remodeling characteristic of PAH. DCA is a selective PDK inhibitor and does not affect other mechanisms of PDH inhibition. DCA may inhibit the feedback loop that links PDK-driven mitochondrial suppression and secondary normoxic activation of HIF1α to HIF-induced PDK induction. ETC, electron transport chain; Ca++mito, intramitochondrial calcium; acetyl-CoA, acetyl–coenzyme A. (B) Confocal immunohistochemistry of a PAH and a non-PAH control lung with parallel staining for PDK1, PDK2, smooth muscle actin (SMA), and 4ʹ,6-diamidino-2-phenylindole (DAPI, a nuclear marker). Yellow boxes on differential interference contrast (DIC) images focus on small resistance PAs, shown at higher magnification (right). An example of the stain only with secondary antibody is shown at the bottom. See Table 1 for patient information. (C) Immunoblots of peripheral lung tissues from PAH and control (non-PAH) lungs (for patient information, see Table 1) probed for p-PDH-E1α, PDH-E1α, PDK1, and PDK2. Quantification of the PDK1 or PDK2 over actin and p-PDH-E1α/PDH-E1α (normalized to actin) in each gel is shown. *P < 0.05, Mann-Whitney U test. ATP, adenosine triphosphate.

  • Fig. 2. DCA activates PDH and increases respiration in human PAH EVLP.

    (A) Photograph and schematic of the custom-made EVLP system with a human lung (see Materials and Methods). ICU, intensive care unit. (B to C) PDH activity measured from lung biopsies before and after 1 hour of DCA perfusion (0.7 mg/ml). (B) PDH activity dipstick assay, measuring NADH production from the PDH reaction (see box on top), quantitation [AU (arbitrary units)], and SNP scores. (C) Immunoblots for p-PDH-E1α and quantitation [(p-PDH-E1α/actin)/(total PDH-E1α/actin)]. Lungs 1, 2, and 3 showed an increase in PDH activity by DCA, whereas lung 4 was resistant to DCA (for patient information, see Table 1). The control lung 5 (see Table 1) was perfused with vehicle (veh.) (perfusate) for the same time as lungs 1 to 4. (D) Mitochondrial respiration (oxygen consumption rate) measured using a Seahorse protocol from pre- and post-perfusion biopsies. Top: An example of oxygen consumption rate (slope of each line) of a pre- and post-DCA perfusion biopsy from lung 3. Bottom: The % change in oxygen consumption rate after DCA for all lungs. Perfusion of lung 5 with vehicle (perfusate) was a time control experiment.

  • Fig. 3. The effects of DCA on hemodynamic and functional end points and their association with genetic factors (variants of the SIRT3 and UCP2 genes) that cause resistance to DCA.

    (A) mPAP, PVR, and 6-min walk performance of DCA-treated patients. Statistical significance was assessed using before-after paired t test (P < 0.05). The means ± SEM before and after DCA treatment and the P values are shown in each graph. (B) Scatter plots of the DCA-induced changes in mPAP, PVR, and 6-min walk, separated by the SNP score status of each patient. Statistical significance was assessed using a Spearman’s correlation test and rS, and P values are shown in each graph. Highlighted region identifies seven patients who exhibited a significant decrease in the mPAP (>5 mmHg decrease) associated with an increase or no change in the cardiac output, suggestive of a clinically meaningful response. Plots of the DCA-induced change in mPAP, PVR, and 6-min walk (means ± SEM) over the SNP score are shown on the right.

  • Fig. 4. Trough serum DCA concentrations.

    Top: DCA concentration (means ± SEM) in patients exposed to 6.25 mg/kg b.i.d. compared to 3 mg/kg b.i.d. Bottom: DCA concentration (means ± SEM) in responder patients (decrease in mPAP by more than 5 mmHg) compared to nonresponder patients.

  • Fig. 5. Examples of functional imaging biomarkers in two DCA responders.

    (A) MR images showing gadolinium transit time before and after DCA and resolution of the D-shaped septum (arrow) in the heart of patient 3. Note the color scale of the gadolinium transit time: Blue indicates short transit time and increased perfusion; yellow/red indicates long transit time and little/no perfusion at baseline. (B) PA pressure recordings and 18FDG uptake pre- and post-DCA from right heart catheterization and 18FDG-PET-CT, respectively, of patient 10. A shift to the left of the distribution of the 18FDG uptake suggests a decrease in glucose uptake due to decreased glycolysis. Color heat maps show Patlak slope per gram of tissue (score) per voxel in lung sections, indicating the density of high-value voxels (colored in yellow and red, indicating areas of high glucose uptake), overlaid on corresponding CT images.

  • Table 1. Characteristics of patients offering lung tissues.

    C, control tissue; P, patient tissue; aPAH, associated PAH (collagen vascular disease); CTEPH, chronic thromboembolic pulmonary hypertension; PDE5i, phosphodiesterase type 5 inhibitor; ERA, endothelin receptor antagonist; Prostn, parenteral prostanoid; sGC, soluble guanylate cyclase; WHO, World Health Organization.

    PatientDiagnosisAgeSexWHO classPAH therapy
    Histology/immunoblotsC1Lobectomy (tumor)56MIINone
    C2Unused transplant donor25MINone
    C3Unused transplant donor38FINone
    C4Unused transplant donor41FINone
    P1aPAH34FIIIPDE5i + ERA
    P2iPAH18MIVPDE5i + Prostn
    P3iPAH39FIVERA + Prostn
    P4iPAH31FIIIERA + Prostn
    P5iPAH42FIIIPDE5i + ERA + Prostn
    P6aPAH40MIVPDE5i + ERA + ProstnSIRT3 variantUCP2 variant
    EVLP lung1aPAH62FIVPDE5i + ERA + ProstnG/GA/A
    2aPAH61FIVPDE5i + ERA + ProstnG/AA/A
    3iPAH32FIVPDE5i + ERA + ProstnG/GA/A
    4iPAH43FIVPDE5i + ERA + ProstnG/AG/G
    5CTEPH60MIVsGC stim + ProstnG/AA/A
  • Table 2. Baseline characteristics of enrolled patients.

    Pt, patient; RAP, right atrial pressure; PAWP, PA wedge pressure; CO, cardiac output; Tx, therapy; P, phosphodiesterase type 5 inhibitor; E, endothelin receptor antagonist; Pros, parenteral prostanoid. All patients diagnosed with iPAH. Patients 17 to 20 were withdrawn from the protocol due to a grade II peripheral neuropathy, which was eventually improved (rows shaded in gray). Patients 1 to 16 completed the protocol and are all included in the Fig. 3 data analysis. The “risk variant” for each of the two alleles of the SIRT3 and UCP2 genes (the variant associated with decreased SIRT3 activity and UCP2 expression) is shown in bold. The number of risk variants in bold between the two genes corresponds to the SNP score of each patient.

    PtAgeSexRAP
    (mmHg)
    mPAP
    (mmHg)
    PAWP
    (mmHg)
    CO
    (liter/min)
    6-min
    walk
    WHO
    class
    PAH
    Tx
    SIRT3
    variant
    UCP2
    variant
    SNP
    score
    DCA (mg/kg
    b.i.d.)
    167F54368.9298IIIPG/GG/A13
    254F35053.4450IIP + EG/GG/A13
    343F205595.6366IIIP + EG/GA/A03
    460F462113.5409IIP+EG/GA/A06.25
    527F85674.3533IIP + EA/AA/A26.25
    663F633106.1385IIIP + EG/GG/G26.25
    754F746114.7407IIP + EG/AA/A16.25
    875M1348136.7262IIIPG/GA/A06.25
    973M845146223IIIPG/GA/A03
    1058F1164144.8242IIIP + EG/GA/A03
    1141F634124392IIPG/AG/A23
    1264F646104393IIIP + EG/AA/A13
    1324M95095.3660IIP + EG/AA/A16.25
    1437M64884.9399IIP + EA/AG/A36.25
    1535F1672152.2465IIPG/GG/A16.25
    1662F83584.9525IIIP + EG/GG/A16.25*
    1726F105587.9465IIIPG/GA/A012.5
    1838F1046133418IIIP + EG/GG/A112.5
    1941F76883538IIP + EG/GG/A112.5
    2021M1440114412IIP + E + ProsG/AA/A112.5

    *Dose decreased from 12.5 mg/kg.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/413/eaao4583/DC1

      Fig. S1. Confocal immunohistochemistry of six PAH lungs and three non-PAH control lungs.

      Fig. S2. Relationship between risk variants (SNP scores) and decrease in mPAP in patients treated with DCA.

      Fig. S3. Relationship between the combined SNP score of both SIRT3 and UCP2 risk variants and the change in mPAP in patients treated with DCA.

      Fig. S4. The effects of DCA on RV size and function.

      Fig. S5. The effects of DCA on lung perfusion (MRI) and glucose uptake (18FDG-PET-CT).

      Table S1. Gene variants score (SNPs) for DCA resistance.

    • Supplementary Material for:

      Inhibition of pyruvate dehydrogenase kinase improves pulmonary arterial hypertension in genetically susceptible patients

      Evangelos D. Michelakis,* Vikram Gurtu, Linda Webster, Gareth Barnes, Geoffrey Watson, Luke Howard, John Cupitt, Ian Paterson, Richard B. Thompson, Kelvin Chow, Declan P. O'Regan, Lan Zhao, John Wharton, David G. Kiely, Adam Kinnaird, Aristeidis E. Boukouris, Chris White, Jayan Nagendran, Darren H. Freed, Stephen J. Wort, J. Simon R. Gibbs, Martin R. Wilkins*

      *Corresponding author. Email: em2{at}ualberta.ca (E.D.M.); m.wilkins{at}imperial.ac.uk (M.R.W.)

      Published 25 October 2017, Sci. Transl. Med. 9, eaao4583 (2017)
      DOI: 10.1126/scitranslmed.aao4583

      This PDF file includes:

      • Fig. S1. Confocal immunohistochemistry of six PAH lungs and three non-PAH control lungs.
      • Fig. S2. Relationship between risk variants (SNP scores) and decrease in mPAP in patients treated with DCA.
      • Fig. S3. Relationship between the combined SNP score of both SIRT3 and UCP2 risk variants and the change in mPAP in patients treated with DCA.
      • Fig. S4. The effects of DCA on RV size and function.
      • Fig. S5. The effects of DCA on lung perfusion (MRI) and glucose uptake (18FDG-PET-CT).
      • Table S1. Gene variants score (SNPs) for DCA resistance.

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