Research ArticleLung Disease

Hypercapnia increases airway smooth muscle contractility via caspase-7–mediated miR-133a–RhoA signaling

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Science Translational Medicine  05 Sep 2018:
Vol. 10, Issue 457, eaat1662
DOI: 10.1126/scitranslmed.aat1662
  • Fig. 1 High CO2 increases mouse ASM cell contractility via miR-133a–mediated RhoA signaling.

    (A) ACh-induced cell contraction in mouse ASM cells exposed to different conditions. Left: Representative images from 7-day exposure conditions. Scale bars, 50 μm. Right: Time course quantification of ACh-induced cell contraction (n = 10 cells). (B) Representative Western blot (top) and quantification (bottom) of RhoA in ASM cells exposed to 5% (Ctrl) or 20% CO2 for the indicated times (Ctrl, n = 10; 20% CO2, n = 5 per group). (C) Representative Western blot (top) and quantification (bottom) of phosphorylation of Mlc in ASM cells exposed to Ctrl or 20% CO2 for 2 days in the presence or absence of 1 μM Y-27632 (n = 3). (D) Representative images (top) and quantification (bottom) of ACh-induced cell contraction in ASM cells exposed to Ctrl or 20% CO2 for 2 days in the presence or absence of 1 μM Y-27632 (n = 10 cells). Scale bars, 50 μm (top). (E) Time course quantification of miR-133a expression in ASM cells exposed to Ctrl or 20%CO2 (n = 6). (F) Representative Western blot (top) and quantification (bottom) of RhoA in ASM cells transfected with mimic miR-133a and exposed to Ctrl or 20% CO2 for 2 days (n = 7). (G) Representative Western blot (top) and quantification (bottom) of RhoA in ASM cells transfected with antagomiR-133a and exposed to Ctrl for 2 days (n = 6). (H) Representative images (top) and quantification (bottom) of ACh-induced cell contraction in ASM cells with lentiviral overexpression of control miR (Ctrl miR) or miR-133 exposed to Ctrl or 20% CO2 for 2 days (n = 10 cells). Scale bars, 50 μm (top). All data are expressed as means ± SEM. Statistical analysis was performed by two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (A), one-way ANOVA with Dunnett’s post hoc test (B), Tukey’s post hoc test (C, D, F, and H), or one-sample t test with Bonferroni adjustment (E). For (A), (B), and (E), all comparisons were made with the control group of “5% CO2 pHe7.4” (A) or “Ctrl” (B and E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 High CO2 increases ACh-induced cell contraction via miR-133a–RhoA axis signaling in human ASM cells.

    All experiments were performed in human ASM (hASM) cells exposed to 5% (Ctrl) or 20% CO2 for 2 days. (A) Representative images (top) and quantification (bottom) of ACh-induced cell contraction (n = 8 cells). Scale bars, 50 μm (top). (B) Representative images (top) and quantification (bottom) of ACh-induced cell contraction in human ASM cells treated with or without 1 μM Y-27632 (n = 8 cells). Scale bars, 50 μm (top). (C) Representative images (top) and quantification (bottom) of ACh-induced cell contraction in human ASM cells with lentiviral overexpression of control miR (Ctrl miR) or miR-133 (n = 8 cells). Scale bars, 50 μm (top). All data are expressed as median with interquartile range. Statistical analysis was performed by Kruskal-Wallis with Dunn’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 High CO2 induces Caspase-7–dependent cleavage of Mef2D and miR-133a down-regulation.

    (A) Image of computational prediction analysis of transcription factor binding sites of miR-133a. Primer positions for chromatin immunoprecipitation (ChIP) assay in the promotor region of miR-133a are indicated as Primer #1 or Primer #2. (B) Expression of miR-133a (top) and representative Western blot (bottom) of Yy1 and Mef2D in mouse ASM cells transfected with siRNAs of scrambled (si-Scr; n = 5), Yy1(si-Yy1; n = 4), or Mef2d (si-Mef2d; n = 5) and exposed to 5% CO2 (Ctrl) for 2 days. (C) Representative Western blot (top) and quantification (bottom) of RhoA in ASM cells transfected with si-Mef2d and exposed to Ctrl for 2 days (n = 4). (D) Immunocytochemical images of ASM cells exposed to Ctrl or 20% CO2 for 2 days showing localization of Mef2D (green; middle), nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue; top], and overlay (bottom; n > 50 cells). Scale bars, 20 μm. (E) Representative Western blot (top) and quantification (bottom) of Mef2D in nuclear fractions from ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 4). Arrowheads indicate cleaved Mef2D. (F) Representative images (top) and quantification (bottom) of ChIP assay for Mef2D binding to miR-133a promoter in ASM cells exposed to Ctrl or 20% CO2 for 2 days using two different primer sets (n = 3). IgG, immunoglobulin G. (G) Representative Western blots (left) and quantification (right) of procaspase-7 in whole-cell lysates (n = 4) and cleaved Caspase-7 in nuclear fractions (n = 4) from ASM cells exposed to Ctrl or 20% CO2 for 2 days. (H) Representative Western blot (left) and quantification (right) of cleaved Mef2D in nuclear fractions (top) and procaspase-7 in whole-cell lysates (bottom) from ASM cells transfected with si-Scr or Caspase-7 siRNA (si-Casp7) and exposed to Ctrl or 20% CO2 for 2 days (n = 3). Arrowheads indicate cleaved Mef2D. (I) Expression of miR-133a in ASM cells transfected with si-Scr or si-Casp7 and exposed to Ctrl or 20% CO2 for 2 days (n = 4). (J) Potential Caspase-7 cleavage sites of Mef2D (top) and representative Western blot of cleaved Mef2D (bottom) in nuclear fractions from ASM cells infected with lentiviral wild-type (WT) or mutant Mef2D and exposed to Ctrl or 20% CO2 for 2 days (n = 3). Flag was used as a marker of lentiviral infection. Arrowheads indicate cleaved MEF2D. (K and L) Quantification of cleaved Mef2D (K; n = 3) and expression of miR-133a (L; n = 4) in ASM cells infected with lentiviral WT or mutant Mef2D (D478A) and exposed to Ctrl or 20% CO2 for 2 days. Data are expressed as means ± SEM (B to F and H to L) or median with interquartile range (G). Statistical analysis was performed by one-sample t test with Bonferroni adjustment (B), Mann-Whitney U test (G), or one-way ANOVA with Dunnett’s post hoc test (H to L). For (B), all comparisons were made with the control group of “si-Scr”. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., nonsignificant.

  • Fig. 4 High CO2 induces CASPASE-7–dependent cleavage of MEF2D in human ASM cells.

    Representative Western blot (top) and quantification (bottom) of cleaved CASPASE-7 (n = 4) and MEF2D (n = 4) in nuclear fraction (A) and procaspase-7 (n = 3) in whole-cell lysate (B) in human ASM cells. All data are expressed as median with interquartile range. Statistical analysis was performed by Mann-Whitney U test. *P < 0.05.

  • Fig. 5 High CO2 activates calcium-calpain signaling, leading to Caspase-7 activation.

    (A) Time course of calpain activity in mouse ASM cells exposed to 5% (Ctrl) or 20% CO2 (Ctrl, n = 30; 20% CO2, n = 6 per group). (B) Representative Western blot (top) and quantification (bottom) of Calpain-1 and Calpain-2 in ASM cells exposed to Ctrl or 20% CO2 for 6 hours (Ctrl, n = 8; 20% CO2, n = 4 per group). (C) Representative Western blot (top) and quantification (bottom) of cleaved Caspase-7 in ASM cells transfected with si-Scr, Calpain-1 (si-Capn1), or Calpain-2 (si-Capn2) and exposed to Ctrl or 20% CO2 for 2 days (Ctrl si-Scr, n = 8; others, n = 4 per group). (D) Intracellular Ca2+ in ASM cells exposed to Ctrl or 20% CO2 for 6 hours (n = 7 cells). (E) Representative Ca2+ oscillation induced by ACh in ASM cells exposed to Ctrl or 20% CO2 for 6 hours (n = 7 cells). (F) Calpain activity in ASM cells treated with or without 1 μM BAPTA-AM and exposed to Ctrl or 20% CO2 for 6 hours (n = 6). All data are expressed as means ± SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc test (A and B) or with Tukey’s post hoc test (C and F). For (A) and (B), all comparisons were made with the control group. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Chronic hypercapnia induces airway contractility, which is prevented in Caspase-7–null mice.

    C57BL/6J (WT) and B6.129S6-Casp7tm1Flv/J (Casp7−/−) mice were exposed to 21% O2 and 10% CO2 [hypercapnia (HC)] or maintained in room air [normocapnia (NC)] for up to 21 days. Some Casp7−/− mice were infected by intratracheal administration of the lentivirus particles for miR inhibitor–scrambled control (Lv_ctrl miR) or antagomiR-133a (Lv_antagomiR) before HC exposure. (A) Representative images (top) and quantification (bottom) of ACh-induced airway contraction in PCLSs (n = 8 airways from four mice). Scale bars, 100 μm (top). (B) Total resistance of the respiratory system (Rrs) at baseline (top) and after methacholine challenge (bottom) measured on a flexiVent instrument (n = 6 mice). (C) Expression of miR-133a in left main bronchial rings (n = 6 mice). (D) Representative Western blot (left) and quantification (right) of RhoA and Mlc phosphorylation in whole lungs (n = 4 mice). (E) Intracellular Ca2+ in Casp7−/− ASM cells exposed to 5% (Ctrl) or 20% CO2 for 6 hours (n = 6 cells). (F) Calpain activity in Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 6 hours (n = 3). (G) Representative Western blot of Mef2D in nuclear fractions from Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 5). (H) Expression of miR-133a in Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 5). (I) Representative Western blot (left) and quantification (right) of RhoA and Mlc phosphorylation in Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 5). (J) Representative images (top) and quantification (bottom) of ACh-induced cell contraction in Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 10 cells). Scale bars, 50 μm (top). (K) Dose-dependent curve of ACh-induced cell contraction in WT and Casp7−/− ASM cells exposed to Ctrl or 20% CO2 for 2 days (n = 10 cells). Data at 1 μM ACh condition in Casp7−/− ASM cells are from (J). All data are expressed as means ± SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc test [A, B (top), and D (right)], one-sample t test (H), with Bonferroni adjustment (C), or two-way ANOVA with Dunnett’s post hoc test [B (bottom) and K]. All comparisons were made with the group of “WT NC” (A to D) and “WT ASM cells Ctrl” (K). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 Airway/respiratory resistance is increased in hypercapnic patients with COPD.

    (A) Comparison of sRtot measured by plethysmographic assessment between normocapnic (n = 13) and hypercapnic (n = 17) patients with chronic stable COPD in the cohort study #1. (B) Comparison of respiratory resistance measured by IOS between normocapnic (n = 13) and hypercapnic (n = 19) patients with chronic stable COPD in the cohort study #2. Values of R5, R20, and R5-R20 indicate total, proximal, and peripheral respiratory resistance, respectively. (C) Changes of respiratory resistance in hypercapnic patients from the cohort study #2 (n = 6). Data are expressed as median with interquartile range (A and B). Statistical analysis was performed by Mann-Whitney U test (A and B) or paired t test (C).

  • Table 1 Characteristics of the study population.

    Data are presented as means (range). Two-tailed unpaired t test except where stated otherwise. PaO2, partial pressure of O2 in arterial blood; PaCO2, partial pressure of CO2 in arterial blood; FVC, force vital capacity; FEV1, forced expiratory volume in 1 s; TLC, total lung capacity.

    Cohort study #1: Measurement of airway resistance
    COPD patients
    NormocapniaHypercapniaP value
    No. of subjects1317
    Men/women8/53/140.013*
    Age, year66 (50–83)67 (58–77)0.726
    Body surface
    area, m2
    25 (17–31)23 (18–27)0.226
    Blood gas
    results
    pH7.42 (7.33–7.45)7.41 (7.38–7.49)0.702
    PaO2, mmHg68 (58–76)62 (49–81)0.039
    PaCO2, mmHg41 (36–44)49 (45–55)<0.0001
    Pulmonary
    function
    results
    FVC, % predicted63 (46–78)59 (41–86)0.459
    FEV1, %
    predicted
    30 (16–37)25 (13–36)0.094
    FEV1/FVC35 (21–48)33 (22–45)0.467
    TLC, % predicted116 (76–156)116 (96–145)0.938
    Cohort study #2: Measurement of respiratory resistance
    COPD patients
    NormocapniaHypercapniaP value
    No. of subjects1319
    Men/women10/311/80.266*
    Age, year71 (57–81)73 (47–91)0.436
    Body surface
    area, m2
    22 (17–29)21 (14–33)0.735
    Blood gas
    results
    pH7.44 (7.41–7.49)7.40 (7.32–7.47)0.002
    PaO2, mmHg79 (59–113)77 (55–103)0.644
    PaCO2, mmHg39 (32–43)54 (45–88)<0.0001
    Pulmonary
    function
    results
    FVC, % predicted71.4 (41.8–95.4)61.4 (35.3–84.1)0.106
    FEV1, %
    predicted
    42.1 (22.8–65.5)36.0 (13.7–72.5)0.323
    FEV1/FVC51.1 (20.3–82.2)47.9 (23.0–87.8)0.713

    2 test.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/457/eaat1662/DC1

      Materials and Methods

      Fig. S1. Normoxic hypercapnia alters gene expression in mouse lung.

      Fig. S2. Quantification of Mef2D-positive cell in mouse ASM cells.

      Fig. S3. High CO2 causes caspase-dependent cleavage of Mef2D protein, resulting in down-regulation of miR-133a, but does not induce apoptosis in mouse ASM cells.

      Fig. S4. Acute hypercapnia causes ASM relaxation due to hypercapnia-associated acidosis.

      Fig. S5. Central respiratory resistance in WT and Caspase-7–null mice.

      Fig. S6. RhoA protein abundance and Mlc phosphorylation in mouse primary alveolar type II cells.

      Fig. S7. Quantification of cleaved Mef2D in ASM cells from Caspase-7–null mice.

      Fig. S8. Schematic of nonapoptotic role of Caspase-7 in ASM contractility during hypercapnia.

      Fig. S9. Same signaling pathways activated in mouse ASM cells when exposed to similar pCO2 values to the COPD patients.

      Table S1. Processed data from mRNA and miR microarray analysis of lungs isolated from C57BL/6J mice exposed to normoxic hypercapnia or room air for 3 or 7 days (Excel file).

      Table S2. Primary data (Excel file).

      References (42, 43)

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Normoxic hypercapnia alters gene expression in mouse lung.
      • Fig. S2. Quantification of Mef2D-positive cell in mouse ASM cells.
      • Fig. S3. High CO2 causes caspase-dependent cleavage of Mef2D protein, resulting in down-regulation of miR-133a, but does not induce apoptosis in mouse ASM cells.
      • Fig. S4. Acute hypercapnia causes ASM relaxation due to hypercapnia-associated acidosis.
      • Fig. S5. Central respiratory resistance in WT and Caspase-7–null mice.
      • Fig. S6. RhoA protein abundance and Mlc phosphorylation in mouse primary alveolar type II cells.
      • Fig. S7. Quantification of cleaved Mef2D in ASM cells from Caspase-7–null mice.
      • Fig. S8. Schematic of nonapoptotic role of Caspase-7 in ASM contractility during hypercapnia.
      • Fig. S9. Same signaling pathways activated in mouse ASM cells when exposed to similar pCO2 values to the COPD patients.
      • Legends for tables S1 and S2
      • References (42, 43)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S1. Processed data from mRNA and miR microarray analysis of lungs isolated from C57BL/6J mice exposed to normoxic hypercapnia or room air for 3 or 7 days (Excel file).
      • Table S2. Primary data (Excel file).

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