Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma

Abstract

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by different patterns of airway remodeling, which all include an increased mass of bronchial smooth muscle (BSM). A remaining major question concerns the mechanisms underlying such a remodeling of BSM. Because mitochondria play a major role in both cell proliferation and apoptosis, we hypothesized that mitochondrial activation in BSM could play a role in this remodeling. We describe that both the mitochondrial mass and oxygen consumption were higher in the BSM from asthmatic subjects than in that from both COPD and controls. This feature, which is specific to asthma, was related to an enhanced mitochondrial biogenesis through up-regulation of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, nuclear respiratory factor-1, and mitochondrial transcription factor A. The priming event of such activation was an alteration in BSM calcium homeostasis. BSM cell apoptosis was not different in the three groups of subjects. Asthmatic BSM was, however, characterized by increased cell growth and proliferation. Both characteristics were completely abrogated in mitochondria-deficient asthmatic BSM cells. Conversely, in both COPD and control BSM cells, induction of mitochondrial biogenesis reproduced these characteristics. Thus, BSM in asthmatic patients is characterized by an altered calcium homeostasis that increases mitochondrial biogenesis, which, in turn, enhances cell proliferation, leading to airway remodeling.

Figures

Figure 1.

BSM remodeling in both asthma and COPD. Representative optic microscopic images from bronchial sections stained with HES were obtained from an asthmatic (A), a COPD (B), or a control subject (C) and observed at 200× magnification. Smooth muscles were visualized (SM). Bars, 50 μm. (D) The normalized smooth muscle area was assessed from microscopic images. Bronchial specimens were obtained from asthmatic (black column; n = 10), COPD (gray column; n = 7), and control subjects (white column; n = 6). Data are the mean ± the SEM. *, P < 0.05 between populations using ANOVA with the use of Bonferroni's test.

Figure 2.

Increased mitochondrial mass and activity in asthmatic BSM. Representative electronic microscopic images from bronchial sections were obtained from an asthmatic (A), a COPD (B), or a control subject (C) and observed at 26,000× magnification. Some smooth muscle mitochondria were visualized (arrows). Bars, 0.2 μm. The number (D) and the density (E) of mitochondria were assessed from electronic microscopic images (n = 4 for each population). Mitochondrial mass was assessed by the porin content using Western blot (F; n = 8 for asthmatics, n = 5 for COPD, and n = 7 for controls). Endogenous cellular oxygen consumption was evaluated by oxygraphy (G; n = 5 for asthmatics, n = 4 for COPD, and n = 4 for controls). BSM cells (BSMC) were obtained from asthmatic (black columns), COPD (gray columns), and control subjects (white columns). Data are the mean ± the SEM. *, P < 0.05 between populations using ANOVA with the use of Bonferroni's test.

Figure 3.

Increased mitochondrial biogenesis in asthmatic BSM. Representative confocal images of the mitochondrial network after three dimensional reconstruction were obtained from asthmatic (A), COPD (B), or control (C) BSM cells. Bars, 10 μm. mtTFA, NRF-1, and PGC-1α levels were assessed by both Western blot (D) and quantitative RT-PCR (E). BSM cells were obtained from asthmatic (black columns; n = 6), COPD (gray columns; n = 7), and control subjects (white columns; n = 6). Data are the mean ± the SEM. *, P < 0.05 between populations using ANOVA with the use of Bonferroni's test.

Figure 4.

Altered cell calcium homeostasis in asthmatic BSM. (A) Phosphorylated CaMK-IV (P-CaMK-IV) levels were assessed by Western blot. Representative intracellular calcium responses after stimulation for 30 s by 10−5 M acetylcholine (ACh) are presented in BSM cells from asthmatic (B), COPD (C), or control subjects (D). As a reference, response from the control cell (D) is presented as a gray line (B and C). The area under the curve was assessed from the calcium response (E). BSM cells were analyzed in the absence (-) or presence (+) of 2 mM extracellular Ca2 + or 1 μM methoxyverapamil (D600). Cells were obtained from asthmatic (black columns; n = 5), COPD (gray columns; n = 4), and control subjects (white columns; n = 4). Data are the mean ± the SEM. *, P < 0.05 between populations within an experimental condition using ANOVA with the use of Bonferroni's test. †, P < 0.05 between experimental conditions versus 2 mM Ca2+ without D600 within a population using paired Student's t tests.

Figure 5.

Effect of methoxyverapamil (D600) on mitochondrial biogenesis and content. PGC-1α (A), NRF-1 (B), mtTFA (C), and porin (D) levels were assessed by Western blot in BSM cells cultured in the absence (-) or presence (+) of 1 μM D600 for 48 h. Cells were obtained from asthmatic (black columns; n = 5), COPD (gray columns; n = 4), and control subjects (white columns; n = 4). Data are the mean ± the SEM. *, P < 0.05 between populations within an experimental condition using ANOVA with the use of Bonferroni's test. †, P < 0.05 between the absence and the presence of D600 within a population using paired Student's t tests.

Figure 6.

Asthmatic BSM cell proliferation is mitochondria dependent. BSM cell proliferation curves were obtained using either glucose (A) or galactose (B) in the culture medium. The doubling times of cell growth (C) were obtained from the proliferation curves. (D) BrdU incorporations were measured. BSM cells were cultured in various experimental conditions, i.e., glucose, galactose, glucose + ethidium bromide (Et Br), or glucose + cyclic GMP (cGMP). BSM cells were obtained from asthmatic (black symbols and columns; n = 4), COPD (gray symbols and columns; n = 4), and control subjects (white symbols and columns; n = 4). Data are the mean ± the SEM. *, P < 0.05 between populations within an experimental condition using ANOVA with the use of Bonferroni's test. †, P < 0.05 between experimental conditions versus glucose within a population using paired Student's t tests.

Figure 7.

Effect of ethidium bromide and cyclic GMP on the porin content. Mitochondrial mass was assessed by the porin content using Western blot. BSM cells were obtained from asthmatic (black columns; n = 4), COPD (gray columns; n = 4), and control subjects (white columns; n = 4) and were cultured in the absence (-) or presence (+) of ethidium bromide (Et Br) or cyclic GMP (cGMP) for 6 d before the experiments. Data are the mean ± the SEM. *, P < 0.05 between populations within an experimental condition using ANOVA with the use of Bonferroni's test. †, P < 0.05 between experimental conditions versus glucose within a population using paired Student's t tests.

Figure 8.

Effect of methoxyverapamil (D600) on BSM cell proliferation. BSM cell proliferation was measured using BrdU incorporations. Cells were cultured in the absence (-) or presence (+) of 1 μM D600 for 48 h. BSM cells were obtained from asthmatic (black columns; n = 4), COPD (gray columns; n = 4), and control subjects (white columns; n = 4). Data are the mean ± the SEM. *, P < 0.05 between populations within an experimental condition using ANOVA with the use of Bonferroni's test. †, P < 0.05 between the absence and the presence of D600 within a population using paired Student's t tests.