1. Cell Biology
Download icon

NADPH oxidase mediates microtubule alterations and diaphragm dysfunction in dystrophic mice

  1. James Anthony Loehr
  2. Shang Wang
  3. Tanya R Cully
  4. Rituraj Pal
  5. Irina V Larina
  6. Kirill V Larin
  7. George G Rodney  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. University of Houston, United States
  3. Tomsk State University, Russia
Research Article
Cite this article as: eLife 2018;7:e31732 doi: 10.7554/eLife.31732
5 figures, 2 tables and 1 additional file

Figures

Eliminating Nox 2 ROS production prevents alterations in tubulin content and the microtubule network.

(A) Representative western blot images of α-, β-, and DT-tubulin content in all three genotypes. (B–D) Eliminating Nox2 ROS production decreases absolute α-, β- and DT-tubulin content in dystrophic diaphragm muscle. (E) The relative amount of DT-/α-tubulin is not different between groups. (F) Representative images of diaphragm myofibers stained with α-tubulin. (G–I) The lack of Nox 2 ROS prevents microtubule disorganization and the increase in microtubule density seen in mdx muscle. p≤0.05 *Significant difference between groups in at least (A–E) nanimals = 6 and (F–I) nanimals = 3 and nfibers = 15.

https://doi.org/10.7554/eLife.31732.002
Genetic deletion of Nox2 ROS production reduced fibrosis.

(A) Representative trichrome images of fibrosis in all three genotypes. Eliminating Nox2 ROS production in dystrophic muscle reduced fibrosis compared with mdx mice. (B) Hydroxyproline levels were elevated in dystrophic muscle and eliminating Nox2 ROS reduced hydroxyproline content compared with mdx mice. (C) Representative western blot images for fibronectin and collagen I content in all three genotypes. Fibronectin and collagen I content were elevated in mdx diaphragm and eliminating Nox2 ROS reduced both toward WT levels. p≤0.05 * Significant difference between groups in at least nanimals = 6 for trichrome and hydroxyproline and nanimals = 3 for fibronectin and collagen I.

https://doi.org/10.7554/eLife.31732.003
Figure 3 with 1 supplement
The lack of Nox2 ROS reduces muscle stiffness and stretch-induced ROS.

(A) Image of the passive stretch experimental set-up. (B) Average passive diaphragm force recorded during stretch for each genotype. (C) Eliminating Nox2 ROS production reduced diaphragm tissue stiffness. (D) Stretch induced ROS in mdx muscle was elevated above WT levels and eliminated in p47-/-/mdx diaphragm. (E) Image of the OCE experimental set-up. (F) Representative OCT image of the diaphragm taken prior to OCE experiments. (G) Transverse diaphragm muscle stiffness increased in mdx compared with WT mice; eliminating Nox2 ROS resulted in a decrease toward WT (p=0.09). (H) Genetic inhibition of Nox2 ROS reduced longitudinal diaphragm stiffness to WT values. (I) Muscle function was not altered following OCE measurements. p≤0.05 *Significant difference between groups in at least nanimals = 6 per group.

https://doi.org/10.7554/eLife.31732.004
Figure 3—video 1
Longitudinal.

Following the application of the air puff (<1 ms in duration), the displacement of the diaphragm tissue was monitored as the wave propagated down longitudinal axis while imaged at 62.5 kHz with OCE. Visualization is 5000 times slower than the actual speed.

https://doi.org/10.7554/eLife.31732.005
Figure 4 with 2 supplements
Eliminating Nox2 ROS protects against muscle and respiratory dysfunction.

(A) WT was significantly different from mdx and p47-/-/mdx animals at all stimulation frequencies. The p47-/-/mdx animals were different from mdx at 60–100 Hz and trended toward significance at 40 Hz (p=0.098). (B) Fibrosis significantly correlated with muscle force. p≤0.05 *Significant difference between groups in at least nanimals = 6.

https://doi.org/10.7554/eLife.31732.007
Figure 4—figure supplement 1
Eliminating Nox2 ROS protects against phenotypic alterations in dystrophic diaphragm muscle.

(A–B) Eliminating Nox2 ROS increased median cross sectional area compared with mdx diaphragm. (C) In dystrophic diaphragm lacking Nox2 ROS production the number of centralized nuclei were reduced compared with mdx diaphragm tissue. (D) Representative hematoxylin and eosin stained images of diaphragm cross-section showing central nuclei (arrow head) and smaller fibers (arrow). (E) Fiber type distribution was maintained by eliminating Nox2 ROS production in dystrophic diaphragm muscle. (F) Representative immunofluorescently labeled diaphragm cross-sectional images showing fiber type distribution. Type I (red), IIA (green), IIB/IIX (white x, unstained and viewed from bright field overlay). p≤0.05 *Significant difference between groups in at least nanimals = 3.

https://doi.org/10.7554/eLife.31732.008
Figure 4—figure supplement 2
Linear correlation of stiffness measured by OCE and the peak force.

There was a significant correlation between peak force and transverse as well as peak force and longitudinal diaphragm stiffness.

https://doi.org/10.7554/eLife.31732.009
Taxol-induced MT polymerization has no effect on tissue stiffness but induced ROS production.

(A) Representative images of MT network in control (DMSO) and Taxol-treated diaphragm (20 μM for 2 hr). (B–D) Taxol induced MT disorganization and increased microtubule density compared with control. (E) Average passive diaphragm force recorded during stretch was not affected by Taxol. (F) Polymerizing the MT network had no effect on diaphragm tissue stiffness. (G) MT network polymerization enhanced stretch-induced ROS in Taxol-treated diaphragm. p≤0.05 *Significant difference between groups in at least (A–D) nanimals = 3 and nfibers = 15 and (E–G) nanimals = 5.

https://doi.org/10.7554/eLife.31732.012

Tables

Table 1
Tubulin and stiffness correlations.
https://doi.org/10.7554/eLife.31732.006
Adj R2Fibrosisα-tubulinβ-tubulinDT-tubulinDT-/α-tubulinMLR (fibrosis/DT)MLR (fibrosis/ratio)
Transverse0.69 *0.46 *0.51 *0.51 *0.100.690.67
Longitudinal0.44 *0.20 *0.40 *0.41 *0.19 *0.440.49
  1. Most variables significantly correlated with both transverse and longitudinal stiffness. MLR revealed fibrosis accounted for the majority of the variance observed in either stiffness measure. p≤0.05 *Significant correlation in at least nanimals = 6.

Table 2
Force and stiffness correlations
https://doi.org/10.7554/eLife.31732.010
Adj R2FibrosisMLR (fibrosis/trans)MLR (fibrosis/long)MLR (fibrosis/long/trans)
Force0.570.520.520.49
  1. MLR revealed fibrosis accounted for a majority of the variance observed in diaphragm muscle function. p≤0.05 *Significant difference between groups in at least nanimals = 6.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)