Cervical spinal cord injury exacerbates ventilator-induced diaphragm dysfunction

Abstract

Cervical spinal cord injury (SCI) can dramatically impair diaphragm muscle function and often necessitates mechanical ventilation (MV) to maintain adequate pulmonary gas exchange. MV is a life-saving intervention. However, prolonged MV results in atrophy and impaired function of the diaphragm. Since cervical SCI can also trigger diaphragm atrophy, it may create preconditions that exacerbate ventilator-induced diaphragm dysfunction (VIDD). Currently, no drug therapy or clinical standard of care exists to prevent or minimize diaphragm dysfunction following SCI. Therefore, we first tested the hypothesis that initiating MV acutely after cervical SCI will exacerbate VIDD and enhance proteolytic activation in the diaphragm to a greater extent than either condition alone. Rats underwent controlled MV for 12 h following acute (∼24 h) cervical spinal hemisection injury at C2 (SCI). Diaphragm tissue was then harvested for comprehensive functional and molecular analyses. Second, we determined if antioxidant therapy could mitigate MV-induced diaphragm dysfunction after cervical SCI. In these experiments, SCI rats received antioxidant (Trolox, a vitamin E analog) or saline treatment prior to initiating MV. Our results demonstrate that compared with either condition alone, the combination of SCI and MV resulted in increased diaphragm atrophy, contractile dysfunction, and expression of atrophy-related genes, including MuRF1. Importantly, administration of the antioxidant Trolox attenuated proteolytic activation, fiber atrophy, and contractile dysfunction in the diaphragms of SCI + MV animals. These findings provide evidence that cervical SCI greatly exacerbates VIDD, but antioxidant therapy with Trolox can preserve diaphragm contractile function following acute SCI.

Keywords: antioxidant; diaphragm; mechanical ventilation; respiratory muscles; spinal cord injury.

Fig. 1.

Spinal cord injury (SCI) exacerbates mechanical ventilation (MV)-induced contractile dysfunction and atrophy in the ipsilateral hemidiaphragm. In vitro diaphragm force-frequency response (A) and cross-sectional area of diaphragm skeletal muscle myofibers (B) expressing myosin heavy chain (MHC) I (Type I), MHC IIa (Type IIa), and MHC IIb/IIx (Type IIb/IIx). Representative fluorescent staining of MHC I (DAPI filter/blue), MHC IIa (FITC filter/green), and dystrophin (Rhodamine filter/red) proteins in diaphragm samples are shown below the graph. Values are means ± SE. ‖Control (CON) significantly different vs. MV and SCI. §SCI + MV significantly different vs. all groups. *Significantly different vs. CON (P < 0.05).

Fig. 2.

SCI and MV increase oxidative stress in the ipsilateral hemidiaphragm. A: mitochondrial hydrogen peroxide emission from permeabilized diaphragm muscle fibers using pyruvate/malate as the substrate. Values are normalized to tissue dry weight. B: Western blot of 4-HNE modified proteins measured in whole diaphragm muscle normalized to tubulin. Representative Western blot images for 4-HNE and tubulin are shown to the right of the graph. Values are means ± SE. §Significantly different vs. all groups. *Significantly different vs. CON (P < 0.05).

Fig. 3.

SCI and MV enhance expression and activity of proteolytic proteins in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for Atrogin-1/MaFbx, MuRF1, and LC3. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. B: Western blot analysis of the ratio of LC3II/LC3I. C: Western blot analysis of the spectrin breakdown product (SBDP) for calpain. D: Western blot analysis of the spectrin breakdown product (SBDP) for caspase-3. All Western blot analyses are presented as corrected for tubulin. Representative Western blot images are shown to the right of the graph. Values are means ± SE. §Significantly different vs. all groups. *Significantly different vs. CON (P < 0.05).

Fig. 4.

SCI augments Fn14 expression and signaling in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for Fn14. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. B: Western blot analysis of Fn14. C: Western blot analysis of the ratio of pJNK/JNK. D: Western blot analysis of the ratio of pERK/ERK. All Western blot analyses are presented as corrected for tubulin. Representative images are shown to the right of the graph. Values are means ± SE. †Significantly different vs. CON and MV. *Significantly different vs. CON (P < 0.05).

Fig. 5.

SCI and MV alter neuromuscular junction protein expression in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for MuSK, Agrin, and LRP4. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. B: Western blot analysis of MuSK. C: Western blot analysis of Agrin. D: Western blot analysis of LRP4. All Western blot analyses are presented as corrected for tubulin. Representative images are shown to the right of the graph. Values are means ± SE. *Significantly different vs. CON (P < 0.05).

Fig. 6.

Trolox administration prevents SCI and MV-induced diaphragm contractile dysfunction and atrophy in the ipsilateral hemidiaphragm. In vitro diaphragm force-frequency response (A) and diaphragm muscle fiber cross-sectional area in diaphragm skeletal muscle myofibers (B) expressing myosin heavy chain (MHC) I (Type I), MHC IIa (Type IIa), and MHC IIb/IIx (Type IIb/IIx). Representative fluorescent staining of MHC I (DAPI filter/blue), MHC IIa (FITC filter/green), and dystrophin (Rhodamine filter/red) proteins in diaphragm samples are shown below the graph. Dotted lines on graphs represent control values. Values are means ± SE. @SCI + Trolox significantly different vs. SCI. ^ΔSCI + Trolox and SCI + MV + Trolox significantly different vs. SCI. *Significantly different vs. CON (P < 0.05).

Fig. 7.

Trolox administration reduces SCI-induced diaphragm oxidative stress in the ipsilateral hemidiaphragm. A: mitochondrial hydrogen peroxide emission from permeabilized diaphragm muscle fibers using pyruvate/malate as the substrate. Values are normalized to tissue dry weight. Dotted line represents control values. B: Western blot of 4-HNE modified proteins measured in whole diaphragm muscle normalized to tubulin. Representative Western blot images for 4-HNE and tubulin are shown to the right of the graph. Values are means ± SE. #Significantly different vs. SCI (P < 0.05).

Fig. 8.

Trolox administration reduces SCI-induced diaphragm proteolytic protein expression and activity in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for Atrogin-1/MaFbx, MuRF1, and LC3. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. Dotted line represents control values. B: Western blot analysis of the ratio of LC3II/LC3I. C: Western blot analysis of the spectrin breakdown product (SBDP) for calpain. D: Western blot analysis of the spectrin breakdown product (SBDP) for caspase-3. All Western blot analyses are presented as corrected for tubulin. Representative Western blot images are shown to the right of the graph. Values are means ± SE. #Significantly different vs. SCI (P < 0.05).

Fig. 9.

Trolox administration attenuates SCI-induced Fn14 signaling in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for Fn14. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. Dotted line represents control values. B: Western blot analysis of Fn14. C: Western blot analysis of the ratio of pJNK/JNK. D: Western blot analysis of the ratio of pERK/ERK. All Western blot analyses are presented as corrected for tubulin. Representative images are shown to the right of the graph. Values are means ± SE. ^Significantly different vs. SCI and SCI + Trolox. #Significantly different vs. SCI (P < 0.05).

Fig. 10.

Trolox does not affect the expression of neuromuscular junction proteins in the ipsilateral hemidiaphragm. A: real-time RT-PCR analysis for MuSK, Agrin, and LRP4. Gene expression is presented as corrected for β-glucoronidase and normalized to control values. Dotted line represents control values. B: Western blot analysis of MuSK. C: Western blot analysis of Agrin. D: Western blot analysis of LRP4. All Western blot analyses are presented as corrected for tubulin. Representative images are shown to the right of the graph. Values are means ± SE.