Diaphragm Atrophy and Weakness in the Absence of Mitochondrial Dysfunction in the Critically Ill

Abstract

Rationale: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency and increases morbidity, duration of hospital stay, and health care costs. The mechanisms underlying diaphragm weakness are unknown, but might include mitochondrial dysfunction and oxidative stress.

Objectives: We hypothesized that weakness of diaphragm muscle fibers in critically ill patients is accompanied by impaired mitochondrial function and structure, and by increased markers of oxidative stress.

Methods: To test these hypotheses, we studied contractile force, mitochondrial function, and mitochondrial structure in diaphragm muscle fibers. Fibers were isolated from diaphragm biopsies of 36 mechanically ventilated critically ill patients and compared with those isolated from biopsies of 27 patients with suspected early-stage lung malignancy (control subjects).

Measurements and main results: Diaphragm muscle fibers from critically ill patients displayed significant atrophy and contractile weakness, but lacked impaired mitochondrial respiration and increased levels of oxidative stress markers. Mitochondrial energy status and morphology were not altered, despite a lower content of fusion proteins.

Conclusions: Critically ill patients have manifest diaphragm muscle fiber atrophy and weakness in the absence of mitochondrial dysfunction and oxidative stress. Thus, mitochondrial dysfunction and oxidative stress do not play a causative role in the development of atrophy and contractile weakness of the diaphragm in critically ill patients.

Keywords: critically ill; diaphragm weakness; mechanical ventilation; mitochondrial dysfunction; oxidative stress.

Figures

Figure 1.

Atrophy and contractile weakness of diaphragm muscle fibers in critically ill patients. (A and D) The cross-sectional area of diaphragm muscle fibers was significantly smaller for both slow- and fast-twitch fibers in critically ill patients than in control patients, indicating atrophy. (B and E) Maximal absolute force was lower in both slow- and fast-twitch fibers from critically ill patients, indicating contractile weakness. (C and F) Maximal normalized force, that is, maximal absolute force normalized to fiber cross-sectional area, was lower in slow-twitch but not in fast-twitch diaphragm muscle fibers from critically ill patients. Each data point represents the mean of all slow-twitch or fast-twitch muscle fibers measured from a diaphragm biopsy of one patient. Data in A, B, D, and E are presented as median and interquartile range, and data in C and F are presented as mean ± SEM. Crit. ill = critically ill; CSA = cross-sectional area; Fmax = maximal force; NS = not significant. **P ≤ 0.01, ***P ≤ 0.001.

Figure 2.

Maintained mitochondrial respiration in diaphragm muscle fibers. (A) Example of experimental recording of mitochondrial respiration in diaphragm muscle fibers. (Top) Oxygen concentration, which was maintained above 300 μM to avoid limitations in oxygen supply. (Bottom) Oxygen consumption per milligram wet weight muscle mass. Leak respiration is a measure of oxygen flux compensating for proton leakage from the intermembranous space through the inner membrane to the matrix. Maximal complex I–stimulated respiration is measured after addition of glutamate, malate and pyruvate, and ADP. Maximal uncoupled respiration is measured after adding succinate and FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone] to dissipate the mitochondrial membrane potential. Uncoupled complex II–stimulated respiration is the oxygen consumption of complex II by blocking complex I with rotenone. No differences were observed in (B) leak respiration, (C) complex I–stimulated respiration, (D) maximal uncoupled respiration, and (E) uncoupled complex II–stimulated respiration between critically ill patients and control patients. Each data point represents the mean of two mitochondrial respiration measurements in a diaphragm biopsy of one patient. Data in B–E are presented as median and interquartile range. CI = complex I; CII = complex II; Crit. ill = critically ill; NS = not significant.

Figure 3.

Maintained succinate dehydrogenase (SDH) activity in diaphragm muscle fibers. (A) Typical examples of diaphragm cryosections stained for fast-myosin heavy chain (MyHC, top) and for SDH activity (bottom). Blue, fast-MyHC; red, wheat germ agglutinin, indicating cell membranes. The circles represent corresponding slow-twitch (open) and fast-twitch (solid) fibers in control patients, and the squares represent corresponding slow-twitch (open) and fast-twitch (solid) fibers in critically ill patients. (B and C) SDH activity of diaphragm muscle fibers was comparable in both slow- and fast-twitch fibers in critically ill patients compared with control patients. Each data point represents the mean SDH activity of all slow-twitch or fast-twitch fibers measured in cryosections of a diaphragm biopsy of one patient. Data are presented as median and interquartile range. (D and E) The area of the cryosections covered by slow- and fast-twitch fibers (fiber area fraction) and the distribution of slow- and fast-twitch fibers (fiber type fraction) were preserved, suggesting that maintained respiration in critically ill patients was not caused by a fiber type shift toward slow-twitch muscle fibers. Data are presented as mean ± SEM. Crit. ill = critically ill; NS = not significant.

Figure 4.

Preserved content of mitochondrial complexes in diaphragm muscle fibers. (A) Typical example of a Western blot with MitoProfile antibody cocktail of the five complexes. Lanes 1 and 2, diaphragm homogenates of critically ill patients; lane 3, rat heart homogenate (loading control); lanes 4 and 5, diaphragm homogenates of control patients. (B) Quantification of protein content of mitochondrial complexes. Protein content was not lower in diaphragm muscle fibers of critically ill patients (analysis of variance, interaction effect P = 0.56). Data are presented as mean ± SEM. AU = arbitrary units; CI = complex I; CII = complex II; CIII = complex III; CIV = complex IV; Crit. ill = critically ill; CV = complex V.

Figure 5.

No major alterations of mitochondrial structure in diaphragm muscle fibers. (A) Electron micrograph of diaphragm biopsy from a control patient. Mitochondria are well aligned and have a normal general appearance. (B) Electron micrograph of a diaphragm biopsy from a critically ill patient. In A and B, the arrow indicates an example of a mitochondrion with a normal appearance. (C–E) Quantification of mitochondria in the micrographs revealed no differences in mitochondrial size, number, and density. Each data point represents the mean mitochondrial size/number/volume density as measured or counted in micrographs of a diaphragm biopsy of one patient. Data are presented as median and interquartile range. Crit. ill = critically ill; NS = not significant.

Figure 6.

Down-regulation of master regulator of biogenesis PGC-1α in diaphragm muscle fibers from critically ill patients. (A) Typical example of Western blot with antibodies for master regulator of biogenesis PGC-1α; fission proteins Mfn1, Mfn2, and OPA1; and fusion protein DRP1 from control and critically ill patients. (B) Compared with control subjects, PGC-1α protein content was lower in critically ill patients. (C–E) In line with lower PGC-1α, pro-fusion proteins Mfn1, Mfn2, and OPA1 were also lower in critically ill patients. This suggests that mitochondrial fusion dynamics are impaired in diaphragm fibers of critically ill patients, but without major changes in mitochondrial structure and function (seeFigure 5). (F) Finally, content of pro-fusion protein DRP1 was comparable in critically ill patients and control patients. Each data point represents the mean protein content per group of diaphragm biopsies of three to four patients. Data are presented as median and interquartile range. AU = arbitrary units; Crit. ill = critically ill; DRP1 = dynamin-related protein 1; Mfn1 = mitofusin 1; Mfn2 = mitofusin 2; NS = not significant; OPA1 = optic atrophy 1; PGC-1α = peroxisome proliferator–activated receptor γ coactivator 1α. *P ≤ 0.05, **P ≤ 0.01.

Figure 7.

Absence of metabolic oversupply in diaphragm muscle fibers. (A) Compared with control patients, the ATP concentration was not changed in diaphragm muscle fibers of critically ill patients, suggesting the absence of metabolic oversupply in critically ill patients. Each data point represents the mean ATP concentration of a diaphragm biopsy of one patient. (B) Typical example of Western blot with antibodies for metabolic stress markers pAMPK and AMPK. (C) The ratio of pAMPK to AMPK was higher in diaphragm muscle fibers of critically ill patients than in those of control patients. Each data point represents the mean protein content per group of diaphragm biopsies from three to four patients. Data are presented as median and interquartile range. AMPK = AMP-activated protein kinase; AU = arbitrary units; Crit. ill = critically ill; NS = not significant; pAMPK = phosphorylated AMPK. **P ≤ 0.01.

Figure 8.

Lower oxidative stress levels in diaphragm muscle fibers from critically ill patients. (A) Typical example of a Western blot stained for nitrosative stress marker nitrotyrosine (NT)-specific antibodies, indicating a footprint of nitrosative stress. (E) Typical example of a Western blot with 4-hydroxy-2-nonenal (HNE)-specific antibodies, a measure for lipid peroxidation. (I) Typical example of an OxyBlot, showing carbonylated proteins. (B, F, and J) NT and HNE contents were lower in diaphragm muscle fibers of critically ill patients than in those of control subjects, whereas the oxidation index was comparable. (C, G, and K) NT content, HNE content, and the oxidation index were not higher in critically ill patients mechanically ventilated for less than 72 hours than in critically ill patients mechanically ventilated longer than 72 hours. (D, H, and L) No correlation between the duration of ventilation and NT content, HNE content, and oxidation index was observed, suggesting that oxidative stress was not an early phenomenon in critically ill patients. Each data point represents the NT content, HNE content, or oxidation index in a diaphragm biopsy of one patient. Data in B, C, F, G, J, and K are presented as median and interquartile range. AU = arbitrary units; Crit. ill = critically ill; MV = mechanical ventilation; NS = not significant. *P ≤ 0.05, ***P ≤ 0.001.

Figure 9.

Antioxidant response in diaphragm muscle fibers from critically ill patients. (A) Expression of Nrf2, a master regulator of antioxidant pathways, was increased in critically ill patients, indicating activation of antioxidant pathways. (B) Typical example of a Western blot stained for antioxidant enzymes SOD1 and catalase, and loading control tubulin. (C and D) Antioxidant enzymes SOD1 and catalase were comparable in critically ill patients and control patients. Each data point represents the Nrf2 expression or SOD1 or catalase content in a diaphragm biopsy of one patient. Data in A and D are presented as median and interquartile range, and data in C are presented as mean ± SEM. AU = arbitrary units; Crit. ill = critically ill; Nrf2 = nuclear erythroid 2–related factor 2; NS = not significant; SOD1 = superoxidase dismutase-1. ***P ≤ 0.001.