Reduction in Ventilation-Induced Diaphragmatic Mitochondrial Injury through Hypoxia-Inducible Factor 1α in a Murine Endotoxemia Model

Abstract

Mechanical ventilation (MV) is essential for patients with sepsis-related respiratory failure but can cause ventilator-induced diaphragm dysfunction (VIDD), which involves diaphragmatic myofiber atrophy and contractile inactivity. Mitochondrial DNA, oxidative stress, mitochondrial dynamics, and biogenesis are associated with VIDD. Hypoxia-inducible factor 1α (HIF-1α) is crucial in the modulation of diaphragm immune responses. The mechanism through which HIF-1α and mitochondria affect sepsis-related diaphragm injury is unknown. We hypothesized that MV with or without endotoxin administration would aggravate diaphragmatic and mitochondrial injuries through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. MV with endotoxemia augmented VIDD and mitochondrial damage, which presented as increased oxidative loads, dynamin-related protein 1 level, mitochondrial DNA level, and the expressions of HIF-1α and light chain 3-II. Furthermore, disarrayed myofibrils; disorganized mitochondria; increased autophagosome numbers; and substantially decreased diaphragm contractility, electron transport chain activities, mitofusin 2, mitochondrial transcription factor A, peroxisome proliferator activated receptor-γ coactivator-1α, and prolyl hydroxylase domain 2 were observed (p < 0.05). Endotoxin-stimulated VIDD and mitochondrial injuries were alleviated in HIF-1α-deficient mice (p < 0.05). Our data revealed that endotoxin aggravated MV-induced diaphragmatic dysfunction and mitochondrial damages, partially through the HIF-1α signaling pathway.

Keywords: autophagy; biogenesis; dynamics; hypoxia-inducible factor-1α; mitochondria; ventilator-induced diaphragm dysfunction.

Figure 1

Suppression of endotoxin-enhanced mechanical ventilation-induced diaphragm and mitochondrial injuries in HIF-1α-deficient mice. Representative micrographs of the longitudinal sections of diaphragm (×20,000: left panel; ×40,000: right panel) were from the same diaphragms of nonventilated control mice and mice ventilated at a tidal volume (V_T) of 10 mL/kg (V_T 10) for 8 h (n = 3 per group). (A,B) Nonventilated control wild-type mice with or without LPS treatment: normal sarcomeres with clear A bands, I bands, and Z bands; (C) 10 mL/kg wild-type mice without LPS treatment (normal saline): increase of diaphragmatic disarray; (D) 10 mL/kg wild-type mice with LPS treatment: disruption of sarcomeric structure, mitochondrial swelling with a vacuole-like structure, streaming of Z bands, and collection of lipid droplets; (E) 10 mL/kg HIF-1α deficient mice: reduction of diaphragmatic disruption. (F) Diaphragm muscle-specific force production was measured as described in Methods. (G) Cross-sectional area of diaphragm muscle fiber was measured as described in Methods (n = 5 per group). Mitochondrial swelling with concurrent formation of vacuoles and autophagosomes containing heterogeneous cargo are identified by arrows. * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. Scale bar represents 500 nm. CSA = cross-sectional area; HIF^−/− = hypoxia-inducible factor-1α-deficient mice; Hz = hertz; LPS = lipopolysaccharide; N = newton.

Figure 2

Inhibition of endotoxin-augmented mechanical ventilation-induced oxidative load, mitochondrial DNA, and mitochondrial transcription factor A in HIF-1α-deficient mice. (A) Protein carbonyl groups and (B) SOD were from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). (C) Real-time PCR performed for mitochondrial DNA expression was from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as the ratio of mitochondrial DNA to nuclear DNA (n = 5 per group). (D) Western blots were performed using antibodies that recognize mitochondrial transcription factor A and GAPDH expression from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative Tfam activation (n = 5 per group). * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. GAPDH = glyceraldehydes-phosphate dehydrogenase; mt = mitochondria; nDNA = nuclear DNA; SOD = superoxide dismutase; Tfam = mitochondrial transcription factor A.

Figure 3

Reduction of endotoxin-aggravated mechanical ventilation-mediated mitochondrial dynamics in HIF-1α-deficient mice. Western blots were performed using antibodies that recognize Drp1 (A), Mfn2 (B), and GAPDH expression from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative Drp1 and Mfn2 activation (n = 5 per group). * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. Drp1 = dynamin-related protein 1; Mfn2 = mitofusion 2.

Figure 4

Abrogation of endotoxin-enhanced mechanical ventilation-mediated mitochondrial biogenesis in HIF-1α-deficient mice. (A) SDH (complex II)) and (B) COX (complex IV) were from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Western blots were performed using antibodies that recognize mitochondrial cytochrome C (C), PGC-1α (D), and GAPDH expression from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative mitochondrial cytochrome C and PGC-1α activation (n = 5 per group). * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. COX = cytochrome-c oxidase; Mito-Cyt C = mitochondrial cytochrome C; PGC-1α = peroxisome proliferator activated receptor-γ coactivator; SDH = succinate dehydrogenase.

Figure 5

Amelioration of endotoxin-augmented mechanical ventilation-mediated HIF-1α and PHD2 protein expression in HIF-1α-deficient mice. Western blots were performed using antibodies that recognize HIF-1α (A), PHD2 (B), and GAPDH expression from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative HIF-1α and PHD2 activation (n = 5 per group). * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. PHD2 = prolyl hydroxylase domain 2.

Figure 6

Inhibition of endotoxin-aggravated mechanical ventilation-induced diaphragm injury and autophagy in HIF-1α deficient mice. (A–D) Representative micrographs of the longitudinal sections of diaphragm (×40,000) were from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with LPS administration (n = 3 per group). Mitochondrial damage with coexisting autophagosomes containing heterogeneous cargo, loss of cristae, and vacuole formation is identified by arrows. Western blots were performed using antibodies that recognize LC3-II (E), P62 (F), and GAPDH expression from the diaphragms of nonventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative LC3-II and P62 activation (n = 5 per group). * p < 0.05 versus the nonventilated control mice with LPS treatment; † p < 0.05 versus all other groups. LC3-II = light chain 3-II.

Figure 7

Schematic figure illustrating the signaling pathway activation with mechanical ventilation and endotoxemia. Endotoxin-induced augmentation of mechanical stretch-mediated oxidative stress, mitochondrial dynamics and biogenesis, and autophagy, and diaphragm damage were alleviated with HIF-1α homozygous knockout. Drp1 = dynamin-related protein 1; ETC = electron transport chain; HIF = hypoxia-inducible factor; LC3-II = light chain 3-II; LPS = lipopolysaccharide; Mfn2 = mitofusion 2; Mt = mitochondria; PGC-1α = peroxisome proliferator activated receptor-γ coactivator-1α; PHD2 = prolyl hydroxylase domain 2; ROS = reactive oxygen species; Tfam = mitochondrial transcription factor A; VIDD = ventilator-induced diaphragm dysfunction.