Suppression of Hypoxia-Inducible Factor 1α by Low-Molecular-Weight Heparin Mitigates Ventilation-Induced Diaphragm Dysfunction in a Murine Endotoxemia Model

Abstract

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α-deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.

Keywords: endotoxemia; hypoxia-inducible factor-1α; low-molecular-weight heparin; mitochondria; ventilator-induced diaphragm dysfunction.

Figure 1

Electron microscopy, excursion, and thickening of the diaphragm. Representative micrographs of the longitudinal sections of diaphragm (×20,000: upper panel; ×40,000: lower panel) were from the same diaphragms of non-ventilated control mice and mice ventilated at a tidal volume (V_T) of 6 mL/kg (V_T 6) or 10 mL/kg (V_T 10) for 8 h with or without LPS administration (n = 3 per group). (A,B) Non-ventilated control wild-type mice with or without LPS treatment: normal sarcomeres with distinct A bands, I bands, and Z bands; (C) 6 mL/kg wild-type mice with LPS treatment: reduction of diaphragmatic disruption compared to that of 10 mL/kg groups; (D) 10 mL/kg wild-type mice without LPS treatment (normal saline): increase of diaphragmatic disarray; (E) 10 mL/kg wild-type mice with LPS treatment: disruption of sarcomeric structure with loss of streaming of Z bands, mitochondrial swelling, and accumulation of lipid droplets (asterisks); (F) 10 mL/kg wild-type mice pretreated with enoxaparin: attenuation of diaphragmatic disruption. (G) Injury scores of mitochondria were from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 3 per group). (H,I) Excursion and thickness variation of diaphragm. Mitochondrial swelling with concurrent loss of cristae and autophagosomes containing heterogeneous cargo are identified by arrows. Enoxaparin, 4 mg/kg, was given subcutaneously 30 min before mechanical ventilation. * p < 0.05 versus the non-ventilated control mice with LPS treatment; † p < 0.05 versus all other groups. Scale bar represents 500 nm. LPS = lipopolysaccharide; LMWH = low-molecular-weight heparin.

Figure 2

Inhibition of endotoxin-aggravated mechanical ventilation-enhanced oxidative stress, inflammatory cytokines production, and calpain expression by enoxaparin. (A) MDA (diaphragm), (B) total antioxidant capacity (diaphragm), (C) BAL fluid MIP-2, and (D) BAL fluid IL-6 were from the non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Western blots were performed using antibodies that recognize calpain (E) and GAPDH expression from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative calpain activation (n = 5 per group). Enoxaparin, 4 mg/kg, was given subcutaneously 30 min before mechanical ventilation. * p < 0.05 versus the non-ventilated control mice with LPS treatment; † p < 0.05 versus all other groups. BAL = bronchoalveolar lavage; GAPDH = glyceraldehydes-phosphate dehydrogenase; IL = interleukin; MIP-2 = macrophage inflammatory protein-2; MDA = malondialdehyde.

Figure 3

Reduction of endotoxin-augmented mechanical ventilation-mediated atrogin-1, MuRF-1, LC3-II, and Beclin expression by enoxaparin. Western blots were performed using antibodies that recognize atrogin-1 (A), MuRF-1 (B), LC3-II (C), beclin (D), and GAPDH expression from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative atrogin-1, MuRF-1, LC3-II, and beclin activation (n = 5 per group). Enoxaparin, 4 mg/kg, was given subcutaneously 30 min before mechanical ventilation. * p < 0.05 versus the non-ventilated control mice with LPS treatment; † p < 0.05 versus all other groups. LC3-II = light chain 3-II; MuRF-1 = muscle ring finger-1.

Figure 4

Suppression of endotoxin-augmented mechanical ventilation-induced HIF-1α mRNA activation and HIF-1α protein expression by enoxaparin. (A) Real-time PCR performed for HIF-1α mRNA expression was from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as the ratio of HIF-1α mRNA to GAPDH (n = 5 per group). (B) Western blots from the same animals were conducted using antibodies that recognize HIF and GAPDH expression from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as the ratio of HIF-1α to GAPDH (n = 5 per group). (C and D) Representative micrographs (×400) with HIF-1α staining of paraffin lung sections and quantification were from non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Enoxaparin, 4 mg/kg, was given subcutaneously 30 min before mechanical ventilation. Scale bars represent 20 μm. * p < 0.05 versus the non-ventilated control mice with LPS treatment; † p < 0.05 versus all other groups.

Figure 5

Abrogation of endotoxin-stimulated mechanical ventilation-mediated diaphragm dysfunction in HIF-1α deficient mice. (A) MDA (diaphragm), (B) total antioxidant capacity (diaphragm), (C) BAL fluid MIP-2, and (D) BAL fluid IL-6 were from the non-ventilated 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 calpain (E), atrogin-1 (F), MuRF-1 (G), LC3-II (H), and GAPDH expression from the diaphragms of non-ventilated 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 calpain, atrogin-1, MuRF-1, and LC3-II activation (n = 5 per group). (I) Real-time PCR performed for HIF-1α mRNA expression was from the diaphragms of non-ventilated 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 HIF-1α mRNA to GAPDH (n = 5 per group). * p < 0.05 versus the non-ventilated control mice with LPS; † p < 0.05 versus HIF-1α-deficient mice. HIF^−/− = hypoxia inducible factor-1α-deficient mice.

Figure 6

Reduction of endotoxin-exacerbated mechanical ventilation-induced diaphragm and mitochondrial injury in HIF-1α deficient mice. (A–D) Representative micrographs of the longitudinal sections of diaphragm (×20,000: upper panel; ×40,000: lower panel) were from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 10 mL/kg for 8 h with LPS administration (n = 3 per group). Mitochondrial swelling with coexisting vacuole formation, loss of cristae, and autophagosomes containing heterogeneous cargo are identified by arrows. Mitochondrial swelling with concurrent loss of cristae and autophagosomes containing heterogeneous cargo are identified by arrows. (E,F) Excursion and thickness variation of diaphragm. * p < 0.05 versus the non-ventilated control mice with LPS treatment; † p < 0.05 versus HIF-1α-deficient mice. Scale bars represent 500 nm.

Figure 7

Suppression of endotoxin-augmented mechanical ventilation-induced expression of caspase-3 and BNIP-3, and muscle fiber apoptosis by enoxaparin and in HIF deficient mice. Caspase-3 (A), BNIP-3 (B), and GAPDH expression from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Arbitrary units were expressed as relative cleaved caspase-3 and BNIP-3 activation (n = 5 per group). (C) Representative micrographs (×400) with TUNEL staining of paraffin diaphragm sections and quantification were from the diaphragms of non-ventilated control mice and mice ventilated at a tidal volume of 6 mL/kg or 10 mL/kg for 8 h with or without LPS administration (n = 5 per group). Enoxaparin, 4 mg/kg, was given subcutaneously 30 min before mechanical ventilation. Apoptotic cells are identified by arrows. A bright green signal indicates positive staining of apoptotic cells, and shades of dull green signify non-reactive cells. * p < 0.05 versus the non-ventilated control mice with room air; † p < 0.05 versus all other groups. Scale bars represent 20 μm. BNIP-3 = BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.

Figure 8

Schematic figure illustrating the signaling pathway activation with. mechanical ventilation and endotoxemia. Endotoxin-induced augmentation of mechanical stretch-mediated cytokine production and diaphragm damage were attenuated by the administration of enoxaparin and with HIF-1α homozygous knockout. HIF = hypoxia-inducible factor; IL = interleukin; LC3-II = light chain 3-II; LMWH = low-molecular-weight heparin; LPS = lipopolysaccharide; MIP-2 = macrophage inflammatory protein-2; MuRF-1 = muscle ring finger-1; ROS = reactive oxygen species; BNIP-3 = BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; VIDD = ventilator-induced diaphragm dysfunction.