Inhibition of IP3R/Ca2+ Dysregulation Protects Mice From Ventilator-Induced Lung Injury via Endoplasmic Reticulum and Mitochondrial Pathways

Abstract

Rationale: Disruption of intracellular calcium (Ca2+) homeostasis is implicated in inflammatory responses. Here we investigated endoplasmic reticulum (ER) Ca2+ efflux through the Inositol 1,4,5-trisphosphate receptor (IP3R) as a potential mechanism of inflammatory pathophysiology in a ventilator-induced lung injury (VILI) mouse model.

Methods: C57BL/6 mice were exposed to mechanical ventilation using high tidal volume (HTV). Mice were pretreated with the IP3R agonist carbachol, IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) or the Ca2+ chelator BAPTA-AM. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected to measure Ca2+ concentrations, inflammatory responses and mRNA/protein expression associated with ER stress, NLRP3 inflammasome activation and inflammation. Analyses were conducted in concert with cultured murine lung cell lines.

Results: Lungs from mice subjected to HTV displayed upregulated IP3R expression in ER and mitochondrial-associated-membranes (MAMs), with enhanced formation of MAMs. Moreover, HTV disrupted Ca2+ homeostasis, with increased flux from the ER to the cytoplasm and mitochondria. Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects. HTV activated the IRE1α and PERK arms of the ER stress signaling response and induced mitochondrial dysfunction-NLRP3 inflammasome activation in an IP3R-dependent manner. Similarly, disruption of IP3R/Ca2+ in MLE12 and RAW264.7 cells using carbachol lead to inflammatory responses, and stimulated ER stress and mitochondrial dysfunction.

Conclusion: Increase in IP3R-mediated Ca2+ release is involved in the inflammatory pathophysiology of VILI via ER stress and mitochondrial dysfunction. Antagonizing IP3R/Ca2+ and/or maintaining Ca2+ homeostasis in lung tissue represents a prospective treatment approach for VILI.

Keywords: calcium; endoplasmic reticulum stress; inositol 1,4,5-trisphosphate receptor; mitochondrial dysfunction; ventilator-induced lung injury.

Figure 1

MV with HTV induces IP3R1 activation in lungs. Immunofluorescence photomicrographs (A) and quantification analysis (B) of IP3R1 in lung tissues of mice with spontaneous breathing (CON group) or mechanical ventilation at high tidal volume (HTV group). Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. Western blot (C) and quantification analysis (D) of IP3R1 protein expression in lung extracts. (E) Western blot analysis of the indicated subcellular fractions for IP3R1, calnexin (CNX; ER and MAM marker), FACL-4 (MAM marker) and tubulin (cytosolic marker). (F) Quantification of IP3R1 protein expression in the ER and MAM was performed by normalizing to CNX. Data are expressed as means ± SD (n = 6 animals per group). *P < 0.05 vs. CON group.

Figure 2

MV with HTV induces MAM formation and changes in ER and mitochondrial morphology in lung cells. Representative TEM images of lung sections derived from group CON and HTV mice at ×10,000 (top images) or ×40,000 magnifications of the boxed areas (bottom images) in (A) alveolar type II epithelial cells (AT-II) and (B) alveolar macrophage (AM). The red stars indicate mitochondria, and the yellow curve indicate the ERs which around the mitochondria (Mito). (C) Quantitation of ER length adjacent to mitochondria normalized to total ER length. (D) Quantitation of width between ER and mitochondrial. Data are expressed as means ± SD (n = 3 per group), *P < 0.05 vs. CON group.

Figure 3

Effects of carbachol and 2-APB on HTV-induced Ca2+ homeostasis in lungs. Single-cell suspensions from lung tissues were preloaded with Fluo-4 AM, Rhod-2 and Mag-Fluo-AM, respectively. (A) Fluo-4 AM labeling the cytoplasmic Ca2+ in lung tissues from CON group, HTV group, IP3R agonist carbachol pretreatment upon HTV stimulation group (HTV+Carbachol group) and IP3R inhibitor 2-APB pretreatment upon HTV stimulation group (HTV+2-APB group). An inset picture was employed to show the indicated area at 4X magnification. (B) Fluorescence quantification analysis of Fluo-4 AM. (C) Rhod-2 and Mag-Fluo-AM co-dyeing to label Ca2+ in the mitochondria and ER in different groups. An inset picture was employed to show the indicated area at 4X magnification. (D) Fluorescence quantification analysis of Rhod-2. (E) Fluorescence quantification analysis of Mag-Fluo-AM. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group; ^#P < 0.05 vs. HTV group.

Figure 4

Effects of carbachol, 2-APB and BAPTA-AM on HTV-induced pathological lung injury and inflammation. (A) H&E staining the histology of lung tissues from group CON, HTV, HTV+Carbachol, HTV+2-APB mice, and the HTV-treated mice pretreated with Ca2+ chelator BAPTA-AM (HTV+BAPTA-AM group). Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. (B) Pathological scores were assessed by results of H&E staining. (C) Lung edema was assessed by determining the weight ratio between wet and dry lungs. (D) Total protein concentration in BALF. (E) Infiltrated cell counts in BALF. (F–H) Levels of IL-1β (F), IL-6 (G) and TNF-α (H) in BALF. Data are expressed as means ± SD (n = 8 per group except for group HTV+ Carbachol (n=7), which has 1 mouse died unexpectedly and was removed from analysis). *P < 0.05 vs. CON group; ^# P < 0.05 vs. HTV group.

Figure 5

2-APB inhibits ER stress in HTV-treated mice. (A) Immunofluorescence photomicrographs of GRP78 (green) and CHOP (red) in lung tissues of group CON, HTV, and HTV+2-APB mice. Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. (B, C) Graphic presentations of fluorescence mean densities of GRP78 and CHOP. (D) Levels of GRP78, CHOP, p-IRE1α, t-IRE1α, TRAF2, XBP-1s, p-PERK, t-PERK, p-eIF2α, t-eIF2α, t-ATF6, β-actin proteins by Western blot. (E) Relative protein expression of GRP78, CHOP, TRAF2, XBP-1s and t-ATF6 relative to β-actin. (F) The relative ratio of p-IRE1α protein was presented to t-IRE1α. (G) The relative ratio of p-PERK protein was presented to t-PERK. (H) The relative ratio of p-eIF2α protein was presented to t-eIF2α. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group. ^# P < 0.05 vs. HTV group.

Figure 6

2-APB inhibits activation of NF-κB signaling pathway in HTV-treated mice. (A, B) Immunofluorescence photomicrographs and quantification analysis of p-NF-κB p65 in lung tissues of group CON, HTV and HTV+2-APB mice. Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. (C, D) Representative immunoblots of IκBα in lung extracts and densitometric analyses of IκBα. (E–G) Representative immunoblots of NF-κB p65 in nucleus and cytoplasm, and densitometric analyses of NF-κB p65. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group. ^# P < 0.05 vs. HTV group.

Figure 7

2-APB improves mitochondrial dysfunction in HTV-treated mice. (A, B) Fluorescence microscopy and the ratio of JC-1 staining (red, J-aggregates; green, monomer) in lung tissues from CON, HTV and HTV+2-APB group, Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. (C) Levels of ATP. (D, E) Levels of ROS were determined by flow cytometry. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group. ^# P < 0.05 vs. HTV group.

Figure 8

2-APB inhibits NLRP3 inflammasome activation in HTV-treated mice. (A, B) Representative immunoblots of NLRP3, cleaved caspase-1and Asc in lung extracts from CON, HTV and HTV+2-APB group and densitometric analyses of NLRP3, cleaved caspase-1and Asc. (C) Levels of NLRP3, caspase-1 and Asc mRNA. Data are expressed as means ± SD (n = 6 per group). *P < 0.05 vs. CON group. ^# P < 0.05 vs. HTV group.

Figure 9

Carbachol facilitates inflammatory response in lung epithelial cells and macrophage. (A–C) Levels of IL-1β (A), IL-6 (B) and TNF-α (C) in MLE12 and RAW264.7 cell supernatants. Data are expressed as means ± SD from 3 independent experiments. *P < 0.05 vs. Mock group.

Figure 10

Carbachol stimulates ER stress in lung epithelial cells and macrophage. (A, B) Representative immunoblots of GRP78, CHOP, p-NF-κB p65 and IκBα in MLE12 cells and densitometric analyses of GRP78, CHOP, p-NF-κB p65 and IκBα. MLE12 cells were treated with 50 μM carbachol or non-treated for 24h. (C) Levels of GRP78, CHOP mRNA in MLE12 cells. (D, E) Representative immunoblots of GRP78, CHOP, p-NF-κB p65 and IκBα in RAW264.7 cells and densitometric analyses of GRP78, CHOP, p-NF-κB p65 and IκBα. RAW264.7 cells were treated with 50 μM carbachol or non-treated for 24h. (F) Levels of GRP78, CHOP mRNA in RAW264.7 cells. Data are expressed as means ± SD from 3 independent experiments. *P < 0.05 vs. Mock group.

Figure 11

Carbachol stimulates mitochondrial dysfunction and NLRP3 inflammasome activation in lung epithelial cells and macrophage. (A, B) Fluorescence microscopy and the ratio of JC-1 staining (red, J-aggregates; green, monomer) in MLE12 and RAW264.7 cells after treating with 50 μM carbachol or non-treated for 24h, Scale bar: 200μm. (C) Levels of ATP. (D, E) Levels of ROS were determined by flow cytometry. (F) Levels of NLRP3, caspase-1and Asc mRNA in MLE12 cells. (G) Levels of NLRP3, caspase-1 and Asc mRNA in RAW264.7 cells. Data are expressed as means ± SD from 3 independent experiments. Mann-Whitney U test was used for caspase-1 mRNA comparison between two groups from MLE12 cells, because the data were non-normally distributed. *P < 0.05 vs. Mock group.

Figure 12

Working model detailing the role of IP3R/Ca2+ in VILI. Mechanical overstretch induces cell inflammation through an accelerated ER-to-cytosolic and ER-to-mitochondria efflux of Ca2+ through IP3R, followed by ER stress and mitochondrial dysfunction.