Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells

Abstract

Rationale: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation.

Objective: This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells.

Methods and results: We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs.

Conclusions: These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

Figure 1

Hypoxia shifts the cytosol to a more oxidized state in PASMC. (A) Combined results from multiple experiments in PASMC expressing Cyto-RoGFP and superfused with either normoxic (21% O₂) or hypoxic (1.5% O₂) media. (B-E) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Cyto-RoGFP sensor. PASMC expressing Cyto-RoGFP were superfused with normoxic (21% O₂) media (B), then with hypoxic (1.5% O₂) media for 30 min (C), followed by media containing DTT to fully reduce the sensor (D), and then with media containing tBH to fully oxidize the sensor (E). (F) Representative time course, quantitative analysis of the fluorescence intensity ratio from representative individual PASMC imaged in B-E. ‘Img Fig’ denotes the time at which the images B-E were captured during the experiment. (G) Representative time course, quantitative analysis of the reciprocal changes in Cyto-RoGFP fluorescence intensity at the two excitation maxima (484 and 400 nm) for a single PASMC. Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 1

Hypoxia shifts the cytosol to a more oxidized state in PASMC. (A) Combined results from multiple experiments in PASMC expressing Cyto-RoGFP and superfused with either normoxic (21% O₂) or hypoxic (1.5% O₂) media. (B-E) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Cyto-RoGFP sensor. PASMC expressing Cyto-RoGFP were superfused with normoxic (21% O₂) media (B), then with hypoxic (1.5% O₂) media for 30 min (C), followed by media containing DTT to fully reduce the sensor (D), and then with media containing tBH to fully oxidize the sensor (E). (F) Representative time course, quantitative analysis of the fluorescence intensity ratio from representative individual PASMC imaged in B-E. ‘Img Fig’ denotes the time at which the images B-E were captured during the experiment. (G) Representative time course, quantitative analysis of the reciprocal changes in Cyto-RoGFP fluorescence intensity at the two excitation maxima (484 and 400 nm) for a single PASMC. Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 1

Hypoxia shifts the cytosol to a more oxidized state in PASMC. (A) Combined results from multiple experiments in PASMC expressing Cyto-RoGFP and superfused with either normoxic (21% O₂) or hypoxic (1.5% O₂) media. (B-E) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Cyto-RoGFP sensor. PASMC expressing Cyto-RoGFP were superfused with normoxic (21% O₂) media (B), then with hypoxic (1.5% O₂) media for 30 min (C), followed by media containing DTT to fully reduce the sensor (D), and then with media containing tBH to fully oxidize the sensor (E). (F) Representative time course, quantitative analysis of the fluorescence intensity ratio from representative individual PASMC imaged in B-E. ‘Img Fig’ denotes the time at which the images B-E were captured during the experiment. (G) Representative time course, quantitative analysis of the reciprocal changes in Cyto-RoGFP fluorescence intensity at the two excitation maxima (484 and 400 nm) for a single PASMC. Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 1

Hypoxia shifts the cytosol to a more oxidized state in PASMC. (A) Combined results from multiple experiments in PASMC expressing Cyto-RoGFP and superfused with either normoxic (21% O₂) or hypoxic (1.5% O₂) media. (B-E) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Cyto-RoGFP sensor. PASMC expressing Cyto-RoGFP were superfused with normoxic (21% O₂) media (B), then with hypoxic (1.5% O₂) media for 30 min (C), followed by media containing DTT to fully reduce the sensor (D), and then with media containing tBH to fully oxidize the sensor (E). (F) Representative time course, quantitative analysis of the fluorescence intensity ratio from representative individual PASMC imaged in B-E. ‘Img Fig’ denotes the time at which the images B-E were captured during the experiment. (G) Representative time course, quantitative analysis of the reciprocal changes in Cyto-RoGFP fluorescence intensity at the two excitation maxima (484 and 400 nm) for a single PASMC. Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 2

Hypoxia shifts the mitochondrial IMS to a more oxidized state in PASMC. (A-C) Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for cytochrome c (B), and demonstrating co-localization in mitochondria (C). (D) IMS-RoGFP targeted to the mitochondrial inter-membrane space of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). Note the restriction of labeling to the mitochondrial membrane on the mitochondrial periphery. (E) Combined results from multiple experiments in PASMC expressing IMS-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the IMS-RoGFP sensor. PASMC expressing IMS-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 2

Hypoxia shifts the mitochondrial IMS to a more oxidized state in PASMC. (A-C) Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for cytochrome c (B), and demonstrating co-localization in mitochondria (C). (D) IMS-RoGFP targeted to the mitochondrial inter-membrane space of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). Note the restriction of labeling to the mitochondrial membrane on the mitochondrial periphery. (E) Combined results from multiple experiments in PASMC expressing IMS-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the IMS-RoGFP sensor. PASMC expressing IMS-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 2

Hypoxia shifts the mitochondrial IMS to a more oxidized state in PASMC. (A-C) Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for cytochrome c (B), and demonstrating co-localization in mitochondria (C). (D) IMS-RoGFP targeted to the mitochondrial inter-membrane space of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). Note the restriction of labeling to the mitochondrial membrane on the mitochondrial periphery. (E) Combined results from multiple experiments in PASMC expressing IMS-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the IMS-RoGFP sensor. PASMC expressing IMS-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 3

Hypoxia shifts the mitochondrial matrix to a more reduced state in PASMC. Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for MnSOD (B), and demonstrating co-localization in mitochondria (C). (D) Mito-RoGFP targeted to the mitochondrial matrix of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). (E) Combined results from multiple experiments in PASMC expressing Mito-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Mito-RoGFP sensor. PASMC expressing Mito-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 3

Hypoxia shifts the mitochondrial matrix to a more reduced state in PASMC. Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for MnSOD (B), and demonstrating co-localization in mitochondria (C). (D) Mito-RoGFP targeted to the mitochondrial matrix of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). (E) Combined results from multiple experiments in PASMC expressing Mito-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Mito-RoGFP sensor. PASMC expressing Mito-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 3

Hypoxia shifts the mitochondrial matrix to a more reduced state in PASMC. Confocal images of PASMC expressing IMS-RoGFP (A), immunostained for MnSOD (B), and demonstrating co-localization in mitochondria (C). (D) Mito-RoGFP targeted to the mitochondrial matrix of rat PASMC as determined by electron microscopy. Primary antibody: mouse monoclonal anti-GFP; secondary antibody: goat anti-mouse IgG, conjugated to 10 nm gold particles (arrows). (E) Combined results from multiple experiments in PASMC expressing Mito-RoGFP and superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (F-I) False-color ratiometric images of PASMC demonstrate the dynamic redox range of the Mito-RoGFP sensor. PASMC expressing Mito-RoGFP were superfused with normoxic (21% O₂) media (F), then with hypoxic (1.5% O₂) media for 30 min (G), followed by media containing DTT to fully reduce the sensor (H) and then with media containing tBH to fully oxidize the sensor (I). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to normoxia.

Figure 4

Catalase attenuates hypoxia-induced oxidation of Cyto -RoGFP in PASMC. Cytosolic catalase was over-expressed in cells expressing Cyto-RoGFP (A) while mitochondrial catalase was over-expressed in cells expressing Mito-RoGFP (B). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia.

Figure 4

Catalase attenuates hypoxia-induced oxidation of Cyto -RoGFP in PASMC. Cytosolic catalase was over-expressed in cells expressing Cyto-RoGFP (A) while mitochondrial catalase was over-expressed in cells expressing Mito-RoGFP (B). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia.

Figure 5

Hypoxia shifts the redox status of subcellular compartments in SASMC. SASMC expressing Cyto-RoGFP (A), IMS-RoGFP (B), or Mito-RoGFP (C) were superfused under controlled O₂ conditions. Effects of hypoxia on [Ca²⁺]_i in PAMSC and SASMC assessed by YC2.3 (D). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia (A-C) and *p<0.05 compared to normoxic baseline (D).

Figure 5

Hypoxia shifts the redox status of subcellular compartments in SASMC. SASMC expressing Cyto-RoGFP (A), IMS-RoGFP (B), or Mito-RoGFP (C) were superfused under controlled O₂ conditions. Effects of hypoxia on [Ca²⁺]_i in PAMSC and SASMC assessed by YC2.3 (D). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia (A-C) and *p<0.05 compared to normoxic baseline (D).

Figure 5

Hypoxia shifts the redox status of subcellular compartments in SASMC. SASMC expressing Cyto-RoGFP (A), IMS-RoGFP (B), or Mito-RoGFP (C) were superfused under controlled O₂ conditions. Effects of hypoxia on [Ca²⁺]_i in PAMSC and SASMC assessed by YC2.3 (D). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia (A-C) and *p<0.05 compared to normoxic baseline (D).

Figure 5

Hypoxia shifts the redox status of subcellular compartments in SASMC. SASMC expressing Cyto-RoGFP (A), IMS-RoGFP (B), or Mito-RoGFP (C) were superfused under controlled O₂ conditions. Effects of hypoxia on [Ca²⁺]_i in PAMSC and SASMC assessed by YC2.3 (D). Values are means ± S.E., n=6 cover slips, 4-10 cells/cover slip. *p<0.05 compared to hypoxia (A-C) and *p<0.05 compared to normoxic baseline (D).

Figure 6

Model depicting spatial differences in hypoxia-induced ROS signaling.