Hypoxia induces pulmonary artery smooth muscle dysfunction through mitochondrial fragmentation-mediated endoplasmic reticulum stress

Figures

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  Figure 1. Hypoxia-induced PASMC dysfunction. (A) Hypoxia increased mean pulmonary artery pressure (mPAP) in rats. (B) Hypoxia increased pulmonary vessel resistance (PVR) in rats. (C) PE-induced vasoconstriction presented as percentage of maximal contraction. (D) PE-induced vasoconstriction presented as contraction force per tissue weight (g/mg tissue). (E, F) Both endothelium-dependent (E) and endothelium-independent (F) vasodilation was impaired in hypoxic rats. *, p<0.05, **, p<0.01. n=8.

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  Figure 2. Hypoxia induced mitochondrial fragmentation and ER stress in PASMCs. (A) Ultrastructural alteration of the mitochondria and ER in pulmonary artery smooth muscle of normoxic and hypoxic rats. Scale bar, 500 nm. (B) Expression of mitochondrial dynamics-related proteins in endothelium-denuded pulmonary arteries. Twenty micrograms of protein was loaded in each lane. (C) Mitochondrial ROS detected by mitoSOX in isolated pulmonary arteries. Scale bar, 20 μm. (D) ER stress as assessed by CHOP expression in endothelium-denuded pulmonary arteries. Twenty micrograms of protein was loaded for each lane. *, p<0.05, **, p<0.01. n=8.

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  Figure 3. Mitochondrial fragmentation enhanced ER stress in cultured PASMCs under hypoxia. (A) Mdivi-1 treatment decreased Drp1 and Drp1 phosphorylation at serine 616 in PASMCs under hypoxia. Twenty micrograms of protein was loaded for each lane. (B) Mdivi-1 treatment inhibited mitochondrial fragmentation in PASMCs under hypoxia. Scale bar, 20 μm. (C) Mdivi-1 treatment inhibited ER stress as evidenced by the decreased CHOP expression in PASMCs under hypoxia. Twenty micrograms of protein was loaded for each lane. (D) Mdivi-1 treatment improved PASMC function as evidenced by increased PE/SNP-induced MLC phosphorylation/dephosphorylation in PASMCs under hypoxia. Twenty micrograms of protein was loaded for each lane. *, p < 0.05, **, p < 0.01. n = 8.

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  Figure 4. Drp1 overexpression induced ER stress. (A) Drp1 was overexpressed by adenovirus in PASMCs. Twenty micrograms of protein was loaded for each lane. (B) Overexpression of Drp1 induced mitochondrial fragmentation as detected by mitoTracker. Scale bar, 20 μm. (C) Overexpression of Drp1 increased mitochondrial ROS as detected by mitoSOX. Scale bar, 100 μm. (D) Drp1 overexpression increased CHOP expression in PASMCs. Twenty micrograms of protein was loaded for each lane. (E) Drp1 overexpression decreased PASMCs function as evidenced by decreased PE/SNP-induced MLC phosphorylation/dephosphorylation in PASMCs. Twenty micrograms of protein was loaded in each lane. *, p < 0.05, **, p < 0.01. n = 8.

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  Figure 5. Inhibition of ER stress showed little effects on mitochondrial morphology but improved cell function in PASMCs under hypoxia. (A) PBA and TUDCA decreased CHOP expression in PASMCs under both normoxia and hypoxia. Twenty micrograms of protein was loaded in each lane. (B) PBA and TUDCA improved PASMC function as evidenced by increased PE/SNP-induced MLC phosphorylation in PASMCs under hypoxia. Twenty micrograms of protein was loaded in each lane. (C, D) PBA and TUDCA showed little effects on mitochondrial morphology (C) and mitochondrial ROS (D) in PASMCs under hypoxia. Scale bar, 20 μm in C and 100 μm in D. *, p < 0.05, **, p < 0.01. n = 8.

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  Figure 6. Mitochondrial ROS mediated the interaction between mitochondria and ER. (A) SS31 and mitoTEMPO scavenged mitochondrial ROS as detected by mitoSOX. Scale bar, 100 μm. (B) SS31 and mitoTEMPO inhibited hypoxia-induced mitochondrial fragmentation in PASMCs. Scale bar, 20 μm. (C) SS31 and mitoTEMPO showed little effects on Drp1 expression and Drp1 phosphorylation at serine 616 in PASMCs under hypoxia. Twenty micrograms of protein was loaded in each lane. (D) SS31 and mitoTEMPO decreased CHOP expression in PASMCs under hypoxia. Twenty micrograms of protein was loaded in each lane. (E) SS31 and mitoTEMPO improved PASMC function as evidenced by increased PE/SNP-induced MLC phosphorylation/dephosphorylation in PASMCs under hypoxia. Twenty micrograms of protein was loaded in each lane. (F) Inhibition of mitochondrial fragmentation using Mdivi-1 decreased mitochondrial ROS in PASMCs in hypoxia. *, p < 0.05, **, p < 0.01. n = 8.

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  Figure 7. Inhibition of mitochondrial fragmentation using Mdivi-1 improved pulmonary artery smooth muscle function in response to hypoxia in vivo. (A) Mdivi-1 treatment decreased Drp1 and Drp1 phosphorylation at serine 616 in PASMCs of hypoxic rats. Twenty micrograms of protein was loaded in each lane. (B, C) Mdivi-1 treatment decreased both mPAP and PVR in hypoxia rats. (D) Mdivi-1 treatment deceased ER stress as detected by CHOP in isolated endothelium-denuded pulmonary arteries from hypoxic rats. Twenty micrograms of protein was loaded in each lane. (E, F) Mdivi-1 treatment improved PE-induced vasoconstriction (E) and ACh/SNP-induced vasodilation (F) in isolated pulmonary arteries from hypoxic rats. *, p < 0.05, **, p < 0.01. n = 8.