Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 1;317(5):L639-L652.
doi: 10.1152/ajplung.00396.2018. Epub 2019 Aug 28.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension

Karthik Suresh  1 , Laura Servinsky  1 , Haiyang Jiang  1 , Zahna Bigham  1 , Joel Zaldumbide  1 , John C Huetsch  1 , Corrine Kliment  1 , Michelle G Acoba  2 , Brian J Kirsch  3 , Steven M Claypool  2 , Anne Le  3 , Mahendra Damarla  1 , Larissa A Shimoda  1
Affiliations

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension

Karthik Suresh et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Keywords: PAH; calcium; endothelium; mitochondria.

PubMed Disclaimer

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
Hemodynamics and microvascular endothelial cell (MVEC) mitochondrial fragmentation in the Sugen/hypoxia (SuHx) model of pulmonary arterial hypertension (PAH). A: representative tracings of closed-chest right ventricular systolic pressure (RVSP) measurement in normoxic (N) and SuHx rats. Scatter plots showing means ± SE for RVSP (B), right ventricle/left ventricle+septal weight (RV/LV+S; C), and RV/body weight in N- and SuHx rats; D). *Significant difference from normoxic animals (t test). E: representative photomicrographs and reconstructed mitochondrial network images for N- and SuHx-MVECs. F: length distribution curves for N- and SuHx-MVEC mitochondria. *Significant difference from N-MVECs (ANOVA); n = 5/group.
Fig. 2.
Fig. 2.
Effect of mitochondrial reactive oxygen species (mtROS) quenching on mitochondrial fragmentation. A: Representative mitochondrial network images in N- and SuHx-MVECs in the absence and presence of MitoQ (MQ; 1µM – 1 h). B and C: length distribution curves (with SE at each distribution length) for mitochondria from N- and SuHx-MVECs with and without MQ treatment; n = 5 per/group (ANOVA). D and E: Western blot and densitometry showing pSer616Drp1 and total Drp1 protein levels in N- and SuHx-MVECs with and without MQ treatment, each n from a different animal. *Significant difference from normoxic control. **Significant difference from SuHx-MVECs (ANOVA).
Fig. 3.
Fig. 3.
mtROS production in N-MVECs. A: scatter plot showing means ± SE for roGFP ratios in N-MVECs with and without treatment with antimycin A (AA; 10 µM). B: representative mitochondrial network images in untreated, AA-, and HC+AA-treated N-MVECs. C: length distribution curves for N-MVEC mitochondria in untreated, AA-, and HC+AA-treated N-MVECs. *Significant difference from normoxic control (ANOVA); n = 5/group.
Fig. 4.
Fig. 4.
Induction of fusion in N- and SuHx-MVECs. Representative images (A and C) and length distribution curves (B and D) in N- and SuHx-MVECs in basal (5% FBS) serum and after incubation in serum-free media for 2 h.*Significant difference from untreated SuHx control; n = 10–15 images from 5 different animals. E and F: representative immunoblots and densitometry showing mitofusin-2 (Mfn-2) protein levels in N- and SuHx-MVECs. *Significant difference from N-MVEC control. **Significant difference from SuHx control (ANOVA); n = 5/group.
Fig. 5.
Fig. 5.
Transient receptor potential vanilloid-4 (TRPV4) and mitochondrial fragmentation in SuHx-MVECs. A: scatter plot showing means ± SE for roGFP ratios in N- and SuHx-MVECs with and without treatment with GSK2193874 (GSK2; 30 nM); n = 5–6 (from different animals)/group. Representative network images (B) and length distribution curves (C–F) for N- and SuHx-MVEC mitochondria with and without treatment with 2 TRPV4 inhibitors: HC-067047 (HC; 10 µM) and GSK2 (30 nM). *Significant difference from SuHx-MVECs (ANOVA).
Fig. 6.
Fig. 6.
GSK1016790A (GSKA)-induced Ca2+ influx in SuHx-MVECs. A and B: representative traces showing intracellular Ca2+ concentration ([Ca2+]i) in N- and SuHx-MVECs with and without MQ treatment at baseline and following perfusion with TRPV4 agonist GSKA (1.5 µM). C: scatter plot showing means ± SE baseline and GSKA-induced changes in [Ca2+]i in N- and SuHx-MVECs in the absence and presence of MQ. D: scatter plot showing means ± SE change in [Ca2+]i (nM) in N-MVECs before and after GSKA (N vs. N+GSKA), untreated SuHx-MVECs before and after GSKA (S vs. S+GSKA), and MQ-treated SuHx-MVECs before and after GSKA (S+MQ vs. S+MQ+GSKA). *Significant difference from N-MVECs. **Significant difference from SuHx-MVECs (ANOVA).
Fig. 7.
Fig. 7.
Effect of mtROS and Ca2+ inhibition on mitochondrial respiration. Curves of mitochondrial oxygen consumption rate (OCR) in N- and SuHx-MVECs at baseline and following treatment with MQ (A and B) and HC (C and D). Scatter plots showing means ± SE for basal OCR (E) and extracellular acidification rate (ECAR; F) in N-MVECs normalized to untreated N-MVEC controls. *Significant difference from N-MVEC control. **Significant difference from SuHx-MVECs (ANOVA).
Fig. 8.
Fig. 8.
A: heat map showing fold-change differences in glycolysis and TCA metabolites in N- and SuHx-MVECs (n = cells isolated from 8 individual animals). B: table showing median fold-change (and IQR) for metabolite levels; n = 8 biological replicates. C: scatter plot showing fold-change in ATP levels in SuHx-MVECs. D: scatter plot showing fold-change in extracellular lactate at baseline and following treatment (24 h) with MitoQ (MQ) or HC-067047 (HC). *Significant difference from normoxic control (t test). **Significant difference from untreated SuHx-MVEC control (ANOVA).
Fig. 9.
Fig. 9.
Schematic describing our proposed pathway of interaction between TRPV4 and mitochondrial fragmentation in SuHx-MVECs (solid lines) as well as other established pathways linking mitochondrial dysfunction to EC migration/proliferation (dashed lines). aIncreased ROS production, decreased basal/maximal respiration, increased fission, and evidence of glycolytic shift. bDirect effects of mtROS on transcription (3). cChanges in fuel utilization following glycolytic shift providing carbons (i.e., anaplerosis) for generation of metabolites essential for biosynthetic activities such as proliferation (79).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adapala RK, Talasila PK, Bratz IN, Zhang DX, Suzuki M, Meszaros JG, Thodeti CK. PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells. Am J Physiol Heart Circ Physiol 301: H757–H765, 2011. doi:10.1152/ajpheart.00142.2011. - DOI - PMC - PubMed
    1. Aggarwal S, Gross CM, Sharma S, Fineman JR, Black SM. Reactive oxygen species in pulmonary vascular remodeling. Compr Physiol 3: 1011–1034, 2013. doi:10.1002/cphy.c120024. - DOI - PMC - PubMed
    1. Al-Mehdi AB, Pastukh VM, Swiger BM, Reed DJ, Patel MR, Bardwell GC, Pastukh VV, Alexeyev MF, Gillespie MN. Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Sci Signal 5: ra47, 2012. doi:10.1126/scisignal.2002712. - DOI - PMC - PubMed
    1. Alvarez DF, King JA, Weber D, Addison E, Liedtke W, Townsley MI. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier: a novel mechanism of acute lung injury. Circ Res 99: 988–995, 2006. doi:10.1161/01.RES.0000247065.11756.19. - DOI - PMC - PubMed
    1. Archer SL. Mitochondrial dynamics—mitochondrial fission and fusion in human diseases. N Engl J Med 369: 2236–2251, 2013. doi:10.1056/NEJMra1215233. - DOI - PubMed

Publication types

MeSH terms