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Biochem Biophys Res Commun. Author manuscript; available in PMC 2010 October 16.
Published in final edited form as:
PMCID: PMC2746082
NIHMSID: NIHMS137835

Pulsatile Shear Stress Increased Mitochondrial Membrane Potential: Implication of Mn-SOD

Abstract

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (ΔΨm) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on ΔΨm and the role of Mn-SOD expression on ΔΨm. Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and ΔΨm was monitored using tetramethylrhodamine methyl ester (TMRM+), a mitochondrial membrane potential probe. PSS significantly increased ΔΨm and the change in ΔΨm was a dynamic process. ΔΨm returned to baseline level after PSS for 2 hours followed by static state for 4 hours. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated ΔΨm increase while adding Mn-SOD mimetic, MnTMPyP, increased ΔΨm to the similar extent as induced by PSS. Our findings suggest that PSS increased mitochondrial ΔΨm, in part, via Mn-SOD up-regulation.

Keywords: Shear stress, mitochondria, membrane potential, Mn-SOD, endothelial cells

Introduction

Vascular oxidative stress plays an important role in atherosclerosis1-3. Hemodynamics, particularly fluid shear stress, regulates the generation of vascular nitrogen (RNS) and reactive oxygen species (ROS)4-6. While eNOS is the major source of RNS, NADPH oxidase system is considered as a major source of ROS in vascular endothelial cells4, 7. Mitochondria are also an important source of cellular superoxide anion (O2·-) and H2O22. Mitochondrial function is relevant to metabolic homeostasis8. Oxidative phosphorylation in the mitochondria drives the proton translocation across the mitochondrial inner membrane to intermembrane space9, generating an electrochemical proton gradient that is expressed as mitochondrial membrane potential (ΔΨm)10.

Mitochondrial ΔΨm is an important indicator of mitochondrial energetic state and cell viability10. ΔΨm is coupled with oxidative phosphorylation to drive ATP synthesis11, 12. During myocardial reperfusion injury, opening of the mitochondrial permeability transition pore (MPTP) collapses ΔΨm and uncouples oxidative phosphorylation, resulting in ATP depletion and apoptosis13, 14. Fluid shear stress is reported to influence mitochondrial ATP synthesis, which is coupled with ΔΨm15.

The formation of mitochondrial ROS (mtROS) is dependent on ΔΨm16, and mtROS level increases exponentially as ΔΨm is hyperpolarized above -140 mV17. In response to oxidative stress, mitochondrial manganese superoxide dismutase (Mn-SOD) is up-regulated18, leading to dismutation of O2·- anion to H2O2. In response to laminar shear stress, cytosolic CuZn-SOD expression is also up-regulated19. However, the potential mechanism whereby shear stress modulates ΔΨm via Mn-SOD remains unknown.

We hypothesized that pulsatile shear stress (PSS) increased ΔΨm via an up-regulation of Mn-SOD expression. In this paper, we provided the following new insights: (1) PSS increased ΔΨm in HAEC, (2) PSS up-regulated Mn-SOD mRNA and protein expression, and (3) adding Mn-SOD mimetic, MnTMPyP, increased ΔΨm to the similar extent as induced by PSS while silencing Mn-SOD attenuated PSS-induced increase in ΔΨm. Our findings suggest Mn-SOD up-regulation represents a potential pathway whereby shear stress influenced ΔΨm.

Materials and Methods

Methods

Endothelial Cell Culture

Human aortic endothelial cells (HAEC) were purchased from Cell Applications (San Diego, CA) and cultured in endothelial growth medium (Cell Applications). Endothelial cells (ECs) between passages 4 and 7 were seeded on Cell-Tak cell adhesive (Becton Dickson Labware, Bedford, MA) and Collagen Type I (BD bioscience, San Jose, CA) coated glass slides at 1.5 × 105 cells per slide (5 cm2). ECs were grown to confluent monolayers in endothelial growth medium for 48 hours in 5% CO2 at 37°C before being set up into the dynamic flow system.

Dynamic Flow System

Two-dimensional dynamic flow channels were used to implement shear stress simulating physiologic flow profiles in human carotid arterial bifurcation. Please see supplemental materials and methods for details.

Measurement of Mitochondrial Membrane Potential (ΔΨm)

After exposing the cells to flow conditions, mitochondrial membrane potential was measured with a cationic fluorescent dye, tetramethylrhodamine methyl ester (TMRM+) (Molecular probes, Carlsbad, CA). For details, please see supplemental materials and methods.

Conversion of TMRM+ Fluorescent Intensity to Voltage

Calculation of ΔΨm from fluorescence intensity was performed essentially as described by Nicholls20. To account for the plasma membrane potential (ΔΨp) in the intact cells, we monitored ΔΨp by “Membrane Potential Assay kit” (Molecular Devices, Sunnyvale, CA). The fluorescence intensity was captured at an excitation wavelength of 488 nm and emission of 527 nm (Olympus IX70). Calibration curve for ΔΨp was established by probing the fluorescent intensity at the potassium concentration, ranging from 3.9 mM to 80 mM, followed by application of the Nernst equation20.

Measurement of Mitochondrial Superoxide Production

Mitochondrial superoxide was measured with flow cytometry (FACS) after staining with mitochondrial superoxide specific dye MitoSOX Red (Invitrogen) as described21.

Quantitative RT-PCR

Mn-SOD mRNA expression is measured with quantitative RT-PCR. Please see supplemental materials and methods for details.

Mn-SOD Activity Assay

Cells exposed to static condition or shear stress were scrapped into PBS and spun down. The cells were lysed in Mn-SOD assay sample buffer (10mM HEPES, pH7.9, 420mM NaCl, 1.5mM MgCl2, 0.5mM EDTA, 0.1% Triton X-100) for 20 minutes in ice. After spin at 12000g for 5 minutes, the supernatant was collected for Mn-SOD activity assay. Mn-SOD activities were measured using the SOD assay kit from Cayman Chemicals (#706002 Ann Arbor, MI) in the presence of 2mM potassium cyanide, which inhibited the activities of Cu-Zn-SOD and EC-SOD but not Mn-SOD. Relative SOD activities were expressed as absorbance at 450 nm in the absence SOD less absorbance with sample or SOD standard. Reading in the presence of 1U/ml of SOD standard was used as blank. Relative Mn-SOD activities were normalized to protein concentration of samples.

Transfection of siRNA in Human Aortic Endothelial Cells

Silencer siRNAs for Mn-SOD were custom-designed by Ambion (Austin, TX). The sequences were as follows: sense sequence: GGC CUG AUU AUC UAA AAG Ctt, anti-sense sequence: GCU UUU AGA UAA UCA GGC Ctg. Confluent human aortic endothelial cells (HAEC) were trypsinized and re-suspended to 100,000 cells/mL in standard growth medium. siPORT NeoFX transfection reagent (Ambion, Austin, TX) was diluted in OPTI-MEM I (Invitrogen, CA) medium and incubated at room temperature for 10 minutes. Mn-SOD siRNA at a final concentration of 30 nM was diluted in OPTI-MEM I and mixed with diluted siPORT NeoFX reagent at room temperature for additional 10 minutes. The transfection solution was dispensed into the 6-well plates, followed by adding HAEC in suspension. The medium was changed to standard growth medium after 24 hours. The medium was replaced every other day until confluent HAEC monolayer developed. The cells were cultured for another 24hours and then used for experiments. Quantitative RT-PCR and Western blot were performed to assess the levels of silencing. Scrambled siRNA were used as the control.

Western Blot

Cells were harvested, washed with phosphate-buffered saline and lysed with RIPA buffer. The lysate was centrifuged at 12,000g for 10 minutes, and the resulting supernatants were used as the whole cell lysate. 15μg protein samples were size-separated in 10% SDS BioRad polyacrylamide electrophoresis gel (BioRad, CA). Mn-SOD and β-tubulin (loading control) were detected with rabbit Mn-SOD antibody (Upstate), mouse anti-β-tubulin antibody (Millipore). Chemiluminescence signal was developed with Supersignal Western Pico (Pierce) and recorded with FluorChem FC2 (Alpha Inotech Inc). Densitometry scan of western blot was done with the software come with FlorChem FC2 machine.

Statistical Analysis

Data are expressed as mean ± SD and compared among separate experiments. For comparisons between two groups, two-sample independent-groups t-test was used. Comparisons of multiple values were made by one-way analysis of variance (ANOVA), and statistical significance among multiple groups determined using the Tukey test (for pairwise comparisons of means between static-like and pulsatile flow conditions). p-values of < 0.05 were considered statistically significant.

Results

Characterization of Mitochondrial Membrane Potential (ΔΨm) in Human Aortic Endothelial Cells (HAEC)

We employed TMRM+ dye to measure mitochondrial membrane potential (ΔΨm). Mitochondrial respiratory chain inhibitors and uncouplers were used to validate the method in intact HAEC. Addition of FCCP, a protonophore to uncouple oxidative phosphorylation, depolarized ΔΨm in the intact HAEC and decreased the TMRM+ intensity by 30% compared to the control (P< 0.05, n=5). Treatment with rotenone, a NADH dehydrogenase inhibitor, depolarized ΔΨm and decreased the TMRM+ intensity by 56% (P < 0.05, n=5). Treatment with oligomycin, an ATP synthase inhibitor, hyperpolarized ΔΨm and increased TMRM+ intensity by 2.5-fold (P< 0.05, n=5). Furthermore, cyclosporine-A, an inhibitor of mitochondrial permeability transition pores (MPTP), increased the TMRM+ intensity by 2.6-fold (p< 0.05, n=5, supplemental Fig. 1A). The change in TMRM+ intensity were converted to ΔΨm (mV) as described in materials and methods with background level of ΔΨm set at -140mV. In control condition, ΔΨm was -140mV ± 6.9. In the presence of FCCP, Rotenone, Oligomycin and cyclosporine A, ΔΨm was -118.6mV ±5, -107.8mV± 7.3, -165mV± 4.2 and -165.3mV ±9.8, respectively (supplemental Fig.1B). These data were in agreement with published results 22, 23. Hence, the dynamic range of TMRM+ provided a basis to characterize shear stress-induced ΔΨm.

Pulsatile Shear Stress Increased Mitochondrial Membrane Potential (ΔΨm)

In response to pulsatile shear stress (PSS) simulating arterial flow at a time-averaged shear stress (τave) of 23 dyn·cm-2 for 2 and 4 hours, endothelial cells became energized as evidenced by an increase in ΔΨm (Fig. 1A. static: -140 mV±4.9; PSS 2 hours: -170.5±1.5 mV, P<0.05, n=4; PSS 4 hours: -187.5±5.6 mV, P<0.05, n=4). The dynamic changes in ΔΨm were consistent with those of physiological range in mammalian cells between -90 and -180 mV17. Fluorescence microscopy indicated that PSS increased TMRM+ uptake by HAEC (Fig. 1B), consistent with an increase in mitochondrial membrane potential. In parallel, PSS increased mitochondrial superoxide production (Fig. 1C). PSS is considered to be cardioprotective24 and our observations suggest that one of the possible mechanisms whereby PSS confers cardioprotection may be due to an increase in ΔΨm that is important for oxidative phosphorylation and ATP synthesis10.

Fig. 1
Pulsatile shear stress (PSS) increased mitochondrial membrane potential (ΔΨm)

Pulsatile Shear Stress Up-regulated Mn-SOD Expression in HAEC

In response to PSS for 4 hours, Mn-SOD mRNA level was up-regulated by 3.6-fold as compared to that of static condition (P <0.01, n=3) (Fig. 2A). Mn-SOD protein expression was also increased by 1.9-fold (Fig. 2B). In parallel, Mn-SOD activities were increased by 40% (Fig. 2C). Next, HAEC were subjected to PSS for 2 hours, followed by static condition for 4 hours. Mitochondrial ΔΨm was increased to -160±5.3 mV in response to PSS for 2 hours (Fig. 3A), a similar trend shown in Fig.1A. ΔΨm returned to near baseline value at -145±6.4 mV (baseline was -140 mV) after 4 hours in static condition. This finding suggests that changes in ΔΨm are a dynamic process in response to shear stress. Despite the static condition, the Mn-SOD protein and activity levels remain elevated (Fig. 3B & 3C), consistent with Mn-SOD mRNA up-regulation at 4 hours in response to PSS as previously reported in bovine aortic endothelial cells25.

Fig. 2
Pulsatile shear stress up-regulated Mn-SOD expression and activities
Fig. 3
Changes of mitochondrial membrane potential in response to PSS were a dynamic process

Mn-SOD Expression Modulated PSS-Mediated Mitochondrial Membrane Potential

To test the role of Mn-SOD on ΔΨm, we first treated HAEC with Mn-SOD mimetic, MnTMPyP, for 4 hours. MnTMPyP treatment resulted in an increase in ΔΨm to a similar extent as induced by PSS (Fig. 4A, p<0.05, n=3). To examine the effect of Mn-SOD level on PSS-mediated ΔΨm change, we transfected HAEC with Mn-SOD siRNA (siMn-SOD). siMn-SOD decreased Mn-SOD mRNA expression by 80% compared to scrambled siRNA in HAEC (Fig. 4B). siMn-SOD significantly attenuated PSS-mediated ΔΨm (Fig. 4C, p<0.05, n=3). Taken together, our findings support the notion that PSS-induced Mn-SOD expression plays a role on changes in ΔΨm.

Fig. 4
Mn-SOD mediated PSS-increased mitochondrial membrane potential

Discussion

In this study, we employed a dynamic fluorescent technique to monitor mitochondrial membrane potential (ΔΨm). We demonstrated that pulsatile shear stress (PSS) increased ΔΨm in the intact aortic endothelial cells (HAEC). PSS also up-regulated mitochondrial Mn-SOD expression. Treatment of HAEC with Mn-SOD siRNA attenuated PSS-mediated increase in ΔΨm while Mn-SOD mimetic, MnTMPyP, increased ΔΨm to similar extent as PSS. Our findings suggest that PSS hyperpolarized ΔΨm via Mn-SOD up-regulation.

Maintenance of ΔΨm at physiologic range (~ -90mV to -150mV) is critical for cellular function 17. Important factors that influence ΔΨm include (1) proton leakage across the inner membrane, (2) ATP synthesis or hydrolysis, (3) substrate availability, (4) electron flux through the respiratory chain, and (5) ion transport26. The methyl esters of tetramethylrhodamine (TMRM+) is useful for monitoring mitochondrial depolarization relevant to cytosolic Ca++ transient and for imaging time-dependent mitochondrial membrane potential27. The precise concentration of TMRM+ was critical to detect step changes in ΔΨm. At a low concentration (10 nM), TMRM+ operates at the distribution/redistribution mode. When the mitochondrial membrane is depolarized, TMRM+ in mitochondria undergoes dequenching, rendering a decrease in fluorescent intensity. Distribution mode is applicable to our experiments that involved slow step changes in ΔΨm at a low TMRM+ concentration27.

Several lines of evidence support that shear stress increases exogenous ATP release. ATP production is a process dependent on ΔΨm8. Kudo et al. demonstrated an increase in ΔΨm and ATP production in endothelial cells exposed to laminar shear stress for 48 hours in a parallel plate flow system15. Our data showed a hyperpolarized ΔΨm in response to pulsatile shear stress within 2 hours. An increase in ΔΨm is accompanied with an increase of ATP release and the generation of mitochondrial reactive oxygen species (mtROS) when ΔΨm is above -140mV11, 12. In response to PSS, mitochondrial superoxide generation in HAEC was increased compared to static condition (Fig. 1C). Thus PSS can modulate ΔΨm and mitochondrial redox state with an implication for endothelial function.

Over-expression of Mn-SOD or SOD mimetic has been shown to protect against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function28. Mn-SOD was also reported to suppress selenite-induced decrease in mitochondrial membrane potential29. Furthermore, over-expression of Mn-SOD reduced polarization of colorectal cancer cells30. In this study, we demonstrated that PSS increased ΔΨm in endothelial cells, which was at least partially mediated via up-regulation of Mn-SOD expression. Hence, Mn-SOD is important in maintaining homeostasis of endothelial cell function as an antioxidant31, 32. Although the precise mechanisms remain to be identified, Mn-SOD-mediated membrane potential increase is likely related to its antioxidant activity since the activity of proton pumps depends on the reductive power at the respiratory chain of mitochondria.

A host of factors modulates mitochondria membrane potential. While Mn-SOD may contribute to PSS-mediated increase in ΔΨm, other factors such as metabolic status and ATP level may dominate. Our data showed that ΔΨm returned to baseline level (-145mV) after 2 hours of PSS followed by 4 hours of static condition. At the same time, both Mn-SOD protein levels and activities remained elevated. These data are consistent with shear stress-induced Mn-SOD mRNA at 4 hours and the ensuing protein expression observed in bovine aortic endothelial cells25. While membrane potential returned to baseline after shear stress exposure, the persistent elevated Mn-SOD may plays an important role in dismutating mitochondrial superoxide production to maintain mitochondrial homeostasis8.

In summary, PSS favors mitochondrial energetic state with an implication for mitochondrial function in endothelial cells15. In this paper, we linked pulsatile shear stress with mitochondrial ΔΨm in the intact vascular endothelial cells via Mn-SOD regulation. Regulation on ΔΨm may represent one of the mechanisms whereby pulsatile shear stress confers a cardioprotective effect. The precise mechanisms will be of important interest for future investigation.

Supplementary Material

02

Acknowledgments

The authors were grateful for the scientific advice from Dr. Enrique Cadenas of the Department of Molecular Pharmacology & Toxicology at the University of Southern California. The authors also appreciate the technical advice from David G. Nicholls of Buck Institute for Age Research, Novato, California. This work was supported by AHA Pre-Doctoral Fellowship 0615063Y (MR), AHA GIA 0655051Y (TKH), NIH HL 83015 (TKH), and HL NIH HL068689 (TKH).

Footnotes

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