Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells

Cell culture

HCASMCs (Cat no. C-12511) were obtained from Promocell and maintained in smooth muscle growth medium 2 supplemented with smooth muscle growth medium 2 supplement mix (Promocell, Heidelberg, Germany) with a final supplement concentration of (per ml): 5% v/v fetal calf serum, 0.5 ng epidermal growth factor (recombinant human), 2 ng basic fibroblast growth factor (recombinant human) and 5 µg insulin (recombinant human). Penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) was added to cell culture medium with a final concentration of 100 U/mL and 100 µg/mL, respectively. Cells were used between P7-P13. The expression of signature smooth muscle proteins, SMC α-actin, calponin and myosin heavy chain (MHC) was maintained for the duration of culture as detected by Western blot (WB) (Fig. S1) and immunocytochemistry (ICC) (Fig. S2).

Subcultivation of HCASMCs

HCASMCs were grown in T75 cm² flasks with 15 ml fully supplemented smooth muscle growth medium 2 in a 5% CO₂ humidified incubator at a temperature of 37 °C. Cells were routinely passaged every 48–72 h when reaching about 70–80% confluency. Cell culture medium was aspirated, and the cells were washed with 15 ml pre-warmed Dulbecco’s Phosphate Buffered Saline (DPBS, no Ca²⁺/Mg²⁺, Cat no. 14190-144, Gibco) or HEPES Buffered Saline Solution (HepesBSS). Cells were then trypsinised with 1.5 ml versene (Gibco) containing 0.025% trypsin (Gibco) or 100 µl per cm² Trypsin/EDTA Solution (0.04%/0.03%) for approximately 1 min. Enzyme was neutralized by addition of 8 ml fully supplemented cell culture medium or 100 µl per cm² Trypsin Neutralizing Solution (TNS). HepesBSS, Trypsin/EDTA Solution and TNS were all contained in Promocell DetachKit (Cat no. C-41200). Cells were then re-suspended in fully supplemented fresh cell culture medium and cultured in T75 cm² flasks at a volume of 15 ml.

Western blotting

Proteins were separated according to their size by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred from polyacrylamide gel onto nitrocellulose membrane (Amersham Hybond ECL, Cat no. RPN303D) for western blot analyses. Ponceau S solution (Sigma, Cat no. P7170) for 5 min to check the transfer efficiency of the protein. Membranes were blocked in 1% w/v non-fat powdered milk (Marvel) before incubation with the following primary and secondary antibodies: anti α-actin (Sigma-Aldrich, Cat no. A2547) at 500 fold dilution, anti calponin (Sigma-Aldrich, Cat no. C2687) at 500 fold dilution, anti actin (Sigma-Aldrich, Cat no. A4700) at 500 fold dilution and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Fitzgerald Industries International, Cat no. 43C-CB1569-FIT) at 10,000 fold dilution. Protein bands were detected with ECL western blotting detection reagents (Amersham, Cat no. RPN2109). Bands were sized by referencing to the rainbow protein mass marker (Amersham full-range rainbow MW marker, Cat no. RPN800E).

Immunocytochemistry

HCASMCs were fixed with 2% w/v paraformaldehyde in PBS (in mM: 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8.0 Na2HPO4, pH 7.4) for 10 min and then incubated in permeabilisation solution (0.1% v/v Triton X-100 in PBS, pH adjusted to 7.4 using 1 M NaOH) for 10 min at room temperature. Next, the cells were incubated with 200 µl antibody diluting solution (in mM: SSC containing 250 NaCl and 15 Na₃Citrate, 2% v/v goat serum, 0.05% v/v Triton X-100, 1% w/v bovine serum albumin, pH 7.2) for 30 min at room temperature to block non-specific binding. Cells were then incubated overnight at 4 °C with the following primary antibodies: anti α-actin (Sigma-Aldrich, Cat no. A2547) at 300 fold dilution, anti calponin (Sigma-Aldrich, Cat no. C2687) at 300 fold dilution and anti MHC (Novocastra, Cat no. NCL-MHCs) at 300 fold dilution. On the following day, primary antibodies were indirectly stained using fluorescent Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Molecular probes, Cat no. A-11029) at the dilution of 1:500 for 2 h at room temperature. A LSM510 multiphoton laser-scanning confocal microscope was used to visualize the labelled cells. Alexa Fluor 488 was excited at 488 nm using an argon-ion laser, and emission light was collected at 500–550 nm.

Cell transfection

Plasmids encoding Perceval, PercevalHR and pHRed were obtained from Addgene (Cambridge, MA). HCASMCs expressing PercevalHR or pHRed were transiently transfected using either FuGENE6 (Promega UK, Southampton, UK) or Promofectin (Promocell, Heidelberg, Germany) following manufacturer’s instruction. HCASMCs were seeded 24 h prior to transfection at a density of 1.25 × 10⁵ per well in 35 mm glass-bottom dishes (Greiner Bio-One, Gloucestershire, UK). The cells expressing Perceval and PercevalHR were used after 24–48 h of transfection, and cells expressing pHRed were also used after 24-48 h of transfection. The cells expressing PercevalHR based on a conventional 3rd generation lentivirus packaging system which has been described previously were used in PercevalHR in vitro calibration experiments (Yang et al., 2017).

Confocal imaging

Experiments were carried out using either culture medium or physiological saline solution (PSS) containing [in mM]: 120 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 24 NaHCO₃, and 10 Glucose. Fluorescence signal of Perceval/PercevalHR and pHRed was measured with LSM510 (Carl Zeiss AG, Oberkochen, Germany), which allowed control of CO₂, humidity and temperature while environmental O₂ can also be changed to create hypoxic conditions. Manipulating O₂ tension in the custom made micro-imaging chamber (atmospheric gas) was achieved by an O₂ controller (# 0508.000, PeCon GmbH, Germany). This system has been specifically designed to image cultured cells under conditions of varying O₂ tension, achieved by changing the O₂ level in the atmosphere of the micro-imaging chamber. Images in the experiments of PercevalHR calibration were taken using LSM510 high speed multiphoton confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany). Software analysis of the data from LSM510 and LSM510 Multiphoton were carried out by region of interest analysis using AIM software, version 3.2 SP2 (Carl Zeiss AG).

Measurement of intracellular ATP:ADP ratio, pH and membrane potential

Cells expressing Perceval or PercevalHR were excited with 488 nm light from an argon laser, and emission was collected at 500–550 nm. Cells expressing pHRed was excited at 458 nm, and emission signal over 575 nm was collected. To examine change in membrane potential, HCASMCs were plated into 35 mm glass-bottom dishes a day before experiments. Next day, cells were washed twice with 2 ml PSS, then incubated with 1 µM DiBAC₄(3) dissolved in PSS for 15 min in a 37 °C/5%CO₂ incubator. Cells were then used without washing the dye as the continued presence of the dye in the extracellular media was required for this experiment. Cells were excited with 488 nm and the emission signal was captured at 500–550 nm. The photon counts (F) from Perceval/PercevalHR, pHRed and DiBAC₄(3) were normalized against the initial reading (F₀) at the start of experiments and expressed as a ratio (relative fluorescence (F/F₀)).

Permeabilisation of HCASMCs with Staphylococcus aureus α-toxin

It is of high importance that the signals from biosensors are calibrated in vitro to validate that sensitivity and range are satisfactory for planned experiments. Furthermore, although Perceval and PercevalHR are supposed to detect ATP:ADP ratio, it has been also reported that their signals are independent of ADP (Li et al., 2013). Thus, attempts were made to calibrate the PercevalHR using solutions containing known concentration of ATP and ADP. To achieve this, HCASMCs have to be permeabilised in such a way that hydrophilic nucleotides can be introduced into the cells without loss of biosensor. Thus, permeabilisation with α-toxin was carried out for PercevalHR in vitro calibration. Preliminary experiments resulted in, however, loss of HCASMCs during solution exchange due to cell detachment from the glass. This was due to the excessive HCASMC contraction in the solution once cells were permeablised. The reason for this is unknown, but in order to minimize loss of cells, 35 mm glass-bottom dishes were coated with Poly D-Lysine and 10 µM wortmannin was added to solutions.

HCASMCs were washed with calibration solution A containing (mM) 110 KCl, 30 NaOH, 10 KCl, 10 HEPES, 10 EGTA, and 0.05 EDTA (pH adjusted to 7.2) to remove Ca²⁺. Cells were then further washed with calibration solution B containing (in mM) 140 KCl, 10 NaCl, 10 HEPES, 2 EGTA, and 0.05 EDTA. Next, Cells were permeabilised with calibration solution B containing 200 µg/ml α-toxin for about 1 h at room temperature. Finally, cells were washed with calibration solution B and in vitro calibration was carried out using calibration solution B containing required concentrations of nucleotides while keeping free Mg²⁺ constant at 0.5 mM (All calculations were done using MAXCHELATOR: http://maxchelator.stanford.edu/).

Drugs

DiBAC₄(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K⁺ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).

Statistical analysis

When appropriate, values are expressed as a mean ± standard error of mean (SEM). N indicates number of cells unless otherwise stated. Statistical significance was evaluated with a paired Student’s t-test or with ANOVA with Tukey’s test for post hoc analysis using SPSS software package (version 20.0, IBM, New York, NY).

In vitro calibration using α-toxin permeablized HCASMCs confirms PercevalHR detects ATP concentration over physiological range and is sensitive to intracellular ADP

Perceval is a fluorescent biosensor of adenylate nucleotides (Berg, Hung & Yellen, 2009) with T loop of ATP binding bacterial protein GlnK1 containing a yellow cpFP and circularly permuted monomeric Venus (cpmVenus). The ATP/ADP binding site is always occupied due to very high affinity, and the competition between ATP and ADP makes the sensor a reporter of intracellular ATP:ADP ratio (Berg, Hung & Yellen, 2009). PercevalHR is an improved fluorescent biosensor and is better tuned to sense the ATP:ADP ratios expected in healthy mammalian cells (Tantama et al., 2013; Tantama & Yellen, 2014).

First, the sensitivity of PercevalHR to ATP over a physiological range and the effect of ADP on signal were assessed using α-toxin permeabilized HCASMCs. Figure 1A indicates that relative fluorescence (F/F₀), determined in the presence of 10 mM ATP at the start of the experiment was reduced by a third with ATP-free solution. Upon the re-introduction of 10 mM ATP, relative fluorescence recovered to the previous level (Fig. 1A). The response of fluorescence probe to 10 mM and 0 mM ATP was stable and repeatable over 100 min, establishing that PercevalHR can report ATP level for a prolonged period.

Next, experiments were carried out to determine the K_D of PercevalHR in vitro using extracellular solutions containing 10, 3, 1 and 0 mM ATP. Figure 1B depicts a representative result showing confocal images of α-toxin-permeabilized HCASMCs expressing PercevalHR (Figs. 1B–1F) and the time-course of relative fluorescence change with varying concentrations of extracellular ATP (Fig. 1G). The original data show that the signal strength of PercevalHR was proportional to the concentration of extracellular ATP. When concentration–response curve was constructed, curve fitting showed a half maximal effect occurring at ∼3.3 mM of ATP (Fig. 1H).

As described earlier, one publication reported that Perceval is insensitive to ADP (Li et al., 2013). This is an important unresolved issue as some metabolically sensitive detectors such as K_ATP channels are regulated by not only absolute amount of ATP but also ADP (Quayle et al., 1994). We therefore studied the effect of intracellular ADP on PercevalHR fluorescence measured using 1 mM ATP in the presence of either 0.1 or 1 mM ADP. As shown in Fig. 1I, increasing ADP concentration from 0.1 mM to 1 mM reduced relative fluorescence significantly (p < 0.01) indicating that PercevalHR reports change in ATP:ADP ratio. Taken together, results obtained with permeabilised HCASMCs showed that PercevalHR can be used as a reporter of physiological ATP:ADP change.

Removal of glucose from extracellular solution reduces the ATP:ADP ratio in HCASMCs

Figures 2A–2C shows that Perceval fluorescence remained stable over a period of 40 min when the cell was perfused continuously with PSS. However, upon the removal of 10 mM extracellular glucose, the fluorescent signal dimmed (Figs. 2D–2E) and relative fluorescence decreased by almost 50% over 30 min (Fig. 2F). Removal of glucose significantly reduced relative fluorescence by 25.7 ± 8.3% (p < 0.05, n = 6, Fig. 2G). This reduction was not due to the bleaching of biosensor or loss of focus as the re-introduction of 10 mM extracellular glucose restored the signal (original trace, Fig. 2H; summary of 3 cells, Fig. 2I). When the glucose analogue, 2-deoxyglucose (2-DG, 5mM) that cannot be metabolized by the cell, was used, fluorescent signal became weaker (Figs. 2J–2L). Mean results from 3 cells showed that Perceval fluorescence signal was reduced by 54.0 ±15.3% after the application of 2-DG (n = 3, Fig. 2M). These results indicate that removal of extracellular glucose affects ATP:ADP ratio to a degree that is detectable by Perceval.

Inhibition of oxidative phosphorylation causes a measurable decrease in cellular ATP:ADP ratio

ATP can be generated using extracellular glucose by oxidative phosphorylation and glycolysis pathways. Therefore, whether inhibition of oxidative phosphorylation affects PercevalHR signal was examined next in the cell culture medium. Oxidative phosphorylation was blocked by a number of approaches: inhibiting the electron transport chain with rotenone (1 µM), an inhibitor of mitochondrial complex I, or antimycin (1 µM), an inhibitor of mitochondrial complex III; inhibiting ATPase activity with oligomycin (6 µM), or through use of the uncoupling agent CCCP (1 µM). As shown in Figs. 3B–3E, application of metabolic inhibitors leads to a decrease in relative fluorescence. The effects of metabolic inhibitors, along with that of vehicle (DMSO), are summarized in Fig. 3F. When compared to the fluorescence ratio just before the administration of metabolic inhibitors, application of antimycin and CCCP induced significant reduction in ATP:ADP ratio signal (both p < 0.05).

Like other biosensors, Perceval may respond to changes in intracellular pH, which may accompany hypoxia (Berg, Hung & Yellen, 2009). Thus, the effect of hypoxia on the fluorescence of a pH sensor, pHRed, was also examined (Tantama, Hung & Yellen, 2011). pHRed responds robustly to intracellular alkalinization and acidification induced by the addition and subsequent removal of extracellular NH₄Cl (20 mM) (Dart & Vaughan-Jones, 1992) (Fig. S3). There was little change in intracellular pH in response to metabolic interventions, as assessed by pHRed in cell culture medium (Fig. S4).

Effect of hypoxia on cellular ATP:ADP ratio and pH

Figure 4A-C show confocal images of HCASMC transiently transfected with Perceval under normoxia (20% oxygen), hypoxia (1% O₂), and following the restoration of oxygen (20%). Fractional ATP:ADP ratio was reduced by about 10% by hypoxia and recovered upon oxygen reintroduction (Fig. 4D). Exposure to hypoxia significantly reduced Perceval signal by 10.3 ± 2.1%, and the change was reversible (p < 0.05, n = 5, Fig. 4E). There was no change in Perceval signal when mild hypoxia (10% and 5% O₂) was tested (Fig. S5). pHRed however detected no change in intracellular pH after exposure to hypoxia (Figs. 4F–4I), suggesting that the fluorescence change induced in Perceval was due to changes in the ATP:ADP ratio and not an artefact caused by shift in intracellular pH. All the experiments were conducted in cell culture medium.

Identification of K⁺ channels setting resting membrane potential of HCASMCs

DiBAC₄(3) is a slow-response potential-sensitive fluorescent probe (Apell & Bersch, 1987; Mustafa et al., 2011), and thus a useful tool for HCASMCs where changes in membrane potential are generally slow and steady. It is permeable to the plasma membrane, accumulating in the cytoplasm following a Nernst equilibrium distribution (Apell & Bersch, 1987). The anionic dye in the cell binds to intracellular proteins or to membranes, resulting in an enhanced fluorescence and also a red spectral shift (Epps, Wolfe & Groppi, 1994; Klapperstuck et al., 2009). Depolarization of membrane potential causes entry of the anionic dye into the cell, increasing the fluorescence signal. Hyperpolarization, on the other hand, results in a decrease in the signal. Compared to cationic carbocyanines, DiBAC₄(3) is largely excluded from mitochondria due to their overall negative charge, and so largely measures changes in the plasma membrane potential (Apell & Bersch, 1987; Baczkó, Giles & Light, 2004). To establish that DiBAC₄(3) can detect moderate changes in membrane potential in HCASMCs, initial experiments were designed to characterize the changes in the relative fluorescence (F/F₀) using the extracellular solutions containing various concentrations of [K⁺]_o (5, 30, and 80 mM). Resting membrane potential, assumed to mainly reflect K⁺ equilibrium potential, can be calculated with the Nernst equation. A representative characterization experiment is depicted in supplementary data, showing that the relationship of relative fluorescence and predicted membrane potential, note that the cultured HCASMCs have a more depolarized membrane potential at rest (Fig. S6).

Like other excitable cells, HCASMCs express multiple types of K⁺ channels (Nelson & Quayle, 1995; Standen & Quayle, 1998). In order to investigate which K⁺ channel(s) are important in setting the resting membrane potential, a series of K⁺ channel inhibitors were applied. These included glibenclamide (10 µM), penitrem A (200 nM), tram34 (1 µM), apamin (200 nM), BaCl₂(25 µM) and XE991 (10 µM) which are inhibitors of K_ATP channels, calcium-activated large, intermediate and small conductance potassium (BK_Ca, IK_Ca, SK_Ca) channels, inward rectifying potassium (K_ir) channels and voltage-gated potassium (K_v7) channels respectively (Asano et al., 2012; Blatz & Magleby, 1987; Gluais et al., 2005; Ko et al., 2008; Mackie & Byron, 2008; Nelson & Quayle, 1995; Wulff & Castle, 2010). These inhibitors were used at concentrations thought to be selective for their respective class of channel (Alexander, Mathie & Peters, 2009). An increase in DiBAC₄(3) signal (membrane depolarization) upon inhibition of a particular class of K⁺ channel would indicate that this channel is important in maintaining the resting membrane potential. Figs. 5A–5F show the representative time-course of relative fluorescence observed with different K⁺ channel inhibitors. The fractional fluorescence, determined as the difference between the relative fluorescence of DiBAC₄(3) before and after drug application and expressed as percentage change is summarized in Fig. 5G. Application of glibenclamide and BaCl₂ caused a significant change in fractional fluorescence (p < 0.001), suggesting that K_ATP and K_ir channels play major roles in setting resting membrane potential of HCASMCs.

Effects of metabolic inhibitors and hypoxia on membrane potential

Of the K⁺ channels involved in setting the resting membrane potential of HCASMCs, K_ATP channels seem a likely candidate responsible for hypoxic vasodilation. Initial experiments using the K_ATP channel opener, pinacidil (10 µM), caused a significant decrease in DiBAC₄(3) fluorescence (Fig. S7), suggesting that K_ATP channels have spare capacity to increase their open probability and/or active channel number from the resting condition. Therefore, the effect of metabolic insult and hypoxia on the DiBAC₄(3) signal was examined.

Inhibiting mitochondrial complex III with antimycin (1 µM) induced a significant decrease in DiBAC₄(3) fluorescence (Figs. 6A–6B), which was blocked by glibenclamide (10 µM) or 60 K⁺ (Figs. 6C–6D), an indication that K_ATP channels may be involved. The effect induced by hypoxia on DiBAC₄(3) fluorescence was highly significant (Figs. 6E–6F). The effect from hypoxia was blocked by 60 K⁺ (Fig. 6H), suggesting the involvement of K⁺ channels. However, exposure to hypoxia resulted in significant decrease in DiBAC₄(3) fluorescence when in the presence of 10 µM pinacidil (n = 5), 10 µM glibenclamide (n = 36), 25 µM BaCl₂ (n = 14), 100 nM IbTX (n = 17), 200 nM penitrem A (n = 14), and 10 µM glibenclamide plus 25 µM BaCl₂(n = 25) (Fig. S8). These results indicate that K⁺ channels are likely to be involved in hypoxia-induced membrane hyperpolarization reported by DiBAC₄(3), but classic K⁺ channel inhibitors, when applied singly or in combination in the case of glibenclamide plus BaCl₂, failed to block the effect of hypoxia.