Dynamic Responses of Single Cardiomyocytes to Graded Ischemia Studied by Oxygen Clamp in On-Chip Picochambers
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Vladimir Ganitkevich, Sibylle Reil, Brig
参见附件。
the Institut für Physiologie II (V.G., S.R., K.B.), Friedrich-Schiller-Universitt
Institut für Fügetechnik und Werkstoffprüfung GmbH (B.S., T.S.), Jena, Germany.
Abstract
Single mouse cardiomyocytes were exposed to defined ischemia. We designed chambers on glass chips with a volume of 192 pL (picochambers). After a picochamber was loaded with a single cardiomyocyte, PO2 in the picochamber was equilibrated with that in the headspace, where it was controlled in the critical range between <0.2 and 10 mm Hg. Because the extracellular fluid volume in a picochamber was restricted, these conditions are close to tissue ischemia. Responses of the sarcolemmal KATP-channel current (IKATP), the production of reactive oxygen species (ROS), and the mitochondrial membrane potential () of single cardiomyocytes to graded ischemia and, in particular, to rapid changes of the ischemic grade by defined oxygen steps were studied. The results show that IKATP is readily activated during ischemia and that the grade of ischemia tightly controls the amplitude of IKATP. Furthermore, maximal ischemia-induced IKATP was similar when it followed either reoxygenation or reperfusion, suggesting that there is no major autocrine modulation of maximal IKATP during ischemia. A PO2 staircase from <0.2 to 10 mm Hg increased the ROS signal, starting already at a PO2 of 0.3 mm Hg. With a similar PO2 staircase, first hyperpolarized and then, above 1 mm Hg, depolarized. The depolarizing response of at a PO2 of >1 mm Hg could be blocked by increasing the antioxidant defense with glutathione–monoethyl ester. It is concluded that in an ischemic cardiomyocyte IKATP is essentially controlled by PO2 and that at low PO2 is balanced by oxygen-induced hyperpolarization and ROS-induced depolarization.
Key Words: single cardiomyocytes ischemia reactive oxygen species mitochondrial membrane potential ATP-sensitive K+ channels
Introduction
The myocardium depends critically on sufficient blood perfusion. If blood perfusion is interrupted, ischemia develops which is characterized by a deprivation of oxygen and substrates and an accumulation of waste. Depending on the intensity of the injury, the cells in the tissue die by either apoptosis or necrosis, in particular if ischemia and reperfusion follow each other.1–3 Responses of the myocardium to acute ischemia were extensively studied at the level of the whole organ by ligation or obstruction of coronary arteries.4–6 However, such macroscopic approaches are severely limited to study functional parameters of the injured cells, such as the mitochondrial membrane potential (), the production of reactive oxygen species (ROS), or transmembrane ion currents. Instead, these parameters can be studied with much higher precision in isolated cells. Unfortunately, no satisfying experimental approach has been developed to make single cells ischemic, and studies in isolated cardiomyocytes therefore simulated only some aspects of ischemia, eg, mitochondrial blockade by either various metabolic poisons7,8 or severe hypoxia.9–11 In particular, cellular responses to defined graded ischemia, including any accumulation of released signal molecules in the confined extracellular space, could not be investigated because experimental conditions for single-cell analysis are usually performed in bath volumes exceeding that of the cell by 5 or more orders of magnitude. As such signal molecules are considered eg, adenosine,12 opioids (for review, see Barron13), bradykinin,14 and tumor necrosis factor .15 Experiments in restricted picoliter-sized volumes can overcome this limitation, as shown for the release of lactate16 or purine17 from a single cardiomyocyte. However, in these studies the fluid had to be sealed to the headspace by mineral oil. This would neither allow to control the oxygen tension efficiently nor to replace the extracellular fluid for reperfusion.
We developed a technique to investigate isolated cells under conditions similar to ischemia. Microscopically small open chambers with a volume of 192 pL (picochambers) were constructed on glass chips. A picochamber was loaded with a single cardiomyocyte, the open tiny fluid volume was kept constant in time, and the oxygen tension in the picochamber was rapidly equilibrated with that in the gas phase, where it was controlled at sufficiently low levels.18 We studied responses of the sarcolemmal KATP-channel current (IKATP), the production of ROS, and the to graded ischemia and to rapid changes of the ischemic grade.
Materials and Methods
Preparation of Mouse Ventricular Cardiomyocytes
Mouse ventricular cardiomyocytes were prepared as described previously.19 All procedures were performed in accordance with institutional guidelines. The cells were stored in Kraftbrühe (KB) solution.
Picochambers and Experimental Setup
Picochambers were constructed on glass wafers of 170-μm thickness. They were coated on 1 side with a 32-μm thick layer of a negative photoresist in which cuboid-like pits of 150-μm length, 40-μm width, and 32-μm depth were created, resulting in a geometric volume of 192 pL (see the online data supplement, available at http://circres.ahajournals.org). Thus, the walls and the bottom of the picochambers were formed by the photoresist and the glass of the wafer, respectively. For experimental use, chips of 12x12 mm were sawed from the wafer (Figure 1A). The photoresist was transparent and its surface was hydrophobic.
The experimental chamber was mounted on the stage of an inverted microscope (Axiovert 135M, Zeiss, Germany). Argon was established above the site where measurements were performed to insulate this site from the atmospheric air while having free access for glass pipettes (supplemental Figure I). A chip carrying the picochambers formed the bottom of the experimental chamber. Humidified argon or air entered the funnel of the experimental chamber via 2 inlets and left the funnel via the top aperture. The oxygen tension was measured in the headspace directly above the surface of the chip by a Clark electrode.
Up to 4 glass pipettes were positioned for electrophysiological measurements in a single picochamber (Figure 1C and supplemental Figure I). These pipettes were used as patch pipette, ion sensitive electrode, and the respective references. To minimize equilibration of the solutions in the reference pipette for patch clamp with that in the picochamber, it was mounted on a piezo translator and dipped only in the solution of the picochamber when the current was measured.
A picochamber was loaded with a cardiomyocyte as follows. At the beginning the experimental chamber was filled with 200 μL of Tyrode solution. A cardiomyocyte was transferred into the solution of the experimental chamber. The cardiomyocyte was patch clamped in the whole-cell configuration and then lowered into the picochamber (Figure 1B). The solution of the experimental chamber was removed, leaving only solution in the picochambers resulting from the capillary forces. After this step, the surface of the solution in the picochambers was plain, which could be judged from light diffraction. Therefore the filling volume of the picochamber was assumed to be 192 pL. By means of the humidified gas streaming above the filled picochambers, the fluid volume of the picochambers could be kept constant for hours (online data supplement). The reference pipette and, optionally, other pipettes were positioned in the picochamber. These pipettes did not severely alter the plain surface profile. When establishing an atmosphere of humidified air above the picochamber, the cell within the picochamber could be voltage clamped in the usual way.
When changing the oxygen tension in the headspace above the picochamber, the oxygen tension in the solution equilibrates within 2 seconds (supplemental Figure II). The flow rate of the humidified argon in the funnel determined how much oxygen from the atmospheric air above the argon diffused to the picochamber against the argon stream. Rapid change of the argon flow rate by an electronic flow-controller allowed steps to another oxygen tension in a few seconds.
Patch-Clamp Technique
Transmembrane currents of cardiomyocytes were recorded with the patch-clamp technique in the whole-cell configuration. The pipettes were pulled from borosilicate glass tubing (outer diameter, 1.0 mm; inner diameter, 0.7 mm) using a conventional 2-step puller (PP-83, Narishige). When filled with intracellular solution, the resistance of both the patch pipette and the reference pipette was 1 to 2 M. Recording was performed with a patch-clamp amplifier (VE-2, Alembic Instruments). The records were filtered at a cut-off frequency of 10 kHz. If not otherwise indicated, the holding potential was –80 mV and the cells were depolarized to +40 mV for 100 ms. All experiments were performed at room temperature (22±1°C).
K+ and pH Measurement With Ion-Sensitive Microelectrodes
Ion-sensitive microelectrodes were pulled from the same glass tubing as patch-clamp electrodes. They were silanized at 200°C with N-N dimethyltrimethylsilamine or bis(dimethylamino)dimethylsilane (Fluka) as described.20 Electrode tips were filled with hydrogen ionophore II (Cocktail A, Fluka) or potassium ionophore I (Cocktail A, Fluka), and the electrodes were backfilled with Tyrode solution. The slope was larger than 54 mV per p[K] or pH unit. Reference electrodes were filled with Tyrode solution and they had a resistance of 8 to 10 M. The differential signals were measured with the amplifier FD-223, World Precision Instruments. The signals were sampled at 1 kHz.
Fluorescence Optical Measurements
ROS production and the were measured optically by using the fluorescence dyes 5-(-6)-chloromethyl-2',7'-dichlorohydrofluorescein diacetate (CM-H2DCFDA) and tetramethylrhodamine ethyl ester (TMRE), respectively. For ROS measurements, cardiomyocytes were incubated for 20 minutes in KB solution containing 5 μmol/L of the cell-permeable CM-H2DCFDA, which, after deesterification, resulted in accumulation of CM-H2DCFH within the cell. On oxidation CM-H2DCFH yields the highly fluorescent CM-DCF. For measuring , cardiomyocytes were incubated for 10 minutes in KB solution containing 2 μmol/L TMRE. Mitochondrial depolarization, which liberates TMRE from the mitochondria, resulted in an increase of the total fluorescence attributable to TMRE "dequenching." Hyperpolarization produced opposite effects. For details, see the expanded Materials and Methods section in the online data supplement.
Data Acquisition and Analysis
Measurements were controlled and data were collected and analyzed with the ISO2 soft- and hardware (12-bit resolution; MFK Niedernhausen, Germany) running on a Pentium personal computer. The sampling rate was 1 kHz.
Solutions and Chemicals
All chemicals were of analytical grade. Tyrode solution contained (in mmol/L): 140 NaCl, 5.4 KCl, 1.2 CaCl2, 0.5 MgCl2, 0 glucose if not otherwise indicated, 10 HEPES, pH=7.4 (NaOH). For measuring extracellular pH changes, HEPES was reduced to 1 mmol/L. Intracellular solution in the patch pipette contained (in mmol/L): 140 KCl, 10 NaCl, 10 HEPES, 5 EGTA if not otherwise indicated, pH=7.3 (KOH). The cells were stored for experimental use in KB medium containing (mmol/L): 30 KCl, 30 KH2PO4, 50 glutamine acid, 20 taurine, 20 HEPES, 10 glucose, 3 MgSO4 0.5 EGTA, pH 7.3 (KOH). For scavenging ROS, the cells were incubated for at least 20 minutes in KB medium containing 1 mmol/L of the membrane permeable glutathione–monoethyl ester (GME). TMRE and CM-H2DCFDA were purchased from Molecular Probes; GME from Calbiochem. Other chemicals were of analytical grade and were purchased from Sigma.
Results
IKATP in Ischemic Cardiomyocytes
Freshly isolated mouse ventricular cardiomyocytes were subjected to ischemia. Figure 2A shows an experiment in which the transmembrane current and the oxygen tension were measured simultaneously. At an oxygen tension (PO2) of <0.4 mm Hg, an extra outward current developed, first slowly, then with increasing speed. Stepping to PO2=3 mm Hg removed the extra current within seconds. From the rapid removal of the extra current by increasing oxygen and the fact that it did not appear in the presence of 10 μmol/L glibenclamide (n=4; not shown), we concluded that it was generated by KATP channels (IKATP10). After repeating the maneuver, reperfusion was performed by flooding the experimental chamber for 20 seconds with normoxic solution, causing washout of all accumulated extracellular ions and molecules from the picochamber. Then the volume of the extracellular solution was reduced to that of the picochamber again. The following third period at low oxygen produced an IKATP with roughly similar amplitude, as did the second period of low oxygen. The similarity of the amplitude of IKATP at low PO2 before and after reperfusion suggests that under these conditions, there is no major modulating effect of any accumulated substance on the maximal activation of KATP channels. Similar results were obtained in 4 other experiments.
Figure 2B shows an experiment in which ischemia was graded by clamping the oxygen tension to different defined levels. IKATP developed at a PO2 of <0.2 mm Hg. Setting PO2 to 8 mm Hg and back to <0.2 mm Hg abolished and induced IKATP rapidly. Clamping PO2 to increasing levels led to a progressive disappearance of IKATP with a half maximum current near 1 mm Hg. This result suggests that IKATP rapidly and gradually responded to graded ischemia and that cytoplasmic ATP levels seen by KATP channels could be graded by PO2.
Extracellular Accumulation of K+ and H+ Ions During Ischemia
To verify that under our experimental conditions molecules accumulate in the extracellular fluid, as typical for ischemia, we recorded [K+]e and [H+]e by means of ion-sensitive microelectrodes (for example, Figure 1C). These ions are known to accumulate in the extracellular space during ischemia,4,21 although it is still controversial on which pathway they leave the cells.22–24 Figure 2C shows the ischemia-induced changes of [K+]e in conjunction with the transmembrane ionic current of a representative experiment from a total of 8 experiments. Initially, [K+]e rises because pulsing to +40 mV for 100 ms at a frequency of 2 Hz caused extracellular K+ accumulation. At the time of appearance of IKATP, the rise of [K+]e accelerates. An increase of PO2 to 4 mm Hg reverses the increase of [K+]e and the protocol could be repeated.
Changes of [H+]e were recorded with a pH-sensitive microelectrode in the picochamber. The pH drop started much earlier than IKATP appeared (Figure 2D). An increase of the oxygen tension rapidly removed IKATP and initiated a slow decrease of [H+]e. The high resolution of [H+]e revealed that ischemia-induced acidosis was consistently preceded by a short period of alkalinization (n=9), a phenomenon known from neurons during spreading depression25 or repetitive electrical stimulation,26 but so far not from ischemic cardiomyocytes. Hence, substances released by an ischemic cell accumulate in the extracellular fluid of the picochamber, similarly as observed in the interstitial space of the ischemic myocardium, and this accumulation can be measured if appropriate sensor techniques are available.
ROS Production at Graded Ischemia
In the myocardium, ROS contribute to reperfusion injury following ischemia,27,28 but are produced already during ischemia before reperfusion.29 We applied the oxygen-clamp technique in the picochambers to assess the dependency of the ROS production on the oxygen tension. The production of ROS was monitored by means of the fluorescent dye CM-H2DCFDA (Figure 3A), being aware that there are limitations with the use of this compound.30–32 The CM-DCF fluorescence decreased at a PO2 of <0.2 mm Hg, which is likely attributable to photoreduction of the dye in the presence of cellular reducing agents at very low PO2.30 Repeated oxygen steps from <0.2 mm Hg to 3 mm Hg induced an increase of the CM-DCF fluorescence. Stepping back to <0.2 mm Hg resulted in a decrease of the fluorescence, however, only after passing a transient increase of fluorescence. We attribute this transient fluorescent signal to a burst of ROS generation. The following PO2 staircase up to 10 mm Hg shows that already the very low PO2 of 0.3 mm Hg sufficed to produce an increase in the CM-DCF signal. KATP channels started to close only at higher PO2 compared with the increase of CM-DCF signal. Subsequently, larger pulses to PO2=10 mm Hg were applied repeatedly, and the result was that the steps to the high PO2 evoked a faster and overshooting increase of the CM-DCF fluorescence. Figure 3B substantiates this response for a smaller (single asterisk) and a larger oxygen pulse (double asterisk). The observation that the CM-DCF fluorescence burst does not cause a respective overshoot in IKATP suggests that under our ischemic conditions, there is no relevant opening effect of ROS on maximally activated sarcolemmal KATP channels (for example, Tokube and colleagues33,34).
The Mitochondrial Membrane Potential at Graded Ischemia
The fluorescent dye TMRE was used to study the response of the to step-like changes of the oxygen tension. Figure 4A shows an experiment in which during the phase of ischemia-induced rise of IKATP at a PO2 of <0.4 mm Hg, 2 oxygen pulses of small but different amplitude concomitantly affected and IKATP. The decrease of IKATP and the hyperpolarization of were both graded and fully reversible. The time relation between the changes of PO2, , and IKATP are shown in Figure 4B. Whereas the response of follows the PO2 pulse immediately, the decrease of IKATP follows the hyperpolarization of only after a delay and with slower kinetics. This delay includes ATP production in the mitochondria, translocation to the cytosol, diffusion to the sarcolemma, binding to the sarcolemmal KATP channels, and channel closing. The mean half-maximum delay was 3.8±0.4 seconds (n=5).
We then extended the oxygen range for graded ischemia. A representative experiment from a total of 12 experiments is shown in Figure 4C. Switching from 10 to <0.2 mm Hg rapidly induced IKATP and depolarization of . As before, a staircase of oxygen pulses to increasing values progressively abolished IKATP. In contrast, the response of was biphasic. It first hyperpolarized until PO2 reached 1 mm Hg and then depolarized at higher PO2, suggesting that is controlled by 2 oxygen-controlled processes: hyperpolarization in the lower PO2 range could be explained by an increasing turnover of the electron transport chain (ETC), whereas depolarization at the higher PO2 could be explained by an additional increase of ROS inducing opening of permeability transition pores (PTP35), inner membrane anion channels (IMAC36), or mitochondrial KATP channels (mitoKATP37,38). To test this hypothetic effect of ROS, we loaded the cells with the membrane-permeable reduced glutathione (GME), which constitutes an important part of the cellular antioxidant defense,39 and repeated the experiments. The result was that for increasing oxygen tension, the glutathione loading selectively removed the depolarizing phase of at PO2 >1 mm Hg but did not affect the hyperpolarizing phase at a PO2 of <1 mm Hg (Figure 4D). The same result was obtained in 5 other experiments with GME. We propose that increasing PO2 to values <1 mm Hg leads to hyperpolarization of because the turnover of the ETC is oxygen controlled, whereas further increasing PO2 leads to depolarization of because an increased ROS production progressively opens PTP, IMAC, and/or mitoKATP channels, shunting the inner mitochondrial membrane. Under this condition the mitochondrial ATP production must be sufficiently high to keep the KATP channels closed. Because the sarcolemmal IKATP was not affected by application of GME, these results further substantiate that at sufficiently high ATP, there is no major effect of ROS on the activity of sarcolemmal KATP channels.
Discussion
In this report, we describe experiments on single isolated cardiomyocytes performed under conditions of restricted extracellular volume and defined limited oxygen supply, allowing metabolites to accumulate during ischemia in a confined extracellular space. Because of the small picochamber volume and the rapid equilibration with the gas phase, the oxygen tension in the solution surrounding the cell could be controlled via the composition of the gas in the headspace. This combination of features mimics relevant aspects of tissue ischemia for an isolated cell, including possible autocrine feedback mechanisms involved. Moreover, the possibility to change the oxygen tension around the cell rapidly allows studies of the dynamic cellular responses to graded ischemia.
Our results showed that an ischemic cardiomyocyte developed IKATP similarly as observed previously under conditions of anoxia.10,11 Furthermore, administration of oxygen by reperfusion, and thus replacement of the confined extracellular solution by fresh solution, or by reoxygenation only, leaving the confined extracellular solution unchanged, led to subsequent ischemia-induced IKATP of similar maximal amplitude. This result shows that under ischemic conditions, the oxygen tension tightly controls the activity of the KATP channels. Therefore, possible autocrine mechanisms do not essentially modulate the amplitude of maximal IKATP, which, however, does not exclude that the sensitivity of the KATP channels is modulated. Our results also provide novel information on the relationship between oxygen tension around a cardiomyocyte and the activity of KATP channels (Figure 2B). An oxygen tension of a few millimeters of mercury is necessary to produce enough ATP in the mitochondria to keep the channels closed. Because the oxygen tension around a cardiomyocyte in an operating heart is thought to be only 2.5 mm Hg, and thus in the same range,18 it is therefore likely that in the working heart the portion of open KATP channels is bigger than negligible.
The measurements of ROS production with CM-DCF as a tool also showed graded responses to changes of the oxygen tension in the relevant low range between several millimeters of mercury and 0 mm Hg. ROS production, assessed by the CM-DCF signal, was minimal at a PO2 of <0.2 mm Hg and increased already at a slightly higher PO2 of 0.3 mm Hg (Figure 3A). This suggests that an only very low oxygen tension is required for notable ROS production. Oxygen pulses revealed that steps in both directions induce an increase of the CM-DCF signal. In the myocardium, ROS bursts at reperfusion following ischemia are well established,40 and our CM-DCF signals for oxygen steps from <0.2 mm Hg to 10 mm Hg (Figure 3B bottom) correspond to these observations. Interestingly, smaller oxygen steps to 3 mm Hg resulted in slower increases of the CM-DCF signal (Figure 3B, top). This suggests that at the higher PO2 of 10 mm Hg, the ETC reduced during ischemia can transiently transfer more electrons to oxygen. In contrast, at the lower PO2 of 3 mm Hg, the equally reduced ETC cannot transfer so many electrons to oxygen because its availability is lower.
More surprising was that switching from a PO2 of several millimeters of mercury to <0.2 mm Hg consistently led to a pronounced transient increase of the CM-DCF signal. This result can be explained as follows. The formation of superoxide from oxygen requires a sufficient PO2 and a sufficiently reduced ETC. When PO2 drops, then a time interval is passed in which the effect of the reduced ETC overcomes that of the reduced availability of oxygen, resulting in an increased production of superoxide and subsequent ROS by further electron transfers. This result fits to the observation that notable ROS production, assessed by CM-DCF signals, appears already at the low PO2 of 0.3 mm Hg. Furthermore, it also fits to a recent result in the myocardium that perfusion with 2% O2 generates more ROS on reperfusion than perfusion with 20% or even 100% O241 because 2% O2 corresponds to 15 mm Hg, and only a small portion of this oxygen should reach the cardiomyocytes because of consumption of oxygen by the endothelium of the capillaries.18
The dependence of the on the oxygen tension in the range between <0.2 and 10 mm Hg suggests that at least 2 oppositely directed processes are involved. At a PO2 of <1 mm Hg, increasing PO2 hyperpolarizes , whereas at a PO2 of >1 mm Hg, increasing PO2 depolarizes . Hyperpolarization for PO2 increase at a PO2 of <1 mm Hg finds a plausible explanation by assuming that below this value, the turnover of the ETC is predominantly controlled by PO2, ie, that ROS-induced depolarization of is less relevant. We propose that at PO2 near 1 mm Hg, the ROS production counterbalances the turnover of the ETC, and, at higher PO2, it drives into depolarizing direction. Our argument that at a PO2 of >1 mm Hg ROS mediates depolarization with increasing PO2 is based on the results that ROS production, assessed by CM-DCF signals, increases with increasing PO2 and that the ROS scavenger GME abolishes depolarization. If so, then oxygen controls by 2 oppositely directed forces, of which 1 is the concentration of ROS.
In conclusion, we studied essential aspects of cardiac ischemia at the level of a single cell, including extracellular acidosis and potassium accumulation, activation of IKATP, production of ROS and changes of . We show that the degree of ischemia tightly controls both metabolic and electric responses. A cell can be kept in the picochamber for an hour or even longer. Control of oxygen supply during this time can help, eg, to disentangle single-cell responses involved in the phenomenon of preconditioning, which is the increased tolerance of the myocardium to ischemia following a short conditioning ischemia and recovering period.42,43
Acknowledgments
We thank G. Ditze, G. Sammler, A. Hertel, F. Horn, S. Bernhardt, and B. Tietsch for excellent technical assistance.
Source of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant BE1250/15-1 to K.B).
Disclosures
None.
Footnotes
Original received February 1, 2006; revision received May 31, 2006; accepted June 5, 2006.
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Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning. Ionic alterations. Circ Res. 1993; 72: 112–125.
the Institut für Physiologie II (V.G., S.R., K.B.), Friedrich-Schiller-Universitt
Institut für Fügetechnik und Werkstoffprüfung GmbH (B.S., T.S.), Jena, Germany.
Abstract
Single mouse cardiomyocytes were exposed to defined ischemia. We designed chambers on glass chips with a volume of 192 pL (picochambers). After a picochamber was loaded with a single cardiomyocyte, PO2 in the picochamber was equilibrated with that in the headspace, where it was controlled in the critical range between <0.2 and 10 mm Hg. Because the extracellular fluid volume in a picochamber was restricted, these conditions are close to tissue ischemia. Responses of the sarcolemmal KATP-channel current (IKATP), the production of reactive oxygen species (ROS), and the mitochondrial membrane potential () of single cardiomyocytes to graded ischemia and, in particular, to rapid changes of the ischemic grade by defined oxygen steps were studied. The results show that IKATP is readily activated during ischemia and that the grade of ischemia tightly controls the amplitude of IKATP. Furthermore, maximal ischemia-induced IKATP was similar when it followed either reoxygenation or reperfusion, suggesting that there is no major autocrine modulation of maximal IKATP during ischemia. A PO2 staircase from <0.2 to 10 mm Hg increased the ROS signal, starting already at a PO2 of 0.3 mm Hg. With a similar PO2 staircase, first hyperpolarized and then, above 1 mm Hg, depolarized. The depolarizing response of at a PO2 of >1 mm Hg could be blocked by increasing the antioxidant defense with glutathione–monoethyl ester. It is concluded that in an ischemic cardiomyocyte IKATP is essentially controlled by PO2 and that at low PO2 is balanced by oxygen-induced hyperpolarization and ROS-induced depolarization.
Key Words: single cardiomyocytes ischemia reactive oxygen species mitochondrial membrane potential ATP-sensitive K+ channels
Introduction
The myocardium depends critically on sufficient blood perfusion. If blood perfusion is interrupted, ischemia develops which is characterized by a deprivation of oxygen and substrates and an accumulation of waste. Depending on the intensity of the injury, the cells in the tissue die by either apoptosis or necrosis, in particular if ischemia and reperfusion follow each other.1–3 Responses of the myocardium to acute ischemia were extensively studied at the level of the whole organ by ligation or obstruction of coronary arteries.4–6 However, such macroscopic approaches are severely limited to study functional parameters of the injured cells, such as the mitochondrial membrane potential (), the production of reactive oxygen species (ROS), or transmembrane ion currents. Instead, these parameters can be studied with much higher precision in isolated cells. Unfortunately, no satisfying experimental approach has been developed to make single cells ischemic, and studies in isolated cardiomyocytes therefore simulated only some aspects of ischemia, eg, mitochondrial blockade by either various metabolic poisons7,8 or severe hypoxia.9–11 In particular, cellular responses to defined graded ischemia, including any accumulation of released signal molecules in the confined extracellular space, could not be investigated because experimental conditions for single-cell analysis are usually performed in bath volumes exceeding that of the cell by 5 or more orders of magnitude. As such signal molecules are considered eg, adenosine,12 opioids (for review, see Barron13), bradykinin,14 and tumor necrosis factor .15 Experiments in restricted picoliter-sized volumes can overcome this limitation, as shown for the release of lactate16 or purine17 from a single cardiomyocyte. However, in these studies the fluid had to be sealed to the headspace by mineral oil. This would neither allow to control the oxygen tension efficiently nor to replace the extracellular fluid for reperfusion.
We developed a technique to investigate isolated cells under conditions similar to ischemia. Microscopically small open chambers with a volume of 192 pL (picochambers) were constructed on glass chips. A picochamber was loaded with a single cardiomyocyte, the open tiny fluid volume was kept constant in time, and the oxygen tension in the picochamber was rapidly equilibrated with that in the gas phase, where it was controlled at sufficiently low levels.18 We studied responses of the sarcolemmal KATP-channel current (IKATP), the production of ROS, and the to graded ischemia and to rapid changes of the ischemic grade.
Materials and Methods
Preparation of Mouse Ventricular Cardiomyocytes
Mouse ventricular cardiomyocytes were prepared as described previously.19 All procedures were performed in accordance with institutional guidelines. The cells were stored in Kraftbrühe (KB) solution.
Picochambers and Experimental Setup
Picochambers were constructed on glass wafers of 170-μm thickness. They were coated on 1 side with a 32-μm thick layer of a negative photoresist in which cuboid-like pits of 150-μm length, 40-μm width, and 32-μm depth were created, resulting in a geometric volume of 192 pL (see the online data supplement, available at http://circres.ahajournals.org). Thus, the walls and the bottom of the picochambers were formed by the photoresist and the glass of the wafer, respectively. For experimental use, chips of 12x12 mm were sawed from the wafer (Figure 1A). The photoresist was transparent and its surface was hydrophobic.
The experimental chamber was mounted on the stage of an inverted microscope (Axiovert 135M, Zeiss, Germany). Argon was established above the site where measurements were performed to insulate this site from the atmospheric air while having free access for glass pipettes (supplemental Figure I). A chip carrying the picochambers formed the bottom of the experimental chamber. Humidified argon or air entered the funnel of the experimental chamber via 2 inlets and left the funnel via the top aperture. The oxygen tension was measured in the headspace directly above the surface of the chip by a Clark electrode.
Up to 4 glass pipettes were positioned for electrophysiological measurements in a single picochamber (Figure 1C and supplemental Figure I). These pipettes were used as patch pipette, ion sensitive electrode, and the respective references. To minimize equilibration of the solutions in the reference pipette for patch clamp with that in the picochamber, it was mounted on a piezo translator and dipped only in the solution of the picochamber when the current was measured.
A picochamber was loaded with a cardiomyocyte as follows. At the beginning the experimental chamber was filled with 200 μL of Tyrode solution. A cardiomyocyte was transferred into the solution of the experimental chamber. The cardiomyocyte was patch clamped in the whole-cell configuration and then lowered into the picochamber (Figure 1B). The solution of the experimental chamber was removed, leaving only solution in the picochambers resulting from the capillary forces. After this step, the surface of the solution in the picochambers was plain, which could be judged from light diffraction. Therefore the filling volume of the picochamber was assumed to be 192 pL. By means of the humidified gas streaming above the filled picochambers, the fluid volume of the picochambers could be kept constant for hours (online data supplement). The reference pipette and, optionally, other pipettes were positioned in the picochamber. These pipettes did not severely alter the plain surface profile. When establishing an atmosphere of humidified air above the picochamber, the cell within the picochamber could be voltage clamped in the usual way.
When changing the oxygen tension in the headspace above the picochamber, the oxygen tension in the solution equilibrates within 2 seconds (supplemental Figure II). The flow rate of the humidified argon in the funnel determined how much oxygen from the atmospheric air above the argon diffused to the picochamber against the argon stream. Rapid change of the argon flow rate by an electronic flow-controller allowed steps to another oxygen tension in a few seconds.
Patch-Clamp Technique
Transmembrane currents of cardiomyocytes were recorded with the patch-clamp technique in the whole-cell configuration. The pipettes were pulled from borosilicate glass tubing (outer diameter, 1.0 mm; inner diameter, 0.7 mm) using a conventional 2-step puller (PP-83, Narishige). When filled with intracellular solution, the resistance of both the patch pipette and the reference pipette was 1 to 2 M. Recording was performed with a patch-clamp amplifier (VE-2, Alembic Instruments). The records were filtered at a cut-off frequency of 10 kHz. If not otherwise indicated, the holding potential was –80 mV and the cells were depolarized to +40 mV for 100 ms. All experiments were performed at room temperature (22±1°C).
K+ and pH Measurement With Ion-Sensitive Microelectrodes
Ion-sensitive microelectrodes were pulled from the same glass tubing as patch-clamp electrodes. They were silanized at 200°C with N-N dimethyltrimethylsilamine or bis(dimethylamino)dimethylsilane (Fluka) as described.20 Electrode tips were filled with hydrogen ionophore II (Cocktail A, Fluka) or potassium ionophore I (Cocktail A, Fluka), and the electrodes were backfilled with Tyrode solution. The slope was larger than 54 mV per p[K] or pH unit. Reference electrodes were filled with Tyrode solution and they had a resistance of 8 to 10 M. The differential signals were measured with the amplifier FD-223, World Precision Instruments. The signals were sampled at 1 kHz.
Fluorescence Optical Measurements
ROS production and the were measured optically by using the fluorescence dyes 5-(-6)-chloromethyl-2',7'-dichlorohydrofluorescein diacetate (CM-H2DCFDA) and tetramethylrhodamine ethyl ester (TMRE), respectively. For ROS measurements, cardiomyocytes were incubated for 20 minutes in KB solution containing 5 μmol/L of the cell-permeable CM-H2DCFDA, which, after deesterification, resulted in accumulation of CM-H2DCFH within the cell. On oxidation CM-H2DCFH yields the highly fluorescent CM-DCF. For measuring , cardiomyocytes were incubated for 10 minutes in KB solution containing 2 μmol/L TMRE. Mitochondrial depolarization, which liberates TMRE from the mitochondria, resulted in an increase of the total fluorescence attributable to TMRE "dequenching." Hyperpolarization produced opposite effects. For details, see the expanded Materials and Methods section in the online data supplement.
Data Acquisition and Analysis
Measurements were controlled and data were collected and analyzed with the ISO2 soft- and hardware (12-bit resolution; MFK Niedernhausen, Germany) running on a Pentium personal computer. The sampling rate was 1 kHz.
Solutions and Chemicals
All chemicals were of analytical grade. Tyrode solution contained (in mmol/L): 140 NaCl, 5.4 KCl, 1.2 CaCl2, 0.5 MgCl2, 0 glucose if not otherwise indicated, 10 HEPES, pH=7.4 (NaOH). For measuring extracellular pH changes, HEPES was reduced to 1 mmol/L. Intracellular solution in the patch pipette contained (in mmol/L): 140 KCl, 10 NaCl, 10 HEPES, 5 EGTA if not otherwise indicated, pH=7.3 (KOH). The cells were stored for experimental use in KB medium containing (mmol/L): 30 KCl, 30 KH2PO4, 50 glutamine acid, 20 taurine, 20 HEPES, 10 glucose, 3 MgSO4 0.5 EGTA, pH 7.3 (KOH). For scavenging ROS, the cells were incubated for at least 20 minutes in KB medium containing 1 mmol/L of the membrane permeable glutathione–monoethyl ester (GME). TMRE and CM-H2DCFDA were purchased from Molecular Probes; GME from Calbiochem. Other chemicals were of analytical grade and were purchased from Sigma.
Results
IKATP in Ischemic Cardiomyocytes
Freshly isolated mouse ventricular cardiomyocytes were subjected to ischemia. Figure 2A shows an experiment in which the transmembrane current and the oxygen tension were measured simultaneously. At an oxygen tension (PO2) of <0.4 mm Hg, an extra outward current developed, first slowly, then with increasing speed. Stepping to PO2=3 mm Hg removed the extra current within seconds. From the rapid removal of the extra current by increasing oxygen and the fact that it did not appear in the presence of 10 μmol/L glibenclamide (n=4; not shown), we concluded that it was generated by KATP channels (IKATP10). After repeating the maneuver, reperfusion was performed by flooding the experimental chamber for 20 seconds with normoxic solution, causing washout of all accumulated extracellular ions and molecules from the picochamber. Then the volume of the extracellular solution was reduced to that of the picochamber again. The following third period at low oxygen produced an IKATP with roughly similar amplitude, as did the second period of low oxygen. The similarity of the amplitude of IKATP at low PO2 before and after reperfusion suggests that under these conditions, there is no major modulating effect of any accumulated substance on the maximal activation of KATP channels. Similar results were obtained in 4 other experiments.
Figure 2B shows an experiment in which ischemia was graded by clamping the oxygen tension to different defined levels. IKATP developed at a PO2 of <0.2 mm Hg. Setting PO2 to 8 mm Hg and back to <0.2 mm Hg abolished and induced IKATP rapidly. Clamping PO2 to increasing levels led to a progressive disappearance of IKATP with a half maximum current near 1 mm Hg. This result suggests that IKATP rapidly and gradually responded to graded ischemia and that cytoplasmic ATP levels seen by KATP channels could be graded by PO2.
Extracellular Accumulation of K+ and H+ Ions During Ischemia
To verify that under our experimental conditions molecules accumulate in the extracellular fluid, as typical for ischemia, we recorded [K+]e and [H+]e by means of ion-sensitive microelectrodes (for example, Figure 1C). These ions are known to accumulate in the extracellular space during ischemia,4,21 although it is still controversial on which pathway they leave the cells.22–24 Figure 2C shows the ischemia-induced changes of [K+]e in conjunction with the transmembrane ionic current of a representative experiment from a total of 8 experiments. Initially, [K+]e rises because pulsing to +40 mV for 100 ms at a frequency of 2 Hz caused extracellular K+ accumulation. At the time of appearance of IKATP, the rise of [K+]e accelerates. An increase of PO2 to 4 mm Hg reverses the increase of [K+]e and the protocol could be repeated.
Changes of [H+]e were recorded with a pH-sensitive microelectrode in the picochamber. The pH drop started much earlier than IKATP appeared (Figure 2D). An increase of the oxygen tension rapidly removed IKATP and initiated a slow decrease of [H+]e. The high resolution of [H+]e revealed that ischemia-induced acidosis was consistently preceded by a short period of alkalinization (n=9), a phenomenon known from neurons during spreading depression25 or repetitive electrical stimulation,26 but so far not from ischemic cardiomyocytes. Hence, substances released by an ischemic cell accumulate in the extracellular fluid of the picochamber, similarly as observed in the interstitial space of the ischemic myocardium, and this accumulation can be measured if appropriate sensor techniques are available.
ROS Production at Graded Ischemia
In the myocardium, ROS contribute to reperfusion injury following ischemia,27,28 but are produced already during ischemia before reperfusion.29 We applied the oxygen-clamp technique in the picochambers to assess the dependency of the ROS production on the oxygen tension. The production of ROS was monitored by means of the fluorescent dye CM-H2DCFDA (Figure 3A), being aware that there are limitations with the use of this compound.30–32 The CM-DCF fluorescence decreased at a PO2 of <0.2 mm Hg, which is likely attributable to photoreduction of the dye in the presence of cellular reducing agents at very low PO2.30 Repeated oxygen steps from <0.2 mm Hg to 3 mm Hg induced an increase of the CM-DCF fluorescence. Stepping back to <0.2 mm Hg resulted in a decrease of the fluorescence, however, only after passing a transient increase of fluorescence. We attribute this transient fluorescent signal to a burst of ROS generation. The following PO2 staircase up to 10 mm Hg shows that already the very low PO2 of 0.3 mm Hg sufficed to produce an increase in the CM-DCF signal. KATP channels started to close only at higher PO2 compared with the increase of CM-DCF signal. Subsequently, larger pulses to PO2=10 mm Hg were applied repeatedly, and the result was that the steps to the high PO2 evoked a faster and overshooting increase of the CM-DCF fluorescence. Figure 3B substantiates this response for a smaller (single asterisk) and a larger oxygen pulse (double asterisk). The observation that the CM-DCF fluorescence burst does not cause a respective overshoot in IKATP suggests that under our ischemic conditions, there is no relevant opening effect of ROS on maximally activated sarcolemmal KATP channels (for example, Tokube and colleagues33,34).
The Mitochondrial Membrane Potential at Graded Ischemia
The fluorescent dye TMRE was used to study the response of the to step-like changes of the oxygen tension. Figure 4A shows an experiment in which during the phase of ischemia-induced rise of IKATP at a PO2 of <0.4 mm Hg, 2 oxygen pulses of small but different amplitude concomitantly affected and IKATP. The decrease of IKATP and the hyperpolarization of were both graded and fully reversible. The time relation between the changes of PO2, , and IKATP are shown in Figure 4B. Whereas the response of follows the PO2 pulse immediately, the decrease of IKATP follows the hyperpolarization of only after a delay and with slower kinetics. This delay includes ATP production in the mitochondria, translocation to the cytosol, diffusion to the sarcolemma, binding to the sarcolemmal KATP channels, and channel closing. The mean half-maximum delay was 3.8±0.4 seconds (n=5).
We then extended the oxygen range for graded ischemia. A representative experiment from a total of 12 experiments is shown in Figure 4C. Switching from 10 to <0.2 mm Hg rapidly induced IKATP and depolarization of . As before, a staircase of oxygen pulses to increasing values progressively abolished IKATP. In contrast, the response of was biphasic. It first hyperpolarized until PO2 reached 1 mm Hg and then depolarized at higher PO2, suggesting that is controlled by 2 oxygen-controlled processes: hyperpolarization in the lower PO2 range could be explained by an increasing turnover of the electron transport chain (ETC), whereas depolarization at the higher PO2 could be explained by an additional increase of ROS inducing opening of permeability transition pores (PTP35), inner membrane anion channels (IMAC36), or mitochondrial KATP channels (mitoKATP37,38). To test this hypothetic effect of ROS, we loaded the cells with the membrane-permeable reduced glutathione (GME), which constitutes an important part of the cellular antioxidant defense,39 and repeated the experiments. The result was that for increasing oxygen tension, the glutathione loading selectively removed the depolarizing phase of at PO2 >1 mm Hg but did not affect the hyperpolarizing phase at a PO2 of <1 mm Hg (Figure 4D). The same result was obtained in 5 other experiments with GME. We propose that increasing PO2 to values <1 mm Hg leads to hyperpolarization of because the turnover of the ETC is oxygen controlled, whereas further increasing PO2 leads to depolarization of because an increased ROS production progressively opens PTP, IMAC, and/or mitoKATP channels, shunting the inner mitochondrial membrane. Under this condition the mitochondrial ATP production must be sufficiently high to keep the KATP channels closed. Because the sarcolemmal IKATP was not affected by application of GME, these results further substantiate that at sufficiently high ATP, there is no major effect of ROS on the activity of sarcolemmal KATP channels.
Discussion
In this report, we describe experiments on single isolated cardiomyocytes performed under conditions of restricted extracellular volume and defined limited oxygen supply, allowing metabolites to accumulate during ischemia in a confined extracellular space. Because of the small picochamber volume and the rapid equilibration with the gas phase, the oxygen tension in the solution surrounding the cell could be controlled via the composition of the gas in the headspace. This combination of features mimics relevant aspects of tissue ischemia for an isolated cell, including possible autocrine feedback mechanisms involved. Moreover, the possibility to change the oxygen tension around the cell rapidly allows studies of the dynamic cellular responses to graded ischemia.
Our results showed that an ischemic cardiomyocyte developed IKATP similarly as observed previously under conditions of anoxia.10,11 Furthermore, administration of oxygen by reperfusion, and thus replacement of the confined extracellular solution by fresh solution, or by reoxygenation only, leaving the confined extracellular solution unchanged, led to subsequent ischemia-induced IKATP of similar maximal amplitude. This result shows that under ischemic conditions, the oxygen tension tightly controls the activity of the KATP channels. Therefore, possible autocrine mechanisms do not essentially modulate the amplitude of maximal IKATP, which, however, does not exclude that the sensitivity of the KATP channels is modulated. Our results also provide novel information on the relationship between oxygen tension around a cardiomyocyte and the activity of KATP channels (Figure 2B). An oxygen tension of a few millimeters of mercury is necessary to produce enough ATP in the mitochondria to keep the channels closed. Because the oxygen tension around a cardiomyocyte in an operating heart is thought to be only 2.5 mm Hg, and thus in the same range,18 it is therefore likely that in the working heart the portion of open KATP channels is bigger than negligible.
The measurements of ROS production with CM-DCF as a tool also showed graded responses to changes of the oxygen tension in the relevant low range between several millimeters of mercury and 0 mm Hg. ROS production, assessed by the CM-DCF signal, was minimal at a PO2 of <0.2 mm Hg and increased already at a slightly higher PO2 of 0.3 mm Hg (Figure 3A). This suggests that an only very low oxygen tension is required for notable ROS production. Oxygen pulses revealed that steps in both directions induce an increase of the CM-DCF signal. In the myocardium, ROS bursts at reperfusion following ischemia are well established,40 and our CM-DCF signals for oxygen steps from <0.2 mm Hg to 10 mm Hg (Figure 3B bottom) correspond to these observations. Interestingly, smaller oxygen steps to 3 mm Hg resulted in slower increases of the CM-DCF signal (Figure 3B, top). This suggests that at the higher PO2 of 10 mm Hg, the ETC reduced during ischemia can transiently transfer more electrons to oxygen. In contrast, at the lower PO2 of 3 mm Hg, the equally reduced ETC cannot transfer so many electrons to oxygen because its availability is lower.
More surprising was that switching from a PO2 of several millimeters of mercury to <0.2 mm Hg consistently led to a pronounced transient increase of the CM-DCF signal. This result can be explained as follows. The formation of superoxide from oxygen requires a sufficient PO2 and a sufficiently reduced ETC. When PO2 drops, then a time interval is passed in which the effect of the reduced ETC overcomes that of the reduced availability of oxygen, resulting in an increased production of superoxide and subsequent ROS by further electron transfers. This result fits to the observation that notable ROS production, assessed by CM-DCF signals, appears already at the low PO2 of 0.3 mm Hg. Furthermore, it also fits to a recent result in the myocardium that perfusion with 2% O2 generates more ROS on reperfusion than perfusion with 20% or even 100% O241 because 2% O2 corresponds to 15 mm Hg, and only a small portion of this oxygen should reach the cardiomyocytes because of consumption of oxygen by the endothelium of the capillaries.18
The dependence of the on the oxygen tension in the range between <0.2 and 10 mm Hg suggests that at least 2 oppositely directed processes are involved. At a PO2 of <1 mm Hg, increasing PO2 hyperpolarizes , whereas at a PO2 of >1 mm Hg, increasing PO2 depolarizes . Hyperpolarization for PO2 increase at a PO2 of <1 mm Hg finds a plausible explanation by assuming that below this value, the turnover of the ETC is predominantly controlled by PO2, ie, that ROS-induced depolarization of is less relevant. We propose that at PO2 near 1 mm Hg, the ROS production counterbalances the turnover of the ETC, and, at higher PO2, it drives into depolarizing direction. Our argument that at a PO2 of >1 mm Hg ROS mediates depolarization with increasing PO2 is based on the results that ROS production, assessed by CM-DCF signals, increases with increasing PO2 and that the ROS scavenger GME abolishes depolarization. If so, then oxygen controls by 2 oppositely directed forces, of which 1 is the concentration of ROS.
In conclusion, we studied essential aspects of cardiac ischemia at the level of a single cell, including extracellular acidosis and potassium accumulation, activation of IKATP, production of ROS and changes of . We show that the degree of ischemia tightly controls both metabolic and electric responses. A cell can be kept in the picochamber for an hour or even longer. Control of oxygen supply during this time can help, eg, to disentangle single-cell responses involved in the phenomenon of preconditioning, which is the increased tolerance of the myocardium to ischemia following a short conditioning ischemia and recovering period.42,43
Acknowledgments
We thank G. Ditze, G. Sammler, A. Hertel, F. Horn, S. Bernhardt, and B. Tietsch for excellent technical assistance.
Source of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant BE1250/15-1 to K.B).
Disclosures
None.
Footnotes
Original received February 1, 2006; revision received May 31, 2006; accepted June 5, 2006.
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