AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 280: H42-H50, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H42-H50, January 2001

Short-term hibernation in adult cardiomyocytes is PO2 dependent and Ca2+ mediated

Thomas Stumpe and Jürgen Schrader

Department of Physiology, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of myocardial hibernation, the reversible downregulation of contractile activity on reduction of coronary flow with unchanged cardiac energetics, is presently not understood. The oxygen consumption (VO2), shortening fraction (Delta L), energy status [phosphocreatine (PCr), ATP, and adenosine and lactate release], and free intracellular Ca2+ concentration ([Ca2+]i) were measured in isolated rat cardiomyocytes at precisely controlled ambient PO2 (Oxystat). When PO2 was reduced from 25 to 6 mmHg, VO2 decreased by 50%, while Delta L was downregulated from 11.2 ± 4.1 to 7.6 ± 4.0%, and energy status was unchanged in the steady state (observation time 12 min). Only transiently PCr decreased, and lactate and adenosine release increased. Further reduction of PO2 (to 3 mmHg) reduced VO2 by 80%, decreased PCr by 35%, moderately increased adenosine and lactate release, and progressively reduced Delta L by 50% (to 5.6 ± 3.3%). All parameters fully recovered during reoxygenation. PO2-dependent downregulation of Delta L was accompanied by a progressive reduction in systolic [Ca2+]i (from 512 ± 110 to 357 ± 91 nmol/l at 6 mmHg and to 251 ± 69 nmol/l at 3 mmHg), whereas diastolic free [Ca2+]i remained unchanged. Therefore, the mechanism of the reversible, PO2-dependent downregulation of contractile activity (myocardial hibernation) involves a substantial reduction of systolic calcium.

Oxystat system; oxygen; myocardial hibernation; energy status; partial pressure of oxygen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A MODERATE REDUCTION OF CORONARY BLOOD FLOW in an acute setting results in a sustained impairment of baseline contractile function, whereas high-energy phosphates only transiently decrease (1, 25, 29, 30, 40). Apparently, in the steady state, a new match between ATP formation and ATP consumption was established, however, at a reduced level of energy turnover. This process is reversible because normalization of myocardial blood flow causes contractile function to fully recover (1). Such an acute adaptive reduction of energy expenditure by reduced contractile activity in a situation of decreased energy supply has been termed short-term hibernation (10). This phenomenon must be clearly distinguished from the chronic situation in long-term hibernation (28). A chronically decreased coronary flow at rest not only induces a persistently impaired myocardial function but also elicits changes in cardiac morphology, protein expression, and calcium handling (2, 6, 14, 33, 35). The depressed contractile force of this "hibernating myocardium," originally described by Rahimtoola (26) in 1985 in a clinical setting, also recovers after restoration of myocardial blood flow. Short-term hibernation must also be differentiated from the ischemia-induced "early contractile failure" (13, 16). This situation is characterized by a rapid reduction in high-energy phosphates, nonsteady-state conditions, and a lack of reversibility on reperfusion after an ischemic duration of ~15 min (27).

Although it is most likely that metabolic signals are responsible for the acute downregulation of cardiac energy consumption during short-term hibernation, the mechanism linking energy consumption to myocardial activity is largely unclear. Both inorganic phosphate and free ADP may contribute to the control of cardiac function when the energy status becomes severely compromised, such as in early contractile failure (13, 16, 27). Whether these parameters govern cardiac function when ATP formation and energy status are only moderately impaired or even remain unchanged, such as in short-term hibernation, is uncertain. To date, all available studies on potential mechanisms of short-term hibernation provided negative results: density or affinity of beta -adrenoceptor appears to be unchanged (31); similarly, the activation of ATP-dependent potassium (KATP) channels and increases in the concentration of interstitial adenosine are most likely unimportant (32) as well as changes in calcium responsiveness (11, 15). A decrease in systolic calcium, as measured by 5F-labeled 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (5F-BAPTA), was shown to occur when coronary perfusion pressure was lowered in the isolated ferret heart; however, there was a simultaneous decrease in intracellular pH and an increase in Pi (15). On the other hand, measurements of cytosolic calcium by surface fluorescence using indo 1 observed an increase in systolic and diastolic calcium immediately on onset of ischemia (22). It is therefore unknown whether, in the situation of short-term hibernation, changes in calcium transients trigger the adaptive reduction in contractile activity when cardiac energetics are not compromised.

In a previous study (36), we have defined a PO2 range in isolated contracting rat cardiomyocytes in which oxygen consumption (VO2) is reduced while energy status remains constant. Here, we further investigate whether isolated contracting cardiomyocytes serve as a suitable cellular model for short-term hibernation. Furthermore, we explored the role of calcium as a potential intracellular mechanism that might reduce contractile activity at oxygen-limited ATP formation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxystat

Cardiomyocytes of ether-anesthetized Wistar rats (250-350 g) were prepared as described previously (36). To exactly control ambient PO2, cells were incubated in a continuously stirred chamber [in 10 ml of incubation buffer containing (in mmol/l) 137 NaCl, 5.4 KCl, 1.0 NaH2PO4, 0.8 Mg2SO4, 2.0 CaCl2, 5.5 glucose, and 5.0 Tris; pH 7.4 at 37°C], the so-called Oxystat system (36) (Hugo Sachs Electronics, March-Hugstetten, Germany; Fig. 1). The building blocks of the Oxystat form a feedback-controlled system containing a PO2 electrode as the sensor, a control unit (which compares the sensor signal to a preselected value), and a motor-driven burette (which pumps oxygen-rich, air-bubbled medium into the chamber). Thereby, ambient chamber PO2 is maintained in a steady state close to the preselected PO2.


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  		Fig. 1.   Schematic outline of the experimental setup. The Oxystat (top) is a feedback-controlled perfusion system in which isolated cardiomyocytes in suspension can be maintained at exactly controlled ambient PO2. Connected to the outlet of the Oxystat is a perfusion chamber for microscopic investigation of single cardiomyocytes at an ambient PO2, which is identical to that in the Oxystat. Both cardiomyocytes in suspension and single cells were paced electrically by platinum electrodes.

Continuous recordings of the chamber PO2 and volume of the medium, supplied to maintain the PO2, were used for the calculation of VO2 of the isolated cardiomyocytes and were correlated with the actual protein content in the chamber (36). Increases in VO2 were achieved by electrical pacing (stimulator G270, Strotmann, Aachen, Germany; 9 Hz, 20 µA, biphasic impulses) of the suspended cells using platinum nets positioned at the top and bottom of the incubation chamber.

In the Oxystat system, fluorescence was measured using a modular fluorescence detection system (Oriel, Stratford, CT). A light guide with a superior ultraviolet transmittance was used to illuminate the cardiomyocytes directly in the Oxystat chamber. Light source was a 100-W Hg Arc lamp with a condenser system to focus the light in the Oxystat chamber. Another light guide was used to measure the fluorescence with an end-on photomultiplier tube. An interference filter of 360 nm defined the wavelength of the excitation light, and a filter of 410 or 480 nm defined the excitation wavelength (band width 10 nm). Data from the photomultiplier were recorded by a personal computer using the program Chart (Power-Lab 800, ADInstruments, Sydney, Australia).

Single Cardiomyocytes Perfusion Chamber

To investigate single cardiomyocytes at defined PO2 values, the outlet of the Oxystat system was connected to a perfusion chamber (Fig. 1, bottom). The volume of medium supplied to maintain the Oxystat chamber PO2 leaves the chamber while a filter (pore size 5 µm) prevents cell loss. Because of the effective stirring inside the Oxystat chamber, the PO2 of the outflowing medium is exactly the same as inside the Oxystat chamber. This medium enters the single cell perfusion chamber by a gas-tight connection line [steel canules and Tygon connections (wall 1/16 in.), all lines optimized in length]. Cardiomyocytes were allowed to immobilize for 15 min on glass slides (diameter 30 mm) coated with Cell-Tak (Becton-Dickinson Labware, Bedford, MA). A glass slide with immobilized cardiomyocytes was fixed in the perfusion chamber by an opaque cover. An inlet and outlet and two platinum electrodes for perfusion and electrical pacing (stimulator G270, Strotmann; 3 Hz, 5-20 µA, biphasic impulses) were included in this cover. Cells were illuminated from above through the opaque cover, and cells were visualized by an inverse microscope (Axiovert 35, Carl Zeiss, Germany) through the glass slide. With the use of a video camera (F3012C, Pieper, Germany), the contractions of the single cardiomyocytes were recorded on a video tape for subsequent analysis of cell length.

Fluorescence measurements of single cardiomyocytes were obtained by illuminating the cells with a Hg lamp (HBO 100 W, Carl Zeiss) through an objective with a superior ultraviolet transmittance (Fluar ×40, 1.30 oil; Carl Zeiss). An excitation-interference filter of 340 and 380 nm, respectively, defined the excitation wavelength. Emitted fluorescence light was measured in an end-on photomultiplier tube (Oriel). A beam splitter at 425 nm blocked the excitation light, and an interference filter of 510 nm defined the measured fluorescence light. Data were recorded as described above (data accumulation rate 1,000 Hz).

Protocols

In the first protocol, ambient PO2 was switched from 25 to 6 mmHg (n = 5) while VO2 was monitored, and, each minute, samples were withdrawn. After 1-bromododecane centrifugation (36), we analyzed lactate and adenosine (17) in the extracellular medium. Protein (Lowry), ATP, and phosphocreatine (PCr) were determined in the intracellular volume after separation with ion-pairing HPLC (36) by a bioluminescence method (Biolumat LB9500T, Berthold, Bad Wildbach, Germany). Contraction was then inhibited by wortmannin (23) (10 µmol/l) at an ambient PO2 of 25 and 6 mmHg (each n = 4) while monitoring VO2. Furthermore, ambient PO2 was switched from 6 to 3 mmHg (n = 5) while VO2 and energy status were measured as described above.

To measure the free intracellular Ca2+ concentration ([Ca2+]i) (n = 5) or autofluorescence (n = 8) in the Oxystat, the fluorescence of indo 1-acetoxymethyl ester (AM)-loaded (5 µmol/l, 10 min, Molecular Probes) or unloaded cardiomyocytes was measured when ambient PO2 was reduced from 25 to 6 mmHg. For calibration of Ca2+ fluorescence, Triton X-100 (0.1%) and EGTA (100 mmol/l) were applied. [Ca2+]i was calculated (dissociation constant 230 nmol/l) according to Grynkiewicz et al. (7).

In single cell measurements, the fluorescence of fura 2-AM-loaded (5 µmol/l, 10 min, Molecular Probes, n = 7) and autofluorescence (n = 4) or video recordings (n = 8) of unloaded cardiomyocytes were measured at 25 mmHg. Electrical stimulation was then started, and ambient PO2 was reduced to 6, 3, and 1 mmHg. Subsequently, PO2 was switched back to 25 mmHg. At each PO2 level, steady-state conditions were achieved, and contractions or fluorescence were recorded. For calibration of Ca2+ fluorescence, cardiomyocytes were perfused with Triton X-100 (0.1%) plus 20 mmol/l butanedione monoxime and 100 mmol/l EGTA. Calculation of free [Ca2+]i was as described above, taking a dissociation constant of 150 nmol/l into account.

Analytic Procedures

1-Bromododecane centrifugation. To analyze the cellular content of PCr and ATP in isolated cardiomyocytes, samples of the cardiomyocyte suspension were centrifuged through 1-bromododecane into 100 µl of 2 mol/l HClO4 as previously described (36). For determination of release rates, the supernatant of the 1-bromododecane centrifugation was deproteinized with 0.5 mol/l HClO4, neutralized with 1 mol/l K3PO4, and then used for HPLC or biochemical analysis.

Bioluminescence method. Because of the small amount of cells in each sample obtained in the Oxystat experiments, a bioluminescence method was developed to measure ATP and PCr. Therefore, ATP and PCr were separated by HPLC using the ion-pairing substance tetrabutylammonium sulfate (TBAS). The HPLC system consisted of an ultraviolet detector measuring at 210 nm and a µ-Bondapak C18 5-µm column (Waters, Eschborn, Germany). A linear gradient, created in a low-pressure gradient mixer, was used, and we changed (within 5 min) from KH2PO4/TBAS (37/3 mM) with pH 3.0 to KH2PO4/TBAS buffer (15/3 mM) with pH 5.0 to separate PCr (elution at 4.5 min). Subsequently, 70% methanol was added in a linear gradient until at 25 min, when ATP was eluted. Chromatogram peaks were identified by external and internal standards, and PCr and ATP fractions were sampled. In a second step, both ATP and PCr were quantitated using ATP bioluminescence measured by a luminescence detector (Biolumat LB 9500T, Berthold). A sample (200 µl) of the ATP fraction was mixed with 600 µl of distilled water and 200 µl luminol (CLS, Boehringer, Mannheim, Germany). For the measurement of PCr, a 200-µl sample of the PCr fraction (containing 0.05-0.2 µmol/l PCr) was mixed with 590 µl of ADP solution (50 µmol/l; pH 9) and incubated at 37°C for 30 min with creatine kinase (350 U/mg, 0.3 mg) to quantitatively dephosphorylate PCr. The reaction was stopped using 10 µl of perchloric acid (2 mol/l). After centrifugation and addition of 200 µl of luminol, we measured the amount of ATP produced using the bioluminescence test. Quantification of the light emission was performed with internal and external standards that were treated similar to the samples. Standard curves were linear in a range from 2 to 25 pmol PCr. The content of PCr and ATP in the samples was usually between 8 and 25 pmol.

The concentration of cytosolic free ADP ([ADP]) was calculated using the following formula
[ADP] = ([ATP] × [Cr])/([PCr] × [ H<SUP>+</SUP>] × K<SUB>CK</SUB>) (1)
where [ATP], [PCr], and [Cr] are the concentrations of ATP, PCr, and creatine, respectively, and KCK is the equilibrium constant of the creatine kinase reaction. The dependence of KCK on H+ and Mg2+ was appropriately taken into account (Bünger et al., Ref. 4).

Statistical Analysis

To compare groups of experimental data (means ± SD), Student's t-test and analysis of variance (ANOVA) with post hoc test (Bonferroni) were used where appropriate. P values <0.05 were considered to be significantly different.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiomyocytes incubated at an ambient PO2 of 25 mmHg in the Oxystat were metabolically stable for an incubation period of at least 30 min. The PCr and ATP content were 27.8 ± 5.6 and 22.5 ± 4.4 nmol/mg protein, respectively. The PCr-to-ATP ratio was 1.43 ± 0.47, and free cytosolic ADP was calculated to be 53.4 µmol/l. The adenosine and lactate release were 0.23 ± 0.15 and 3.70 ± 0.50 nmol · min-1 · mg protein-1 (each n = 5), respectively.

As shown in Fig. 2 (left), lowering the ambient PO2 from 25 to 6 mmHg reduced VO2 by 50% from 27.8 ± 5.6 to 13.5 ± 0.7 nmol · min-1 · mg protein-1 (n = 5; P < 0.001). Having attained this lower level, VO2 remained unchanged thereafter. Two minutes after switching to a PO2 of 6 mmHg, PCr was reduced by 55% to 11.4 ± 7.8 nmol/mg protein (n = 5; P < 0.01). Despite the continuing reduction in oxygen supply, PCr recovered within the following 3 min, ultimately attaining control values again (25.7 ± 5.7 nmol/mg protein). Similarly, adenosine and lactate concentrations increased almost linearly for ~5 min. Thereafter, the respective concentrations remained unchanged, indicating a transient increase in adenosine and lactate release as well.


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  		Fig. 2.   Time course of hypoxia-induced changes in oxygen consumption (VO2) and energy status [phosphocreatine (PCr), ATP, lactate, and adenosine (AR)] of contracting cardiomyocytes. To achieve different degrees of hypoxia, the ambient PO2 was switched from 25 to 6 mmHg (left) and from 6 to 3 mmHg (right). Values are means ± SD of 5 experiments each. *P < 0.05; **P < 0.01; ***P < 0.001.

Further reduction of ambient PO2 (from 6 to 3 mmHg) in a separate experimental series (Fig. 2, right) induced a stable decrease of VO2 to 5.5 ± 0.9 nmol · min-1 · mg protein-1 (n = 5; P < 0.001). This corresponds to a 80% inhibition of VO2 compared with normoxic control values. Under these conditions, PCr fell to 16.8 ± 5.3 nmol/mg protein (n = 5; P < 0.05) and remained stable at this lower level. Adenosine and lactate concentration increased steadily for 12 min. Despite a fivefold decrease in VO2, ATP content of the isolated cells remained unchanged (ambient PO2 25 mmHg, 22.5 ± 4.4 nmol/mg protein; 6 mmHg, 22.4 ± 4.4 nmol/mg protein; and 3 mmHg, 22.3 ± 2.0 nmol/mg protein; each n = 5).

Figure 3 summarizes the energy status of isolated cardiomyocytes under steady-state conditions at an ambient PO2 of 25, 6, and 3 mmHg. With decreasing O2 supply, VO2 progressively decreased. Despite a 50% reduction in VO2 (PO2 from 25 to 6 mmHg), PCr, ATP, and adenosine and lactate release remained unchanged. A further reduction to 3 mmHg resulted in a significant decrease in PCr by ~30%, a stable rise in lactate and adenosine release, but no change in ATP. Free cytosolic ADP did not change in the PO2 range from 25 to 6 mmHg. It was, however, nearly doubled (53.4 vs. 127.0 µmol/l) when PO2 was further reduced to 3 mmHg.


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  		Fig. 3.   Steady-state VO2 (A) and energy status [calculated free ADP concentration (B), PCr (C), ATP (D), and adenosine (E) and lactate (F) release] of contracting cardiomyocytes at an ambient PO2 of 25, 6, and 3 mmHg, respectively. Values are means ± SD; each n = 5. *P < 0.05; **P < 0.001 vs. PO2 25 mmHg.

To demonstrate that the stimulation-dependent increase in VO2 is due to contractile activity of cardiomyocytes, experiments with wortmannin were performed in the Oxystat at different ambient PO2. Wortmannin is an effective inhibitor of the myosin light-chain kinase in the used concentration range and, therefore, inhibits calcium-induced contraction (23). As shown in Fig. 4, VO2 of quiescent cardiomyocytes (PO2 25 mmHg, 6.8 ± 1.9 nmol · min-1 · mg protein-1, n = 4) was not affected by wortmannin, although it is shown that wortmannin unspecifically inhibits many other kinases, e.g., the phosphatidylinositol-3 kinase (37). Electrical pacing forced the cells to contract and stimulated VO2 threefold to 27.0 ± 3.1 nmol · min-1 · mg protein-1 (n = 4). Under these conditions, wortmannin reduced VO2 to 12.0 ± 1.5 nmol · min-1 · mg protein-1 by 75% (n = 4; P < 0.001). Lowering the ambient PO2 to 6 mmHg decreased VO2 by ~50% to 12.4 ± 1.5 nmol · min-1 · mg protein-1 (n = 4). However, under these conditions, wortmannin did not further decrease VO2 (n = 4; Fig. 4), suggesting that, at an ambient PO2 of 6 mmHg, contraction was already substantially inhibited.


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  		Fig. 4.   Effect of inhibition of contraction by wortmannin (10 µmol/l) on cellular VO2 of quiescent and electrically paced cardiomyocytes at 25 and 6 mmHg ambient PO2. Values are means ± SD; n = 4-5. ***P < 0.001 wortmannin vs. control.

To determine whether [Ca2+]i, as a main regulator of contractile activity, is altered at a PO2 of 6 mmHg, the fluorescence of indo 1-loaded cardiomyocytes was measured via a light guide directly in the Oxystat at an excitation wavelength of 360 nm and emission wavelengths of 410 and 480 nm. Figure 5 summarizes the calculated [Ca2+]i values of quiescent and stimulated cardiomyocytes at different ambient PO2. [Ca2+]i of quiescent cardiomyocytes was 94.5 ± 7.8 nmol/l. Electrical stimulation increased mean [Ca2+]i to 358.6 ± 32.3 nmol/l. A reduction in ambient PO2 from 25 to 6 mmHg caused a significant decrease in [Ca2+]i by 30% to 235.3 ± 76.5 nmol/l (P < 0.01).


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  		Fig. 5.   Intracellular Ca2+ concentration ([Ca2+]i) of isolated quiescent and contracting cardiomyocytes at different ambient PO2 directly monitored in the Oxystat. The excitation wavelength was 360 nm, fluorescence of indo 1-loaded cardiomyocytes was monitored at the emission wavelength 410 and 480 nm, autofluorescence was subtracted, and the ratio was calculated. After the calibration using Triton (0.1%) and EGTA (100 mmol/l), we calculated [Ca2+]i. Values are means ± SD; n = 5. **P < 0.01; ***P < 0.001.

To exclude changes in autofluorescence as a possible source of error, respective control experiments were carried out. The autofluorescence of quiescent cardiomyocytes at an excitation wavelength of 360 nm and an emission wavelength of 480 nm was 6.7 ± 0.8 mV/mg protein and decreased significantly upon electrical stimulation to 3.2 ± 1.1 mV/mg protein (n = 5, P < 0.001). Similar results were obtained at an emission wavelength of 410 nm, so that the ratio of both emission wavelengths remained almost unchanged. Reduction of ambient PO2 to 6 mmHg did not alter autofluorescence (n = 8) and, therefore, did not interfere with determination of the [Ca2+]i in this experimental setup. However, an increase in autofluorescence was detected when ambient PO2 of quiescent cardiomyocytes was lowered to 0.2 mmHg (at 480 nm, increase to 12.1 ± 1.0 mV/mg protein, n = 5; P < 0.01), suggesting that mitochondrial NADH increased under these conditions.

The perfusion chamber connected to the Oxystat enabled a direct observation of the contraction of single cardiomyocytes at exactly controlled PO2 (Fig. 6). Diastolic cell length did not change when ambient PO2 was progressively reduced (Fig. 6, top, n = 8). However, cell shortening decreased from 11.2 ± 4.1 to 7.6 ± 4.0% of diastolic cell length (-30%) when oxygen supply was reduced from 25 to 6 mmHg (n = 8). Additional reduction of ambient PO2 further decreased cell shortening, attaining a contraction amplitude of only 4.0 ± 2.7% of diastolic cell length (-60%) at an ambient PO2 of 1 mmHg (P < 0.01 vs. 25 mmHg). The reduction in contraction amplitude was almost fully reversible on reoxygenation (9.4 ± 4.4% of diastolic cell length).


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  		Fig. 6.   Diastolic cell length (top) and cell shortening (bottom) of isolated quiescent and contracting cardiomyocytes at 25, 6, 3, and 1 mmHg. Subsequently, cardiomyocytes were reperfused with 25 mmHg. Values are means ± SD; n = 8. *P < 0.05; **P < 0.01.

Figure 7 depicts the Ca2+ transients of single cardiomyocytes in a representative experiment when ambient PO2 was stepwise decreased from 25 to 6, 3, and 1 mmHg. As is clearly visible, there is a major decrease in the systolic [Ca2+] on reduction of ambient PO2. Diastolic [Ca2+]i remained unchanged but was slightly increased compared with [Ca2+]i of the quiescent cell. The autofluorescence at an excitation wavelength of 340 and 380 nm and an emission wavelength of 510 nm was measured in unloaded cardiomyocytes and was found not to change when PO2 was reduced from 25 to 6, 3, and 1 mmHg (n = 4).


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  		Fig. 7.   Ca2+ transients of a single cardiomyocyte in a representative experiment when ambient PO2 was reduced from 25 to 6, 3, and 1 mmHg. Subsequently, cell was reperfused with 25 mmHg, and electrical pacing was stopped. Fluorescence was monitored with a data accumulation rate of 1,000 Hz. Each data point was standardized over 5 single transients, autofluorescence was subtracted, the ratio was formed, and [Ca2+]i was calculated using the appropriate calibration.

The protocol shown in Figs. 6 and 7 was carried out in 32 single cardiomyocytes from 21 individual cardiomyocyte preparations. About 30% of cardiomyocytes hypercontracted due to hypoxia or during reoxygenation. Furthermore, 20% of cells investigated showed a major increase in diastolic calcium at low ambient PO2, causing a decoupling between electrical stimulation and contraction frequency. Only cells that remained viable and rod-shaped during the entire protocol were included in the statistical analysis. Figure 8 summarizes the statistical analysis of the Ca2+ transients obtained (n = 7). The mean [Ca2+]i of quiescent cardiomyocytes determined in single cell experiments was 85.4 ± 7.5 nmol/l and increased in the contracting cell to 230.4 ± 53.2 nmol/l. Reduction of ambient PO2 from 25 to 6 mmHg decreased [Ca2+]i by 20% (P < 0.05). Further reduction of the oxygen supply to 1 mmHg progressively reduced [Ca2+]i (1 mmHg, 172.5 ± 39.0 nmol/l, ~50% reduction; P < 0.001). This reduction in [Ca2+]i was reversible on reperfusion. The decrease in mean [Ca2+]i, which is similar to data obtained in the Oxystat using cardiomyocytes in suspension (Fig. 5), is predominantly due to a decrease in systolic but not diastolic [Ca2+]i (Fig. 8, bottom). Systolic [Ca2+]i decreased from 512 ± 110 to 357 ± 91 nmol/l (-30%) when ambient PO2 was switched from 25 to 6 mmHg and to 251 ± 69 nmol/l (-50%; P < 0.01) when ambient PO2 was lowered to 1 mmHg. Reperfusion with 25 mmHg normalized systolic [Ca2+]i to basal values (417 ± 222 nmol/l).


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  		Fig. 8.   [Ca2+]i of single, fura 2-loaded cardiomyocytes at different ambient PO2. Top, mean [Ca2+]i; bottom, systolic and diastolic [Ca2+]i. Values are means ± SD; n = 8. *P < 0.05; **P < 0.01; ***P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The concept of hibernation implies a reversible downregulation of contractile function as an adaptive process induced by a reduction in myocardial blood flow; it serves to maintain myocardial energetics and viability during periods of limited oxygen supply (10). Whereas in previous studies hibernation was only shown in intact perfused hearts, this study demonstrates short-term hibernation at the level of single cardiomyocytes. We found significant downregulation of contractile activity when oxygen supply was reduced at an unchanged cellular ATP and provided evidence that short-term hibernation is mediated by a PO2-dependent decrease in intracellular Ca2+ transients.

Short-Term Hibernation in Isolated Cardiomyocytes

Short-term hibernation has been characterized by a sustained balance between the reduced myocardial oxygen supply and reduced contractile function. Perfusion-contraction matching has therefore become a hallmark of short-term hibernation (10). The present study demonstrates that the same phenomenon can be demonstrated at the level of single cardiomyocytes. A reduction in ambient PO2 from 25 to 6 and 3 mmHg reduces cellular VO2 by 50 and 80%, respectively (Fig. 2), and this effect was associated by a 30 and 50% decrease of cell shortening (Fig. 6). Another feature of short-term hibernation is also fulfilled: contractile function fully recovered during reperfusion (Fig. 6).

The cellular model of hibernation also resembles the in vivo situation with respect to cardiac energetics. In blood-perfused pig hearts, PCr and lactate exhibited transient changes when myocardial oxygen supply was acutely reduced, whereas no changes were observed in the steady state (1, 25). Similarly, isolated contracting cardiomyocytes showed a transient decrease in PCr and a transient increase in the release of lactate when the ambient PO2 was lowered from 25 to 6 mmHg. The same holds true for adenosine, a very sensitive index of the cellular energy status. In the steady state, all measured metabolic parameters were back to control, suggesting a precise match between oxygen supply and demand, however, at a drastically reduced level. Interestingly, cellular ATP remained unchanged during the entire adaptive process.

From a metabolic point of view, different stages of short-term hibernation can now be differentiated in the given cellular model. As discussed above, no metabolic changes are observed in the steady state with moderate hypoxia (6 mmHg). At more severe hypoxia (3 mmHg), however, PCr remains constantly reduced, and lactate and adenosine release remain elevated. Still, there are only minor changes in cellular ATP (Fig. 2), indicating a new stable match between ATP formation and consumption. Therefore, the situation at more severe hypoxia can also be regarded as hibernation. Obviously, there appears to be a certain regulatory range in which contractile function is depressed in a PO2-dependent manner without any measurable changes in cellular energetics. In this regulatory range, high-energy phosphates are unlikely to be involved in sustaining the depressed contractile function. By the same argument, this excludes the activation of KATP channels (24) and a change in the free energy of ATP hydrolysis (Delta GATP) (20) as potential mechanisms. However, at more severe hypoxia, where we found PCr to be depressed, Delta GATP, KATP channels, inorganic phosphate (21), or intracellular pH (18) may play a role in depressing contractile activity.

It was shown that hypoxic endothelial cells release an as yet unidentified factor that inhibits the contraction of isolated adult rat cardiomyocytes (34). This endothelium-derived factor was proposed as a potential mechanism for matching oxygen supply and demand (34). Because the present study demonstrates all features of acute hibernation in isolated cardiomyocytes without endothelial cells being present, the proposed factor appears not to be essential for hibernation to occur. Similarly, other vascular factors, such as a collapse of coronary arteries (16), are unlikely to be important. Rather, it is the cellular oxygen supply that triggers the metabolic and functional downregulation of individual cardiomyocytes. This rules out the possibility that a reduction in substrate supply or washout of bioactive metabolites normally associated with a reduced coronary flow in the in vivo heart is crucial for the adaptive process.

The precise match between ATP formation and consumption is not only a feature of cardiomyocytes but also occurs in many other cell types. For example, a remarkable regulatory precision is achieved in skeletal muscle between pathways of ATPase and ATP synthesis, when ATP turnover varies over a broad dynamic work range (12). The precise mechanism, however, is presently only poorly understood. Also, nonmuscle cells, such as endothelial cells, have the remarkable ability to reversibly downregulate their energy requirements within minutes when glucose as a substrate is omitted (5). Whether substrate deprivation induces active downregulation of ATP consumption in cardiomyocytes also has not been explored. Together, these findings suggest that short-term hibernation as a result of limited substrate or oxygen supply appears to be a general regulatory feature of many eukariotic cells that is aimed to preserve cellular energetics despite fluctuating demand.

Limitations of Cellular Model for Myocardial Hibernation

Isolated perfused hearts and isolated cardiomyocytes are characterized by a comparable rate of ATP formation and a similar energy status. In isolated contracting cardiomyocytes cellular VO2 was 27.8 ± 5.6 nmol · min-1 · mg protein-1 and the PCr-to-ATP ratio was 1.43 ± 0.47, and this compares well with data reported for the isolated heart (8). Somewhat higher PCr-to-ATP ratios have been observed in the in situ rat heart (9), which may be due to a more complex substrate supply in the in vivo situation (4).

A major limitation of cardiomyocytes is that they are rather fragile and are metabolically stable for only 30 min in a stirred suspension, such as our incubation system (36). This limited stability precludes the study of maintained viability and long-term processes involving, e.g., changes in expression pattern. Furthermore, isolated cardiomyocytes do not perform external work and cannot be readily characterized by a defined pre- and afterload. On the other hand, the isolated cell model allows precise environmental control of substrates, particularly of ambient PO2. Because oxygen supply was demonstrated in the present study to be a crucial factor for the induction of hibernation, isolated cardiomyocytes appear to be well suited to study the molecular mechanisms of this phenomenon in more detail.

Mechanisms of Acute Hibernation

The mechanism responsible for the maintenance of short-term hibernation do not involve alterations in beta -receptor density (31) or changes in the expression pattern, i.e., of sarco(endo)plasmatic Ca2+-ATPase and phospholamban (19). The postulate of KATP channels to be important (24) is equally unlikely in our cellular model because ATP did not sufficiently change. It is conceivable, however, that due to the observed decrease in PCr, there is a respective increase in cellular inorganic phosphate, which is known to inhibit contractile activity (16, 21). According to the data presented in this study, acute changes in calcium handling are likely to be a key factor in the downregulation of contractile activity. We observed a pronounced decrease in intracellular Ca2+ transients, which paralleled the decrease in myocyte shortening. Systolic Ca2+ decreased, whereas diastolic Ca2+ remained largely unchanged (Fig. 8).

The acute downregulation of contractile activity and calcium release into the cytoplasm suggests the existence of a signal transduction pathway linking oxygen supply to the functional response. The nature of this mechanism is presently not clear. Mitochondrial cytochrome oxidase has been proposed as an oxygen sensor directly coupled to oxidative phosphorylation (3). Cytochromes in the mitochondria show an apparent Michaelis-Menten constant for oxygen below 1 µM (38), and it was assumed that cytochromes remain independent of oxygen until near-anoxic conditions. However, studies on isolated cardiomyocytes (36, 39) demonstrate a pronounced oxygen gradient between the cell surface and mitochondria, suggesting that mitochondrial PO2 may be well below 1 µM when extracellular PO2 is in the range of 1-6 mmHg.


    ACKNOWLEDGEMENTS

This study was supported by the Deutsche Forschungsgemeinschaft (Schr 154/6-2).


    FOOTNOTES

Address for reprint requests and other correspondence: J. Schrader, Dept. of Physiology, Heinrich-Heine-Univ. Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany (E-mail: schrader{at}uni-duesseldorf.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 May 2000; accepted in final form 14 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(1):H42-H50
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