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Physiologisches Institut, Justus-Liebig-Universität, D-35392 Giessen, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 µmol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 µmol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na+/Ca2+ overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.
cellular calcium; heart function; ischemia; reperfusion
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SEVERAL STUDIES (18, 25, 32) have demonstrated that protection of the myocardium against ischemia-reperfusion injury caused by a brief preceding ischemia (ischemic preconditioning) is due to the release and accumulation of endogenous mediators, such as adenosine or noradrenaline, during ischemic preconditioning and a subsequent activation of protein kinase C (PKC). There are, however, data (20, 23, 27) indicating that protection by ischemic preconditioning can also be achieved independent of PKC activation. The mechanism of this PKC-independent protection could not be explained. Brief periods of hypoxia, i.e., hypoxic preconditioning (HP), which do not allow accumulation of ischemic mediators, can also provide protection against ischemia-reperfusion injury (17, 19, 30, 32). Previous studies (14, 26) of isolated hearts demonstrated that protection induced by HP does not involve the activation of adenosine receptors or Gi proteins, suggesting a protective signaling different from well-known mechanisms of ischemic preconditioning. A protective role of PKC in HP was suggested for cultured embryonic cardiac myocytes (6, 29), but this was not demonstrated in adult cardiac myocytes or the adult whole heart. In general, the mechanisms of HP-induced protection are still not fully understood. For the present study, we hypothesized that HP leads to protection due to a PKC-independent mechanism. We (1) tested whether protection induced by HP is PKC independent and (3) which other type of signaling is responsible for HP-induced protection. Motivated by reports (2, 3) that inhibitors of these protein phosphatases (PP) can imitate ischemic preconditioning in some experimental models, we focused our study on the role of serine threonine PP1 and PP2A.
We used the experimental model of isolated Langendorff-perfused rat
hearts exposed to 60 min of global ischemia and 60 min of
reperfusion. To test for implication of PKC we applied the PKC
inhibitor bisindolylmaleimide (BIM), and to test for implication of
PP1/PP2A we applied the PP inhibitors cantharidin or okadaic acid. To
analyze the mechanism of HP protection on the cellular level,
experiments were performed on isolated adult ventricular cardiomyocytes
exposed to 60-min simulated ischemia (hypoxia at extracellular
pH 6.4) and 20-min reoxygenation. Effects of HP on homeostasis of
cations (Ca2+, Na+, and H+) and
cell length were analyzed. We used cantharidin or okadaic acid to
differentiate between the roles of PP1 and PP2A (9, 10).
Cantharidin has an IC50 for PP1 of 10
6 M, for
PP2A of 107 M. To inhibit both isoforms we used 20 or 5 µM. Okadaic acid has an IC50 for PP1 of 107
M, for PP2A of 109 M. To inhibit only PP2A we used 5 nM.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The investigation conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996).
Experiments on isolated perfused hearts. Hearts from adult male Wistar rats (250-300 g) were mounted on a Langendorff system in a temperature-controlled chamber (37°C) and perfused for a 20-min stabilization period at constant flow of 10 ml/min with solution A containing (in mmol/l) 120.0 NaCl, 3.5 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.0 CaCl2, 22.0 NaHCO3 and 11.0 glucose, pH 7.35, at 37°C. The perfusate was saturated with 95% O2-5% CO2. These conditions are called normoxic standard perfusion. Left ventricular pressure was monitored with the use of a water-filled latex balloon connected to a pressure transducer. A second pressure transducer was used to monitor pressure in the aorta, i.e., the coronary perfusion pressure.
For ischemia-reperfusion controls, hearts were subjected for another 20 min to normoxic standard perfusion. This was followed by 60 min of global ischemia (37°C) and 60 min of normoxic standard perfusion (reperfusion). In the HP group, hearts were first subjected to 10 min of perfusion (10 ml/min) with glucose-free hypoxic solution A saturated with 95% N2-5% CO2, followed by 10 min of normoxic standard perfusion (HP). Thereafter, ischemia-reperfusion was performed as described earlier. Drugs (BIM, 1 µmol/l; cantharidin, 20 or 5 µmol/l; okadaic acid, 5 nmol/l) were applied alone or in combination during 20-min preischemic normoxia or 20-min preischemic HP protocol.Experiments on isolated cardiomyocytes. Ventricular heart muscle cells were isolated from adult male Wistar rats (250-300 g) as previously described (21) and were used in experiments 5 to 10 h after being plated on glass coverslips.
To measure cytosolic Ca2+, Na+, or H+ concentrations, cardiomyocytes were loaded in medium 199 at 35°C for 30 min with acetoxymethyl esters of 2.5 µmol/l fura 2, 5.0 µmol/l sodium-binding benzofuran isophthalate (SBFI), or 1.5 µmol/l 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), respectively. After being loaded, cells were washed twice with medium 199, followed by incubation for 30 min to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 30-40 times higher than background fluorescence from unloaded cells. Dye compartmentation was assessed by using digitonin (14) and did not exceed 10% for fura 2, 15% for SBFI, and 12% for BCECF. Fluorescence ratios of fura 2, SBFI, and BCECF were calibrated with 5 µmol ionomycin (14), 6 µmol/l gramicidin D (8), and 10 µg/ml nigericin (13), respectively. Simultaneously to the fluorescence measurement, the microscopic image of the cells was recorded with a video camera. The extent of cell hypercontracture was expressed as a percentage, relating the cell shortening to cell length before anoxia. Cardiomyocytes were superfused at a flow rate of 0.6 ml/min with solution B containing (in mmol/l) 140.0 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, 5.0 glucose, and 25.0 HEPES (pH 7.4, at 37°C). Normoxic medium was equilibrated with air. The medium was made anoxic by autoclavation as previously described (1). The anoxic medium was glucose-free and equilibrated with 100% N2. Under all protocols, cells were exposed to 60-min anoxia at pH 6.4 and 20-min reoxygenation at pH 7.4. To exclude PKC-dependent effects, all experiments were performed in the presence of 1 µmol/l BIM. For anoxia-reoxygenation controls, cells were superfused for 20 min with normoxic medium (pH 7.4). HP was induced by 10-min superfusion of cells with anoxic medium (pH 6.4), followed by 10 min of reoxygenation before sustained anoxia. PP inhibitors were applied during the initial 20 min of normoxic superfusion or 20-min HP protocol and during sustained anoxia.Statistics. Data are given as means ± SE. For each experimental protocol, 20-40 individual cells were used, with not >6 cells from the same cell isolate. Comparisons between groups were performed by one- or two-way analysis of variance, followed by the Student-Newman-Keuls test where appropriate. Statistical significance was accepted when P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of HP in isolated hearts.
To produce HP, hearts were perfused for 10 min with hypoxic medium,
followed by reoxygenation for another 10 min (20-min HP protocol)
before 60 min of global ischemia. At the end of the hypoxic
period, left ventricular developed pressure (LVdevP) was reduced to
39 ± 5% (n = 15, P < 0.05) of
its normoxic control value, but contractile performance rapidly
recovered thereafter. HP did not alter hemodynamic parameters
immediately before 60 min of ischemia (not shown). In contrast,
HP significantly improved postischemic recovery of left
ventricular function. After 60 min of reperfusion, LVdevP was 40 ± 7 mmHg in HP-treated hearts (n = 9) versus 16 ± 5 mmHg in control hearts (n = 15, P < 0.05) (Fig. 1A). The
rate-pressure product showed similar differences (Table 1). This was associated with reduced left
ventricular end-diastolic pressure (LVEDP), i.e., contracture in
HP-treated hearts. After 60 min of reperfusion, LVEDP was 39 ± 6 mmHg after HP (n = 9) versus 59 ± 6 mmHg in
non-HP controls (n = 15, P < 0.05)
(Fig. 1B).
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Effects of HP in isolated cardiomyocytes.
To analyze the cellular mechanism of HP-induced PKC-independent
protection, cytosolic ion homeostasis (Ca2+,
Na+, pH) and cell length were investigated in isolated
cardiomyocytes exposed to simulated ischemia (anoxia at pH
6.4). For this purpose, all experiments with isolated cardiomyocytes
were performed in the presence of 1 µmol/l BIM. In the control group,
a 60-min superfusion of cardiomyocytes with anoxic glucose-free medium
at pH 6.4 (simulated ischemia) caused an accumulation of
Ca2+ in the cytosol as indicated by an increase in the fura
2 ratio (Fig. 4). Calibration of the fura
2 ratio showed that cytosolic intracellular Ca2+
(Cai) concentration was 2.14 ± 0.09 µmol/l
(n = 18) at the end of anoxia. The HP protocol was
simulated by preexposure of the cells to 10 min of these anoxic
conditions, followed by 10 min of normoxia. HP significantly reduced
Cai overload at the end of sustained anoxia
(Cai: 0.75 ± 0.06 µmol/l, n = 37, P < 0.05 vs. control).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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There were several findings of this study. First, in isolated ischemic-reperfused hearts, HP caused a significant improvement of functional recovery and a reduction of contracture development during reperfusion. This effect was not altered by PKC inhibition. Second, the protective effect could be abolished by the presence of cantharidin, an inhibitor of PP1 and PP2A. The result of experiments with a low dose of okadaic acid suggests that PP1 plays a beneficial role in HP-mediated protection. Third, in isolated cardiomyocytes HP provided PKC-independent protection against Nai and Cai overload during simulated ischemia and against hypercontracture during reoxygenation. This protection had the same responsiveness to PP inhibitors as HP protection in whole hearts.
Ischemic preconditioning represents a powerful procedure to protect ischemic-reperfused myocardium in most investigated animal species as well as in humans. It is now clear that numerous triggers and mediators can elicit this protective mechanism. It is still not clear whether all forms of protection elicited by an ischemic-preconditioning protocol have a common final end point (effector) within the protective signaling. As mentioned above, in some models ischemic preconditioning provides protection independent of PKC and exchanging preconditioning ischemia for brief hypoxic perfusion can also provide protection. We found in a rat model that hypoxic preconditioning provides protection independent of PKC inhibition. To our knowledge others have not investigated this aspect of HP in a rat model. In mouse neonatal cell cultures, HP protection was found to be PKC dependent (6), but this may be due to species or developmental differences. In rabbit cardiomyocytes and hearts, Armstrong et al. (3) reported that application of a PP2A inhibitor provides protection in a PKC-independent manner. Therefore, we tested the role of PP1/PP2A using appropriate inhibitors. Inhibition of PP1/PP2A with a dose of cantharidin (20 µmol/l) well above either of the respective IC50, abolished HP protection. Inhibition of PP2A with a dose of okadaic acid two orders below the IC50 of PP1 but above that of PP2A augmented the protective effects of HP on LVdevP and the rate-pressure product in reperfused hearts. These observations were made in hearts as well as in cardiomyocytes. They indicate that PP1 activation represents a mediator of HP protection in this rat model, whereas PP2A activation is adverse to this beneficial effect. In this latter aspect, our results resemble the findings of Armstrong et al. (2, 3). In contrast to their observations, however, we did not see a protective effect of PP2A inhibition alone. The difference may be related to differences in model ischemia (dense cell pellet vs. cells superfused with anoxic acidotic medium) or in species (rabbit or pig vs. rat).
A word of caution seems appropriate: as in all studies that are based on the use of pharmacological inhibitors, our experiments and those described by Armstrong's group (2, 3) could be affected by unknown side effects of these inhibitors. This consideration may apply particularly to the use of cantharidin at a concentration (20 µmol/l) well above the IC50 of PP1 and PP2A. Therefore, we also performed experiments with 5 µmol/l. Under normoxic conditions, 20 µmol/l cantharidin also affected contractile performance and coronary resistance, as previously described (12). These hemodynamic effects, however, are not accountable for the HP-antagonistic effects of cantharidin. First, because hearts were perfused at a constant flow rate, an increase in coronary resistance did not infringe on coronary flow. Second, the increase in contractility observed before ischemia cannot explain the reduction of contractile recovery in the reperfused hearts because these effects are opposite. Third, these preischemic hemodynamic effects were not seen with 5 µmol/l cantharidin. Fourth, the finding that cantharidin interferes with HP protection also in isolated cells provides another argument that its effect on HP protection is not due to its hemodynamic side effects. Okadaic acid (5 nmol/l) had no such side effect on normoxic hemodynamics.
To analyze the downstream cellular mechanisms of PKC-independent HP protection in isolated hearts, a model of isolated cardiomyocytes was used. Cells were exposed to simulated ischemic conditions (anoxia, extracellular pH 6.4) and subsequent reoxygenation. This model was characterized before in detail (14-16). The alteration of ion homeostasis, especially Cai overload, during ischemia was shown to play an important role in the development of ischemic injury of hearts (26). The effect of HP on Cai homeostasis in isolated cardiomyocytes during simulated ischemia was analyzed under conditions excluding the role of PKC, i.e., in the presence of the PKC inhibitor BIM. HP led to a significant reduction of anoxic Cai overload. The mechanism of this HP-induced protection in isolated cardiomyocytes seems similar to that found in whole hearts. Treatment with cantharidin completely abolished protection in either model, whereas treatment with okadaic acid partly augmented it. Taken together, these data indicate that attenuation of Cai overload in cardiomyocytes represents the cellular basis of the HP-induced improvement of postischemic heart function.
An important consequence of Cai overload is the development of hypercontracture in reoxygenated cardiomyocytes (15). Similar to Cai overload, hypercontracture was reduced by HP in a PP-sensitive manner. In whole hearts, hypercontracture of cardiomyocytes seems to be the basis for the observed rise of LVEDP during reperfusion (4). Indeed, in the present study, the effects of HP with or without cantharidin or okadaic acid treatment were similar in respect to LVEDP in whole hearts and or hypercontracture in cardiomyocytes.
Koop and Piper (13) showed that the main cause for Cai accumulation in metabolically inhibited cardiomyocytes consists in the influx of extracellular Ca2+ through the reverse mode of the Na+/Ca2+ exchanger. The rise of Nai is one of the driving forces for the activation of the reverse mode of Na+/Ca2+ exchanger in cardiomyocytes (5, 28). Consistent with this causal sequence is the finding that the changes in Nai overload are perfectly matched with changes in Cai overload under corresponding experimental conditions. These data indicate that the mechanism leading to Nai overload is indeed the primary target of HP-induced action. The mechanism of ischemic Nai overload is not yet fully understood. It has been speculated that the noninactivating Na+ current (also called sustained or persistent Na+ current) is responsible for Nai overload under ischemic conditions (7, 11). Recent studies (24) demonstrated that the Na+ channel represents a target molecule for PP1. It may, therefore, be suggested that the observed changes in Nai overload in HP-treated cardiomyocytes under phosphatase inhibition are due to a change in the phosphorylation of Na+ channels.
It is noteworthy that the phosphatase-dependent hypoxic preconditioning effect is found in the same experimental model of isolated cardiomyocytes from the adult rat as a previously described PKC-dependent mechanism of protection (15). It shows that these cells contain multiple pathways that may confer signaling of ischemic preconditioning.
In conclusion, the results of this study indicate that HP provides protection of isolated hearts and isolated cardiomyocytes against ischemic injury by a mechanism endogenously antagonized by PP2A activation and supported by activation of PP1. It may, therefore, be suggested that PP are targets for new strategies to protect the ischemic-reperfused heart. Because hypoxia-reoxygenation transition is also part of the protocol of ischemic preconditioning, it may be supposed that the described mechanism of protection is contained within the complex protective mechanism of ischemic preconditioning. It is possibly responsible for the finding in some biological models that ischemic preconditioning can provide myocardial protection independent of PKC activation (20, 21, 23, 27).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical help of H. Holzträger, D. Schreiber, and P. Volk.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grant LA 1159/2-1. Part of this study was used for a Doctor of Medicine thesis (H. Maxeiner), Justus-Liebig-Universität, Giessen, Germany.
Address for reprint requests and other correspondence: H. M. Piper, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany (E-mail: michael.piper{at}physiologie.med.uni-giessen.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.
10.1152/ajpheart.00318.2001
Received 27 April 2001; accepted in final form 2 May 2002.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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(n = 15); hypoxic preconditioning (HP), 
(n = 9); bisindolylmaleimide (BIM), 
(n = 5); HP + BIM, 
(n = 5). Data are means ± SE.
* P < 0.05 vs. control.
(n = 6); HP, 
(n = 5). Data are
means ± SE. * P < 0.05 vs. control.
(n = 5); HP, 
(n = 5). Data are means ± SE. * P < 0.05 vs. control; #P < 0.05 vs. HP.
