AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H1092-H1098, 2002; doi:10.1152/ajpheart.00318.2001
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ladilov, Y.
Right arrow Articles by Piper, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ladilov, Y.
Right arrow Articles by Piper, H. M.
Vol. 283, Issue 3, H1092-H1098, September 2002

Role of protein phosphatases in hypoxic preconditioning

Yury Ladilov, Hagen Maxeiner, Christopher Wolf, Claudia Schäfer, Karsten Meuter, and H. Michael Piper

Physiologisches Institut, Justus-Liebig-Universität, D-35392 Giessen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-7 M. To inhibit both isoforms we used 20 or 5 µM. Okadaic acid has an IC50 for PP1 of 10-7 M, for PP2A of 10-9 M. To inhibit only PP2A we used 5 nM.


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

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   A: left ventricular (LV) developed pressure (LVdevP) before 60-min global ischemia (C) and during reperfusion. B: LV end-diastolic pressure (LVEDP) before 60 min of global ischemia and during reperfusion. The following protocols are shown: control,  (n = 15); hypoxic preconditioning (HP), open circle  (n = 9); bisindolylmaleimide (BIM), black-triangle (n = 5); HP + BIM, triangle  (n = 5). Data are means ± SE. * P < 0.05 vs. control.


                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Developed pressure, heart rate, aortic pressure, and rate pressure product in ischemic-reperfused hearts

It was analyzed whether the protective effect of HP could be affected by PKC inhibition. For this purpose, PKC blockade with 1 µmol/l BIM was applied during the 20-min normoxic reperfusion or the 20-min HP protocol before ischemia. In either case, it had no significant effect on the functional recovery of hearts during reperfusion (Fig. 1). We tested previously whether this concentration of BIM could abolish a PKC-dependent preconditioning effect. By following previous experiments (14), we exposed the cardiomyocytes to the PKC activator 1,2-dioctanoyl glycerol (DOG; 20 µmol/l) under normoxic conditions instead of the anoxic conditions in the HP protocol. DOG-treated cells showed less reoxygenation-induced hypercontracture than control cells not treated with DOG (11.8 ± 2.0% vs. 27.9 ± 2.1%, n = 29, P < 0.05). The copresence of BIM (1 µmol/l) during the DOG-preconditioning protocol abolished this protection of cells (hypercontracture: 28.4 ± 1.8% vs. 27.1 ± 3.0%; n = 34, not significant).

To analyze the role of PP in the protective effect of HP, hearts were treated during the 20-min preischemic period (either normoxic perfusion or HP protocol) with 20 µmol/l cantharidin. This concentration is one order of magnitude above the IC50 of PP1 and two orders above the IC50 of PP2A, i.e., it blocks both isoforms. Because this represents a high-pharmacological concentration with the possibility of side effects, we also used cantharidin at 5 µmol/l. Under normoxic control conditions, cantharidin (20 µmol/l) exerted a small positive contractile effect, shown by a rise in LVdevP, and a rise in coronary resistance as shown by an increase in aortic pressure (Table 1). Neither of these normoxic effects was seen at 5 µmol/l (not shown). Cantharidin treatments did not alter the transient depression of contractile performance during the HP period. On reperfusion, the elevation of aortic pressure was still apparent under treatment with 20 µmol/l cantharidin, but LVdevP was no longer higher but lower than in the ischemic-reperfused controls (Fig. 2A). This lowering of LVdevP was also apparent at 5 µmol/l (7.7 ± 2.2 vs. 17.3 ± 5.1 mmHg at 60-min reperfusion, n = 5, P < 0.05). More importantly, the enhanced contractile recovery in HP-treated hearts was abolished after cantharidin pretreatment. The development of contracture, i.e., LVEDP, in reperfused control hearts was not affected by pretreatment with cantharidin (Fig. 2B). The reduction of contracture seen after HP, however, was abolished in presence of cantharidin 20 or 5 µmol/l (with or without 5 µmol/l: 48.8 ± 4.7 vs. 38.6 ± 5.7 mmHg at 60-min reperfusion, n = 5, P < 0.005). The rate-pressure product of the postischemic hearts could not be determined because the cardiac excursions were too small to allow undisturbed monitoring of the beating frequency (Table 1).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   A: LVdevP before 60 min of global ischemia (C) and during reperfusion. B: LVEDP before 60 min of global ischemia and during reperfusion. The following protocols are shown: control,  (n = 15); control + cantharidin, black-lozenge  (n = 6); HP, open circle  (n = 9); HP + cantharidin, diamond  (n = 5). Data are means ± SE. * P < 0.05 vs. control.

To further analyze the role of PPs, treatment with 5 nmol/l okadaic acid was combined with the HP protocol. At this concentration, okadaic acid has been shown to selectively inhibit PP2A but not PP1 (9, 10). In contrast to cantharidin, okadaic acid led to improved functional recovery of the reperfused HP-treated hearts; LVdevP and the rate-pressure product were significantly increased (Fig. 3A, Table 1). No significant changes were seen for LVEDP (Fig. 3B). In normoxic controls, treatment with 5 nmol/l okadaic acid had no effect. The transient contractile depression during the HP period and the rapid return to control values were the same as in HP protocols without okadaic acid. Aortic pressure was not increased in the presence of okadaic acid. In summary, the experiments showed that the HP protocol provides protection in a PKC-independent but PP-dependent manner.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3.   A: LVdevP before 60 min of global ischemia (C) and during reperfusion. B: LVEDP before 60 min of global ischemia and during reperfusion. The following protocols are shown: control,  (n = 15); control + okadaic acid,  (n = 5); HP, open circle  (n = 9); HP + okadaic acid,  (n = 5). Data are means ± SE. * P < 0.05 vs. control; #P < 0.05 vs. HP.

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).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Fura 2 ratio (arbitrary units) after 60-min anoxia in control (solid bars) and HP-treated (open bars) cardiomyocytes. Under all protocols, treatment with 1 µmol/l BIM was applied. The following experimental protocols are shown: control (n = 18); control + cantharidin (n = 17); control + okadaic acid (n = 25); HP (n = 37); HP + cantharidin (n = 27); HP + okadaic acid (n = 18). The initial value of fura 2 ratio was equal to 0.47. Data are means ± SE. * P < 0.05 vs. control; #P < 0.05 vs. HP.

To investigate the role of PP in HP-induced protection against Cai overload, experiments were performed in the presence of 20 µmol/l cantharidin or 5 nmol/l okadaic acid, 20 min before and 60 min during anoxia. Treatment with cantharidin completely abolished the protective effect of HP against Cai overload, but treatment with okadaic acid further enhanced the reduction of Cai overload by HP (Cai: 0.55 ± 0.05 µmol/l, n = 18, P < 0.05 vs. HP). Treatment of non-HP control cells with cantharidin or okadaic acid had no effects on Ca2+ overload during sustained anoxia.

Accumulation of cytosolic intracellular Na+ (Nai) can play an important role for Cai overload through the activation of the reverse mode of the Na+/Ca2+ exchanger in anoxic or ischemic myocardial cells (16). To analyze whether an alteration in the Nai accumulation is the cause of the HP effect on Cai overload, Nai concentration was monitored by the SBFI fluorescence ratio. Under control conditions, the SBFI ratio rose during 60 min of anoxia from 1 to 1.45 (Fig. 5), indicating an increase in Nai concentration from 5.0 ± 0.7 to 85 ± 6 mmol/l (n = 38). HP significantly reduced the rise of the SBFI ratio. This corresponds with a reduction of Nai overload under this condition to 41 ± 6 mmol/l, n = 39, P < 0.05 vs. control. Treatment with 20 µmol/l cantharidin abolished this reduction of Nai overload at the end of anoxia (Nai: 80 ± 7 mmol/l, n = 27, P > 0.05 vs. control). In contrast to cantharidin, treatment with 5 nmol/l okadaic acid enhanced the HP-induced protection against Nai overload. Treatment of non-HP control cells with cantharidin or okadaic acid had no effects on Nai overload during sustained anoxia.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5.   Sodium-binding bensofuran isophthalate (SBFI) ratio (arbitrary units) after 60 min of anoxia normalized to initial values in control (solid bars) and HP-treated (open bars) cardiomyocytes. Under all protocols treatment with 1 µmol/l BIM was applied. The following experimental protocols are shown: control (n = 29), control + cantharidin (n = 16), control + okadaic acid (n = 22), HP (n = 39), HP + cantharidin (n = 27), and HP + okadaic acid (n = 21). Data are means ± SE. * P < 0.05 vs. control. #P < 0.05 vs. HP.

In the present study, hypoxia was combined with low extracellular pH (6.4) to simulate ischemic acidosis. Under these conditions, cytosolic pH (pHi) was reduced from 7.15 ± 0.02 to 6.47 ± 0.02 (n = 42) in control cells during 60 min of anoxic incubation. HP alone (6.49 ± 0.03, n = 29) or in combination with cantharidin (6.45 ± 0.04, n = 21) or okadaic acid (6.50 ± 0.03, n = 19) did not affect pHi at the end of anoxia. During reoxygenation, pHi recovered to the preanoxic level with similar rapidity under all experimental conditions.

It has been shown previously (15) that the degree of cytosolic Ca2+ overload is an important determinant for the reoxygenation-induced hypercontracture of isolated cardiomyocytes. Similarly to this effect on Ca2+ overload, HP produced a significant reduction of reoxygenation-induced hypercontracture. Hypercontracture was 10.1 ± 1.1% (n = 37) vs. 27.4 ± 1.8% (n = 18, P < 0.05) in ischemic-reoxygenated control cells. The protection by HP could be abolished by treating the cells with cantharidin (25.5 ± 1.5%, n = 27) but not with okadaic acid (8.5 ± 1.1%, n = 18, P < 0.05 vs. control). Treatment of ischemic-reoxygenated control cells with cantharidin or okadaic acid had no effect on hypercontracture during reoxygenation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical help of H. Holzträger, D. Schreiber, and P. Volk.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allshire, A, Piper HM, Cuthbertson KS, and Cobbold PH. Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation. Biochem J 244: 381-385, 1987[Web of Science][Medline].

2.   Armstrong, SC, Gao W, Lane JR, and Ganote CE. Protein phosphatase inhibitors calyculin A and fostriecin protect rabbit cardiomyocytes in late ischemia. J Mol Cell Cardiol 30: 61-73, 1998[Web of Science][Medline].

3.   Armstrong, SC, Kao R, Gao W, Shivell LC, Downey JM, Honkanen RE, and Ganote CE. Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. J Mol Cell Cardiol 29: 3009-3024, 1997[Web of Science][Medline].

4.   Barrabes, JA, Garcia-Dorado D, Ruiz-Meana M, Piper HM, Solares J, Gonzalez MA, Oliveras J, Herrejon MP, and Soler Soler J. Myocardial segment shrinkage during coronary reperfusion in situ relation to hypercontracture and myocardial necrosis. Pflügers Arch 431: 519-526, 1996[Web of Science][Medline].

5.   Doering, AE, and Lederer WJ. The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. J Physiol 466: 481-499, 1993[Abstract/Free Full Text].

6.   Gray, MO, Karliner JS, and Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 30945-30951, 1997[Abstract/Free Full Text].

7.   Hammarstrom, AK, and Gage PW. Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons. J Physiol 510: 735-741, 1998[Abstract/Free Full Text].

8.   Harootunian, AT, Kao JP, Eckert BK, and Tsien RY. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J Biol Chem 264: 19458-19467, 1989[Abstract/Free Full Text].

9.   Herzig, S, and Neumann J. Effects of serine/threonine protein phosphatases on ion channels in excitable membranes. Physiol Rev 80: 173-210, 2000[Abstract/Free Full Text].

10.   Ishihara, H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y, Fusetani N, Watabe S, Hashimoto K, Uemura D, and Hartshorne DJ. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 159: 871-877, 1989[Web of Science][Medline].

11.   Ju, YK, Saint DA, and Gage PW. Hypoxia increases persistent sodium current in rat ventricular myocytes. J Physiol 497: 337-347, 1996[Abstract/Free Full Text].

12.   Knapp, J, Boknik P, Huke S, Gombosova I, Linck B, Lüss H, Müller FU, Müller T, Nacke P, Schmitz W, Vahlensieck U, and Neumann J. Contractility and inhibition of protein phosphatases by cantharidin. Gen Pharmacol 31: 729-733, 1998[Web of Science][Medline].

13.   Koop, A, and Piper HM. Protection of energy status of hypoxic cardiomyocytes by mild acidosis. J Mol Cell Cardiol 24: 55-65, 1992[Web of Science][Medline].

14.   Ladilov, YV, Balser C, and Piper HM. Protection of rat cardiomyocytes against simulated ischemia and reoxygenation by treatment with protein kinase C activator. Circ Res 82: 451-457, 1998[Abstract/Free Full Text].

15.   Ladilov, YV, Balser-Schafer C, Haffner S, Maxeiner H, and Piper HM. Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload. Cardiovasc Res 43: 408-416, 1999[Abstract/Free Full Text].

16.   Ladilov, Y, Haffner S, Balser-Schafer C, Maxeiner H, and Piper HM. Cardioprotective effects of KB-R7943: a novel inhibitor of the reverse mode of Na+/Ca2+ exchanger. Am J Physiol Heart Circ Physiol 276: H1868-H1876, 1999[Abstract/Free Full Text].

17.   Lasley, RD, Anderson GM, and Mentzer RM, Jr. Ischaemic and hypoxic preconditioning enhance postischaemic recovery of function in the rat heart. Cardiovasc Res 27: 565-570, 1993[Abstract/Free Full Text].

18.   Mitchell, MB, Meng X, Ao L, Brown JM, Harken AH, and Banerjee A. Preconditioning of isolated rat heart is mediated by protein kinase C. Circ Res 76: 73-81, 1995[Abstract/Free Full Text].

19.   Moolman, JA, Genade S, Winterbach R, and Lochner A. Preconditioning with hypoxia versus global ischemia in the isolated rat heart: effect on function and metabolism. Cardioscience 5: 73-80, 1994[Web of Science][Medline].

20.   Moolman, JA, Genade S, Tromp E, and Lochner A. No evidence for mediation of ischemic preconditioning by alpha 1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart. Cardiovasc Drugs Ther 10: 125-136, 1996[Web of Science][Medline].

21.   Piper, HM, Probst I, Schwartz P, Hütter FJ, and Spieckermann PG. Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol 14: 397-412, 1982[Web of Science][Medline].

22.   Przyklenk, K, Sussman MA, Simkhovich BZ, and Kloner RA. Does ischemic preconditioning trigger translocation of protein kinase C in the canine model? Circulation 92: 1546-1557, 1995[Abstract/Free Full Text].

23.   Sandhu, R, Diaz RJ, Mao GD, and Wilson GJ. Ischemic preconditioning: differences in protection and susceptibility to blockade with single-cycle versus multicycle transient ischemia. Circulation 96: 984-995, 1997[Abstract/Free Full Text].

24.   Schiffmann, SN, Desdouits F, Menu R, Greengard P, Vincent JD, Vanderhaeghen JJ, and Girault JA. Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase. Eur J Neurosci 10: 1312-1320, 1998[Web of Science][Medline].

25.   Speechly-Dick, ME, Mocanu MM, and Yellon Protein kinase C DM Its role in ischemic preconditioning in the rat. Circ Res 75: 586-590, 1994[Abstract/Free Full Text].

26.   Steenbergen, C, Fralix TA, and Murphy E. Role of increased cytosolic free calcium concentration in myocardial ischemic injury. Basic Res Cardiol 88: 456-470, 1993[Web of Science][Medline].

27.   Vahlhaus, C, Schulz R, Post H, Onallah R, and Heusch G. No prevention of ischemic preconditioning by the protein kinase C inhibitor staurosporine in swine. Circ Res 79: 407-414, 1996[Abstract/Free Full Text].

28.   Vanden Hoeck, TL, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res 86: 541-548, 2000[Abstract/Free Full Text].

29.   VerDonck, L, Borgers M, and Verdonck F. Inhibition of sodium and calcium overload pathology in the myocardium: a new cytoprotective principle. Cardiovasc Res 27: 349-357, 1993[Web of Science][Medline]

30.   Yabe, K, Nasa Y, and Takeo S. Hypoxic preconditioning in isolated rat hearts: non-involvement of activation of adenosine A1 receptor, Gi protein, and ATP-sensitive K+ channel. Heart Vessels 10: 294-303, 1995[Web of Science][Medline].

31.   Ytrehus, K, Liu Y, and Downey JM. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol Heart Circ Physiol 266: H1145-H1152, 1994[Abstract/Free Full Text].

32.   Zhai, X, Lawson CS, Cave AC, and Hearse DJ. Preconditioning and post-ischaemic contractile dysfunction: the role of impaired oxygen delivery vs extracellular metabolite accumulation. J Mol Cell Cardiol 25: 847-857, 1993[Web of Science][Medline].

Am J Physiol Heart Circ Physiol 283(3):H1092-H1098
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
P. C. Beguin, M. Joyeux-Faure, D. Godin-Ribuot, P. Levy, and C. Ribuot
Acute intermittent hypoxia improves rat myocardium tolerance to ischemia
J Appl Physiol, September 1, 2005; 99(3): 1064 - 1069.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
M. M. Kittleson, S. Q. Ye, R. A. Irizarry, K. M. Minhas, G. Edness, J. V. Conte, G. Parmigiani, L. W. Miller, Y. Chen, J. L. Hall, et al.
Identification of a Gene Expression Profile That Differentiates Between Ischemic and Nonischemic Cardiomyopathy
Circulation, November 30, 2004; 110(22): 3444 - 3451.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ladilov, Y.
Right arrow Articles by Piper, H. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ladilov, Y.
Right arrow Articles by Piper, H. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online