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Am J Physiol Heart Circ Physiol 288: H2594-H2602, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00926.2004
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Role of dual-site phospholamban phosphorylation in intermittent hypoxia-induced cardioprotection against ischemia-reperfusion injury

Yan Xie,1 Yi Zhu,1 Wei-Zhong Zhu,2 Le Chen,1 Zhao-Nian Zhou,2 Wen-Jun Yuan,3 and Huang-Tian Yang1

1Laboratory of Molecular Cardiology, Health Science Center, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Second Medical University, Shanghai; 2Physiological Laboratory of Hypoxia, SIBS, CAS, Shanghai; and 3Department of Physiology, Second Military Medical University, Shanghai, China

Submitted 7 September 2004 ; accepted in final form 3 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca2+ overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca2+-pump ATPase, a critical regulator in intracellular Ca2+ cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser16 (PKA site) and Thr17 (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca2+-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser16 phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr17 phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser16 phosphorylation, Ca2+-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr17 phosphorylation, Ca2+-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca2+-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.

phospholamban phosphorylation residues; cardiac sarcoplasmic reticulum Ca2+-pump ATPase; cardiac contractile function

ADAPTATION TO INTERMITTENT HIGH-ALTITUDE (IHA) hypoxia increases cardiac tolerance to subsequent severe hypoxic or ischemic injury in animal models (1, 17, 24, 26, 37). Some clinical and epidemiological observations also have shown that the cardiac tolerance to acute oxygen deprivation is increased and that the incidence of myocardial infarction and mortality from coronary heart disease is reduced in people living at high altitude (8). In addition, this form of protection persists longer in comparison with the temporal character of classic preconditioning (2, 36) and has fewer side effects such as right ventricular hypertrophy compared with the adaptation to continuously chronic hypoxia (1, 25, 35). Therefore, obtaining a better understanding of the mechanisms underlying such long-term protection might be of clinical importance. However, unlike brief hypoxic or ischemic preconditioning, adaptation to IHA hypoxia has attracted much less attention, and its cardioprotective mechanisms are far from clear.

Several mechanisms have been proposed to explain the IHA hypoxia-induced cardiac protection, such as increases in oxygen uptake and antioxidative ability (36), activation of protein kinase C (PKC) isoforms (5), and opening of mitochondrial ATP-sensitive K+ channel (mitoKATP) (1, 37). Investigators in our laboratory have recently demonstrated that IHA hypoxia significantly protects the heart against lethal Ca2+ overload injury induced by Ca2+ paradox (34) and eliminates cytosolic intracellular Ca2+ overload induced by ischemia-reperfusion (I/R) (37). This effect also may play a critical role in the cardioprotection of IHA hypoxia. However, the precise mechanisms against intracellular Ca2+ overload by IHA hypoxia are not clear.

Intracellular Ca2+ overload is suggested to be one of the main factors involved in I/R injury. Because the sarcoplasmic reticulum (SR) plays a critical role in regulating cytosolic Ca2+ concentration and subsequent cardiac contractility and relaxation, the dysfunction of cardiac SR Ca2+-pump ATPase (SERCA2a) during I/R may contribute to Ca2+ overload and thereby affect the extent of contractile dysfunction that follows ischemia (23, 38). It is conceivable that one of the key factors affecting SR Ca2+-pump ATPase function and its ability to offset Ca2+ overload is phospholamban (PLB), an important phosphorylation protein of the cardiac SR that reversibly inhibits SR Ca2+-pump ATPase activity and SR Ca2+ uptake. Phosphorylation of PLB by cAMP-dependent protein kinase (PKA) at the Ser16 residue or by Ca2+-calmodulin-dependent protein kinase (CaMKII) at the Thr17 residue removes this inhibition, thereby increasing SR Ca2+-pump ATPase activity and the rate of SR Ca2+ uptake (12, 28). Thus an alteration in the phosphorylation status of PLB residues during I/R should influence the consequent changes in SR Ca2+-pump ATPase activity and SR Ca2+ uptake (20, 23). There is evidence that ischemic preconditioning, which is associated with cardioprotection against I/R injury, attenuates I/R-induced depression in cardiac SR function and gene/protein expression including SERCA2a and PLB (22, 30). Recently, Wang et al. (33) demonstrated that IHA hypoxia prevents an I/R-induced decrease in cardiac SR Ca2+ release channel. However, it is unknown whether IHA hypoxia alters PLB phosphorylation status and SR Ca2+-pump ATPase activity in I/R hearts and, if it does, whether this alteration contributes to the cardioprotection of IHA hypoxia.

In the present study, we have compared the time-dependent changes in PLB phosphorylation status at Ser16 and Thr17 in normoxic and IHA hypoxic rat hearts during I/R and determined the possible role of such changes on IHA hypoxia-induced contractile recovery after ischemia.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and all procedures were approved by the Institutional Review Board of the Health Science Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Second Medical University.

Animals. Male Sprague-Dawley rats were exposed to IHA hypoxia in a hypobaric chamber for one 6-h period each day for 42 days. Their body weights during this period rose from 100–130 g to 290–310 g. Barometric pressure (PB) was lowered to the level equivalent to that at an altitude of 5,000 meters (PB = 404 mmHg, PO2 = 84 mmHg). Age-matched control (normoxic) animals were kept under the normoxic environment for a corresponding time period. Their body weights and gains were identical to those in the group exposed to the IHA hypoxia. All animals had free access to water and a standard laboratory diet.

Heart perfusions. Rats were anesthetized with pentobarbital sodium (60 mg/kg ip). The hearts were rapidly excised and perfused with Krebs-Henseleit solution at 37°C by using the Langendorff technique at a constant pressure of 80 mmHg, as previously described (33, 37). The perfusion solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4·7H2O, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 0.026 Na2-EDTA, and 11.1 glucose was gassed with 95% O2-5% CO2 (pH 7.4). For measuring the mechanical activity of the heart, a latex balloon connected to a pressure transducer (Gould model P23 Db, AD Instrument, Castle Hill, Australia) was inserted into the left ventricle. The balloon was filled with aqueous solution to achieve a left ventricular end-diastolic pressure (LVEDP) of 5–7 mmHg during initial equilibration. Contractile performance of the left ventricle was evaluated on the basis of its developed pressure (LVDP), the LVEDP, the maximal rate of pressure development (+dP/dt), and the maximal rate of pressure decay (–dP/dt). These parameters were monitored and analyzed with PowerLab (AD Instrument).

Experimental protocol. After stabilization, hearts were perfused for 10 additional minutes (preischemia) and were then subjected to 30 min of no-flow global ischemia (I30) followed by 30 min of reperfusion (R30), as shown in Fig. 1A. For the nonischemic control groups, the hearts were perfused for a comparable period with the oxygenated medium and then freeze-clamped at different times to match either the different ischemic or reperfusion periods with the studies on the association between PLB phosphorylation status and Ca2+-pump ATPase activity. Because basal values of PLB phosphorylation status and Ca2+-pump ATPase activity did not change with time, they were considered as a single group for statistical analysis. To test whether inhibitors of either PKA or CaMKII attenuate the beneficial effects of IHA hypoxia against I/R, we used H-89 (Calbiochem) and KN-93 (Sigma), respectively. When such reagents were used, either their corresponding vehicle or each inhibitor (H-89, 20 µM or KN-93, 1 µM) was delivered by an infusion pump into the perfusion stream above the aortic cannula at 1 ml/min (11, 32). Inhibitors were perfused for 5 min before ischemia, ending at the first minute of reperfusion (H-89) or during reperfusion (KN-93). At the end of the experiments, the hearts were freeze-clamped and stored at –80°C until used in assays performed later.



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  		Fig. 1. Experimental protocol and left ventricular (LV) function during ischemia-reperfusion (I/R) in normoxic and intermittent high-altitude (IHA) hypoxic rat hearts. A: experimental protocol. After stabilization, rat hearts were perfused for 10 additional minutes (Pre) and then subjected to 30 min of no-flow global ischemia followed by 30 min of reperfusion. Arrows indicate time points at which hearts were removed for assays. B: cardiac performance during I/R in normoxic (n = 5) and IHA hypoxic hearts (n = 6). LVDP, LV developed pressures; LVEDP, LV end-diastolic pressure; +dP/dt, maximal rate of LV pressure development; –dP/dt, maximal rate of LV pressure decline. C: maximal extent of ischemic contracture during ischemia in normoxic and IHA hypoxic hearts in the presence or absence of H-89 or KN-93. Values are means ± SE (n = 4–14). *P < 0.05; **P < 0.01 vs. corresponding normoxia values.

 
Preparation of SR vesicles. The SR vesicles were prepared as described previously (20, 22, 23). Briefly, the left ventricular tissue was homogenized in an extraction buffer containing (in mM) 10 NaHCO3, 5 NaN3, and 15 Tris·HCl (pH 6.8) with a Polytron PT 3100 homogenizer. The homogenate was centrifuged for 10 min at 11,000 g to remove cellular debris. The supernatant was centrifuged at 45,000 g for 45 min. The pellet was suspended in 8 ml of 600 mM KCl and 10 mM Tris·HCl (pH 6.8) and centrifuged at 45,000 g for 45 min. The final pellet was resuspended in 1 ml of 250 mM sucrose and 10 mM histidine buffer. The concentration of the SR membrane proteins was determined using the Bradford method. The yield was 1–2 mg membrane vesicles protein/g cardiac tissue.

Determination of SR Ca2+-pump ATPase activities. The SR Ca2+-pump ATPase activities were determined colorimetrically by measuring the inorganic phosphate (Pi) liberated from ATP hydrolysis according to a previously described method (6, 22). Briefly, total (Mg2+ and Ca2+) and basal (Mg2+) Ca2+-pump ATPase activities were determined in the presence or absence of Ca2+ by using a Ca2+-pump ATPase enzyme assay kit (Jiancheng Bioengineering Institute, Nanjing, China). Mg2+-dependent (basal) ATPase activity was assayed in a reaction medium containing (in mM) 20 Tris·HCl, 5 MgCl2, 100 KCl, 5 NaN3, and 1 EGTA. Total Ca2+-pump ATPase activity was assayed in a similar medium, except that EGTA was replaced with 0.05 mM CaCl2. The reaction was started by the addition of 5 mM Tris·ATP in the presence of 20 µg of SR protein and was terminated with 12% trichloroacetic acid. Pi liberated during the reaction was estimated in the protein-free supernatant. The Ca2+-stimulated, Mg2+-dependent ATPase (Ca2+-pump ATPase) activity was calculated as the difference between the total and basal ATPase activities.

Western blot analysis. The detection of PLB was performed as described previously (14). Briefly, ~100 mg of freeze-clamped left ventricular tissue were homogenized at 4°C with a homogenizer in 10 vols of buffer containing (in mM) 5 histidine-HCl (pH 7.4), 10 EDTA, 50 Na4P2O7, 25 NaF, 0.2 dithiothreitol, and 0.1 phenylmethylsulfonyl fluoride. The concentration of protein was determined using the Bradford method. The homogenates were stored at –80°C. Thirty micrograms of homogenate protein per lane were resolved by performing urea SDS-PAGE with 7.5% (wt/vol) acrylamide gels. Proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad) and probed with the total PLB antibody anti-phospholamban A1 (1:2,000 dilution; Upstate Biotechnology) or the phosphorylation site-specific PS-16 and PS-17 PLB antibodies (1:10,000 dilution; Phosphoprotein Research, Leeds, UK). The membranes were then incubated with peroxidase-conjugated goat anti-mouse IgG (1:2,000 dilution; Sigma) or peroxidase-conjugated goat anti-rabbit IgG (1:15,000 dilution; Jackson ImmunoResearch). The immunoreaction was visualized using an enhanced chemiluminescent detection kit (Amersham Pharmacia Biotech, Amersham, UK), exposed to X-ray film, and quantified with a video documentation system (Gel Doc 2000; Bio-Rad). After exposure to a second antibody and luminescent detection of PLB phosphorylation status, the membrane was then stripped and exposed to anti-phospholamban A1 to detect total PLB to ensure equivalent protein loading. The signal intensity of the bands was quantified using optical densitometry analysis. Total PLB was expressed as a percentage of control densitometry. The phosphorylation of PLB was expressed as a percentage of isoproterenol-induced Ser16 or Thr17 phosphorylation at 30 nM, a concentration that produces maximal phosphorylation of PLB residues and mechanical response (19), running in parallel with each experimental series.

Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined using ANOVA or repeated ANOVA for multiple comparisons or repeated measurements. Significant differences between the two mean values were estimated using Student's t-test (SPSS 11.5). A P value <0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of IHA hypoxia on left ventricular function during I/R. The time courses of changes in left ventricular function of the normoxic and IHA hypoxic groups with or without I/R are shown in Fig. 1B. During the preischemic phase, IHA hypoxia did not affect the LVDP, LVEDP, +dP/dt, and –dP/dt. During no-flow ischemia, LVDP, +dP/dt, and –dP/dt declined to zero, whereas LVEDP increased in both normoxic and IHA hypoxic hearts. However, in IHA hypoxic hearts, the maximum extent of ischemic contracture was significantly lower compared with that in normoxic hearts (74.1 ± 1.4 vs. 83.1 ± 2.2 mmHg, P < 0.01). During the reperfusion phase, IHA hypoxia significantly improved recovery of cardiac contractility. After 30 min of reperfusion of the ischemic hearts, the recovery of LVDP, +dP/dt, and –dP/dt in each group (normoxia vs. IHA hypoxia) was 19.6 ± 1.8 vs. 37.6 ± 2.7%, 18.4 ± 0.5 vs. 40.1 ± 3.9%, and 20.4 ± 0.8 vs. 39.9 ± 3.0% of preischemic values (P < 0.01 each), respectively. Concomitantly, this was associated with a marked decrease of LVEDP in the IHA hypoxic group (50.6 ± 3.9 mmHg) compared with that in the normoxic group (73.2 ± 4.6 mmHg, P < 0.01).

Time course of PLB protein content and Ser16/Thr17 phosphorylation of PLB during I/R. To gain insight into the effect of IHA hypoxia on PLB, we determined the time course of the total PLB expression and its phosphorylation status during I/R in normoxic and IHA hypoxic left ventricles. The data correspond to the same hearts in which cardiac function was evaluated. Figure 2 represents immunoblots and densitometric evaluations of total protein content of PLB. No significant difference in total PLB was detected between normoxic and IHA hypoxic hearts with or without I/R. Figure 3 shows the time course of changes in the phosphorylation status of PLB at Ser16 and Thr17 during I/R in normoxic and IHA hypoxic hearts. In normoxic hearts, Ser16 phosphorylation significantly increased at I30 by 188.8 ± 12.6% and after 1 min of reperfusion (R1) by 220.1 ± 10.9% of preischemic values, respectively (P < 0.01), but decreased to a level lower than that during preischemia by R30 (P < 0.01). In IHA hypoxic hearts, Ser16 phosphorylation in the preischemic phase was unchanged compared with that in normoxic hearts. However, I/R-induced increases in the phosphorylation status of this residue in hypoxic hearts were significantly higher (increased at I30 by 252.1 ± 19.9% and at R1 276.6 ± 30.4% of preischemic values, respectively, P < 0.05) compared with normoxic values, whereas I/R-induced depression at R30 remained unchanged (Fig. 3A).



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  		Fig. 2. Time course of phospholamban (PLB) content during I/R in normoxic and IHA hypoxic hearts. A: representative original immunoblots of PLB protein levels. B: average densitometric results of immunoblotting. Total PLB contents were determined before ischemia (Pre), at 2 (I2) and 30 min of ischemia (I30) and at 1 (R1) and 30 min of reperfusion (R30) and were expressed as a percentage of normoxic densitometry units at preischemia. Values are means ± SE of 4 different freeze-clamped left ventricles at each time point.

 


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  		Fig. 3. Time course of Ser16 and Thr17 PLB phosphorylation during I/R in normoxic and IHA hypoxic hearts. A: representative original immunoblots of Ser16 and total PLB (top) and average densitometric results (bottom) of phosphorylation of Ser16 PLB (PSer16-PLB). B: representative original immunoblots (top) and average densitometric results (bottom) of phosphorylation of Thr17 PLB (PThr17-PLB). Phosphorylation of PLB was determined before ischemia (Pre), at I2 and I30, and at R1 and R30. Results are expressed as percentages of the phosphorylation of PLB sites induced by 30 nM isoproterenol (Iso) in nonischemic hearts. Values are means ± SE; n = 4 hearts at different time points in each group. **P < 0.01 vs. corresponding preischemia values. #P < 0.05 vs. corresponding normoxia values.

 
In normoxic hearts, phosphorylation of PLB at Thr17 significantly decreased at I30, R1, and R30 by 74.4 ± 8.8, 63.0 ± 9.3, and 79.5 ± 8.6% of preischemic values, respectively (P < 0.01 each). IHA hypoxia did not significantly affect preischemic phosphorylation of Thr17 but markedly attenuated the I/R-induced decrease in Thr17 phosphorylation (at R1 and R30, 30.7 ± 8.1 and 41.2 ± 4.2% of preischemic values, respectively, P < 0.01) compared with corresponding normoxic values (Fig. 3B).

Time course of SR Ca2+-pump ATPase activities during I/R. Because under normoxic and hypoxic conditions SR Ca2+-pump ATPase activity is tightly controlled by PLB phosphorylation status, we tested the hypothesis that IHA hypoxia alters the activity of Ca2+-pump ATPase during I/R. Figure 4 shows the time course of SR Ca2+-pump ATPase activities during I/R in normoxic and IHA hypoxic groups. The data correspond to the same hearts in which cardiac function and PLB phosphorylation were evaluated. Relative to preischemic values, SR Ca2+-pump ATPase activities decreased significantly at the end of ischemia and during postischemic reperfusion by 54.3 ± 4.2% (I30), 38.4 ± 2.9% (R1), and 55.4 ± 2.8% (R30), respectively (P < 0.01) in normoxic hearts. The depression was more significant at I30 and R30. However, these changes were significantly attenuated in hearts exposed to IHA hypoxia (34.3 ± 5.3% at I30, 26.7 ± 4.7% at R1, and 44.1 ± 1.3% at R30, P < 0.05; Fig. 4).



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  		Fig. 4. Time course of sarcoplasmic reticulum (SR) Ca2+-pump ATPase activities during I/R in normoxic and IHA hypoxic rat left ventricles. SR Ca2+-pump ATPase activities were examined before ischemia (Pre), at I2 and I30, and at R1 and R30. Values are means ± SE of 4 hearts in each group. **P < 0.01 vs. corresponding preischemia values. #P < 0.05 vs. corresponding normoxia values. {dagger}P < 0.05; {dagger}{dagger}P < 0.01 vs. corresponding R1 values.

 
Relative contribution of PKA to IHA hypoxia-induced protection against I/R injury. Because it is well established that the phosphorylation status of PLB at Ser16 is exclusively mediated by the PKA pathway, we used the PKA inhibitor H-89 to study the relationship between phosphorylation of PLB at Ser16 and the dependence of mechanical recovery on exposure to IHA hypoxia during I/R. H-89 completely abolished the increased phosphorylation of PLB Ser16 at R1 in both normoxic and IHA hypoxic hearts (Fig. 5, A and B). Consistently, H-89 inhibited SR Ca2+-pump ATPase activities and postischemic recovery of cardiac relaxation in normoxic hearts and abolished the beneficial effect of IHA hypoxia on these parameters (Fig. 5, C and D), although it did not affect preischemic contractile and relaxation parameters in both groups (data not shown). No significant difference in the maximum extent of ischemic contracture was detected with or without H-89 in both normoxic and IHA hypoxic hearts (Fig. 1C).



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  		Fig. 5. Effects of PKA inhibitor H-89 on phosphorylation of Ser16 PLB, activities of Ca2+-pump ATPase, and cardiac relaxation after ischemia in normoxic and IHA hypoxic left ventricles. A: representative original immunoblots of PLB Ser16 phosphorylation. B: average densitometric results of immunoblotting. C: effects of H-89 on Ca2+-pump ATPase activities. D: effects of H-89 on cardiac relaxation. Results are means ± SE of n = 4–6 hearts at different time points in each group. *P < 0.05; **P < 0.01 vs. corresponding control values. ##P < 0.01 vs. IHA hypoxia values.

 
Relative contribution of CaMKII to IHA hypoxia-induced protection against I/R injury. Site-specific phosphorylation of PLB at Thr17 is mediated by the CaMKII pathway. To evaluate the contribution by this pathway to inducement of the beneficial effects of IHA hypoxia, we determined in I/R hearts the effects of KN-93, a CaMKII-specific inhibitor, on Thr17 phosphorylation of PLB, SR Ca2+-pump ATPase activity, and cardiac function. KN-93 did not affect preischemic contractile and relaxation in normoxic and IHA hypoxic hearts (data not shown). In contrast, Fig. 6 shows that KN-93 completely abolished the IHA hypoxia-induced increase in Thr17 phosphorylation of PLB and SR Ca2+-pump ATPase activity at R30 but had no effect in normoxic hearts (Fig. 6, A–C). These inhibitory effects were associated with an abolishment by KN-93 of the improved recovery of cardiac relaxation induced by IHA hypoxia, although KN-93 did not affect cardiac relaxation in normoxic hearts (Fig. 6D). No significant difference in the maximum extent of ischemic contracture was detected with or without KN-93 in both normoxic and IHA hypoxia hearts (Fig. 1C).



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  		Fig. 6. Effects of CaMKII inhibitor KN-93 on phosphorylation of Thr17 PLB, activities of Ca2+-pump ATPase, and cardiac performance after ischemia in normoxic and IHA hypoxic left ventricles. A: representative original immunoblots of PLB Thr17 phosphorylation. B: average densitometric results of immunoblotting. C: effects of KN-93 on Ca2+-pump ATPase activities. D: effects of KN-93 on cardiac relaxation. Results are means ± SE of n = 4–6 hearts at different time points in each group. *P < 0.05; **P < 0.01 vs. corresponding control values. #P < 0.05 vs. IHA hypoxia values.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We evaluated changes in the phosphorylation status of PLB Ser16 and Thr17 during I/R in normoxic and IHA hypoxic rat hearts to determine whether IHA hypoxia-induced upregulation of PLB Ser16 and Thr17 phosphorylation improved contractile recovery after ischemia.

Effects of IHA hypoxia on Ser16 and Thr17 phosphorylation of PLB during I/R. The phosphorylation status of PLB in normoxic hearts is generally similar to that reported earlier (20, 32). We report here for the first time the effect of IHA hypoxia on the time course of the phosphorylation of PLB Ser16 and Thr17 during I/R. The phosphorylation of PLB at Ser16 and Thr17 decreased at the end of reperfusion. However, at the end of ischemia and at the onset of reperfusion, there is dissociation between the changes in the phosphorylation status of these two residues, i.e., an increase in Ser16 phosphorylation associated with a decrease in Thr17 phosphorylation. The trend at the onset of reperfusion is contrary to that described by Vittone et al. (32). They found, instead, an opposite dissociation between the phosphorylation of the two residues at the onset of reperfusion. This discrepancy may be caused by a difference in the duration of ischemia and/or experimental condition, because the ischemic time was 30 min in our protocol, whereas it was 20 min in that of Vittone et al. (32). IHA hypoxia did not significantly affect total PLB protein levels or alter the pattern of changes in dual-site phosphorylation of PLB in I/R. However, it significantly enhanced the upregulation of PLB Ser16 phosphorylation at the end of ischemia and at the onset of reperfusion and attenuated the decreased PLB Thr17 phosphorylation during I/R.

The observed dissociated regulation of Ser16 and Thr17 phosphorylation during I/R in either normoxic or IHA hypoxic hearts suggests that different mechanisms are involved. Intracellular Ca2+ overload is known to occur under severe I/R. A recent study (21) demonstrated that intracellular Ca2+ overload depressed SR CaMK activity and reduced CaMK phosphorylation of PLB. Thus the decrease in phosphorylation of Thr17 during I/R may be due to a depression in CaMK activity caused by intracellular Ca2+ overload. This is supported by our observation that whereas CaMKII inhibitor did not affect depressed Thr17 phosphorylation further in the normoxic heart, it completely inhibited the IHA hypoxia-improved Thr17 phosphorylation. It also has been well documented that ischemia induces intracellular acidosis (3). Acidosis reduced PLB Ser16 phosphorylation in the absence of isoproterenol, a {beta}-adrenergic agonist, but did not alter the isoproterenol-induced Ser16 phosphorylation (10). Acidosis also increased intracellular Ca2+ levels (7) and inhibited activity of protein phosphatase 1 (PP1), a major phosphatase that dephosphorylates PLB in the rat myocardium (18). In addition, ischemia induced the increases in the activation of the {beta}-adrenergic system and norepinephrine level (32). This is consistent with our observation that increased Ser16 phosphorylation could be completely inhibited by PKA inhibitor in either normoxic or IHA hypoxic hearts. Thus the dissociation between the Ser16 and Thr17 phosphorylation of PLB during I/R is the consequence of different mechanisms triggered by Ca2+ overload, acidosis, and the {beta}-adrenergic system. It seems that the increase in Ser16 phosphorylation may be induced mainly by enhanced PKA activity as well as an inhibition in PP1 caused by acidosis. The decrease in Thr17 phosphorylation is more likely caused by a depression in CaMKII activity, a consequence of Ca2+ overload and acidosis. However, increased PP1 activity also may be involved in the reduction of dual-site PLB phosphorylation during reperfusion in the prolonged ischemia myocardium (9). Therefore, further studies are required to clarify these issues by determining the alteration of the balance between the kinases (PKA and CaMKII) and PP1 during I/R in normoxic and IHA hypoxic heart.

Effects of IHA hypoxia on inhibited SR Ca2+-pump ATPase activity during I/R. Cytosolic Ca2+ concentration in cardiomyocytes is dependent on the ability of SR Ca2+-pump ATPase to mediate Ca2+ uptake into the SR lumen. Dysfunctional transport activity is considered to be one of the major determinants of I/R injury (4). It has been observed that ischemic preconditioning exerts beneficial effects on I/R-induced alterations in SR Ca2+ handling abilities. This can occur through prevention of changes in SR gene/protein expression and activities (22, 23). A previous study in our laboratory (37) demonstrated that IHA hypoxia-induced mitigating effects on I/R-induced alterations in postischemic contractility are accompanied by a significant decrease in intracellular Ca2+ overload during I/R. Wang et al. (33) reported that IHA hypoxia prevents I/R-induced suppression of maximal binding sites of cardiac SR Ca2+ release channels/ryanodine receptors. We thus proposed that IHA hypoxia may attenuate I/R-induced Ca2+ overload by reducing alterations in SR function. The present data show that IHA hypoxia adaptation did not change the activity of SR Ca2+-pump ATPase in the preischemic period but attenuated I/R-induced reduction in its enzymatic activity via PKA and CaMKII pathways.

Importantly, we have identified the relationship between the IHA hypoxia-induced alteration of PLB phosphorylation and SR Ca2+-pump ATPase activity during I/R. The function of SR Ca2+-pump ATPase is critically regulated by the PLB phosphorylation status. Dephosphorylated PLB inhibits SR Ca2+-pump ATPase activity, whereas phosphorylation of PLB by either cAMP-dependent PKA at Ser16 residue or by CaMKII at Thr17 residue relieves this inhibition, resulting in an increase in Ca2+-pump ATPase (12, 28, 29). It has been proposed that I/R-induced phosphorylation of PLB and a consequent decrease in PLB inhibition of Ca2+-pump ATPase serve to decrease cytosolic Ca2+ overload by increasing sequestration of Ca2+ into the SR, which might be a protective mechanism (27, 32). This is supported by the observation that both of the aforementioned PLB phosphorylation sites are involved in the mechanical recovery after ischemia (32). In the present study, we have demonstrated that the transient increases in phosphorylation of PLB Ser16 alone at the end of ischemia and at the beginning of reperfusion occurred simultaneously with a decrease in SR Ca2+-pump ATPase activities. However, the IHA hypoxia-induced increase in the upregulation of PLB Ser16 phosphorylation at the end of ischemia is associated with an attenuation of a decline in SR Ca2+-pump ATPase activity, although IHA hypoxia did not alter the status of PLB Thr17 phosphorylation at this time point. Moreover, inhibiting PKA blocked the beneficial effect of IHA hypoxia on Ser16 phosphorylation and SR Ca2+-pump ATPase activity. On the other hand, the depression in PLB Thr17 phosphorylation during I/R was associated with similar changes in SR Ca2+-pump ATPase activities. IHA hypoxia-attenuated depression of PLB Thr17 phosphorylation status during reperfusion is associated with improved SR Ca2+-pump ATPase activities. Inhibiting CaMKII abolished both of these effects. Taken together, the data show that during I/R, the transient increase in Ser16 phosphorylation status induced by hypoxic adaptation may account for the attenuated depression of SR Ca2+-pump ATPase activity. These changes may enable cardiomyocytes to better cope with I/R-induced intracellular Ca2+ overload. However, the increased phosphorylation of Ser16 alone is not enough to counteract the depression in the SR Ca2+-pump ATPase activity caused by a marked decline in the Thr17 phosphorylation status during I/R. Therefore, the decrease in SR Ca2+-pump ATPase activity during I/R is a result of the net balance between the phosphorylation status of PLB at Ser16 and Thr17 residues. Phosphorylation of PLB Ser16 may have more effects at the end of the ischemic period as well as at the onset of reperfusion, whereas Thr17 plays more significant roles during reperfusion. IHA hypoxia may improve the SR Ca2+-pump ATPase activity by increasing dual-site PLB phosphorylation during I/R through PKA and CaMKII pathways.

Role of Ser16 and Thr17 PLB phosphorylation on IHA hypoxia-induced mechanical recovery of I/R heart. PLB is a key regulator of basal contractility in the mammalian heart (15). Recent studies (27) demonstrated that changes in the phosphorylation status of PLB at both Ser16 and Thr17 are involved in the contractile recovery after ischemia. However, the functional significance of dual-site PLB phosphorylation in the mechanism of IHA hypoxia-induced cardioprotection of contractility after ischemia has not been previously evaluated. The present studies were therefore designed to address this issue. The results demonstrate for the first time that in IHA hypoxic hearts, both upregulated Ser16 and Thr17 phosphorylation of PLB contribute to the improved functional recovery after ischemia, but Thr17 phosphorylation plays a major role throughout the reperfusion period.

It is noteworthy that IHA hypoxia-increased Thr17 phosphorylation of PLB is associated with improved recovery of mechanical function at R30, but IHA hypoxia-increased Ser16 and Thr17 phosphorylation of PLB at R1 had no direct and immediate impact on the mechanical function, although SR Ca2+-pump ATPase activity improved. Moreover, at R30 there were significant decreases in SR Ca2+-pump ATPase activities compared with those at R1 in both groups, whereas myocardial function was still improving in the IHA hypoxia group. This indicates that mechanisms other than SR Ca2+-pump ATPase also underlie the improved recovery of function after an ischemic insult in IHA hypoxic animals, but preservation of SR function during ischemia and early reperfusion seems important for the later functional recovery. As has been suggested by others (32), it could be that enhanced SR calcium uptake allows I/R myocytes to deal with transiently increased cytosolic Ca2+ during the early reperfusion period, which would allow the myocytes to remain intact and allow other recovery mechanisms to occur. In this case, increased PLB phosphorylation would be indirectly associated with improved recovery. This is supported by the observation that inhibition of the transient increase in the Ser16 phosphorylation status in the presence of H-89, used to inhibit PKA, was associated with significant inhibitions of Ca2+-pump ATPase activity and functional recovery during reperfusion.

Recently reported data (3) also have shown that ischemic injury is increased in PLB knockout mice. This suggests that PLB phosphorylation during I/R might be detrimental, because phosphorylation and ablation of PLB both result in increased SR Ca2+-pump ATPase activity. However, the present results indicate that abolishment of increased Ser16 phosphorylation by the PKA-specific inhibitor H-89 was associated with an inhibition of the contractile recovery after ischemia in either the normoxic or IHA hypoxic hearts. Similarly, inhibition of IHA hypoxia-induced upregulated Thr17 phosphorylation by the CaMKII-specific inhibitor KN-93 was accompanied by an abolishment of functional recovery. These results are consistent with the observation that mutation of either Ser16 or Thr17 phosphorylation diminished the contractile recovery after the ischemia (27). Therefore, the presence of PLB phosphorylation sites improves, rather than impairs, the contractile recovery after ischemia, but permanent enhancement of SR Ca2+-pump ATPase activity in PLB knockout mice may impair the recovery. H-89 and KN-93 also could have inhibited the phosphorylation of other substrates of PKA and CaMKII, such as SR Ca2+-pump ATPase or ryanodine receptors; thus the possible involvement of other substrates of PKA and CaMKII in providing the protective effects of IHA hypoxic hearts needs to be considered. However, a previous study (16) in permeabilized myocytes has shown that the declines in SR Ca2+ uptake induced by PKA and CaMKII inhibitors could be prevented by an antibody to PLB, suggesting that PLB is the critical substrate for PKA and CaMKII regulation of cardiac contractility. Taken together, these findings indicate that dual-site PLB phosphorylation is necessary for the contractile recovery after ischemia, because its regulation of Ca2+-pump ATPase activity plays an important role in the cardioprotection of IHA hypoxia via PKA and CaMKII pathways.

Interestingly, neither PKA nor CaMKII inhibition hindered the ability of IHA hypoxia to protect against the maximal ischemic contracture. Therefore, the protective effects of IHA hypoxia on this response are not mediated by PKA and CaMKII pathways. It has been proposed (31) that ischemic contracture may be caused by a lock in the rigor state as a result of a decrease in myocardial ATP or an increase in ADP. Because IHA hypoxia significantly accelerated the restoration of creatine phosphate, ATP, and creatine phosphokinase activity in reoxygenated myocardium following acute anoxia (13), IHA hypoxic hearts may preserve cytosolic ATP content at higher levels than those in normoxic hearts. Accordingly, bioenergetics may be less impacted in IHA hypoxia, which could explain the decreased contracture response to ischemia and also the better recovery of postischemic contractile function in IHA hypoxic hearts.

Recent studies have shown that ATP-sensitive K+ channels, especially mitoKATP, are involved in the cardioprotective effects of IHA hypoxia (1, 37). It is possible that mitoKATP may mediate the beneficial effects of IHA hypoxia by lessening Ca2+ overload induced by I/R (37). However, such a mechanism cannot explain the beneficial effects of IHA hypoxia against I/R-induced changes in SR function and in phosphorylation of PLB because the relationship between them is not understood. Therefore, the possible involvement of yet other protective mechanisms of IHA hypoxia against ischemia warrants future studies.

In conclusion, the present results provide the first evidence that IHA hypoxia upregulates the level of Ser16 and Thr17 phosphorylation of PLB in I/R hearts via activation of PKA and CaMKII pathways. These effects are beneficial because they lessen the declines in SR Ca2+-pump ATPase activity during I/R and, in turn, may contribute to the IHA hypoxia-induced contractile recovery after ischemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants from Major and General Programs of the National Natural Sciences Foundation of China (30393133 and 30370536), Major State Basic Research Development Program of People's Republic of China (G2000056905), and Science & Technology Committee of Shanghai Municipality (02JC14038).


   ACKNOWLEDGMENTS
 
We thank Drs. R.-P. Xiao and W.-Z. Zhu for constructive discussion of the phospholamban assay, Prof. P. Reinach for important comments on paper writing, and Dr. J.-W. Dong, J. Chen, and Z.-H. Qu for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H.-T. Yang, Laboratory of Molecular Cardiology, Health Science Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Second Medical Univ., 225 Chong Qing Nan Rd., Bldg. 1, Rm. 613, Shanghai 200025, China (E-mail: htyang{at}sibs.ac.cn)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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