Studies on the kinetics and the molecular mechanism of Ca2+ transport by the cardiac sarcoplasmic reticulum (SR) have indicated that phospholamban is a key phosphoprotein involved in the regulation of Ca2+-ATPase (1). The dephosphorylated phospholamban functions as an inhibitor of the Ca2+-ATPase (2,3). Activation of β-adrenergic receptor (βAR) on the sarcolemma of the cardiac cell leads to the phosphorylation of phospholamban (4). The phosphorylated phospholamban relieves the inhibition of Ca2+-ATPase and thus leads to an increase in the Ca2+-ATPase activity and consequently, the Ca2+ uptake into the SR (2–4). Because of its role in regulating SR Ca2+ uptake, the altered phospholamban-calcium ATPase interaction has been suggested to be one of the etiological factors contributing to the cardiac dysfunction under various pathological conditions (5–8).
Clinically, sepsis is a two-phase process in which patients initially go through a hyperdynamic phase and, subsequently, a hypodynamic phase (9). In endotoxin shock model in which the animal exhibits only the hypodynamic phase, the phosphorylation of phospholamban by the exogenous protein kinase was impaired (10), and furthermore, the ATP-dependent Ca2+ uptake by cardiac SR was depressed (11). Although these findings provide a basis of involvement of the SR protein phosphorylation in mediating the altered Ca2+ uptake in the endotoxin shock, the direct evidence for changes of phospholamban phosphorylation in relationship to alterations in SR Ca2+-ATPase as well as Ca2+ uptake during the progression of sepsis is lacking. Therefore, the present study was undertaken to evaluate alteration in the phosphorylation of phospholamban, and its relationship with changes in SR Ca2+ transport and cardiac contractility in the rat heart during the two hemodynamically distinct phases of sepsis.
MATERIALS AND METHODS
All animal experiments in this study were performed with the approval of the Animal Care Committee of St. Louis University School of Medicine, and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing from 275 to 300 g were used. All animals were fasted overnight with free access to water. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al. (12) with minor modification (13). Under anesthesia with halothane, a laparotomy was performed and the cecum was ligated just distally to the ileocecal valve to avoid any intestinal obstruction and was punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers. Control rats were sham-operated (a laparotomy was performed and the cecum was manipulated but neither ligated nor punctured). All animals were resuscitated subcutaneously with 4 mL/100 g body wt of normal saline at the completion of surgery and also at 7 h post-surgery. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The mortality rates were 0% for control, 3.3% for early sepsis, and 28.9% for late sepsis.
Phosphorylation of phospholamban
Hearts removed from the control and the septic rats under urethane anesthesia were retrogradely perfused by the Langendorff technique (14) under a constant aortic pressure (80 cm H2O) at 37°C with Krebs-Henseleit buffer (in millimoles: 118.0 NaCl, 4.8 KCl, 1.2 MgSO4, 2.0 CaCl2, 27.5 NaHCO3, pH 7.4, 11.1 glucose, and 0.1 KH2PO4) for 10 min (first perfusion). The perfusion circuit was then switched to a recirculating system containing the same buffer in the presence of [32P]H3PO4 (2.0 mCi/50 mL) and this perfusion continued for 30 min to label the tissue ATP pool (second perfusion). After completion of the second perfusion, the hearts were perfused for 5 min with nonradioactive buffer to wash out radioactivity (third perfusion). When the effects of isoproterenol or propranolol were studied in some experiments, atropine (0.1 μM) and prazosin (0.1 μM) were present in the perfusion media to block muscarinic and α1-adrenergic receptors, respectively. All of the perfusion solutions were saturated with 95% O2-5% CO2. At the end of the third perfusion, the hearts were freeze-clamped with aluminum clamps pre-cooled in liquid nitrogen and pulverized. The pulverized myocardium was used for isolation and purification of SR as described previously (13). Cardiac SR proteins were separated by SDS-PAGE (10%–17% acrylamide gradient gels). The phosphorylated proteins were identified by autoradiography, cut into 3-mm sections from the gel, and the radioactivity was counted by a liquid scintillation counter (14). Myocardial ATP content was quantified by the method of Adams (15). The specific radioactivity of the γ-phosphate group of 32P-labeled ATP was determined by the method of Cogoli and Dobson (16). 32P incorporation into phospholamban was expressed as picomoles of Pi per milligram of protein.
Measurement of cardiac contractility
Hearts from the control and the septic rats were perfused with nonradioactive buffer by the same protocol as described above. A saline-filled latex balloon was inserted into left ventricle through left atrium and was then connected to a pressure transducer (Statham P23 DB) for left ventricular pressure measurement. The left ventricular end-diastolic pressure was adjusted at 6 mmHg at the beginning of the experiment. Left ventricular developed pressure (LVDP, the difference between systolic and diastolic pressures), the maximal rates of left ventricular pressure development (+dP/dtmax) and decline (−dP/dtmax), were monitored on a multichannel polygraph (Grass Instruments Model 79D). At the end of each experiment, the heart was freeze-clamped, stored at −70°C, and then used for measurements of SR Ca2+ uptake and Ca2+-ATPase activities, as well as the determination of cAMP content. Results obtained from preliminary experiments indicate that SR Ca2+ uptake and Ca2+-ATPase activities were almost identical between the perfused and nonperfused hearts.
Assays of Ca2+-ATPase activity and Ca2+ uptake
Ca2+-ATPase activity was measured as described by Jones and Besch (17). SR vesicles (20 μg of protein) were preincubated at 37°C for 10 min in a medium containing 50 mM histidine, pH 7.4, 5 mM MgCl2, 110 mM KCl, 5 mM NaN3, 1 mM EGTA, and various concentrations of CaCl2 to yield 0.01 to 10 μM of free Ca2+. The free Ca2+ concentrations were calculated as described by Fabiato (18). Phosphoenolpyruvate (3 mM) and pyruvate kinase (3 units) was added as an ATP-regenerating system. The reaction was initiated by the addition of 3 mM ATP, allowed to proceed for 20 min at 37°C, and was terminated by the addition of 2.5 mL of stop solution (31 mM sodium bisulfate, 8.3 mM p-methylaminophenol sulfate, 2.9 mM molybdic acid, and 350 mM H2SO4). The amount of inorganic phosphate liberated from ATP was determined with a spectrophotometer.
The ATP-dependent, oxalate-facilitated 45Ca2+ uptake of the cardiac SR was measured by the Millipore filtration technique as described previously (13). SR vesicles (20 μg of protein) were preincubated for 1 min at 37°C in 0.2 mL of reaction medium containing 120 mM KCl, 20 mM HEPES, pH 7.2, 3 mM MgCl2, 5 mM NaN3, 5 mM potassium oxalate, and various concentrations of free Ca2+. The Ca2+ transport reaction was initiated by the addition of 3 mM ATP and was allowed to proceed for 1 min at 37°C. At the end of each incubation, the reaction mixture was diluted with 3 mL of ice-cold washing solution (120 mM KCl, 20 mM Tris, pH 7.2, and 1 mM EGTA) and filtered immediately through a 0.45-μm filter paper under suction. The filter paper was washed three times with 3 mL of washing solution to remove nonspecifically bound calcium. After drying, the quantity of 45Ca2+ taken up by SR was determined by counting the washed filters with a liquid scintillation counter.
Other assays and the statistical analysis
For measurement of heart cAMP content, 100 mg of frozen tissue sample from nonradioactive perfused heart was homogenized at 4°C in 1 mL of 50 mM Tris, pH 7.5, 4 mM EGTA. The homogenates were placed in a boiling water bath for 10 min, then cooled down and centrifuged at 3,000 g for 20 min. The cAMP content in the supernatants was determined by radioimmunoassay with a commercial [3H]cAMP assay kit (Amersham). The protein content of SR vesicles was determined by the method of Lowry et al. (19) using bovine serum albumin as a standard. Data are presented as means ± SEM. Statistical analysis of the data was performed by using ANOVA procedure followed by Student-Newman-Keuls test. P < 0.05 was considered as statistically significant.
[32P]H3PO4, [γ-32P]ATP, and 45CaCl2 were purchased from ICN Biomedicals Inc. (Costa Mesa, CA). Na2ATP, isoproterenol, prazosin, propranolol, atropine, and molecular weight standard proteins were purchased from Sigma Chemical (St. Louis, MO). Other chemicals and reagents were of analytical grade.
Figure 1 depicts a representative experiment of the autoradiography from SDS-PAGE of phospholamban labeled with 32P in the cardiac SR. Without boiling, the major phosphorylated proteins were found to have an apparent molecular weight of approximately 27 kDa both in the control and the septic groups (Fig. 1, lanes 1, 3, and 5). Only small amount of the lower molecular weight form of phosphorylated phospholamban could be observed when SR membrane sample was not boiled. Similar findings were reported by other investigators (8,10). When SR membrane samples were boiled for 3 min prior to the electrophoresis, the high molecular weight forms of phospholamban were completely dissociated into the low molecular weight forms (approximately 11 kDa) in all three groups of experiment (Fig. 1, lanes 2, 4, and 6). These data are consistent with those reported previously that phospholamban is a temperature-sensitive pentamer and that boiling dissociates phospholamban from the pentameric form to the monomeric form (20).
Figure 2 shows the quantitative analysis of phospholamban phosphorylation in the control, early septic, and late septic rat hearts. Phospholamban phosphorylation was increased by 153% (P < 0.01) during the early hyperdynamic phase of sepsis, while it was decreased by 51% (P < 0.01) during the late hypodynamic phase of sepsis. These findings indicate that phospholamban phosphorylation in the cardiac SR exhibits a biphasic change, i.e., an initial increase followed by a decrease, during the progression of sepsis.
Figure 3 shows the effect of sepsis on the activities of Ca2+-ATPase in the cardiac SR as a function of different concentrations of Ca2+. A sigmoid relationship between Ca2+-ATPase activities and Ca2+ concentrations was observed both in the control and the septic groups (Fig. 3a). ATP hydrolysis catalyzed by SR Ca2+-ATPase was relatively unchanged during early sepsis, but was inhibited at all concentrations of Ca2+ during late sepsis. Analysis of data using Eadie-Hofstee plots (Fig. 3b) indicates that the Vmax for Ca2+ was not affected during the early phase, but it was significantly decreased by 33.3% (P < 0.05) during the late phase of sepsis (0.39 ± 0.01, 0.35 ± 0.01, and 0.26 ± 0.04 μmol/mg/min for control, early sepsis, and late sepsis, respectively). The Km for Ca2+ remained unchanged during the early and the late phases of sepsis (0.16 ± 0.03, 0.15 ± 0.03, and 0.18 ± 0.01 μM for control, early sepsis, and late sepsis, respectively). These results suggest that cardiac SR Ca2+-ATPase activity was relatively unaffected during early sepsis, but it was impaired during late sepsis. Furthermore, the impairment in the Ca2+-ATPase during the late stage of sepsis was associated with a mechanism not affecting the affinity of the enzyme moiety toward Ca2+.
Figure 4 depicts the effect of sepsis on the ATP-dependent Ca2+ uptake in the cardiac SR as a function of different concentrations of Ca2+. There was a sigmoid relationship between Ca2+ uptake activities and Ca2+ concentrations in the control and the septic groups (Fig. 4a). Analysis of data using Eadie-Hofstee plots (Fig. 4b) indicates that the Vmax for Ca2+ was not significantly affected during the early phase, but it was decreased by 37.4% (P < 0.01) during the late phase of sepsis (77.9 ± 5.9, 69.3 ± 2.5, and 48.8 ± 2.6 nmol/mg/min for control, early sepsis, and late sepsis, respectively). The Km for Ca2+ remained unchanged during the early and the late phases of sepsis (0.11 ± 0.02, 0.11 ± 0.02, and 0.10 ± 0.02 μM for control, early sepsis, and late sepsis, respectively). These data, together with those presented in previous figure, indicate that changes in SR Ca2+-ATPase activities were paralleled by altered SR Ca2+-transport. Furthermore, the mechanism of impairment in the SR Ca2+ uptake during late sepsis was similar to that of Ca2+-ATPase, i.e., not affecting the Ca2+ affinity.
Figure 5 depicts changes in various contractile parameters in the rat hearts during different phases of sepsis. As shown in Figure 5a, +dP/dtmax was increased by 17.3% (P < 0.05) during the early phase, but it was reduced by 46.7% (P < 0.01) during the late phase of sepsis. Unlike +dP/dtmax, −dP/dtmax was relatively unaffected during early sepsis, but it was decreased by 51.5% (P < 0.01) during late sepsis (Fig. 5b). These data demonstrate that the rate of contraction was increased during the early hyperdynamic phase, while the rates of both the contraction and the relaxation were reduced during the late hypodynamic phase of sepsis. These findings demonstrate that myocardial function was elevated during the initial phase, while it was depressed during the late phase of sepsis.
Table 1 depicts the effect of sepsis on phospholamban phosphorylation, Ca2+ transport (Ca2+-ATPase activity and Ca2+ uptake), contractile parameters (LVDP and ±dP/dtmax), and cAMP content in the intact beating hearts perfused in the absence or the presence of βAR agonist or antagonist. It should be mentioned that phospholamban phosphorylation and ±dP/dtmax on columns 1, 3, and 6 represent the values from Figures 2 and 5. As shown on columns 2 and 1, isoproterenol was capable of stimulating phospholamban phosphorylation, Ca2+ transport, contractile parameters, and cAMP content in the control rat hearts. When hearts were perfused in the absence of βAR agonist or antagonist, phospholamban phosphorylation was significantly increased during the early phase of sepsis, and the increase in phospholamban phosphorylation was accompanied by increases in +dP/dtmax and tissue cAMP content (comparison between columns 3 and 1). When hearts were perfused in the presence of isoproterenol, the increases in phospholamban phosphorylation, +dP/dtmax, and cAMP content during early sepsis were further elevated (comparison between columns 4 and 3). It should be noted that LVDP and −dP/dtmax, which were unaffected during early sepsis, were also elevated by isoproterenol (comparison between columns 4 and 3). The isoproterenol-induced increases in the phospholamban phosphorylation, LVDP, ±dP/dtmax, and cAMP content were completely blocked by propranolol (comparison between columns 5 and 4). These findings indicate that during the early phase of sepsis, the increase in the phospholamban phosphorylation and its associated increase in cardiac contractility were mediated via βAR pathway. It should be pointed out that Ca2+-ATPase and Ca2+ uptake activities remained unchanged during early sepsis (comparison between columns 3 and 1), and furthermore, they were unaffected by either isoproterenol or propranolol (comparison among columns 5, 4, and 3). These findings suggest that during early sepsis, SR Ca2+ transport was dissociated with increased phospholamban phosphorylation. Unlike early sepsis, phospholamban phosphorylation, Ca2+-ATPase and Ca2+ uptake activities, LVDP and ±dP/dtmax, and tissue cAMP content during late sepsis were all decreased (comparison betweens columns 6 and 1). These reductions induced by the late sepsis were ameliorated by isoproterenol, except that Ca2+ transport activities remained unaffected (comparison between columns 7 and 6). These findings indicate that during the late phase of sepsis, the observed decrease in myocardial contractility was due to the decrease in phospholamban phosphorylation, which resulted in decreased Ca2+ transport across the SR. Furthermore, the alterations observed during late sepsis were mediated through βAR pathway.
In the present study, the phosphorylation of phospholamban was found to increase during the early hyperdynamic phase, and to decrease during the late hypodynamic phase of sepsis. In the hearts isolated from the control group, phospholamban phosphorylation, Ca2+-ATPase and Ca2+ uptake activities, relaxation of the heart (−dP/dtmax), and tissue cAMP content were collectively stimulated by βAR agonist, isoproterenol. In the hearts isolated from the early sepsis group, phospholamban phosphorylation and cAMP content were increased, while Ca2+ transport activities and −dP/dtmax remained unchanged. Under such conditions, phospholamban phosphorylation, −dP/dtmax and tissue cAMP content were responsive, whereas Ca2+ transport activities were unresponsive to isoproterenol stimulation. In the hearts isolated from the late sepsis group, phospholamban phosphorylation, Ca2+ transport activities, −dP/dtmax, and cAMP content were all reduced. However, these reductions, except for Ca2+ transport activities, were ameliorated by isoproterenol. These findings demonstrate that during the late hypodynamic phase of sepsis, the interaction between phospholamban phosphorylation and Ca2+ transport activity appears to be intact. The decrease in myocardial contractility observed during the late phase of sepsis can be explained by the decrease in phospholamban phosphorylation, which results in decreased Ca2+ transport across the SR. In contrast, during the early hyperdynamic phase of sepsis, the increase in phospholamban phosphorylation did not correlate with an increase in Ca2+-ATPase activity. Thus, the interaction between phospholamban phosphorylation and Ca2+ transport across the SR was disrupted during the early phase of sepsis.
Active Ca2+ transport into the SR of cardiac muscle is catalyzed by Ca2+-ATPase and is regulated by phospholamban. The mechanism of regulation of Ca2+-ATPase by phospholamban involves the phosphorylation and dephosphorylation of phospholamban protein. The dephosphorylated phospholamban is an inhibitor of the SR Ca2+-ATPase activity (2,3). The inhibition has been suggested to involve direct protein-protein interactions followed by conformational changes in the Ca2+-ATPase, resulting in a decrease in the affinity of the Ca2+ pump for Ca2+ (21,22). Phosphorylation of phospholamban by various protein kinases results in stimulation of SR Ca2+-ATPase (2–4). The stimulation involves the deinhibition of Ca2+-ATPase, thereby increasing the affinity of the Ca2+-ATPase for Ca2+ without any change in the Vmax (22). In cardiac muscle, phospholamban phosphorylation has been shown to correlate temporally, over a time course of seconds, with the ability of the βAR agonist isoproterenol to accelerate the rate of relaxation (−dP/dtmax) of the heart (2). Although the physiological role of phospholamban phosphorylation has been widely investigated, the molecular mechanism of phospholamban and Ca2+-ATPase interaction under various pathological conditions has not been fully explored. In the present study we reported that the phosphorylation of phospholamban in intact beating rat hearts underwent a biphasic change during the progression of sepsis, i.e., an increase during the early hyperdynamic phase followed by a decreased during the late hypodynamic phase of sepsis. The observed biphasic change is most likely a result of the altered βAR expression because the density of βAR in the rat heart was found to be increased during the early hyperdynamic phase, and it decreased during the late hypodynamic phase of sepsis (14). It should be noted that during the early phase of sepsis, the increase in phospholamban phosphorylation was not accompanied by increases in Ca2+-ATPase and Ca2+ uptake activities. Thus, the interaction between phospholamban phosphorylation and Ca2+ transport across the SR appeared to be disrupted during early sepsis. Ca2+-ATPase has been suggested to be a marker of the transition from compensated hypertrophy to failure because the altered expression of Ca2+-ATPase occurred in failing animals. but not in nonfailing hypertrophied heart (21,23,24). In addition, change in Ca2+-ATPase gene expression has been reported to precede that of phospholamban and the development of cardiac dysfunction in volume-overloaded cardiac hypertrophy (25). It has been reported that cAMP-dependent phosphorylation of phospholamban was increased at the compensatory stage of hypertrophy, but decreased with the decompensated failing hearts (26). Although the enhanced phospholamban phosphorylation at the compensatory stage was associated with the increase in LVDP, the relaxation parameter was reduced at the compensatory stage of hypertrophy and it was further decreased in the failing hearts (26). The underlying mechanisms in regard to the dissociation among phospholamban phosphorylation, Ca2+ transport across the SR, and cardiac contractility during the early hyperdynamic phase of sepsis requires further investigation.
During the late phase of sepsis in which cardiac function has shown signs of deterioration (decreases in ±dP/dtmax and LVDP), the phosphorylation of phospholamban was decreased and the decrease in phospholamban phosphorylation was accompanied by reductions in Ca2+-ATPase and Ca2+ uptake activities. The pathological manifestations observed during the late phase of sepsis were strikingly similar to those found in end-stage cardiac failure (6,8,25). Phospholamban knockout hearts exhibit significant increases in Ca2+-ATPase affinity for Ca2+ and in the basal contractile parameters (±dP/dt, the time to peak pressure, and the time to half-relaxation) as compared to the wild-type littermates (27). Overexpression of phospholamban in the heart results in a decrease in SR Ca2+ uptake and a depression of cardiac contractile function (LVDP and ±dP/dt) (22,28,29). Overexpression of Ca2+-ATPase in mammalian heart is associated with significant increases in the rate of Ca2+ uptake and cardiac contractile parameters (±dP/dt) (30). Our findings that the decrease in phospholamban phosphorylation that was coupled by the decrease in Ca2+ transport across the SR may have a pathophysiological significance in contributing to the understanding of the depressed cardiac function during the end stage of sepsis. It is noteworthy that other factors such as altered ryanodine receptor function (31) or altered Ca2+ binding to contractile proteins (32) may also account for the depressed myocardial contractility during late sepsis.
This work was supported by HL-30080 from National Heart, Lung, and Blood Institute, and GM-31664 from National Institute of General Medical Sciences, National Institutes of Health.
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