Articles in PresS. Am J Physiol Heart Circ Physiol (May 22, 2003). 10.1152/ajpheart.00209.2003 H-00209-2003 R1 1
Role of dual site phospholamban phosphorylation in the stunned heart: Insights from phospholamban-site specific mutants
Said M 1, Vittone L1, Mundiña-Weilenmann C 1, Ferrero P1, Kranias E G2, Mattiazzi A1
1
Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 60 y 120, (1900) La Plata, Argentina. 2 Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575, USA.
Correspondence to: Dr. Alicia Mattiazzi Centro de Investigaciones Cardiovasculares Facultad de Ciencias Médicas 60 y 120, (1900) La Plata Argentina Tel/Fax: 54-221-483 4833 email:
[email protected]
Running title: PLB and stunning in transgenic mouse hearts.
Copyright (c) 2003 by the American Physiological Society.
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ABSTRACT Phosphorylation of phospholamban (PLB) at Ser16 (PKA site) and at Thr17 (CaMKII site), increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation which was dependent on β-adrenergic stimulation, and an ischemia and reperfusion-induced Thr17 phosphorylation which was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and post-ischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and the presence of the CaMKII inhibitor KN-93 (1 µM), or the ßadrenergic blocker dl-propranolol (1 µM). KN-93 diminished the reperfusioninduced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in the post-ischemic mechanical recovery, transgenic mice expressing wild type PLB (PLB-WT), or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLBS16A, respectively) into the PLB null background, were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared to PLB-WT. These findings indicate that although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.
Key words: phospholamban phosphorylation residues, phospholamban mutants, ischemia-reperfusion.
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INTRODUCTION Myocardial stunning describes the reversible myocardial dysfunction that follows a brief ischemic insult, clinically manifested as sluggish recovery of the pump function after revascularization (3, 22). Experimental evidence indicates that the function of the sarcoplasmic reticulum (SR) is altered both, in the reversible as well as in the irreversible ischemia-reperfusion injury (21, 23, 32, 41, 43). In particular, in the case of myocardial stunning, a decrease in the activity of the SR Ca2+.pump (SERCA2) and/or in the rate of Ca2+ reuptake by the SR have been described in several species, including rats, mice, dogs and humans, submitted to moderate and reversible injury during cardiac surgery (21, 23, 41, 43). A decrease in intracellular Ca2+ transient has indeed been described in stunned myocytes isolated from chronically instrumented pigs (18), but was not detected in stunned rat myocytes and ventricular trabeculae or in isolated perfused ferret and dog hearts (5, 9, 11, 18). Thus, an intriguing question is: Why does the intracellular Ca2+ transient remain unaltered in species in which the SR function is depressed? A possible explanation is that compensatory mechanisms can overcome the depressed SERCA2 activity. The function of SERCA2 is regulated by the state of phosphorylation of another SR protein, named phospholamban (PLB) (4). In the dephosphorylated state, PLB inhibits the activity of SERCA2 and SR Ca2+ transport (15). Phosphorylation of PLB by PKA at Ser16 residue or CaMKII at Thr17 residue removes its inhibitory effect on SERCA2, thereby accelerating Ca2+ uptake by the SR and increasing the Ca2+ available for being released (36, 37). Thus, the status of phosphorylation of PLB residues should influence SR function during ischemia and reperfusion. Moreover, the fact that intracellular Ca2+ appears to be normalized before complete recovery of myocardial performance in several species (5, 9, 11) does not necessarily mean that Ca2+ homeostasis is not altered during ischemia and reperfusion. There is a compelling body of evidence, which indicates that in all species studied, cytosolic Ca2+ concentration rises during ischemia (2, 27, 31). Furthermore, reperfusion induces a sudden Ca2+ overload, which appears as a major player in determining the abnormal contractile behavior of the stunned heart (5, 27, 31). In a previous study, we showed that phosphorylation of both PLB
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residues increased in rat hearts submitted to an ischemia-reperfusion protocol (39). Whereas this study suggested a possible functional role of the phosphorylation of the Thr17 site at the beginning of reperfusion, the role of the phosphorylation of the Ser16 residue remained elusive. The availability of transgenic mouse models expressing wild-type PLB, the Ser16 →Ala mutant or the Thr17 →Ala mutant in the cardiac compartment of the PLB knockout mouse, provides a unique tool to delineate the role of each PLB phosphorylation site during ischemia-reperfusion. These new models prompted us to re-examine the possible role of PLB phosphorylation residues on the contractile recovery after ischemia.
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METHODS
Animals. Experiments were performed in Wistar male rats (200-300 g body wt), in Balb-c mice (25-30 g body wt) and in transgenic mice (25-30 g body wt) expressing wild type PLB (PLB-WT), a PLB mutant in which Thr17 was replaced by Ala (PLB-T17A), or a PLB mutant in which Ser16 was replaced by Ala (PLB-S16A) into the PLB null background (SvJ129/CF1). The mouse transgenic models were developed as previously described (6, 25). 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). Heart perfusions. Isolated rat hearts (wet weight between 0.90-1.10 g) were perfused according to the Langendorff technique at constant temperature (37°C), flow (10 ml/min) and heart rate (250 beats/min), as previously described (30, 38). The composition of the physiological salt solution was (in mM): 128.3 NaCl, 4.7 KCl, 1.35 CaCl2, 20.2 NaHCO3, 0.4 NaH2PO4, 1.1 MgCl2, 11.1 glucose and 0.04 Na2EDTA; this solution was equilibrated with 95% O2 – 5% CO2 to give a pH of 7.4. Perfusion of the isolated mouse hearts (wet weight between 0.25-0.35 g) was modified as follows: flow 4 ml/min, heart rate 360 beats/min and 2.5 mM CaCl2 in the perfusate. The mechanical activity of the heart was assessed by passing into the left ventricle a latex balloon connected to a pressure transducer (Namic, perceptor DT disposable transducer). The balloon was filled with aqueous solution to achieve a left ventricular end-diastolic pressure of approximately 10 mmHg (rat) and 20 mmHg (mouse). Contractile performance of the left ventricle was evaluated by the developed pressure (LVDP) and the maximal rate of pressure development (+dP/dt). Relaxation was assessed by the time constant Tau, obtained by fitting the time course of LVDP fall with a monoexponential function assuming zero pressure asymptote. Experimental fitting was performed from the time of the maximal rate of pressure decline to a level of 5 mmHg above the end diastolic pressure (28). LVDP and +dP/dt were expressed as a percentage of preischemic values. Tau was expressed as differences from preischemic values. Tables 1 and 2 show preischemic contraction and relaxation parameters of the rat and mouse heart, respectively. The basal mechanical data obtained in the mouse heart appear relatively low compared to a previous report in
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the literature (26), which may be due to the different mouse strain used, as it has been previously reported (17).
Experimental protocol. After stabilization, hearts were perfused for 10 min (preischemia) and then, normothermic global ischemia was produced by interruption of the coronary flow for a period of 20 min in the rat and 12 min in the mouse (ischemia). Although the term ischemia refers to the interruption of blood flow, it is used in the present experiments to refer to the interruption of coronary perfusion of saline solution, consistent with previous reports (5, 7, 27, 43). Previous experiments, in the isolated rat heart, have shown that a 20 min period of ischemia did not produce irreversible damage (29). In the mouse, a 12 min period of ischemia was chosen from preliminary experiments, which indicated that longer ischemic periods resulted in negligible recovery of contractile function. After ischemia, coronary perfusion was restored for 30 min, unless otherwise indicated (reperfusion) (See Results). Electrical stimulation was stopped after 1 min of ischemia and resumed upon reperfusion. Nonischemic hearts, freeze-clamped at different times to match either the ischemic or reperfusion periods, were used as controls for phosphorylation studies. Since these basal phosphorylation values did not change with time, they were considered as a single group for statistical analysis. When drugs were used, they were perfused during the pre-ischemic period or during the pre-ischemic period and the onset of reperfusion (See Results). In some parallel experiments, non-ischemic-reperfused hearts were perfused with 30 nM (rat) or 300 nM (mouse) isoproterenol to produce the maximal inotropic and PLB-Ser16 or Thr17 phosphorylation responses (30, 34). At the end of the experimental period, the hearts were freeze-clamped and stored at 80°C until biochemical assays were performed.
Preparation of mouse heart homogenates and of rat heart SR membranes. The pulverized ventricular tissue from mouse hearts was homogenized in 5 volumes of the homogenization buffer containing: 5 mM Na2EDTA, 25 mM NaF, 300 mM sucrose, 1 mM PMSF, 1 mM benzamidine and 30 mM KH2PO4 (pH 7 at 4ºC). The homogenate was then centrifuged at 16,000 x g for 20 min and the supernatant obtained was subjected to SDS-PAGE. Rat SR membrane vesicles were prepared as previously described (30, 38). Briefly, the pulverized ventricular tissue was
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homogenized in 6 volumes of the same homogenization buffer described above, and the homogenate was centrifuged twice at 14,000 x g and 16,000 x g for 20 min. The resulting supernatant was centrifuged at 45,000 x g for 45 min. The pellet obtained was suspended in 3 volumes of buffer containing 10 mM Na2EDTA, 25 mM NaF, 600 mM KCl, and 50 mM KH2PO4 (pH 7 at 4ºC) and recentrifuged as in the previous step. The resulting pellet was suspended in 10 mM Na2EDTA, 10 mM NaF, 250 mM sucrose and 30 mM histidine (pH 7 at 4ºC), and then subjected to SDS-PAGE. In both cases, protein was measured by the method of Bradford using bovine serum albumin as standard.
Electrophoresis and Western Blot analysis. For immunological detection of PLB phosphorylation sites, 25 µg of mouse homogenate proteins or 20 µg of rat SR membrane proteins were electrophoresed per gel lane, as previously described (30, 38). Proteins were transferred to PVDF membranes (Immobilon-P, Millipore) and probed with polyclonal antibodies raised to a phospholamban peptide (residues 9-19) phosphorylated at Ser16 or at Thr17 (1:5000) (Cyclacel Ltd., UK). Immunoreactivity was visualized by peroxidase-conjugated antibodies using a peroxidase-based chemiluminescence detection kit (Boehringer Mannheim, Germany). The signal intensity of the bands on the film was quantified using Scion Image software (based on NIH Image). Phosphorylation results were expressed as percentage of Ser16 and Thr17 phosphorylation induced by isoproterenol in non-ischemic-reperfused hearts.
Statistics. Data are expressed as means ± SE. Statistical significance was determined by Student's t-test for unpaired observations. A P value
