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1Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 1900 La Plata, Argentina; and 2Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575
Submitted 6 March 2003 ; accepted in final form 15 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-adrenergic
stimulation and an ischemia and reperfusion-induced Thr17
phosphorylation that was dependent on Ca2+ influx. To
elucidate the relationship between these two PLB phosphorylation sites and
postischemic mechanical recovery, rat hearts were submitted to
ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93
(1 µM) or the -adrenergic blocker dl-propranolol (1 µM).
KN-93 diminished the reperfusion-induced 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 postischemic 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 PLB-S16A, 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 with 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.
phospholamban phosphorylation residues; phospholamban mutants; ischemia-reperfusion
Ala mutant (PLB-S16A), or the Thr17 Ala mutant (PLB-T17A) in
the cardiac compartment of the PLB knockout (KO) mouse provides a unique tool
to delineate the role of each PLB phosphorylation site during
ischemia-reperfusion. These new models prompted us to reexamine the possible
role of PLB phosphorylation residues on the contractile recovery after
ischemia. |
METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Heart perfusions. Isolated rat hearts (wet weight between 0.90 and
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,
39). 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 and 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 
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 
, obtained by fitting the time course of
LVDP fall with a monoexponential function assuming a 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 the preischemic values; was expressed
as differences from preischemic values. Tables
1 and
2 show preischemic contraction
and relaxation parameters of the rat and mouse hearts, respectively. The basal
mechanical data obtained in the mouse heart appear relatively low compared
with a previous report in the literature
(26), which may be due to the
different mouse strain used, as previously reported
(16).
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Experimental protocol. After stabilization, hearts were perfused for 10 min (preischemia), and normothermic global ischemia was then 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, 41). 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 that 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. Because 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 preischemic period or during the preischemic period and the onset of reperfusion (see RESULTS). In some parallel experiments, nonischemia-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 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 (pH7at4°C). The homogenate was then centrifuged at 16,000 g for 20 min, and the supernatant obtained was subjected to SDS-PAGE. Rat SR membrane vesicles were prepared as previously described (30, 39). Briefly, the pulverized ventricular tissue was homogenized in 6 volumes of the same homogenization buffer described above, and the homogenate was centrifuged twice at 14,000 and 16,000 g for 20 min. The resulting supernatant was centrifuged at 45,000 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 4°C) 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 4°C) and then subjected to SDS-PAGE. In both cases, protein was measured by the method of Bradford using BSA as the 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, 39). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore) and probed with polyclonal antibodies raised to a PLB peptide (residues 9–19) phosphorylated at Ser16 or at Thr17 (1:5,000, Cyclacel). Immunoreactivity was visualized by peroxidase-conjugated antibodies using a peroxidase-based chemiluminescence detection kit (Boehringer Mannheim). The signal intensity of the bands on the film was quantified using Scion Image software (based on NIH Image). Phosphorylation results were expressed as the percentage of Ser16 and Thr17 phosphorylation induced by isoproterenol in nonischemia-reperfused hearts.
Statistics. Data are expressed as means ± SE. Statistical significance was determined by Student's t-test for unpaired observations. A P value of <0.05 was considered as statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-adrenergic cascade (Fig.
1, C and D). These concentrations of KN-93 and
dl-propranolol did not affect basal contractile and relaxation
parameters (Table 1).
Figure 1A shows that
the recovery of LVDP of hearts treated with KN-93 was significantly lower than
that of nontreated hearts. The impairment of the contractile recovery was
associated with a decrease in the reperfusion-induced increase in
Thr17 phosphorylation (Fig.
1B) and a prolongation of , both of which occurred
at the beginning of reperfusion. changed from 24.9 ± 2.9 ms
(n = 10) to 32.7 ± 2.0 ms (n = 4) in the absence and
presence of KN-93, respectively. In contrast, -blockade, although
significantly decreasing the phosphorylation of Ser16 at the end of
the ischemia, failed to modify the contractile and relaxation recoveries
throughout reperfusion (Fig. 1, C
and D). These findings would suggest that, whereas the
phosphorylation of Thr17 participates in the recovery of
contractile performance that follows the ischemic episode, the phosphorylation
of Ser16 is not involved. However, either inhibition of CaMKII or
blockade of the -adrenergic cascade may alter the phosphorylation of
several proteins other than PLB
(8,
10,
12,
20,
35,
40), which might also
influence the recovery of contraction after the ischemia and therefore mask
the actual role of PLB residues.
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Precise knowledge of the role of PLB phosphorylation on the recovery of contraction and relaxation after a period of ischemia and discrimination of the relative participation of each PLB phosphorylation residue on this recovery requires the use of transgenic models with selective mutation of each phosphorylation site. We first examined the status of phosphorylation of PLB residues induced by ischemia-reperfusion in the mouse heart. Figure 2 shows immunoblots and overall results of the phosphorylation of Thr17 and Ser16 in control experiments using BALB/c mice. Phosphorylation of both PLB residues decreased during the ischemic period with respect to control values. Furthermore, Thr17 phosphorylation increased at the beginning of reperfusion and then returned to control values, whereas Ser16 phosphorylation remained near control levels all along reperfusion. In additional experiments performed in transgenic mice, it was found that phosphorylation of Ser16 was not different from control levels after ischemia or during reperfusion, whereas Thr17 phosphorylation significantly increased at the beginning of reperfusion (76.3 ± 12.2%, n = 3) in PLB-WT mice. The reperfusion-induced Thr17 phosphorylation was also found in PLB-S16A mice (157.1 ± 63.0%, n = 3). Thus the increase in Thr17 phosphorylation at the beginning of reperfusion appears to be a common finding in all species and models studied (Ref. 38 and the present results).
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The mechanical response to ischemia-reperfusion injury was then evaluated
in the three groups of transgenic mice. PLB-WT, PLB-T17A, and PLB-S16A mice,
expressing similar levels of wild-type or mutant PLB in the heart, exhibited
similar contraction and relaxation values under basal (preischemic)
conditions, indicating that both mutant PLB were capable of modulating
contraction and relaxation in a manner similar to that for PLB-WT
(Table 2). Figure 3 shows the time course
of contraction (A and B) and relaxation (C)
parameters during ischemia and reperfusion in PLB-WT and PLB-T17A mutant
hearts. Mechanical activity decreased to nondetectable levels after the
cessation of flow. In PLB-WT hearts, a partial contractile recovery and a
slight slow down of relaxation, which did not attain statistical significance,
occurred during reperfusion. After 30 min of reperfusion, LVDP and
+dP/dt recovered to 37.2 ± 8.1% and to 35.1 ± 8.4% of
preischemic values, respectively. In PLB-T17A mutant hearts, the recovery of
LVDP and +dP/dt was significantly lower than that of PLB-WT hearts
throughout the reperfusion period, whereas was significantly prolonged
at the beginning of reperfusion and then recovered toward control values.
Figure 4 compares the
mechanical activity of PLB-WT and PLB-S16A mutant hearts submitted to the
ischemia-reperfusion protocol. The recovery of LVDP and +dP/dt was
also lower in PLB-S16A mutant hearts than in PLB-WT hearts. However, this
impaired contractile recovery attained statistical significance only at the
beginning of reperfusion. Moreover, there were no differences in the recovery
of relaxation between both groups.
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These findings strongly support the idea of a functional relationship between both phosphorylatable sites of PLB and the mechanical recovery that follows the ischemic period. Although both sites appear to be involved, the role of Thr17 seems to be more important than that of Ser16 in the recovery of contraction and relaxation parameters of the stunned heart.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-agonists in the mammalian heart
(13,
17,
25,
30,
34). The functional role of
single- and dual-site PLB phosphorylation during -adrenergic stimulation
has been elucidated through the use of genetically altered mouse models
(6,
24,
25). Recent studies have
demonstrated that ischemic injury increased in PLB-KO mice compared with
wild-type mice (7). However,
the functional significance of specific-site PLB phosphorylation on the
mechanical recovery after ischemia has not been previously evaluated. The
present work was designed to address this issue. The results demonstrate, for
the first time, that both Ser16 and Thr17 residues of
PLB are involved in the contractile recovery of the stunned heart. Moreover,
Thr17 phosphorylation seems to be the only phosphorylation site
necessary for the recovery of relaxation in the stunned heart. Our previous study (38) in isolated rat hearts has demonstrated that both PLB phosphorylation residues become phosphorylated during ischemia-reperfusion injury. However, the role of ischemia-reperfusion-induced PLB phosphorylation on the mechanical recovery of the stunned heart has not been previously explored. Ca2+ overload appears to be a major player in determining the abnormal contractile behavior of the stunned heart (5, 27, 31). PLB phosphorylation could modify Ca2+ overload in two opposite ways. The increased phosphorylation state of PLB increases the Ca2+ sensitivity of SERCA2, and more Ca2+ is pumped into the SR (36, 37). This would decrease Ca2+ overload in the cytosol. However, the increase in the SR Ca2+ uptake would produce an enhanced SR Ca2+ load and Ca2+ release (1), which would contribute to the cytosolic Ca2+ overload and contractile dysfunction. Moreover, the increased cytosolic Ca2+ would stimulate SR Ca2+ release, triggering a self-perpetuating cycle, which would further increase cytosolic Ca2+. However, accumulation of Mg2+ and H+ and alterations in SR Ca2+ channel protein tend to reduce the SR Ca2+ release capability during ischemia-reperfusion injury (42). The association of an impairment of the SR Ca2+ release with an improvement of the SR Ca2+ uptake, induced by PLB phosphorylation, would supply a mechanism that breaks the vicious cycle, helping to cope with the cytosolic Ca2+ overload produced by ischemia-reperfusion. Thus phosphorylation of PLB sites may be critically involved in the mechanical recovery after ischemia.
The present data show that in the rat, the decrease of reperfusion-induced
Thr17 phosphorylation produced by inhibiting CaMKII by KN-93 was
associated with an impaired contraction and relaxation recoveries. In
contrast, the decrease of ischemia-induced Ser16 phosphorylation
produced by dl-propranolol administration did not alter these
recoveries. Taken together, these experiments indicate that, whereas the
phosphorylation of Thr17 of PLB and/or some other CaMKII-dependent
protein phosphorylation (10,
35,
40) are functionally involved
in the mechanical recovery of the stunned heart, the phosphorylation of
Ser16 is not. However, the effects of -blockade on the
phosphorylation of proteins other than PLB may mask a possible favorable
effect of Ser16 phosphorylation on the recovery of LVDP.
PKA-dependent phosphorylation of the L-type Ca2+ channel
has been reported to increase Ca2+ influx through the
sarcolemma (35). Moreover, it
has been suggested that PKA-dependent phosphorylation of SR
Ca2+ channels increases Ca2+
release from the SR (33).
Inhibition of the phosphorylation of these proteins by -adrenergic
blockade might contribute to decrease cytosolic Ca2+
overload and to improve the contractile recovery. These effects of
-adrenergic blockade would counteract the negative effect that the
inhibition of Ser16 phosphorylation might have. Another mechanism
that may be playing a role in the contractile recovery produced by
dl-propranolol is the reported cardioprotective effect of the drug,
attributed to its membrane-stabilizing action
(14). This possibility seems
unlikely, however, because the dl-propranolol concentration used in
the present experiments was lower than that reported to produce stabilizing
membrane effects (14).
The availability of transgenic mouse models expressing unique PLB mutants in the cardiac compartment of the null background provided an excellent tool to unequivocally determine the contribution of each PLB phosphorylation site in the ability of the heart to recover upon ischemia-reperfusion. Mutation of either Thr17 or Ser16 sites to Ala diminished the contractile recovery after the ischemic episode. However, only the ablation of the Thr17 site had a negative impact on the contractile recovery all along the reperfusion period and on the recovery of relaxation. Although we did not assess intracellular Ca2+ dynamics, it is tempting to speculate that the phosphorylation of Thr17 produces an increase in the SR Ca2+ uptake and diminishes the Ca2+ overload, resulting in an improvement of relaxation and contractile recovery. Dephosphorylation of Thr17 by KN-93 or mutation of this residue would preclude this mechanism.
In contrast to the results in the rat, suggesting that Thr17 is the only site able to provide a mechanism to enhance the contractile recovery after ischemia, the results from transgenic mice indicate that Ser16 also plays a role. An intriguing finding of the present results is that phosphorylation of Ser16 did not increase at any time during the ischemia-reperfusion in the mouse heart, despite the fact that the absence of this residue impairs the mechanical recovery. The presence of Ser16 may be necessary to handle Ca2+ overload at the basal phosphorylation state. Another possible explanation to these findings is that compensatory mechanisms developed in these transgenic models contribute to the contractile recovery after ischemia. However, the fact that preischemic mechanical parameters of PLB mutants did not differ from PLB-WT mice makes this possibility unlikely.
Recent data from Cross et al. (7) showed that the injury produced by ischemia-reperfusion is increased in PLB-KO mice. Because phosphorylation and ablation of PLB both result in enhanced SERCA2 activity, it might be concluded from these experiments that PLB phosphorylation during ischemia-reperfusion has a detrimental effect in the stunned heart. The present results indicate that the presence of PLB phosphorylation sites improves, rather than impairs, the recovery of contractility and relaxation after ischemia. Thus, although a permanent enhancement of SERCA2 activity (as it is the case of PLB-KO mice) may impair the contractile recovery, the presence of PLB phosphorylation sites, which provide the adequate mechanism for a fine regulation of SERCA2 activity, seems to be necessary for the contractile recovery after ischemia. Besides, the energetic alterations described in the PLB-KO mice (19) may play a direct deleterious role on the mechanical recovery during stunning.
In summary, the present results provide the first evidence that both PLB phosphorylation sites are involved in the functional recovery after ischemia, elucidating the role of PLB site-specific phosphorylation in the altered cardiac function during stunning. It was shown that Ser16 influences in the contractile recovery only immediately after ischemia, whereas the presence of Thr17 has a major beneficial impact all along the reperfusion period and on the recovery of relaxation in the stunned heart. These findings suggest that dualsite PLB phosphorylation offers a mechanism that helps to limit intracellular Ca2+ overload induced by ischemia-reperfusion. Experimental data indicate that Ca2+ overload at the onset of reperfusion would lead to troponin I proteolysis, which is considered one of the main mechanisms responsible for the mechanical dysfunction of the stunned heart in rodents (9). The susceptibility of the stunned myocardium harboring the mutated PLB phosphorylation sites, which are key regulators of Ca2+ handling in the heart (4), suggests that PLB phosphorylation may play a protective role by limiting Ca2+ overload and thus interfering with the cascade of events that lead to abnormal function of the stunned heart.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-agonists. J Biol
Chem 275:
38938–38943, 2000.-agonist stimulation.
Circ Res 75:
401–409, 1994.-adrenergic stimulation.
Pflügers Arch 444:
801–809, 2002.[ISI][Medline]
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; n = 13) or
presence (
; n = 8) of the CaMKII inhibitor KN-93 (KN). KN (1
µM) was perfused for 10 min before ischemia and for the first 3 min upon
reperfusion. Hearts treated with KN showed a significantly lower recovery of
LVDP than nontreated hearts. B: the increase in Thr17
phosphorylation (PThr17-PLB) detected at 1 min of reperfusion (R;
n = 5) was diminished by KN (RKN; n = 4). C: time
course of LVDP of rat hearts submitted to ischemia-reperfusion protocols in
the absence (
; n = 9) of
the 
