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Cardiovascular Research 2007 73(3):560-567; doi:10.1016/j.cardiores.2006.11.020
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Copyright © 2006, European Society of Cardiology

The inotropic adaptation during late preconditioning against myocardial stunning is associated with an increase in FKBP12.6

Laurence Lucatsa,b,c, Laurent Vinete, Alain Bizéa,b,c, Xavier Monneta,b,c, Didier Morina,b,c, Jin Bo Sua,b,c, Patricia Rouet-Benzinebe, Olivier Cazorlah, Jean-Jacques Mercadiere,f,g, Luc Hittingera,b,c,d, Alain Berdeauxa,b,c,d,* and Bijan Ghaleha,b,c,d

aINSERM, U 660, Créteil, F-94010, France
bUniversité Paris XII, Faculté de Médecine, Laboratoire de Pharmacologie, Créteil, F-94000, France
cEcole Nationale Vétérinaire d'Alfort, INSERM U 660, Maisons-Alfort, F-94700, France
dAP-HP, Groupe Hospitalier Henri Mondor, Fédération de Cardiologie, Créteil, F-94000, France
eINSERM U 698, Paris, F-75018, France
fUniversité Paris VII-Denis Diderot, Faculté de Médecine, site Xavier Bichat, Département de Physiologie
gAP-HP, Groupe Hospitalier Xavier Bichat, Service d'explorations Fonctionnelles, Paris, F-75018, France
hINSERM, U 637, Université Montpellier 1, UFR de Médecine, F-34925 Montpellier, France

* Corresponding author. Laboratoire de Pharmacologie, INSERM U 660, Faculté de Médecine Paris XII, 8, rue du Général Sarrail, 94010 CRETEIL Cedex, France. Tel.: +33 1 49 81 36 51; fax: +33 1 49 98 17 77. Email address: alain.berdeaux{at}creteil.inserm.fr

Received 16 August 2006; revised 30 October 2006; accepted 16 November 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The inotropic adaptation during late preconditioning against myocardial stunning is associated with an increase in FKBP12.6. by Laurence Lucats, Laurent Vinet, Alain Bizé, Xavier Monnet, Didier Morin, Jin Bo Su, Patricia Rouet-Benzineb, Olivier Cazorla, Jean-Jacques Mercadier, Luc Hittinger, Alain Berdeaux, Bijan Ghaleh.

Objectives: Late preconditioning reduces contractile dysfunction during myocardial stunning. Mechanisms involving adaptation of calcium handling during excitation–contraction coupling to late preconditioning remain to be established. Thus, we investigated whether the late preconditioned myocardium is associated with contractile adaptation and changes in the cardiac ryanodine receptor (RyR2) and its regulatory protein FKBP12.6.

Methods: Chronically instrumented conscious dogs (coronary occluder, ultrasonic crystals for sonomicrometry) underwent a 10-min coronary artery occlusion followed by reperfusion. They were studied 24 h later in the late preconditioned state (day 1).

Results: Maximal velocity of wall thickening at day 1 was increased as compared to corresponding baseline at day 0 (39±4 vs. 30±3 mm/s, p<0.05) although systolic wall thickening was similar (2.8±0.2 vs. 2.9±0.2 mm), demonstrating a significant change in left ventricular inotropic state. Intracoronary infusion of ryanodine (0.5–6 µg) induced a dose-dependent decrease in wall thickening. In the late preconditioned state, this negative inotropic response was significantly reduced vs. control state, suggesting changes in sarcoplasmic reticulum (SR) Ca2+-release through RyR2. Immunoquantification of FKBP12.6 revealed a 2.8 fold ventricular increase after late preconditioning as compared to the control state. The amount of RyR2 and its phosphorylated state were similar and binding experiments did not reveal any alterations in Bmax or KD for RyR2. Calsequestrin, SERCA2a and phospholamban levels were not altered by late preconditioning.

Conclusions: The late preconditioned myocardium is characterized by an adaptation of regional function associated with an increased expression of FKBP12.6. This demonstrates an adaptation of the SR Ca2+-release through RyR2 during late preconditioning.

KEYWORDS Stunning; Contractile function; Preconditioning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Late preconditioning is well known to protect the heart against myocardial stunning, i.e., it reduces the duration and severity of the contractile dysfunction resulting from a brief ischemic episode [1]. The signaling pathways leading to this cardioprotective phenomenon have been studied extensively [2] but comparatively little is known about the mechanisms potentially involving adaptation of calcium handling during excitation–contraction coupling [3]. In the normal heart, the Ca2+ needed for contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor 2 (RyR2) [4]. This is a finely regulated process that involves not only RyR2 but also several accessory proteins modulating its activity [5]. Among these proteins, the FK506-binding protein FKBP12.6 stabilizes the coupled gating between RyR2 channels, providing coordinated activation and inactivation of Ca2+-release by RyR2 during excitation–contraction coupling [6]. It has been reported that FKBP12.6 dissociation from RyR2 occurs in the failing heart [7] whereas FKBP12.6 overexpression in adult rabbit cardiomyocytes enhances Ca2+ transient amplitude [8] and improves cell shortening [9]. Therefore, our goal was to investigate whether the protection afforded by late preconditioning against myocardial stunning involves contractile adaptations of the myocardium and whether changes in RyR2 and/or FKBP12.6 occur.

Accordingly, we first analyzed regional systolic contractile function of the late preconditioned myocardium in conscious chronically instrumented dogs. Then we investigated whether systolic contractile adaptation during late preconditioning against myocardial stunning was associated with in vivo changes in RyR2 function. For this purpose, we delivered the alkaloid ryanodine through an intracoronary catheter in order to explore RyR2 function without inducing significant systemic hemodynamic changes [10]. Then we quantified myocardial RyR2 ex vivo using binding and Western blot experiments. As FKBP12.6 is one of the major proteins regulating RyR2 function, we next determined whether RyR2 adaptation was associated with changes in FKBP12.6. Finally, we investigated whether these adaptations in calcium handling during late preconditioning were associated with changes in calsequestrin, SR Ca2+-ATPase (SERCA2a) and its regulatory protein, phospholamban.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996).

2.1. Instrumentation
A left thoracotomy was performed in dogs as previously described [11]. Fluid-filled Tygon catheters were placed in the descending thoracic aorta and the left atrium for measurement of blood pressure. A solid-state pressure transducer (P7A, Konigsberg Instruments, Pasadena, CA, USA) was introduced into the apex of the left ventricle (LV). A pneumatic occluder was placed around the left circumflex coronary artery (LCX). Two pairs of ultrasonic crystals were used for measurement of LV wall thickening in the distribution of LCX (posterior zone) and left anterior descending (anterior zone) vascular beds. One crystal was implanted in the endocardium and the other was sutured to the epicardium. Proper alignment of the crystals was ensured by visualizing the signal on an oscilloscope. An additional indwelling silastic catheter was inserted in the LCX [10]. All catheters and wires were exteriorized between the scapulae and the pneumothorax was evacuated. Cefazolin (1 g iv) and gentamicin (40 mg iv) were administered before and during the first week after surgery. Post-operative analgesia was provided with morphine. The correct position of the crystals was verified after sacrifice of the animals.

2.2. Hemodynamic measurements
All hemodynamic data were recorded, digitized at 500 Hz and analyzed using the data acquisition software HEM v3.5 (Notocord Systems, Croissy sur Seine, France). Aortic and left atrial pressures were measured with a Statham P23 ID strain-gauge transducer (Gould-Nicolet, Courtaboeuf, France). LV pressure was measured using the Konigsberg gauge and the change in LV pressure over time (LV dP/dt) was computed from the LV pressure signal. LV pressure was calibrated in vitro with a mercury manometer and in vivo with the left atrial and aortic pressures.

2.3. Measurements of regional function
Wall thicknesses were obtained by using an ultrasonic transit-time dimension gauge (Module 201, System 6, Triton Technology Inc., San Diego, CA, USA). To determine wall thickening, end-diastolic wall thickness was measured at the initiation of the upstroke of LV pressure tracing and the end-systolic wall thickness was measured within 20 ms before peak negative LV dP/dt. Systolic wall thickening was defined as the difference between end-diastolic and end-systolic wall-thicknesses, i.e., the wall thickening (expressed in mm) that occurs during the ejection period. The velocity of wall thickening was computed from the wall thickness signal and its maximal value was measured during systole (dW/dtmax). This parameter is a very sensitive index of inotropism.

2.4. Preparation of homogenates
At sacrifice, the hearts were excised and placed in ice-cold saline. LV samples of approximately 1 g obtained from the anterior (control zone) and posterior (late preconditioned zone) walls were trimmed of fat and connective tissue. Homogenates were prepared as follows: samples (1 g/10 ml) were homogenized in ice-cold buffer (10 ml of 0.3 M sucrose, 30 mM Tris–malate at pH=7) with a polytron (four times for 15 s). The homogenate was filtered through a layer of cheesecloth and the protein concentration was determined by the method of Lowry et al. [12].

2.5. Receptor binding studies
High affinity ryanodine binding sites were measured as follows: myocardial homogenates (1 mg/ml) were incubated with 12 concentrations (0.5–40 nM) of [3H]ryanodine (50 Ci/mmol) for 120 min at 37 °C in 250 µl of a buffer containing 10 mM HEPES, 1 M KCl, pH=7.4 at 37 °C. Binding was stopped by the addition of ice-cold buffer. Bound and free ligands were separated by rapid filtration through Whatman GF/B glass fiber filters (presoaked in 0.1% polyethylenimine). Each filter was washed twice with an additional 5 ml of ice-cold Tris buffer (50 mM) and counted in a liquid scintillation counter Packard 1600 TR. Non-specific binding was defined using 10 µM ryanodine. In these conditions, specific binding was higher than 90% at KD (dissociation constant) value. The equilibrium binding parameters, KD and Bmax (maximal density of binding sites), were calculated by means of a non-linear regression method using a commercially available software (Micropharm, INSERM 1990 [13]) as described previously [14].

2.6. Western blot assays
Myocardial samples were ground to a fine powder under nitrogen liquid using a mortar and pestle, homogenized in protein buffer (0.05 M Tris–HCl [pH 7.4], NaCl 0.9%, 0.1% Triton X100, protease inhibitor cocktail [Complete, Roche Diagnostic, Meylan, France]) and centrifuged for 5 min at 10,000 g. Crude homogenates were separated by SDS-Page, transferred onto nitrocellulose membranes. Equal protein loading was checked by staining membrane with Ponceau S solution (0.1% PonceauS (w/v) in 5% (v/v) acetic acid) before protein immunodetection. Membrane were probed with primary antibodies against SERCA2a (1/200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or phospholamban (1/5000, Affinity Bioreagents, Golden, CO, USA) or calsequestrin (1/2500, Affinity Bioreagents, Golden, CO, USA) or RyR2 (1/5000, Affinity Bioreagents, Golden, CO, USA) or P-S2809 RyR2 (1/2000, Badrilla, Leeds, UK) or FKBP12/12.6 (1/1000, Affinity Bioreagents, Golden, CO, USA). All dilutions were performed in Tris buffered saline with Tween 20 (TBS-T: 0.05 M Tris [pH 8,0], 0.138 M NaCl, 0.0027 KCl, 0.05% Tween 20) supplemented with 1% non-fat milk. After washing, membranes were incubated with secondary antibodies (IgG) against rabbit, mouse or goat immunoglobulin conjugated to horseradish peroxidase (1:50,000 dilution in TBS-T 1% non-fat milk). Ternary immune complexes were revealed by chemiluminescence using ECL plus and Hyperfilm ECL (GE Healthcare, Orsay, France). Densitometric analysis was performed using a Biorad GS800 densitometer calibrated scanner (Biorad, Marnes La Coquette, France). The relative amount of positive protein was quantified with ImageJ and calsequestrin was used as standard for controlling equal loading. The band intensity of the late preconditioned samples was compared to those of corresponding control samples (normalized to 100%) as previously described [15].

2.7. Experimental protocol
Three weeks after instrumentation, dogs were submitted in the conscious state to one of the three following protocols (Fig. 1). In Protocol I, in order to confirm late preconditioning in our experimental conditions and to analyze the regional systolic contractile function of the preconditioned myocardium, dogs were submitted to two episodes of 10-min coronary artery occlusion (CAO) followed by reperfusion performed 24 h apart. In Protocol II, in order to investigate the in vivo changes in RyR2 function in the late preconditioned heart, dogs were submitted to intracoronary infusion of increasing doses of ryanodine (0.5 to 6 µg) in the control state and in the late preconditioned state, i.e. 24 h after a 10-min CAO as previously described [10]. In Protocol III, dogs were sacrificed for myocardial sampling in the late preconditioned state, i.e., 24 h after a 10-min CAO followed by reperfusion. Late preconditioned and control samples were taken from the posterior and anterior walls, respectively.


Figure 1
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  		Fig. 1 A – Experimental protocol. A first group of dogs underwent two 10-min coronary artery occlusions (CAO) followed by reperfusion 24 h apart (Protocol I). A second group of dogs underwent two intracoronary (ic) ryanodine infusions, i.e., one in the control state and another one in the late preconditioned state (24 h after a 10-min CAO) (Protocol II). Finally in Protocol III, dogs were sacrificed in the late preconditioned state, i.e., 24 h after a 10-min CAO followed by reperfusion. Late preconditioned and control samples were taken from the left ventricular posterior and anterior walls, respectively. B – Representative recordings of aortic and left ventricular pressures (AO and LVP, respectively), the first derivative over time of left ventricular pressure (LV dP/dt), posterior wall thickness and its first derivative over time (dW/dt) at baseline and 2 h after reperfusion (stunning).

 
2.8. Statistical analysis
Data are reported as means±S.E.M. Comparisons were performed using two-way ANOVA for repeated measures. Individual comparisons were conducted using a paired Student t-test. The ic ryanodine responses were analyzed by comparing regression lines using ANCOVA. A value of p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Contractile adaptation in the late preconditioned heart (Protocol I)
As shown in Table 1, heart rate, mean arterial pressure, LV pressure and LV dP/dtmax were not significantly different at baseline, during CAO and reperfusion between day 0 and day 1 (n=6).


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  		Table 1 Hemodynamic measurements performed at baseline, during CAO and myocardial stunning at day 0 and at day 1 in Protocol I

 
At day 0, systolic wall thickening in the ischemic zone was dramatically reduced during CAO (–108±8% from baseline) as illustrated in Fig. 2A. During reperfusion, systolic wall thickening remained depressed and returned progressively to its corresponding baseline value, indicating myocardial stunning. Concomitantly, the maximal velocity of wall thickening (dW/dtmax) was depressed during reperfusion as compared to baseline and progressively tended to return to its corresponding baseline value as shown in Fig. 2B.


Figure 2
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  		Fig. 2 A. Time–course of systolic wall thickening (expressed in % change from baseline) measured at baseline, during coronary artery occlusion (CAO) and reperfusion at day 0 and 24 h later (day 1) in Protocol I (n=6). The duration and the severity of myocardial stunning were reduced at day 1 vs. day 0, indicating late preconditioning. *, p<0.05 vs. day 0. B. Time–course of maximal velocity of wall thickening (expressed in mm/s) measured at baseline and reperfusion following a 10-min coronary artery occlusion at day 0 and 24 h later (day 1) in Protocol I (n=6). At day 1, this parameter was significantly greater at baseline and throughout recovery as compared to day 0. *, p<0.05 vs. day 0.

 
At day 1, systolic wall thickening in the ischemic zone was similarly reduced during CAO as compared to day 0 but during reperfusion, it was significantly greater as compared to day 0, demonstrating late preconditioning (Fig. 2A). Interestingly, at baseline, dWT/dtmax was significantly increased at day 1 as compared to day 0 (39±4 mm/s vs. 30±3 mm/s, respectively) although systolic wall thickening was similar (2.9±0.3 mm and 2.8±0.3 mm at day 1 and day 0, respectively), demonstrating contractile adaptation for similar thickening. At dWT/dtmax, wall thickness and LV pressure were not significantly different between days 0 and 1.

3.2. Inotropic responses to intracoronary ryanodine infusion (Protocol II)
Intracoronary infusion of ryanodine dose-dependently reduced systolic wall thickening in the control state (n=4) as shown in Fig. 3. In the late preconditioned state, i.e., 24 h after 10 min CAO, this negative inotropic effect of ryanodine was still observed but it was of lower magnitude. The dose–response curve was significantly shifted rightward in the late preconditioned state as compared to control conditions leading to a significant increase in ED40 (effective dose inducing a 40% decrease in systolic wall thickening) (2.6±0.5 µg vs. 1.2±0.1 µg in the late preconditioned and control states, respectively, p<0.05). Analysis of regression lines confirmed that the dose–response curves were significantly different. Systemic hemodynamic in the late preconditioned state was similar to control conditions (Table 2).


Figure 3
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  		Fig. 3 Wall thickening response to intracoronary (ic) infusion of increasing ryanodine doses (0.5 to 6 µg) in the control state and in the late preconditioned state (24 h after a 10-min coronary artery occlusion) in Protocol II (n=4). The ED40 (effective dose of ryanodine inducing a 40% decrease in systolic wall thickening) was significantly greater in the late preconditioned state as compared to control (2.6±0.5 µg vs. 1.2±0.1 µg, respectively, p<0.05).

 

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  		Table 2 Hemodynamic measurements performed at baseline and during increasing doses of intracoronary ryanodine (µg) infusion in Protocol II

 
3.3. RyR2 and FKBP12.6
Fig. 4A shows a representative experiment of [3H]ryanodine binding to control myocardium. As shown in Fig. 4B, the amount of ryanodine receptor (Bmax) and the dissociation constant (KD) were not different between the late preconditioned and control states (n=6). These results were confirmed by Western blot of RyR2 as illustrated in Fig. 5. Late preconditioning did not alter the amount of phosphorylated RyR2 (Fig. 5). The amount of FKBP12.6 was significantly increased by 184% in the late preconditioned as compared to control myocardium (Fig. 5).


Figure 4
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  		Fig. 4 A. Representative direct (left) and Scatchard (right) plots of [3H]ryanodine equilibrium binding to myocardial homogenate prepared from a control sample. [3H]ryanodine labelled one class of binding sites of high affinity (KD=3.295 nM) with a Bmax value of 482.8 fmol/mg proteins, as attested by the linear Scatchard plot. B. The receptor density (Bmax) and dissociation constant (KD) (Protocol III, n=6) were similar in late preconditioned (LPC) and control myocardium.

 

Figure 5
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  		Fig. 5 Ryanodine receptor (RyR2), phosphorylated ryanodine receptor (P-RyR2) and FKBP12.6 density in control and late preconditioned myocardium (LPC). To characterize FKBP expression in dog myocardium, two recombinant proteins (RP), FKBP12 (25 ng) and FKPB12.6 (100 ng), were also used as control of immunoreactivity. These Western blot experiments demonstrated a significant increase in FKBP12.6 in the late preconditioned state as compared to control (Protocol III, n=6). *, p<0.05 vs. day 0.

 
3.4. Calsequestrin, SERCA2a and phospholamban
Fig. 6 shows Western blots of control and late preconditioned myocardium probed with antibodies against calsequestrin, SERCA2a and phospholamban (n=6). As shown by the bands and quantified by the column graphs, none of these proteins were altered by late preconditioning.


Figure 6
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  		Fig. 6 Calsequestrin, SERCA2a and phospholamban density in late preconditioned (LPC) and control myocardium (Protocol III, n=6).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study demonstrates that the late preconditioned myocardium is characterized by a systolic contractile adaptation with a change in the inotropic state associated with molecular changes in calcium handling proteins in a pre-emptive way before any new ischemic insult. In fact, 24 h after the late preconditioning stimulus, maximal velocity of wall thickening was increased although systolic wall thickening during ejection was unchanged. In vivo responses to intracoronary ryanodine infusion demonstrated that this contractile adaptation was associated with in vivo functional changes of RyR2 suggesting changes in SR Ca2+-release through RyR2. In vitro experiments revealed that RyR2 adaptation was associated with an increased in FKBP12.6 level.

As previously described [1], we observed that late preconditioning protected the myocardium against myocardial stunning as demonstrated by the improved recovery in systolic wall thickening at day 1 as compared to day 0 (Protocol I). In these conditions, we focused our study on the contractile phenotype of the late preconditioned heart at day 1 and demonstrated that for similar baseline systolic wall thickening at days 0 and 1, the maximal velocity of wall thickening, a sensitive index of inotropy, was increased in the late preconditioned state at day 1. This demonstrates that the late preconditioning stimulus induces adaptation of regional function with a change in the inotropic state in a pre-emptive way, i.e., before any additional ischemic episode. The interpretation of maximal velocity of wall thickening needs careful analysis as this parameter is load dependent. In the present study, increase in the maximal velocity of wall thickening was unlikely related to changes in loading conditions between days 0 and 1 as end-diastolic wall thicknesses and LVP at maximal dW/dt were similar. Interestingly, we previously reported that late preconditioning induces changes in the metabolic phenotype [11], i.e., myocardial oxygen consumption of the late preconditioned myocardium is reduced. All these results suggest a major contractile adaptation associated with an improved cardiac efficiency that could contribute to the protection against myocardial ischemia and stunning.

We next focused on RyR2 which releases the calcium needed for contraction during systole from sarcoplasmic reticulum. For this purpose, we evaluated the in vivo regional contractile response to intracoronary administration of increasing doses of ryanodine. This selective infusion to the posterior wall of the left ventricle was devoid of significant changes in heart rate or arterial pressure [16], which could have polluted the observations [10]. This experimental approach in chronically instrumented conscious dogs has been shown to be a valuable tool to investigate excitation-coupling [16] and to detect changes in RyR2 function [10]. In agreement with these previous studies, intracoronary infusion of ryanodine induced a dose-dependent decrease in systolic wall thickening. In the preconditioned state, this negative inotropic effect was of lower magnitude as the dose–response curve to ryanodine infusion was rightward significantly shifted. This result demonstrates functional adaptation of RyR2 during late preconditioning suggesting changes in SR calcium release. The relationship between modulation of SR activity and preservation of contractility is an active area of research [17] and has been demonstrated for other cardioprotective strategies such as early preconditioning [18,19]. Accordingly, we then investigated whether these changes in response to ryanodine were related to molecular alterations in RyR2. In our study, adaptation during late preconditioning did not result from a change in the number or affinity of RyR2, as shown by our binding experiments. This result was also confirmed by Western blot experiments which furthermore did not reveal any change in the serine-2809 RyR2 phosphorylation state. Importantly, we found a significant increase in the amount of FKBP12.6 with late preconditioning without any changes in calsequestrin, phospholamban and SERCA2a. Our results are in agreement with a previous study demonstrating no changes in mRNA and protein levels of phospholamban, SERCA, calsequestrin, and TnI during myocardial stunning [20]. It must be stressed that analysis of the degree of phosphorylation of phospholamban is missing in the present study. To our knowledge, this study is the first to describe an increase in FKBP12.6 with a cardioprotective strategy.

Our data suggests that the increase in FKBP12.6 was responsible for the accelerated rate in wall thickening and indicative of the change in inotropic state observed in the late preconditioned state. Previous studies have demonstrated that FKBP12.6 modulates cardiac excitation–contraction coupling through its binding to RyR2 [8,9]. FKBP12.6 is thought to mediate the coupled gating observed in RyR2 channel clusters [6] and its deficiency participates to the pathophysiology of heart failure [7] and arrhythmia [21]. Conversely using adenoviral-mediated gene transfer, Loughrey et al. [8] showed that overexpression of FKBP12.6 in cardiomyocytes increases systolic Ca2+ transients amplitude secondary to higher degree of excitation–contraction coupling synchrony, independently from SR Ca2+ content. These effects were accompanied by a significant increase in cell shortening [9].

Increased FKBP12.6 density must also take part to the cardioprotection afforded by late preconditioning. On the one hand, contractile dysfunction during myocardial stunning is characterized by a decrease in overall myocardial calcium responsiveness without significant myocardial desensitization to calcium [22]. Other studies have also demonstrated an impaired calcium handling with depressed SR function [23] and potentially reduced calcium transient [24]. On the other hand, this contractile dysfunction can be reduced by the administration of JTV-519, a drug that stabilizes RyR2–FKBP12.6 interaction [25]. Therefore, one can speculate that the protection against myocardial stunning during late preconditioning involves a preserved RyR2 function secondary to an improved RyR2–FKBP12.6 interaction. This does not exclude other changes in calcium handling or adaptations of contractile proteins and thus further studies are needed.

In conclusion, late preconditioning is characterized by an adaptation of regional function associated with increased FKBP12.6. This involves adaptations of the SR Ca2+-release through RyR2. This study provides new insights into the mechanisms of late preconditioning by giving evidences that the control of calcium release by the SR represents a promising target for future pharmacological development.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Laurence Lucats was supported by INSERM ("poste d'accueil") and Académie Nationale de Médecine. Laurent Vinet was supported by Ministère de l'Enseignement Supérieur et de la Recherche and by Institut de Recherche Servier (IDRS, Suresnes, France). Laurent Vinet, Patricia Rouet-Benzineb and Jean-Jacques Mercadier are supported in part by an EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart. The authors wish also to thank Guillermo Salazar for his technical assistance.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Sun J.Z., Tang X.L., Knowlton A.A., Park S.W., Qiu Y., Bolli R. Late preconditioning against myocardial stunning. An endogenous protective mechanism that confers resistance to postischemic dysfunction 24 h after brief ischemia in conscious pigs. J Clin Invest (1995) 95:388–403.[ISI][Medline]
  2. Bolli R. The late phase of preconditioning. Circ Res (2000) 87:972–983.[Abstract/Free Full Text]
  3. Bers D.M. Cardiac excitation–contraction coupling. Nature (2002) 415:198–205.[CrossRef][Medline]
  4. Meissner G. Molecular regulation of cardiac ryanodine receptor ion channel. Cell Calcium (2004) 35:621–628.[CrossRef][ISI][Medline]
  5. Bers D.M. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol (2004) 37:417–429.[CrossRef][ISI][Medline]
  6. Marx S.O., Gaburjakova J., Gaburjakova M., Henrikson C., Ondrias K., Marks A.R. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ Res (2001) 88:1151–1158.[Abstract/Free Full Text]
  7. Marx S.O., Reiken S., Hisamatsu Y., Jayaraman T., Burkhoff D., Rosemblit N., et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][ISI][Medline]
  8. Loughrey C.M., Seidler T., Miller S.L., Prestle J., MacEachern K.E., Reynolds D.F., et al. Over-expression of FK506-binding protein FKBP12.6 alters excitation–contraction coupling in adult rabbit cardiomyocytes. J Physiol (2004) 556:919–934.[Abstract/Free Full Text]
  9. Prestle J., Janssen P.M., Janssen A.P., Zeitz O., Lehnart S.E., Bruce L., et al. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility. Circ Res (2001) 88:188–194.[Abstract/Free Full Text]
  10. Hittinger L., Ghaleh B., Chen J., Edwards J.G., Kudej R.K., Iwase M., et al. Reduced subendocardial ryanodine receptors and consequent effects on cardiac function in conscious dogs with left ventricular hypertrophy. Circ Res (1999) 84:999–1006.[Abstract/Free Full Text]
  11. Monnet X., Ghaleh B., Lucats L., Colin P., Zini R., Hittinger L., et al. Phenotypic adaptation of the late preconditioned heart: myocardial oxygen consumption is reduced. Cardiovasc Res (2006) 70:391–398.[Abstract/Free Full Text]
  12. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  13. Urien S. MicroPharm-K, a microcomputer interactive program for the analysis and simulation of pharmacokinetic processes. Pharm Res (1995) 12:1225–1230.[CrossRef][ISI][Medline]
  14. Morin D., Elimadi A., Sapena R., Crevat A., Carrupt P.A., Testa B., et al. Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore. Br J Pharmacol (1998) 123:1385–1394.[CrossRef][ISI][Medline]
  15. Jiang M.T., Lokuta A.J., Farrell E.F., Wolff M.R., Haworth R.A., Valdivia H.H. Abnormal Ca2+ release, but normal ryanodine receptors, in canine and human heart failure. Circ Res (2002) 91:1015–1022.[Abstract/Free Full Text]
  16. Kalthof B., Sato N., Iwase M., Shen Y.T., Mirsky I., Patrick T.A., et al. Effects of ryanodine on cardiac contraction, excitation–contraction coupling and "Treppe" in the conscious dog. J Mol Cell Cardiol (1995) 27:2111–2121.[CrossRef][ISI][Medline]
  17. Zucchi R., Ronca F., Ronca-Testoni S. Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection? Pharmacol Ther (2001) 89:47–65.[CrossRef][ISI][Medline]
  18. Domenech R.J., Sanchez G., Donoso P., Parra V., Macho P. Effect of tachycardia on myocardial sarcoplasmic reticulum and Ca2+ dynamics: a mechanism for preconditioning? J Mol Cell Cardiol (2003) 35:1429–1437.[CrossRef][ISI][Medline]
  19. Sanchez G., Pedrozo Z., Domenech R.J., Hidalgo C., Donoso P. Tachycardia increases NADPH oxidase activity and RyR2 S-glutathionylation in ventricular muscle. J Mol Cell Cardiol (2005) 39:982–991.[CrossRef][ISI][Medline]
  20. Luss H., Boknik P., Heusch G., Muller F.U., Neumann J., Schmitz W., et al. Expression of calcium regulatory proteins in short-term hibernation and stunning in the in situ porcine heart. Cardiovasc Res (1998) 37:606–617.[Abstract/Free Full Text]
  21. Lehnart S.E., Wehrens X.H., Laitinen P.J., Reiken S.R., Deng S.X., Cheng Z., et al. Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak. Circulation (2004) 109:3208–3214.[Abstract/Free Full Text]
  22. Heusch G., Rose J., Skyschally A., Post H., Schulz R. Calcium responsiveness in regional myocardial short-term hibernation and stunning in the in situ porcine heart. Inotropic responses to postextrasystolic potentiation and intracoronary calcium. Circulation (1996) 93:1556–1566.[Abstract/Free Full Text]
  23. Valdivia C., Hegge J.O., Lasley R.D., Valdivia H.H., Mentzer R. Ryanodine receptor dysfunction in porcine stunned myocardium. Am J Physiol (1997) 273:H796–H804.[ISI][Medline]
  24. Kim S.J., Kudej R.K., Yatani A., Kim Y.K., Takagi G., Honda R., et al. A novel mechanism for myocardial stunning involving impaired Ca(2+) handling. Circ Res (2001) 89:831–837.[Abstract/Free Full Text]
  25. Ito K., Shigematsu S., Sato T., Abe T., Li Y., Arita M. JTV-519, a novel cardioprotective agent, improves the contractile recovery after ischaemia–reperfusion in coronary perfused guinea-pig ventricular muscles. Br J Pharmacol (2000) 130:767–776.[CrossRef][ISI][Medline]

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