PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion

Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c

Original article

cGMP/PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion Javier Inserte, Ignasi Barba, Marcos Poncelas-Nozal, Victor Hernando, Luís Agulló, Marisol Ruiz-Meana, David Garcia-Dorado ⁎ Laboratory of Experimental Cardiology, Vall d'Hebron University Hospital and Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 14 January 2011 Received in revised form 14 February 2011 Accepted 22 February 2011 Available online 26 February 2011 Keywords: Ischemia Postconditioning Acidosis Myocardial infarction

a b s t r a c t Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague–Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by 31P-NMR spectroscopy (3.6 ± 0.2 min and 3.5 ± 0.2 min respectively vs. 1.4 ± 0.2 min in control group, P b 0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P b 0.01), but only postconditioning increased myocardial cGMP levels (P = 0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na+/H+-exchange mediated pHi normalization after acidification induced by a NH4Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na+/H+-exchanger. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Ischemic postconditioning (PoCo), defined as rapid sequential intermittent interruption of blood flow applied at the onset of reperfusion, effectively reduces myocardial infarct size in all animal species tested. In contrast to the limited clinical applicability of preconditioning, PoCo represents a feasible approach to attenuate reperfusion injury in patients. Proof-of-concept studies indicate that PoCo enhances myocardial salvage and improves clinical outcomes in patients with on-going acute myocardial infarction receiving primary percutaneous coronary intervention [1–3]. However, although various signaling elements involved in the protection have been identified, our Abbreviations: GC, guanylyl cyclase; KHB, Krebs–Henseleit bicarbonate buffer; LDH, lactate dehydrogenase; LVEDP, left ventricular end-diastolic pressure; LVdevP, left ventricular developed pressure; mPTP, mitochondria permeability transition pore; + + NBC, Na+/HCO− 3 symport; NHE, Na /H -exchanger; PKG, protein kinase G; PoCo, ischemic postconditioning; ROS, reactive oxygen species. ⁎ Corresponding author at: Institut de Recerca-Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119–129, 08035 Barcelona, Spain. Tel.: +34 93 4894038; fax: +34 93 4894032. E-mail address: [email protected] (D. Garcia-Dorado). 0022-2828/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2011.02.013

understanding of the mechanisms responsible for the beneficial effects of PoCo remains far of being complete [4]. Elucidation of these mechanisms could lead to the development of new pharmacological strategies to limit infarct size in patients with acute coronary syndromes receiving reperfusion therapy. The role of delayed recovery of intracellular acidosis was proposed as a hypothesis to explain PoCo protection [5] early after the initial description of PoCo. Experimental evidence supporting this hypothesis was provided by a seminal study by Cohen et al. [6]. Later observations involving monitoring of intracellular pH (pHi) with NMR spectroscopy [7] confirmed that delayed normalization of pHi during initial reperfusion plays a determinant role among the mechanisms leading to the cardioprotective effects of PoCo. Moreover, perfusion of isolated hearts with an acidic buffer during the first minutes of reperfusion mimicked the protection obtained with PoCo [6,8]. On the other hand, the use of either the nitric oxide synthase inhibitor L-NAME or the guanylyl cyclase (GC) inhibitor ODQ blocked the infarct-sparing effect of PoCo in different studies [9,10], suggesting that cGMP-mediated signaling is also involved in the mechanisms responsible for the cardioprotection induced by PoCo. Both delayed normalization of acidosis and activation of the cGMP/PKG pathway can modulate several targets implicated in

904

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

reperfusion injury including mitochondria permeability transition pore (mPTP), Ca2+ homeostasis and development of hypercontracture [11]. However, although it has been suggested that PKG negatively modulates the Na+/H+-exchanger (NHE) in different cell lines including cardiomyocytes [12–14], a direct link between these two proposed mechanisms of PoCo protection has not been established. The aim of our study was to determine whether the activation of the cGMP/PKG pathway and slowed normalization of pHi are interconnected mechanisms of PoCo protection and to identify the link between them. To this purpose ischemia/reperfusion studies were conducted in isolated hearts in which correction of acidosis and activation of the cGMP/PKG pathway during initial reperfusion were modified and pHi, myocardial cGMP content and cell death were monitored. 2. Methods The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85–23, revised 1996), and were approved by the Research Commission on Ethics of the Hospital Vall d'Hebron. 2.1. Experimental protocol Male Sprague–Dawley rats (300–350 g) were anesthetized with sodium pentobarbital (100 mg/kg). Hearts were excised and mounted on a Langendorff apparatus as previously described [15]. The different experimental protocols are illustrated in Fig. 1. In control group, hearts were subjected to global ischemia for 40 min followed by reperfusion of different duration (n = 8). Postconditioning was achieved with a previously established protocol consisting of 6 cycles of 10-second reperfusion/10-second occlusion with flow rate adjusted to 50% of basal value during the reperfusion cycles (PoCo baseline 30 min

ischemia

reperfusion

40 min

10min

group, n = 8) [7]. In an acidic reperfusion group, hearts were perfused with KHB adjusted at pH 6.4 for the first 2 min of reperfusion (pH6.4 group, n = 8). The pH of the acidic perfusate was lowered by reducing bicarbonate concentration and increasing sodium chloride to keep osmolarity constant while maintaining the same gassing mixture containing 5% CO2 [16]. To determine whether cGMP/PKG pathway plays a role in the cardioprotective effects of PoCo and acidic reperfusion, hearts were perfused with the GC inhibitor ODQ (Sigma-Aldrich, 3 μM) or the PKG inhibitor KT-5823 (Sigma-Aldrich, 1 μM) during the first 10 min of reperfusion (n = 6–8 per group). 2.2. Quantification of cell death Lactate dehydrogenase (LDH) activity was spectrophotometrically measured in the coronary effluent throughout the reperfusion period. After 60 min of reperfusion, heart slices were incubated in 1% triphenyltetrazolium chloride to outline the area of necrosis as previously described [17]. 2.2.1. Measurement of myocardial cGMP content The effect of each treatment on myocardial cGMP content and PKG activation was determined in hearts immediately frozen in liquid nitrogen after 10 min of reperfusion (n = 4–5 per group). The contractile inhibitor blebbistatin at 10 μM was added to KHB buffer at the onset of reperfusion [18]. Inhibition of the contractile machinery prevents sarcolemmal rupture and cell death by reducing the mechanical stress caused by the excessive contractile activation occurring at the onset of reperfusion [19,20]. It was used to ensure that any difference between groups was not a mere consequence of the differences in sarcolemmal rupture and secondary massive Ca2+ entry. cGMP was measured in extracts from heart samples with a commercial cyclic GMP EIA kit (Cayman Chemical Co) following manufacturer's instructions. 2.3. NMR spectroscopy

60min

IR IR+ODQ ODQ

IR+KT

Intracellular pH was measured by 31P-NMR in additional series of hearts subjected to the same study protocols described above but perfused with KHB free of phosphate (n= 5–6 hearts per group). Spectroscopy was performed on a Bruker Avance 400 spectrometer as previously described [7]. Time to pHi recovery was defined as the interval from the onset of reperfusion to the time to reach pHi 6.9.

KT5823 6 x (10”/10”)

PoCo PoCo+ODQ ODQ

PoCo+KT KT5823

2 min

pH6.4 pH6.4+ODQ ODQ

Fig. 1. Experimental protocol. Isolated rat hearts were subjected to 40 min of ischemia and 10 min of reperfusion (measurement of cGMP) or 60 min of reperfusion. PoCo group: hearts were exposed to a protocol of postconditioning consisting in 6 cycles of 10-seconds of ischemia and 10-seconds of reperfusion with flow rate reduced to 50%. pH 6.4 group: hearts were perfused with buffer adjusted at 6.4 for the first 2 min of reperfusion. ODQ: 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 μM) was added to the perfusion buffer during the first 10 min of reperfusion. KT: KT-5823 (1 μM) was added 5 min before ischemia and during the first 10 min of reperfusion.

2.4. Correction of intracellular acidosis in adult cardiac myocytes Cardiomyocytes from adult Sprague–Dawley male rats (300 g) were isolated as previously described [21]. Changes in intracellular H+ concentration were monitored using a fast speed monochromator (Visitech) in individual cells loaded for 30 min with 3 μM of 2,7-bis(2carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester (Molecular Probes) in control HEPES buffer at pH 7.4. Intracellular acidosis was induced after 1 min incubation in control HEPES buffer by transient exposure to 30 mM pulse of NH4Cl for 3 min, as previously described [22], and subsequent washout using control buffer, with or without 100 μM 8-pCPT-cGMP or 7 μM cariporide (NHE inhibitor, a gift from Sanofi-Aventis). Changes in intracellular BCECF fluorescence were continuously monitored during 10 min at 1-second intervals and were subsequently calibrated to pH units with nigericin (10 μg/ml). 2.5. Statistical analysis Data analysis was performed using SPSS for Windows. Results are expressed as mean ± S.E.M. Repeated measures ANOVA was used to compare temporal differences in pHi and LVEDP values. In all other cases means between groups were compared by one-way ANOVA. When significant differences were observed least significant square

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

A LDH (U/gdw/60min)

test was applied as post hoc test. P b 0.05 was considered to be statistically significant. 3. Results 3.1. Reperfusion-induced cell death No differences among groups were observed in LV function during the equilibration and the ischemic period. At the end of the equilibration period LVEDP and LVdevP were, respectively, 7.1 ±0.8 and 94.6 ± 6.2 mm Hg, perfusion pressure was 62 ± 4.8 mm Hg and coronary flow 11.8 ± 1.7 ml/min. LVEDP increased during ischemia with a peak of 59.6 ± 4.7 mm Hg at 13.6 ± 1.2 min after the onset of ischemia. On the contrary, during the reperfusion period hearts subjected to PoCo (PoCo group) or acidic perfusion (pH6.4 group) modified LVEDP (Figs. 2A–B), showed delayed (Pb 0.001, Fig. 2C) and attenuated (Pb 0.001, Fig. 2D) hypercontracture, and markedly reduced LDH (P b 0.001, Fig. 3A) and infarct size (Pb 0.001; Fig. 3B). Treatment of hearts with either the GC inhibitor, ODQ , or the PKG blocker, KT5823, abolished the effects of PoCo on hypercontracture (Fig. 2) and cell death (Fig. 3). No effect of ODQ or KT5823 treatment on hypercontracture or cell death was observed in control or pH6.4 groups.

Myocardial cGMP content was measured after 10 min of reperfusion in the presence of the contractile inhibitor blebbistatin. Perfusion with buffer containing blebbistatin prevented the development of hypercontracture and abolished the differences in LDH release during initial reperfusion between the experimental groups.

400

*

*

*

200 0

Infarct size (%)

60

* *

40

*

20

0

IR

PoCo

pH6.4

In accordance with previous studies [23], cGMP content was severely reduced in control myocardium reperfused after 40 min of ischemia. The reduction in cGMP levels was attenuated in the PoCo 160 140

140

120

*$

$

100 80

$

$

*

*

$

*

*

60

*

40

IR PoCo PoCo+ODQ PoCo+KT

*

20

* *

0 0

2

LVEDP (mmHg)

120

LVEDP (mmHg)

600

B

B

160

4

6

100 80

*

* *

40

*

20

IR pH6.4 pH6.4+ODQ

*

0

8

C

*

*

60

0

Time (min of reperfusion)

2

4

6

8

Time (min of reperfusion)

D 8

Control ODQ KT5823

6

*

* * *

$

*$

4

2

0

IR

Control ODQ KT5823

160

PoCo

pH6.4

Hypercontracture (mmHg)

Time to hypercontracture (min)

Control ODQ KT-5823

800

Fig. 3. Quantification of reperfusion induced cell death in the different groups of treatment. (A) Total LDH released during reperfusion and (B) infarct size measured by triphenyltetrazolium chloride and expressed as percentage of ventricular mass. *Pb 0.05 vs. control group. Data are mean± SEM.

3.2. Myocardial cGMP and PKG activation

A

905

120

*

*

*

80

40

0

IR

PoCo

pH6.4

Fig. 2. Effect of the different interventions on the development of hypercontracture calculated as the difference between maximal LVEDP measured at reperfusion and at the end of ischemia. (A) Time course of changes in LVEDP (mm Hg) during the first minutes of reperfusion expressed as increment with respect to LVEDP at the end of ischemia in ischemia/ reperfusion control group (IR) vs. postconditioned groups (PoCo) reperfused with and without ODQ or KT5823 (KT) and (B) the same control group vs. acidic perfusion groups (pH 6.4) with and without ODQ during reperfusion. (C) Time from the onset of reperfusion to the development of hypercontracture (minutes) and (D) magnitude of hypercontracture (mm Hg) in the different groups of treatment. *P b 0.05 vs. control group. Data are mean ± SEM.

906

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

1.5

significantly shortened the delay in pHi normalization caused by PoCo (Pb 0.001 and P=0.015 respectively compared to PoCo group) (Fig. 5A). However, ODQ did not modify the kinetics of pHi recovery in the pH6.4 group or in the control group (Fig. 5B).

1.0

3.4. Effect of cGMP on pHi recovery in isolated cardiomyocytes

0.5

Isolated cardiac myocytes subjected to a transient NH4Cl pulse in bicarbonate-free buffer experienced an initial intracellular alkalinization followed by a rapid acidification (up to pH 6.6–6.8). This maneuver of induction of intracellular acidosis activated a mechanism of slow and progressive intracellular H+ extrusion after NH4Cl washout under control conditions (Fig. 6A). However, when the cGMP analog 8-pCPTcGMP at 100 μM was present in the extracellular buffer, cells failed to recover from NH4Cl-induced intracellular acidosis (Fig. 6B). Inhibition of NHE with 7 μM cariporide completely prevented pHi correction (Fig. 6C). The effect of 8-pCPT-cGMP on pHi reached at different time points after the induction of acidosis is shown in Fig. 6D.

*

control ODQ

cGMP (pmol/mg)

2.0

0.0

Nx

IR

PoCo

pH6.4

Fig. 4. Myocardial cGMP content in the different groups of treatment. cGMP was measured in hearts perfused under normoxic conditions (Nx) and after 10 min of reperfusion in hearts subjected to 40 min of ischemia and control reperfusion (IR), postconditioning (PoCo) and acidic perfusion (pH6.4) with and without the guanylyl cyclase inhibitor ODQ. *P= 0.021 vs. control group. Data are mean± SEM.

group (P = 0.021) while no differences with respect to control group were observed in the pH6.4 group. Addition of ODQ during reperfusion decreased cGMP content and eliminated the differences among the groups (Fig. 4).

4. Discussion In the present study both PoCo and acidic reperfusion delayed pHi normalization and limited cell death, but only PoCo increased the myocardial levels of cGMP during reperfusion to preischemic values. Inhibition of the cGMP/PKG pathway accelerated the normalization of pHi during reperfusion and abolished cardioprotection in PoCo but not in acidic reperfusion. These results demonstrate a link between two previously described mechanisms of PoCo protection, and that the protective effect of activation of the cGMP/PKG pathway is due in part to maintenance of intracellular acidosis during the first minutes of reperfusion. Our studies in isolated cardiomyocytes suggest that the

3.3. Effect of cGMP/PKG pathway on intracellular pH Myocardial pHi was 7.04 ±0.05 before ischemia and decreased progressively during ischemia reaching its minimum (6.44±0.15) after 20 min without differences between groups. In control group, reperfusion resulted in a rapid rise in pHi, with a normalization within 1.4±0.2 min (Fig. 5). In accordance with previous studies [7], both, PoCo and acidic perfusion delayed pHi normalization (3.6±0.2 min in PoCo group and 3.5±0.2 min in pH6.4 group, Pb 0.001). Perfusion with ODQ or KT5823

A

B 7.0

7.0

$

*

6.8

*$

6.6

*

pHi

pHi

$

* *

* * *

6.4 0

1

6.8

* *

IR PoCo PoCo+ODQ PoCo+KT5823

6.6

*

*

IR pH 6.4 pH 6.4+ODQ

6.4

2

3

4

5

6

0

Time (min of reperfusion)

pHi (time to recovery, min.)

C

1

2

3

4

5

6

Time (min of reperfusion)

5

4

Control ODQ KT5823

3

*

* *$

*

*$

2

1

0

IR

PoCo

pH6.4

Fig. 5. Time course of changes in intracellular pH (pHi) measured by 31P-RMN during the first minutes of reperfusion in the different groups of treatment. (A) Ischemia/reperfusion control group (IR) vs. postconditioned groups (PoCo) reperfused with and without ODQ or KT5823 (KT). (B) Ischemia/reperfusion control group (IR) vs. acidic perfusion groups (pH 6.4) with and without ODQ during reperfusion. (C) Time (in minutes) from the onset of reperfusion to pHi normalization. *P b 0.05 vs. IR group. $P b 0.05 vs. PoCo control group. Data are presented as mean ± SEM.

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

A

7.6

907

Control

pHi

7.4 7.2 7.0 washout

6.8

NH4Cl

6.6 0

100 200 300 400 500 600

D

NH4Cl 7.6

B

8-pCPT-cGMP

7.6

pHi

7.4 7.2 7.0

washout

6.8

NH4Cl

6.6 0

100 200 300 400 500 600

Time (s)

C

7.6

Cariporide

pHi (490/450 BCECF ratio)

Time (s) 7.4

7.2

7.0

* 6.8

6.6

baseline

0

3

10 min

7.4 Control (n= 12 cells) 8-pCPT-cGMP (n= 8 cells)

pHi

7.2 7.0 washout

6.8

NH4Cl

6.6 0

100 200 300 400 500 600

Time (s) Fig. 6. Representative recordings of pHi in adult rat cardiac myocytes subjected to transient intracellular acidosis by external NH4Cl pulse of 3 min duration (A) under control (bicarbonatefree) conditions, (B) in the presence of GMPc analog 8-pCPT-cGMP (100 μM) and (C) in the presence of Na+/H+−exchanger inhibitor cariporide (7 μM). (D) Intracellular pH obtained at baseline, immediately after the addition of NH4Cl, at 3 min post-NH4Cl pulse and at 10 min, both in control conditions and when 8-pCPT-cGMP was present during recovery from intracellular acidosis. While pHi reached after the addition of NH4Cl was similar in both groups, recovery from intracellular acidosis was significantly decreased in the presence of 8-pCPTcGMP. *P= 0.02. Data are expressed as mean± SEM of 8–12 independent cells per group.

effect of cGMP/PKG activation on pHi recovery is mediated through inhibition of NHE. These findings provide an important clue for the understanding of the mechanism of PoCo protection. 4.1. Intracellular pH normalization modulates reperfusion induced cell death Analysis of the extensive available data shows that the conditions for a protocol to be optimally effective vary with the species and the experimental model, suggesting that a gradual washout of metabolites is determinant for the cardioprotective effects of PoCo [24–26]. Based on this concept, it has been proposed that PoCo protects because it slows the normalization of pHi during the first minutes of reperfusion. Initial evidence supporting this hypothesis demonstrated that brief acidic perfusion (2 min) was as protective as a PoCo protocol while perfusing with an alkalotic buffer completely abolished infarct size reduction by PoCo in anesthetized rabbits [6]. In a more recent study using NMR spectroscopy we demonstrated that only those PoCo algorithms able to induce a significant prolongation of acidosis were cardioprotective in isolated rat hearts. Moreover, a close correlation between delay in pHi recovery and the magnitude of protection was observed [7]. It is thus clear that delayed recovery of pHi by more than 2 min is protective, and this has been confirmed in pigs submitted to transient coronary occlusion [27]. The mechanisms by which delayed pHi recovery could protect against reperfusion injury have been reviewed recently [11]. It has been

postulated that prolongation of acidosis prevents mPTP formation long enough to allow the activation of endogenous protective signaling pathways which will keep mPTP in a closed state even after correction of myocardial pHi [28]. Low pHi also reduces Ca2+ overload and myofibrillar contractility that causes the Ca2+-dependent hypercontracture during the initial minutes of reperfusion in cardiomyocytes [8]. In our study both PoCo and acidic perfusion resulted in a pronounced attenuation of LVEDP, similar to that observed in different studies with the use of 2,3-butanedione monoxime, a reversible inhibitor of actomyosin-ATPase [19,20]. Acidosis maintains gap-junctions in a closed state impairing the prolongation of cell death [29] and, recently, it has been demonstrated that prolongation of acidosis prevents activation of calpains during PoCo and that inhibited calpain activity contributes to PoCo protection [7]. However, the relative importance of each mechanism to the potential cardioprotective effects of delayed pHi recovery has not been established yet. 4.2. cGMP/PKG pathway contributes to the cardioprotective effects of postconditioning The myocardial cGMP/PKG signaling is severely depressed in cardiomyocytes and endothelial cells after prolonged ischemia [30]. The reduced myocardial cGMP content measured after 15 min of reperfusion in our study is in agreement with these previous data. A large body of experimental evidence indicates that preservation of this pathway, either with NO donors, natriuretic peptides or cGMP

908

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

analogs, ameliorates irreversible injury associated to reperfusion [23,31,32]. More recently, the use of either the NOS inhibitor L-NAME or ODQ blocked the infarct-sparing effects of PoCo [9,10], suggesting that cGMP-mediated signaling is involved in the mechanisms responsible for PoCo induced cardioprotection. In our study, both the inhibition of GC with ODQ or PKG with KT5843 blocked the cardioprotective effects of PoCo. In contrast, ODQ was ineffective in preventing the beneficial effects obtained after transient acidic reperfusion. To rule out the possibility that the observed differences in cGMP and PKG activation are just a reflection of differences between groups in the extent of cell death, hearts were reperfused in the presence of blebbistatin [18]. The mechanisms by which preservation of cGMP levels induces cardioprotection are not fully elucidated. Through the activation of PKG, cGMP has been suggested to participate in different molecular mechanisms implicated in reperfusion injury. It has been described that PKG activation correlates with increased phosphorylation of phospholamban at Ser16 and enhances SERCA activity, accelerating the normalization of cytosolic Ca2+ concentration and attenuating Ca2+ oscillations. Sarcoplasmic reticulum-induced Ca2+ oscillations can trigger cardiomyocyte hypercontracture [31] and through close communication between sarcoplasmic reticulum and mitochondria can favor mitochondria permeability transition during the initial phase of reperfusion [21]. Other proposed mechanisms for cGMP mediated cardioprotection involve a role of PKG in the transfer of the signals triggering protection from the cytosol to the mitochondria. PKG dependent phosphorylation has been shown to induce opening of mKATP evoking K+ entry into the mitochondria and generation of ROS, being PKCε the intermediate step between cytosol and the mitochondrial inner membrane [33]. In addition, PKG dependent phosphorylation of TnI and/or direct or indirect effects on myosin light chain phosphatase, attenuates contractility during reperfusion and has been proposed to prevent hypercontracture [23,34].

permeable cGMP analog, was blocked. Jointly considered, these results strongly suggest that PKG-induced inhibition of NHE contributes to the prolongation of acidosis observed in PoCo. Extensive experimental data have solidly demonstrated that inhibition of NHE before ischemia delays the progression of myocardial injury by attenuating intracellular Na+ accumulation and markedly reduces infarct size [36,37]. In contrast, many studies indicate that the efficacy of NHE inhibitors administered during reperfusion is very limited [37,38]. Although its inhibition at the onset of reperfusion was expected to prolong acidosis, the different NHE inhibitors tested only resulted in a small effect on kinetics of pHi recovery probably due to the compensatory effect of lactate washout and the action of bicarbonate transporters [38]. In contrast, simultaneous inhibition of both, NHE and NBC significantly delayed recovery of pHi and attenuated reperfusioninduced cell death [37]. Our results suggesting that NHE modulates pHi recovery do not disagree with these previous studies. Since in hearts subjected to PoCo the contribution of H+-coupled lactate efflux to pHi correction is severely limited due to the reduced metabolite washout [7], the effect of NHE inhibition on the kinetics of pHi recovery is expected to be higher than in normally reperfused hearts due to the reduced capacity of the heart to compensate the blockade of NHE. 4.4. Study limitations This study was performed in isolated, perfused rat hearts, a model that does not reproduce all the aspects of reperfusion in patients with acute coronary syndrome. However, the use of this model was necessary to monitor the kinetics of pHi during ischemia and reperfusion. 4.5. Implications

4.3. cGMP-PKG pathway contributes to delayed pHi in postconditioned hearts

Limitation of reperfusion injury is now recognized as a promising strategy in patients with acute coronary syndrome receiving reperfusion therapy. Both PoCo [1] and stimulation of the cGMP/PKG pathway [39] have been successfully tested in patients, but none of them has been established yet in standard clinical practice. Other approaches

In the present study both PoCo and acidic perfusion delayed pHi recovery and produced reductions of cell death of similar magnitude while only the cardioprotective effects of PoCo were blocked with ODQ and KT-5823. Our data showing that inhibition of GC or PKG accelerates pHi recovery in hearts subjected to PoCo but not in those reperfused with acidic buffer could explain this different response to the blockade of cGMP/PKG pathway. It has been demonstrated in isolated hearts that 1 min of acidic reperfusion or PoCo protocols that prolonged acidosis by only 1 min with respect to control hearts fail to induce protection [6,7] and that at least 2 min are necessary to be clearly protective. The loss of protection associated to the significant reduction in the time to pHi recovery observed in PoCo hearts treated with inhibitors of cGMP/PKG signaling is in agreement with these previous studies. Normalization of pHi during reperfusion is the consequence of the combined action of different transport systems including H+-coupled lactate efflux, NHE and Na+/HCO− 3 symport (NBC) [35]. In a previous study, measurement of lactate in the coronary effluent demonstrated that the reduced metabolite washout in hearts subjected to PoCo protocol contributes to the rate of pHi recovery during reperfusion [7]. The present study demonstrates that, in addition to reduced lactate washout, PKG signaling participates in the prolongation of acidosis and that its contribution is essential for PoCo protection. Previous studies have demonstrated that PKG modulate negatively NHE [12,13]. In isolated cardiomyocytes, the activation of PKG by a NO donor or cGMP analog caused intracellular acidification by inhibiting NHE [14]. In our study, NHE activity, measured as pHi recovery from acidosis induced with a NH4Cl-HEPES pulse in isolated cardiomyocytes pretreated with a

Fig. 7. Scheme showing the proposed link between two mechanisms of postconditioning protection: delayed pHi normalization and activation of cGMP/PKG pathway. Our data suggest that cGMP/PKG pathway contributes to the delay of pHi normalization via PKG dependent inhibition of NHE. ●: represents other cardioprotective effects associated to PKG activation. The cardioprotective effects of acidic reperfusion are independent of cGMP signaling. Thickness of the arrows is in accordance with the relative contribution of the different mechanisms to the maintenance of acidosis during the first minutes of reperfusion.

J. Inserte et al. / Journal of Molecular and Cellular Cardiology 50 (2011) 903–909

also found useful in proof of concept trials, as cyclosporine [40] or remote conditioning [41], have not been incorporated either to common practice. A better understanding of the mechanism of PoCo protection could be of great help in the design of new trials in which the effect of these strategies, separately or in combination will be assessed. In conclusion, the current study demonstrates that cGMP/PKG signaling contributes to the delay of pHi recovery, probably via PKG dependent inhibition of NHE. This finding provides a new mechanism by which cGMP/PKG pathway could contribute with other cardioprotective effects of PKG activation to the infarct size sparing effect of PoCo (Fig. 7). Funding sources This study was supported by the Redes Temáticas de Investigación Cooperativa Santinaria (RETICS-RECAVA RD06/0014/0025); Comisión Interministerial en Ciencia y Tecnología (CICYT SAF/2008-03067) and Fondo de Investigación Sanitaria (FIS-PI080238 and FIS-PS09/02034). References [1] Thibault H, Piot C, Staat P, Bontemps L, Sportouch C, Rioufol G, et al. Long-term benefit of postconditioning. Circulation 2008;117:1037–44. [2] Darling CE, Solari PB, Smith CS, Furman MI, Przyklenk K. ‘Postconditioning’ the human heart: multiple balloon inflations during primary angioplasty may confer cardioprotection. Basic Res Cardiol 2007;102:274–8. [3] Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, et al. Postconditioning the human heart. Circulation 2005;112:2143–8. [4] Ovize M, Baxter GF, Di Lisa F, Ferdinandy P, Garcia-Dorado D, Hausenloy DJ, et al. Postconditioning and protection from reperfusion injury: where do we stand? * Position Paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res 2010;87:406–23. [5] Garcia-Dorado D, Piper HM. Postconditioning: reperfusion of “reperfusion injury” after hibernation. Cardiovasc Res 2006;69:1–3. [6] Cohen MV, Yang XM, Downey JM. The pH hypothesis of postconditioning: staccato reperfusion reintroduces oxygen and perpetuates myocardial acidosis. Circulation 2007;115:1895–903. [7] Inserte J, Barba I, Hernando V, Garcia-Dorado D. Delayed recovery of intracellular acidosis during reperfusion prevents calpain activation and determines protection in postconditioned myocardium. Cardiovasc Res 2009;81:116–22. [8] Inserte J, Barba I, Hernando V, Abellan A, Ruiz-Meana M, Rodriguez-Sinovas A, et al. Effect of acidic reperfusion on prolongation of intracellular acidosis and myocardial salvage. Cardiovasc Res 2008;77:782–90. [9] Penna C, Cappello S, Mancardi D, Raimondo S, Rastaldo R, Gattullo D, et al. Postconditioning reduces infarct size in the isolated rat heart: role of coronary flow and pressure and the nitric oxide/cGMP pathway. Basic Res Cardiol 2006;101:168–79. [10] Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 2004;44:1103–10. [11] Inserte J, Ruiz-Meana M, Rodriguez-Sinovas A, Barba I, Garcia-Dorado D. Contribution of delayed intracellular pH recovery to ischemic postconditioning protection. Antioxid Redox Signal 2011;14:923–39. [12] Schulte EA, Hohendahl A, Stegemann H, Hirsch JR, Saleh H, Schlatter E. Natriuretic peptides and diadenosine polyphosphates modulate pH regulation of rat mesangial cells. Cell Physiol Biochem 1999;9:310–22. [13] Fidzinski P, Salvador-Silva M, Choritz L, Geibel J, Coca-Prados M. Inhibition of NHE-1 Na+/H + exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium. Am J Physiol Cell Physiol 2004;287:C655–63. [14] Ito N, Bartunek J, Spitzer KW, Lorell BH. Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes. Circulation 1997;95:2303–11. [15] Inserte J, Garcia-Dorado D, Ruiz-Meana M, Agullo L, Pina P, Soler-Soler J. Ischemic preconditioning attenuates calpain-mediated degradation of structural proteins through a protein kinase A-dependent mechanism. Cardiovasc Res 2004;64:105–14. [16] Avkiran M, Ibuki C, Shimada Y, Haddock PS. Effects of acidic reperfusion on arrhythmias and Na(+)-K(+)-ATPase activity in regionally ischemic rat hearts. Am J Physiol 1996;270:H957–64.

909

[17] Ferrera R, Benhabbouche S, Bopassa JC, Li B, Ovize M. One hour reperfusion is enough to assess function and infarct size with TTC staining in Langendorff rat model. Cardiovasc Drugs Ther 2009;23:327–31. [18] Dou Y, Arlock P, Arner A. Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle. Am J Physiol Cell Physiol 2007;293:C1148–53. [19] Inserte J, Garcia-Dorado D, Hernando V, Soler-Soler J. Calpain-mediated impairment of Na+/K+−ATPase activity during early reperfusion contributes to cell death after myocardial ischemia. Circ Res 2005;97:465–73. [20] Garcia-Dorado D, Theroux P, Duran JM, Solares J, Alonso J, Sanz E, et al. Selective inhibition of the contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion. Circulation 1992;85:1160–74. [21] Ruiz-Meana M, Abellan A, Miro-Casas E, Agullo E, Garcia-Dorado D. Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death during reperfusion. Am J Physiol Heart Circ Physiol 2009;297: H1281–9. [22] Haworth RS, McCann C, Snabaitis AK, Roberts NA, Avkiran M. Stimulation of the plasma membrane Na+/H + exchanger NHE1 by sustained intracellular acidosis. Evidence for a novel mechanism mediated by the ERK pathway. J Biol Chem 2003;278:31676–84. [23] Inserte J, Garcia-Dorado D, Agullo L, Paniagua A, Soler-Soler J. Urodilatin limits acute reperfusion injury in the isolated rat heart. Cardiovasc Res 2000;45:351–9. [24] Iliodromitis EK, Downey JM, Heusch G, Kremastinos DT. What is the optimal postconditioning algorithm? J Cardiovasc Pharmacol Ther 2009;14:269–73. [25] Penna C, Mancardi D, Raimondo S, Geuna S, Pagliaro P. The paradigm of postconditioning to protect the heart. J Cell Mol Med 2008;12:435–58. [26] Skyschally A, van Caster P, Iliodromitis EK, Schulz R, Kremastinos DT, Heusch G. Ischemic postconditioning: experimental models and protocol algorithms. Basic Res Cardiol 2009;104:469–83. [27] Rodriguez-Sinovas A, Cabestrero A, Garcia del Blanco B, Inserte J, Garcia A, GarciaDorado D. Intracoronary acid infusion as an alternative to ischemic postconditioning in pigs. Basic Res Cardiol 2009;104:761–71. [28] Cohen MV, Yang XM, Downey JM. Acidosis, oxygen, and interference with mitochondrial permeability transition pore formation in the early minutes of reperfusion are critical to postconditioning's success. Basic Res Cardiol 2008;103: 464–71. [29] Peracchia C. Chemical gating of gap junction channels; roles of calcium, pH and calmodulin. Biochim Biophys Acta 2004;1662:61–80. [30] Agullo L, Garcia-Dorado D, Escalona N, Ruiz-Meana M, Inserte J, Soler-Soler J. Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes. Am J Physiol Heart Circ Physiol 2003;284:H2170–6. [31] Abdallah Y, Gkatzoflia A, Pieper H, Zoga E, Walther S, Kasseckert S, et al. Mechanism of cGMP-mediated protection in a cellular model of myocardial reperfusion injury. Cardiovasc Res 2005;66:123–31. [32] Padilla F, Garcia-Dorado D, Agullo L, Inserte J, Paniagua A, Mirabet S, et al. LArginine administration prevents reperfusion-induced cardiomyocyte hypercontracture and reduces infarct size in the pig. Cardiovasc Res 2000;46:412–20. [33] Costa AD, Pierre SV, Cohen MV, Downey JM, Garlid KD. cGMP signalling in pre- and post-conditioning: the role of mitochondria. Cardiovasc Res 2008;77:344–52. [34] Shah AM, Spurgeon HA, Sollott SJ, Talo A, Lakatta EG. 8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes. Circ Res 1994;74: 970–8. [35] Vaughan-Jones RD, Spitzer KW, Swietach P. Intracellular pH regulation in heart. J Mol Cell Cardiol 2009;46:318–31. [36] Karmazyn M. The role of the myocardial sodium-hydrogen exchanger in mediating ischemic and reperfusion injury. From amiloride to cariporide. Ann NY Acad Sci 1999;874:326–34. [37] Garcia-Dorado D, Gonzalez MA, Barrabes JA, Ruiz-Meana M, Solares J, Lidon RM, et al. Prevention of ischemic rigor contracture during coronary occlusion by inhibition of Na(+)-H + exchange. Cardiovasc Res 1997;35:80–9. [38] Ten Hove M, Van Echteld CJ. Limited effects of post-ischemic NHE blockade on [Na+]i and pHi in rat hearts explain its lack of cardioprotection. Cardiovasc Res 2004;61: 522–9. [39] Kitakaze M, Asakura M, Kim J, Shintani Y, Asanuma H, Hamasaki T, et al. Human atrial natriuretic peptide and nicorandil as adjuncts to reperfusion treatment for acute myocardial infarction (J-WIND): two randomised trials. Lancet 2007;370: 1483–93. [40] Piot C, Croisille P, Staat P, Thibault H, Rioufol G, Mewton N, et al. Effect of cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J Med 2008;359:473–81. [41] Botker HE, Kharbanda R, Schmidt MR, Bottcher M, Kaltoft AK, Terkelsen CJ, et al. Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet 2010;375:727–34.