Feedback Control Through Cgmp-Dependent Protein Kinase Contributes to Differential Regulation and Compartmentation of Cgmp in Rat Cardiac Myocytes Liliana R.V

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Feedback Control Through cGMP-Dependent Protein Kinase Contributes to Differential Regulation and Compartmentation of cGMP in Rat Cardiac Myocytes Liliana R.V. Castro, Julia Schittl, Rodolphe Fischmeister

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Liliana R.V. Castro, Julia Schittl, Rodolphe Fischmeister. Feedback Control Through cGMP- Dependent Protein Kinase Contributes to Differential Regulation and Compartmentation of cGMP in Rat Cardiac Myocytes. Circulation Research, American Heart Association, 2010, 107 (10), pp.1232- 1240. 10.1161/CIRCRESAHA.110.226712. hal-02940481

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Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of cGMP in rat cardiac myocytes

Liliana R. V. Castro1,2, Julia Schittl1,2 & Rodolphe Fischmeister1,2

1INSERM, UMR-S 769, Châtenay-Malabry, France; 2Univ Paris-Sud, Faculté de Pharmacie, IFR141, Châtenay-Malabry, France;

Running title: PKG controls cGMP compartmentation

Correspondence to: Rodolphe FISCHMEISTER INSERM UMR-S 769 Faculté de Pharmacie 5, Rue J.-B. Clément F-92296 Châtenay-Malabry Cedex France Tel. +33-1-46 83 57 57 Fax +33-1-46 83 54 75 E-mail: [email protected] Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 2

Rationale: We have shown recently that particulate (pGC) and soluble guanylyl cyclases

(sGC) synthesize cGMP in different compartments in adult rat ventricular myocytes

(ARVMs). We hypothesized that cGMP-dependent protein kinase (PKG) exerts a feedback control on cGMP concentration contributing to its intracellular compartmentation.

Methods and Results: Global cGMP levels, cGMP-phosphodiesterase (PDE) and pGC enzymatic activities were determined in purified ARVMs. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (ICNG) in myocytes expressing the wild type rat olfactory cyclic nucleotide-gated (CNG) channel. While the NO- donor S-nitroso-N-acetyl-penicillamine (SNAP, 100 µmol/L) produced per se little effect on

ICNG, the response increased 2-fold in the presence of the PKG inhibitors KT5823 (KT, 50 nmol/L) or DT-2 (2 µmol/L). The effect of KT was abolished in the presence of the non selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxantine

(IBMX, 100 µmol/L) or the selective cGMP-PDE5 inhibitor sildenafil (100 nmol/L). PKG inhibition also potentiated the effect of SNAP on global cGMP levels and fully blocked the increase in cGMP-PDE5 activity. In contrast, PKG inhibition decreased by ~50% the ICNG response to ANP (10 and 100 nmol/L) even in the presence of IBMX. Conversely, PKG activation increased the ICNG response to ANP and amplified the stimulatory effect of ANP on pGC activity.

Conclusion: PKG activation in adult cardiomyocytes limits the accumulation of cGMP induced by NO-donors via PDE5 stimulation, but increases that induced by natriuretic peptides. These findings support the paradigm that cGMP is not uniformly distributed in the cytosol, and identifies PKG as a key component in this process.

Key words: cGMP  cGMP-dependent protein kinase  nitric oxide  natriuretic peptides  phosphodiesterases  sildenafil Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 3

Non-standard Abbreviations and Acronyms:

ARVM, adult rat ventricular myocyte CNG, cyclic nucleotide gated GC, guanylyl cyclase IBMX, isobutylmethylxanthine

ICNG, CNG current NP, natriuretic peptides PDE, phosphodiesterase PDE5, cGMP-specific PDE pGC, particulate GC PKA, cAMP-dependent protein kinase PKG, cGMP-dependent protein kinase sGC, soluble GC SNAP, S-nitroso-N-acetyl-penicillamine Sp-8, 8-(4-chlorophenylthio) guanosine-3',5'-cyclic monophosphorothioate, Sp-isomer Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 4

Introduction cGMP synthesis is controlled by two types of guanylyl cyclases (GC) that differ in their cellular location and activation by specific ligands: a particulate GC (pGC) present at the plasma membrane, which is activated by natriuretic peptides (NPs) such as atrial (ANP), brain

(BNP) and C-type natriuretic peptide (CNP),1 a soluble guanylyl cyclase (sGC) present in the cytosol and activated by nitric oxide (NO).2 The physiological effects of cGMP are largely mediated by phosphorylation of various effectors via cGMP-dependent protein kinase

(PKG).3 PKG phosphorylation regulates major components of the excitation-contraction coupling, such as the L-type Ca2+ channels,4,5 phospholamban6 and troponin I.7-9

Although both pGC and sGC synthesize cGMP, there is ample evidence that the outcome of the cGMP produced differs depending on which GC is activated.9 For instance, in frog ventricular myocytes, sGC activation causes a pronounced inhibition of L-type Ca2+ current upon cAMP stimulation,10 while pGC activation has little effect.11 Similarly, in intact mouse heart and isolated myocytes, sGC activation blunted the β-adrenergic cardiac response, while pGC activation did not.9,12 In the same preparation, pGC activation decreased Ca2+ transients, whereas sGC activation had marginal effects,13 similarly to what was found in pig airway smooth muscle,14 suggesting that pGC signaling works mainly to decrease intracellular Ca2+ level, whereas sGC-signaling mainly decreases Ca2+ sensitivity. In rabbit atria, pGC activation caused a larger cAMP accumulation (via PDE3 inhibition), cGMP efflux and ANP release than activation of sGC.15

In a recent study, we proposed a rationale for the different functional effects of pGC and sGC activators by demonstrating that pGC and sGC synthesize cGMP in different compartments in adult rat ventricular myocytes (ARVMs).16 These compartments appear to be mediated by reaction-diffusion processes, rather than physical barriers, involving cGMP Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 5 hydrolysis by specific cyclic nucleotide phosphodiesterases (PDEs): the ‘particulate’ cGMP pool, which is readily accessible at the plasma membrane, is under the exclusive control of the cGMP-stimulated PDE2; the ‘soluble’ cGMP pool, which is not accessible to the plasma membrane, is controlled by both PDE2 and the cGMP-specific PDE5.16 When PDEs are functional, their activity limits the spread of cGMP, and the nucleotide can affect only a limited number of effectors via locally available PKG molecules and substrates; when PDE activity is blocked (e.g. by IBMX), cGMP compartmentation is totally abrogated and cGMP is free to diffuse inside the cell.16

The organization of intracellular cGMP in cardiac myocytes is reminiscent of that described for intracellular cAMP.17,17 One characteristic feature of the cAMP compartmentation in ARVMs is a negative feedback control of cAMP concentration by cAMP-dependent protein kinase (PKA) which involves PKA stimulation of PDE activity.18

Biochemical evidence also demonstrates that PDE5 is activated by PKG phosphorylation in several tissues providing a putative negative feedback mechanisms by which cGMP might regulate its own level.19-21 For these reasons we hypothesized that cGMP signaling might also be regulated by feedback mechanisms in cardiac myocytes.

Here, we used complementary biochemical and electrophysiological techniques to measure changes in cGMP concentration at the cellular and subsarcolemmal level in ARVMs upon sGC or pGC activation. RIA experiments provided an estimate of the global intracellular cGMP level in cell extracts; adenovirus expression of the wild-type (WT) α-subunit of the rat olfactory cyclic nucleotide-gated channel (CNGA2) allowed real-time measurement of cGMP at the sarcolemmal membrane in intact cells.16 We demonstrate that PKG controls both the soluble and particulate pools of cGMP, yet in an opposite manner.

Methods Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 6

All experiments performed conform to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines and the French decree n°87-848 of

October 19, 1987 (J Off République Française, 20 October 1987, pp. 12245–12248).

Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture, de la Pêche et de l'Alimentation (nº92-283, June 27,

2007). Detailed methods are included in the online data supplement at http://circres.ahajournlas.org.

Results

Negative feedback of PKG on sGC-cGMP signaling

To explore the role of PKG in the regulation of cGMP homeostasis in cardiac myocytes, subsarcolemmal cGMP changes were monitored using CNGA2 channels. These channels provide a reliable cGMP readout because they are directly opened by cGMP, time- independent and do not desensitize.22 In ARVMs infected with the CNGA2 adenovirus, the

CNG current (ICNG) was measured upon sGC stimulation in the absence and presence of two

PKG inhibitors, KT582323 (KT, 50 nmol/L), a derivative of staurosporine isolated from

Nocardiopsis, and DT-2 (2 µmol/L), a membrane-permeant peptide blocker.24 Although the efficacy and selectivity of KT to inhibit PKG has been questioned in few studies,25,26 this drug has been found to efficiently block PKG-mediated inhibition of L-type Ca2+ current by NO- donors in ARVMs.27 Figure 1A shows a typical experiment in which the NO-donor SNAP

(100 µmol/L) had little effect on ICNG under control conditions, but induced a major activation of the current in the presence of KT. The effect of KT was mimicked by DT-2 (Figure 1B).

On average, KT or DT-2 had no effect per se on ICNG but increased ~2.5-fold the effects of Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 7

SNAP to values ranging from 5% to 15% of the maximal response induced by 100 µmol/L of the non-hydrolysable membrane-permeable cGMP analog 8-(4-chlorophenylthio) guanosine-

3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8) (Figure 1B). This analog activates

CNGA2 channels with an absolute efficacy which is similar to cGMP28 and acts as an internal control for CNGA2 expression. Altogether, these results suggest that PKG limits the increase of ICNG upon sGC activation.

Role of PDE activity in the negative feedback of PKG on sGC-cGMP signaling

Because the soluble cGMP compartment is controlled by the activity of cGMP PDEs,16 we examined whether the inhibitory effect of PKG was due to activation of a PDE. As previously

16 reported, IBMX (100 µmol/L) strongly potentiated the ICNG response to SNAP (Figure 2A).

However, addition of KT (50 nmol/L) under these conditions had no additional effect (Figure

2A). The absence of a stimulatory effect of KT on ICNG in the presence of IBMX was not due to a saturation of the CNG channels because a subsequent application of Sp-8 (100 µmol/L) was still able to increase the current amplitude further. On average (Figure 2B), IBMX, which had no effect on basal ICNG when used alone, enhanced ~8-fold the subsarcolemmal cGMP signal induced by SNAP (to ~60% of the maximal Sp-8 response) but KT had no significant additional effect. These results demonstrate that functioning PDEs are required for KT to increase sGC-cGMP signaling, which suggests that PKG might cause a decrease in sGC- cGMP signaling via activation of a PDE.

PKG negative feedback on sGC-cGMP signaling is due to PDE5 activation

Earlier studies in non-cardiac myocytes or in vitro using purified enzymes have demonstrated that the activity of PDE5 is increased by PKG phosphorylation.19,21,29-31 If such regulation also takes place in ARVMs, this could account for the observed negative feedback of PKG on Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 8

sGC-induced subsarcolemmal cGMP signal. To test this hypothesis, the effect of KT on ICNG was examined in the presence of the PDE5 inhibitor sildenafil (100 nmol/L). As shown

16 earlier, sildenafil dramatically increased ICNG in the presence of SNAP (Figure 3A) and the effect averaged ~5-fold stimulation (Figure 3B). However, under such conditions, KT had no additional effect (Figure 3), although Sp-8 (100 µmol/L) was still able to increase the current

~2-fold further.

The above results support the notion that PKG limits the sGC-cGMP signal via activation of PDE5. Biochemical assays were designed to further test this hypothesis. First, radioimmunoassay (RIA) experiments were performed to measure global cGMP levels in

ARVMs under similar conditions as used in the single cell experiments. Figure 4A shows that neither IBMX, nor KT nor both drugs used in combination induced any significant effect on basal cGMP level. SNAP alone increased cGMP level by ~2.5-fold (to 185 fmol/50,000 cells, n=6) and this effect was further increased ~50% by KT and ~140% by IBMX. As expected,

KT had no effect on cGMP after all PDEs were blocked by IBMX. Second, cGMP-PDE activity was measured in freshly isolated ARVMs using a modification of the two-step assay method described by Thompson & Appleman32 (see Methods in the online data supplement).

PDE5 activity was determined as the fraction of cGMP-PDE activity inhibited by 100 nmol/L sildenafil. As shown in Figure 4B, at a substrate concentration of 1 µmol/L, basal PDE5 activity was on average 1.2±0.4 pmol/min/mg protein (n=4) and represented 10.4±2.6% of total cGMP-PDE. A 15 min exposure of the cells to SNAP (100 µmol/L) increased PDE5 twofold. However, this increase was not observed when the cells were pre-incubated during

15 min with 2 µmol/L DT-2 and exposed to SNAP in the presence of the PKG inhibitor. DT-2 alone (30 min incubation) had no effect on basal PDE5 activity (Figure 4B). Altogether, these results strongly support our hypothesis that PKG causes a decrease in sGC-cGMP signaling via activation of PDE5. Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 9

Positive feedback of PKG on pGC-cGMP signaling

Our previous study demonstrated that pGC activation by ANP or BNP in ARVMs leads to activation of a pool of cGMP that is insensitive to PDE5 hydrolysis.16 Therefore, based on the above results, we anticipated that PKG inhibition should be without effect on the pGC-cGMP signal. To test this hypothesis, the response of ICNG to ANP was examined both in the absence and presence of KT or DT-2. As shown earlier,16 ANP alone (100 nmol/L) induced a large increase in ICNG (Figure 5A). However, addition of KT (50 nmol/L) or DT-2 (2 µmol/L) reduced this effect 2-fold (Figure 5A & B). The inhibitory effect of KT observed both at low

(10 nmol/L) and high (100 nmol/L) ANP concentration (Figure 5B) and was not due to a direct inhibition of the CNGA2 channel by KT, because the drug had no inhibitory effect on the channel stimulated by Sp-8 (n=5, data not shown).

Role of PDE activity in the positive feedback of PKG on pGC-cGMP signaling

Particulate GC activation by ANP or BNP in ARVMs leads to activation of a pool of subsarcolemmal cGMP that is exclusively hydrolyzed by PDE2.16 Therefore, inhibition of

PDE2 by PKG could account for some of the effects seen above. To test this hypothesis, we examined whether KT had any effect on the response of ICNG to ANP in the presence of

IBMX. In the typical experiment shown in Figure 6A, KT (50 nmol/L) still reduced ICNG activated by ANP (100 nmol/L) + IBMX (100 µmol/L). On average (Figure 6B), KT reduced by ~50% the response of ICNG to ANP+IBMX. These results suggest that the positive control of PKG on pGC-cGMP signaling is not due to PDE inhibition.

PKG amplifies pGC stimulation by ANP

To complement the results obtained with pharmacological inhibition, the role of PKG in Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 10 controlling pGC-cGMP pools was also examined by increasing its activity. First, we used a constitutively active PKGI-β mutant lacking the autoinhibitory N-terminus (PKG+). PKG+ is a monomer and lacks the sequences that are implicated in anchoring PKG. ARVMs were co- infected with the CNGA2 adenovirus and an adenovirus encoding PKG+. In a first series of experiments we investigated the immunofluorescence of PKG expression in ARVMs after

24 h of culture. Native or infected myocytes were labeled with primary antibody against PKG and visualized with fluorophore conjugated secondary antibody (Alexa 488). Figure 7A shows confocal images of representative cells in native or infected cardiac myocytes. Control cells (Figure 7Aa and b) showed a fluorescent signal generated at the sarcolemmal membrane, with a somewhat stronger signal at the non-tubular vs. tubular membrane. The fluorescent signal was strongly increased in cells expressing PKG+ (Figure 7Ac). Cells not incubated with the PKG antibody showed no fluorescence signal with the secondary antibody (Figure 7Aa).

These results demonstrate that the infection of the ARVMs with the adenovirus encoding

PKG+ led to a detectable level of protein expression.

The effect of ANP was subsequently tested in these cells and the results are summarized in Figure 7B. The basal current density and the effect of Sp-8 in co-infected myocytes were not significantly different from those obtained in myocytes infected by the

Ad-CNGA2 alone. However, PKG+ expression increased by ~40% the response to ANP (10 nmol/L). Furthermore, the stimulatory effect of PKG+ was blocked by addition of KT (50 nmol/L). These results suggest that PKG amplifies the stimulation of pGC by ANP. To further test this hypothesis, pGC activity was measured in a crude membrane fraction of freshly isolated ARVMs (see Methods in the online data supplement) either in the absence or presence of recombinant PKGI-α. As shown in Figure 7C, PKGI-α alone (at a concentration of 0.1 or 0.2 µg/mg protein) had no significant effect on basal pGC activity. As expected, exposure of the membrane fraction to ANP strongly increased pGC activity (Figure 7C). Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 11

Particulate GC activity increased on average by 54% and 134% when the membranes were exposed during 15 min to 10 or 100 nmol/L ANP, respectively. However, when PKGI-α was present during the ANP stimulation, the stimulation of pGC activity was twice stronger

(respectively, 116% and 300%, Figure 7C). Therefore, we conclude that PKG is a positive regulator of pGC-cGMP signaling in ARVMs.

Discussion

While cardiac cAMP activates PKA to control many cellular functions, PKA also prevents excessive accumulation of the nucleotide by reducing its synthesis, via inhibition of adenylyl cyclase type 5 and 6,33 and activation of its hydrolysis, via stimulation of PDE3 and

PDE4.18,34,35 Our goal here was to examine whether the control of cardiac cGMP concentration shares some similarity with its cAMP counterpart and to investigate the role of the cGMP-dependent protein kinase (PKG) in this process. For this, we used complementary biochemical and electrophysiological techniques to measure changes in cGMP concentration at the cellular and subsarcolemmal level in ARVMs. RIA experiments provided an estimate of the global intracellular cGMP level in cell extracts while CNGA2 channel current (ICNG) allowed a real-time measurement of cGMP concentration at the sarcolemmal membrane in intact cells.16,16,18,36 Using either the PKG inhibitors, KT5823 (KT) and DT-2, or increasing

PKG activity by overexpression of a constitutively active PKG-Iβ mutant,37 we found that

PKG controls both the soluble and particulate pools of cGMP, yet in an opposite manner. Our findings led us to propose a model for intracellular cGMP compartmentation which is illustrated in Figure 8 and will be discussed below.

Activation of sGC by the NO-donor SNAP had limited effect on cGMP near the plasma membrane, although it strongly increased global cGMP. However, when PKG was blocked, cGMP produced by sGC increased further and the nucleotide reached the membrane. Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 12

Therefore, PKG limits the accumulation and intracellular diffusion of cGMP. Two possible mechanisms may account for this effect: a PKG-inhibition of cGMP synthesis or a PKG- activation of cGMP hydrolysis. Evidence for the former comes from earlier studies in smooth muscle19 and chromaffin cells38 showing that PKG-phosphorylation of sGC leads to inhibition of the enzyme. However, this hypothesis was discarded in our study because KT had no effect on ICNG in the presence of IBMX, although the current was still not maximally stimulated

(Figure 2A & B). Evidence for the latter comes from earlier work on either recombinant

PDE5,21,29,31 PDE5 isolated from gastric smooth muscle19 or platelets,39 or endogenous PDE5 in intact vascular40 or uterine smooth muscle cells,30 showing that PDE5 is a substrate for

PKG and its hydrolytic activity is increased by PKG phosphorylation. PDE5 is expressed in cardiac myocytes, both at the mRNA41 and protein level,42,43 and plays a role in the control of the β-adrenergic stimulation of cardiac systolic and diastolic function in dog,42 mouse,43 and human.44 Moreover, chronic PDE5 inhibition was found to prevent and reverse cardiac hypertrophy in mouse hearts exposed to sustained pressure overload.45 We now found that the

PDE5 inhibitor sildenafil (Viagra ®) produced a similar effect on SNAP-induced ICNG as

PKG inhibition, and PKG inhibition had no further effect when PDE5 was blocked (Figure

3A and B). Furthermore, PKG inhibition not only enhanced membrane cGMP but also caused a global rise in intracellular cGMP as revealed by RIA experiments (Figure 4A). Finally, PKG inhibition totally blunted the stimulatory effect of SNAP on PDE5 activity (Figure 4B). Based on this, we conclude that PKG exerts a negative feedback on the ‘soluble’ cGMP pool by activating cGMP hydrolysis via PDE5 (Figure 8). This PKG-mediated control mechanism is reminiscent of the control of cAMP hydrolysis via PKA activation of PDE3 and PDE4.34

Our finding that PKG inhibition produced a similar effect as sildenafil on SNAP-induced

ICNG and totally blunted the stimulatory effect of SNAP on PDE5 activity suggests that no other mechanism besides PKG is responsible for PDE5 activation upon cGMP production by Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 13 sGC. This is somewhat surprising since PDE5 contains two N-terminal domains (GAF A and

GAF B), and cGMP binding to the GAF A domain was shown previously to strongly increase

PDE5 actvity.39,46 However, allosteric activation by cGMP at the GAF A domain and stimulation by PKG phosphorylation are interdependent processes. Indeed, phosphorylation of PDE5 by PKG was shown to facilitate the cGMP-induced activation, allowing it to occur at a cGMP concentration not sufficient to directly induce activation.21,39 It is therefore possible that, under the experimental conditions used here, both cGMP binding to the GAF A domain and PKG phosphorylation are necessary for PDE5 activation upon sGC-cGMP stimulation, so that blocking one of these processes is sufficient to fully block the response.

On the contrary to the sGC-cGMP signaling, when cGMP was generated by pGC, PKG inhibition by either KT or DT-2 led to a reduction of the membrane cGMP signal (Figure 5 and 6). Moreover, this effect remained even when PDEs were blocked by IBMX (Figure 6) which suggests that PKG regulates the membrane pool of cGMP by acting on the production of cGMP rather than on its degradation. The natriuretic peptide (NP) receptors (type A and B) are composed of four structural domains: an extracellular ligand binding domain, a single transmembrane-spanning region, an intracellular kinase homology domain, and a cyclase- catalytic domain.1 Previous studies have demonstrated that phosphorylation of NP receptors is required for their activation;47,48 conversely, phosphatase treatment of crude membranes has been shown to dephosphorylate and desensitize NP receptors.49,50 Here, we found that treatment of crude membranes with recombinant PKG had no effect on basal pGC activity, but doubled the stimulatory effect of ANP (Figure 7C). Therefore, the PDE-independent stimulatory effect of PKG on pGC-cGMP levels (Figure 8) may involve PKG phosphorylation of the NP receptor, or of a protein that impacts pGC catalytic function, ANP binding, or transmission of the ANP signal to the pGC. In HEK cells, PKG was shown to interact with NP type A receptors (NPR-A).51 Moreover, cotransfection of PKG with NPR-A Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 14 increases ANP-dependent pGC activity.51 However, a recent study using the Pro-Q Diamond phosphoproteins dye technology demonstrated that PKG does not regulate pGC activity.52

Although the Pro-Q Diamond technique is a more sensitive method than the classical metabolic labeling used in previous studies, the latter stains all phosphorylated NP receptors in the cell, whereas the former only assesses the phosphorylation of the receptors that were synthesized during the labeling period. This difference may limit the ability of Pro-Q

Diamond to detect small (but physiologically relevant) changes in NP receptor phosphorylation.52

We previously showed that the membrane cGMP pool produced by pGC is under the exclusive control of PDE2,16 a PDE isoform mainly located near the plasma membrane.53,54

However, this does not exclude the possibility that spillover cGMP generated at the membrane diffuses to the cytosol where it becomes hydrolyzed by PDE5, located preferentially in Z-bands.42,43 Whether PKG is active or not must impact on the distribution of cGMP, with a strong cGMP gradient from membrane to cytosol when PKG is active, and a more uniform distribution of the nucleotide when PKG is blocked. Our ongoing efforts aim at addressing this question using single-cell fluorescence imaging of ARVMs expressing a

FRET-based cGMP fluorescent probe, such as Cygnet55 or cGES-DE2.56

In conclusion, PKG activation in adult cardiomyocytes limits the accumulation of cGMP induced by NO-donors via PDE5 stimulation, but increases that induced by NPs (Figure 8).

These findings support the paradigm that cGMP is not uniformly distributed in the cytosol, and identifies PKG as a key component in this process. Distinct spatiotemporal dynamics of the nucleotide may generate distinct functional responses upon activation of soluble vs. particulate guanylate cyclases. Clinically, NPs mediate natriuresis, inhibition of renin and aldosterone, as well as vasorelaxant, anti-fibrotic, anti-hypertrophic, and lusitropic effects, all of which are mediated by the cGMP/PKG signaling cascade.57 The NP system thus serves as Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 15 an important compensatory mechanism against neurohumoral activation in heart failure.

Despite an increase in the circulating level of NPs in heart failure, their beneficial effects are blunted by progressive receptor desensitization and pGC down regulation.58,59 Our results indicate that PKG may serve not only to activate downstream effectors that regulate cardiac contractility, but also to limit the desensitization process and increase the therapeutic utility of

NPs in heart failure.

Acknowledgements

We thank Florence Lefebvre and Patrick Lechêne for excellent technical assistance. We are grateful to Valérie Nicolas, Plateforme Imagerie Cellulaire IFR141, for confocal analysis, to

Valérie Domergue-Dupont and the animal core facility of IFR141 for efficient handling and preparation of the animals, to Dr Dermot Cooper (University of Cambridge, UK) for the adenovirus encoding CNGA2, and to Dr Albert Smolenski (University of Frankfurt,

Germany) for the adenovirus encoding the PKG+ mutant.

Sources of Funding

L.R.V.C. was a recipient of doctoral of a grant from “Fundação para a Ciência e Tecnologia”

(Portugal). This work was supported by grants from the Fondation Leducq 06CVD02 cycAMP, EU contract LSHM-CT-2005-018833/EUGeneHeart and a grant from Inserm

“Programme National de Recherche sur les Maladies Cardiovasculaires”.

Disclosures

None.

Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 16

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1999;45:H1935-H1942. Figure 1. Negative feedback of PKG on sGC-cGMP signaling. A, Time course of CNGA2 current (ICNG) amplitude at -50 mV in an Ad-CNGA2 infected myocyte. The cell was superfused with control external Ringer solution and challenged with different drugs during the periods indicated by the solid lines. Activation of sGC was achieved by the NO donor SNAP (100 µmol/L) alone or in the presence of the PKG inhibitor KT5823 (KT, 50 nmol/L). At the end, the cell was challenged with Sp-8 (100 µmol/L), a cGMP analog. B, Summary of the results of several similar experiments as in A, including the results obtained with the PKG inhibitor, DT-2 (2 µmol/L). The bars show the mean ± S.E.M.. Statistical significance is indicated as:*, p<0.05 vs. basal; ***, p<0.005 vs. basal; ###, p<0.005 vs SNAP. A Sp-8 5 (100 µmol/L)

3 SNAP 50 mV (nA) (nA) mV 50 - 2 SNAP KT5823 at at (100 µmol/L) (100 nmol/L)

CNG I

0 5 10 15 20 25 30 35 40 45 Time (min) B *** 26 8 24

22 ###

20 ### 6 7 8 4 * 14 2 8 12 0 KT DT2 SNAP SNAP SNAP Sp-8 +KT +DT-2

Figure 1 Figure 2. Role of PDE activity in the negative feedback of PKG on sGC-cGMP signaling. A,

Time course of ICNG amplitude in an Ad-CNGA2 infected myocyte. The cell was superfused with control external Ringer solution and challenged with different drugs during the periods indicated by the solid lines. Activation of sGC was achieved by the NO donor SNAP (100 µmol/L) in the presence of 100 µmol/L IBMX, a broad spectrum PDE inhibitor. The PKG inhibitor KT5823 (KT, 50 nmol/L) had no effect. At the end, the cell was challenged with Sp-8 (100 µmol/L) as an internal control for WT-CNGA2 channel expression. B, Summary of the results of several similar experiments as in A. The bars show the mean  S.E.M.. Statistical significance is indicated as:*, p<0.05 vs. basal; ***, p<0.005 vs. basal; non significant, ns. A 7 Sp-8 (100 µmol/L) 6

5 KT5823 (50 nmol/L)

4 SNAP (100 µmol/L) 50 mV (nA) mV 50

- 3 IBMX (100 µmol/L) at at

CNG I 1

0 0 5 10 15 20 25 30 35 Time (min) B 30 + KT (50 nmol/L) ***12 25 ns 20 8 8 15

5 *7 13 0 IBMX SNAP SNAP Sp-8 + IBMX

Figure 2 Figure 3. Role of PDE5 activity in the negative feedback of PKG on sGC-cGMP signaling. A,

Time course of ICNG amplitude in an Ad-CNGA2 infected myocyte. Activation of sGC was achieved by the NO donor SNAP (100 µmol/L) in the presence of either 100 nmol/L sildenafil, a PDE5 inhibitor, or sildenafil + KT5823 (50 nmol/L). At the end, the cell was challenged with Sp- 8 (100 µmol/L) as an internal control for WT-CNGA2 channel expression. B, Summary of the results of several similar experiments as in A. The bars show the mean  S.E.M.. Statistical significance is indicated as:*, p<0.05 vs. basal; ***, p<0.005 vs. basal; non significant, ns. A 5 Sp-8 (100 µmol/L) SNAP Sildenafil 4 SNAP Sildenafil (100 nmol/L) KT5823

3 (50 nmol/L) 50 mV (nA) mV 50 - 2 at at SNAP

(100 µmol/L) CNG

I 1

0 0 10 20 30 40 50 60 Time (min) B *** 25 + KT (50 nmol/L) 17

20 ns 15 9 8 10

5 11* 3 0 Sildenafil SNAP SNAP Sp-8 +Sildenafil

Figure 3 Figure 4. PKG inhibition increases cellular sGC-cGMP content via PDE5 inhibition. A, Radioimmunoassay (RIA) measurements of cellular cGMP content. The cells were stimulated (5 min) with SNAP (100 µmol/L) alone or in the presence of KT5823 (KT, 50 nmol/L), IBMX (100 µmol/L), or both, which were applied to the cells 10 min before SNAP stimulation. The bars show the mean  s.e.m. (n=6). Statistical significance is indicated as: **, p<0.01 vs. basal; #, p<0.05 vs. SNAP; non significant, ns. B, PDE5 activity was measured in freshly isolated ARVMs. PDE5 activity was determined as the fraction of cGMP-PDE activity inhibited by 100 nmol/L sildenafil, at a cGMP concentration of 1 µmol/L, in control cells or in cells exposed to SNAP (100 µmol/L) and/or DT-2 (2 µmol/L). The bars show the mean  s.e.m. of 3 to 4 experiments, each measure obtained in duplicate. Statistical significance is indicated as: *, p<0.05 vs. basal; #, p<0.05 vs. SNAP alone; non significant, ns. A B # 500 ns 3.0 + KT + DT-2 400

cells) 2.5 # 4 * 300 2.0 ns 1.5 200 **

1.0 PDE5 activity PDE5 100 (pmol/min/mg)

0.5 cGMP (fmol/5x10 cGMP 0 0 Basal IBMX SNAP IBMX control SNAP + SNAP

Figure 4 Figure 5. Positive feedback of PKG on pGC-cGMP signaling. A, Time course of ICNG amplitude in an Ad-CNGA2 infected myocyte. The cell was superfused with control external Ringer solution and challenged with different drugs during the periods indicated by the solid lines. Activation of pGC was achieved by the natriuretic peptide ANP (100 nmol/L) alone or in the presence of KT5823 (50 nmol/L). At the end of the experiment the cell was challenged with Sp-8 (100 µmol/L) as an internal control for WT-CNGA2 channel expression. B, Summary of the results of several similar experiments as in A, including the results obtained with another PKG inhibitor DT-2 (2 µmol/L). Statistical significance is indicated as: #, p<0.05 vs. ANP; ###, p<0.005 vs. ANP; ***, p<0.005 vs. basal. A 6 Sp-8 (100 µmol/L)

5 ANP (100 nmol/L) 4 KT5823 (50 nmol/L)

3 50 mV (nA) mV 50

- ANP

at at 2 CNG I 1

0 10 20 30 40 50 Time (min) B 30 *** ### 18 25 + KT (50 nmol/L) + DT-2 (2 µmol/L) ### 20 8 15 # *** 8 10 8 6 7 5 12 12 0 KT DT -2 ANP ANP Sp-8 (10 nmol/L) (100 nmol/L)

Figure 5 Figure 6. Role of PDE activity in the positive feedback of PKG on pGC-cGMP signaling. A,

Time course of ICNG amplitude in an Ad-CNGA2 infected myocyte. The effect of ANP (100 nmol/L) was tested in the presence of IBMX (100 µmol/L) and KT5823 (50 nmol/L) was further applied. At the end of the experiment the cell was challenged with Sp-8 (100 µmol/L) as an internal control for WT-CNGA2 channel expression. B, Summary of the results of several similar experiments as in A. Statistical significance is indicated as: ###, p<0.005 vs. ANP; ***, p<0.005 vs. basal. A 6 IBMX (100 µmol/L) Sp-8 (100 µmol/L)

5 ANP (100 nmol/L) 4

50 mV (nA) mV 50 -

at at 2 KT5823

(50 nmol/L) CNG

I 1

0 5 10 15 20 25 30 35 Time (min) B

30 + KT (50 nmol/L) ***20 ### 25 9 20

15 9 10 ***7

5 10 0 IBMX ANP ANP+IBMX Sp-8

Figure 6 Figure 7. PKG increases pGC-cGMP signaling. A, Immunocytochemical detection of a recombinant PKGIβ mutant. Myocytes were either not infected (a, b) or infected with an adenovirus encoding a constitutively active PKGI-β mutant (c), truncated at the N-terminus (PKGI-∆N1-92, PKG+). All cells but (a) were incubated with anti-PKG antibody after 24h of culture, and all cells were labeled with secondary antibody. Images were produced using a laser scanning confocal microscope. Each image represents one optical slice of a stack of images collected every 0.5 µm. B, Effect of ANP (10 or 100 nmol/L) and Sp-8 in CNGA2 infected myocytes co-expressing the active (PKG+) PKG mutant. The bars show the mean  S.E.M. of the number of cells indicated. Statistical significance is indicated as: #, p<0.05 vs. ANP alone; ***, p<0.005 vs. basal. C, pGC activity was measured in a crude membrane fraction prepared from freshly isolated ARVMs. Cyclic GMP concentration was measured by ELISA either in control membranes, or in membranes exposed during 30 min to recombinant PKGI-α, in the absence or presence of ANP (10 or 100 nmol/L) added during the last 15 min. The bars show the mean  s.e.m. of 3 to 6 experiments, each measure obtained in duplicate. Statistical significance is indicated as: *, p<0.05 vs. control; #, p<0.05 vs. ANP alone; ##, p<0.01 vs. ANP alone; non significant, ns. A a B

*** 30 + + PKG 13 *** 25 11 b 20 #

15 *** 10 *** 10 8 6 c

5 + KT + 0 ANP Sp-8 (10 nmol/L) (100 µmol/L)

C # 1.6 + PKGI-α 1.4 1.2 * 1.0 ##

0.8 * ns

0.6 pGC activity pGC

0.4 (pmol cGMP/min/mg) (pmol 0.2

0 control ANP ANP (10 nM) (100 nM)

Figure 7 Figure 8. PDE- and PKG-dependent cGMP compartmentation. The diagram represents the sarcolemmal membrane, with CNG channels and NP receptor type A inserted. Activation of the receptor by ANP or BNP leads to pGC activity and cGMP production. cGMP binds to CNG channel to produce a ICNG current. Cyclic GMP also activates PKG which exerts a positive feedback on the NP receptor or pGC function to increase production of cGMP. A diffusion barrier mediated by PDE2 limits the diffusion of cGMP, so that not all CNG channels are being activated. Activation of sGC by NO in the cytosol leads to cGMP generation in a different compartment. Cyclic GMP hydrolysis in this compartment is mainly due to PDE5. PKG activation by this pool of cGMP phosphorylates PDE5 to increase its activity and further limit the diffusion of cGMP. ICNG out ANP, BNP

CNG

CNG CNG in + pGC PKG GTP PDE2 cGMP

NO P

P P

PDE5 P sGC P

GTP P P cGMP

P P PKG

Figure 8 Castro et al. PKG controls cGMP compartmentation in cardiac myocytes 27

What Is Known?  In the heart, cyclic GMP (cGMP) is synthesized by two different guanylyl cyclases (GCs), a particulate one (pGC) activated by natriuretic peptides and a soluble one (sGC) activated by nitric oxide (NO), and mediates its physiological effects by activation of cGMP- dependent protein kinase (PKG).  cGMP is not uniformly distributed inside the cytosol of cardiac myocytes, but accumulates in different subcellular compartments upon activation of pGC or sGC.  This cGMP compartmentation is largely mediated by cGMP hydrolysis via two different phosphodiesterases (PDEs), the cGMP-specific PDE5 controlling the sGC pool, and the cGMP-stimulated PDE2 controlling the pGC pool.

What New Information Does This Article Contribute?  In adult rat cardiomyocytes, PKG exerts opposite effects on the accumulation of cGMP generated by sGC and pGC.  PKG decreases the accumulation of cGMP generated by sGC by activation of PDE5.  PKG increases the accumulation of cGMP generated by pGC by stimulating pGC activity.  In the presence of a NO donor, inhibition of PKG increases cGMP level to the same extent as sildenafil, a PDE5 inhibitor.

Cyclic GMP and its primary effector PKG regulate many proteins involved in blood pressure homeostasis and cardiac contraction. Although both pGC and sGC synthesize cGMP, there is ample evidence that the outcome of the cGMP produced differs. Previous work from this laboratory suggested that this is due to intracellular cGMP compartmentation, a process that involves the activity of PDE2 and PDE5. This study was designed to determine whether PKG exerts a feedback control on cGMP level. We show that PKG limits the accumulation and diffusion of cGMP in the ‘soluble’ pool, by activating its hydrolysis via PDE5. Hence, blocking PKG increases cGMP concentration to a similar extent as sildenafil ((Viagra ®), a PDE5 inhibitor. On the contrary, PKG increases cGMP accumulation in the ‘particulate’ pool, by increasing cGMP synthesis via pGC. These findings support the paradigm that cGMP is not uniformly distributed in the cytosol, and identifies PKG as a key component in this process. The opposite effect of PKG on cGMP produced by sGC and pGC may help understanding the differential regulation of heart function by natriuretic peptides and NO- donors.

Castro et al. SUPPLEMENT MATERIAL 1

ONLINE DATA SUPPLEMENT

Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of cGMP in rat cardiac myocytes

Liliana R. V. Castro1,2, Julia Schittl1,2 & Rodolphe Fischmeister1,2

1INSERM, UMR-S 769, Châtenay-Malabry, France; 2Univ Paris-Sud 11, Faculté de Pharmacie, IFR141, Châtenay-Malabry, France;

Running title: PKG controls cGMP compartmentation

Correspondence to: Rodolphe FISCHMEISTER INSERM U769 Faculté de Pharmacie 5, Rue J.-B. Clément F-92296 Châtenay-Malabry Cedex France Tel. +33-1-46 83 57 57 Fax +33-1-46 83 54 75 E-mail: [email protected] Castro et al. SUPPLEMENT MATERIAL 2

Materials and Methods

Isolation, culture and infection of rat ventricular myocytes

Ventricular myocytes from adult male Wistar rats (160-180 g) were obtained using an enzymatic technique as previously described.1 Freshly isolated cells were suspended in minimal essential medium (MEM: M4780; Sigma) containing 1.2 mM Ca2+, 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillin-streptomycin and 2%

HEPES (pH 7.6) and plated on laminin-coated culture dishes (10 µg/ml, 2h) at a density of

104 cells per dish. After one hour of plating, the medium was replaced by 200 µl of FBS-free

MEM containing WT CNGA2 encoding adenovirus (Ad-CNGA2), used at a multiplicity of infection (MOI) of 3000 plaque forming units per cell (pfu/cell). In co-infection experiments the Ad-CNGA2 (MOI 2000) was used in the presence of another adenoviral vector (MOI

1000) containing a constitutively active PKGI-β mutant, truncated at the N-terminus (PKGI-

∆N1-92, PKG+, generous gift from Dr Albert Smolenski, Frankfurt, Germany).2 After infection, the cells were placed overnight in an incubator.

Electrophysiological experiments

The whole cell configuration of the patch-clamp technique was used to record the CNGA2 current (ICNG). The cells were maintained at 0 mV holding potential and routinely hyperpolarized every 8 s to -50 mV test potential during 200 ms. ICNG was recorded in the absence of divalent cations in the extracellular solution (see below) allowing monovalent cations to flow through the channels in a non specific manner.3 Currents were not compensated for capacitance and leak currents. All experiments were done at room temperature.

Castro et al. SUPPLEMENT MATERIAL 3 cGMP measurements by radioimmunoassay

ARVMs attached onto coverslips were incubated for 5 min in the presence of different stimuli. After stimulation, the cells were disrupted with HClO4 at 4ºC. The acidic supernatants were recovered and neutralized with KOH solution. The samples were centrifuged for 1 min at 10,000 g and the supernatants were collected. Increase in sensitivity was achieved by succinylation of the samples with succinic anhydride and the cGMP levels were quantified in cell extracts by radioimmunoassay (RIA) using the commercially available

Kit (Beckman-Coulter, Immunotech SA, Marseille, France). The results were expressed in fmoles per 5×104 cells.

PDE Assay

Freshly isolated ARVMs were seeded at a density of 250,000 cells per dish. After 24h the cells were washed with PBS and exposed to either control solution or SNAP (100 µmol/L) for 15 min at 37°C in the absence or presence of 2 µmol/L DT-2 (added 15 min prior to

SNAP application). After stimulation, the cells were homogenized in ice-cold buffer containing (in mmol/L): NaCl 150, Hepes 20 (pH 7.4), EDTA 2, Glycerol 10%, NP40 0.5%, microcystin-LR 1 µmol/L and Roche complete protease inhibitors (Roche Diagnostics, Basel;

Switzerland). Cell lysates were centrifuged at 13,000 rpm at 4°C for 10 min and protein concentrations in the supernatants were determined by bicinchonic acid assay. Seventy-five micrograms of protein was used for the PDE assay.

Cyclic GMP-PDE activity was measured according to the method of Thompson and

Appleman4 as described in detail previously.5 In brief, samples were assayed in a reaction mixture of 200 µl containing 30 mmol/L Tris-HCl (pH 8.0), 10 mmol/L MgCl2, 5 mmol/L β- mercaptoethanol, 1 µmol/L cGMP, 1 mg/mL bovine serum albumin and 0.17 μCi of Castro et al. SUPPLEMENT MATERIAL 4

[3H]cGMP for 25 min at 33°C. The reaction was terminated by adding 200 μL of 10 mmol/L

EDTA in 40 mmol/L Tris-HCl (pH 7.4) followed by heat inactivation in a boiling water bath for 1 min. The PDE reaction product 5′-GMP was then hydrolyzed with 50 μg of Crotalus atrox snake venom (Sigma) for 20 min at 33°C, and the resulting guanosine was separated by anion exchange chromatography using 1 ml of AG1-X8 resin (BioRad) and quantitated by scintillation counting. PDE5 activity was determined as the fraction of cGMP-PDE activity inhibited by 100 nmol/L sildenafil.

Preparation of membrane fraction

Freshly isolated ARVMs were seeded at a density of 500,000 cells per dish. After 24h, the cells were homogenized in ice-cold buffer containing 10 mmol/L Tris-HCl (pH 7.4) and 5 mmol/L EDTA. The cell lysate was then centrifuged at 20,000 g for 20 min at 4°C. The supernatant was removed, and the pellet was resuspended in 1 mL ice-cold buffer A composed of (in mmol/L): Hepes 30 (pH 7.4), MgCl2 5, EGTA 1, NaCl 5, creatine phosphate

10, GTP 1, ATP 1, IBMX 1 and Roche complete protease inhibitors. Total protein concentration was determined by bicinchonic acid assay, normalized to 0.5 mg/mL and then used for guanylyl cyclase determinations without freezing.

Guanylyl cyclase assay

Membrane fractions containing 100 µg protein were first centrifuged at 20,000 g for 15 min at 4°C and then assayed for guanylyl cyclase activity by resuspension in buffer A (for basal determination) or in buffer A containing either ANP (10 or 100 nmol/L), recombinant PKG type Iα isoform (0.1 or 0.2 µg/mg protein, Biaffin GmbH & Co, Kassel, Germany), or

ANP+PKG. Membranes were assayed for 15 min at 37°C. The reaction was stopped by Castro et al. SUPPLEMENT MATERIAL 5 addition of 20 µL ice-cold 1 mol/L HCl. One tenth of the reaction was then removed and assayed for cGMP concentrations by direct cGMP ELISA kit from New East Biosciences

(Malvern, Pennsylvania, USA).

Solutions and Drugs

Control zero Ca2+/Mg2+ extracellular Cs+-Ringer solution contained (in mM): HEPES 10,

NaCl 107.1, CsCl 20, NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, adjusted to pH 7.4 with NaOH. Control and drug-containing solutions were applied to the exterior of the cell by placing the cell at the opening of a 250 µm (inner diameter) capillary tube from which the solutions were flowing at a rate of about 50 µl/min. Patch electrodes (0.7-1.2 MΩ) were made of soft glass (Drummond, Broomall, PA, USA) and filled with control internal solution containing (in mM): HEPES 10, CsCl 118, EGTA 5, MgCl2 4, sodium phosphocreatine 5,

Na2ATP 3.1, Na2GTP 0.42, CaCl2 0.0062 (pCa 8.5), adjusted to pH 7.3 with CsOH. S- nitroso-N-acetyl-penicillamine (SNAP) was from Tocris-Cookson (Bristol, UK); 8-(4- chlorophenylthio) guanosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8) and DT-2 were from Biolog L.S.I. (Bremen, Germany). Sildenafil was a generous gift from Dr Claire

Lugnier (Strasbourg, France); all other drugs were from Sigma-Aldrich (Saint Quentin

Fallavier, France).

Immunocytochemistry

Cardiomyocytes attached onto coverslips were rinsed with phosphate-buffered saline solution

(PBS), fixed in paraformaldehyde 4% (5 min) and washed in PBS. The cells were then permeabilized in Triton X-100 0.5% (15 min), washed in PBS-BSA 1% and incubated with antibody against PKG (dilution 1:800, 1h, 37ºC). This antibody was a generous gift from Dr Castro et al. SUPPLEMENT MATERIAL 6

Albert Smolenski (Germany). After three additional washes in PBS-BSA 1%, the cells were revealed with Alexa fluor 488 anti-rabbit antibody (30 min, 37ºC) and washed in PBS. Next, the coverslips were mounted in Mowiol antifadent mounting medium (France Biochem) and examined under confocal scanning laser microscope. Optical section series were obtained with a Plan Apochromat ×63 objective (NA 1.4 oil immersion). The fluorescence was observed with a LP 505-nm emission filter under 488-nm laser illumination.

Data Analysis

ICNG amplitude is time-independent and was measured at the end of the 200 ms pulse. In a total of 90 cells, mean membrane capacitance (Cm) was 147.7±2.9 pF. ICNG density (dICNG) which represents the ratio of current amplitude to Cm was on average 1.78±0.09 pA/pF. In each experimental condition, the response of dICNG to a drug was expressed relative to the

‘basal current’ obtained in control extracellular solution following the relation:

‘response’=(ICNG-‘basal current’)/Cm. All the data are expressed as mean ± S.E.M.. When appropriate, the Student’s t-test was used for statistical evaluation of ICNG variation induced by the different drugs and a p value <0.05 was considered statistically significant.

References

1. Verde I, Vandecasteele G, Lezoualc'h F, Fischmeister R. Characterization of the cyclic

nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+

current in rat ventricular myocytes. Br J Pharmacol. 1999;127:65-74.

2. Fiedler B, Feil R, Hofmann F, Willenbockel C, Drexler H, Smolenski A, Lohmann SM,

Wollert KC. cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated

protein kinase apoptosis signaling in cardiac myocytes. J Biol Chem. 2006;281:32831-40. Castro et al. SUPPLEMENT MATERIAL 7

3. Castro LRV, Verde I, Cooper DMF, Fischmeister R. Cyclic guanosine monophosphate

compartmentation in rat cardiac myocytes. Circulation. 2006;113:2221-2228.

4. Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase activities

from rat brain. Biochemistry. 1971;10:311-316.

5. Richter W, Conti M. Dimerization of the type 4 cAMP-specific phosphodiesterases is

mediated by the upstream conserved regions (UCRs). J Biol Chem. 2002;277:40212-

40221.