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Cardiovascular Research Advance Access originally published online on November 21, 2008
Cardiovascular Research 2009 81(2):294-300; doi:10.1093/cvr/cvn320
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

ERM proteins mediate the effects of Na+/H+ exchanger (NHE1) activation in cardiac myocytes

Amaria Darmellah, Catherine Rücker-Martin and Danielle Feuvray*

University of Paris-Sud 11 and CNRS UMR 8162, Marie Lannelongue Hospital, 133 avenue de la Résistance, 92350 Le Plessis Robinson, France

* Corresponding author. Tel: +33 1 40 94 25 21; fax: +33 1 40 94 25 22. E-mail address: danielle.feuvray{at}u-psud.fr

Received 19 March 2008; revised 6 November 2008; accepted 18 November 2008

Time for primary review: 36 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Ezrin, radixin, and moesin (ERM) proteins have been implicated in regulating signalling molecules. The aim of the present study was to investigate the activity and subcellular distribution of ERM proteins in cardiac myocytes from both Wistar and diabetic Goto-Kakizaki (GK) rats, and the role of these proteins in mediating the downstream effects of the cardiac sarcolemmal Na+/H+ exchanger (NHE1) activation in response to cell acidification.

Methods and results: Immunofluorescence microscopy revealed that activated ERM proteins were localized predominantly at the intercalated disc regions in left ventricular (LV) myocytes of both Wistar and GK rats under basal conditions. After acid loading, profound changes in activated ERM distribution were observed in both groups of myocytes, with immunolabelling detected in regions corresponding to the transverse tubules. This correlated with a marked increase in phospho-ERM levels in both groups, which was higher in GK myocytes and blocked by NHE1 inhibitor treatment. Levels of phospho-Akt paralleled those of phospho-ERM under the various experimental conditions used; in particular, the marked acid-induced increase in both phospho-ERM and phospho-Akt in GK myocytes was abolished by an NHE1 inhibitor treatment. Moreover, the pattern of glycogen synthase kinase-3β (GSK-3β) phosphorylation in these myocytes was strikingly similar to that observed for Akt activity under the conditions used.

Conclusion: Activated ERM proteins mediate the effects of acid-induced NHE1 activation in LV myocytes. Akt is a downstream effector in the cascade activated by NHE1–ERM interaction. In addition, GSK-3β phosphorylation is required for downstream effects of NHE1/ERM-Akt signalling.

KEYWORDS Cardiac myocytes; ERM; NHE1; Akt; GSK-3β; Intracellular acidification; Hypertrophy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Previous studies suggest that cardiac sarcolemmal Na+/H+ exchanger (NHE1) activity plays a causal or permissive role in the development of cardiac hypertrophy.1–4 We recently demonstrated that left ventricular (LV) myocytes from the Goto-Kakizaki (GK) rat model of type 2 diabetes are exquisitely sensitive to a small decrease in intracellular pH (pHi = 6.8–6.9) such as that observed during mild ischaemia, exhibiting a marked increase in NHE1 activity.5 Elevated NHE1 activity was associated with activation of the Akt pathway and coupled with the development of LV myocyte hypertrophy.5 Akt activation and myocyte hypertrophy were both blocked by chronic administration of an NHE1 inhibitor, suggesting a major role for the Akt pathway in mediating the hypertrophic effect of increased NHE1 activity. However, molecular links between the ion exchanger and Akt activity in cardiac myocytes remain unknown.

Ezrin, radixin, and moesin (ERM) proteins are closely related proteins originally identified as cytoskeleton cross-linkers and important components of cell structure.6 Inactive ERM proteins are retained in the cytoplasm in a closed conformation that masks transmembrane protein-binding sites in their N-terminal (FERM) domain and F-actin-binding sites in the C-terminal region.7 Thus, the intramolecular interaction must be disrupted to allow these proteins to bind to their partners. The mechanism of activation (i.e. open conformation) requires the binding of ERM proteins to phosphatidylinositol 4,5-biphosphate (PIP2) followed by phosphorylation at conserved C-terminal threonine residues.8,9 Active ERM proteins assemble and integrate signalling molecules to exert diverse downstream effects.10–12 In this respect, a direct binding of ERM proteins with NHE1 has been observed in fibroblasts.13 In epithelial cells, NHE1 is primarily present at the basolateral surface14–16 and its association with active ERM proteins regulate Akt-dependent cell survival.12 To the best of our knowledge, the subcellular distribution of ERM proteins in cardiac myocytes is not known. As to the transmembrane protein NHE1, it was shown to be localized predominantly in the intercalated disc regions of cardiac myocytes in ventricular sections of normal rat hearts.17 NHE1 immunolabelling was also observed, although less intense, in the transverse tubular system of cardiac myocytes in that study.17 If the association between NHE1 and active ERM proteins does occur in cardiac myocytes, active ERM (i.e. phosphorylated) proteins should be present at the same specific sites of the sarcolemmal membrane as NHE1. Therefore, our first aim in this study was to investigate ERM protein activity and localization in cardiac myocytes under basal conditions with low activity of NHE1. Secondly, we examined the hypothesis that ERM protein activity and distribution are affected under intracellular acidification, which is the primary trigger for NHE1 activation. In addition, we explored the signalling cascade downstream from NHE1/ERM activation likely to be involved in the development of LV myocyte hypertrophy. This study was carried out in LV myocytes from adult Wistar and GK rats, the latter being highly sensitive to mild acidification,5 as mentioned above.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animals
Male diabetic GK rats were obtained from the Paris colony.18,19 Age-matched Wistar rats were used as control animals. Wistar and GK rats were assigned to two groups: animals were fed with a standard diet (untreated Wistar and GK, both n = 22) or a diet containing 6000 parts per million of the selective NHE1 inhibitor cariporide (Aventis Pharma, Frankfurt, Germany) beginning at 4 weeks of age for 10–12 weeks (both n = 20).2,5 Animal characteristics have been described previously.5 In particular, the hypertrophic phenotype in diabetic GK rat hearts was determined at ventricular level from the increased biventricular weight to tibial length ratio (by approximately 22%), and at LV myocyte level from the increase (approximately 50%) in whole-cell membrane capacitance. Animal care and procedures were in accordance with the guidelines formulated by the European convention for the protection of vertebrate animals used for experimental purposes, and institutional guidelines no. 86/609/CEE November 24, 1986.

2.2 Isolated myocytes
To assess cellular kinase activity, individual myocytes were isolated from the LV wall of perfused hearts. Rats were anaesthetized with thiopenthal sodium (5 mg/100 g body weight, i.p.). Hearts were quickly excised from the chest, cannulated via the aorta, and perfused at 37°C, at a rate of 5 mL/min, with a Hanks–Hepes buffer solution. After 5 min of washing, enzymatic digestion was achieved by recirculating fresh buffer containing collagenase (0.28 mg/mL, Yakult, Tokyo, Japan) and protease type XIV (0.05 mg/mL, Sigma, St Louis, MO, USA) for 45–60 min. The heart was then removed from the perfusion apparatus and the LV was cut into small pieces. Individual cells were obtained by gentle agitation of the chunks in enzyme-free buffer. Rod-shaped LV myocytes were used on the day of isolation.5,20,21 NHE1 is nearly quiescent at resting pHi≥7.1; therefore we induced intracellular acidification to activate NHE1 by addition and then removal of NH4Cl (10 mmol/L) in bicarbonate-free HEPES-buffered Tyrode's solution.20 All the solutions had a pH of 7.4 at 37°C. Where indicated, Rho-kinase (ROCK) inhibitor II, Akt inhibitor API-2 (Merck Biosciences, Beeston/Nottingham, UK) or hydroxyfasudil monohydrochloride (Sigma) was added 20 min before the NH4Cl prepulse (all inhibitors dissolved in dimethyl sulphoxide, final concentration <0.1%, except for hydroxyfasudil monohydrochloride which was dissolved in H2O). All the experiments were performed at 36 ± 1°C.

2.3 Western blot analysis
Whole-cell lysates (20–40 µg protein) were obtained under basal pHi conditions and following intracellular acidification, as previously described.5 Lysates were resolved by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis and immunoblotted with specific antibodies to phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558), ezrin, phospho-Akt (Ser473), Akt, phospho-mTOR (mammalian target of rapamycin) (Ser2448), mTOR, phospho-p70S6K (Thr389, Thr421/424), p70S6K, phospho-GSK-3β (Ser9) (Cell Signaling Technology, Beverly, MA, USA), and GSK-3β (Santa Cruz Biotechnology, CA, USA) and secondary antibody goat anti-rabbit horseradish peroxidase conjugate (Amersham Biosciences, Freiburg, Germany). To quantify the extent of phosphorylation of each kinase, the phosphorylated band density was expressed relative to the intensity of the total protein band.

2.4 Immunolabelling
Freshly isolated myocytes, obtained under basal pHi conditions and following intracellular acidification,5 were fixed with paraformaldehyde for 10 min and treated as described.22 They were incubated overnight with antibody directed against phospho-ERM (pERM, 1:50 dilution) and then with anti-rabbit IgG coupled to Alexa Fluor 488 (green, 1:100 dilution; Invitrogen, Paisley, UK). Incubations involving both the antibody directed against RyR2 (1:50 dilution; Affinity BioReagents, Golden, CO, USA) and the antibody directed against pERM were done simultaneously overnight. Secondary antibodies were anti-mouse IgG coupled to Alexa Fluor 594 (red, 1:100 dilution) and anti-rabbit IgG coupled to Alexa Fluor 488 (green, 1:100 dilution), respectively. Incubation with primary antibodies was omitted for negative controls. Samples were analysed either using an ApoTome imaging system based on an Axio Observer microscope or by confocal scanning laser microscopy (LSM-510) (both systems from Carl Zeiss, Inc.) using X63 oil-immersion objectives (numerical aperture 1.4). Three-dimensional reconstruction images were obtained using the processing software AxioVision 4.6 (Carl Zeiss, Inc.).

2.5 Statistical analysis
Data are presented as means ± SEM. Results were analysed using Student's t-test or analysis of variance followed by the Tukey post hoc test to identify differences between groups. Values of P < 0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 ERM activation and subcellular localization in cardiac myocytes
Inactive, cytosolic ERM proteins require phosphorylation at conserved C-terminal threonine residues8 for unfolding, translocation to the plasma membrane, and cross-linking between integral membrane proteins and cytoskeleton.7 We used fluorescence microscopy to detect the immunocytochemical localization of phospho-ERM in single cardiac myocytes under basal conditions. Phospho-ERM signal was predominant and intense at the intercalated disc regions in both Wistar and GK LV myocytes (Figure 1A). Sparse labelling was occasionally detected along the lateral sarcolemma. However, phospho-ERM was detected at intercalated disc regions in approximately 99% and 98% of the myocytes isolated from Wistar and GK rat hearts, respectively (Figure 1C).


Figure 1
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  		Figure 1 (A) Images illustrating phospho-ERM immunolabelling (green) in left ventricular (LV) myocytes of Wistar (W) and Goto-Kakizaki (GK) rats under basal conditions (first row), and of W and GK rats 1 min after acidification (acid, second row). The right-hand column shows differential interference contrast views with superimposed phospho-ERM immunolabelling (green) in GK LV myocytes. (B) The left-hand image shows a high magnification view of dual labelling of phospho-ERM (green) and RyR2 (red) in a GK LV myocyte after acidification. The image corresponds to one optical section in the z-axis (1 µm beneath the membrane surface; thickness: 200 nm). Horizontal bars = 10 µm. On the right-hand side is a three-dimensional image of phospho-ERM (green) and RyR2 (red) immunolabelling in a GK LV myocyte after acidification. Arrows indicate the entrance of T-tubules at the membrane surface. (C) Bar graph gives the percentage of LV myocytes with specific phospho-ERM staining at the intercalated disc regions under basal conditions, and in regions corresponding to the transverse tubules 1 min after acidification, for Wistar (open bars) and GK myocytes (black bars). At least 70 myocytes were analysed in at least four hearts for each group. #P < 0.001 vs. Wistar after acidification, ***P < 0.001 and *P < 0.05 vs. untreated Wistar or GK. CARI, cariporide; Rho-kII, Rho-kinase-inhibitor II.

 
We then determined whether phospho-ERM distribution is affected by intracellular acidification. We found profound changes in phospho-ERM subcellular distribution in both groups of cells after acid loading (Figure 1A). There was clear dotted immunolabelling along the lateral sarcolemma. Furthermore, we observed strong dotted staining at regions probably corresponding to transverse tubules in the myocytes. These changes were more marked in GK LV myocytes than in Wistar LV myocytes, being detected in approximately 98% of GK cells and 60% of Wistar cells (Figure 1C). Figure 1C also shows that phospho-ERM immunolabelling near the T-tubules was present in a smaller number of cells acidified in the presence of the NHE1 inhibitor cariporide (1 µM). To establish phospho-ERM protein distribution at the transverse tubular system in acidified cells, we co-labelled the well-characterized ryanodine receptor 2 (RyR2), located in the vicinity of T-tubules. Dual-labelling experiments of activated ERM and RyR2 confirmed that activated ERM proteins following acidification are localized close to RyR2 (Figure 1B), but are not uniformly distributed inside the cells.

Therefore, we measured the levels of phospho-ERM proteins under basal conditions and after intracellular acidification. Cell lysates obtained under basal conditions and after acidification were assayed for the active ERM conformation by immunoblotting with anti-phospho-ERM antibody. Basal ERM phosphorylation was similar in the two groups of myocytes, and ERM phosphorylation was increased after induction of acidification in both Wistar and GK LV myocytes (Figure 2A). Although significant (P = 0.026), this increase was less marked in Wistar myocytes than in GK LV myocytes (P < 0.001 compared with basal level). Furthermore, phospho-ERM stimulation in acidified GK myocytes was blocked by NHE1 inhibitor treatment, suggesting a role for the exchanger in ERM activation. Several kinases phosphorylate ERM family proteins at threonine residues in the C-terminal domain, including ROCK.8 GK myocytes treated with ROCK inhibitor II (1 µM)23 during acidification had a lower level of ERM phosphorylation than that observed in these cells without ROCK inhibitor II. This difference in phosphorylation levels (approximately 26% decrease) was smaller than that observed in myocytes from GK rats treated with the selective NHE1 inhibitor (approximately 46% decrease) (Figure 2A). This is in agreement with the small reduction in the percentage of cells showing phospho-ERM immunolabelling near the T-tubules in the presence of ROCK inhibitor II (1 µM) (Figure 1C). Similar results were obtained in the presence of the ROCK inhibitor fasudil (30 µM, data not shown). These findings indicate that acidification stimulates ERM phosphorylation and leads to significant changes in the phospho-ERM distribution pattern in cardiac myocytes.


Figure 2
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  		Figure 2 Phosphorylation status for ERM (A) and Akt (B) under basal conditions and 1 min after acidification. Autoradiograms show representative western blots. Bar graphs show the ratio of phosphorylated to total protein for ERM (A) and Akt (B) in left ventricular (LV) myocytes from untreated Wistar rats (open bars) and from untreated Goto-Kakizaki (GK) rats (black bars) and cariporide-treated Wistar (hatched bars) and GK rats (grey bars). Data are from between six and nine experiments with cells from Wistar (n = 5 or 6) and GK hearts (n = 5 or 6) for each condition. *P < 0.05 vs. basal Wistar, #P < 0.001 vs. basal GK, §P < 0.05, §§§P < 0.001.

 
3.2 Signalling cascade downstream from NHE1/ERM activation
We recently showed that Akt is activated following a marked acid-induced increase in NHE1 activity.5 We determined whether or not this occurs through an ERM-dependent mechanism. Under experimental conditions similar to those in our previous study, Akt phosphorylation (Figure 2B) was increased in GK LV myocytes following acidification, and showed a tendency to increase in Wistar myocytes. Phospho-Akt stimulation in acidified GK myocytes was completely blocked by NHE1 inhibitor treatment. We observed a significant decrease in phospho-Akt when the ROCK inhibitor was applied to these acidified myocytes, although this decrease was smaller than that in acidified myocytes from GK rats treated with the NHEI inhibitor cariporide. To further clarify the role of the Akt pathway, we examined ERM phosphorylation in acidified GK LV myocytes exposed to the Akt inhibitor, API-2 (10 µM).5,24 The level of ERM phosphorylation was unchanged (Figure 2A), thus indicating that Akt is clearly downstream from ERM activation.

Akt phosphorylates multiple substrates, thereby regulating the apoptotic response12 as well as cell hypertrophy.25,26 Akt activation is coupled to the development of hypertrophy in GK LV myocytes.5 The regulation of cell size by Akt seems to be mediated by Akt phosphorylation and by subsequent downstream phosphorylation of glycogen synthase kinase-3β (GSK-3β)27,28 and the mTOR.29–31 The phosphorylation of mTOR leads to increased protein synthesis via phosphorylation of downstream targets such as the p70 ribosomal S6 protein kinase (p70S6K).30 We demonstrated that the levels of phosphorylation of mTOR and its downstream target p70S6K, which were the same under basal conditions in both Wistar and GK LV myocytes, remained unchanged after acidification in these two groups of myocytes (Figure 3A and B). These observations suggest that the mTOR/p70S6K pathway does not contribute to Akt-mediated hypertrophy in GK LV myocytes.


Figure 3
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  		Figure 3 Phosphorylation status for mammalian target of rapamycin (mTOR) (A), p70S6K (B), and glycogen synthase kinase-3β (GSK-3β) (C) under basal conditions and 1 min after acidification. Autoradiograms show representative western blots. Bar graphs show the ratio of phosphorylated to total protein for mTOR (A), p70S6K (B), and GSK-3β (C) in left ventricular (LV) myocytes from untreated Wistar (open bars) and Goto-Kakizaki (GK) rats (black bars) and from cariporide-treated GK rats (grey bars). Data are from between six and nine experiments with cells from Wistar (n = 5 or 6) and GK hearts (n = 5 or 6) for each condition. #P < 0.001 vs. all other groups.

 
In contrast, we detected an increase in GSK-3β phosphorylation in acidified GK LV myocytes, that paralleled the marked acid-induced increase in Akt activity (Figure 3C). Unlike most protein kinases, GSK-3β remains active in the resting state. Upon phosphorylation by upstream kinase such as Akt, it becomes inactive, thereby removing its negative constraints on hypertrophy; this represents a unique mechanism of cardiac hypertrophy.27,28 Phosho-GSK-3β levels were nearly doubled after acidification in GK LV myocytes. NHE1 inhibitor treatment reversed the change in phospho-GSK-3β activity (P < 0.001). Phospho-GSK-3β stimulation was similarly blunted in GK myocytes exposed to the Akt inhibitor, API-2, during acidification. The acid-induced increase in GSK-3β phosphorylation was also significantly reduced in GK myocytes treated with Rho-kinase-inhibitor II. Thus, NHE1–ERM signalling may be transmitted through Akt to the downstream effector GSK-3β.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
In this study we report: (i) The subcellular distribution of activated ERM proteins in cardiac LV myocytes; (ii) activated ERM subcellular distribution was markedly changed in cardiac myocytes following intracellular acidification; (iii) activated ERM proteins mediate the effects of acid-induced NHE1 activation; (iv) Akt is a downstream effector in the NHE1/ERM-mediated signalling cascade; (v) GSK-3β phosphorylation is a downstream effector of activated Akt signalling in LV myocytes of type 2 diabetic GK rat hearts.

Activated ERM proteins are predominantly located at the intercalated discs in cardiac LV myocytes under basal conditions. Phosphorylation of ERM proteins at the conserved threonine residue in the actin-binding site (T567 in ezrin, T564 in radixin, T558 in moesin) results in the localization of these proteins to actin-rich membrane regions,6,8,32 such as those represented by fasciae adhaerentes in the intercalated discs.33 Specific polyclonal antibodies directed against the Na+/H+ exchanger NHE1 in rat myocardium have previously revealed predominant localization of this protein at the intercalated disc regions.17 NHE1 has been shown to directly interact with phospho-ERM proteins in fibroblasts13 and more recently in a human renal epithelial cell line;12 thus, this direct interaction may also exist in cardiac LV myocytes. We also observed marked changes in the subcellular distribution of phospho-ERM in response to acidification in LV myocytes for both normal and diabetic rat hearts; in particular, immunostaining was detected in regions probably corresponding to transverse tubules. Although this staining was less intense than in the intercalated discs, NHE1 immunolabelling has previously been detected along the transverse tubular system in longitudinal sections of rat ventricles.17 Furthermore, we showed that acidification is associated with a specific phospho-ERM distribution pattern in LV myocytes of both Wistar and diabetic GK rat hearts; this pattern was observed in a greater number of GK rat cells than Wistar rat cells (Figure 1C). ERM distribution in acidified myocytes is most likely to be related to the degree of phosphorylation of ERM proteins which we found to be increased in both groups of myocytes, with a greater increase observed in GK myocytes. Increased phospho-ERM in these myocytes was reversed to basal level by NHE1 inhibitor treatment. Furthermore, isolated myocytes exposed to the NHE1 inhibitor (1 µM) during acidification were less susceptible to changes in phospho-ERM distribution. Thus, it seems likely that activated NHE1 interacts with phosphorylated ERM proteins. Our findings are consistent with previous observations in renal tubular epithelial cell lines, demonstrating that NHE1 stimulation leads to ERM activation.12 In addition, these previous observations suggested involvement of a direct NHE1–ERM interaction. It remains to be determined whether acid-induced activated ERM at regions corresponding to the transverse tubules was derived from a pool of newly phosphorylated ERM or whether previously activated ERM was translocated from the intercalated disc regions. However, phospho-ERM content was significantly higher after acidification than at basal levels in both groups of myocytes; it therefore seems likely that ERM proteins were newly phosphorylated in these cells. Phospho-ERM content may also depend on the activity of several kinases such as ROCK.8,10 Indeed, ROCK activity has been used in previous studies to evaluate the extent of phosphorylation of ERM family proteins.34,35 We found that acidified GK LV myocytes exposed to the selective ROCK-inhibitor II had lower levels of phospho-ERM than acidified cells in the absence of this inhibitor. This effect, however, was less marked (by approximately 50%) than that observed with NHE1 inhibitor treatment, again highlighting the major role of NHE1 in ERM activation during cell acidification.

Our study also shows that phospho-Akt levels parallel those of phospho-ERM under the various experimental conditions used; in particular, the marked acid-induced increase in phospho-ERM and phospho-Akt levels was abolished by treatment with NHE1 inhibitor in GK myocytes. Our findings for LV myocytes are therefore consistent with a previous study by Wu et al.12 on renal cell lines, showing that activated NHE1 associates with ERM proteins, resulting in the activation of the kinase Akt. We previously demonstrated5 that marked Akt activation associated with enhanced NHE1 activity in GK LV myocytes is coupled with the development of hypertrophy. Although several studies have demonstrated that NHE1 plays a role in the signalling cascade leading to cardiac hypertrophy,1–4 the molecular events involved are poorly understood. Only one of these previous studies showed that Akt activity is decreased upon reversal of hypertrophy by NHE1 inhibitor treatment,4 consistent with our results.5 We found that the activity of mTOR, one of the possible pro-hypertrophic pathways downstream of Akt,36 remains unchanged in acidified GK LV myocytes when Akt is markedly activated. In contrast, we observed changes in GSK-3β phosphorylation that paralleled Akt activity, revealing increased phosphorylation when Akt activity was markedly stimulated or, conversely, reduced phosphorylation when Akt activity was reduced. GSK-3β is among the best characterized Akt substrate and much attention has been focussed on its role in cardiac myocyte hypertrophy.36 GSK-3β activity is inhibited upon phosphorylation by Akt. GSK-3β inhibits a key component of the protein translation machinery, as well as a number of transcription factors believed to be involved in the induction of the hypertrophic gene expression programme; thus, inhibition of GSK-3β promotes both protein synthesis and gene transcription.27,28,36,37

The hypertrophic effect of increased NHE1 activity was observed only in GK LV myocytes in our previous study and this increased activity was revealed at mildly acidic pHi values (pHi 6.8–6.9).5 Under basal conditions, NHE1 activity was indeed the same in both GK and Wistar myocytes when no change in steady-state pHi values was observed.5 Interestingly, phospho-ERM levels determined in the present work were similar in both groups of myocytes under basal conditions, whereas their increase following intracellular acidification paralleled that in NHE1 activity,5 the increase being less in Wistar myocytes compared with GK myocytes. This seems to indicate that a certain degree of NHE1 activity and of ERM activation is required in order to trigger the hypertrophic process. Our data may appear to be somewhat at variance with those obtained in the hypertrophic myocardium of spontaneously hypertensive rats (SHR) exhibiting increased Na+/H+ exchanger activity, in which basal pHi was reported to be higher than in normotensive rats.38 However, the pHi sensitivity of NHE1 is largely determined by inhibitory and excitatory phoshorylation,39 and the balance of these two factors may be somewhat different in SHR myocytes and in GK myocytes. It would therefore be important to explore the role of phospho-ERM in other models of cardiac hypertrophy in which the link between NHE1 activity and myocardial growth has been established.1–4,38 Although there was a tendency for an increase in phospho-Akt in acidified Wistar myocytes, it was less marked than the increase in phospho-ERM. The signal downstream from Akt was attenuated to an even greater extent: no change was detected in GSK-3β phosphorylation upon acidification. It is therefore possible that a certain degree of NHE1 activation has to be reached in cardiac myocytes to promote sufficient ERM phosphorylation and its associated changes in subcellular distribution, and to promote sufficient levels of downstream Akt activation and its inhibitory phosphorylation of GSK-3β. Alternatively, or in addition, the duration of the signal, i.e. a sustained increase in NHE1 activity, may also be an important determinant of ERM activation. GSK-3β phosphorylation may also be mediated via ERK1/2 signalling through p90RSKs,40 suggesting that other environmental factors may affect GSK-3β phosphorylation status. Indeed, we previously reported5 a less marked acid-induced increase in ERK phosphorylation in Wistar myocytes than in GK myocytes. Further work will be required to clarify this point.

This study is the first to demonstrate the subcellular distribution of phospho-ERM proteins in cardiac myocytes and the role of these proteins in mediating the downstream effects of NHE1 activation in response to cell acidification. Such changes in pHi resemble conditions associated with impaired myocardial perfusion.41 In addition, our findings provide further insight into mechanisms explaining the role of NHE1 activation in cardiac hypertrophy.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was in part supported by a grant from the Foundation of France (to D.F.). A.D. was supported by a fellowship from the French Ministry for Education and Research.


   Acknowledgements
 
We wish to thank Valérie Nicolas (The Cell Imaging facility of the Federative Research Institute 141, Châtenay-Malabry, France) for expert assistance with confocal microscopy. We also wish to thank Mrs C. Martens for critical reading of the manuscript.

Conflict of interest: none declared.


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

  1. Kusumoto K, Haist JV, Karmazyn M. Na+/H+ exchange inhibition reduces hypertrophy and heart failure after myocardial infarction in rats. Am J Physiol (2001) 280:H738–H745.[Web of Science]
  2. Engelhardt S, Hein L, Keller U, Klämbt K, Lohse MJ. Inhibition of Na+-H+ exchange prevents hypertrophy, fibrosis and heart failure in β1-adrenergic receptor transgenic mice. Circ Res (2002) 90:814–819.[Abstract/Free Full Text]
  3. Camilion de Hurtado MC, Portiansky EL, Perez NG, Rebolledo OR, Cingolani HE. Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na+/H+ exchanger. Cardiovasc Res (2002) 53:862–868.[Abstract/Free Full Text]
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  5. Darmellah A, Baetz D, Prunier F, Tamareille S, Rücker-Martin C, Feuvray D. Enhanced activity of the myocardial Na+/H+ exchanger contributes to left ventricular hypertrophy in the Goto-Kakizaki rat model of type 2 diabetes: critical role of Akt. Diabetologia (2007) 50:1335–1344.[CrossRef][Web of Science][Medline]
  6. Niggli V, Rossy JR. Ezrin/radixin/moesin: versatile controllers of signaling molecules and of the cortical skeleton. Int J Biochem Cell Biol (2008) 40:344–349.[CrossRef][Web of Science][Medline]
  7. Hamada K, Shimizu T, Matsui T, Tsukita S, Hakoshima T. Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J (2000) 19:4449–4462.[CrossRef][Web of Science][Medline]
  8. Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol (1998) 140:647–657.[Abstract/Free Full Text]
  9. Nakamura F, Huang L, Pestonjamasp K, Luna EJ, Furthmayr H. Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides. Mol Biol Cell (1999) 10:2669–2685.[Abstract/Free Full Text]
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