Published online before print September 5, 2008, doi: 10.1161/CIRCRESAHA.108.175141
© 2008 American Heart Association, Inc.
Integrative Physiology |
Activation of Na+/H+ Exchanger 1 Is Sufficient to Generate Ca2+ Signals That Induce Cardiac Hypertrophy and Heart Failure
From the Departments of Molecular Physiology (T.Y.N., Y.I., S.W.), Bioscience (Y.A.), and Cardiovascular Dynamics (K.K.), National Cardiovascular Center Research Institute, Osaka, Japan.
Correspondence to Shigeo Wakabayashi, Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan. E-mail wak{at}ri.ncvc.go.jp
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Abstract |
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Activation of the sarcolemmal Na+/H+ exchanger (NHE)1 is increasingly documented as a process involved in cardiac hypertrophy and heart failure. However, whether NHE1 activation alone is sufficient to induce such remodeling remains unknown. We generated transgenic mice that overexpress a human NHE1 with high activity in hearts. The hearts of these mice developed cardiac hypertrophy, contractile dysfunction, and heart failure. In isolated transgenic myocytes, intracellular pH was elevated in Hepes buffer but not in physiological bicarbonate buffer, yet intracellular Na+ concentrations were higher under both conditions. In addition, both diastolic and systolic Ca2+ levels were increased as a consequence of Na+-induced Ca2+ overload; this was accompanied by enhanced sarcoplasmic reticulum Ca2+ loading via Ca2+/calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force–frequency dependence was observed with preservation of high Ca2+, suggesting a decrease in myofibril Ca2+ sensitivity. Furthermore, the Ca2+-dependent prohypertrophic molecules calcineurin and CaMKII were highly activated in transgenic hearts. These effects observed in vivo and in vitro were largely prevented by the NHE1 inhibitor cariporide. Interestingly, overexpression of NHE1 in neonatal rat ventricular myocytes induced cariporide-sensitive nuclear translocation of NFAT (nuclear factor of activated T cells) and nuclear export of histone deacetylase 4, suggesting that increased Na+/H+ exchange activity can alter hypertrophy-associated gene expression. However, in transgenic myocytes, contrary to exclusive translocation of histone deacetylase 4, NFAT only partially translocated to nucleus, possibly because of marked activation of p38, a negative regulator of NFAT signaling. We conclude that activation of NHE1 is sufficient to initiate cardiac hypertrophy and heart failure mainly through activation of CaMKII–histone deacetylase pathway.
Key Words: Na+/H+ exchanger • Na+ and Ca2+ overload • cardiac remodeling • CaMKII-HDAC pathway • calcineurin–NFAT pathway
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Introduction |
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Intracellular Na+ levels are regulated by a network of ion channels and transporters.1 In the myocardium, Na+ homeostasis is closely linked to intracellular Ca2+ handling via the Na+/Ca2+ exchanger (NCX), the principal mechanism for Ca2+ efflux from cardiomyocytes. Na+ dysfunction alters Ca2+ homeostasis, thereby contributing to the pathogenesis of heart failure (HF) in animal models and in humans. The sarcolemmal Na+/H+ exchanger (NHE)1 is a major Na+ influx pathway that also serves as a powerful acid extrusion system. NHE1 couples H+ efflux to Na+ influx in a 1:1 stoichiometry under the driving force of a Na+ gradient formed by the Na+ pump. Thus, enhanced NHE activity leads to elevated intracellular Na+ concentration ([Na+]i) and cytoplasmic alkalinization. NHE1 activity is controlled by intracellular pH (pHi) and numerous other factors, such as hormones, catecholamines, and mechanical stimuli, known to be associated with a failing heart. NHE1 activity has been implicated in myocardial ischemia and reperfusion injury,2 and NHE1 inhibition was reported to protect against these injuries in animal models and in patients undergoing coronary interventions.3 However, in other cardiac disease states such as cardiac remodeling process, the role of NHE1 is not fully understood. It has been proposed that enhanced myocardial NHE1 activity is partially responsible for the cardiac remodeling observed in animal models such as guanylyl cyclase-A knockout (GC-A KO) mice,4 β1-adrenergic receptor–overexpressing transgenic (Tg) mice,5 and spontaneously hypertensive rats.6 However, many signal transduction pathways are activated in these models; therefore, whether activation of NHE1 alone is sufficient to induce hypertrophy remains unknown. We also lack detailed understanding of the molecular and cellular events, including altered intracellular Na+ and/or Ca2+ handling and the activation of signaling pathways that result from NHE1 activation.
To directly address these questions, we generated Tg mice that overexpress an activated form of human NHE1 that lacks the calmodulin-binding inhibitory domain (
637 to 656)7 under the control of cardiac 
-myosin heavy chain promoter. Previously, we reported that deletion of this domain resulted in constitutive elevation of pHi sensitivity in quiescent cells.7,8 Here, we asked whether overexpressing this mutant form of NHE1 would affect development of cardiac hypertrophy and/or HF in vivo and investigated which signal transduction pathways were involved in NHE1-dependent cardiac pathogenesis. We present evidence that NHE1 serves as an important signal mediator in initiating cardiac remodeling and HF via activation of a Ca2+-dependent hypertrophic pathway.
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Materials and Methods |
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All animal experiments were carried out according to guidelines of the Animal Welfare Committee of the National Cardiovascular Center Research Institute. Tg mice that overexpressed an activated form of NHE1 (human NHE1 with amino acids 637 to 656 deleted) were generated from C57BL/6J mice according to standard procedures; the expression of the transgene was under control of the murine cardiac
-myosin heavy chain promoter. The pHi, [Na+]i, and Ca2+ transients in isolated adult myocytes were measured in Hepes- or bicarbonate-buffered Tyrode solutions by epifluorescent analysis [acetoxymethyl forms of 2',7'-bis (carboxy-ethyl)-5- (and -6)-carboxyfluorescein (BCECF), sodium-binding benzofuran isophtalate (SBFI), and Indo-1, respectively] using an imaging system (AQUACOSMOS, Hamamatsu Photonics). Data are presented as means±SD or SEM of at least 3 determinations. We used the paired or unpaired t test for statistical analyses. Values of P<0.05 were considered statistically significant (indicated as * versus wild-type [WT] or 
versus no-cariporide control, or as otherwise indicated). An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. |
Results |
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Overexpression of Activated NHE1 Induces Cardiac Hypertrophy and HF In Vivo
We confirmed a marked increase (
5-fold) in NHE1 protein levels in Tg hearts by immunoblot analysis (Figure 1A). Immunofluorescence analysis of tissue sections revealed that whereas WT hearts expressed low but detectable levels of NHE1 localized to intercalated discs, Tg hearts expressed much higher levels of NHE1 in both intercalated discs and sarcolemma (Figure 1B).
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Overexpression of NHE1 in vivo resulted in cardiac hypertrophy and dilated cardiomyopathy; the Tg mouse hearts had thinner ventricular walls and greater chamber dilation accompanied by ventricular fibrosis (Figure 2A, supplemental Figure IB and the Table). We detected the onset of progressive hypertrophy in Tg hearts beginning at the age of 20 days (Figure 2B, a), with outstanding enlargement of the atria (supplemental Figure I A). Cardiomyocyte diameters in Tg mice were significantly larger than those in WT mice (Figure 2B, b). These results suggest that each myocyte became bigger at approximately 40 days of age, but they progressively died, as evidenced by chamber dilation. Echocardiography of Tg mice at 40 days of age showed increased diastolic and systolic ventricular diameters and decreased systolic function, which was demonstrated by a decrease in the percentage of fractional shortening (Figure 2C and the Table). Markers of HF, atrial natriuretic peptide, and cardiac troponin I in serum were also increased in Tg mice (supplemental Figure IC and ID), indicating that their hearts had developed HF. Tg mice had a high mortality rate (Figure 2D) and showed cardiac arrhythmia; this was particularly so for older mice (data not shown). However, all cardiac remodeling events observed in Tg mice were largely prevented by IP administration of cariporide for 20 days (from the age of 20 to 40 days; Figure 2, supplemental Figure I, and the Table) without significant change in NHE1 protein levels (Figure 1A), suggesting that these phenomena resulted from elevated NHE1 activity.
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Overexpression of NHE1 did not significantly change the expression levels of other Ca2+ regulatory proteins (NCX1, sarcoplasmic reticulum Ca2+-pump [SERCA2a], or calsequestrin [CsQ]) with or without cariporide treatment (Figure 1A). In addition, expression of Na+/K+-ATPase was also unchanged (WT, 0.36±0.04; Tg, 0.35±0.05; and Tg+cariporide, 0.40±0.07; n=3 hearts). As predicted from our previous work,9 we detected a marked increase in the expression of calcineurin homologous protein (CHP)1, an obligatory subunit of NHE family members (supplemental Figure IIA),10 concomitant with the high level of NHE1 expression in Tg mice.
Increased Exchange Activity and [Na+]i in Cardiomyocytes from Tg Mice
We measured pHi and [Na+]i in freshly isolated ventricular myocytes from WT and Tg hearts. Tg myocytes appeared to be more vulnerable to collagenase treatment and mechanical stress than WT myocytes. Surviving Tg myocytes were larger than WT myocytes (Figure 3A), similar to our observations in whole-heart sections (Figure 2B). In addition, the pHi recovery of Tg myocytes after acid loading that was cariporide-inhibitable was faster than that of WT myocytes (Figure 3B). Consistent with our previous data using this NHE1 mutant,7,8 the pHi dependence of the H+ efflux rate (JH) was alkaline-shifted in Tg myocytes (Figure 3C) after correction for the intracellular H+ buffering power, which was similar between WT and Tg myocytes (supplemental Figure III). In addition, the resting pHi was significantly higher in Tg myocytes under bicarbonate-free conditions (pHi 7.40 versus pH 7.23 in WT; Figure 3D), indicating that overexpressing the active NHE1 mutant led to elevated NHE activity. However, under bicarbonate-buffered conditions, the pHi was not significantly different between WT and Tg myocytes (Figure 3D, see also supplemental Figure IV), suggesting that pHi was compensated by bicarbonate-dependent mechanisms. According to the pH dependence of JH, we can assume that the steady-state exchange activity would be higher in Tg than WT myocytes at the given resting pHi under physiologically relevant bicarbonate conditions. Indeed, fluorescence measurements using the null-point method11 revealed that [Na+]i was almost 1.5-fold higher in Tg myocytes (17 to 21 mmol/L) than in WT myocytes (12 to 13 mmol/L) under both bicarbonate- and Hepes-buffered conditions (Figure 3E). Treatment with cariporide resulted in a rapid reduction of [Na+]i in Tg myocytes, which reached the value close to the WT myocytes (supplemental Figure V). Thus, elevation in [Na+]i rather than pHi would be the important event caused by NHE1 activation under physiological conditions.
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Tg Myocytes Exhibited Increased Ca2+ Levels in the Cytoplasm and Sarcoplasmic Reticulum
As shown in Figure 4A, at 1-Hz stimulation, both diastolic and systolic Ca2+ levels were significantly higher in Tg than in WT cardiomyocytes (179±22.7 and 486±18.4 nmol/L in WT and 263±15.8 and 1142±41 nmol/L in Tg myocytes, respectively); Ca2+ amplitude (difference between the diastolic and systolic Ca2+ levels) was also 2-fold higher in Tg myocytes. Treatment with 1 µmol/L cariporide for 2 minutes, which led to 70% to 80% inhibition of NHE1 activity (data not shown), had little effect on Ca2+ transients in WT cardiomyocytes, whereas all parameters (diastolic, systolic, and Ca2+ amplitude) were at least partly decreased in Tg cardiomyocytes. Two-minute washout reversed the effects of cariporide, suggesting that the increase in intracellular Ca2+ levels in Tg myocytes was attributable to increased NHE1 activity. Furthermore, we also detected marked elevations in diastolic and systolic Ca2+ levels at more physiologically relevant stimulation frequencies (up to 3.3 Hz), especially in Tg myocytes; Ca2+ amplitude was also significantly higher in Tg myocytes at all stimulation frequencies tested (supplemental Figure VI).
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Furthermore, the peak amplitude of the caffeine-induced Ca2+ transient, which we used as an index of sarcoplasmic reticulum (SR) Ca2+ content, was 2-fold higher in Tg versus WT myocytes (Figure 4B, a through c), suggesting that this is one of the mechanisms of increased [Ca2+]i in Tg myocytes. The rate of decline in the field stimulation-elicited Ca2+ transient was significantly faster in Tg than in WT myocytes (Figure 4B, d): the time constant (tau) was reduced to 60% (inset), indicating accelerated Ca2+ removal, mainly by SR Ca2+ pumping. In addition, the Ca2+ transient upstroke was faster in Tg myocytes than in WT myocytes (supplemental Figure VII), suggesting that the rate of Ca2+ release from the SR may also be higher in Tg myocytes. Ca2+/calmodulin-dependent protein kinase (CaMK)II-dependent Thr17 phosphorylation of phospholamban (PLB), a Ca2+ pump regulatory protein, was almost 5-fold greater in Tg hearts than in WT hearts, yet no difference was detected at Ser16, a protein kinase (PK)A phosphorylation site (Figure 4B, e and f), strongly suggesting that CaMKII, but not PKA, is activated in Tg hearts and induces PLB phosphorylation, leading to enhanced SR Ca2+ loading and subsequent increase in cytoplasmic Ca2+ levels.
Effects on Single-Cell Shortening
We measured single-cell shortening in the same myocyte from which the Ca2+ transient was obtained, stimulated over a range of frequencies (0.5–2.0 Hz). Interestingly, we observed a clear frequency-dependent decrease in contractility in Tg myocytes after normalizing to the maximal value, although the actual single-cell shortening (percentage) was greater in the Tg than in the WT group at low stimulation frequency (0.5 Hz; Figure 4C). In addition, we observed that Tg myocytes were more susceptible to high frequency stimulation-induced cytotoxicity (supplemental Figure VIIIA). These results indicate that contractile dysfunction occurs at the cellular level in Tg myocytes.
Activation of Prohypertrophic Molecules in Tg Mice
We assessed the activity of 2 Ca2+-dependent prohypertrophic signaling molecules, CaMKII and calcineurin,12 in Tg hearts. The level of phosphorylated CaMKII was markedly increased (Figure 5A), consistent with increased CaMKII-dependent phosphorylation of PLB (Figure 4B, e and f). In addition, the amounts of mRNA (Figure 5B) and protein (supplemental Figure IIB) of MCIP1 (modulatory calcineurin inhibitory protein-1), which was used as a sensitive indicator of calcineurin activity, was significantly elevated in Tg hearts. Cariporide largely prevented both effects, suggesting that both CaMKII and calcineurin activation were NHE1-dependent. Furthermore, we detected a marked increase in the phosphorylation of p38, as well as a slight but significant increase in the phosphorylated active forms of extracellular signal-regulated kinase (ERK)42/44 mitogen-activated protein kinases (MAPKs), which were both largely reversed by cariporide treatment. Akt also appeared to be slightly activated in Tg hearts, although this was not statistically significant (Figure 5C).
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NHE1-Dependent Translocation of HDAC and NFAT
Calcineurin is known to induce nuclear translocation of the NFAT family of transcription factors during hypertrophy via its dephosphorylation,13 whereas CaMKII has been reported to induce nuclear export of histone deacetylase (HDAC)4 by phosphorylating it and thereby promote cardiomyocyte hypertrophy by blocking HDAC-dependent inhibition of target gene transcription.14 To determine whether these pathways were activated by NHE1 overexpression, we treated primary cultured neonatal rat ventricular myocytes (NRVMs), which are often used as a model system of pathological hypertrophy, with a hypertrophic stimulus from phenylephrine and found that it induced a clear translocation of HDAC4 from the nucleus to the cytoplasm in these cells (supplemental Figure IX). Strikingly, overexpression of NHE1 also triggered translocation of HDAC4–green fluorescent protein (HDAC4-GFP) from the nucleus to the cytoplasm (Figure 6A, a) to an extent similar to that detected in phenylephrine-treated myocytes (Figure 6A, c) and redistributed from the cytoplasm to the nucleus on treatment with cariporide (10 µmol/L for at least 3 hours; Figure 6A shows the same myocyte before and after cariporide treatment). Similar NHE1-dependent cytoplasmic accumulation of HDAC4 was observed for endogenous HDAC4 (supplemental Figure IXB). These results strongly suggest that overexpression of NHE1 promotes CaMKII-HDAC signaling. Furthermore, overexpression of NHE1 resulted in nuclear import of NFAT-GFP from the cytoplasm, as observed in the phenylephrine-treated group; this was partially reversed by cariporide treatment (Figure 6A, d and e). These results suggest that overexpression of NHE1 activated the calcineurin–NFAT pathway.
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20 cells from 3 independent preparations). Scale bar=20 µm.We performed similar experiments in primary cultured mouse ventricular myocytes from WT and Tg hearts. As shown in Figure 6B, HDAC4-GFP accumulated in the cytoplasm of Tg myocytes, whereas it was localized to the nucleus in WT myocytes, as was the case for the NHE1-overexpressing NRVMs (see also supplemental Figure IXC). On the other hand, NFAT-GFP localized to the cytoplasm in WT cells, but in Tg myocytes, its localization was somewhat intermediate: some localized to the nucleus and some to the cytoplasm (Figure 6B, b and c, for averaged data). These results indicate that the CaMKII-HDAC pathway is fully activated but that the calcineurin–NFAT pathway is only partially activated in Tg myocytes, suggesting that a more complex regulatory mechanism exists in Tg hearts.
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Discussion |
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Despite accumulating evidence of a pathological role for NHE1 in cardiac hypertrophy, remodeling, and HF, it was not known whether direct activation of NHE1 itself could induce these states. Here, we demonstrated that NHE1 activation is sufficient to initiate these cardiac disease states via activation of Ca2+-dependent prohypertrophic signaling pathways and possibly via acceleration of Ca2+-induced cell death. This conclusion is supported by a number of novel findings from our mouse model: (1) overexpression of an activated form of NHE1 (
637 to 656) resulted in cardiac hypertrophy followed by dilated cardiomyopathy in vivo; (2) in isolated Tg myocytes, [Na+]i was increased because of enhanced Na+/H+ exchange activity, and both diastolic and systolic Ca2+ levels were significantly elevated but with less contractility at higher stimulation frequencies; (3) such remodeling was accompanied by activation of Ca2+-dependent prohypertrophic pathways CaMKII-HDAC and calcineurin–NFAT, as well as p38 and ERK42/44 MAPKs; and (4) these effects observed in vivo and in vitro were largely prevented by cariporide. Because NHE1 is activated by numerous extracellular stimuli, these findings support the view that NHE1 plays an important role in transducing extracellular stimuli into intracellular Ca2+ signals, and therefore has a great impact on cardiac pathogenesis.
Elevated [Ca2+]i in Tg Myocytes: Mechanisms and Consequences
The increased resting pHi and accelerated net H+ efflux under Hepes-buffered conditions provided direct evidence for enhanced Na+/H+ exchange activity in Tg myocytes. Furthermore, we observed that [Na+]i were elevated in Tg myocytes under both bicarbonate-buffered and bicarbonate-free conditions, although no significant changes in pHi were detected between WT and Tg myocytes under bicarbonate buffer. These findings suggest that the increased [Na+]i rather than pHi would be an initial signal to trigger the phenotypic changes in myocytes, consistent with previous reports demonstrating the relative importance of increased [Na+]i in cardiac hypertrophy and HF.15–18 Although the expression level of NCX1 protein was unchanged in our Tg hearts, increased [Na+]i would have influenced the driving force and hence the activity of the NCX by decreasing the forward (Ca2+ efflux) and/or increasing the reversed mode (Ca2+ influx), resulting in increased [Ca2+]i, as reported previously.18 In fact, we observed that both systolic and diastolic Ca2+ levels were significantly increased in Tg myocytes and that these levels were further pronounced at higher stimulation frequencies. Previous reports showed that [Na+]i in failing myocardium, which is significantly higher than that in nonfailing myocardium, increases more in response to a higher stimulation frequency in both rats and humans.19,20 Rapid stimulation-induced membrane depolarization can further activate the reverse mode of NCX1 and promote diastolic Ca2+ overload.
Furthermore, we present evidence of altered SR Ca2+ handling in Tg hearts, which involved CaMKII-dependent phosphorylation of PLB and subsequent activation of SERCA2a, leading to a higher rate of SR Ca2+ pumping. In addition to the higher SR Ca2+ content, CaMKII-induced phosphorylation and activation of the ryanodine receptor21 might also be involved in the accelerated Ca2+ release from the SR in Tg myocytes. Thus, overexpression of NHE1 results in both (1) increased diastolic Ca2+ levels and (2) altered SR Ca2+ handling. A continuous increase in diastolic Ca2+ could activate critical hypertrophic signaling mechanisms and could trigger some death signals. Although reduced SR Ca2+ handling is often reported in failing hearts,22 in the case of NHE1 Tg myocytes, the combination of Na+-induced diastolic Ca2+ overload and enhanced SR Ca2+ pumping would ultimately lead SR Ca2+ overload, which itself has been reported to activate some Ca2+-dependent protease calpains and induce apoptosis through the mitochondrial death pathway involving BAD, Bid, and caspase1223; we observed marked degradation of cardiac troponin I in Tg heart (supplemental Figure IIC), which might be a consequence of such an event. Thus, it is plausible that both Na+-induced diastolic Ca2+ overload and enhanced SR Ca2+ pumping promote HF in Tg hearts.
As has been observed in human failing hearts,1 we observed negative force–frequency dependence in Tg myocytes (see Figure 4C) without any change in the diastolic cell length (data not shown). Preservation of the high Ca2+ amplitude relative to diminished contractility observed in Tg mice at high stimulation frequency within the same myocyte suggests that myofibril Ca2+ sensitivity might be decreased. Possible mechanisms of this phenomenon are presently unknown. However, a recent report demonstrated that a reduction in myofilament Ca2+ sensitivity was introduced by PKD-dependent cardiac troponin I phosphorylation,24 which may occur in cardiac disease states.25 We also detected a slight but significant increase in the phosphorylation of troponin I in Tg hearts (supplemental Figure VIIIB).
Overall, the results of the single-cell experiments suggest the cellular mechanisms of hypertrophy/HF observed in vivo. However, we must consider that in vivo hearts consist of a heterogeneous population of cardiomyocytes that includes dead cells that are not available for in vitro experiments after cell isolation.
Downstream Signaling Pathways Leading to Cardiac Hypertrophy and HF
Numerous signaling pathways are known to coordinate pathological hypertrophy and HF, including calcineurin and CaMKII, PKC, and MAPKs (ERK42/44, ERK5, p38, and JNK).12 We found that the Ca2+-dependent signaling molecules CaMKII and calcineurin are both highly activated in Tg hearts and that this activation was reversed by cariporide treatment. These findings suggest that NHE1, coupled with NCX1, can supply a Ca2+ source for activating both the CaMKII and calcineurin pathways. Indeed, recent reports have suggested that both NCX18 and calcineurin26 are involved in endothelin-1–induced hypertrophy in rat myocytes, secondary to NHE1 activation. Furthermore, overexpression of NHE1 triggered nuclear export of HDAC and nuclear import of NFAT in a cariporide-sensitive manner, providing direct evidence that alterations in the sarcolemmal Na+/H+ exchange activity can modulate hypertrophy-associated gene expression in cardiomyocytes.
In contrast to the exclusive NHE1-dependent translocation patterns of NFAT and HDAC in NRVMs, NFAT was incompletely nuclear translocated in Tg myocytes, suggesting that activation of NFAT was partially blocked in Tg myocytes despite calcineurin activation. In addition to Ca2+-dependent pathways, we found that p38 and ERK42/44 MAPKs were significantly activated in Tg hearts. Because p38 (as well as GSK3β, the downstream molecule of Akt) is known to negatively regulate calcineurin–NFAT signaling via phosphorylation of NFAT,12 we assume that p38 activation would be one of the mechanisms for partial activation of the NFAT signal in Tg myocytes. This is consistent with the recent finding that CaMKII rather than calcineurin might serve as a major NHE1-dependent hypertrophy molecule in GC-A KO mice.4 Indeed, overexpression of activated CaMKII itself results in pronounced hypertrophy and dilated cardiomyopathy.27 Because p38 activation could have been caused by stress and/or receptor activation secondary to enhanced mechanical load, we do not exclude the possibility that the calcineurin pathway predominates in the initial period of hypertrophy, as previously reported.26
Pathological Implications of NHE1 in Cardiac Hypertrophy and HF
NHE1 activity is upregulated in several in vivo and in vitro models of cardiac hypertrophy/HF,4,5,28 and NHE1 inhibitors prevent the detrimental effects,2 suggesting that NHE1 contributes to cardiac remodeling. Consistent with these reports, we directly demonstrated that NHE1 activation is sufficient to induce hypertrophy and HF; however, there are also some differences between our model and those used in other studies. For example, hearts of GC-A KO mice exhibit hypertrophy but do not develop HF.4 This may be a matter of the degree of NHE1 activation, because the expression level of NHE1 protein was unchanged in GC-A KO mice. In addition, there was a significant increase in p38 phosphorylation in our Tg hearts. Because it has been shown that p38 activation in MKK3 or MKK6 Tg mice induced dilated cardiomyopathy and HF without causing hypertrophy,29 it is possible that there are independent mechanisms for inducing hypertrophy and HF, both of which might be activated in our mouse model (supplemental Figure X).
Our data suggest that NHE1-induced Na+ overload and altered Ca2+ handling are sufficient to introduce cardiac hypertrophy mainly through activation of the CaMKII-HDAC4 pathway. Whether or not this is "the necessary event" remains to be determined. However, our results do demonstrate that NHE1 activation can be an initiation signal to promote cardiac remodeling.
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Acknowledgments |
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We thank Dr Munekazu Shigekawa (Senri Kinran University) and Dr William Coetzee (New York University School of Medicine) for fruitful discussions about the manuscript. We also thank Hitomi Otake and Madoka Hirayama for technical assistance.
Sources of Funding
This work was supported by Grants-in-Aid for Priority Areas 18077015 and 18059053; grants-in-aid 19390080, 19590220, 18590796; a grant for the Cooperative Link for Unique Science and Technology for Economy Revitalization from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a grant for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO); research grants for Cardiovascular Diseases (17A-1) and for Nervous and Mental Disorders (16B-2 and 19B-1) from the Ministry of Health, Labor, and Welfare; Salt Science Research Foundation grant 0737; and a grant from the Takeda Science Foundation.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received March 5, 2008; revision received August 21, 2008; accepted August 25, 2008.
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