Published online before print March 6, 2008, doi: 10.1161/CIRCRESAHA.107.164947
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© 2008 American Heart Association, Inc.
Integrative Physiology |
Epac Mediates β-Adrenergic Receptor–Induced Cardiomyocyte Hypertrophy
From Inserm (M.M., A.L., M.G., E.M., F.L.), U769, Signalisation et Physiopathologie Cardiaque, Châtenay-Malabry; Univ Paris-Sud (M.M., A.L., M.G., E.M., F.L.), Faculté de Pharmacie, IFR141, UMR-S769, Châtenay-Malabry; Inserm (J.-L.S., C.H.), U689, Centre de Recherche Cardiovasculaire, Paris; and Université D. Diderot (J.-L.S., C.H.), Paris, France.
Correspondence to Frank Lezoualc’h, Inserm, U769, Faculté de Pharmacie, 5 rue JB Clément, Châtenay-Malabry, F-92296, France. E-mail: Frank.Lezoualch{at}u-psud.fr
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Abstract |
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Cardiac hypertrophy is promoted by adrenergic overactivation and can progress to heart failure, a leading cause of mortality worldwide. Although cAMP is among the most well-known signaling molecules produced by β-adrenergic receptor stimulation, its mechanism of action in cardiac hypertrophy is not fully understood. The identification of Epac (exchange protein directly activated by cAMP) proteins as novel sensors for cAMP has broken the dogma surrounding cAMP and protein kinase A. However, their role and regulation in the mature heart remain to be defined. Here, we show that cardiac hypertrophy induced by thoracic aortic constriction increases Epac1 expression in rat myocardium. Adult ventricular myocytes isolated from banded animals display an exaggerated cellular growth in response to Epac activation. At the molecular level, Epac1 hypertrophic effects are independent of its classic effector, Rap1, but rather involve the small GTPase Ras, the phosphatase calcineurin, and Ca2+/calmodulin-dependent protein kinase II. Importantly, we find that in response to β-adrenergic receptor stimulation, Epac1 activates Ras and induces adult cardiomyocyte hypertrophy in a cAMP-dependent but protein kinase A–independent manner. Knockdown of Epac1 strongly reduces β-adrenergic receptor–induced hypertrophic program. Finally, we report for the first time that Epac1 is mainly expressed in human heart as compared with Epac2 isoform and is increased in heart failure. Taken together, our data demonstrate that the guanine nucleotide exchange factor Epac1 contributes to the hypertrophic effect of β-adrenergic receptor in a protein kinase A–independent fashion and may, therefore, represent a novel therapeutic target for the treatment of cardiac disorders.
Key Words: G protein–coupled receptor • small G protein • cardiac hypertrophy
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Introduction |
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The β-adrenergic receptor (β-AR) is a prototypical member of the G protein–coupled receptor superfamily and plays a central role in sympathetic regulation of cardiac function.1 Although acute stimulation of β-ARs has beneficial effects on heart function, accumulating evidence suggests that their chronic activation causes progressive cardiac dysfunction, cell loss, and cardiac chamber remodeling in human.2,3 Consistent with this notion, it has been demonstrated that chronic stimulation of β-ARs causes hypertrophy in cardiac myocytes.4 Adult myocyte hypertrophy is the compensatory response of the heart to stress and is characterized by nonmitotic growth, addition of new sarcomeres, fetal gene expression, and specific changes in ion channel properties.5 Maladaptive cardiac hypertrophy can progress to heart failure (HF), a leading cause of morbidity and mortality in industrialized countries. Thus, understanding the signaling mechanisms that mediate the growth of adult cardiac myocytes by β-AR stimulation may lead to better treatment for patients with HF.
Although cAMP is among the most well-known signaling molecules produced by β-AR stimulation, its mechanism of action in cardiac growth is not fully understood. Protein kinase A (PKA) has been regarded as the main effector of cAMP in most eukaryotic cells. In cardiomyocytes, PKA phosphorylates and activates key proteins of the excitation–contraction coupling, such as L-type calcium channels, phospholamban, or ryanodine receptors, as well as troponin I, a regulatory thin filament protein.6 Apart from PKA, cAMP can also activate phosphodiesterases or hyperpolarization-activated cyclic nucleotide–gated channels. More recently, a novel family of proteins directly activated by cAMP has been discovered.7 These proteins, called Epac (exchange protein directly activated by cAMP) proteins, activate the Ras-like small G proteins Rap1 and Rap2. There are 2 subtypes of Epac, Epac1 and Epac2, both characterized by a regulatory domain which binds directly cAMP and a catalytic region containing an exchange factor motif that catalyzes the exchange of GDP from GTP on Rap GTPases.7 Epac2 also possesses a second, lower-affinity cAMP-binding domain at its N terminus, and its function is still unknown. Recent studies indicate that Epac is involved in diverse cAMP-dependent processes,8 such as insulin secretion9 or the amyloid precursor protein processing.10 With respect to the heart, Epac induces gap junction neoformation and expression of fetal phenotype gene markers, such as atrial natriuretic factor (ANF), in neonatal rat cardiac myocytes.11–13
To date there is no information on the expression levels of Epac isoforms in normal and pathological human hearts. In addition, the function of Epac, as well as its downstream effectors and their neurohormonal regulation, has not been yet studied in mature cardiac myocytes. Here, we show that (1) Epac1 is increased at the onset of rat cardiac hypertrophy and its activation exacerbates cellular growth; (2) β-AR–induced cardiac myocyte hypertrophy involves endogenous Epac; (3) Epac hypertrophic effect is PKA-independent and involves the small GTPase Ras, the phosphatase calcineurin and Ca2+/calmodulin-dependent protein kinase II (CaMKII) but not Rap1; and (4) Epac 1 is upregulated in HF.
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Materials
All media, sera, and antibiotics used in the cell culture were purchased from Invitrogen and Sigma-Aldrich. 8-(4-Chloro-phenylthio)-2'-O-methyladenosine-3'-5'cyclic monophosphate (8-CPT) was from Biolog Life Science Institute (Bremen, Germany). Phenylephrine, isoprenaline (ISO), cyclosporin A, and KN-93 were obtained from Sigma-Aldrich.
Thoracic Aortic Constriction
Thoracic aortic constriction was performed as previously described.14 Briefly, 25-day-old Wistar male rats (Iffa Credo, L’Arbresle, France) were anesthetized (intraperitoneal injection xylazine 50 mg/kg and ketamine 100 mg/kg), and the ascending aorta was partially occluded with a hemoclip (Atrau clip, PlilingÒ). Sham-operated animals were submitted to a similar protocol without the clip. After 5 days, heart weights increased by 34±5% in thoracic aortic constriction as compared with sham animals. Body weights were similar in both groups.
Human Heart Tissues
All studies are conformed to the Declaration of Helsinki and institutional ethical regulations. Explanted failing hearts were obtained from patients undergoing cardiac transplantation for end-stage cardiac HF secondary to idiopathic dilated cardiomyopathy. All patients had New York Heart Association class IV HF, with a mean pretransplant left ventricular ejection fraction of 22±4%. None had received chronic intravenous inotropic support over at least 7 days immediately before transplantation. HF therapy consisted of angiotensin-converting enzyme inhibitors and diuretics in all patients. Nonfailing hearts were obtained from prospective multiorgan donors who had died from head trauma or intracranial bleeds; these hearts were unsuitable for transplantation for technical reasons. All tissues were stored at –80°C until further analyses.
For a description of other methods, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
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Epac1 Is Upregulated in Cardiac Hypertrophy and Promotes Adult Rat Ventricular Myocyte Cell Growth
To assess the expression levels of Epac in cardiac hypertrophy, we performed immunoblot analysis against Epac1 on left ventricular myocardium of rats subjected to pressure overload induced by thoracic aortic constriction. We found that Epac1 protein was significantly upregulated at the early phase of cardiac hypertrophy development when compared with specimen from sham operated animals (Figure 1A). Activation of endogenous Epac with the Epac selective cAMP analog, 8-CPT (10–6 mol/L),15 increases cell surface area of adult rat ventricular myocytes (ARVMs) isolated from sham or stenosed animals (Figure 1B). 8-CPT at 10–6 mol/L had no effect on PKA activity in cardiac myocytes (Figure I in the online data supplement). This is consistent with a previous study performed in noncardiac cells.15 Interestingly, the hypertrophic effect of Epac was significantly stronger in myocytes isolated from banded rats than in those isolated from sham-operated animals, indicating that elevation of Epac1 expression may contribute to the progression of pathophysiologic hypertrophy (Figure 1B). Consistent with this finding, upregulation of Epac1 in ARVMs with an adenovirus encoding the wild-type form of human Epac1 (Ad.Epac1) increased the effect of 8-CPT on cell surface area compared with ARVMs infected with control Ad.GFP (Figure 1C and 1D). The effect of Ad.Epac1 on cell growth was also confirmed by protein synthesis measurement, which is another marker of cardiac hypertrophy (data not shown).
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-actinin was visualized by immunocytochemistry as described in Materials and Methods. Pictures show sarcomeric
Ras but Not Rap1 Is Involved in the Hypertrophic Effect of Epac
Because the primary function of Epac proteins is to act as guanine nucleotide exchange factors (GEFs) for Rap GTPases,7 we next asked whether Rap1 was involved in Epac1-induced ARVM hypertrophy. Although 8-CPT and recombinant Epac1 significantly enhanced Rap1 activation (Figure 2A), adenoviral infection of ARVMs with a dominant positive form of Rap1 (Ad.Rap1Q63E) did not influence protein synthesis (Figure 2B). In addition, a Rap1 GTPase-activating protein (RapGAP) failed to inhibit the hypertrophic effect of Epac1 (Figure 2B) despite the fact that Ad.RapGAP was effective in ARVMs. Indeed, it completely blocked Epac1-induced Rap1 activation (supplemental Figure II). Hence, Rap1 is not involved in the Epac-induced myocyte hypertrophy.
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Ras GTPases have been reported to induce cardiac hypertrophy.16 Moreover, Ras activation has been shown to be controlled by Epac in HEK and neuroblastoma cell lines.17 Thus, we hypothesized that a dominant negative form of Ras (RasS17N) may blunt Epac1-induced myocyte hypertrophy. Figure 2C shows that Epac activation increased the amount of Ras-GTP. In addition, RasS17N completely blocked the stimulating effect of Epac activation on [3H]-leucine incorporation (Figure 2D). Taken together, these data demonstrate that Ras but not Rap1 is involved in Epac1-induced myocyte hypertrophy.
Epac Activates Calcineurin and CaMKII Signaling Pathways
The Ser/Thr protein phosphatase calcineurin and CaMKII are 2 prominent Ca2+-dependent pathways that play a crucial role in cardiomyocyte hypertrophy.5,18 To characterize further the Epac hypertrophic signaling pathway in a more relevant cell model, we investigated the effect of Epac1 stimulation on calcineurin and CaMKII activation in adult cardiomyocytes. Figure 3A shows that calcineurin activity was increased in cells infected with Ad.Epac1 and treated with 8-CPT (10–6 mol/L), suggesting that the phosphatase is a downstream target of Epac1. When we measured CaMKII activation using an antibody against the autophosphorylation site of CaMKII, Thr-286, we found that active CaMKII increased significantly on Epac1 activation, although total CaMKII expression was not altered (Figure 3B). The increase in active CaMKII level induced by 10–6 mol/L 8-CPT was inhibited by a selective CaMKII inhibitor, KN-93 (10–6 mol/L) (Figure 3B). Interestingly, we found that 8-CPT–induced calcineurin and CaMKII activities were inhibited by Ad.RasS17N (Figure 3A and 3C). Consistent with these findings, KN-93 significantly blocked Epac1-induced increase in [3H]-leucine incorporation (Figure 3D). Similarly, pharmacological inhibition of calcineurin with cyclosporin A (5x10–7 mol/L) prevented the hypertrophic effects of Epac1 (Figure 3D). Taken together, these data show that Epac1 activates a prohypertrophic signaling pathway that involves the Ca2+-sensitive proteins calcineurin and CaMKII. In response to Epac activation, the small GTPase Ras regulates calcineurin and CaMKII.
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Epac1 Is Involved in the Induction of Myocyte Hypertrophy Induced by β-ARs
Because β-ARs are positively coupled to adenylyl cyclase resulting in cAMP production and promote cardiac hypertrophy, we tested whether β-ARs activation may regulate Epac effects. To explore the role of native Epac1 in the hypertrophic effect of β-AR, we used a short hairpin (sh)RNA targeting Epac1 (shEpac1) to knockdown its expression. ARVM cultures are less suitable than neonatal rat ventricular myocyte (NRVM) cultures for this purpose because of their known fragility and dedifferentiation after only few days. NRVM culture is a well-proven model to study hormonal and gene transfer effects in cell growth and was therefore chosen as test system for experiments using shRNA. As expected, NRVMs transfected with shEpac1 showed a decreased level of Epac1 compared with shRNA sequence control (shCT) transfected cells (Figure 4A). Activation of β-ARs with the nonselective β-AR agonist ISO (10–5 mol/L) for 2 days induced cytoskeletal reorganization (Figure 4B) and increased cell surface area (Figure 4B and 4C) in NRVMs transfected with shCT. These effects of ISO were impaired in cardiomyocytes expressing shEpac1 (Figure 4B and 4C). Similar findings were obtained on another marker of myocyte hypertrophy, ANF expression. Indeed, silencing Epac1 expression significantly inhibited ISO-induced ANF-Luc gene transcriptional activity (Figure 4D). Taken together, these data support the hypothesis that Epac participates in β-AR-induced cellular hypertrophy.
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Epac1 Mediates β-ARs Induced Myocyte Hypertrophy in a cAMP-Dependent but PKA-Independent Manner
To investigate the cellular localization of Epac and its possible regulation by β-ARs in ARVMs, we constructed an adenovirus encoding human Epac1–green fluorescent protein (GFP) fusion protein (Ad.Epac1-GFP). Epac1-GFP showed sarcolemmal and perinuclear linear fluorescence, as well as small fluorescent dots organized in a transverse striated pattern, likely T tubule in ARVMs (Figure 5A). To explore the effect of β-ARs activation on adult cardiac myocyte hypertrophy, ARVMs were treated with 5x10–7 mol/L ISO. Higher concentrations of ISO (10–5 or 10–6 mol/L) have indeed been previously shown to induce apoptosis of ARVMs.19 As shown in Figure 5B, ISO (5x10–7 mol/L) significantly increased [3H]-leucine incorporation in ARVMs infected with Ad.Epac1 as compared with control cells. This process was independent of PKA because an adenovirus encoding the substrate inhibitor PKI (Ad.PKI)20 failed to block the hypertrophic effect of ISO in Ad.Epac1-infected cells (Figure 5B). Moreover ISO-induced ARVM hypertrophy was also observed in the absence of Ad.Epac1 and PKA activity (supplemental Figure III). The PKA-independent effects of ISO on cell growth were comparable to those of 8-CPT (supplemental Figure III). Ad.PKI was effective in our cellular system because it completely inhibited ISO-induced PKA activity in ARVMs (Figure 5C).
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To further show that Epac1 activation by β-ARs induced myocyte hypertrophy in a cAMP-dependent but PKA-independent manner, ARVMs were coinfected with a mutated form of Epac1 (Ad.EpacR279K) unable to bind cAMP21 and Ad.PKI to block endogenous PKA activity. Figure 5D shows that ISO (5x10–7 mol/L) failed to induce hypertrophy of cells infected with Ad.EpacR279K in contrast to those infected with the wild-type form of Epac1. Furthermore, we found that ISO and Epac1 activated Ras in a PKA-independent manner (supplemental Figure IVA), and RasS17N prevented the Epac hypertrophic effect induced by ISO (supplemental Figure IVB). Altogether, these data indicate that β-ARs activate the cAMP/Epac1 signaling pathway to induce myocyte hypertrophy in a PKA-independent manner.
Epac1 Is Mainly Expressed in Human Myocardial Tissue and Is Upregulated in HF
Finally, we analyzed the expression levels of Epac1 and Epac2 in human cardiac tissues using quantitative RT-PCR in nonfailing and failing left ventricular myocardial samples (HF). We found there was a 2-fold increase in Epac1 mRNA in HF tissues (Figure 6A). Although Epac2 mRNA was not significantly different between the 2 groups, it tended to decrease in HF (Figure 6B). As shown by the Epac1/Epac2 ratio, Epac1 was predominant in nonfailing tissues as compared with Epac2 (Figure 6C). This predominance of Epac1 over Epac2 is largely increased in HF (Figure 6C). Furthermore, we found by Western blot that Epac1 protein was significantly upregulated in left ventricular samples from patients with HF (Figure 6D). Altogether, our results provide evidence that Epac1 is expressed in human ventricular tissue and is upregulated in HF.
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Discussion |
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cAMP is among the most important second messengers, and its cardiac effects are classically attributed to the PKA signaling pathway. The identification of Epac proteins that directly bind cAMP raises the question of the functional role of these GEFs in the heart. We report for the first time that (1) in vivo, Epac1 is upregulated at the onset of cardiac pressure overload-induced hypertrophy; and (2) in vitro, its activation mediates ARVM hypertrophy in response to increased cAMP. Interestingly, the hypertrophic effects of Epac were more pronounced in ARVMs isolated from banded rats than in those isolated from sham-operated animals. This can be explained by the high expression level of Epac in stress condition. Consistent with our data, Ulucan et al22 have recently shown that Epac expression is upregulated in mice hearts during chronic ISO infusion, which is another model of left ventricular hypertrophy. Therefore, one can imagine a scenario in which increased Epac1 expression in response to stress (ie, high levels of catecholamine) contributes to the progression of pathological cardiac growth. In addition, our findings that Epac1 is increased in human failing heart suggest a possible role of this cAMP-GEF during the late cardiac remodeling.
The hypertrophic effects of Epac in ARVMs extend our recent data showing that Epac1 induces expression of hypertrophic gene markers in neonatal cardiomyocytes.11 However, contrary to neonatal cells, overexpression of Epac1 in ARVMs failed to spontaneously induce cell growth in the absence of any cAMP analog treatment. This suggests that basal concentrations of cAMP are not sufficient to induce Epac-dependent hypertrophy in ARVMs, in contrast to neonatal cardiac myocytes. These data indicate that the cellular response to a stimulus varies according to the differentiation stage, as shown for several hypertrophic stimuli. Alternatively, Epac activity could also be influenced by molecular partners. For instance, because cyclic nucleotide phosphodiesterases are key enzymes controlling the concentration and diffusion of cAMP in cardiac cells,23 one can speculate that they may influence Epac signaling pathway. Of note, a cAMP-responsive signaling complex that includes Epac1 and phosphodiesterase 4D3 has recently been identified in neonatal cardiac myocytes.24 Also of interest, the light chain 1 of microtubule-associated protein 1B (LC1) has been shown to act as a molecular chaperone of Epac1, increasing the binding of cAMP to this GEF and consequently increasing Epac1 signaling in PC12 cells.25
The results of the present study reveal a key contribution of Epac1 in β-AR–induced myocyte hypertrophy. Indeed, silencing Epac1 expression blocked the hypertrophic effect of ISO. In addition, we showed that sustained β-ARs stimulation with ISO induced hypertrophy of ARVMs when Epac1 was expressed independently of PKA activity as shown with the peptide inhibitor PKI (Figure 5). This is consistent with previous in vitro and in vivo experiments showing the hypertrophic effect of β-ARs stimulation on ventricular cardiomyocytes.26–28 Finally, a dominant negative form of Epac1, EpacR279K, that is unable to bind cAMP inhibited the hypertrophic effect of β-AR activation. Together, these results indicate that following stimulation of β-ARs, cAMP binds to Epac1, which then triggers a hypertrophic program in a PKA-independent manner. The persistence of Epac-induced cell growth despite a PKA inhibition extends previous work showing that ISO-induced cardiac myocyte fetal gene expression (ie, ANF) was PKA-independent.29,30 Therefore, in addition to the classic cAMP/PKA pathway, β-AR stimulation under certain pathophysiological circumstances, such as during the onset of pressure overload–induced cardiac hypertrophy and/or late phase of HF, may switch on the Epac signaling pathway. These lines of evidence raise the question of the identification of Epac downstream effectors involved in this process.
One of our major findings is that in adult cardiomyocytes, Rap1 signaling was not involved in Epac1-dependent cell growth, although Epac1 was able to activate Rap1. Indeed, activated Rap1 (Ad.RapQ63E) failed to induce ARVM hypertrophy, and expression of RapGAP, which resulted in a general blockade of Rap1 signaling, did not alter Epac1-induced ARVM growth (Figure 2). These results are apparently opposed to data indicating that functional effects of Epac are Rap1-dependent.7 Indeed, Epac1 inhibits the ERK5 pathway by a mechanism involving Rap1 in neonatal cardiomyocytes.24 Similarly, Epac-Rap1 signaling regulates the assembly of gap junction.12 This apparent discrepancy likely reflects the existence of spatiotemporal dynamics of Epac signaling, which determines its coupling to different effectors.
Because recent studies have identified Epac as a key regulator of cAMP-dependent activation of Ras,17 we examined whether this small GTPase was involved in the β-ARs/cAMP/Epac hypertrophic signaling pathway. We showed that a dominant negative form of Ras, RasS17N, blunted the trophic effect of Epac in ARVMs treated with ISO. These results are in agreement with the involvement of Ras in hypertrophic cardiomyopathy.16 Thus, we identified an entirely new hypertrophic signaling pathway that is initiated by β-ARs and involves cAMP/Epac/Ras. The previous observation showing the absence of any GEF activity of Epac on Ras31 suggests that Epac-mediated Ras activation is indirect. By analogy with our data, Shi et al32 recently showed that Epac promotes activation of Rit, a small GTPase of the Ras family, in a manner that does not appear to rely on Rap signaling or the direct regulation of Rit by Epac. Thus, further investigation will be required to identify the steps linking Epac to Ras activation in cardiac myocytes.
Epac has been previously shown to influence Ca2+ release in cardiac myocytes in a PKA independent but CaMKII-dependent manner.33–35 Accordingly, we found that prolonged activation of Epac1 by 8-CPT increased the activity of 2 Ca2+-dependent prohypertrophic proteins, calcineurin and CaMKII. Most importantly, pharmacological inhibition of 1 of these 2 signaling pathways was sufficient to block Epac-induced hypertrophy (Figure 4C). This indicates that CaMKII and calcineurin pathways converge on common downstream target genes in the hypertrophic signaling pathway initiated by Epac and cAMP. In line with this hypothesis, transcriptional activation of some muscle-specific genes appears to be mediated by a combinatorial mechanism involving downstream effectors of calcineurin and CaMKII such as NFAT and MEF2.36 Thus, we could hypothesize that a sustained activation of Epac leads to a sustained increase in [Ca2+]i, which then activates CaMKII and calcineurin. This scenario is supported by the findings that calcineurin and CaMKII inhibitors suppress expression of cardiac hypertrophic markers induced by ISO in vitro.37,30 Another interesting question raised by our work concerns the interaction of Ras with calcineurin and CaMKII and their effectors, such as the transcription factors NFAT and MEF2. Indeed, we found that Ras was also involved in Epac-induced ARVM hypertrophy. This raises the question of how this small GTPase may interfere with CaMKII and calcineurin signal transduction pathways to regulate cell growth. Interestingly, the small GTPase Rad has been shown to directly interact with CaMKII to regulate its activity.38 Therefore, as a next step, it would be interesting to test the potential interaction of Ras with calcineurin and CaMKII in response to Epac activation.
In conclusion, we have provided in vivo and in vitro evidence that Epac proteins play a key role in the development of β-AR–induced cardiac hypertrophy in adult mammals. Any dysregulation of cAMP compartmentation and/or concentration may contribute to enhance Epac signaling and, in turn, cardiac hypertrophy. Thus, our data open a new avenue for the treatment of cardiac disorders such as chronic HF associated with a high concentration of catecholamine.
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Acknowledgments |
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We thank the Production and Control department of Genethon, which is supported by the Association Française contre les Myopathies, in the frame of the GVPN network (http://www.gvpn.org) for providing us with Ad.GFP, Ad.Epac1, Ad.Epac1-GFP, and Ad.Rap1GAP. We are grateful to the vector core of the University Hospital of Nantes, which is supported by the Association Française contre les Myopathies, for the construction of Ad.RasS17N and Ad.Rap1Q63E. We are grateful to Valérie Nicolas, Claudine Deloménie, and Anne Garnier for confocal analysis and quantitative PCR and to Françoise Marotte and Valérie Domergue for animal surgery. We acknowledge Bertrand Crozatier for critical reading of the manuscript.
Sources of Funding
This work was supported by grants from Inserm "Programme National de Recherche sur les Maladies Cardiovasculaires" (to F.L, JL.S, C.H), Agence Nationale de la Recherche (Physio 2006) (to F.L), and the Fondation pour la Recherche Médicale (équipe FRM) (to F.L.)
Disclosures
None.
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Footnotes |
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Original received February 21, 2007; resubmission received September 26, 2007; revised resubmission received January 22, 2008; accepted February 27, 2008.
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