Published online before print August 2, 2007, doi: 10.1161/CIRCRESAHA.107.154252
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© 2007 American Heart Association, Inc.
Cellular Biology |
Connexin43 Remodeling Caused by Inhibition of Plakophilin-2 Expression in Cardiac Cells
From the Departments of Pharmacology (E.M.O., H.M., K.M., W.C., M.D.) and Microbiology and Immunology (S.M.T.), State University of New York, Upstate Medical University, Syracuse.
Correspondence to Mario Delmar, MD, PhD, Department of Pharmacology, SUNY Upstate Medical University, 766 Irving Ave, Syracuse NY 13210. E-mail delmarm{at}upstate.edu
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Abstract |
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Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.
Key Words: adenovirus • arrhythmogenic right ventricular cardiomyopathy • connexin43 • gap junction • plakophilin 2
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Introduction |
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Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease that presents with sustained monomorphic ventricular tachycardia and sudden cardiac death. The disease is characterized by progressive fibrofatty infiltration of the myocardium, most prominent in the free wall of the right ventricle.1 Recent studies have linked ARVC with mutations in proteins of the cardiac desmosome,2 a component of the intercalated disc essential for mechanical coupling between cardiac cells.3 It is estimated that as many as 70% of the mutations linked to familial ARVC are in the gene coding for plakophilin (PKP)2,4 a 98-kDa desmosomal protein. PKP2 interacts with plakoglobin, desmoplakin, and the desmosomal cadherins via its amino terminal ("head") domain.5–6 Loss of PKP2 destabilizes the desmosome,7 and its genetic deletion in mice leads to rupture of the myocardial wall during the embryonic stage.7
Loss of desmosomal integrity could lead to disruption of mechanical function in hearts afflicted with ARVC; yet, the latter does not directly explain the highly arrhythmogenic nature of the disease, particularly in cases in which life-threatening arrhythmias occur in the absence of severe displacement of myocardium with fatty or fibrous tissue.8 Recently, Saffitz and colleagues proposed that disruption of mechanical coupling may lead to loss of gap junction–mediated electrical communication between cells.8–10 This hypothesis awaits confirmation in a cellular model in which protein expression can be manipulated and intercellular communication can be assessed directly.
Here, we used small interfering (si)RNA technology to silence PKP2 expression in neonatal cardiac cells, and we explored the effect of loss of PKP2 expression on the distribution and function of gap junctions. Our studies focused primarily on 2 cell populations: cardiac myocytes and epicardium-derived cells (EPDCs). Although the importance of cardiac myocytes in the context of ARVC and arrhythmias seems self-evident, a possible role for EPDCs in ARVC has not been described. Yet, as progenitors of the cardiac fibroblast cell lineage, the function of EPDCs deserves attention. Our results show that loss of PKP2 leads to a redistribution of connexin (Cx)43 inside the cell, loss of gap junction plaques detectable by immunofluorescence, and reduction in lucifer yellow (LY)-permeable gap junctions between cells. Additional studies show that Cx43 and PKP2 can coexist in a common macromolecular complex. This is the first demonstration of a link between PKP2 disruption and loss of Cx43-mediated cell–cell communication. Our data open a new avenue for the understanding of the molecular mechanisms that may be responsible for ARVC.
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Materials and Methods |
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All details for methods are provided in the online data supplement at http://circres.ahajournals.org. Brief descriptions are presented below.
Cell Culture and PKP2 Silencing
Experiments were conducted in neonatal rat primary cell cultures.11 After dissociation, cells were resuspended in supplemented M199 media and preplated for 2 hours to allow for nonmuscle cells to attach to the plate. These dishes were used for experiments conducted in "preplated" cells (see under Results). After preplating, myocytes were plated to 70% confluence and maintained in supplemented DMEM media at 37°C.
Preparation of epicardial–mesenchymal cells (EPDCs) followed the method of Chen et al6 Hearts from 1- to 4-day-old rats were excised, and ventricles were dissected. Each ventricular section was cut into 4 pieces, and each piece was placed epicardium-side down onto a 60-mm dish coated with 0.1% gelatin. Ventricular pieces were covered with supplemented DMEM media. Ventricular sections were removed after 4 days of culture. After 3 additional days, cells were trypsinized and plated to 70% confluence.
We used viral transfer technology to silence the expression of PKP2 in neonatal rat ventricular myocytes (NRVMs). Cells in culture were infected with adenovirus containing short hairpin (sh)RNA for PKP2 (shRNA-PKP2). Cells untreated or treated with virus coding for green fluorescent protein (GFP) were used as controls. An alternative construct, predicted as a potential PKP2 silencer but shown not to interfere with PKP2 expression, was used as an additional control (shRNA-PKP2Ø). Unless otherwise indicated, experiments were conducted at 100 multiplicities of infection (mois) for shRNA-PKP2, 25 mois for GFP, and 100 mois for RNA-PKP2Ø and were performed 5 days after infection.
For PKP2 silencing in EPDCs, plates were separated in 3 groups: untreated, treated with Lipofectamine and the silencing construct (stealth RNAi interference [RNAi]; Invitrogen), or treated with Lipofectamine and a scrambled oligonucleotide. The scrambled construct corresponded to a series of the same bases organized to avoid any existing coding sequence. Experiments were performed 72 hours after completion of the PKP2 silencing protocol. Silencing of PKP2 was confirmed on every experiment by Western blot.
Immunochemical and Functional Assays
All immunochemical protocols, as well as the production of recombinant glutathione S-transferase (GST) fusion proteins and the GST pull-down assays, followed standard techniques.12,13 Details are provided in the online data supplement.
Dye transfer through gap junctions in NRVM cell pairs followed the method described by Valiunas et al.14 Dye transfer in monolayers of EPDCs followed conventional methods, and details are provided in the online data supplement.
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Results |
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Loss of PKP2 Expression in NRVMs After shRNA-PKP2 Treatment
To characterize the effect that loss of PKP2 expression has on function and distribution of Cx43, NRVMs in culture were infected with adenovirus containing a PKP2-silencing sequence (shRNA-PKP2). Figure 1 shows an example. PKP2 was present in control conditions and after transfer of cDNA coding for enhanced GFP (control) but decreased significantly after shRNA exposure in a manner that was dependent on the viral concentration used. Subsequent experiments were conducted using 100 mois for shRNA-PKP2. For quantification, each band density was measured relative to its corresponding actin control. Treatment with 100 mois shRNA-PKP2 caused a decrease in the density of the PKP2 signal recorded 5 days after exposure to virus to a level that was 10.80±2.23% of control (mean±SEM; n=8). In contrast, the actin-calibrated PKP2 signal obtained in cells treated with an enhanced GFP virus was not different from that recorded from untreated cells (101.36±5.23%; n=4). We used this experimental model to assess the effect of PKP2 silencing on the distribution of Cx43.
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Effect of PKP2 Silencing on Content and Distribution of Cx43
Figure 2A shows the immunolocalization of Cx43 and PKP2 in NRVMs. Colocalization to the site of cell–cell contact was apparent in untreated conditions (Figure 2A, left images) and when cells were infected with virus coding for a construct that failed to silence PKP2 (ie, shRNA-PKP2Ø; middle images; see also Figure 2B). Yet, loss of PKP2 expression induced by shRNA-PKP2 correlated with a drastic redistribution of Cx43 (Figure 2A, right images); few gap junction plaques were detectable, and, instead, Cx43 was found mostly within the intracellular space. Similar results were obtained in 4 experiments. Consistent with previous observations, loss of PKP2 expression also led to significant remodeling of desmoplakin and of desmin in NRVMs (Figure I in the online data supplement). Separately, we measured Cx43 protein content by Western blot. As shown in Figure 2B, the density of the Cx43 signal recorded from cells treated with shRNA-PKP2 was 48.56±6.04% of that recorded from untreated cells (n=4). Additional controls were obtained from cells treated with a virus containing a construct that did not silence PKP2 (lanes labeled RNA-PKP2Ø). In that case, the density of the Cx43 signal was 97.54±5.60% of that recorded from untreated cells (n=4). Although PKP2 silencing led to a decrease in total Cx43 content, we did not observe a shift in the ratio of low versus high-mobility bands. This ratio was 1.91±0.07 in untreated cells, 1.91±0.15 in cells treated with shRNA-PKP2, and 1.89±0.10 in cells treated with shRNA-PKP2Ø (n=4 for all experiments). Overall, these data show that loss of PKP2 leads to a decrease in total Cx43 content and a significant disruption of the structural integrity of Cx43 gap junction plaques. As a next step, we asked whether loss of PKP2 expression affected the function of gap junctions.
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Loss of Dye Transfer in PKP2-Silenced NRVMs
Transfer of LY across gap junctions allows for assessment of the extent of functional coupling between cells.14 Here, we determined whether loss of PKP2 expression correlated with changes in the extent of dye transfer between cell pairs. Figure 3A shows fluorescent images of dye transfer in a cell pair maintained in control conditions. An LY-filled patch pipette was used to gain access to the intracellular space of one cell in the pair (cell 1) and the extent, and the time course of diffusion into the partner cell (cell 2) was recorded. Fluorescent images in Figure 3A were obtained 1, 5, and 9 minutes after patch break, respectively. Dye diffused from the pipette into cell 1 and from there into cell 2. Yet, as shown in Figure 3B, dye transfer was significantly decreased in cells treated with shRNA-PKP2. The plot in Figure 3C shows the average fluorescence intensity recorded from both cell 1 and cell 2 (relative to the maximum fluorescence in cell 1 for each individual experiment) as a function of time after patch break. Data for fluorescence intensity in cell 2 are depicted for control (open squares) and cells treated with shRNA-PKP2 (open circles). The time course and extent of diffusion observed in control was similar to that previously reported.14 In contrast, dye diffusion was severely interrupted after PKP2 silencing, and the fluorescence intensity in cell 2 measured 10 minutes after patch break was significantly different from that observed in control (P<0.005).
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and 
) and in recipient cells (
and 
) as a function of time. Notice the reduction in dye transfer in cells silenced for PKP2. LY diffusion was measured in 10 untreated and 8 shRNA-treated myocyte pairs.
PKP2 Expression and Cx43 Distribution in Nonmyocyte Cardiac Cells
Loss of gap junction plaques in cardiac myocytes may result from the mechanical strain imposed on the beating myocytes in the absence of proper mechanical junctions. As a first approach to the study of an alternative nonbeating cardiac cell population, we used cells that were retained in the preplating step during dissociation of neonatal rat hearts. These cultures contained cells of at least 3 different morphologies:
- Triangular or polygonal shape with clear cytoplasm and a "bulk" volume that allowed the membrane to raise slightly over the bottom of the dish; these cells clustered in monolayers, reminiscent of those seen in cultures of epithelial cells, and they were positive for E-cadherin (see supplemental Figure II).
- Cells of bigger dimensions and clear cytoplasm that laid flat over the bottom of the dish; consistent with the morphology of fibroblasts.
- Contractile cells of smaller dimension and darker cytoplasm (cardiac myocytes). The latter category represented not more than 5% of the total cell population.
Cells described in no. 1 were intensely positive (by immunofluorescence) for both PKP2 and Cx43 and amenable to Lipofectamine-mediated transfection. Silencing of PKP2 was confirmed by Western blot (supplemental Figure III). Drastic changes were observed on Cx43 distribution after PKP2 silencing. Results are shown in Figure 4. Figure 4A through 4C shows microscopic images of cultured cells in control conditions. Cells were labeled with antibodies detecting PKP2 (red; Figure 4A) and Cx43 (green; Figure 4B). An overlay image is shown in Figure 4C. As expected, both proteins preferentially localized at, or near, the cell membrane, with little or no signal originating from the intracellular space. Similar results were obtained when a scrambled RNAi sequence was transfected (Figure 4D through 4F). This pattern was significantly altered by pretreating cells with RNAi for PKP2. As shown in Figure 4G, PKP2 signal was minor or absent in cells treated with PKP2 RNAi, and loss of PKP2 signal correlated with drastic redistribution of Cx43 (Figure 4H and 4I). In this case, there was a significant increase in the amount of signal localized to the intracellular space, at the expense of the membrane-selective localization seen under control conditions. Overall, these results indicate that loss of PKP2 leads to significant remodeling of Cx43 in this cell population.
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Dye Transfer in Nonmyocyte Cardiac Cells
Loss of PKP2 expression (and Cx43 redistribution) correlated with changes in the extent of dye transfer in a cell cluster. A patch pipette filled with LY was used to enter the intracellular space of a cell in the center of a cluster (Figure 5A through 5C). The extent and time course of dye diffusion into surrounding cells was recorded. Figure 5D shows a fluorescent image of dye transfer in a group of cells maintained in untreated conditions (10 minutes after patch break; exposure time, 2 seconds). The dye diffused from the impaled cell (asterisk) into the cells in the outer layers, thus demonstrating the presence of dye-permeable gap junctions. A similar recording was obtained from a cell cluster pretreated with scrambled RNAi (Figure 5E). However, a different picture emerged from cells where PKP2 expression had been abolished. In that case (Figure 5F), the impaled cell filled rapidly with the dye but the number of cells receiving the dye was significantly reduced, and the average intensity of the fluorescent signal decreased significantly with distance. A summary of data is presented in Figure 5G and 5H. Two parameters were measured: mean fluorescence intensity per cell layer (Figure 5G) and percentage of dye-positive cells within each successive layer (Figure 5H). A partial effect of the scrambled construct was observed. The reasons for this effect are unclear; the transfection procedure may have caused a certain degree of damage (perhaps intracellular acidification or a rise in intracellular calcium) that reflected in dye coupling. Yet, a much larger effect was observed in the PKP2-silenced cultures, and the effect was significant when compared with either scrambled or control. Overall, the data show that loss of PKP2 expression led to a decrease in the ability of LY to diffuse between cells.
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PKP2-Cx43 Crosstalk in EPDCs
The phenotypic characteristics of the cells presented in Figures 4 and 5
were consistent with those of EPDCs.15 To obtain a less heterogeneous cell population, we cultured EPDCs directly from explants of neonatal rat hearts using the technique of Chen et al16 After 8 days in culture, cells were transfected with either siRNA-PKP2 or the scrambled construct. Figure 6A through 6C (untreated) and Figure 6D through 6F (scrambled) show the characteristic colocalization of PKP2 and Cx43. In contrast, cells treated with siRNA-PKP2 revealed the loss of detectable PKP2 and a significant redistribution of Cx43 (Figure 6G through 6I), mostly to compartments within the perinuclear space (similar pattern also observed in Figure 4H and 4I). Consistent with observations in cardiac myocytes, Western blot for Cx43 in EPDCs demonstrated a reduction in total Cx43 protein content and a preservation of the ratio of low versus high-mobility bands (Figure 6H). It is worth noting that loss of PKP2 expression did not cause significant remodeling of the adherens junction protein cadherin (see supplemental Figure IV). Overall, these data confirm that PKP2 silencing leads to Cx43 remodeling, even in the absence of mechanical strain imposed by regular beating. Whether Cx43 and PKP2 are able to interact, directly or indirectly, was assessed by the experiments described below.
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PKP2 and Cx43 As Part of a Common Macromolecular Complex
Figure 7, left, shows that a recombinant protein corresponding to the head domain of PKP2 (PKP2H) can pull down Cx43 out of a rat heart lysate. GST-fused PKP2H was bound to glutathione beads and incubated either in the absence (lanes marked "minus") or presence of a cell lysate obtained from an adult rat heart (lanes marked "plus"). Two controls were used: a GST protein (left 2 lanes) and GST fused to the cytoplasmic loop domain of Cx43 (GST–loop). GST alone failed to pull down Cx43 whereas, as expected,17 GST–loop brought down cardiac Cx43. More importantly, a Cx43-immunoreactive protein of the appropriate size was recovered from the precipitate of beads coated with GST–PKP2-H, indicating an interaction between the 2 proteins. The reverse pull down is shown at right in Figure 7, where GST-Cx43CT (a recombinant protein corresponding to the CT domain of Cx43) pulled down PKP2 out of both a mouse heart (last lane) and a rat heart extract (second-to-last lane). Additional studies showed that wild-type PKP2 and Cx43 were coimmunoprecipitated from rat heart lysate (see supplemental Figure V). Overall, the results indicate that Cx43 and PKP2 are part of a common macromolecular complex and that the presence of PKP2 is necessary for the proper distribution and function of Cx43 channels.
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Discussion |
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In recent years, investigators have proposed that structures involved in mechanical coupling may crosstalk with those involved in maintaining electrical synchrony.18–19 This concept, interesting from the point of view of basic sciences, has gained relevance after the discovery that a number of cases of ARVC may be linked to mutations in desmosomal proteins2 and that in those cases studied, diseased hearts showed remodeling of gap junction plaques.8–9 Here, we demonstrate that loss of PKP2 leads to redistribution of Cx43 protein inside the cell, loss of gap junction plaques detectable by conventional immunofluorescence, and a reduction in the ability of LY to diffuse from cell to cell. This is the first demonstration of a link between PKP2 disruption and partial loss of Cx43-mediated cell–cell communication. Our studies further show that crosstalk between desmosomes and gap junctions is not necessarily caused by the mechanical strain imposed by the contractile forces in the beating heart. The data suggest that PKP2 may regulate (and/or coordinate) the formation and interaction of mechanical and electrical junctional complexes. The mechanism through which these 2 molecular entities (PKP2 and Cx43) interact is likely indirect and remains to be determined. Yet, our data show that PKP2 and Cx43 can be present within a common macromolecular complex. As such, our data are consistent with the possibility of a molecular crosstalk mediating gap junction remodeling subsequent to disruption of the desmosome.
The possible interaction of gap junctions with other intercalated disc structures is further emphasized by the recent studies of Shaw et al.20 These authors showed that Cx43 is targeted for delivery near adherens junctions in the membrane through microtubule plus ends. Their studies further demonstrated that siRNA-mediated silencing of ß-catenin (an integral protein of the adherens junction) compromised this interaction and resulted in diminished gap junction plaque size.20 Interestingly, both PKP2 and Cx43 have been shown to interact with ß-catenin.6,21 Although further experiments are required, it is tempting to speculate that ß-catenin may be an important link in the crosstalk between Cx43 and PKP2 and in the integration of gap junctions, adherens junctions, and desmosomes at the intercalated disc.
We have conducted experiments using not only cardiac myocytes but also EPDCs. This cell system allowed us to study the fate of Cx43 after PKP2 silencing in a nonbeating preparation. Yet, we should note that EPDCs are not completely void of mechanical strain, because loss of desmosomes in epithelia may be accompanied by a certain degree of cell retraction. Although our results indicate that cadherin remained present at (or near) the cell membrane after PKP2 silencing (see the online data supplement), preservation (or not) of adherens junctions after PKP2 silencing remains to be determined. On the other hand, the coexistence of PKP2 and Cx43 in heart precipitates strongly suggests that the fate of Cx43 could be linked to the presence of PKP2 through an intermolecular crosstalk between the 2 molecules, likely mediated through common molecular partners.
In addition to their utility as an experimental cell system, the study of EPDCs may bear relevance to the understanding of ARVC. These cells are progenitors of the cardiac fibroblast cell lineage.22 Recent studies show that in the presence of appropriate agonists, EPDCs retain their ability to proliferate and differentiate not only during fetal and neonate stages but also during adult life.23–24 The hypothesis emerges as to whether ARVC-relevant mutations can alter the balance of epicardial–mesenchymal transformation in EPDCs, and, if so, whether this disruption is a factor in the fibrofatty infiltration that characterizes the disease. Previous studies show that a reduction in Cx43 expression significantly affects motility and proliferation of proepicardial cells,25 and other investigators have documented the importance of cell–cell adhesion in the control of epithelial–mesenchymal transformation.26–27 As such, a role for PKP2 and/or Cx43 on the migratory, proliferative, and metaplastic behavior of the epicardium seems plausible.
Our studies show that loss of PKP2 expression leads to Cx43 remodeling. We speculate that in the setting of ARVC, reduced expression of wild-type PKP2 (as it occurs in patients afflicted with dominant mutations) could in itself alter the integrity of the junctional structures. In that regard, it is worth noting that heterozygous deletion of desmoplakin leads to fibrofatty infiltration in mice.28 Whether a similar disruption (and/or arrhythmogenesis) occurs in PKP2 heterozygous mice remains to be determined.
Although the actual intracellular compartment hosting Cx43 after PKP2 silencing remains to be defined, our images suggest that Cx43 is more concentrated in the perinuclear region. Future studies will characterize the specific intracellular compartment, and the coexistence of other intercalated disc proteins in the same compartment, following loss of PKP2. Similarly, it will be interesting to determine whether expression of ARVC-relevant PKP2 mutants, rather than silencing of the wild-type protein, leads to a similar redistribution of Cx43.
Our data indicate that loss of PKP2 expression leads to a decrease in the total content of Cx43. Future studies will address whether the decrease in Cx43 content is attributable to changes in gene transcription, protein synthesis, protein degradation or a complex combination of these factors. Moreover, it is surprising that despite the loss of gap junction plaques, the relative density of low versus high mobility bands remained constant. In fact, previous studies have indicated that the mobility of Cx43 in SDS-PAGE correlates with its phosphorylation state29 and that internalization of Cx43 associates with a decrease in the phosphorylated form of the protein.30 Further studies will be necessary to address this apparent inconsistency. One possibility is that the internalized (presumably dephosphorylated fraction) is also degraded at a faster rate. The latter could explain the combination of a total decrease in Cx43 content but an apparent preservation of the high- versus low-mobility ratio.
Our data show a drastic loss of Cx43 gap junction plaques and yet only a decrease of cell–cell dye coupling. It is important to note that gap junction plaques do not define the location of all gap junctions, because the optical resolution of our system is incapable of discerning for the presence of isolated areas where functional gap junctions may remain. Furthermore, previous studies have shown that Cx43 is not the only connexin expressed in heart cells. Expression of Cx45 has been reported both for ventricular myocytes and for cardiac fibroblasts,31 and changes in Cx45 content in human failing hearts have been reported.32 Additional studies in our laboratory indicated that Cx45 is present in our preparations, and its expression may be slightly increased after PKP2 silencing, in a manner similar to that detected by Yamada et al32 (supplemental Figure VI). It is indeed possible that Cx45 provides a pathway for dye coupling in our preparations, which may remain available after PKP2 silencing.
The preservation of dye coupling suggests that electrical coupling may be maintained (although to a lesser extent than in control), even after total loss of PKP2. Whether the decrease in functional gap junctions is enough to explain arrhythmogenesis remains unclear. Previous studies show that a significant reduction in Cx43 content is necessary to modify conduction velocity,33–34 although even a 50% reduction in Cx43 content can increase the susceptibility to arrhythmias under conditions such as ischemia.35 Our experiments have only explored the presence and the function of gap junctions. Other ion channel proteins, particularly those with preference for localization at the intercalated disc36 may also be affected by the loss of desmosomal coupling. Cx43 remodeling may be one of a number of factors that provide a substrate for arrhythmias during ARVC. Finally, it is worth noting that gap junctions allow not only for passage of electrical currents but of molecular signals as well.37 Thus, loss of functional gap junctions may play an important role in the coordination of cellular events (such as apoptosis or fibrofatty infiltration) that are fundamental to the natural history of ARVC.
In summary, this is the first demonstration of an intermolecular crosstalk between PKP2 and Cx43. The limitations of our experimental system notwithstanding, our results support the idea that changes in gap junctions may participate in the pathophysiological processes leading to ARVC in a subgroup of the afflicted population. The exact mechanisms mediating this molecular crosstalk and its consequences to the synchronization of cellular events within the heart will be determined in future studies.
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Acknowledgments |
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We thank Li Gao and April Lazarus for generating the viruses and Halina Chkourko and Stephanie Boeckmann for assistance with experiments.
Sources of Funding
This work was supported by NIH grants HL39707 and GM057691. E.O. was supported by a Predoctoral Fellowship from the American Heart Association.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received November 8, 2006; resubmission received April 18, 2007; revised resubmission received July 3, 2007; accepted July 24, 2007.
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