A Connexin43-Binding Peptide That Prevents Action Potential Propagation Block
Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP–RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing ≈20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP–RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pHi 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP–RXP-E–treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.
Connexins (Cxs) are integral membrane proteins that oligomerize to form intercellular channels called gap junctions (GJs). The most abundant GJ protein in a number of mammalian systems is Cx43. GJs allow passage of ions and small molecules between cells and are regulated by a variety of chemical interactions between the Cx molecule and the microenvironment. As such, GJs act as active filters to control passage of intercellular messages and modulate function.
Our previous work has suggested that regulation of Cx43 results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the Cx molecule acting as a receptor for the gating particle.1,2 Additional studies have shown that this intramolecular particle–receptor interaction can be modulated by other intermolecular interactions in the microenvironment of the GJ plaque.3–5 Based on the particle–receptor model, we reasoned that regulation of Cx43 could be disrupted by the binding of exogenous molecules to regions of the gating particle required for its interaction with the receptor. The latter rationale led us to the identification of a 34-aa peptide dubbed “RXP-E.”6 This peptide bound in vitro to Cx43CT with an apparent KD of 3.9 μmol/L, modified the structure of Cx43CT, partially prevented octanol-induced and acidification-induced uncoupling of N2a cells transfected with exogenous Cx43, and caused a significant prolongation of the single channel open time.6 Accordingly, we proposed that RXP-E could be developed as a tool to address the relevance of preserved intercellular communication in the maintenance of electric synchrony in the heart. Here, we show that RXP-E binds to native cardiac Cx43, prevents the closure of cardiac GJs, can be introduced into multicellular preparations, and can prevent heptanol- and acidification-induced action potential propagation block in monolayers of neonatal cardiac myocytes. Our results suggest that RXP-E can be used as a platform for development of a pharmacophore that could interfere with Cx43 regulation. The potential of GJ modifiers as antiarrhythmic agents has been described before.7–10 However, the identity of the molecular target for existing compounds is unknown, thus making them unsuitable for optimization by targeted drug design. Our results identify RXP-E as the first molecule known to bind Cx43 and, in doing so, preserve action potential propagation in cardiac myocytes under conditions otherwise expected to induce propagation block.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. Brief descriptions are presented below.
Recombinant Protein Production and Immunochemical Assays
All immunochemical protocols, as well as production of recombinant glutathione S-transferase (GST) fusion proteins and the GST pull-down assays, followed standard techniques.1,11 Details are provided in the online data supplement.
Myocyte Isolation and Culture
Primary cultures of neonatal rat ventricular myocytes (NRVMs) for patch-clamping, immunofluorescence, and monolayers were obtained using established procedures.12–14 After dissociation, cells were resuspended in supplemented medium 199 and preplated for 2 hours to reduce the presence of noncardiomyocytes. Cells were plated on 35-mm tissue culture dishes at a density of 1.2×106 cells per dish for monolayers and at low density on 22-mm coverslips for patch-clamp experiments. For immunofluorescence, cells were seeded onto 22-mm coverslips at a density of 5.0×105 cells per coverslip.
Translocation of RXP-E Into NRVMs
Peptides were concatenated with a cytoplasmic transfer peptide (CTP)15 sequence for translocation into the cytoplasm of NRVMs. Peptide synthesis was carried out by a commercial supplier (Anaspec Inc). Purity of peptide products was >85%.
Determination of GJ Currents
Experiments were performed in NRVM pairs endogenously expressing Cx43. The dual whole-cell voltage clamp technique was used to record GJ currents, as previously described.6,16,17
Determination of Nav1.5 Currents
Voltage clamp experiments were conducted in HEK293 cells (ATCC no. CRL1573) transfected (Effectene Transfection Kit, Qiagen) with human SCN5A cDNA subcloned in a pcDNA3.1 vector. The protocol and solutions used for recording sodium current from HEK293 cells stably expressing the SCN5A gene are described in the online data supplement.
Determination of Kir2.1 and Kir2.3 Currents
Kir2.1 and Kir2.3 currents were recorded from HEK293 cells (ATCC no. CRL1573) using established procedures.18,19
Effect of Heptanol Superfusion on Ionic Currents
The effect of heptanol on cardiac GJ currents, as well as in the amplitude of Nav1.5 and Kir2.1 to 2.3 currents, was assessed in the presence and absence of RXP-E in the internal pipette solution using methods previously described.6
Effect of Intracellular Acidification on Junctional Currents
Experiments were conducted to assess the effect of RXP-E on acidification-induced uncoupling of NRVMs. Intracellular acidification was induced by filling the patch pipettes with a solution buffered to pH 6.2 (see the online data supplement). Junctional conductance (Gj) was measured immediately after patch break and for the following 10 minutes.
Optical mapping was used to study action potential propagation in monolayers of NRVMs. Measurements were performed in cells kept for 3 to 4 days in culture. Preparations were stained for 15 minutes with a voltage-sensitive dye (di-8-ANEPPS) (40 μmol/L; Molecular Probes) to optically determine the characteristics of impulse propagation. A custom-made setup was used to assess changes in fluorescence that correspond to transmembrane voltage changes. Details are described elsewhere.20
Heptanol superfusion was initiated after 3 minutes of successful pacing. Heptanol (1 to 2 mmol/L, as specified) was prepared in HBSS solution. Heptanol perfusion was continued for 5 minutes, followed by washout. All monolayers demonstrated complete washout of heptanol, as established by return to paced propagation.
Intracellular Acidification in Monolayers of NRVMs
Intracellular acidification was induced by modifying the proton concentration of a Na-acetate superfusing solution. Intracellular pH calibration was carried out as described in the online data supplement (Figure I in the online data supplement).
We have previously shown RXP-E binding to recombinant Cx43CT.6 Here, we explored whether RXP-E is able to interact with cardiac Cx43. A GST–RXP-E recombinant protein was bound to glutathione beads and used as a bait to pull-down Cx43 from rat heart lysate. Parallel negative control experiments were conducted using either a GST protein (without RXP-E) or a construct coding for a “scrambled” version of RXP-E (GST-Scr) (see Materials and Methods and elsewhere6). The presence or absence of Cx43 in the lysate or in the precipitate was assessed by Western blot using an antibody against the N-terminal domain of Cx43. Results are shown in Figure 1A. A Cx43-immunoreactive band of the appropriate size was recovered from the precipitate of beads coated with GST–RXP-E and exposed to heart lysate (fourth lane from the left). On the other hand, no signal was obtained from samples obtained after exposure of heart lysate to either GST, or GST-Scr, or from coated beads that were not exposed to heart lysate. Additionally, the density of the Cx43-immunoreactive band was found to be dependent on the concentration of the heart lysate, as demonstrated in Figure 1B. Overall, the results indicate that RXP-E can interact with native cardiac Cx43.
Effect of RXP-E on Heptanol-Induced Uncoupling of NRVMs
The ability of RXP-E to bind cardiac Cx43 led us to propose that this peptide may also alter the behavior of cardiac GJs. Whole cell GJ currents were recorded from NRVMs via dual-whole-cell voltage clamp. Junctional current amplitude was measured every 20 seconds. All cell pairs showed an initial Gj value of <35 nS. Ten minutes after patch break, heptanol superfusion (1 mmol/L) was initiated. The time course of heptanol-induced changes in Gj is shown in Figure 2A. Data were obtained from pairs recorded in control conditions (black squares; n=10) or when the internal pipette solution contained either RXP-E (red circles; n=10), or the scrambled version of the peptide (green triangles; n=10). Peptide concentration in all cases was 0.05mmol/L. The plot correlates percentage of Gj (relative to control) as a function of time after onset of heptanol superfusion. Clearly, heptanol exposure led to a rapid drop in electric coupling either in control, or in the presence of the scrambled RXP-E peptide. However, all cell pairs recorded in the presence of RXP-E remained electrically coupled (ie, Gj did not reach 0) throughout continuous heptanol superfusion. The average Gj decreased only to 71.8% of control, and the average Gj measured 10 minutes after onset of heptanol superfusion from cells kept in control was significantly different from that measured from cells dialyzed with RXP-E (P<0.05).
RXP-E Partially Prevented Acidification-Induced Uncoupling in NRVMs
Next, we assessed whether RXP-E can interfere with the extent and time course of uncoupling induced by reduced intracellular pH (pHi). Patch pipettes were filled with a 2(N-morpholino) ethanesulfonic acid (MES)-containing solution, buffered to a pH of 6.2. Junctional current was measured immediately after patch break and every 20 seconds thereafter. Figure 2B shows the results. In the absence of RXP-E, Gj decreased progressively, reaching 1.3% of control within 10 minutes after patch break (black squares; n=10). In the presence of RXP-E (red circles; n=10), a decrease in Gj was also observed, but it was significantly dampened; after 10 minutes, average Gj decreased only to 71.2% of the initial value. This value was significantly different from that recorded in control (P<0.05). Interestingly, scrambled RXP-E did not disrupt acidification-induced uncoupling (green triangles; n=10; P>0.05 when compared to control). Overall, the data show that RXP-E partially prevented closure of Cx43 channels consequent to a reduction in pHi.
Does RXP-E Prevent the Effect of Heptanol on Nav1.5 Currents?
Our results show that RXP-E prevents heptanol-induced closure of GJ channels. Heptanol is known to affect sodium currents as well.21 Here, we asked whether RXP-E can interfere with the effect of heptanol on the ionic current obtained after expression of the SCN5A gene in transfected HEK293 cells. Figure 3A shows the results. Peak current amplitude was plotted as a function of the test voltage (see Materials and Methods for details). From each cell, a complete current–voltage relation was obtained before and 10 minutes after onset of superfusion with heptanol (2mmol/L). To minimize variability between experiments, the amplitude of the peak current recorded at each voltage step was normalized to maximum peak current amplitude obtained from the same cell before heptanol superfusion. As expected, heptanol caused a drastic reduction in the amplitude of the Nav1.5 current. Yet, as opposed to what we observed in the case of GJs, the effect of heptanol was the same regardless of whether or not RXP-E was present in the pipette solution. Peak current amplitude was observed at a command potential of −35±5 mV for control (black symbols; n=12), and −30±5 mV in the presence of RXP-E (red symbols; n=12; P>0.05). After heptanol, the command voltage that elicited maximum peak current amplitude was −30±5 mV for control and −30±5 mV in the presence of RXP-E (P>0.05 when values before and after heptanol were compared). Moreover, the relative reduction of maximum peak current amplitude was 36.6±4.8% for control and 30.5±5.1% in the presence of RXP-E (green and blue symbols, respectively; P>0.05). Overall, the data show that RXP-E does not interfere with heptanol-induced reduction of sodium current amplitude.
RXP-E and Inward Rectifier Currents
Further assessment of RXP-E specificity involved measurements of Kir2.1 and Kir2.3 currents recorded from double stably transfected HEK293 cells. Figure 3B shows the results. Current–voltage relations were generated as detailed in Materials and Methods. In this case, currents were normalized to the amplitude measured at −100 mV. Kir2.1 and Kir2.3 currents were not affected by heptanol, and no significant shifts in the current–voltage relation were recorded in the presence of RXP-E.
From Cell Pairs to Multicellular Preparations: Transfer of RXP-E to the Intracellular Space in Multicellular Preparations
Our results show that RXP-E can prevent closure of GJs in cardiac cell pairs. As a next step, we developed an assay for introducing RXP-E into multicellular preparations. The latter was necessary for assessment of the effect of RXP-E on action potential propagation in cardiac cells. Monolayers of NRVMs were exposed to synthetic fusion peptides containing a cytoplasmic transduction peptide (CTP512 domain; sequence, YGRRARRRRRR15). A cysteine residue at the C terminus allowed us to conjugate fluorescein isothiocyanate (FITC) to the peptides for detection by fluorescence microscopy. Synthetic peptides were created for both RXP-E and its scrambled version. Figure 4A shows an example of peptide transfer into NRVMs. Monolayers were prepared from the hearts of neonates (1-day old); cells were cultured for 6 days. The top image was obtained from cells maintained in control conditions (no CTP–RXP-E). The image in the bottom was obtained from cells previously incubated with CTP–RXP-E (0.1 mmol/L) for 2 hours. Cx43 was immunolabeled (red) and nuclei were stained (Hoechst33528; blue) for easier identification of individual cells. No FITC signal was present in control; however, intense signal was recorded from all cells that had been preincubated with CTP–RXP-E. These experiments were repeated, this time with the scrambled CTP–RXP-E, as shown in Figure 4B. Top images correspond to cells not presented with the peptide. The bottom images were recorded after cells were incubated for 30 minutes with a solution containing 0.1 mmol/L of scrambled CTP–RXP-E. Right images show the overlay of the blue (nuclei) green (fluorescein-labeled RXP-E) and red (Cx43) signals. Left images show the DIC image of the myocyte monolayers. Clearly, a bright green fluorescein signal was detected from all cells within the field, indicating successful peptide translocation. Signal was detected up to 18 hours after peptide incubation (not shown). Further confirmation of successful transfer into almost 100% of the cells was obtained by flow-cytometric detection of the FITC signal from NRK cells (not shown). Similarly timed transduction experiments were repeated in NRK cells (supplemental Figure II). Overall, the data show that CTP allows for efficient transfer of FITC-labeled peptides into the cytoplasm of rat neonatal cardiac myocytes. We used this translocation protocol to test for the effect of RXP-E on action potential propagation in monolayers of NRVMs.
Effect of RXP-E on Action Potential Propagation
The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping. Monolayers of NRVMs were labeled with a voltage-sensitive dye (di-8-ANEPPS; 40 μmol/L) and a high-resolution, high-speed camera was used to record the electric activity in the 35-mm monolayer (see elsewhere20; see also Materials and Methods for details). A stimulating electrode was placed in the center of the preparation (see the left images in Figure 5). The electric activity propagated from the center toward the periphery of the preparation, as shown by the activation map displayed in the right images of Figure 5A. (Notice, however, the “shadow” of the stimulus electrode, preventing view of activity at the site of stimulation.) Overall, conduction velocity in normal saline solution was unaffected by the presence or absence of the RXP-E peptide. Indeed, the average conduction velocity of all experiments in monolayers not treated with CTP peptides was 164±8 mm/sec (n=12). This value was not statistically different from those obtained from monolayers in which either CTP–Scr–RXP-E or CTP–RXP-E had been translocated into cells (conduction velocity, 158±10 mm/sec, n=6, and 180±7 mm/sec, n=10, respectively). Exposure of nontreated monolayers to 2 mmol/L (n=4) or even 1 mmol/L (n=4) heptanol, caused a total loss of propagated activity (see Figure 5B) that was restored on washout. Similar results were obtained from monolayers treated with CTP–Scr–RXP-E (2 experiments at each heptanol concentration; complete conduction block in all 4 cases). However, a different result was obtained from CTP–RXP-E–treated monolayers. In that case, the propagated activity observed in normal saline solution (Figure 5C) was not interrupted by exposure to the uncoupler (Figure 5D). Indeed, when the higher dose of heptanol (2 mmol/L) was added to the superfusate, action potential propagation was maintained, albeit at a slower velocity. In average, conduction velocity in CTP–RXP-E–treated monolayers exposed to 2 mmol/L of heptanol was 87±5 mm/sec (n=4; P<0.001 by paired t test when compared to conduction velocity before heptanol superfusion in the same preparation). The result was consistent with the ability of RXP-E to preserve GJ communication but not to prevent the effect of heptanol on sodium currents.
RXP-E also prevented action potential propagation failure caused by acidification of the intracellular space. The recordings shown in Figure 6A were obtained from a preparation not treated with peptide. When the bathing solution was kept at normal pH (7.4), action potentials propagated through the preparation with a conduction velocity of 180±18 mm/sec. When the same preparation was exposed to a low pH solution (intracellular pH 6.2, as estimated by the protocol described in Materials and Methods), propagation failure was observed (n=6; see Figure 6B). Propagation failure was also observed in 2 monolayers pretreated with CTP–Scr–RXP-E. Yet, in preparations pretreated with CTP–RXP-E, propagation was maintained despite acidification of the intracellular space (see Figure 6C), although conduction velocity was significantly decreased (average conduction velocity, 93±28 mm/sec; n=4, P<0.05 when compared by paired t test with values at normal pH measured from the same preparation). Overall, these results demonstrate that RXP-E can preserve action potential propagation under conditions which consistently induce propagation block.
Previous work from our laboratory, using high-throughput phage display analysis, led to the identification of the “RXP” sequence as a Cx43CT binding motif.6 Of the sequences identified, a particular 34-aa peptide (RXP-E) was shown to prevent heptanol- and acidification-induced uncoupling of N2a cells exogenously expressing Cx43.6 Here, we show that RXP-E: (1) binds to endogenous cardiac Cx43; (2) prevents heptanol and acidification-induced uncoupling of cardiac GJs; (3) can be introduced into monolayers of cardiac myocytes using a peptide translocator; and (4) preserves action potential propagation among cardiac myocytes. These results strongly suggest that RXP-E can be used as a platform for the development of a pharmacophore that could interfere with Cx43 regulation. Overall, the results identify RXP-E as the first Cx43-binding molecule capable of preserving action potential propagation in cardiac myocytes under conditions otherwise expected to induce propagation block.
The utility of peptidic molecules targeting intracellular domains is limited by the fact that, in general, peptides are not membrane-permeable molecules. Recent advances, however, have made it possible to translocate peptides into the intracellular space. A particular strategy consists of fusing the sequence of interest with a “cell-penetrating peptide” (CPP).22–24 For our studies, we concatenated RXP-E with a cytoplasmic transduction peptide (CTP), as described by Kim et al.15 Our results show that a fluorescein-labeled molecule abundantly localized to the cytoplasm, though, showed no preference for junctional membranes. Whether the CTP–RXP-E–FITC complex remained as concatenated inside the cells remains to be determined. If peptide cleavage occurred, the FITC signal would not pinpoint the exact location of RXP-E but, rather, of the cleaved FITC molecule. If, on the other hand, the complex remained intact, our results suggest that RXP-E distributes diffusely within the cell. This may be a reflection of the limited concentration capacity of the peptide at the site of cell–cell apposition, consistent with its low binding affinity to Cx43.6 What is evident from our data are that the peptide modified GJ function and prevented propagation block, suggesting that, although its affinity for Cx43 may be low (see Shibayama et al6), its efficacy to modify GJ function was preserved. Overall, the CTP system allowed for pharmacological manipulation of a molecular target previously “protected” by the cell membrane. These and other techniques will be useful to determine whether modulation of the pH regulatory mechanism of Cx43 can modify the likelihood of cardiac arrhythmias under conditions that cause acidification of the intracellular space.
Conduction Velocity and Effect of RXP-E
It is worth noting that the propagation velocities measured in our experiments were lower than those reported by other authors.20,25 A likely explanation is that our preplating and culture methods did not completely remove fibroblasts from the cell preparation.26 In fact, ancillary experiments in which we immunolocalized nonmyocyte cells using a α-smooth muscle actin antibody (Anaspec Inc; data not shown) indicated that ≈25% of the cells were nonmyocytes. Our conduction velocities, therefore, correspond well with those measured by Miragoli et al.25 The presence of fibroblasts is unlikely to affect the outcome of our results. In fact, it is interesting to note that a number of cardiac pathologies associate with deposition of fibroid tissue and, likely, fibroblasts electrically coupled to the cardiac myocytes.26,27 It is also of interest that RXP-E by itself did not modify propagation. Conduction velocities measured from samples kept in control conditions were not different from those preloaded with RXP-E. This is similar to results obtained with other GJ openers28,29 and supports the notion that, in a well-coupled system of cells, GJ modification minimally affects propagation.30,31 Yet, once the system was challenged, RXP-E was capable of preserving action potential propagation, albeit at a slower velocity than in control. The latter is consistent with the observation that RXP-E did not prevent heptanol-induced decrease in sodium current amplitude (see Figure 3). In addition, because other Cx isoforms also express in NRVMs, it is possible that the observed decreases in Gj (and conduction velocity) could reflect, at least in part, closure of those channels by heptanol or acidification. The effect (or lack thereof) of RXP-E on other Cxs remains to be determined. Or data do show that RXP-E preserves propagation albeit with a slower conduction velocity. Whether preservation of conduction at a slower velocity is an effective antiarrhythmic strategy, or instead, a proarrhythmic event, remains to be determined.
Pharmacology of GJs
It is unclear whether holding GJs open under pathological conditions such as ischemia would be beneficial or deleterious to the heart, although initial data seem to indicate that preservation of GJ communication may have a powerful antiarrhythmic effect (see studies with rotigaptide, previously known as ZP1238,9). However, among the reasons why such a gap in knowledge exists is that we lack pharmacological agents that can, with at least some partial selectivity, interfere with GJ regulation. (Much has been learned about the functional role of sodium channels, for example, from results obtained in the presence and absence of tetrodotoxin.) Our previous studies show that RXP-E can interfere with chemically induced closure of GJs. In the present study, we sought to determine the extent of selectivity of RXP-E action and its effect on propagation velocity and susceptibility to block.
Although other molecules (eg, cAMP) have been described that increase GJ coupling,28,32–36 their target remains elusive, and studies have not identified the Cx molecule itself as the target.37 Previous studies have determined that a small peptide (AAP10) can facilitate intercellular communication in heart preparations.38 Additional chemical modifications on AAP10 led to a compound initially identified as ZP123 (now rotigaptide). Experiments have suggested a potential therapeutic value to this molecule10 and, in general, to the approach of modifying GJ conductance to prevent or treat cardiac arrhythmias.8 Yet, although rotigaptide-based experiments suggest that GJ openers represent a promising strategy for antiarrhythmic therapy, this particular molecule lacks an identifiable target. (The molecule[s] acting as receptor for rotigaptide is not yet known.)8 The latter limits the possibility of conducting structure-activity studies to optimize the mechanisms of action of rotigaptide through target-based drug design. Moreover, recent studies suggest that rotigaptide may modify the catalytic activity of some kinases.39 Given the broad range of substrates and intracellular signaling pathways that can be affected by kinase modification, secondary effects on other cellular functions are likely. Here, we have used a different approach, that is, to select the target molecule (Cx43CT) as the bait for identification of the ligand (RXP-E). With this knowledge, we now demonstrate that RXP-E acts as a ligand for cardiac Cx43 and prevents the action of at least 2 well-known chemical uncouplers.
In conclusion, we have presented evidence indicating that a molecule derived from the RXP series of Cx43-binding molecules6 is capable of binding to cardiac Cx43, modifying cardiac GJs and preventing action potential propagation block. This is the first Cx43-targetted molecule capable of preserving electric conduction in a preparation of cardiac cells. Much remains to be done to determine the arrhythmogenic versus antiarrhythmic effect of GJ closure/opening under conditions of metabolic stress. Although pharmacological optimization and minimization is necessary, the results presented here suggest that RXP-E can serve as a starting point for the development of a target-based class of drugs centered on the objective of modifying cardiac GJ function.
We thank Viviana Muñoz, David Auerbach, and Sergey Mironov for assistance with optical mapping experiments and analysis.
Sources of Funding
This work was supported by NIH grants HL39707 and GM057691. R.L. was supported by a Predoctoral Fellowship from the American Heart Association. K.P. and M.S.N. were funded by the Danish National Research Foundation and the Danish Cardiovascular Research Academy.
Original received May 6, 2008; revision received July 15, 2008; accepted July 17, 2008.
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