Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
Circulation Research. 2007;101:703-711
Published online before print August 2, 2007, doi: 10.1161/CIRCRESAHA.107.154252
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
101/7/703    most recent
CIRCRESAHA.107.154252v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oxford, E. M.
Right arrow Articles by Delmar, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oxford, E. M.
Right arrow Articles by Delmar, M.
Related Collections
Right arrow Arrythmias-basic studies
Right arrow Cell biology/structural biology
Right arrow Gene expression
Right arrow Myocardial cardiomyopathy disease
Right arrowRelated Article
(Circulation Research. 2007;101:703.)
© 2007 American Heart Association, Inc.


Cellular Biology

Connexin43 Remodeling Caused by Inhibition of Plakophilin-2 Expression in Cardiac Cells

Eva M. Oxford*, Hassan Musa*, Karen Maass, Wanda Coombs, Steven M. Taffet, Mario Delmar

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


*    Abstract
up arrowTop
*Abstract
down arrowIntroduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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.


*    Materials and Methods
up arrowTop
up arrowAbstract
up arrowIntroduction
*Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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.


*    Results
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
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.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

 
Figure 1. Western blot for PKP2 or actin (bottom bands) obtained from NRVMs. Cells were either untreated or infected with a replication-deficient adenovirus containing cDNA coding for GFP or infected with adenovirus containing a construct designed to silence PKP2 expression (shRNA-PKP2). Concentration of viral particles is expressed in multiplicity of infection units. The experiment was conducted 5 days after viral infection (or 5 days after incubation in control [CTRL]). Notice the gradual decrease in PKP2 abundance subsequent to shRNA-PKP2 exposure.

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.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

 
Figure 2. Effect of shRNA-PKP2 on Cx43 content and distribution. A, Immunolocalization of PKP2 (red) or Cx43 (green) in NRVMs that were untreated (UNT) (left images) or treated with 100 mois of a virus containing a construct that did not silence PKP2 (shRNA-PKP2Ø) (middle images) or with 100 mois of shRNA-PKP2 (right images). Notice the loss of PKP2 expression and significant redistribution of Cx43 in RNA-PKP2–treated cells. B, Western blot for PKP2 and Cx43 in the same conditions used for A.

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).


Figure 3
View larger version (38K):
[in this window]
[in a new window]

 
Figure 3. Dye coupling between pairs of rat neonatal cardiac myocytes. Cells were either untreated (A) or infected with 100 mois of virus shRNA-PKP2 (B). Fluorescence micrographs were taken at 1, 5, and 9 minutes after patch break. The graph in C shows the rise in dye intensity in control and silenced donor cells ({blacksquare} and bullet) and in recipient cells ({square} and {circ}) 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:

  1. 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).
  2. Cells of bigger dimensions and clear cytoplasm that laid flat over the bottom of the dish; consistent with the morphology of fibroblasts.
  3. 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.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

 
Figure 4. Effect of siRNA-PKP2 on cardiac cells obtained from preplating dissociated neonatal rat ventricles. Phenotypic characteristics of cells were compatible with those of EPDCs. Cells were untreated (UNT) (A through C), treated with a scrambled oligonucleotide (SCR) that does not lead to PKP2 silencing (D through F), or treated with stealth RNAi-PKP2 (SIL) (G through I). Cells were stained for PKP2 (A, D, and G) and for Cx43 (B, E, and H). Merged images are shown at the bottom (C, F, and I). Notice the loss of PKP2 staining and remodeling of Cx43 subsequent to RNAi-PKP2 treatment.

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.


Figure 5
View larger version (96K):
[in this window]
[in a new window]

 
Figure 5. Intercellular transfer of LY in cells obtained at the preplating step of dissociation. Cell phenotype was consistent with that of EPDCs. A through C, Bright field. The asterisk represents site of dye injection. D through F, Fluorescent images demonstrating dye transfer. Cells were untreated (UN) (A and D), treated with a scrambled oligonucleotide (Scr) (B and E), or treated with RNAi for PKP2 (RNAi) (C and F). Notice the reduction in dye transfer after PKP2 silencing. G, Fluorescence intensity, normalized to intensity in the injected cell, as a function of number of cell layers traversed by the dye. Exponential decay constants were 1.40±0.15, 1.29±0.03, and 0.80±0.06 for untreated, scrambled, and RNAi, respectively. A t test returned the following values for the decay constants calculated: scrambled vs control: P=0.07; silenced vs scrambled: P<0.001; silenced vs control: P<0.001. H, Percentage of dye-positive cells at each cell layer under each condition. Data in layers 4 and 5 were statistically analyzed (ANOVA). **P<0.005; ***P<0.001. Number of experiments: 7, 11, and 14 for untreated, scrambled, and RNAi, respectively.

PKP2-Cx43 Crosstalk in EPDCs
The phenotypic characteristics of the cells presented in Figures 4 and 5Up 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.


Figure 6
View larger version (47K):
[in this window]
[in a new window]

 
Figure 6. Effect of siRNA-PKP2 on EPDCs that were untreated (UNT) (A through C), treated with a scrambled oligonucleotide (SCR) that does not lead to PKP2 silencing (D through F), or treated with stealth RNAi-PKP2 (SIL) (G through I). Cells were stained for PKP2 (A, D, and G) and for Cx43 (B, E, and H). Merged images are shown at the bottom (C, F, and I). Notice the loss of PKP2 staining and remodeling of Cx43 subsequent to RNAi-PKP2 treatment. H, Western blot of PKP2, Cx43, or actin (loading control) in untreated cells or cells treated with the scrambled construct or RNAi-PKP2. Notice the loss of PKP2 expression in the latter group and concurrent decrease in Cx43 content.

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.


Figure 7
View larger version (12K):
[in this window]
[in a new window]

 
Figure 7. Pull down of Cx43 by GST-PKP2H (left) and of PKP2 by GST-Cx43CT (right). – and + refer to the presence or absence of heart lysate, respectively. GST alone was used as negative control. A construct expressing the cytoplasmic loop domain of Cx43 (GST–loop) was used as positive control for the pull down of Cx43.17


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
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.


*    Acknowledgments
 
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.


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received November 8, 2006; resubmission received April 18, 2007; revised resubmission received July 3, 2007; accepted July 24, 2007.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 
1. Corrado D, Basso C, Thiene G, McKenna WJ, Davies MJ, Fontaliran F, Nava A, Silvestri F, Blomstrom-Lundqvist C, Wlodarska EK, Fontaine G, Camerini F. Spectrum of clinicopathologic manifestations of arrhythmogenic right ventricular cardiomyopathy/dysplasia: a multicenter study. J Am Coll Cardiol. 1997; 30: 1512–1520.[Abstract]

2. Sen-Chowdhry S, Syrris P, McKenna WJ. Genetics of right ventricular cardiomyopathy. J Cardiovasc Electrophysiol. 2005; 16: 927–935.[CrossRef][Medline] [Order article via Infotrieve]

3. Hatsell S, Cowin P. Deconstructing desmoplakin. Nat Cell Biol. 2001; 3: E270–E272.[CrossRef][Medline] [Order article via Infotrieve]

4. van Tintelen JP, Entius MM, Bhuiyan ZA, Jongbloed R, Wiesfeld AC, Wilde AA, van der Smagt J, Boven LG, Mannens MM, van Langen IM, Hofstra RM, Otterspoor LC, Doevendans PA, Rodriguez LM, van Gelder IC, Hauer RN. Plakophilin-2 mutations are the major determinant of familial arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circulation. 2006; 113: 1650–1658.[Abstract/Free Full Text]

5. Kowalczyk AP, Hatzfeld M, Bornslaeger EA, Kopp DS, Borgwardt JE, Corcoran CM, Settler A, Green KJ. The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease. J Biol Chem. 1999; 274: 18145–18148.[Abstract/Free Full Text]

6. Chen X, Bonne S, Hatzfeld M, van Roy F, Green KJ. Protein binding and functional characterization of plakophilin 2. Evidence for its diverse roles in desmosomes and beta -catenin signaling. J Biol Chem. 2002; 277: 10512–10522.[Abstract/Free Full Text]

7. Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W. Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation. J Cell Biol. 2004; 167: 149–160.[Abstract/Free Full Text]

8. Kaplan SR, Gard JJ, Carvajal-Huerta L, Ruiz-Cabezas JC, Thiene G, Saffitz JE. Structural and molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol. 2004; 13: 26–32.[Medline] [Order article via Infotrieve]

9. Kaplan SR, Gard JJ, Protonotarios N, Tsatsopoulou A, Spiliopoulou C, Anastasakis A, Squarcioni CP, McKenna WJ, Thiene G, Basso C, Brousse N, Fontaine G, Saffitz JE. Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm. 2004; 1: 3–11.[Medline] [Order article via Infotrieve]

10. Saffitz JE. Adhesion molecules: why they are important to the electrophysiologist. J Cardiovasc Electrophysiol. 2006; 17: 225–229.[CrossRef][Medline] [Order article via Infotrieve]

11. Gaudesius G, Miragoli M, Thomas SP, Rohr S. Coupling of cardiac electrical activity over extended distances by fibroblasts of cardiac origin. Circ Res. 2003; 93: 421–428.[Abstract/Free Full Text]

12. Sorgen PL, Duffy HS, Spray DC, Delmar M. pH-dependent dimerization of the carboxyl terminal domain of Cx43. Biophys J. 2004; 87: 574–581.[CrossRef][Medline] [Order article via Infotrieve]

13. Sobolik-Delmaire T, Katafiasz D, Wahl JK 3rd. Carboxyl terminus of plakophilin-1 recruits it to plasma membrane, whereas amino terminus recruits desmoplakin and promotes desmosome assembly. J Biol Chem. 2006; 281: 16962–16970.[Abstract/Free Full Text]

14. Valiunas V, Polosina YY, Miller H, Potapova IA, Valiuniene L, Doronin S, Mathias RT, Robinson RB, Rosen MR, Cohen IS, Brink PR. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions. J Physiol. 2005; 568: 459–468.[Abstract/Free Full Text]

15. Wada AM, Smith TK, Osler ME, Reese DE, Bader DM. Epicardial/mesothelial cell line retains vasculogenic potential of embryonic epicardium. Circ Res. 2003; 92: 525–531.[Abstract/Free Full Text]

16. Chen TH, Chang TC, Kang JO, Choudhary B, Makita T, Tran CM, Burch JB, Eid H, Sucov HM. Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor. Dev Biol. 2002; 250: 198–207.[CrossRef][Medline] [Order article via Infotrieve]

17. Duffy HS, Sorgen PL, Girvin ME, O’Donnell P, Coombs W, Taffet SM, Delmar M, Spray DC. pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains. J Biol Chem. 2002; 277: 36706–36714.[Abstract/Free Full Text]

18. Saffitz JE. Dependence of electrical coupling on mechanical coupling in cardiac myocytes. In: Thiene G, Pessina AC, ed. Advances in Cardiovascular Medicine. Padova, Italy: Universita degli Studi di Padova; 2003: 15–28.

19. Delmar M. The intercalated disk as a single functional unit. Heart Rhythm. 2004; 1: 12–13.[CrossRef][Medline] [Order article via Infotrieve]

20. Shaw RM, Fay AJ, Puthenveedu MA, von Zastrow M, Jan Y, Jan LY. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell. 2007; 128: 547–560.[CrossRef][Medline] [Order article via Infotrieve]

21. Ai Z, Fischer A, Spray DC, Brown AM, Fishman GI. Wnt-1 regulation of Connexin43 in cardiac myocytes. J Clin Invest. 2000; 105: 161–171.[Medline] [Order article via Infotrieve]

22. Fishman MC, Chien KR. Fashioning the vertebrate heart: Earliest embryonic decisions. Development. 1997; 124: 2099–2117.[Abstract]

23. Smart N, Risebro CA, Melville AA, Moses K, Schwartz RJ, Chien KR, Riley PR. Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization. Nature. 2007; 445: 177–182.[CrossRef][Medline] [Order article via Infotrieve]

24. van Tuyn J, Atsma DE, Winter EM, van der Velde-van Dijke I, Pijnappels DA, Bax NA, Knaan-Shanzer S, Gittenberger-de Groot AC, Poelmann RE, van der Laarse A, van der Wall EE, Schalij MJ, de Vries AA. Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells. 2007; 25: 271–278.[CrossRef][Medline] [Order article via Infotrieve]

25. Li WE, Waldo K, Linask KL, Chen T, Wessels A, Parmacek MS, Kirby ML, Lo CW. An essential role for connexin43 gap junctions in mouse coronary artery development. Development. 2002; 129: 2031–2042.[Medline] [Order article via Infotrieve]

26. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 2006; 7: 131–142.[CrossRef][Medline] [Order article via Infotrieve]

27. Dettman RW, Denetclaw W Jr, Ordahl CP, Bristow J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol. 1998; 193: 169–181.[CrossRef][Medline] [Order article via Infotrieve]

28. Gallicano GI, Kouklis P, Bauer C, Yin M, Vasioukhin V, Degenstein L, Fuchs E. Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage. J Cell Biol. 1998; 143: 2009–2022.[Abstract/Free Full Text]

29. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol. 2004; 36: 1171–1186.[CrossRef][Medline] [Order article via Infotrieve]

30. Musil LS, Goodenough DA. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol. 1991; 115: 1357–1374.[Abstract/Free Full Text]

31. Beauchamp P, Choby C, Desplantez T, de Peyer K, Green K, Yamada KA, Weingart R, Saffitz JE, Kleber AG. Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice. Circ Res. 2004; 95: 170–178.[Abstract/Free Full Text]

32. Yamada KA, Rogers JG, Sundset S, Steinberg TH, Saffitz JE. Up-regulation of connexin45 in heart failure. J Cardiovasc Electrophysiol. 2003; 14: 1205–1212.[CrossRef][Medline] [Order article via Infotrieve]

33. Thomas SP, Kucera JP, Bircher-Lehmann L, Rudy Y, Saffitz JE, Kleber AG. Impulse propagation in synthetic strands of neonatal cardiac myocytes with genetically reduced levels of connexin43. Circ Res. 2003; 92: 1209–1216.[Abstract/Free Full Text]

34. Danik SB, Liu F, Zhang J, Suk HJ, Morley GE, Fishman GI, Gutstein DE. Modulation of cardiac gap junction expression and arrhythmic susceptibility. Circ Res. 2004; 95: 1035–1041.[Abstract/Free Full Text]

35. Lerner DL, Yamada KA, Schuessler RB, Saffitz JE. Accelerated onset and increased incidence of ventricular arrhythmias induced by ischemia in Cx43-deficient mice. Circulation. 2000; 101: 547–552.[Abstract/Free Full Text]

36. Kucera JP, Rohr S, Rudy Y. Localization of sodium channels in intercalated disks modulates cardiac conduction. Circ Res. 2002; 91: 1176–1182.[Abstract/Free Full Text]

37. Goldberg GS, Valiunas V, Brink PR. Selective permeability of gap junction channels. Biochim Biophys Acta. 2004; 1662: 96–101.[Medline] [Order article via Infotrieve]


Related Article:

Molecular Crosstalk Between Mechanical and Electrical Junctions at the Intercalated Disc
Stephan Rohr
Circ. Res. 2007 101: 637-639. [Extract] [Full Text] [PDF]



This article has been cited by other articles:


Home page
Sports Health: A Multidisciplinary ApproachHome page
S. Gupta, T. Baman, and S. M. Day
Cardiovascular Health, Part 1: Preparticipation Cardiovascular Screening
Sports Health: A Multidisciplinary Approach, November 1, 2009; 1(6): 500 - 507.
[Abstract] [Full Text] [PDF]


Home page
Circ Arrhythm ElectrophysiolHome page
M. G.P.J. Cox, J. J. van der Smagt, A. A.M. Wilde, A. C.P. Wiesfeld, D. E. Atsma, M. R. Nelen, L.-M. Rodriguez, P. Loh, M. J. Cramer, P. A. Doevendans, et al.
New ECG Criteria in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy
Circ Arrhythm Electrophysiol, October 1, 2009; 2(5): 524 - 530.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
P. Y. Sato, H. Musa, W. Coombs, G. Guerrero-Serna, G. A. Patino, S. M. Taffet, L. L. Isom, and M. Delmar
Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes
Circ. Res., September 11, 2009; 105(6): 523 - 526.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
V. Verma, B. D. Larsen, W. Coombs, X. Lin, G. Spagnol, P. L. Sorgen, S. M. Taffet, and M. Delmar
Novel Pharmacophores of Connexin43 Based on the "RXP" Series of Cx43-Binding Peptides
Circ. Res., July 17, 2009; 105(2): 176 - 184.
[Abstract] [Full Text] [PDF]


Home page
NEJMHome page
F. Tavora, N. Creswell, A. P. Burke, M. A. Kohn, T. B. Newman, J. E. Saffitz, A. Asimaki, and S. Gautam
Arrhythmogenic right ventricular cardiomyopathy.
N. Engl. J. Med., June 25, 2009; 360(26): 2784 - 2785.
[Full Text] [PDF]


Home page
Circ Arrhythm ElectrophysiolHome page
P. G. Postema, P. F.H.M. van Dessel, J. M.T. de Bakker, L. R.C. Dekker, A. C. Linnenbank, M. G. Hoogendijk, R. Coronel, J. G.P. Tijssen, A. A.M. Wilde, and H. L. Tan
Slow and Discontinuous Conduction Conspire in Brugada Syndrome: A Right Ventricular Mapping and Stimulation Study
Circ Arrhythm Electrophysiol, December 1, 2008; 1(5): 379 - 386.
[Abstract] [Full Text] [PDF]


Home page
Cardiovasc ResHome page
N. J. Severs, A. F. Bruce, E. Dupont, and S. Rothery
Remodelling of gap junctions and connexin expression in diseased myocardium
Cardiovasc Res, October 1, 2008; 80(1): 9 - 19.
[Abstract] [Full Text] [PDF]


Home page
JEMHome page
U. Lisewski, Y. Shi, U. Wrackmeyer, R. Fischer, C. Chen, A. Schirdewan, R. Juttner, F. Rathjen, W. Poller, M. H. Radke, et al.
The tight junction protein CAR regulates cardiac conduction and cell-cell communication
J. Exp. Med., September 29, 2008; 205(10): 2369 - 2379.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
R. Lewandowski, K. Procida, R. Vaidyanathan, W. Coombs, J. Jalife, M. S. Nielsen, S. M. Taffet, and M. Delmar
RXP-E: A Connexin43-Binding Peptide That Prevents Action Potential Propagation Block
Circ. Res., August 29, 2008; 103(5): 519 - 526.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
S. Rohr
Molecular Crosstalk Between Mechanical and Electrical Junctions at the Intercalated Disc
Circ. Res., September 28, 2007; 101(7): 637 - 639.
[Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
101/7/703    most recent
CIRCRESAHA.107.154252v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oxford, E. M.
Right arrow Articles by Delmar, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oxford, E. M.
Right arrow Articles by Delmar, M.
Related Collections
Right arrow Arrythmias-basic studies
Right arrow Cell biology/structural biology
Right arrow Gene expression
Right arrow Myocardial cardiomyopathy disease
Right arrowRelated Article