Cardiac Sodium Channel Nav1.5 Is Regulated by a Multiprotein Complex Composed of Syntrophins and Dystrophin
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Bruno Gavillet, Jean-Sébastien Rougier,
参见附件。
the Department of Pharmacology and Toxicology (B.G., J.-S.R., R.B., C.B., H.A.), University of Lausanne, Switzerland
Service of Cardiology (H.A.), CHUV, Lausanne, Switzerland
Department for Cardiovascular Surgery (P.R.), CHUV, Lausanne, Switzerland
Institute of Pathology (H.-A.L.), CHUV, Lausanne, Switzerland
Deparment of Medicine (A.A.D., T.P.), CHUV, Lausanne, Switzerland.
Abstract
The cardiac sodium channel Nav1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Nav1.5 C-terminus. Pull-down experiments were performed using Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx5cv mice to study the role of this protein complex in Nav1.5 function. Western blot experiments revealed a 50% decrease in the Nav1.5 protein levels in mdx5cv hearts, whereas Nav1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx5cv cardiomyocytes. Finally, ECG measurements of the mdx5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Nav1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.
Key Words: Duchenne dystrophy dystrophin ECG mouse sodium channels syntrophin
Introduction
The main cardiac voltage-gated sodium channel, Nav1.5, generates the fast depolarization of the cardiac action potential, and plays a key role in cardiac conduction. Its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene SCN5A, which encodes Nav1.5, that are linked to various cardiac diseases.1 Among them, the congenital long QT syndrome type-3 and the Brugada syndrome are caused by gain or loss-of-function of Nav1.5, respectively.1 Nav1.5 is the pore-forming -subunit protein of the cardiac sodium channel. It has a molecular weight of 220 kDa, and may be associated with at least 4 types of auxiliary small (30 to 35 kDa) -subunits. Recently, several proteins that bind directly to Nav1.5 have been described.2 However, in most cases the physiological relevance of these interactions remains poorly understood, mainly because of a lack of appropriate animal models. With the exception of ankyrin-G,3 all these partner proteins interact with the 243-residues-long intracellular C-terminal domain of the channel which contains several protein-protein interaction motifs.2 Among them, the last three residues of Nav1.5 (2014-Ser-Ile-Val-2016) constitute a PDZ domain-binding motif to which syntrophins and dystrophin have been shown to interact directly or indirectly, respectively.4–6 However, thus far, the role of these interacting proteins in the heart has never been investigated.
In this study, by performing mass spectrometry-based protein identification using human and rodent cardiac lysates, we could confirm that dystrophin and syntrophin proteins are interacting specifically with the PDZ domain-binding motif of Nav1.5. Biochemical and electrophysiological studies using cardiac tissue and cells of dystrophin-deficient mdx5cv mice (an animal model of Duchenne muscular dystrophy [DMD]) revealed that Nav1.5 protein content as well as Nav1.5-mediated current (INa) were decreased in the absence of dystrophin. Finally, ECGs of mdx5cv mice showed alterations characteristic of conduction defects. These findings suggest that Nav1.5 is part of a multiprotein complex in which dystrophin and syntrophin proteins play an important role in its expression level. Moreover, these results provide the pathophysiological basis for some of the ECG abnormalities seen in DMD patients, and other dystrophinopathy patients, in whom cardiac arrhythmias and conduction defects may lead to sudden death.7
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals
Wild-type C57BL/6 mice, used as control, were purchased at Janvier (Le Genest St Isle, France), and C57BL/6Ros-5Cv (mdx5cv) mice (Jackson laboratories, Bar Harbor, Me) were raised in our department. Male mice at an age of 10 to 14 weeks were used in this study. All animal procedures were performed in accordance with the Swiss laws.
Cardiac Tissue Samples
The experimental procedures were approved by the ethical commission on clinical research of the faculty of medicine of the University of Lausanne. Human right atrial sample was collected from patients in normal sinus rhythm undergoing coronary bypass surgery. Mice were anesthetized and heart ventricles were excised, rinsed with ice cold PBS and frozen in liquid nitrogen. Fresh human atrial appendage or frozen mouse ventricle was transferred into lysis buffer. Tissues were then homogenized using a Polytron and a Teflon homogenizer. Triton Tx-100 was added to a final concentration of 1% and solubilization occurred by rotating for 1 hour at 4°C. The soluble fraction from a subsequent 15-minute centrifugation at 13 000g (4°C) was used for the experiments.
Mass Spectrometry Peptide Analysis
SDS-PAGE gels were stained with SYPRO dye (Molecular Probes, Eugene, Ore). The gel was visualized on a UV-light transilluminator. The bands of interest were excised and sent to the Protein Analysis Facility of Lausanne where they were submitted to trypsin digestion, liquid chromatography and tandem mass spectrometry.
Pull-Down Assays
The cDNAs encoding the 66 last amino acids of Nav1.5 WT, Y1977A, and S2014Stop were cloned into pGEX-4T1 (Amersham Bioscience, Piscataway, NJ). GST-fusion proteins were obtained using Escherichia coli. GST-pull-down assays of soluble fractions of ventricular lysate were performed using GSH-sepharose beads containing either GST or the GST fusion proteins. After 2-hour rotation and washing, bound proteins were detected by Western blot.
Western Blot and Antibodies
Western blotting conditions and the specificity of the polyclonal Nav1.5 antibody (ASC-005; Alomone) has been previously described.9 A control Western blot using the peptide antigen showing the specificity of the signal is presented in the online data supplement. The polyclonal antibody Kir2.1 (APC-026) was purchased from Alomone (Jerusalem, Israel). Polyclonal connexin-43 antibody (71-0700) was obtained from Zymed (San Francisco, Calif). Monoclonal dystrophin antibody (MANDYS8) was from SIGMA (Buchs, Switzerland). Monoclonal pan-syntrophin antibody (MA1–745) was purchased from Affinity Bioreagents (Golden, USA). Polyclonal 1-, 1-, and 2-syntrophin antibodies were kindly provided by M. Schaub (University of Zurich), and 1- and 2-syntrophin antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif). Polyclonal Cav1.2 antibody was a gift from J. Hell (University of Iowa), Na,K-ATPase antibody was a gift from K. Geering (University of Lausanne) and Nedd4–2 antibody was a gift from O. Staub (University of Lausanne).
TaqMan Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA was extracted from whole ventricles according to described protocol in.9 cDNA was synthesized from 1 μg of total RNA using the MU-MLV reverse transcriptase (Q-Biogene EMMLV100; Irvine, Calif). Fifty nanograms of cDNA combined with 1x TaqMan Universal Master Mix (Applied Biosystems, Foster) and 1 μL of either Nav1.5, Kir2.1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Applied Biosystems, respectively, Mm00451971, Mm00434616 and Mm99999915) were loaded into each well. GAPDH was used as reference gene for normalizing the data.
Isolation of Cardiac Myocytes and Patch-Clamp Experiments
Adult mouse ventricular myocytes were isolated as described.9 Whole-cell configuration of the patch-clamp technique was used to record INa. Experiments were performed at room temperature (22°C to 23°C), using a VE-2 (Alembic Instruments) amplifier allowing the recording of large sodium currents.9 Pipettes (tip resistance 1 to 2 M) were filled with a solution containing 60 mmol/L aspartic acid, 70 mmol/L cesium asparte, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES, 11 mmol/L EGTA, and 5 mmol/L Na2ATP (pH was adjusted to 7.2 with CsOH). Myocytes were bathed with a solution containing 10 mmol/L NaCl, 120 mmol/L NMDG-Cl, 2 mmol/L CaCl2, 1.2 mmol/L MgCl2, 5 mmol/L CsCl, 10 mmol/L HEPES, and 5 mmol/L Glucose (pH was adjusted to 7.4 with CsOH).
ECG
The mouse ECG was recorded as previously described.10 Corrected QT interval (QTc) was calculated according to Mitchell et al11: QTc=QT/(RR/100)1/2.
Histology and Image Analysis
Four-μM sections through the subvalvular plane of formaldehyde-fixed paraffin-embedded hearts were prepared and stained with Elastica van Gieson stain according to standard protocols. Three representative 20x fields were photographed, using an digital camera, from the subendocardial, central, and subepicardial region of the left ventricle and imported into Photoshop (version 7.0; Adobe systems Inc). Using the magic wand tool and the select similar command, all dark-stained nuclei were selected and their surface quantified by the histogram tool (given in pixel per inch, and then translated into μm2 according to the total magnification of the image). The mean nuclei size was calculated by the division of the total nuclear area by the number of nuclei as counted using a commercially available plug-in (count marks, The image processing toolkit; Reindeer Games, Charlotte, NC) and given in μm2. In the absence of notable tissue fibrosis, edema, or prominent intra-myocardic vessels, the cytoplasmic area was calculated as the total area of the 20x field minus the nuclear area (see above) then divided by the number of nuclei assessed as described.
Statistical Analyses
Data are represented as mean values±SEM. Two-tailed Student t test was used to compare means. Statistical significance was set at P<0.05.
Results
C-Terminal Domain of Nav1.5 Interacts With Dystrophin and Syntrophin Proteins
Recently, several proteins have been described to interact with specific protein-protein interaction motifs of the C-terminal domain of Nav1.5.2 Using a mass spectrometry-based approach with GST-fusion proteins of the last 66 residues of Nav1.5 C-terminus (Figure 1A), we searched for proteins extracted from human right atrial appendage samples interacting specifically with the PDZ domain-binding consensus motif formed by the last three Ser-Ile-Val (SIV) amino acids.4 The SDS-page presented in Figure 1B shows a high-molecular-weight protein that was reproducibly pulled down using the WT fusion protein, but not with a protein lacking the SIV residues (SIV). This protein was identified as dystrophin in human atrial samples (Table 1) as well as in mouse and guinea pig ventricular samples, with a high degree of confidence. Because dystrophin is known to frequently interact with partner proteins via syntrophin adapter proteins,12 we performed an additional experiment in which we examined by mass spectrometry the 50 to 60 kDa region of the WT lane of the gel: both 1- and 1-syntrophin proteins were identified with high confidence based on the numbers of peptidic fragments (Table 1). To better understand the molecular features of this multiprotein complex that binds to the C-terminal domain of Nav1.5, we performed Western blot analyses of the pulled-down proteins. As a control tissue we used ventricular lysates of dystrophin-deficient mdx5cv mice.8 In this set of experiments, we also performed pull-down experiments using a fusion protein in which the Nav1.5 PY-motif was mutated (Y1977A; Figure 1A) because this motif is known to interact with WW-domains similar to the one present in dystrophin.13 However, as depicted in Figure 1C, dystrophin also interacted with the PY-motif mutated protein, indicating that this motif is not likely to play a key role in the interaction of dystrophin with Nav1.5. Moreover, it is also apparent that neither dystrophin nor syntrophin proteins bind to the SIV-truncated protein (Figure 1C). The endogenous mouse ubiquitin-protein ligase Nedd4–2, previously shown to bind to the PY-motif of Nav1.513 was pulled-down with the WT and the SIV-truncated fusion proteins, which attests for the integrity of the SIV-truncated protein (Figure 1C). Using isoform-specific antibodies recognizing 1-, 1-, and 2-syntrophin proteins, we found that all three were pulled-down with the WT Nav1.5 C terminus fusion protein. We also tested two antibodies recognizing 1- and 2-syntrophin, but these two proteins could not be detected in the mouse cardiac lysates (not shown).
Nav1.5 Protein Is Specifically Decreased in mdx5cv Cardiac Tissue
We then performed Western blot experiments using ventricular lysates of mdx5cv and control mice to study the consequences of the dystrophin deficiency on Nav1.5 protein level. Figure 2A illustrates that when dystrophin was absent, a consistent reduction of the Nav1.5 protein level was detected in four mdx5cv mouse hearts. This phenomenon was specific for Nav1.5 because we did not observe any other obvious decrease in protein levels of major membrane proteins such as the L-type Ca2+ channel (Cav1.2), connexin-43, and -subunit of the Na-K,ATPase. The protein levels of several heart extracts were quantified by digital densitometry, and Figure 2B shows that the Nav1.5 content was decreased by 50±9% in mdx5cv hearts compared with controls. With the exception of a small, albeit statistically significant, 14±4% decrease in the level of Kir2.1 in mdx5cv hearts, the expression of all other tested proteins was not altered in the absence of dystrophin. It could have been hypothesized that this decrease in Nav1.5 amount in mdx5cv hearts may have been caused by a reduction of the SCN5A mRNA level. However, this was not the case because quantitative RT-PCR experiments using a SCN5A TaqMan probe did not reveal any difference between control and mdx5cv hearts (Figure 2C). Similarly, the mean mRNA level of Kir2.1 was not modified in mdx5cv hearts. Thus, the decreased Nav1.5 content may be attributable to either a reduced translation rate or increased turnover of the channel protein in the absence of dystrophin.
Sodium Current Is Decreased in mdx5cv Cardiomyocytes
To investigate whether the decrease of the total cardiac Nav1.5 protein amount seen in mdx5cv hearts alters the cellular sodium current (INa), we performed patch-clamp experiments using freshly isolated ventricular myocytes in the whole-cell configuration. Peak INa density was reduced by 29±2% in mdx5cv myocytes compared with control cells (Figure 3A, 3B). This INa reduction was not caused by alterations of the voltage-dependence of either steady-state activation or inactivation, as illustrated in Figure 3C. Moreover, cell size was not different, as assessed by measuring the electrical capacitance (control cells 149±7 pF and mdx5cv 157±9 pF, not significant, n=17 and 17 cells, respectively).
ECGs of mdx5cv Mice Reveal Conduction Defects
Subsequently, we investigated the consequences of this altered cellular excitability on surface ECGs of control and mdx5cv mice. Figure 4 shows representative ECG recordings (leads I, II, and III) of one control and two mdx5cv mice. The mdx5cv ECGs were characterized by a significant prolongation of the QRS complex (+18±5%), a decrease of the P wave amplitude (–19±6%), and a trend, albeit not significant, to prolonged P wave duration (Table 2). These findings point toward both intra-atrial and intra-ventricular conduction defects. Note that the heart rate and ventricular repolarization, reflected on the mouse ECG by the ST interval (similar, but not equivalent, to the JT interval in humans) were not different between mdx5cv and control mice (Table 2).
Mdx5cv Cardiac Tissues Do Not Show any Sign of Fibrosis or Cellular Hypertrophy
Because the intra-ventricular conduction defects may also have been caused by fibrosis of the mdx5cv ventricular myocardium, a phenomenon described in older mdx mice,14 we (H.A.L.) analyzed, in a blinded fashion, ventricular sections that were prepared with an elastic van Gieson stain. However, there was no increase in interstitial fibrosis neither in mdx5cv nor in control mice (n=4 animals of each group). In addition, we did not find any difference in nuclear and/or cytoplasmic size that may have reflected cardiac hypertrophy (not shown).
Discussion
The principal findings of this study are: (1) that cardiac dystrophin and syntrophin proteins are partners of the main cardiac voltage-gated sodium channel Nav1.5, and that this interaction depends exclusively on the C-terminal PDZ domain-binding motif of Nav1.5; (2) that Nav1.5 protein level is reduced in ventricles of dystrophin-deficient mdx5cv mice (an animal model of DMD); and (3) that this reduced expression of Nav1.5 mdx5cv mice leads to decreased cellular sodium current and conduction defects that could be documented by surface ECGs.
Description of the Protein Complex
Previous studies have already reported that Nav1.5 interacts with syntrophin proteins.4,5 In the present study, we went one step further and characterized this complex by showing that this interaction with syntrophins depends largely on the PDZ domain-binding motif, and that a direct dystrophin-Nav1.5 interaction via the PY-motif of Nav1.5 was unlikely. We also translated this concept into the human situation by reproducing it on human heart protein extracts. Moreover, we obtained evidence suggesting that 1-, 1-, and 2-syntrophins are likely to be the bona fide cardiac partners of Nav1.5, because, in analogy to findings of Iwata et al,15 -syntrophin proteins were not detected in cardiac lysates. Several other ion channels bearing a PDZ domain-binding motif have been reported to be part of the dystrophin protein complex (DPC) located at the plasma membrane of different cell types. Among them, the Kir2.x channels are expressed in cardiac cells,16 Nav1.44,17 and aquaporin-418 are expressed in the skeletal muscle, and Kir4.1 has been localized to glial cells.19 Our present studies have for the first time investigated pathological cardiac phenotypes related to altered function of these channels using the model of mdx mice.
Colocalization of Dystrophin and Nav1.5
Cardiac Nav1.5 channels are localized at the intercalated disk regions of cardiomyoctes,3,20 as well as in the lateral membranes.3,21–23 However, dystrophin has been shown to be absent from the intercalated discs of human24 and rat25 cardiac cells. These findings, in conjunction with the present results, suggest that at least two distinct pools of Nav1.5 channels co-exist in the plasma membrane of cardiac myocytes. One pool, belonging to the DPC, may be localized in lateral membranes, whereas another pool could reside in the intercalated disks. In the latter compartment, the PDZ domain-binding motif of Nav1.5 may be associated to another protein complex that remains to be characterized.
Interestingly, Baba et al22 recently described that dog cardiomyocytes isolated from infarcted zones displayed a reduced INa density (>50%), along with a specific and marked loss of the lateral membrane Nav1.5 staining. In contrast, in the intercalated disk area, the Nav1.5 staining remained unchanged. It is intriguing to speculate whether dystrophin is involved in this phenomenon. Moreover, if this "two Nav1.5 pools" hypothesis is correct, the study of Baba et al,22 in conjunction with our results, would support the importance of these sodium channels in lateral membranes in the conduction of the cardiac impulse.
Is Decreased Expression Level of Channel Proteins a General Phenomenon
Dystrophin is a large protein that exerts multiple roles. Its absence leads to a reduced expression of DPC proteins in skeletal muscle26 and cardiac tissue.27 In skeletal muscle of mdx mice, the expression of Nav1.417 and aquaporin-418 are significantly reduced. In our animal model, a small reduction of Kir2.1 was also observed. The relevance of this phenomenon is most likely marginal since no repolarization abnormality on the ECG was observed. In analogy to our present findings, the reduced protein expression was not attributable to decreased mRNA levels.17,18 In contrast, it should be noted that Nav channel expression in spermatozoa has been shown to be increased when dystrophin is absent.28 Furthermore, in 1-syntrophin-deficient mice, the abundance of Nav channels at the neuromuscular junction was not modified, whereas their localization was altered.29 Altogether, these findings suggest that one of the possible roles of dystrophin is to stabilize specific ion channel proteins by unknown mechanisms (similarly to the other DPC proteins) in the plasma membrane. Whether dystrophin may be important for the trafficking, anchoring, regulation of ion channel protein turnover, or other phenomena remains to be further studied.
ECG Alterations Are Consistent With Previous Studies
In mdx5cv mice, the QRS complex duration, reflecting intra-ventricular conduction, was prolonged by 18%. These data compare to 39% increase in QRS duration in 10-week-old mice expressing only one SCN5A allele,30 where cellular INa is reduced to 50% of control mice.31 These QRS prolongation values are in good agreement with the estimate that can be inferred from the data of Shaw and Rudy32 where a reduction of INa by 29% (this study) or 50%30 would lead to a prolongation in duration for a given distance of 18% and 33%, respectively. Furthermore, the intra-atrial conduction defects seen in mdx5cv mice in our present study are consistent with corresponding alterations reported in the studies mentioned above.30,31 Because connexin-43 levels are not altered, and in the absence of fibrosis, we propose that the cardiac electrical phenotype of mdx5cv mice is a "pure" conduction defect secondary to the reduction in Nav1.5 protein level in absence of dystrophin. Whereas these considerations pertain to young (10 to 12-week old) mdx5cv mice, it is well possible that older mice go onto develop a more complex cardiac phenotype with fibro-fatty degenerative features.
Relevance for the Human Cardiac Dystrophinopathies
DMD and Becker patients frequently show cardiac manifestations secondary to the absence of dystrophin in cardiac tissue. Many of these patients display dilated cardiomyopathy and heart failure.7 In addition, ECG abnormalities can also be detected in up to 60% of 10-year-old DMD patients,7 and among those, conduction defects are frequent.33 Indeed, life-threatening arrhythmia can cause sudden death in these dystrophinopathy patients.34 The relevance of early functional defects involving Nav1.5 in mdx5cv mice on the genesis of cardiac dysfunction in DMD patients is a matter of speculation. Two recent studies have reported that loss-of-function mutations in SCN5A may cause dilated cardiomyopathy35,36 in humans. Whether reduced INa may lead to myocardial degeneration in dystrophinopathies, dilated cardiomyopathies,35,36 or the Brugada syndrome, as recently described,37 remains to be shown. In addition, based on the findings of this study, it could be proposed that genes encoding proteins of the DPC complex, in particular syntrophin proteins, should be screened in patients with Brugada syndrome, because it may be foreseen that absence or loss-of-function of syntrophins may also lead to a reduced function of Nav1.5. Interestingly, a recent abstract38 indicated that mutations in the caveolin-3 gene, a protein that is also part of the DPC,39 may lead to congenital long QT syndrome by altering the function of Nav1.5. This finding supports well the model of Nav1.5 being part of the dystrophin multiprotein complex.
Conclusions
In summary, our work provides evidence that at least one fraction of the Nav1.5 channels is part of the dystrophin protein complex in cardiomyocytes. Absence of dystrophin in young mdx5cv mice leads to reduced Nav1.5 levels and hence reduced INa, associated with cardiac conduction defects. Whether these early functional defects may underlie the more severe morphological and mechanical alterations seen in dystrophinopathy patients warrants further investigation.
Acknowledgments
We thank Drs J.-D. Horisberger, S. Kellenberger, and O. Staub for their comments on this manuscript, and Drs M. van Bemmelen and S. Rohr for helpful suggestions. We are grateful to the staff of the proteomic analysis platform of the University of Lausanne for their work and helpful discussions, as well as the technical staff of the Institute of Pathology.
Source of Funding
This work was supported by grants of the Swiss National Science Foundation (632-66149.01 SNF-professorship to H.A.), Fondation Emma Muschamp, and Fondation Vaudoise de Cardiologie.
Disclosures
None.
Footnotes
Original received March 29, 2006; revision received June 27, 2006; accepted July 11, 2006.
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the Department of Pharmacology and Toxicology (B.G., J.-S.R., R.B., C.B., H.A.), University of Lausanne, Switzerland
Service of Cardiology (H.A.), CHUV, Lausanne, Switzerland
Department for Cardiovascular Surgery (P.R.), CHUV, Lausanne, Switzerland
Institute of Pathology (H.-A.L.), CHUV, Lausanne, Switzerland
Deparment of Medicine (A.A.D., T.P.), CHUV, Lausanne, Switzerland.
Abstract
The cardiac sodium channel Nav1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Nav1.5 C-terminus. Pull-down experiments were performed using Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx5cv mice to study the role of this protein complex in Nav1.5 function. Western blot experiments revealed a 50% decrease in the Nav1.5 protein levels in mdx5cv hearts, whereas Nav1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx5cv cardiomyocytes. Finally, ECG measurements of the mdx5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Nav1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.
Key Words: Duchenne dystrophy dystrophin ECG mouse sodium channels syntrophin
Introduction
The main cardiac voltage-gated sodium channel, Nav1.5, generates the fast depolarization of the cardiac action potential, and plays a key role in cardiac conduction. Its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene SCN5A, which encodes Nav1.5, that are linked to various cardiac diseases.1 Among them, the congenital long QT syndrome type-3 and the Brugada syndrome are caused by gain or loss-of-function of Nav1.5, respectively.1 Nav1.5 is the pore-forming -subunit protein of the cardiac sodium channel. It has a molecular weight of 220 kDa, and may be associated with at least 4 types of auxiliary small (30 to 35 kDa) -subunits. Recently, several proteins that bind directly to Nav1.5 have been described.2 However, in most cases the physiological relevance of these interactions remains poorly understood, mainly because of a lack of appropriate animal models. With the exception of ankyrin-G,3 all these partner proteins interact with the 243-residues-long intracellular C-terminal domain of the channel which contains several protein-protein interaction motifs.2 Among them, the last three residues of Nav1.5 (2014-Ser-Ile-Val-2016) constitute a PDZ domain-binding motif to which syntrophins and dystrophin have been shown to interact directly or indirectly, respectively.4–6 However, thus far, the role of these interacting proteins in the heart has never been investigated.
In this study, by performing mass spectrometry-based protein identification using human and rodent cardiac lysates, we could confirm that dystrophin and syntrophin proteins are interacting specifically with the PDZ domain-binding motif of Nav1.5. Biochemical and electrophysiological studies using cardiac tissue and cells of dystrophin-deficient mdx5cv mice (an animal model of Duchenne muscular dystrophy [DMD]) revealed that Nav1.5 protein content as well as Nav1.5-mediated current (INa) were decreased in the absence of dystrophin. Finally, ECGs of mdx5cv mice showed alterations characteristic of conduction defects. These findings suggest that Nav1.5 is part of a multiprotein complex in which dystrophin and syntrophin proteins play an important role in its expression level. Moreover, these results provide the pathophysiological basis for some of the ECG abnormalities seen in DMD patients, and other dystrophinopathy patients, in whom cardiac arrhythmias and conduction defects may lead to sudden death.7
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals
Wild-type C57BL/6 mice, used as control, were purchased at Janvier (Le Genest St Isle, France), and C57BL/6Ros-5Cv (mdx5cv) mice (Jackson laboratories, Bar Harbor, Me) were raised in our department. Male mice at an age of 10 to 14 weeks were used in this study. All animal procedures were performed in accordance with the Swiss laws.
Cardiac Tissue Samples
The experimental procedures were approved by the ethical commission on clinical research of the faculty of medicine of the University of Lausanne. Human right atrial sample was collected from patients in normal sinus rhythm undergoing coronary bypass surgery. Mice were anesthetized and heart ventricles were excised, rinsed with ice cold PBS and frozen in liquid nitrogen. Fresh human atrial appendage or frozen mouse ventricle was transferred into lysis buffer. Tissues were then homogenized using a Polytron and a Teflon homogenizer. Triton Tx-100 was added to a final concentration of 1% and solubilization occurred by rotating for 1 hour at 4°C. The soluble fraction from a subsequent 15-minute centrifugation at 13 000g (4°C) was used for the experiments.
Mass Spectrometry Peptide Analysis
SDS-PAGE gels were stained with SYPRO dye (Molecular Probes, Eugene, Ore). The gel was visualized on a UV-light transilluminator. The bands of interest were excised and sent to the Protein Analysis Facility of Lausanne where they were submitted to trypsin digestion, liquid chromatography and tandem mass spectrometry.
Pull-Down Assays
The cDNAs encoding the 66 last amino acids of Nav1.5 WT, Y1977A, and S2014Stop were cloned into pGEX-4T1 (Amersham Bioscience, Piscataway, NJ). GST-fusion proteins were obtained using Escherichia coli. GST-pull-down assays of soluble fractions of ventricular lysate were performed using GSH-sepharose beads containing either GST or the GST fusion proteins. After 2-hour rotation and washing, bound proteins were detected by Western blot.
Western Blot and Antibodies
Western blotting conditions and the specificity of the polyclonal Nav1.5 antibody (ASC-005; Alomone) has been previously described.9 A control Western blot using the peptide antigen showing the specificity of the signal is presented in the online data supplement. The polyclonal antibody Kir2.1 (APC-026) was purchased from Alomone (Jerusalem, Israel). Polyclonal connexin-43 antibody (71-0700) was obtained from Zymed (San Francisco, Calif). Monoclonal dystrophin antibody (MANDYS8) was from SIGMA (Buchs, Switzerland). Monoclonal pan-syntrophin antibody (MA1–745) was purchased from Affinity Bioreagents (Golden, USA). Polyclonal 1-, 1-, and 2-syntrophin antibodies were kindly provided by M. Schaub (University of Zurich), and 1- and 2-syntrophin antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif). Polyclonal Cav1.2 antibody was a gift from J. Hell (University of Iowa), Na,K-ATPase antibody was a gift from K. Geering (University of Lausanne) and Nedd4–2 antibody was a gift from O. Staub (University of Lausanne).
TaqMan Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA was extracted from whole ventricles according to described protocol in.9 cDNA was synthesized from 1 μg of total RNA using the MU-MLV reverse transcriptase (Q-Biogene EMMLV100; Irvine, Calif). Fifty nanograms of cDNA combined with 1x TaqMan Universal Master Mix (Applied Biosystems, Foster) and 1 μL of either Nav1.5, Kir2.1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Applied Biosystems, respectively, Mm00451971, Mm00434616 and Mm99999915) were loaded into each well. GAPDH was used as reference gene for normalizing the data.
Isolation of Cardiac Myocytes and Patch-Clamp Experiments
Adult mouse ventricular myocytes were isolated as described.9 Whole-cell configuration of the patch-clamp technique was used to record INa. Experiments were performed at room temperature (22°C to 23°C), using a VE-2 (Alembic Instruments) amplifier allowing the recording of large sodium currents.9 Pipettes (tip resistance 1 to 2 M) were filled with a solution containing 60 mmol/L aspartic acid, 70 mmol/L cesium asparte, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES, 11 mmol/L EGTA, and 5 mmol/L Na2ATP (pH was adjusted to 7.2 with CsOH). Myocytes were bathed with a solution containing 10 mmol/L NaCl, 120 mmol/L NMDG-Cl, 2 mmol/L CaCl2, 1.2 mmol/L MgCl2, 5 mmol/L CsCl, 10 mmol/L HEPES, and 5 mmol/L Glucose (pH was adjusted to 7.4 with CsOH).
ECG
The mouse ECG was recorded as previously described.10 Corrected QT interval (QTc) was calculated according to Mitchell et al11: QTc=QT/(RR/100)1/2.
Histology and Image Analysis
Four-μM sections through the subvalvular plane of formaldehyde-fixed paraffin-embedded hearts were prepared and stained with Elastica van Gieson stain according to standard protocols. Three representative 20x fields were photographed, using an digital camera, from the subendocardial, central, and subepicardial region of the left ventricle and imported into Photoshop (version 7.0; Adobe systems Inc). Using the magic wand tool and the select similar command, all dark-stained nuclei were selected and their surface quantified by the histogram tool (given in pixel per inch, and then translated into μm2 according to the total magnification of the image). The mean nuclei size was calculated by the division of the total nuclear area by the number of nuclei as counted using a commercially available plug-in (count marks, The image processing toolkit; Reindeer Games, Charlotte, NC) and given in μm2. In the absence of notable tissue fibrosis, edema, or prominent intra-myocardic vessels, the cytoplasmic area was calculated as the total area of the 20x field minus the nuclear area (see above) then divided by the number of nuclei assessed as described.
Statistical Analyses
Data are represented as mean values±SEM. Two-tailed Student t test was used to compare means. Statistical significance was set at P<0.05.
Results
C-Terminal Domain of Nav1.5 Interacts With Dystrophin and Syntrophin Proteins
Recently, several proteins have been described to interact with specific protein-protein interaction motifs of the C-terminal domain of Nav1.5.2 Using a mass spectrometry-based approach with GST-fusion proteins of the last 66 residues of Nav1.5 C-terminus (Figure 1A), we searched for proteins extracted from human right atrial appendage samples interacting specifically with the PDZ domain-binding consensus motif formed by the last three Ser-Ile-Val (SIV) amino acids.4 The SDS-page presented in Figure 1B shows a high-molecular-weight protein that was reproducibly pulled down using the WT fusion protein, but not with a protein lacking the SIV residues (SIV). This protein was identified as dystrophin in human atrial samples (Table 1) as well as in mouse and guinea pig ventricular samples, with a high degree of confidence. Because dystrophin is known to frequently interact with partner proteins via syntrophin adapter proteins,12 we performed an additional experiment in which we examined by mass spectrometry the 50 to 60 kDa region of the WT lane of the gel: both 1- and 1-syntrophin proteins were identified with high confidence based on the numbers of peptidic fragments (Table 1). To better understand the molecular features of this multiprotein complex that binds to the C-terminal domain of Nav1.5, we performed Western blot analyses of the pulled-down proteins. As a control tissue we used ventricular lysates of dystrophin-deficient mdx5cv mice.8 In this set of experiments, we also performed pull-down experiments using a fusion protein in which the Nav1.5 PY-motif was mutated (Y1977A; Figure 1A) because this motif is known to interact with WW-domains similar to the one present in dystrophin.13 However, as depicted in Figure 1C, dystrophin also interacted with the PY-motif mutated protein, indicating that this motif is not likely to play a key role in the interaction of dystrophin with Nav1.5. Moreover, it is also apparent that neither dystrophin nor syntrophin proteins bind to the SIV-truncated protein (Figure 1C). The endogenous mouse ubiquitin-protein ligase Nedd4–2, previously shown to bind to the PY-motif of Nav1.513 was pulled-down with the WT and the SIV-truncated fusion proteins, which attests for the integrity of the SIV-truncated protein (Figure 1C). Using isoform-specific antibodies recognizing 1-, 1-, and 2-syntrophin proteins, we found that all three were pulled-down with the WT Nav1.5 C terminus fusion protein. We also tested two antibodies recognizing 1- and 2-syntrophin, but these two proteins could not be detected in the mouse cardiac lysates (not shown).
Nav1.5 Protein Is Specifically Decreased in mdx5cv Cardiac Tissue
We then performed Western blot experiments using ventricular lysates of mdx5cv and control mice to study the consequences of the dystrophin deficiency on Nav1.5 protein level. Figure 2A illustrates that when dystrophin was absent, a consistent reduction of the Nav1.5 protein level was detected in four mdx5cv mouse hearts. This phenomenon was specific for Nav1.5 because we did not observe any other obvious decrease in protein levels of major membrane proteins such as the L-type Ca2+ channel (Cav1.2), connexin-43, and -subunit of the Na-K,ATPase. The protein levels of several heart extracts were quantified by digital densitometry, and Figure 2B shows that the Nav1.5 content was decreased by 50±9% in mdx5cv hearts compared with controls. With the exception of a small, albeit statistically significant, 14±4% decrease in the level of Kir2.1 in mdx5cv hearts, the expression of all other tested proteins was not altered in the absence of dystrophin. It could have been hypothesized that this decrease in Nav1.5 amount in mdx5cv hearts may have been caused by a reduction of the SCN5A mRNA level. However, this was not the case because quantitative RT-PCR experiments using a SCN5A TaqMan probe did not reveal any difference between control and mdx5cv hearts (Figure 2C). Similarly, the mean mRNA level of Kir2.1 was not modified in mdx5cv hearts. Thus, the decreased Nav1.5 content may be attributable to either a reduced translation rate or increased turnover of the channel protein in the absence of dystrophin.
Sodium Current Is Decreased in mdx5cv Cardiomyocytes
To investigate whether the decrease of the total cardiac Nav1.5 protein amount seen in mdx5cv hearts alters the cellular sodium current (INa), we performed patch-clamp experiments using freshly isolated ventricular myocytes in the whole-cell configuration. Peak INa density was reduced by 29±2% in mdx5cv myocytes compared with control cells (Figure 3A, 3B). This INa reduction was not caused by alterations of the voltage-dependence of either steady-state activation or inactivation, as illustrated in Figure 3C. Moreover, cell size was not different, as assessed by measuring the electrical capacitance (control cells 149±7 pF and mdx5cv 157±9 pF, not significant, n=17 and 17 cells, respectively).
ECGs of mdx5cv Mice Reveal Conduction Defects
Subsequently, we investigated the consequences of this altered cellular excitability on surface ECGs of control and mdx5cv mice. Figure 4 shows representative ECG recordings (leads I, II, and III) of one control and two mdx5cv mice. The mdx5cv ECGs were characterized by a significant prolongation of the QRS complex (+18±5%), a decrease of the P wave amplitude (–19±6%), and a trend, albeit not significant, to prolonged P wave duration (Table 2). These findings point toward both intra-atrial and intra-ventricular conduction defects. Note that the heart rate and ventricular repolarization, reflected on the mouse ECG by the ST interval (similar, but not equivalent, to the JT interval in humans) were not different between mdx5cv and control mice (Table 2).
Mdx5cv Cardiac Tissues Do Not Show any Sign of Fibrosis or Cellular Hypertrophy
Because the intra-ventricular conduction defects may also have been caused by fibrosis of the mdx5cv ventricular myocardium, a phenomenon described in older mdx mice,14 we (H.A.L.) analyzed, in a blinded fashion, ventricular sections that were prepared with an elastic van Gieson stain. However, there was no increase in interstitial fibrosis neither in mdx5cv nor in control mice (n=4 animals of each group). In addition, we did not find any difference in nuclear and/or cytoplasmic size that may have reflected cardiac hypertrophy (not shown).
Discussion
The principal findings of this study are: (1) that cardiac dystrophin and syntrophin proteins are partners of the main cardiac voltage-gated sodium channel Nav1.5, and that this interaction depends exclusively on the C-terminal PDZ domain-binding motif of Nav1.5; (2) that Nav1.5 protein level is reduced in ventricles of dystrophin-deficient mdx5cv mice (an animal model of DMD); and (3) that this reduced expression of Nav1.5 mdx5cv mice leads to decreased cellular sodium current and conduction defects that could be documented by surface ECGs.
Description of the Protein Complex
Previous studies have already reported that Nav1.5 interacts with syntrophin proteins.4,5 In the present study, we went one step further and characterized this complex by showing that this interaction with syntrophins depends largely on the PDZ domain-binding motif, and that a direct dystrophin-Nav1.5 interaction via the PY-motif of Nav1.5 was unlikely. We also translated this concept into the human situation by reproducing it on human heart protein extracts. Moreover, we obtained evidence suggesting that 1-, 1-, and 2-syntrophins are likely to be the bona fide cardiac partners of Nav1.5, because, in analogy to findings of Iwata et al,15 -syntrophin proteins were not detected in cardiac lysates. Several other ion channels bearing a PDZ domain-binding motif have been reported to be part of the dystrophin protein complex (DPC) located at the plasma membrane of different cell types. Among them, the Kir2.x channels are expressed in cardiac cells,16 Nav1.44,17 and aquaporin-418 are expressed in the skeletal muscle, and Kir4.1 has been localized to glial cells.19 Our present studies have for the first time investigated pathological cardiac phenotypes related to altered function of these channels using the model of mdx mice.
Colocalization of Dystrophin and Nav1.5
Cardiac Nav1.5 channels are localized at the intercalated disk regions of cardiomyoctes,3,20 as well as in the lateral membranes.3,21–23 However, dystrophin has been shown to be absent from the intercalated discs of human24 and rat25 cardiac cells. These findings, in conjunction with the present results, suggest that at least two distinct pools of Nav1.5 channels co-exist in the plasma membrane of cardiac myocytes. One pool, belonging to the DPC, may be localized in lateral membranes, whereas another pool could reside in the intercalated disks. In the latter compartment, the PDZ domain-binding motif of Nav1.5 may be associated to another protein complex that remains to be characterized.
Interestingly, Baba et al22 recently described that dog cardiomyocytes isolated from infarcted zones displayed a reduced INa density (>50%), along with a specific and marked loss of the lateral membrane Nav1.5 staining. In contrast, in the intercalated disk area, the Nav1.5 staining remained unchanged. It is intriguing to speculate whether dystrophin is involved in this phenomenon. Moreover, if this "two Nav1.5 pools" hypothesis is correct, the study of Baba et al,22 in conjunction with our results, would support the importance of these sodium channels in lateral membranes in the conduction of the cardiac impulse.
Is Decreased Expression Level of Channel Proteins a General Phenomenon
Dystrophin is a large protein that exerts multiple roles. Its absence leads to a reduced expression of DPC proteins in skeletal muscle26 and cardiac tissue.27 In skeletal muscle of mdx mice, the expression of Nav1.417 and aquaporin-418 are significantly reduced. In our animal model, a small reduction of Kir2.1 was also observed. The relevance of this phenomenon is most likely marginal since no repolarization abnormality on the ECG was observed. In analogy to our present findings, the reduced protein expression was not attributable to decreased mRNA levels.17,18 In contrast, it should be noted that Nav channel expression in spermatozoa has been shown to be increased when dystrophin is absent.28 Furthermore, in 1-syntrophin-deficient mice, the abundance of Nav channels at the neuromuscular junction was not modified, whereas their localization was altered.29 Altogether, these findings suggest that one of the possible roles of dystrophin is to stabilize specific ion channel proteins by unknown mechanisms (similarly to the other DPC proteins) in the plasma membrane. Whether dystrophin may be important for the trafficking, anchoring, regulation of ion channel protein turnover, or other phenomena remains to be further studied.
ECG Alterations Are Consistent With Previous Studies
In mdx5cv mice, the QRS complex duration, reflecting intra-ventricular conduction, was prolonged by 18%. These data compare to 39% increase in QRS duration in 10-week-old mice expressing only one SCN5A allele,30 where cellular INa is reduced to 50% of control mice.31 These QRS prolongation values are in good agreement with the estimate that can be inferred from the data of Shaw and Rudy32 where a reduction of INa by 29% (this study) or 50%30 would lead to a prolongation in duration for a given distance of 18% and 33%, respectively. Furthermore, the intra-atrial conduction defects seen in mdx5cv mice in our present study are consistent with corresponding alterations reported in the studies mentioned above.30,31 Because connexin-43 levels are not altered, and in the absence of fibrosis, we propose that the cardiac electrical phenotype of mdx5cv mice is a "pure" conduction defect secondary to the reduction in Nav1.5 protein level in absence of dystrophin. Whereas these considerations pertain to young (10 to 12-week old) mdx5cv mice, it is well possible that older mice go onto develop a more complex cardiac phenotype with fibro-fatty degenerative features.
Relevance for the Human Cardiac Dystrophinopathies
DMD and Becker patients frequently show cardiac manifestations secondary to the absence of dystrophin in cardiac tissue. Many of these patients display dilated cardiomyopathy and heart failure.7 In addition, ECG abnormalities can also be detected in up to 60% of 10-year-old DMD patients,7 and among those, conduction defects are frequent.33 Indeed, life-threatening arrhythmia can cause sudden death in these dystrophinopathy patients.34 The relevance of early functional defects involving Nav1.5 in mdx5cv mice on the genesis of cardiac dysfunction in DMD patients is a matter of speculation. Two recent studies have reported that loss-of-function mutations in SCN5A may cause dilated cardiomyopathy35,36 in humans. Whether reduced INa may lead to myocardial degeneration in dystrophinopathies, dilated cardiomyopathies,35,36 or the Brugada syndrome, as recently described,37 remains to be shown. In addition, based on the findings of this study, it could be proposed that genes encoding proteins of the DPC complex, in particular syntrophin proteins, should be screened in patients with Brugada syndrome, because it may be foreseen that absence or loss-of-function of syntrophins may also lead to a reduced function of Nav1.5. Interestingly, a recent abstract38 indicated that mutations in the caveolin-3 gene, a protein that is also part of the DPC,39 may lead to congenital long QT syndrome by altering the function of Nav1.5. This finding supports well the model of Nav1.5 being part of the dystrophin multiprotein complex.
Conclusions
In summary, our work provides evidence that at least one fraction of the Nav1.5 channels is part of the dystrophin protein complex in cardiomyocytes. Absence of dystrophin in young mdx5cv mice leads to reduced Nav1.5 levels and hence reduced INa, associated with cardiac conduction defects. Whether these early functional defects may underlie the more severe morphological and mechanical alterations seen in dystrophinopathy patients warrants further investigation.
Acknowledgments
We thank Drs J.-D. Horisberger, S. Kellenberger, and O. Staub for their comments on this manuscript, and Drs M. van Bemmelen and S. Rohr for helpful suggestions. We are grateful to the staff of the proteomic analysis platform of the University of Lausanne for their work and helpful discussions, as well as the technical staff of the Institute of Pathology.
Source of Funding
This work was supported by grants of the Swiss National Science Foundation (632-66149.01 SNF-professorship to H.A.), Fondation Emma Muschamp, and Fondation Vaudoise de Cardiologie.
Disclosures
None.
Footnotes
Original received March 29, 2006; revision received June 27, 2006; accepted July 11, 2006.
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