Published online before print April 9, 2009, doi: 10.1161/CIRCRESAHA.108.191809
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© 2009 American Heart Association, Inc.
Cellular Biology |
Ion Channel Subunit Expression Changes in Cardiac Purkinje Fibers
A Potential Role in Conduction Abnormalities Associated With Congestive Heart Failure
From the Department of Medicine (A.M., S.L.B., D.C., L.V., R.W., K.N., S.N.), Department of Physiology (P.C.), and Institute of Biomedical Engineering (P.C.), Montreal Heart Institute and Université de Montréal, Quebec, Canada; Department of Pharmacology and Therapeutics (S.N.), McGill University, Montreal, Quebec, Canada; and Department of Internal Medicine 1 (R.W.), Ludwig-Maximilians University, Munich, Germany.
Correspondence to Stanley Nattel, 5000 Belanger St East, Montreal, Quebec, H1T 1C8, Canada. E-mail Stanley.nattel{at}icm-mhi.org
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Abstract |
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Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing–induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dtmax (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2±0.1 m/s (control) to 1.5±0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5±1.2 (control) to 49.3±3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.
Key Words: heart failure • remodeling • ventricular dyssynchrony • connexins • specialized conducting system
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Introduction |
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Cardiac Purkinje fibers (PFs) play an important role in ensuring rapid and appropriately timed conduction of electric impulses to the ventricles1 and are an important arrhythmogenic source for a variety of cardiac arrhythmias.2–5 Congestive heart failure (CHF) changes ion channel distribution and function, with important electrophysiological consequences,6 and sudden arrhythmic death contributes importantly to CHF-related mortality.7 We previously demonstrated discrete CHF-induced remodeling of repolarizing ion current function in canine cardiac PFs.8 In particular, decreases in transient-outward (Ito) and inward-rectifier (IK1) K+ currents reduce repolarization reserve and enhance the action potential (AP)-prolonging effects of class III antiarrhythmic agents, potentially accounting for the increased Torsades de Pointes risk conferred by CHF.9–11 Virtually no work has been done on CHF-induced remodeling of PF ion channel subunit expression at the molecular level. In the present study, we began by examining CHF-induced changes in PF K+ channel subunit mRNA and protein expression, to understand previously noted repolarization current alterations.8 We then moved on to study effects on molecular determinants of PF impulse propagation: Na+ channel and connexin (Cx) subunits. Substantial changes in these subunits led us to assess associated changes in PF conduction in vitro and in vivo.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animal Model
CHF was induced by 2-week right ventricular tachypacing (VTP) at 240 bpm, as previously described.12 Under anesthesia (morphine, 2 mg/kg SC; 
-chloralose 120 mg/kg IV load, 29.25 mg/kg per hour infusion), hearts were excised and immersed in oxygenated Tyrode solution. Free-running false tendons were removed for study. Animal care procedures were approved by the local animal research ethics committee.
RNA Isolation and Quantitative PCR
RNA was extracted with TRIzol (Invitrogen) and treated with DNaseI to minimize genomic DNA contamination. Quantitative PCR probes and primers are listed in Online Tables I and II. The 18S rRNA housekeeping gene was used as a reference. PCR efficiency was in the range of 95% to 100% for all assays. PCR cycle parameters were: 2 minutes at 50°C; 10 minutes at 95°C initial incubation, followed by 15 seconds at 95°C; and 1 minute at 60°C for 40 cycles. For each construct, analyses were performed separately for n=7 control, n=8 CHF hearts, each in duplicate. Data are expressed as 2–
Ctx107.
Protein Extraction and Immunoblots
Protein-enriched samples were obtained by pooling all usable false tendons from 2 dogs, to provide sufficient protein to load in a well. Results were obtained from 8 dogs per group (n=4 determinations each for control and CHF) unless otherwise indicated. To enrich membrane proteins, snap-frozen free-running false tendons were homogenized in extraction buffer. A preliminary 1000g centrifugation (10 minutes, 4°C) was performed to pellet debris. The supernatant was further ultracentrifuged at 100 000g for 1 hour to pellet membrane-protein fractions. Pellets were resuspended with extraction buffer supplemented with 1% Triton-X100. Protein concentration was determined with Bradford assay. Protein samples (100 µg) were separated with SDS-PAGE. The separated proteins were transferred by electrophoresis to poly(vinylidene fluoride) transfer membranes. Membranes were blocked 2 hours with TTBS solution (Tris-HCl 50-mmol/L, NaCl 500-mmol/L, pH 7.5, 0.1% Tween) containing 5% nonfat dried milk and incubated overnight with primary antibodies (Online Table III) in TTBS with 5% nonfat dried milk. Corresponding secondary antibodies conjugated to horseradish peroxidase were used for detection. Staining was quantified by chemiluminescence. All expression data are provided relative to GAPDH staining for the same samples on the same gels.
AP Recordings and Conduction Velocity Measurements
Preparations containing free-running false tendons were pinned to the floor of a 30-mL Lucite tissue chamber (Figure I in the online data supplement) and superfused with Krebs–Henseleit solution (in mmol/L: NaCl 120, KCl 4, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 1.25, dextrose 1g/L; 95% O2/5% CO2, pH 7.4) at 35°C. Two floating glass microelectrodes with tip resistances of 15 to 20 M
were used to impale cells in the same longitudinal orientation. One microelectrode (E1) was placed at one fiber end near a bipolar platinum stimulation electrode (SE) positioned on ventricular muscle and a second (E2) was placed at various points distal to E1 (Online Figure I). Square wave 2-ms pulses (1.5xlate diastolic threshold current) were delivered with an electronic stimulator. Phase 0 upstroke was analyzed by electronic differentiation. Activation time was defined as the time of dV/dtmax. Conduction velocity was determined by dividing the distance between E1 and E2 by the difference between their respective activation times and averaging conduction velocity estimates at all E2 positions.
Immunohistochemistry
After AP recording and conduction velocity measurement, false tendons were fast-frozen. Cryosections (12-µm thickness) were fixed with a 1x PBS solution containing 4% paraformaldehyde (pH 7.3), blocked, and permeabilized with 1x PBS solution containing 2% normal donkey serum and 0.5%-Triton X-100. Primary antibodies (rabbit anti-Cx40, mouse anti-Cx43, Chemicon; mouse anti–Ser368-phosphorylated Cx43, Cell Signaling) were diluted 1/200 in 1x PBS solution containing 2% normal donkey serum and 0.1% Triton X-100 for overnight incubation with cryosections. (For details regarding antibodies, see Online Table III.) Alexa Fluor–conjugated donkey anti-rabbit (488 nm, Invitrogen) or donkey antimouse (555 nm, Invitrogen) were used as secondary antibodies (1/600 dilution). Alexa Fluor–conjugated phalloidin (647 nm) was used as an actin filament marker (1/600 dilution). Slides were mounted and examined with a laser-scanning confocal microscope. Identical settings were used to image samples from control and CHF dogs. Images were deconvolved with the maximum likelihood estimation algorithm for Cx protein density quantification.
Each intercalated disk was separated into peripheral and central regions and respective Cx density calculated as described in the expanded Materials and Methods section in the online data supplement. Intercalated disk area and plaque numbers were also quantified as specified in Online Materials and Methods. All procedures were performed with MatLab v6.5 (Mathworks).
ECG Recording and AH and HV Interval Measurements
Standard ECG leads were recorded to determine QRS duration. ECGs were recorded once in control dogs and twice (at baseline and after 2 weeks of VTP) in CHF dogs. QRS durations were based on averages of 5 consecutive complexes in sinus rhythm. AH and HV intervals were recorded with intracardiac quadripolar catheters. Bipolar signals were amplified, filtered (40 to 400 Hz), and recorded with a digital recording system.
Statistical Analysis
Average data are expressed as means±SEM. Nonpaired Student’s t tests were used to compare control and CHF groups. Two-tailed P<0.05 indicated statistical significance. When multiple recordings were obtained from individual dogs, test-of-significance statistics were based on average values in each dog, ie, using each dog as a n of 1, to reflect the nonindependence of repeated recordings in each animal.
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Results |
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Hemodynamic Indices
When the chest was opened, moderate to severe pericardial effusions, cardiac enlargement, and pulmonary congestion were evident in all CHF dogs. These were associated with significant hemodynamic impairments (Online Table IV): (1) reduced systolic and mean systemic arterial pressure (by 21% and 16%, respectively); (2) increased left ventricular end-diastolic pressure (to >3-fold control values); and (3) increased right and left atrial pressures (to approximately twice control values each).
Ion Channel Subunit Transcript and Protein Expression
We began by studying ion channel subunits corresponding to transient outward K+ current (Ito), previously shown to be reduced in PFs from CHF dogs.8 CHF decreased expression of all transcripts potentially encoding PF Ito subunits13 (Figure 1A) by 42%, 44%, and 46% for Kv1.4, Kv4.3, and Kv3.4. Overall, KCND3 (Kv4.3) mRNA expression was much stronger than for other subunits, by approximately 20- to 40-fold. The KCNIP2-encoded KChIP2 β subunit was weakly expressed and unchanged in CHF. Corresponding immunoblots are shown in Figure 1B, with mean data in Figure 1C. Changes in Kv4.3 and Kv3.4 protein expression paralleled alterations in mRNA. PF Kv1.4 protein expression was not changed in CHF, unlike mRNA. Like KChIP2 mRNA, KChIP2 protein expression was unchanged.
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We then examined genes encoding proteins underlying outward currents affecting phase 3 repolarization. Transcript levels corresponding to subunits underlying the major delayed-rectifier and inward-rectifier currents (KCNQ1 encoding the KvLQT1 subunit underlying IKs, KCNH2 encoding the ERG subunit of IKr, KCNJ2 encoding the Kir2.1 subunit of IK1 and KCNJ3/KCNJ5 encoding Kir3.1/3.4 IKACh subunits) were unchanged in CHF (Figure 2A). The IKs β subunit (minK)-encoding gene KCNE1 was downregulated at the mRNA level. The putative IKr β subunit MiRP1 was weakly expressed and not altered by CHF. No changes in protein expression were observed for KvLQT1, minK, or MiRP1 (Figure 2B and 2C). Although KCNJ2 mRNA was unchanged, Kir2.1 protein was reduced significantly, by 26%. We noted 4 bands on ERG immunoblots with an antibody directed at the ERG C-terminus. The 160- and 140-kDa isoforms (corresponding to mature and immature forms of ERG 1a respectively)14 were upregulated in CHF, whereas 95- and 83-kDa bands (corresponding to mature and immature ERG 1b14) were unchanged.
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We then turned our attention to ion channel subunits involved in cardiac conduction. The SCN5A gene encoding the predominant Na+ channel subunit Nav1.5 is strongly expressed in cardiac Purkinje cells, along with the β subunit SCN1B (Navβ1) and the more weakly expressed subunit gene SCN1A (Nav1.1).15 Nav1.5 and Navβ1 transcripts were expressed at similar levels, 
4 orders of magnitude greater than Nav1.1 (Figure 3A). Nav1.1 and Navβ1 were unaffected by CHF, but Nav1.5 mRNA was strongly downregulated, by 56%. We also examined expression of genes encoding the hemichannels mediating low-resistance cell-to-cell cardiomyocyte communication, GJA5 (Cx40), GJA1 (Cx43), and GJA7 (Cx45). Cx43 mRNA was very strongly expressed, at an order of magnitude greater than Cx40, Nav1.5, and Navβ1. Cx45 expression was much weaker than Cx40. Cx40 and -43 transcripts were strongly and significantly downregulated in CHF, by 66% and 56%, respectively. Cx45 mRNA was upregulated.
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Western blots (Figure 3B) confirmed 48% downregulation of Nav1.5 protein expression (Figure 3C). Cx40 band intensity was reduced in CHF by 53%. Cx45 bands were too weak for accurate detection. Cx43 migrated as 2 discrete bands, previously identified as nonphosphorylated (smaller-molecular-mass) and phosphorylated (larger-molecular-mass) bands, respectively.16 Estimating total Cx43 protein expression based on the sum of both indicated a 30% reduction. Cx43 dephosphorylation contributes to CHF-induced conduction impairment in ventricular muscle.17 The larger-molecular-mass band intensity decreased as a fraction of the smaller-molecular-mass band in CHF, suggesting reduced phosphorylation. Probing with an antibody specific to Ser368-phosphorylated Cx43 revealed a dominant band at the expected molecular mass (Figure 3D, left), which decreased in CHF by 57% (Figure 3D, right).
Cx Distribution at Intercalated Disks
Changes in Cx expression and phosphorylation may be reflected in changed distribution within intercalated disks, the principal structures governing cardiomyocyte coupling.18 Figure 4A shows representative single en face intercalated disks. Figure 4B shows corresponding lateral views. Cx immunostaining exhibited a typical localization pattern, with small central gap junctions in plicate regions surrounded by larger gap junction plaques located in interplicate regions at the disk periphery.18 In PFs from CHF dogs, peripheral gap junction immunostaining was preserved for Cx40 and total Cx43. However, immunostaining of Ser368-phosphorylated Cx43 was reduced at the periphery of the intercalated disks. Quantitative analyses of CHF-induced changes in Cx distribution are illustrated in Figure 5A (left), which shows representative 3D surfaces of Cx staining at central intercalated disk regions for 1 sample each in control and CHF groups. Quantitative analysis (Figure 5A, right) showed significant 25% reductions in Cx40 density in central intercalated disk regions in CHF dogs, with no significant change in the periphery (Figure 5B). Similarly, Cx43 immunostaining was reduced by 27% in central regions but not significantly altered in the periphery. In contrast, central phosphorylated Cx43 staining was unaffected by CHF, but a 27% decrease was noted in peripheral zones. Average intercalated disk surface areas and numbers of Cx plaques per intercalated disk were not significantly altered in CHF (Online Figure II).
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AP Properties and Conduction
AP properties and conduction velocity were determined at 1 Hz. Resting potentials were not significantly altered by CHF (Table), but AP amplitude, overshoot, and phase 0 upstroke velocity were decreased, consistent with reduced Nav1.5 expression. The slope of phase 1 was also significantly decreased, compatible with reduced Ito subunit expression. Overall, AP duration was not significantly altered, consistent with previous findings.8
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Typical AP recordings used for conduction velocity assessment in a control dog are illustrated in Figure 6A, with the upstrokes shown on an expanded time scale in Figure 6B. Recordings from a CHF dog with a similar E1–E2 distance to the control dog illustrate the longer conduction times that were typically noted in CHF false tendons. Figure 6C provides mean conduction velocity values, with a statistically significant, 30% reduction noted in CHF.
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In Vivo Purkinje System Conduction Changes
To determine whether changes in ventricular conduction properties are altered in vivo in relation to PF INa and Cx remodeling, we measured QRS duration and HV intervals in control and CHF dogs. Typical recordings used for QRS and HV interval measurement are shown in Figure 7A. Because of the noninvasive nature of ECG recordings, we were able to measure QRS duration at baseline and after tachypacing in CHF dogs. QRS durations were very similar in control dogs and at baseline in CHF dogs (Figure 7B). However, with tachypacing, the QRS increased significantly, by a mean of 13.5 ms (30%), in CHF dogs. Similarly, the HV interval (which reflects PF-mediated His-Purkinje conduction) was substantially greater in CHF than in control dogs (Figure 7C), by 13.8 ms (39%). No modification of the AH interval was observed (Figure 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we investigated changes in transcript and protein expression of K+ channel, Na+ channel, and Cx subunits induced by CHF in canine cardiac PFs. We observed decreases in the expression of Ito-related
subunits, little change in inward and delayed-rectifier K+ channel subunits, and concerted downregulation of Na+ channel subunit and Cx40/43 proteins. In addition, Cx43 phosphorylation was reduced, and Cx expression in intercalated disks was altered in quantity and spatial distribution. These changes were associated with in vitro and in vivo evidence of slowed Purkinje system conduction.
PF Electrical Remodeling in CHF
Relatively little work has been performed to address CHF-induced changes in PF electrophysiology. We previously reported an 32% decrease in Ito density and 22% decrease in peak outward IK1 density as a result of tachypacing-induced heart failure in dogs, without any change in delayed-rectifier currents.8 The precise molecular basis of cardiac PF Ito has not been definitively established, but there is evidence supporting a role for Kv4.3 and/or Kv3.4 subunits,13 both of which were downregulated in the present study. Kir2.1 protein expression decreased by 26%, sufficient to account for the previously described IK1 downregulation,8 but mRNA levels were unaffected, suggesting posttranscriptional changes. The lack of change in KvLQT1 and minK protein is consistent with unaltered IKs, but the picture is more complex for IKr. ERG transcript expression was unaltered, as measured by primers directed to a C-terminal sequence shared by both ERG isoforms. Both mature and immature forms of ERG 1a protein were increased in CHF PFs, whereas ERG 1b protein expression was unchanged. Recent evidence points to heteromers containing ERG 1b as a critical contributor to native IKr, with an ERG 1b mutation causing clear long QT syndrome.19 It is therefore possible that ERG 1b is a crucial component of functional IKr and that the lack of change in ERG 1b in canine cardiac PFs explains the unaltered IKr with CHF.
There are no previous studies addressing CHF-induced changes in PF Na+ channel subunit and Cx expression or related function. We found substantial and statistically significant reductions in several of these important determinants of conduction, including the Nav1.5 Na+ channel subunit and Cx43 and Cx40 proteins. The protein expression changes were associated with significant mRNA reductions, suggesting transcriptional downregulation. In addition, Cx43 phosphorylation was decreased. Corresponding functional parameters showed changes consistent with the biochemical alterations: decreased phase 0 upstroke velocity, AP amplitude, AP overshoot, and PF conduction velocity, along with prolonged HV intervals.
Comparison Between CHF-Induced Changes in PFs and Previous Observations in Working Ventricular Muscle
Most previous studies showed that CHF decreases Ito in ventricular muscle, with corresponding changes in subunit mRNA and protein,6 resembling our observations in PFs. Results regarding ventricular Kir2.1 expression are discrepant, with some studies showing mRNA downregulation20,21 and others no change.22,23 Two reports describe no change in Kir2.1 protein expression.20,23 Our findings point to posttranscriptional downregulation as the mechanism of PF IK1 decreases in CHF. Results for IK-related expression in ventricular muscle are consistent in demonstrating no change in ERG20,21,24 (although we are not aware of studies examining both ERG isoforms), but changes in KvLQT1 and minK have varied widely, with increased,21 decreased,24 or unchanged20,23 expression having been reported. The discrepancies may relate to varying experimental conditions, species, duration, and type of disease, etc.
Cx43 is consistently downregulated in failing ventricular muscle, with corresponding changes in mRNA and protein generally reported.18 Cx4025 and Cx4526 have been reported to be upregulated by some heart disease paradigms, possibly as a compensatory response. In the present study, PF Cx40 was downregulated and Cx45 was upregulated. Several studies show reduced INa in failing ventricles, but the molecular mechanisms remain obscure.6 The congruent reduction in Nav1.5 mRNA and protein expression that we observed in PFs has not, to our knowledge, been previously described in working ventricular muscle. Cx43 dephosphorylation is also observed in failing ventricular muscle and is believed to be an important contributor to ventricular conduction slowing.16,17
Novelty and Potential Significance
This study is the first, to our knowledge, to address CHF-induced ion channel subunit remodeling in cardiac PFs. Our results regarding Ito and IK1 subunits are largely consistent with previously reported changes in ventricular muscle and corresponding ion current density alterations in PFs.6,8 We are not aware of previous studies that report differential disease-induced changes in the cardiac expression of ERG 1a versus ERG 1b subunits. In conjunction with our previous observation of unchanged IKr in PFs from CHF dogs, our finding of increased ERG 1a protein with unchanged ERG 1b would suggest that ERG 1b expression is a particularly important determinant of IKr function. These results are consistent with recent observations pointing to an essential role of ERG 1b in IKr.19
Our observations point to particularly important changes in the ion channel subunits determining the primary conduction function of cardiac PFs. Decreases in INa subunits and Cx hemichannel subunits correspond to impairments of the active and passive determinants of PF conduction respectively. These alterations were associated with substantial and statistically significant decreases in directly measured PF conduction velocity in vitro, as well as corresponding His-Purkinje system conduction slowing (as indicated by increased HV interval) in vivo. This novel finding may have important bearing on a clinically significant abnormality frequently observed in CHF patients, dyssynergic ventricular excitation/contraction. Conduction through the His-Purkinje system is an important determinant of the sequence of ventricular activation and contraction.1 Cardiac conduction abnormalities are a strong independent predictor of mortality in CHF patients.27 Evidence of disturbed His-Purkinje system function like bundle–branch block is common in CHF, predicting increased disease progression and mortality.28 Dyssynchronous cardiac contraction has important deleterious effects on cardiac function mediated by adverse remodeling related to deleterious changes in cardiac gene expression.29 Resynchronization therapy improves morbidity and mortality in CHF patients with ventricular activation abnormalities.30 Antiarrhythmic effects of resynchronization therapy31 suggest an important role of dyssynchrony in ventricular arrhythmogenesis, either directly or via ventricular dysfunction-related arrhythmogenic remodeling. Our results point to remodeling of PF ion channel subunits (particularly INa subunits and connexins) as a potentially important contributor to ventricular activation abnormalities in CHF.
Potential Limitations
The canine VTP model of CHF provides a highly reproducible and robust experimental model exhibiting key hemodynamic, electrophysiological, and molecular changes associated with human CHF. However, CHF results from many different etiologies and extrapolation of our results to other forms of CHF should be cautious. Rapid activation of ventricular cardiomyocytes modifies Ito expression in vitro.32 It is therefore possible that some of the changes we observed were attributable to rapid ventricular rates and not CHF per se; however, the ventricular ion channel function changes noted in animals with VTP-induced CHF do correspond closely to results obtained from explanted terminally failing human hearts.6 We investigated modifications in transcript and protein expression of K+ channel, Na+ channel, and Cx subunits induced by CHF in canine cardiac PFs, based on a desire to understand previously observed functional K+ channel changes and to extend our understanding to ion channels governing cardiac conduction. A complete analysis of the molecular basis of CHF-induced PF electric remodeling would require studies of Ca2+ current, Ca2+ handling, HCN, Na+/Ca2+ exchange, and Na+,K+-ATPase gene/protein expression and function,33 work that is beyond the scope of the present article but should be performed in follow-up studies.
We observed decreases in PF Cx40 and -43 expression and increases in Cx45. It was traditionally assumed that all cardiac connexins contribute to conduction and decreases in any Cx isoform would impair cell coupling.18 Recent work has raised questions about this notion. Cx45 overexpression impairs cell coupling and increases ventricular tachyarrhythmia susceptibility in transgenic mice.34 Similarly, pattern-cultured neonatal atrial cardiomyocytes from Cx40-knockout mice show increased conduction velocity.35 Thus, functional effects of changed Cx expression may be difficult to predict directly, particularly when alterations in multiple isoforms are observed. On the other hand, Cx40 knockout clearly slows His-Purkinje system conduction in the mouse,36 and decreases in Cx43 expression slow ventricular conduction.37 There is evidence that colocalization of Cx40 and Cx43 in PFs may be functionally important.38 The mRNA concentrations of Cx43 that we measured in PFs were more than 10-fold greater than those of Cx40 and more than 100-fold greater than Cx45. This observation, combined with decreased Cx43 phosphorylation, an important determinant of Cx43 localization and function,16–18 suggests that Cx43 alterations are likely to have played a particularly important role in the PF conduction slowing that we observed.
Species differences in Cx isoform distribution may be important.18 Therefore our results should be extrapolated with caution and follow-up studies in other species, including humans, would be of interest. Our studies were performed at a particular time point in a particular experimental model of CHF. Remodeling of the determinants of ventricular conduction are time-dependent but are well developed at 2 weeks of VTP and are qualitatively consistent at different time-points.17
We have discussed K+ channel and Cx remodeling as separate entities; however, there is recent thought-provoking evidence that changes in cell coupling may importantly influence remodeling of K+ channels and repolarization properties.39,40 A wide range of ion channel subunits localize to intercalated disks, including Na+ channel subunits.41 There may therefore be coordinate regulation of intercalated disk channel subunits involved in impulse propagation (eg, connexins and Na+ channels) that underlies the changes we observed. This would be a potentially interesting subject for future evaluation.
Conclusions
We report for the first time CHF-induced changes in the expression of K+, Na+, and Cx channel subunits in cardiac PFs. K+ channel subunit expression changes are consistent with and provide mechanistic insights into previously observed alterations in PF K+ channel function. Na+ and Cx subunit expression changes, including downregulation of Nav1.5, Cx40, and Cx43 mRNA and protein expression, decreased Cx43 phosphorylation at Ser368 and altered Cx distribution at intercalated disks, were associated with significant slowing in His-Purkinje conduction. These results may be important for understanding the alterations in ventricular activation and synchrony that contribute to deleterious outcomes in CHF patients.
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Acknowledgments |
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We thank Chantal St-Cyr and Nathalie L’Heureux for technical assistance and France Thériault for secretarial help.
Sources of Funding
Supported by Canadian Institutes for Health Care Research grant MOP 68929 and the MITACS Network.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received November 28, 2008; revision received March 31, 2009; accepted April 1, 2009.
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References |
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