Comparison of Ion-Channel Subunit Expression in Canine Cardiac Purkinje Fibers and Ventricular Muscle
Although Purkinje fibers (PFs) play an important role in cardiac electrophysiology, almost nothing is known about the expression of ion-channel subunits in PFs. We applied competitive reverse transcription–polymerase chain reaction, Western blotting, and immunocytochemistry to compare the expression of ion-channel subunit mRNA and protein in canine PFs versus ventricular muscle (VM). For transient outward current–related subunits, Kv4.2 was not detected, and Kv1.4 expression was extremely low. Kv4.3 expression was of the same order for VM and PFs. The tetraethylammonium chloride–sensitive subunit Kv3.4 was expressed much more strongly in PFs than in VM, and Kv channel–interacting protein transcript expression was 25-fold stronger in VM than in PFs. For delayed rectifiers, ERG and KvLQT1 expression was lower in PFs at both mRNA and protein levels. Although minK transcripts were more numerous in PFs, minK protein was significantly more strongly expressed in VM. L-type Ca2+ current α-subunit (CaV1.2) and Na+-Ca2+ exchanger proteins were more strongly expressed in VM than in PFs. For T-type Ca2+ current, CaV3.1, CaV3.2, and CaV3.3 transcripts were all more strongly expressed in PFs. For the nonselective cation current, hyperpolarization-activated cation channel 1 (HCN1) expression was subquantifiable, HCN2 transcript expression was comparable in PFs and VM, and HCN4 mRNA expression was strong in PFs but below the detection threshold in VM. HCN2 and HCN4 protein expression was much stronger in PFs than in VM. We conclude that ion-channel subunit expression in PFs differs from that in VM in ways that are consistent with, and shed light on the molecular basis of, well-recognized fundamental PF ionic properties.
Cardiac Purkinje fibers (PFs) play an important role in cardiac conduction and arrhythmogenesis. They are particularly significant in generating early afterdepolarizations and triggering transmural reentry in torsade de pointes arrhythmias associated with long QT syndromes.1–4⇓⇓⇓ PFs also appear important in ventricular tachyarrhythmias that are due to delayed afterdepolarizations,5 intraventricular reentry,6 and ventricular fibrillation.7,8⇓
PF action potentials (APs) have a number of properties that differentiate them from ventricular muscle (VM). They show prominent spontaneous phase-4 depolarization and automaticity9; their AP durations increase much more than those of VM at slow rates,10,11⇓ leading to preferential PF generation of early afterdepolarizations1; they show prominent rate dependence of phase-1 repolarization12; and they have a more negative plateau voltage. These AP differences likely reflect differences in the density and/or composition of ion channels in PFs compared with VM. Although much is known about the expression of ion-channel subunits in mammalian atrium and VM, the published literature contains very limited data regarding ion-channel subunit expression in PFs.13 The present study was designed to determine the expression of transcripts and (to the extent possible) proteins corresponding to ion-channel subunits and transporters in PFs compared with VM.
Materials and Methods
Hearts were removed from mongrel dogs (20 to 30 kg) euthanized with overdoses of pentobarbital. Left ventricular midmyocardium and PFs in free-running false tendons were removed and fast-frozen in liquid N2. For all analyses, comparisons were based on at least 4 hearts, with separate measurements in VM and PF (≈100 mg tissue per heart) samples from each heart included.
Competitive reverse transcription (RT)–polymerase chain reaction (PCR) was used to quantify mRNA expression as previously described in detail.14,15⇓ RNA was isolated14 and quantified spectrophotometrically at a 260-nm wavelength, and integrity was confirmed on a denaturing agarose gel. Total RNA was dissolved in RNA Storage Solution (Ambion) and stored at −80°C.
To synthesize RNA mimics (internal standards), gene-specific primers for RT-PCR (Table) were designed according to previously described or cloned sequences, with specificity confirmed by BLAST and FASTA. A 392-bp β-actin fragment was synthesized with the primers shown in the Table and used to construct mimics for the α1C subunit of the L-type Ca2+ channel (CaV1.2), ERG, KvLQT1, minK, hyperpolarization-activated cation channels (HCN1, HCN2, and HCN4), and α1G and α1H subunits of the T-type Ca2+ channel (CaV3.1 and CaV3.2). A 460-bp α-actin fragment was used to construct mimics for Kv4.3, Kv3.4, and Kv1.4 subunits, Na+-Ca2+ exchanger isoform-1 (NCX1), and the α1I subunit of T-type Ca2+ channel (CaV3.3).
First-strand cDNA was synthesized by RT with canine cardiac RNA and random primers. Chimeric primer pairs were constructed by appending gene-specific primers at the 5′ and 3′ ends of actin primers, and an 8-nucleotide (GGCCGCGG) linker homologous to the 3′ end of the T7-promoter sequence was conjugated to the 5′ end of each gene-specific sense primer. The chimeric primers were used in a PCR reaction (Taq polymerase, annealing temperature 55°C) with the first-strand DNA to generate an actin cDNA sequence flanked by gene-specific primers, with the short T7-promoter sequence at the 5′ end. The product of this PCR was diluted 10- to 100-fold, and 1 μL was used as a template in a second PCR. Primers in the second PCR (annealing temperature 60°C) included a T7-promoter primer (sense) and a gene-specific antisense primer. The resulting product (T7 promoter, gene-specific primers, and an internal α- or β-actin fragment) was gel-purified (QIAquick Gel Extraction Kit, Qiagen) and used as a template for in vitro transcription. In vitro transcription was conducted with mMESSAGE mMACHINE (Ambion) at 37°C for 3 hours. RNase-free DNase I (2 U) was added to a 20-μL reaction mixture and incubated at 37°C for 30 minutes. Mimic RNA was purified with phenol chloroform extraction and RNA inactivation reagent (Ambion). The mimic RNA pellet was dried and dissolved in RNA Storage Solution (Ambion). The quantity of RNA was determined by spectrophotometry and denaturing gel analysis.
Serial dilutions of RNA mimics were added to 1-μg samples of RNA in a series of reaction mixtures. RNA was denatured at 70°C for 10 minutes and ice-chilled for 5 minutes before addition to the reaction mixture. RT was conducted at 25°C for 10 minutes and at 42°C for 60 minutes with a 20-μL first-strand cDNA synthesis mixture (3.2 μg random hexamers, 1 mmol/L deoxynucleotide mixture, 50 U RNase inhibitor, and 20 U MMLV reverse transcriptase). Aliquots of first-strand cDNA (5 μL) were amplified by PCR in a 25-μL solution containing (mmol/L) Tris-HCl 10 (pH 8.3), KCl 50, MgCl2 1.5, deoxynucleotide mixture 0.8, 2.6 U Taq polymerase (PCR High Fidelity Kit, Boehringer-Ingelheim), and 0.2 μmol/L gene-specific primers. The reaction mixture was denatured (94°C, 3 minutes) and subjected to 30 PCR cycles (denaturing [94°C, 30 seconds], annealing [temperatures given in the Table, 30 seconds], and elongation [72°C, 40 seconds]), followed by a final 10-minute extension period at 72°C.
Final PCR products (10-μL aliquot) were subjected to electrophoresis on 1.5% to 2% agarose gels containing Tris-acetate (40 mmol/L), EDTA (1 mmol/L), and ethidium bromide. Ethidium bromide fluorescence images were captured with a Nighthawk camera under ultraviolet light, and band density was determined with Quantity-One software. As mimic concentration in the initial reaction mixture decreases, the mimic band intensity decreases, and target-gene bands increase (Figure 1A). The bands should be of equal intensity when mimic and target mRNA concentrations in the reaction mixture are the same. A DNA mass ladder was used to construct a standard curve for quantification. The logarithm of mimic-to-target intensity ratio was plotted as a function of the logarithm of RNA-mimic concentration. Linear regression was used to determine the point of identity.
The absence of genomic contamination in the RNA samples and of DNA contamination in RNA mimics was confirmed with reverse transcriptase–negative controls for each experiment. Known quantities of target and mimic RNA were coamplified for each construct to confirm that target sequences and corresponding mimics were amplified with similar efficiencies.
To extract membrane protein, tissues were pulverized in liquid N2 and suspended in 500 μL of ice-cold TE buffer (containing Tris 20 mmol/L, EDTA 1 mmol/L, benzamidine 0.1 mg/mL, phenylmethylsulfonyl fluoride 10 mg/mL, leupeptin 5 μg/mL, pepstatin A 5 μg/mL, aprotinin 5 μg/mL, and sodium orthovanadate 100 μmol/L). The suspension was homogenized, and 2% Triton X-100 was added, put on ice (2 hours), and then centrifuged (14 000g, 10 minutes, 4°C). The soluble fraction was retained and stored at −80°C. Protein concentration was determined by the Bradford method. Samples (200 μg protein) were denatured in Laemmli sample buffer, separated on 5% to 10% regular or 4% to 15% precast gradient SDS-polyacrylamide gels (Bio-Rad), and transferred to Immobilon-P polyvinylidene difluoride membranes. Membranes were blocked (2 hours, room temperature) with 5% nonfat milk in 0.1% Tween 80 Tris-buffered saline solution (TTBS) and incubated overnight at 4°C with rabbit polyclonal antibodies against Kv4.3, Kv3.4, minK, CaV1.2, HCN1, HCN2, HCN4 (Alomone Labs), KvLQT1 (Chemicon), and ERG (Santa Cruz) or mouse monoclonal antibody against NCX1 (Bioreagents) and the internal control, GAPDH (Research Diagnostics). We were unable to obtain antibodies against Kv channel–interacting protein (KChIP2) and CaV3.1 to CaV3.3; therefore, analysis of these constructs was limited to mRNA expression. After 3 washes in TTBS, the membranes were incubated in 1:5000 to 1:20 000 dilutions of horseradish peroxidase–conjugated goat anti-rabbit IgG (Santa Cruz Biochemicals), donkey anti-goat IgG (Santa Cruz), or goat anti-mouse IgM (Santa Cruz) in 5% nonfat milk in TTBS (60 minutes, room temperature), followed by 3 additional washes in TTBS. Antibody was detected with Chemiluminescence Reagent Plus (New England Nuclear Life Sciences). Band density was quantified by laser densitometry with Quantity-One software. Densitometric comparison of PF and VM bands was performed on blots processed equally and exposed on the same x-ray film. Samples probed with primary antibody preincubated with antigenic peptides were used as negative controls (NCs). For KvLQT1 and NCX1, antigenic peptide was unavailable, and NCs were obtained by omitting primary antibodies.
Ventricular myocytes and Purkinje cells freshly isolated from canine left midmyocardium and PFs of each dog were plated on laminin (15 μg/mL)–coated coverslips at 37°C and incubated (humidified 95% O2/5% CO2) for 1 hour. Cells were fixed with 2% precooled paraformaldehyde containing 0.2% Triton X-100 for 30 minutes, followed by 3 additional washes with PBS, and blocked (2 hours, room temperature) with 10% donkey serum and 5% BSA in PBS. The cells were incubated overnight with primary antibody in PBS containing 1% BSA and 2% donkey serum. NCs consisted of cells exposed to primary antibody preincubated with the antigen (Kv3.4 and Kv4.3) or cells exposed to secondary antibody without primary antibody (NCX1). After 3 washes, cells were incubated (1 hour) with 1:200 fluorescence-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) or goat anti-mouse IgM (Santa Cruz) and then rinsed 3 times with PBS. Coverslips containing cell aliquots were mounted on slides with 1-mg/mL p-phenylenediamine in 75% glycerol and examined under a Zeiss Axiovert 100-M microscope coupled to a Zeiss LSM-510 laser-scanning confocal system. Identical settings were used for PFs and VM from each heart to obtain a qualitative comparison of relative expression.
All comparisons were performed with paired PF and VM data from each dog, with t tests used for statistical comparison. Average data are presented as mean±SEM.
Ito Subunit Expression
Figure 1B shows the mRNA expression levels of a variety of subunits related to cardiac transient outward current (Ito). Kv4.3 was richly expressed, with similar quantities in PFs and VM. Kv1.4 expression was low and similar in both tissues, and Kv4.2 was not detected. A large quantity of mRNA encoding the tetraethylammonium (TEA)-sensitive subunit Kv3.416 was present in PFs, with significantly lower quantities in VM. In addition, KChIP2, a recently reported β subunit with important interactions with cardiac Kv4 subunits,17 was expressed at 25-fold greater levels in VM than in PFs.
To pursue possible expression differences in Ito-encoding α subunits, we used commercially available antibodies to study protein expression. Figure 2A shows Western blots of Kv4.3 from 2 PF and 2 VM samples, with a 70-kDa band corresponding to the expected size of Kv4.3 (horizontal arrow at the right) and a ≈60-kDa band detected. Both bands were absent on probing with antibody preabsorbed with antigenic peptide and were more intense in VM than in PFs. Mean intensity of the 70-kDa band was about twice as great in VM compared with PFs (Figure 2A, right). The 60-kDa band, which may represent a proteolytic product, was also significantly weaker in PFs, averaging 0.69±0.05 of the intensity in VM (P<0.05 versus VM). Figure 2B shows Kv4.3 immunocytochemistry. On probing with anti-Kv4.3 (Figure 2B, top micrographs), signals were clearly present in both PF and VM myocytes, with a transverse striation pattern consistent with transverse-tubular localization. The middle panels show fluorescent images of cells probed with antibody preincubated with antigenic peptide, with corresponding phase-contrast micrographs of the same cells shown in the bottom panels. Antigen preabsorption clearly eliminated the Kv4.3 signal. Similar immunocytochemical results were seen for 6 PF and VM cell samples from 3 hearts exposed to Kv4.3 antibody alone and for 2 PF and VM cell samples from 2 hearts exposed to Kv4.3 antibody after antigen preincubation.
Figure 3A shows Western blots for Kv3.4. A 110-kDa band corresponding to Kv3.4 (horizontal arrow at the right) was stronger in PF samples and was suppressed by preincubation with the antigenic peptide. A second, lower molecular weight band was present in VM but was also present on probing with antibody preincubated with antigenic peptide. Mean 110-kDa band intensities in 6 hearts were ≈3-fold greater in PFs (right panel). On confocal imaging, clear and strong staining was seen in PF cells, with concentration at cell ends and also at transverse tubules (Figure 3B, left). VM myocyte staining was weak and with less clear subcellular localization. No signal was present in cells exposed to Kv3.4 antibody after preincubation with the antigenic peptide (Figure 3B, middle), with phase-contrast images for the cells exposed to antibody preincubated with antigen shown in right panels. Similar immunocytochemical results were seen for 8 PF and VM cell samples from 3 hearts exposed to Kv3.4 antibody alone and for 2 PF and VM cell samples from 2 hearts exposed to Kv3.4 antibody after antigen preincubation.
IK Subunit Expression
Figure 4A shows the mean mRNA concentrations of K+-activated current (IK) subunits ERG, KvLQT1, and minK. The α subunits ERG and KvLQT1 were significantly less strongly expressed at the mRNA level in PFs than in VM. MinK mRNA was significantly more concentrated in PFs. Typical Western blots for ERG, KvLQT1, and minK are shown in Figure 4B. A single clear ERG band was reproducibly detected at ≈165 kDa in PF and VM samples and disappeared on preincubation with antigen. KvLQT1 bands at ≈78 kDa were strong in VM and much weaker in PFs. Antigenic peptide was not available for KvLQT1, but the ≈78-kDa bands were absent on Western blotting with omission of the primary antibody. A single 16-kDa band detected by the minK antibody was less intense in PFs than in VM and was eliminated by preincubation with antigen. The GAPDH signals obtained on the same gels are shown at the bottom of Figure 4B. A comparison of mean protein band intensities relative to GAPDH for ERG, KvLQT1, and minK in PFs versus VM is shown in Figure 4C. ERG and KvLQT1 bands were significantly less strong in PFs than in VM (ERG, 0.28±0.08 versus 0.70±0.09, respectively [P<0.01]; KvLQT1, 0.26±0.04 versus 0.62±0.03, respectively [P<0.01]). In contrast to its mRNA expression profile, the minK protein signal was also significantly weaker in PFs than in VM (0.38±0.04 versus 2.50±0.25, P<0.001). Overall, GAPDH signal intensity was similar in PFs and VM, averaging 3.74±0.22 and 3.71±0.20, respectively, for each (P=NS).
Expression of CaV1.2 and NCX1
Figure 5A shows mean mRNA concentrations of CaV1.2 and NCX1 genes. CaV1.2 mRNA was significantly more concentrated (≈2-fold greater) in VM than in PFs. NCX1 mRNA was present in very high concentrations that were similar in both VM and PFs. Typical Western blots for CaV1.2 and NCX1 are shown in Figure 5B. A signal at the expected molecular weight for CaV1.2 (just over 200 kDa) was present in VM and was eliminated by preincubation with antigenic peptide (NC, lane at right) but was very faint in PFs. NCX1 antibody detected a strong signal at ≈160 and ≈125 kDa in both tissues, along with very faint bands at ≈70 kDa. The 160- and 125-kDa band levels were consistently stronger in VM. The 70-kDa band was difficult to quantify because of its faintness, and no consistent differences were seen. No NCX1 bands were seen on omission of primary antibody (NC). Mean intensities for the ventricular CaV1.2 and the NCX1 bands are shown in Figure 5C. NCX1 signal was substantially stronger in VM compared with PFs. The CaV1.2 band was below the limit for quantification in PFs. Immunocytochemical studies (Figure 5D) showed NCX1 to be localized at cell membranes in both VM and PFs, but VM also showed a clear transverse-striation pattern, consistent with transverse-tubular localization, which was absent in PFs. Similar immunocytochemical results were obtained with NCX1 antibody in 6 VM and PF cell samples from 2 hearts. NC experiments performed by omitting the primary antibody showed no staining for 2 VM and PF samples from 2 hearts.
HCN Isoform Expression
A very faint band corresponding to HCN1 was seen after RT-PCR in PFs but not in VM, and the HCN1 mRNA concentration in PFs was too low to be quantified. HCN4 mRNA was found at a quantifiable level in PFs but not in VM (Figure 6A). HCN2 mRNA was also quantifiable and at similar concentrations in both VM and PFs but was significantly less strongly expressed than HCN4 in PFs. Western blot analysis of HCN2 (Figure 6B) revealed 2 bands (of ≈55 and ≈90 kDa) in the rat brain. A corresponding 55-kDa band was detected in PFs and VM and was strongly suppressed by antigen preincubation. VM and PF bands at ≈80 and ≈50 kDa did not correspond to the rat-brain bands and were largely unaffected by antigen preexposure. HCN1 antibody did not detect a signal in PFs or VM, but HCN4 antibody detected a strong ≈160-kDa signal in PFs corresponding to the rat brain signal. HCN4 signals were suppressed by antigen preincubation. The mean intensities of the 55-kDa HCN2 and the 160-kDa HCN4 bands are provided in the bottom right panel of Figure 6B. HCN2 was significantly stronger in PFs than VM, and HCN4 was below the threshold for quantification in VM.
CaV3 Subunit Expression
Figure 7A shows representative gels of competitive RT-PCRs to analyze the mRNA concentrations of CaV3.1, CaV3.2, and CaV3.3. Results for VM are at the left, and those for PFs are at the right. The point at which target gene signals were equivalent to those for mimics were in each case further to the left for PFs than VM (ie, they occurred at larger mimic concentrations). Figure 7B shows mean±SEM mRNA concentrations from 6 hearts for each construct. All isoforms were significantly more strongly expressed in PFs.
We have completed a detailed study of the expression of subunits contributing to Ito, IK, L-type Ca2+ current (ICaL), T-type Ca2+ current (ICaT), NCX, and the nonselective cation current (If) in canine cardiac PFs compared with matched VM samples from the same animals. We found significant differences in subunit expression patterns that may contribute importantly to the functional electrophysiological specificity of PFs.
Relationship of Our Findings to Specific Aspects of PF Electrophysiology
Transient Outward Current
Ito was first noted to be a prominent feature of the ionic current profile of PFs in the early 1960s.18 Initial studies of PF Ito suggested it to be a Cl− current,19,20⇓ but subsequent work by Kenyon and Gibbons21,22⇓ demonstrated that the vast majority of PF Ito is carried by a 4-aminopyridine and TEA-sensitive K+ conductance. PF APs show rapid phase-1 repolarization and a subsequent notch at low frequencies, with the notch disappearing on repetitive activation at higher rates.12 This behavior was demonstrated by Hauswirth et al12 to be due to the slow time-dependent reactivation of PF Ito at diastolic potentials. Ito inhibition can prolong PF APD,21,23⇓ and downregulation of PF Ito by congestive heart failure sensitizes PFs to the APD-prolonging effects of the rapid component of the delayed rectifier K+ current (IKr)-blocking class 3 drugs, adding to the proarrhythmic risk of class 3 agents.24
Recent work has pointed out a variety of biophysical and pharmacological properties of PF Ito that differentiate it from Ito in the atrium and VM.23 Like VM Ito, PF Ito displays biexponential recovery from inactivation; however, the slow component in PF recovers an order of magnitude more slowly (time constant 1.4 seconds) than in VM (time constant 180 ms).23 In addition, the slow component constitutes ≈2/3 of overall recovery in PFs versus 1/3 in VM. PF Ito is an order of magnitude more sensitive to 4-aminopyridine compared with VM, and whereas ≈80% of PF Ito is sensitive to TEA, VM Ito is unaffected by TEA.23
The present study demonstrates differences between PFs and VM in the expression of Ito-encoding subunits that may help us to understand the molecular basis for the functional discrepancies between PF and VM Ito. Kv1.4 subunits form slowly reactivating Ito channels on heterologous expression.25 Kv1.4 subunit composition appears to underlie slowly recovering Ito in the rabbit heart15 and is thus a potential candidate to explain the slow recovery of PF Ito. However, we found Kv1.4 mRNA expression to be sparse and equivalent in PFs and VM. On the other hand, we noted a marked difference in the expression of the accessory subunit KChIP2 between VM and PFs, with KChIP2 mRNA abundance in PFs being ≈1/25 that in VM. KChIP2 coexpression substantially accelerates the reactivation of Ito carried by Kv4 subunits,26 and its scarcity likely contributes to the characteristic slow reactivation of PF Ito.23,24⇓ We also observed important differences in the expression of the Kv3.4 subunit. Kv3.4 was much more abundant at the mRNA and protein level in PFs than in VM. Moreover, immunocytochemical results point to specific subcellular localization in PFs compared with a more diffuse pattern in VM. Because Kv3.4 subunits carry a TEA-sensitive Ito,16 our results point to Kv3.4 as a candidate for the large TEA-sensitive component of PF Ito.
Delayed-Rectifier K+ Current
The first detailed description of IK and its 2 kinetic components was obtained in multicellular PF preparations in 1969.27 Although numerous subsequent publications have dealt with IK in multicellular PF preparations, the current has proven much more difficult to demonstrate in single Purkinje cells.28,29⇓ This difficulty is likely due to the great sensitivity of IK to cell isolation30 and the need for prolonged exposure to cell-isolating enzymes by the “chunk” method to isolate Purkinje cells. The biophysical properties of IKr and the slow component of the delayed rectifier K+ current (IKs) in Purkinje cells are generally similar to those reported for ventricular myocytes,24,31⇓ but valid quantitative comparisons between PF and VM IK densities cannot be made because of difference in cell isolation techniques. Our results suggest potentially significant differences in IK subunit expression in PFs versus VM. The expression of all 3 constituent subunit proteins (ERG, KvLQT1, and minK) was significantly less in PF than in VM. This would be expected to result in smaller IKr and IKs densities in PF and could contribute to the long-recognized discrepancy between PF and VM AP duration, with PF AP duration exceeding that in VM, particularly at slow activation rates.10,11⇓ The smaller ERG and KvLQT1 expression in PFs may be due to transcriptional regulation, inasmuch as ERG and KvLQT1 mRNA is sparser in PFs than in VM. Transcriptional regulation cannot explain the lesser abundance of minK protein in PFs, because minK transcript concentrations are greater in PFs than in VM.
We found a number of expression differences in Ca2+-handling elements that may be important for PF properties. PFs are known to have a less positive plateau voltage than VM, 12 which may be due to a smaller ICaL.32 Our finding of substantially weaker expression of the α1C subunit in PFs suggests a potential molecular basis for this difference. In contrast to ICaL, ICaT is prominently expressed in PFs, where it is almost as large as ICaL33,34⇓ and is very small or absent in normal VM.35 We observed much more limited expression of ICaT-encoding transcripts in VM compared with PFs. The NCX functions to remove from the cell Ca2+ that enters on depolarization of voltage-dependent Ca2+ channels. We found that PFs had weaker expression of NCX and lacked the T-tubular NCX distribution of VM. Lower level NCX expression might limit the ability of PFs to extrude Ca2+ under conditions of Ca2+ loading, potentially contributing to the enhanced susceptibility of PFs to digitalis toxicity.36 The lack of T-tubular NCX distribution in PFs (Figure 5), in contrast to the T-tubular distribution of ion channels like Kv4.3 and Kv3.4 in PFs (Figures 2 and 3⇑), may relate to the specialization of PFs for electrical activity in contrast to the crucial contractile function of VM.
The predominant contributor to spontaneous phase-4 depolarization of PFs is the nonselective cation current, If.37 HCN subunits are believed to underlie If.38 In the rabbit, HCN1 and HCN4 constitute the most strongly expressed mRNA forms in PFs, with some HCN2 present, whereas HCN2 is the only isoform in VM.39 We found HCN1 mRNA expression to be very low in dog PFs, whereas HCN4 expression was high, and HCN2 expression was intermediate. Our Western blot studies confirmed the presence of HCN2 and HCN4 protein and their greater expression in PFs compared with VM, consistent with the more important pacemaker function of PFs. To our knowledge, this is the first such demonstration at the protein level.
This is the first research of which we are aware to study in detail the expression of a wide range of ion-channel subunit genes in PFs. Previous data regarding ion-channel subunit expression in PFs have been limited to studies of mRNA expression of HCN39 and to a number of detailed studies of connexin expression (see review13). Given the importance of PFs in a wide range of ventricular tachyarrhythmias,1–8⇓⇓⇓⇓⇓⇓⇓ highlighted by a recent report of malignant arrhythmia prevention in man by ablation of Purkinje tissue,8 a better understanding of the molecular basis of PF electrophysiology would seem to be of great significance. Our results indicate that PFs have a distinct ion-channel subunit expression profile, consistent with electrophysiological evidence of the distinct nature of PF ionic and cellular electrophysiology.
We were unable to obtain antibodies against several ion-channel subunits of interest, limiting our evaluation of the expression of CaV3 and KChIP2 to assessments of transcript abundance. In addition, the antigenic peptide was unavailable for several antibodies (notably, KvLQT1 and NCX1). Our studies were performed in dogs and cannot necessarily be extrapolated to other species. We compared PF tissue from free-running false tendons with left ventricular midmyocardium. Ion-channel expression is known to be regionally determined,13 which must be considered in interpreting our findings.
The authors thank the Canadian Institutes of Health Research, the Québec Heart and Stroke Foundation, and the Mathematics of Information Technology and Complex Systems (MITACS) Network for research funding, Evelyn Landry and Chantal St-Cyr for technical assistance, France Thériault for secretarial help with the manuscript, and Drs Robert Dumaine and Charles Antzelevitch for providing KvLQT1 antibody for preliminary studies.
Original received May 31, 2002; revision received September 4, 2002; accepted September 19, 2002.
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