Cardiovascular Research

Cardiovascular Research 2005 65(1):117-127; doi:10.1016/j.cardiores.2004.08.017

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Copyright © 2004, European Society of Cardiology

Contribution of neuronal sodium channels to the cardiac fast sodium current I_Na is greater in dog heart Purkinje fibers than in ventricles

V. Haufe^a, J.M. Cordeiro^b, T. Zimmer^a, Y.S. Wu^b, S. Schiccitano^b, K. Benndorf^a and R. Dumaine^b,*

^aFriedrich Schiller University Jena, Institute of Physiology II, Teichgraben 8, 07740 Jena, Germany
^bMasonic Medical Research Laboratory, 2150 Bleecker Street Utica NY 13501, USA

^* Corresponding author. Tel.: +315 735 2217; fax: +315 735 5648. Email address: rdumaine{at}mmrl.edu

Received 19 May 2004; revised 29 August 2004; accepted 31 August 2004

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
Objective: To determine the presence and the potential contribution of neuronal sodium channels to dog cardiac function.

Methods: We used a combination of electrophysiological (patch clamp), RT-PCR, biochemical and immunohistochemical techniques to identify and localize neuronal Na⁺ channels in dog heart and determine their potential contribution to the fast sodium current.

Results: In all cardiac tissues investigated, Na_v1.1, Na_v1.2 and Na_v1.3 transcripts were detected. In immunoblots, we found Na_v1.1 and Na_v1.2 proteins in the ventricle (V) and in Purkinje fibers (PF). Na_v1.3 immunoblots suggested strong proteolytic activity against this isoform in the heart. Na_v1.6 was not found in any of the tissues tested. Confocal immunofluorescence on cardiac myocytes showed that Na_v1.1 was predominantly localized at the intercalated disks in V and PF and around the nucleus (V). Na_v1.2 was only present at the Z lines (V). Consistent with the immunoblot data, an intense but diffuse intracellular staining was observed for Na_v1.3. Na_v1.6 fluorescence staining was faint and diffuse. Surprisingly, immunoblots indicated the presence of two Na_vβ2 variants: a 42-kDa protein that co-localized with Na_v1.2 at the Z lines in V and a 34-kDa protein that co-localized with Na_v1.1 at the intercalated disks in PF. In agreement with the biochemical data, electrophysiological results suggest that neuronal sodium channels generate 10 ± 5% and 22 ± 5% of the peak sodium current in dog ventricle and Purkinje fibers, respectively.

Conclusions: Our results suggest that neuronal NaChs are more abundant in Purkinje fibers than in ventricles, and this suggests a role for them in cardiac conduction.

KEYWORDS Ion channels; Gene expression; Purkinje fibers; Na-channel

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
Contraction of the heart is initiated when action potentials (AP) from the atria converge towards the atrioventricular (AV) node and travels down the HIS bundles to the Purkinje fibers (PF) to spread the electrical impulse to the ventricles (V) [1]. In PF and V, voltage-dependent Na⁺ channels (Na_V) generate the sodium current (I_Na) responsible for the AP upstroke.

Na_Vs -subunit consists of four domains each containing six transmembrane segments [2–4], associated to accessory β-subunits [5]. The cardiac-specific -subunit Na_v1.5 is resistant to blockade by tetrodotoxin (TTX) and saxitoxin (STX) [6–8] and is believed to generate the bulk of I_Na.

Early evidences hint at the presence of TTX sensitive neuronal Na⁺ channels (nNa_vs) in the heart. Coraboeuf et al. [9] showed that low concentrations of TTX shortened the AP of PF and slowed their beating rate. Renaud et al. [10] identified TTX sensitive receptors in rat hearts. In the 1990s, nNa_Vs mRNA and proteins were detected in rat and mouse heart [11–15].

Metabolic stress enhances nNa_Vs activity and brain cells excitability [16]. Cardiac Na_Vs on the contrary are inhibited by hypoxia and ischemia [16]. These opposite responses suggest that nNa_Vs contribution to cardiac electrophysiology may be linked to pathophysiological conditions. To test such hypothesis, however, knowledge of nNa_V isoforms present in the heart of species close to humans is needed but currently lacking. This study's aim is to determine the genetic makeup of cardiac I_Na in dog PF and V.

Data regarding Na_V isoforms expressed in the heart of large mammal species closer to human seem at odd with results obtained in rodents. Maier et al. [14] initially found the neuronal subtypes: Na_V1.1, Na_V1.3, and Na_V1.6 in the T-tubules but could not detect the presence of Na_V1.2 in mouse myocytes and recently showed that Na_V1.5 and the Na_Vβ2 subunit co-localize at the intercalar disks while Na_Vβ1 and Na_Vβ3 co-localize with Na_V1.1, Na_V1.3 and Na_V1.6 [17]. In contrast, we could not detect Na_v1.3 or Na_V1.6 proteins but found Na_V1.2 in the plasma membrane of canine myocytes. These discrepancies between dog, rat [13] and mouse [14] suggest species-specific requirements for nNa_Vs.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
2.1. Competitive RT-PCR
Total RNA was isolated using the Total RNA Isolation Kit (Ambion). Reverse transcription (RT) was performed using Superscript II (Invitrogen) with an equimolar mix of anchored oligonucleotides (dTAN, dTCN, dTGN). Reverse transcribed cDNA (RT-cDNA) was digested with RNase H. Parts of the C-terminal region of canine Na⁺ channels: cNa_v1.1 (2.2 kb), cNa_v1.2 (2.2 kb), and cNa_v1.3 (1.7 kb), and the full-length cNa_v1.5 (accession AJ555547 [GenBank] ) were amplified by PCR (Pfu DNA polymerase) and subcloned into pUC119 for sequencing. Homologous regions between the canine nucleotide sequences were used to design primers (Table 1). Ten primer pairs were used to test for the amplification efficiency of each Na⁺ channel isoform in the competitive reaction. The selected primer pair amplified cNa_v1.2, cNa_v1.3, and cNa_v1.5 with the same efficiency but produced about two-fold higher levels of cNa_v1.1. Na_v1.1 data in Fig. 1 are presented as uncorrected intensity values. Individual Na⁺ channel fragments were identified by restriction digests. Densitometric values were obtained with the EASY Win32 system from Herolab (Wiesloch, Germany) coupled to a CCD camera.

View this table: [in this window] [in a new window]   Table 1 Primer pairs used for competitive RT-PCR

 

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Relative mRNA content of Na+ channel isoforms in dog heart determined by competitive RT-PCR. (A) Simultaneous amplification of Nav1.1, Nav1.2, Nav1.3, and Nav1.5 from reverse transcribed cDNA (right ventricle, RV) followed by Nav-specific restriction digest of the original PCR amplicon. Lanes 1: undigested (0.45 kb); 2: BseDI digest (0.24 kb) for Nav1.1; 3: Eco47I (0.20 and 0.25 kb) for Nav1.2; 4: Alw44I (0.20 and 0.25 kb) for Nav1.3; 5: ClaI (0.30 and 0.15 kb) for Nav1.5; 6: Water contro1 (no cDNA). Intensity of each Nav-specific band was expressed as a fraction of the intensity of the undigested amplicon. (B) Simultaneous amplification of Nav1.1, Nav1.2, and Nav1.3 using specific primer pairs and confirmation of the presence of each channel in RV by Nav-specific restriction digest of the initial PCR amplicon (0.71 kb) in lane 1. Lanes 2: Nav1.1 (BseDI, 0.39 kb); 3: Nav1.2 (Eco47I, 0.35 kb and 0.29 kb); 4: Nav1.3 (Alw44I, 0.43 kb and 0.29 kb); 5: Water control. (C–E) Transcriptional levels of brain and cardiac Na+ channels mRNA calculated from relative band intensities, as described in (A). (C) Relative intensity of pooled nNaVs (Nav1.1+Nav1.2+Nav1.3) and Nav1.5 digests expressed as percent of the intensity of the undigested amplicon. SA: Sino-atrial node, AV: Atrio-ventricular node, HIS: His Bundle, RA, LA: Right, left atrium, PF: Purkinje fibers, RV, LV: right, left ventricle. (D) Contribution of individual nNaVs as percent of the intensity of the pooled nNaV amplicon shown in (C). (E) Relative expression of nNaVs in dog brain. Nav1.5 was undetectable in these samples. : DNA ladder, bp: base pairs. Data ± S.E.M. Number of samples: SA: 4; RA: 6; LA: 8; AV: 3; HIS: 5; PF: 7; RV: 5; LV: 6.

 
2.2. Mutagenesis
Mutation C372Y was constructed using the megaprimer method of site-directed mutagenesis on plasmid pcDNA3/hH1a obtained by cloning the sodium channel SCN5A into the vector pcDNA3.1+ (Invitrogen) as previously described [18]. Full-length wild type and mutated pcDNA3/hH1a cDNAs were linearized by digestion with EcoRI and transcribed using the T7 mMessage mMachine transcription kit (Ambion, Austin, TX). RNA was resuspended in 0.1 M KCl and stored at –80 °C. The concentration and quality of cRNA were assessed by optical density (OD₂₆₀) reading and electrophoresis.

2.3. Antibodies
The anti-Pan antibody (SP19, Sigma) targets a conserved region of the intracellular loop between domains III and IV of the Na⁺ channel subunits. Na_v1.1, Na_v1.2, and Na_v1.3, antibodies (Alomone Labs, Israel) target an epitope between domains I and II or II and III (Na_v1.6) of their respective -subunit. Na_vβ₂ antibodies (Alomone Labs) target the intracellular C-terminus of the β₂-subunit. In control experiments, antibodies were pre-absorbed against their respective antigen (2 µg/ml) for 1 h at RT then overnight at 4 °C in 1 ml of blocking solution (5% non-fat dry milk, 5% goat serum).

2.4. Immunoblots
Total proteins were isolated from the organic phase obtained from the RNA isolation procedure (Ambion) as previously described [19]. Membrane proteins were isolated by centrifugation on a sucrose gradient according to the protocol published by Alomone Labs. Proteins were denatured by heating for 30 s at 60 °C and were reduced by application of β-mercaptoethanol (β-ME) where indicated (+) before loading and electrophoresis in SDS polyacrylamide gels. Proteins were blotted on PVDF membranes (Perkin Elmer) using standard methods. Antigen-bond primary antibodies (1:200) were detected with HRP-conjugated goat anti-rabbit antibody (BioRad).

2.5. Cell isolation
Ventricular myocytes were dissociated from adult dog heart left ventricle as previously described [20,21] and resuspended in a sterile solution containing in mM: NaCl: 132, KCl: 5, CaCl₂(2H₂O): 0.5, MgSO₄: 2, HEPES: 20, D-Glucose: 11.1, and 1.5% Bovine Serum Albumin (BSA) Fraction V (Sigma). PF were dissected out and myoytes were dissociated by sequential incubations of 10 min in collagenase (chunk method) as previously described [22].

2.6. Immunocytochemistry
Cells in low-calcium Tyrode solution containing (in mM): NaCl: 130, KCl: 4, MgSO₄: 1.2, HEPES: 10, D-Glucose: 11.1, were cytospun on glass slides and immediately fixed in a solution containing: 5% ethanol, 25% acetone, 70% formaldehyde/ZnCl₂, pH 6 for 15 min at 4 °C and then permeabilized for 10 min in a Ca²⁺-free Tyrode solution containing Saponin (0.25% w/v) and CHAPS (0.5% w/v). Primary antibodies (1:200) were applied overnight at 4 °C and detected with a goat anti-rabbit antibody (1:1000) conjugated to Alexa 488 (Molecular Probes, USA). Propidium iodine (PI) was used to stain nucleotide rich regions (nucleus). Cells mounted with Pro-Long antifade mounting media (Molecular Probe) were visualized on a Olympus Fluoview (Olympus, Japan) confocal microscope, as previously described [21].

2.7. Electrophysiology
Whole-cell voltage clamp was performed on myocytes allowed to adhere to the bottom of polylysine-coated Petri Dish (35 mm) mounted on the stage of a Nikon Diaphot microscope and superfused at a rate of 2–3 ml/min with the extracellular solution. The outflow of a micro-manifold fast perfusion apparatus (ALA Scientific Instruments, Westbury, NY) placed closed to the cell was used to deliver (2-aminoethyl)methanethiosulfonate (MTSEA). MTSEA was prepared fresh before each application. Experiments were performed at room temperature using a VE-2 amplifier (Alembic Instruments, Montreal, Qc) or an Axopatch-2B (Axon Instruments, CA), analysis of the data with the pClamp 9 program suite (Axon Instruments). Patch pipettes with 1.5–2.5 M resistance were pulled from borosilicate glass tubes (1.5 mm o.d. and 1.1 mm i.d.). Tip potentials (9–15 mV) were measured for voltage corrections and series resistance were 85% to 95% compensated. Currents were filtered online at 5 kHz (Bessel filter). Reagents were obtained from regular suppliers (Sigma, Alomone Labs, Fisher, Sardstead), MTS reagents were obtained from Toronto Chemicals. To minimize voltage clamp errors due to large cardiac sodium currents, extracellular sodium concentration was reduced and contained, (in mM)::94 Choline–Cl, 40 NaCl, 2 CaCl₂, 1 MgCl₂, 1 CoCl₂, 10 HEPES, 10 glucose, pH 7.4 (Choline–OH). Pipette solution (mM): 5 NaOH, 145 Cs–aspartate, 1 MgCl₂, 10 HEPES, 4 MgATP, 5 EGTA, pH 7.2 (CsOH). An expected small shift in Na_Vs steady-state gating parameters due to the presence of CoCl₂ in our extracellular solution was observed and taken into consideration in our analysis but did not affect the MTSEA block. Recordings in tsa201 cells were as previously described [23].

Xenopus laevis oocytes were prepared, co-injected with 0.2 to 1.25 ng of sodium channel cRNA, and currents were recorded 4 to 10 days post-injection according to methods previously described [18,24,25]. For electrophysiological recordings at room temperature, external solution flowing at 2 to 3 ml/min contained (mM): 135 NaOH, 130 methanesulfonic acid, 5 NaCl, 5 CsCl, 0.2 CaCl_2, 1.8 MgCl_2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3 (NaOH). Oocyte whole cell currents recorded using the two-electrode voltage-clamp method [25] with beveled micropipettes filled with 1% agar in 3 M KCl [26] were amplified by a Warner Oocyte Clamp 725C amplifier (Warner Instrument, Hamden, CT), low pass-filtered at 5 kHz (–3 dB, 4-pole Bessel filter) and digitized at 100 kHz.

The pCMV/SCN1A, pCD8-IRES-hβ1 and pGFP-IREs-hβ2 cDNA constructs [27] were transfected at a molar ratio of 10:1:1, respectively, into tsa201 cells grown to 60% confluency in 60-mM culturing dishes [23]. Data were analysed using a Student's T-test for paired data and ANOVA for unpaired RT-PCR results.

This investigation conforms with the Guide for Care and Use of Laboratory animals published by the US National Institutes of Health (NIH Pub. No. 85-23, revised 1996).

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
We first determined the abundance of nNa_v mRNA in different regions of the heart. In ventricles, primers (Table 1) designed to simultaneously amplify the nNa_V isofoms Na_v1.1, Na_v1.2, Na_v1.3 and Na_v1.5 generated the expected 0.45-kb PCR amplicon (Fig. 1). Selective restriction digest of this amplicon yielded bands of the expected size for each nNa_Vs and Na_v1.5 (Fig. 1A). In control experiments, primers specifically targeting nNa_vs amplified the expected 0.75-kb amplicon. Restriction digests specific to each nNa_v yielded the expected bands (Fig. 1B). PCR/selective-digest experiments using RT-cDNA from the SA and AV nodes, right and left atria, V, HIS bundle and Purkinje fibers showed the presence of nNa_Vs in each tissue type (Table 2).

View this table: [in this window] [in a new window]   Table 2 Relative transcript levels of Na+ channels in different heart regions

 
RNA from nNa_Vs was more abundant in PF, accounting for 35 ± 7% of the total Na⁺ channel RT-cDNA compared to values between 13 ± 2% and 21 ± 2% in other cardiac tissues (Fig. 1C). When nNa_vs RNA contribution was broken down into its constituents (Fig. 1D), Na_v1.3 was the most abundant transcript except in PF where Na_v1.1 and Na_v1.2 levels were higher.

We next tested for proportional amounts of proteins in V and PF using immunoblots and immunofluorescence assays. The SP19 antibody targets a conserved epitope within the cytosolic III–IV loop of all Na_Vs [28,29] and yielded bands with apparent molecular weight of 250, 150 and 90 kDa in proteins from the cortical region of dog brains (Fig. 2A) We next tested for covalent assembly of nNA_Vs with β-subunits [5,30–32]. Application of the reducing agent β-mercaptoethanol (β-ME) slightly decreased the intensity of the (250 kDa) band suggesting that some nNa_vs are heavily glycosylated or not covalently linked to other subunits. Thus, the 150-kDa band represents the unglycosylated and reduced form of nNa_Vs while the band at 90 kDa and the fainter bands are likely to represent proteolytic fragments. In proteins from right ventricles (RV), 150 and 82 kDa bands were detected with SP19. A 170-kDa band was expected based on the literature provided by the manufacturer (Alomone Labs). Immunoblots of brain proteins with Na_v1.1 antibodies revealed a banding pattern similar to SP19 (Fig. 2B). Application of β-ME abolished the high molecular weight band and increased the intensity of the 150-kDa band suggesting covalent assembly of Na_v1.1 with a β-subunit in dog brain. In RV, a faint band >250 kDa (arrow) was observed in all samples tested (n=4) and disappeared after application of β-ME thus suggesting that some Na_v1.1 channels are associated with a β-subunit.

View larger version (66K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Neuronal Nav1.1 proteins in dog ventricles. (A) Detection of Na+ channels by SP19 antibodies. Left panel: Immunoblot of native proteins (–) from the cortical region of dog brain. SP19 recognized major bands at 250, 150, and 90 kDa. Application of the reducing agent β-mercaptoethanol (β-ME; +; 165 mM) decreased the intensity of the heaviest band. Right panel: In right ventricle (RV) membrane proteins, SP19 recognized a 150-kDa protein and a 90-kDa proteolytic fragment. SP19 antibodies pre-absorbed against their antigen (Pa) did not highlight any band. (B) In brain, Nav1.1 antibodies recognized bands of 250 and 150 kDa. β-ME (+) reduced the intensity of the 250-kDa band and increased the amount of 150-kDa proteins suggesting a covalent link to a β-subunit. In RV proteins Nav1.1 antibodies recognized 150 kDa and, faintly, 250-kDa proteins (arrow). β-ME (+) abolished the 250-kDa band. Pre-absorbed antibodies: Pa.

 
In brain, Na_v1.2 antibodies highlighted bands similar to the ones obtained with the Na_v1.1 antibody (Fig. 3A) and β-ME abolished the 250-kDa band. In RV, only the 150-kDa band was observed, suggesting that Na_v1.2 does not co-assemble with other subunits in this tissue.

View larger version (56K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Nav1.2 and the ancillary β-subunit Navβ2 are present in dog ventricles. (A) Left panel: Immunoblot of native brain proteins (–). Nav1.2 antibodies recognized 250, 150 and 90 kDa proteins. β-ME (+) reduced the intensity of the 250 kDa band. Right panel: In RV membrane proteins, a single 150-kDa protein was detected. Pa: Pre-absorbed Nav1.2 antibodies. (B) Left: Navβ2 antibodies recognized a 34-kDa protein in brain. β-ME (+) treatment revealed a second 42-kDa band previously linked to another protein. Right panel: In RV, Navβ2 antibodies recognized a faint band above 210- and the 42-kDa protein. β-ME (+) had no effects on the size of the 42-kDa band. Pa: Pre-absorbed Navβ2 antibodies.

 
Since nNa_Vs covalently assemble with Na_vβ2, we tested for its presence in brain and RV (Fig. 3B). A 42-kDa protein and a 250-kDa band were detected in both tissues. A third 34-kDa band was present only in brain. β-ME reduced the high MW protein in RV but did not change the size of the 42-kDa protein.

Na_v1.3 and Na_v1.6 antibodies recognized proteins of similar sizes (150 kDA) in brain (Fig. 4A,B). In RV, Na_v1.3 antibodies detected proteins likely to be proteolytic fragments in total protein preparations but none in membrane fractions separated by centrifugation (Fig. 4A) suggesting cytosolic degradation of Na_V1.3 proteins before translocation to the sarcolemma. In RV, Na_v1.6 antibodies recognized unspecific low MW bands also detected by pre-absorbed antibodies.

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Nav1.3 and Nav1.6 are not expressed in the plasma membrane of dog ventricular myocytes. (A) Left: Anti-Nav1.3 antibodies recognized bands at 150 and 70 kDa in dog brain. β-ME (+) had no effects, Right: Nav1.3 antibodies recognized proteolytic fragments in RV total cell lysate but not in isolated plasma membrane proteins (M). Pa: Pre-absorbed Nav1.3 antibodies. (B) Left: Nav1.6 recognized a 150-kDa protein in brain. β-ME (+) had no effects. Right: RV cell lysate and membrane proteins showed only proteolytic fragments (<82 kDa). Pa: Pre-absorbed Nav1.6 antibodies recognized unspecific IgGs around 50 kDa.

 
In ventricular myocytes, SP19 recognized Na_Vs at the intercalated disks, Z lines and perinuclear region (Fig. 5A). A dotted pattern typical of the T-tubule distribution was observed in optical sections (0.4 µm) of the sarcolemma. Na_v1.1 was abundant at the intercalated disks and in the perinuclear region with a faint distribution at the Z lines (Fig. 5B). Na_v1.2 and Na_vβ2 were found only along the Z lines suggesting co-localization of the two proteins in V (Fig. 5C,D). The distribution of Na_v1.3 antigens was highly variable but in most cells appeared as a diffuse signal in the cytoplasm (Fig. 5E). Na_v1.3 staining at the Z lines was observed in only one of the four dogs tested. Na_v1.6 antibodies gave a diffused fluorescent signal through the entire cell thickness (Fig. 5F). In all control experiments, except for Na_v1.6 (Fig. 5I), cells probed with pre-absorbed antibodies did not generate fluorescent signals above background levels (Fig. 5G,H).

View larger version (72K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 In-situ localization of neuronal nNaVs in dog ventricular myocytes. Confocal immunofluorescence assays on freshly dissociated and fixed RV myocytes probed with Nav1.1, Nav1.2, Navβ2, Nav1.3, and Nav1.6 antibodies (Green). Propidium iodine (PI, red) stained nucleotide rich areas (nucleus). (A) Staining by SP19. Left panel: Single optical section from the sarcolemma showing strong intercalated disks, Z lines and dotted surface staining. Right panel: Reconstruction from 15 serial optical sections (OS) showing strong perinuclear staining. (B) Nav1.1 antibody primarily stained the intercalated disks and the perinuclear region. (C–D) Nav1.2 and Navβ2 were detected at the Z lines (25 OS). (E) Nav1.3 antibodies stained the Z lines and intracellular organelles (25 OS). (F) Nav1.6 antibodies show diffused intracellular staining (15 OS). (G–I) Controls: Pre-absorbed Nav1.1, Nav1.3, and Nav1.6 antibodies. Pre-absorbed Nav1.6 antibodies showed unspecific staining. Scale bars: 50 µm.

 
In PF, immunoblots showed a distribution pattern similar to V for Na_V1.1 and Na_V1.2 (Fig. 6A). In contrast to V, Na_vβ2 antibodies recognized only the 34-kDa protein in PF. In-situ, Na_V1.1 and Na_vβ2 co-localized at the PF intercalated disks (Fig. 6B,C). In cryosections taken just below the AV node (Fig. 6D,E) localized Na_v1.2 and Na_V1.1 were more abundant in the left and right bundle branch.

View larger version (60K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 In-situ localization of Nav1.1, Nav1.2, and Navβ2 in PF and conduction system. (A) Nav1.1 and NaV1.2 antibodies recognized a 150-kDa protein in PF. Navβ2 antibodies also recognized primarily a 34-kDa protein and a second band of 70 kDa indicating possible dimers. Pre-absorbed antibodies (Pa) did not detect any band. (B–C) Immunostaining as described in Fig. 5. Nav1.1 (B) and Navβ2 (C) showed a preferential distribution at the intercalated disks in PF cells. No PI was used in these experiments. Scale bars: 50 µm. (D) Anatomical location of the cryosection shown in (E). The plane corresponds to the cut area. RBB: right bundle branch, AVN: atrio-ventricular node. (E) Fifteen-micrometer-thick cryosection probed with anti-Nav1.1 and Nav1.2 antibodies and PI indicating the presence of nNaVs in the left and right bundle branch (RBB). Scale bar: 100 µm.

 
To estimate the contribution of nNa_Vs to I_Na, we took advantage of structural differences in the pore region of Na_Vs. A cysteine at position 372 in Na_V1.5 confers high sensitivity to Zinc and low affinity for TTX [33]. In nNa_Vs of all species studied including dog, a phenylalanine or tyrosine (Na_V1.4) at the corresponding position (Y381 in human) increases Na_V1.1 and Na_V1.2 sensitivity to TTX and reduces sensitivity to zinc. Since the sulfhydryl group of cysteine binds covalently to methanethiosulfonate reagents, we reasoned that MTSEA could be used to specifically block Na_V1.5 current.

In control experiments (Fig. 7), MTSEA irreversibly blocked human Na_V1.5 currents. Replacing the cysteine at position 372 by a tyrosine abolished the covalent block by MTSEA. In native myocytes, MTSEA covalently blocked 90 ± 5% and 78 ± 5% of the peak sodium current within 2 min of application onto V and PF myocytes (Fig. 8A,C). MTSEA weakly and reversibly blocked Na_V1.1 channels expressed in tsa201 cells (Fig. 8B).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 MTSEA specifically targets a cysteine residue in the pore region of NaV1.5. (A) Electrical current recordings from X. laevis oocytes injected with NaV1.5 mRNA. MTSEA (5 mM) covalently blocked NaV1.5 peak current within 10 min. Mutating cysteine at position 372 for a tyrosine (C372Y) abolished NaV1.5 covalent block by MTSEA. (B) Time course of the blockade by MTSEA. In NaV1.5, a irreversible 98% block consistent with a covalent bound between MTSEA and C372 was observed within 10 min. C372Y eliminated the covalent block by MTSEA and reduced the affinity for Zinc. Filled symbols: NaV1.5, Open: NaV1.5/C372Y.

 

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Inhibition of NaV1.5 current by MTSEA in Purkinje fibers (PF) and ventricular (V) cardiomyocytes from adult dogs. (A) Representative current recordings from V and PF in low Na+ (40 mM) elicited by sequential 15-ms steps to 10 mV every 30 s. MTSEA block reached a steady state within 2 min. In these recordings, 8.5% of the V peak current remained after application of MTSEA compared to 20% in PF. Block was not removed by washout of MTSEA. (B) Current recordings from tsa201 cells transfected with NaV1.1, NaVβ1 and NaVβ2 during application and washout of MTSEA. (C) Average MTSEA insensitive current in V and PF (n=7 for both cell types). Statistical significance *p<0.05, double sided Student's T-test V vs. PF.

 
A hallmark of nNa_Vs in most species including dog [34–36] is their high sensitivity to TTX. We next tested if the residual current was TTX-sensitive. Fig. 9 shows that MTSEA insensitive currents in PF and V were abolished by 100 nM TTX, a concentration with minimal effects on Na_V1.5. TTX applied before or after wash in of MTSEA abolished the residual current in four PF and four V cells tested, thus confirming the neuronal nature of the MTSEA insensitive current.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 MTSEA insensitive current in PF and V is tetrodotoxin (TTX) sensitive. (A) Representative traces following sequential application of TTX (100 nM) and MTSEA (2 mM) on a PF myocyte. (B) Current recording following sequential application of MTSEA (2 mM) and TTX on a V myocyte. Residual currents were blocked by low TTX concentrations. (C–D) Time course of the MTSEA and TTX block for experiments illustrated in (A) and (B), respectively. Arrows indicate the time of application of each compound.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
We found Na_v1.1 and Na_v1.2 in V but more abundantly in PF. In V, Na_V1.1 is primarily located at the intercalated disks, an important region for cell-to-cell conduction [37], along the Z lines and in the perinuclear region. These preferential locations suggest that Na_v1.1 may play a role in AP propagation by a mechanism similar to neuronal saltatory conduction.

Despite an abundant amount of mRNA, we did not detect mature Na_v1.3 proteins in immunoblot assays and confocal microscopy revealed a diffused cytosolic distribution. The low density of proteins in the sarcolemma may be due to a rapid turnover of Na_V1.3 or recycling of the proteins in intracellular organelles. Alternatively, ventricular cells may be lacking an accessory protein for proper translocation of Na_v1.3. We could not detect proteins of the expected size for Na_v1.6, suggesting that this protein is not expressed in V and PF.

We previously reported that a fraction of Na_v1.5 channels remains trapped in the ER [21]. Our results showing Na_v1.1 proteins in the perinuclear envelope of V myocytes suggest a similar ER trapping mechanism. Retention and exit from the ER often involve protein phosphorylation and are common mechanism regulating gene expressions [38–41]. Given the strong modulation of nNa_Vs expression and gating by phosphorylation [16,42], we speculate that phosphorylation of Na_v1.1 modulates its trafficking through the ER. This may have important consequences for the surface expression of nNa_Vs during metabolic challenges.

In contrast to our results, Maier et al. [14] did not detect Na_v1.2 in mouse V but found Na_v1.1, Na_v1.3 and Na_v1.6 at the Z lines. Differences in the species studied or in the epitope recognized by the antibodies may account for the discrepancies.

Our experiments revealed that two isoforms of Na_vβ2 (a and b) with MW of 42 and 34 kDa were present in the V and PF, respectively. Na_vβ2a co-localized with Na_v1.2 at the Z lines in V but Na_vβ2b co-localized with Na_v1.1 at the intercalated disks in PF. These results suggest tissue-specific nNa_v/Na_vβ2 complexes. The nature of the two subunits and their potential role in the heart remain to be determined.

Our EP results show 10 ± 5% and 22 ± 5% contribution of nNa_Vs to I_Na in V and PF, respectively, in close agreement with the mRNA data (sum of Na_V1.1 and Na_V1.2 mRNA, Table 2). The residual MTSEA insensitive current was blocked by nanomolar concentrations of TTX thus confirming the neuronal nature of the underlying canine channels [34].

A 10–20% contribution of nNa_Vs, especially Na_V1.1 and Na_V1.2, is physiologically significant. Na_v1.1 and Na_v1.2 display a greater availability than Na_v1.5 at positive potentials making them adequately suited to trigger AP in the more depolarized neuronal cells. Their abundance in PF cells with more positive resting membrane potentials suggests that nNa_Vs provide a conduction safety margin for the triggering of AP in PF and HIS bundle. Such safety margin may become important for survival of cardiac cells depolarized by ischemia. The appearance of a large TTX sensitive current in myocytes from post-infarction remodelled myocardium [43] and PF surviving infarction [44] seems to support this hypothesis. To test this hypothesis, we looked for differences in steady-state inactivation after application of MTSEA (not shown). Unfortunately, the surface charge effects introduced by the positively charged MTSEA rendered the interpretation of the results difficult. The use of bulkier polar MTS reagents with side chains long enough for the sulphydryl group to reach C372 inside the pore of the channel would be more suitable for these measurements but are not available at this time.

In our institution, the cardiac left V is shared by several scientists and used for cell dissociation and tissue studies thus leaving RV and PF tissues readily available. Our EP results from myocytes from the left V are in good agreement with the biochemical and RT-PCR data from RV tissues suggesting that nNa_Vs are similarly expressed in both ventricles.

In conclusion, we demonstrated that Na_v1.1, Na_v1.2 and two isoforms of the ancillary subunit Na_vβ2 are present in the heart. Their cellular location suggests a preferential role for them in cardiac conduction, possibly as a safety margin. Their exact physiological function in the heart however remains to be fully determined.

	Acknowledgments

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
We thank Dr. C. Antzelevitch for giving us access to dog tissues and cells, Dr. Al George for providing us with the SCN1A, Na_Vβ1 and Na_vβ2 constructs, Dr. Arthur Iodice, Ms. J. Hefferon and Mr. Robert Goodrow for the cell dissociation and Ms. K. Schoknecht for her contribution to the cloning of the dog Na⁺ channels. This work was supported by AHAF grant H2004-011 (JMC), DFG grant 1250/9-2 (KB, TZ), BMBF grant 01ZZ0105/IZKF Jena (TZ), grant N13 from the IZKF Jena (VH), and NIH grant HL59449 (RD).

	Notes

 
Time for primary review 26 days

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 

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