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(Circulation Research. 2003;93:189.)
© 2003 American Heart Association, Inc.

Report

Canine Ventricular KCNE2 Expression Resides Predominantly in Purkinje Fibers

Marc Pourrier^*, Stephen Zicha^*, Joachim Ehrlich, Wei Han, Stanley Nattel

From the Departments of Medicine (M.P., S.Z., J.E., W.H., S.N.) and Pharmacology (M.P.), University of Montreal; Department of Pharmacology (S.Z., S.N.), McGill University, Montreal, Quebec, Canada.

Correspondence to Stanley Nattel, Montreal Heart Institute, 5000 Belanger St E, Montreal, Quebec, Canada, H1T 1C8. E-mail nattel{at}icm.umontreal.ca

Abstract

Mutations in minK-related peptide 1 (MiRP1), the product of the KCNE2 gene, have been associated with malignant ventricular arrhythmia syndromes related to impaired repolarization. MiRP1 interacts with a variety of ion-channel -subunits, dysfunction of which could account for arrhythmia syndromes; however, the observation of very low-level expression of MiRP1 in ventricular tissue has led to doubts about its relevance. The specialized His-Purkinje system plays a key role in cardiac electrophysiology and is an important contributor to ventricular arrhythmias related to abnormal repolarization. We examined the relative abundance of MiRP1 in canine Purkinje versus ventricular tissue and found much greater expression at both mRNA and protein levels in Purkinje tissue. Thus, the cardiac Purkinje system is a strong candidate to play a role in arrhythmic syndromes due to MiRP1 abnormalities.

Key Words: long-QT syndromes • ion channel • proarrhythmia

Mutations in minK-related peptide 1 (MiRP1), the KCNE2 gene product, produce malignant ventricular arrhythmia syndromes related to impaired repolarization.^1–3 MiRP1 physically interacts with, and alters the properties of currents carried by, a wide range of -subunit proteins, including delayed-rectifier K⁺-current subunits HERG^1–4 and KvLQT1,⁵ transient-outward K⁺-current subunits Kv4.2/4.3^6,7 and Kv3.4⁸ and hyperpolarization-activated cation subunits.⁹ Functional alterations in these channels could impair repolarization and contribute to arrhythmogenesis; however, MiRP1 mRNA expression is very low in ventricular muscle.⁹ The His-Purkinje tissue of the cardiac specialized conducting system is particularly prone to arrhythmogenic early afterdepolarizations¹⁰ and may have a prime role in polymorphic ventricular tachyarrhythmias.¹¹ The mass of cardiac Purkinje tissue is small; therefore, gene expression in ventricular muscle does not necessarily reflect Purkinje fiber expression. We speculated that MiRP1 expression might be particularly strong in cardiac Purkinje tissue, accounting for a role of MiRP1 protein in long-QT syndromes despite overall low-level ventricular expression. We therefore applied competitive reverse transcription–polymerase chain reaction (RT-PCR), real-time PCR, and Western blot to compare MiRP1 expression in canine ventricular muscle versus Purkinje fiber tissue.

Materials and Methods

After extraction from dog tissues, RNA was quantified and integrity confirmed. RNA mimics included a 460-bp rabbit -actin sequence flanked at either end by gene-specific primer (GSP) sequences and an 8-nucleotide linker homologous to the 3'-end of T7 promoter. Serial dilutions of mimic were mixed with 1-µg samples of RNA in 20-µL reaction mixtures for RT. The RT product was used as a template for subsequent PCR with gene-specific primers. PCR products were visualized under ultraviolet light and MiRP1 transcript concentrations calculated. Two-step real-time PCR was conducted with SYBR green dye. MiRP1 results were normalized to GAPDH data obtained by real-time PCR on the same samples. Duplicate standard curves were run for each experiment.

Membrane protein fractions were isolated with 5 mmol/L Tris-HCl (pH 7.4), 2 mmol/L EDTA, 5 µg/mL leupeptin, 10 µg/mL benzamidine, and 5 µg/mL soybean trypsin inhibitor, followed by tissue homogenization. After centrifugation, membrane pellets were resuspended in 75 mmol/L Tris (pH 7.4), 2 mmol/L EDTA, 12.5 mmol/L MgCl₂ and stored at -80°C. Protein concentration was determined and samples denatured. Membrane proteins were fractionated on 12% SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene fluoride membranes in 25 mmol/L Tris base, 192 mmol/L glycine, and 5% methanol. Membranes were blocked and then incubated with MiRP1 primary antibody for 18 hours. After washing and reblocking, membranes were incubated with horseradish peroxidase–conjugated donkey anti-goat IgG secondary antibody. Antibody was detected with Western-Lightning Chemiluminescence Reagent Plus.

Chinese hamster ovary (CHO) cells were cultured at 37°C with 5% CO₂ in supplemented F12 medium. Cells were transfected with 1 µg of MiRP1 cDNA subcloned into PCI/neo vector. For immunofluorescent studies, CHO cells were passed onto glass coverslips prepared with 15 µg/mL laminin for 1 hour, fixed with 2% paraformaldehyde, and blocked with 2% donkey serum and permeabilized (0.2% Triton X-100). After overnight incubation, cells were incubated with secondary antibody for 1 hour and subjected to confocal microscopy.

Data are mean±SEM. Statistical comparisons were with ANOVA and Bonferroni-corrected Student’s t tests.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Results

Expression of MiRP1 mRNA and Protein in Purkinje Fibers
Figure 1A shows representative competitive RT-PCR gels. Upper bands represent RNA mimic products and lower bands are coamplified target genes (MiRP1). Mimic signals in both ventricle and Purkinje fibers are similar, whereas MiRP1 gene bands are clearly stronger in Purkinje fibers. The lower bands were sequenced (GenBank accession No. AY307952) and showed 100% homology with the human MiRP1 gene.² Target/mimic band-intensity ratios decreased log-linearly with increasing mimic concentration in initial reaction mixtures (Figure 1B). Mean concentrations were 2.6±1.3 amol/µg RNA for Purkinje fibers, 2.7±1.1 amol/µg for sinoatrial node, and 0.19±0.05 amol/µg RNA for ventricular muscle (Figure 1C). Similarly, real-time RT-PCR showed MiRP1 mRNA expression to be substantially greater in Purkinje fibers than in ventricle (Figure 1D). The ratio of Purkinje fiber MiRP1 expression to that in other tissues was greatest for ventricle, intermediate for atrium, and least for sinoatrial node (Figure 1D).

View larger version (41K): [in this window] [in a new window]   Figure 1. A, Ethidium bromide–stained gels with products from competitive RT-PCR reactions using dog ventricular muscle (VM), Purkinje tissue (PF), and sinoatrial node (SAN). Lane M shows the DNA mass ladder, with signals obtained with 200, 120, 80, 60, 40, and 20 ng of mass standards (top to bottom). Lanes 1 through 6, Products from reaction mixtures containing serial 10x dilutions from initial mimic quantity of 45.4 fg (lane 1) to 4.5 · 10-4 fg (lane 6), along with a 1-µg sample of RNA in 25 µL. Lane 7 is an RT-negative control (mimic size=496 bp, target=218 bp). B, Logarithm of amplified target MiRP1/mimic signal ratio versus RNA mimic quantity in initial reaction mixture. C, Mean±SEM MiRP1 transcript concentration (n=5 hearts/determination, *P<0.05 vs PF and SAN). D, Ratio of MiRP1 mRNA expression in Purkinje fiber to that in ventricle (V), atrium (A), and SAN, as determined by real-time RT-PCR (n=12 independent heart samples/determination). Inset, Example of amplification curves for ventricular and Purkinje fiber tissue from one experiment.

Western blots detected a band at 25 kDa (the expected molecular mass for MiRP1) in both Purkinje fibers and ventricle, with a greater intensity in Purkinje tissue (Figure 2A). The 25-kDa bands disappeared after antibody preincubation with antigenic peptide. MiRP1 band intensities (normalized to GAPDH) were substantially stronger in Purkinje fibers than in ventricular muscle (P<0.001, Figure 2B). No equivalent band was present in Western blots of canine aorta or ileum (consistent with previous mRNA data¹²) nor lung (Figure 2C). The MiRP1 antibody detected a 25-kDa band in MiRP1-transfected CHO cells (Figure 2D) but not in nontransfected cells. A clear membrane signal was detected with MiRP1 antibody on confocal imaging of MiRP1-transfected, but not nontransfected, cells (online Figure, available in the data supplement at http://www.circresaha.org).

View larger version (48K): [in this window] [in a new window]   Figure 2. MiRP1 protein expression in Purkinje fibers versus ventricle. A, Western blots using Purkinje fiber (PF) and ventricular muscle (VM) membrane protein extracts, with identical protein quantities (60 µg) from each of 4 different dog hearts (mass markers are at 23 and 34 kDa). A 25-kDa band corresponding to MiRP1 (indicated) was eliminated by antigen-antibody preincubation (right panel). The same samples were probed for GAPDH, and the intensity of the corresponding bands (bottom) was used to normalize each MiRP1 signal. B, Mean±SEM MiRP1 band intensities after normalization to GAPDH (n=6 hearts/determination, ***P<0.001 vs VM). C, No 25-kDa signal corresponding to MiRP1 was detected by the MiRP1 antibody in aorta, ileum, or lung. D, MiRP1 antibody detected a 25-kDa band (lane 1) in MiRP1-transfected CHO cells, but no corresponding signal was detected in nontransfected CHO cells.

Discussion

There is convincing evidence for the involvement of KCNE2 mutations in cardiac arrhythmia syndromes involving abnormal repolarization.^1–3 It is also clear that MiRP1 alters the ion-channel function of a variety of K⁺-channel -subunits.^4–8 KCNE2 mutations disrupt the function of a range of subunits, leading to the suggestion of promiscuous interactions with conserved mechanisms.¹³

A difficulty in rationalizing the role of MiRP1 mutations in cardiac arrhythmia syndromes is the extremely low-level ventricular expression of MiRP1. Yu et al⁹ found MiRP1 mRNA to be barely detectable in the ventricles, <4% of signal intensity in the sinoatrial node. In the present study, we found that mRNA and protein expression of MiRP1 in Purkinje fibers is much stronger than in ventricle. Cardiac Purkinje fibers play a key role in cardiac electrophysiology and arrhythmias.¹⁴ Although Purkinje fibers were widely used for classical cardiac electrophysiology studies, technical difficulties in Purkinje cell isolation and the small Purkinje tissue mass have led to much less work on cardiac Purkinje tissue with modern patch-clamp and molecular biology methods. Evidence for an important role of Purkinje tissue in torsades de pointes arrhythmias^10,11,15 and recent observations that clinical malignant ventricular tachyarrhythmias can be prevented by ablating Purkinje system triggers¹⁶ have led to resurgent interest in the physiology and pathophysiology of Purkinje tissue. Remodeling of Purkinje fiber ionic properties by heart failure sensitizes them to action-potential prolongation by class III antiarrhythmic agents, potentially explaining the predisposition of heart failure patients to torsades de pointes arrhythmias.¹⁷

Virtually all of the ion channels known to interact with MiRP1 are expressed in Purkinje fiber tissue.¹⁸ Further clarification of the specific ion-channel interactions mediating MiRP1 function awaits development of more specific probes. Purkinje fiber repolarization is much more susceptible to disruption than that of ventricular muscle.¹⁰ MiRP1 mutations that impair the function of K⁺-channel -subunits mediating Purkinje cell repolarization could therefore cause excess Purkinje fiber action potential prolongation and arrhythmogenic early afterdepolarizations, leading to arrhythmic syndromes.

Acknowledgments

Funding for research was provided by the Canadian Institutes of Health Research and the Quebec Heart Foundation. The authors thank Chantal St-Cyr and Evelyn Landry for technical assistance and France Thériault for secretarial help.

Footnotes

*Both authors contributed equally to this study.

Original received March 19, 2003; revision received June 24, 2003; accepted June 24, 2003.

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