Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular KV currents and predisposes to ventricular arrhythmia

Abstract

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K_V) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout ( Kcne5^-/0) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5^+/0 mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K_V current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current ( I_to,f). The current increases arose from an apex-specific increase in slow transient outward current-1 ( I_Kslow,1) (conducted by K_V1.5) and I_to,f (conducted by K_V4) and an increase in I_Kslow,2 (conducted by K_V2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K_V2.1 was also detected in both Kcne5^-/0 and Kcne5^+/0 mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K_V2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I_K,slow2 by reducing K_V2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K_V2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K_V2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K_V2.1 in mouse heart. Increased K_V current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.-David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular K_V currents and predisposes to ventricular arrhythmia.

Keywords: MiRP4; potassium channel.

Figure 1

Kcne5 is expressed in mouse and human hearts. A) Quantification of Kcne5 transcript expression in atria and ventricles (n = 5 and n = 6, respectively) for male Kcne5^+/0 (WT) and Kcne5^−/0 (knockout) mice by real-time qPCR, normalized to the reference genes ActB and glyceraldehyde phosphate dehydrogenase. Data are means ± sem. B) Relative KCNE5 regional expression in male human hearts (n = 6): left (LA) and right (RA) atria and the left ventricular epi-, mid- and endomyocardial wall. No significant differences in expression levels between the regions were found. Quantification was by real-time qPCR with ACTB and TOP1 as reference genes. C) Body weight, heart size, and tibia length of ∼8-mo-old male mice (n = 20 in each group; means ± sem shown). Kcne5^−/0 mice showed a significant increase in body weight (44.8 ± 1.0 g) compared to control mice (40.1 ± 1.1 g). P = 0.0030. Heart size (0.147 ± 0.003 vs. 0.143 ± 0.003 g, respectively) and the length of tibia (11.8 ± 0.1 vs. 11.9 ± 0.1 mm, respectively) did not show any differences. Likewise, there was no difference in heart size when normalized to tibia length [(mg/mm) 12.5 ± 0.2 vs. 12.0 ± 0.3, respectively]. **P < 0.01.

Figure 2

Kcne5 deletion causes ventricular arrhythmias. A) Spontaneously occurring monomorphic VPBs were detectable during continuous ECG monitoring in sedated live Kcne5^−/0 mice. No such VPBs were found in Kcne5^+/0 mice. Upper ECG curves represent body surface ECGs, middle ECG curves represent intracardiac ECG from the ventricular catheter, and lower ECG curves represent intracardiac ECG registration from the atrial catheter. Note postextrasystolic compensatory pauses after VPBs are pathognomonic for ventricular extrasystolic beats. B) A spontaneously occurring episode of polymorphic VT was noted in 1 Kcne5^−/0 mouse. The episode could not be terminated by burst pacing (pacing spikes on the left). The VT finally converted into an atrial arrhythmia with a 2:1 and 3:1 atrioventricular conduction. C) Polymorphic VTs were inducible during continuous ECG monitoring of sedated live Kcne5^−/0 mice. After a defined pacing protocol followed by 3 extrastimuli polymorphic VTs were highly inducible in Kcne5^−/0 mice as compared to WT controls. Data for ECG curves were obtained as described in A. D) Percentage of inducible protocols was increased in Kcne5^−/0 mice vs. WT controls. E) The ventricular effective refractory period was reduced in Kcne5^−/0 mice vs. Kcne5^+/0 controls. ***P < 0.001.

Figure 3

Kcne5 gene deletion augments ventricular apical myocyte K­_V currents. A) Representative native whole-cell K_V currents in Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular apex cardiomyocytes (voltage protocol inset) (n = 27–31). B) Mean current density vs. voltage characteristics (I-V curve) from recordings as in A for Kcne5^+/0 (filled squares) and Kcne5^−/0 (open squares) apical myocytes (n = 27–31). ***P < 0.001 at +60 mV. C) Typical digitally subtracted 25 mM TEA-sensitive current (I_K,slow2) for Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular myocytes. D) Mean I-V relationships for TEA-sensitive currents as in C (n = 9–10 cells/genotype, derived from 3 mice/genotype). **P = 0.01 at +60 mV. E) Typical digitally subtracted current traces for 50 μM 4-AP-sensitive current (I_K,slow1) for Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular myocytes. F) Mean I-V relationships for myocytes as in E (n = 9–10 cells/genotype, derived from 5 mice/genotype). P < 0.05 at +60 mV. G) Typical digitally subtracted 500 nM HpTx-sensitive current (I_K,slow2) for Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular myocytes. H) Mean I-V relationships for HpTx-sensitive currents as in G (n = 6 cells/genotype, derived from 4 to 5 mice per genotype). P = 0.45 at +60 mV.

Figure 4

Kcne5 gene deletion augments I_K,slow2 in ventricular septum myocytes lacking I_to,f. A) Typical current traces from Kcne5^+/0 and Kcne5^−/0 adult ventricular septum myocytes (voltage protocol inset). B) Mean I-V relationships for Kcne5^+/0 (solid) and Kcne5^−/0 (open) septal myocytes with (squares) or without (circles) I_to,f (n = 15–19 cells per group). *P < 0.05 at +60 mV. C) Typical digitally subtracted 25 mM TEA-sensitive K_V2.1 current (I_K,slow2) for Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular septum myocytes. D) Mean I-V relationships for TEA-sensitive currents as in C (n = 6 cells/genotype, derived from 2 mice/genotype). **P = 0.009 at +60 mV. E) Typical digitally subtracted current traces for 50 μM 4-AP-sensitive K_V1.5 current (I_K,slow1) for Kcne5^+/0 (left) and Kcne5^−/0 (right) ventricular myocytes. Dashed lines: 0 current level. F) Mean I/V relationships for myocytes as in E (n = 8 cells/genotype, derived from 2 mice/genotype). P = 0.984 at +60 mV. Dashed lines: 0 current level.

Figure 5

Kcne5 and K_V2.1 colocalize in the ICD of mouse ventricular cardiomyocytes. A) Immunofluorescence staining of ventricular heart sections of Kcne5^+/+ and Kcne5^−/− mice with an anti-Kcne5 antibody. Kcne5 is detectable in ICDs of cardiomyocytes (arrows) whereas Kcne5 signal is absent in Kcne5^−/− hearts. Yellow framed areas are shown in higher magnification as indicated. B) Coimmunofluorescence staining with antibodies against Kcne5 (red) and β-Catenin (green) for ICD visualization. No Kcne5 signal was detectable in Kcne5^−/− hearts. C) Coimmunofluorescence staining with antibodies against K_V2.1 (red) and Connexin-43 (green) as ICD marker proteins. Scale bars, 20 µm.

Figure 6

KCNE5 and K_V2.1 colocalize in HL-1 cells. A, B) Confocal images of HL-1 cells heterologously expressing WT KCNE5 (A) or HA-tagged WT KCNE5 (B). Phalloidin (Pha) labeled the submembranous actin cytoskeleton and was used to visualize the cell membrane. C) Nonpermeabilized HA-KCNE5–expressing HL-1 cells stained with a specific HA-tagged antibody (surface staining, green), permeabilized, and then stained with a KCNE5-specific antibody (red) and Phalloidin (blue). D) Colabeling of single transfected K_V2.1 (green) and endogenous Phalloidin (red) in HL-1 cells. E) Colabeling of cotransfected KCNE5 (green) and K_V2.1 (red) in HL-1 cells.

Figure 7

KCNE5 and K_V2.1 colocalize in intracellular compartments in proximity to the lysosomes. A, B) Laser confocal microscopy was used to acquire images of KCNE5 and K_V2.1 subunits singly transfected into HL-1 cells, respectively. Both were found to be located in dense clusters and vesicles for which some appear very close to the lysosomal marker LAMP2. Phalloidin (Pha) was used to identify the cell membrane. C) When K_V2.1 and KCNE5 were coexpressed in the same HL-1 cells, they colocalized in vesicles and dense clusters, similar to the above-mentioned structures, which again were located in proximity to the lysosome maker, LAMP2—possibly indicative of late endosome compartments.

Figure 8

AF-associated KCNE5-L65F causes a gain-of-function of K_V2.1-KCNE5 channels. A–D) Biophysical properties of K_V2.1 coexpressed with WT or mutant human KCNE5. All recordings were performed on cells from 3 to 5 repeated transfections of 2–5 independent cell cultures. Representative current recordings determined the activation (A) and inactivation (B) properties of K_V2.1+KCNE5 and K_V2.1+KCNE5-L65F. The applied pulse protocols are given on top. Voltage-dependence of activation (C) and of inactivation (D) of K_V2.1+KCNE5, K_V2.1+KCNE5-P33S, K_V2.1+KCNE5-L65F, and K_V2.1+KCNE5-R85H. Activation and inactivation curves were obtained by plotting the normalized current amplitudes at −25 and +60 mV as a function of the 500-ms and 5-s prepulse potentials, respectively. Solid lines: Boltzmann fits. Coexpression of KCNE5-L65F shifted both the voltage dependence of activation and of inactivation. P < 0.05. E) Time constants of activation and deactivation of K_V2.1 coexpressed with KCNE5 variants as indicated; symbols as in C. Activation and deactivation constants were derived from single and double exponential fits of the raw current recordings, respectively. F) Current densities at 0 mV of K_V2.1 coexpressed with KCNE5 variants, as indicated. The numbers above each bar represent the number of cells analyzed. The L65F mutation increased current density compared with WT K_V2.1-KCNE5 channels. *P < 0.05.

Figure 9

BrS-associated KCNE5 mutations cause a negative shift in the voltage dependence of K_V2.1-KCNE5 channels. All recordings were performed on cells from 3 to 5 repeated transfections of 2–5 independent cell cultures A–D) Biophysical properties of K_V2.1 coexpressed with WT or mutant human KCNE5. Representative current recordings determined the activation (A) and inactivation (B) properties of K_V2.1+KCNE5-Y81H and K_V2.1+KCNE5- D92E/E93X. The applied pulse protocols are given on top. Voltage-dependence of activation (C) and of inactivation (D) of K_V2.1+KCNE5, K_V2.1+KCNE5-Y81H, and K_V2.1+KCNE5- D92E/E93X. Activation and inactivation curves were obtained as in Fig. 4. Solid lines: the Boltzmann fits. Coexpression of KCNE5-D92E/E93X shifted the voltage dependence of activation. P < 0.05. E) Time constants of activation and deactivation of K_V2.1 coexpressed with KCNE5 variants as indicated; symbols as in C. Activation and deactivation constants as in Fig. 4E. F) Current densities at 0 mV of K_V2.1 coexpressed with KCNE5 variants as indicated. The numbers above each bar represent the number of analyzed cells.

Figure 10

AF-associated KCNE5 mutants locate similarly to WT KCNE5 in HL-1 cells. KCNE5-P33S (A), KCNE5-L65F (B), and KCNE5-R85H (C) expressed in HL-1 cells alone were found in compartments similar to WT KCNE5 (compare to Fig. 6) visualized with confocal microcopy. Thus, the mutations did not seem to confer a trafficking deficiency. Phalloidin (Pha) was used to mark the cell membrane.