Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice

Abstract

Myocardial repolarization capacity varies with sex, age, and pathology; the molecular basis for this variation is incompletely understood. Here, we show that the transcript for KCNE4, a voltage-gated potassium (K_v) channel β subunit associated with human atrial fibrillation, was 8-fold more highly expressed in the male left ventricle compared with females in young adult C57BL/6 mice (P < 0.05). Similarly, K_v current density was 25% greater in ventricular myocytes from young adult males (P < 0.05). Germ-line Kcne4 deletion eliminated the sex-specific K_v current disparity by diminishing ventricular fast transient outward current (I_to,f) and slowly activating K⁺ current (I_K,slow1). Kcne4 deletion also reduced K_v currents in male mouse atrial myocytes, by >45% (P < 0.001). As we previously found for K_v4.2 (which generates mouse I_to,f), heterologously expressed KCNE4 functionally regulated K_v1.5 (the K_v α subunit that generates I_Kslow1 in mice). Of note, in postmenopausal female mice, ventricular repolarization was impaired by Kcne4 deletion, and ventricular Kcne4 expression increased to match that of males. Moreover, castration diminished male ventricular Kcne4 expression 2.8-fold, whereas 5α-dihydrotestosterone (DHT) implants in castrated mice increased Kcne4 expression >3-fold (P = 0.01) to match noncastrated levels. KCNE4 is thereby shown to be a DHT-regulated determinant of cardiac excitability and a molecular substrate for sex- and age-dependent cardiac arrhythmogenesis.—Crump, S. M., Hu, Z., Kant, R., Levy, D. I., Goldstein, S. A. N., Abbott, G. W. Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice.

Cardiac myocyte repolarization is a complex process requiring the concerted action of several different types of potassium channel (1). Voltage-gated potassium (K_v) channels comprise both pore-forming α subunits and regulatory proteins including β subunits such as the single-pass transmembrane KCNE proteins (2) (Fig. 1A).

Figure 1.

Kcne4 deletion sex-specifically impairs mouse ventricular myocyte K⁺ currents. A) Cartoon of KCNE4 subunits in a K_vα-KCNE4 channel complex, with a hypothetical 4:2 stoichiometry. B) Strategy for germ-line Kcne4 deletion from mice (Materials and Methods). C) Genotyping gel of PCR of genomic DNA extracted from tail tip tissue confirming Kcne4 deletion from mice. D) Relative Kcne4 expression by real-time qPCR quantification of sexually mature adult (6 mo old) mouse heart chambers. *P < 0.05 compared with corresponding female ventricles. All values were normalized to one of the 4- to 6-mo-old male Kcne4^+/+ mouse LV Kcne4 expression values. E) Representative whole-cell ventricular septal cardiomyocyte K⁺ currents from male and female sexually mature adult (10 mo old) Kcne4^+/+ and Kcne4^−/− mice (voltage protocol inset); n = 9–26 myocytes from 3 to 6 mice per group. F) Mean peak and steady-state whole-cell ventricular septal cardiomyocyte K⁺ current densities recorded from cells as in C, isolated from male (M) or female (F) mice; genotypes are as shown. *P < 0.05 compared with other peak currents at equivalent voltages; n = 9–26 cells per group, as indicated.

In human ventricular myocardium, the delayed rectifier currents I_Kr and I_Ks are the primary repolarizing currents. Generated by hERG and KCNQ1 K_v α subunits, respectively, these K⁺ currents repolarize ventricular myocytes after a plateau phase lasting several hundred milliseconds (1). In adult mouse ventricles, ventricular myocyte action potentials are much shorter; I_Kr is absent, and I_Ks is, at most, weakly expressed and detectable only after β-adrenergic stimulation (3). Instead, the rapidly activating and inactivating I_to and the rapidly activating, slowly inactivating K⁺ current (I_K,slow) dominate adult mouse ventricular repolarization (4).

It is noteworthy that various members of the KCNE family, of which there are 5 members in mammals, are expressed in both human and mouse heart. The KCNE proteins can each form complexes with 1 or more K_v α subunits, generating heteromeric channel assemblies often with unique gating or other functional properties (2). KCNE subunits can also regulate α subunit trafficking and subcellular localization (5). Although human and mouse hearts exhibit fundamental differences in the K⁺ currents that shape their action potentials, previous studies have demonstrated that the same KCNE isoforms can be influential in myocardial repolarization of either species—at least partly because of their functional versatility (6, 7). KCNE gene variants are linked to human cardiac arrhythmias including Long QT syndrome, atrial fibrillation (AF), and Brugada syndrome; Kcne gene deletion in mice can cause analogous pathologies (8).

An important issue in human cardiac arrhythmia is the variable penetrance of disease between individuals harboring the same gene variant. One of the more important sources of variability is the sex of the individual (9). Brugada syndrome, most commonly linked to loss-of-function SCN5A variants, is 8-fold more common in men than women (10). Reduced repolarization reserve and lower I_Kr density are more commonly observed in women than in men and predispose to drug-induced cardiac arrhythmia (9). Uncovering sex-dependent differences in arrhythmia susceptibility and the molecular mechanisms underlying them is therefore an early step toward therapeutic strategies tailored toward the individual (i.e., precision medicine) (11).

KCNE4, which we originally named MinK-related peptide 3 (6), functionally regulates KCNQ1, K_v1.1, K_v1.3, K_v4.2, K_v4.3, and the Ca²⁺-activated K⁺ channel, in heterologous coexpression experiments (12–18). A single nucleotide polymorphism generating an E145D substitution, positioned in the atypically lengthy cytoplasmic C-terminal end of KCNE4, is an independent risk factor for AF in Han (in 3 separate analyses) and Uygur Chinese (19–21). KCNE4 inhibits KCNQ1 activity, but E145D-KCNE4 augments KCNQ1 current, suggesting a potential mechanistic basis for E145D-associated AF (22). Here, we examined the effects of targeted Kcne4 gene deletion in mice and discovered sex-dependent differences in expression and function that were explained by 5α-dihydrotestosterone (DHT) regulation of cardiac KCNE4 expression.

MATERIALS AND METHODS

Kcne4^─/─ mice

Mice were housed in a pathogen-free facility. The present study was approved by the Animal Care and Use Committees at University of California, Irvine and carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). C57BL/6 Kcne4^−/− mice were generated by replacement of the sole coding exon of Kcne4 with neo and LacZ genes (Fig. 1B) in 129/Sv embryonic stem cells for embryonic injection followed by implantation into a C57 breeder, PCR screening of progeny, and confirmed Southern blot analysis (performed by Lexicon, The Woodlands, TX, USA; and Texas A&M Institute for Genomic Medicine, College Station, TX, USA; data not shown). Kcne4^+/− mice were then backcrossed for at least 10 generations into the C57BL/6 strain. Colony genotyping was performed by conventional PCR of genomic DNA isolated from tail tip tissue (Fig. 1C). The PCR primer sequences were as follows: Kcne4 forward 5′-CAACGACAGCAGTGAAGGC-3′, Kcne4 reverse 5′-GCAGAGCAAAAGCAAAACCC-3′, Neo3a 5′-GCAGCGCATCGCCTTCTATC-3′; Kcne4^+/+ band, 137 bp, Kcne4^−/− band, 229 bp. Castration and DHT pellet implants (7.5 mg DHT, 90-d release; Innovative Research of America, Sarasota, FL, USA) were performed by Charles River Laboratories (Hollister, CA, USA).

Real-time quantitative PCR

Mice were killed by CO₂ asphyxiation. Hearts were quickly harvested, washed, and perfused through the left ventricle with PBS + heparin and then processed or stored at −80°C until use. RNA was extracted using 1 ml Trizol (Invitrogen, Carlsbad, CA, USA)/100 mg tissue and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturers’ protocol. RNA samples with A₂₆₀/A₂₈₀ absorbance ratios between 2.00 and 2.20 were used for further synthesis; 500 ng to 1 μg RNA was used for cDNA synthesis (Quantitect Reverse Transcriptase; Qiagen) and stored at −20°C until use. Primer pairs for target genes Kcne4 (NCBI Gene ID 57814) and GAPDH (NCBI Gene ID: 14433) produced an amplicon of 137 and 123 bp, respectively. Primer sequences were obtained from MGH-ParaBioSys:NHLBI Program for Genomic Applications, Massachusetts General Hospital, and Harvard Medical School (Boston, MA, USA) (http://pga.mgh.harvard.edu). The sequences of primers (0.05 µm scale, HPLC purified; Sigma-Aldrich, St. Louis, MO, USA) were as follows: Kcne4, forward 5′-CTTTGCTCGATGGAAGGGGAC-3′, reverse 5′-GCTGTCGTTGAGAGGCGTC-3′; Gapdh, forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′. Real-time quantitative PCR (qPCR) analysis was performed using the CFX Connect System, iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and 96-well clear plates. Thermocycling parameters were set according to the manufacturer’s protocol for iTaq. Samples were run in triplicate as a quality control measure, and triplicates with an sd of 0.600 or higher were repeated. Melting curves were assessed for verification of a single product. Final analysis measuring statistical significance was calculated measuring ∆∆C_q values normalized to the 6-mo-old male Kcne4^+/+ left ventricle data.

Antibodies

Chicken and rabbit antibodies were generated to human KCNE4 residues 67–138 for Western blot/coimmunoprecipitation (co-IP) and residues 136–150 for immunofluorescence (IF), respectively, as described previously (18). Commercial voltage-gated K⁺ channel antibodies raised against rabbit anti-K_v1.5 (Alomone Laboratories, Jerusalem, Israel) were used for Western blots and IF as described previously (7, 23).

Isolation of adult mouse cardiomyocytes

Single cardiomyocytes were isolated from sexually mature adult (10 mo old) mice that were heparinized using (250 IU, i.p., to minimize blood clotting) for 10 min before CO₂ gas and cervical dislocation. The chest was opened to expose the heart and lungs. Hearts were quickly excised, and cannulation of ascending aorta was perfused on a 37°C warmed Langendorff apparatus (Easy Cell Extraction System Type 803; Hugo Sacks Elektronik, March-Hugstetten, Germany) constantly gassed with 95% O₂–5% CO₂. The hearts were perfused at 3 ml/min with calcium-free 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer containing the following (in mM): 137 NaCl, 5.4 KCl, 1.2 MgSO₄, 15 NaH₂PO₄, 20 glucose, 10 HEPES, and 2 l-glutamine (pH 7.4, buffer A) for 5 min, followed by 10–14 min of 0.36 mg/ml type II collagenase (Worthington, Lakewood, NJ, USA) and 10 μM CaCl₂ (buffer B) or until the hearts were swollen and pale in color. The hearts were transferred to a 10 cm dish containing buffer A supplemented with 5 mg/ml bovine serum albumin (BSA) and 150 μM CaCl₂ (buffer C) and mechanically dissociated using forceps until large pieces were dispersed into the cells suspension. Undigested tissue debris were filtered through a 200 µM nylon mesh into a Falcon tube, and 2 volumes of buffer A supplemented with 5 mg/ml BSA and 1 mM CaCl₂ solution (buffer D) were added and allowed to settle for 5 min. Cells were rinsed and pelleted 2 times at 300 rpm for 3 min and resuspended in buffer D. Cells were resuspended to desired cell concentration using buffer D for electrophysiology or seeded on laminin (Corning, Tewksbury, MA, USA)-coated glass coverslips (15 μg/ml) for IF assay. Only rod-shaped cardiomyocytes with clear striations were used for recording.

Cellular electrophysiology

Whole-cell patch-clamp recordings from dispersed adult cardiomyocytes or transfected Chinese hamster ovary (CHO) cells were performed at room temperature using an IX50 inverted microscope equipped with an FHD chamber from IonOptix (Olympus, Center Valley, PA, USA), a Multiclamp 700A Amplifier, a Digidata 1300 Analog/Digital converter, and PC with pClamp9 software (Molecular Devices, Sunnyvale, CA, USA). For cardiomyocytes, the bath solution contained the following (in mM): 117 NaCl, 4 KCl, 1.7 MgCl₂, 10 HEPES, 1 KH₂PO₄, 4 NaHCO₃, 3 CoCl₂, and 10 d-glucose (pH 7.4 with NaOH). Pipettes were of 2.1–3.2 MΩ resistance when filled with intracellular solution containing the following (in mM): 130 KCl, 2 MgCl₂, 20 HEPES, 11 EGTA, 5 Na₂ATP, 0.4 Na₂GTP, and 5 Na₂CP (pH 7.4 with KOH). Outward K⁺ currents were evoked during 4.5 second voltage steps to test potentials between –60 and +60 mV in 20 mV increments from a holding potential of –70 mV after a 100 ms prepulse to –40 mV. Leak currents were always <100 pA and were not corrected. Data were analyzed using pClamp9.1 software (Molecular Devices, Sunnyvale, CA, USA), and statistical analysis (ANOVA) was performed using Origin 6.1 (Microcal, Northampton, MA, USA) software. The decay phase of I_K was fitted to the sum of 3 exponentials to quantify the τ of decay and current amplitude for I_K,slow, fast transient outward current (I_to,f), and slow transient outward current (I_to,s) as before (7).

For inhibition of K_v1.5 and K_v2.1, respectively, 50 μM 4-aminopyridine (4-AP; ICN Biomedicals, Irvine, CA, USA) and 25 mM tetraethylammonium (TEA; Sigma-Aldrich) bath solutions were applied to the recording bath to achieve at least 4× bulk flow bath displacement after baseline recordings and allowed to equilibrate for 2–3 min before drug recordings.

For whole-cell patch clamp of CHO cells, bath solution contained the following (in mM): 135 NaCl, 5 KCl, 1.2 MgCl₂, 5 HEPES, 2.5 CaCl₂, and 10 d-glucose (pH 7.4). Pipettes were 2.1–3.5 MΩ resistance when filled with intracellular solution containing the following (in mM): 10 NaCl, 117 KCl, 2 MgCl₂, 11 HEPES, 11 EGTA, and 1 CaCl₂ (pH 7.2). K⁺ currents were evoked during a 1 second voltage step in 10 mV increments between −80 and +60 mV from a holding potential of −75 mV. Green fluorescent protein-positive cells were selected for recording 24 h after transfection.

For all current and current density comparisons, currents between 0 and 50 or 60 mV were compared between genotypes, with ANOVA followed by Bonferroni correction for multiple comparisons to determine P values.

Cell culture and transfection

CHO cells were cultured at 37°C and 5% CO₂ in F12K medium (ATCC, Manassas, VA, USA) containing l-glutamine and sodium bicarbonate supplemented with 10% fetal bovine serum (Invitrogen) plus 1% penicillin and streptomycin. CHO cells were cultured in 10 cm tissue culture plates to 80% confluence. Next, CHO cells were washed once with PBS (Ca²⁺ and Mg²⁺ free; Invitrogen) and then incubated with Detachin (Genlantis, San Diego, CA, USA) for 3 min. Fetal bovine serum–supplemented F12K medium was added, and cells were centrifuged for 2 min at 1000 rpm. For transfection, 2 × 10⁵ cells were seeded in 35 mm tissue culture dishes 2 h before transfection. CHO medium was replaced with prewarmed Opti-MEM reduced serum medium (Invitrogen) and transiently transfected using a 2:1 ratio of Lipofectamine 2000 (Invitrogen) to cDNA. Specifically for electrophysiology, cells were transfected with 0.02 μg human-K_v1.5 cDNA, 0.02 μg mouse-Kcne4 cDNA, 2.47 μg pBluescript (cells transfected without Kcne4 used 2.49 μg pBluescript), and 0.147 μg enhanced green fluorescent protein added (for identification of transfected cells) to 250 μl of Opti-MEM, followed by dropwise addition of Lipofectamine 2000 and incubated at room temperature for 20 min. Cell medium was replaced with fresh prewarmed 10% fetal bovine serum–supplemented F12K medium after 3 h of transfection. Whole-cell patch-clamp or IF experiments were conducted 24 h after transfection.

Immunofluorescence

CHO cells (4 × 10⁵ cells per 10 cm dish) were transfected as described above and then rinsed twice in ice-cold PBS before fixation in ice-cold 4% paraformaldehyde for 10 min. All treatments for the rest of the procedure were at room temperature. Following fixation in formaldehyde, cells were permeated with PBS containing 0.25% Triton X-100 in 1% BSA for 10 min and then rinsed 3 times for 5 min with PBS. The cells were blocked with 1% BSA + 10% normal goat serum in PBS/0.25% Triton X-100 for 30 min. Blocking buffer was changed to primary antibody (1:200) and incubated for 3 h followed by 3 × 5 min PBS rinses. Cells were then incubated with secondary Fluor-antibodies (1:600) in 1% BSA/10% normal goat serum/PBS/0.25% Triton X-100 for 1 h. Cells were washed 3 × 5 min with PBS, and then the coverslips were mounted with DAPI-labeled mounting medium and visualized after drying using an Olympus BX51 microscope and CellSens software (Olympus, Waltham, MA, USA). Secondary antibodies used were donkey anti-chicken 488 and goat anti–rabbit-555 (Invitrogen). Effects of KCNE4 on K_v1.5 surface expression in live CHO cells were quantified using Nikon NIS Elements v4.10 software via a primary antibody raised against an extracellular epitope of K_v1.5 (APC-150; Alomone Laboratories); total cell K_v1.5 expression was quantified via a C-terminal cyan fluorescent protein (CFP) tag while maintaining the same time capture exposure for all images. In brief, a circular region of interest was overlaid and adjusted to the exact size of the CFP-K_v1.5 signal boundary to measure fluorescence. Next, the same circular region of interest pixel area and coordinates were used to measure the anti-K_v1.5 cell fluorescence in the same cell. Anti-K_v1.5 fluorescence was normalized to total K_v1.5-CFP fluorescence.

Western blot and co-IP

CHO cells were transiently transfected overnight with a 3:1 ratio of LT1 (Mirus, Madison, WI, USA) to cDNA in an 80% confluent 10 cm plate containing 2 μg human K_v1.5 + 2 μg mouse Kcne4 + 11 μg pBluescript. CHO cells were lysed 24 h after transfection after they were scraped, pelleted at 1000 rpm for 2 min, and then washed once in PBS. The cell pellet was lysed with 450 μl buffer containing 150 mM NaCl, 50 mM Tris, 1% Nonidet P-40, and 0.1% SDS (Sigma-Aldrich) with protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), pH to 7.4, for 1 h at 4°C while rotating. Lysates were pelleted for 10 min at 10,000 rpm, and supernatant was collected for BCA protein quantification (Thermo Fisher Scientific) after preclearing with 40 μl of Pierce agarose A beads (Thermo Fisher Scientific). For Western blot analysis, 15 μg lysate per lane was fractionated by SDS-PAGE, transferred onto PVDF membranes, and probed with anti-Kcne4 or anti-K_v1.5 antibody, followed by horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad) or goat anti-chicken IgY secondary antibody (Aves Laboratories, Tigard, OR, USA) for visualization with fluorography (ECL-plus; Amersham Biosciences/GE Healthcare Life Sciences, Marlborough, MA, USA). For co-IPs, 350 μg lysates was incubated overnight with primary antibody at 4°C, and then 40 μl of Pierce agarose A beads was added for 1 h. Beads were washed 3 × 5 min with lysis buffer and eluted with Tris(2-carboxyethyl)phosphine hydrochloride and NuPage LDS loading buffer (Novex Life Technologies, Carlsbad, CA, USA) at 95°C for 5 min. Elutions (20 μl/lane) were fractionated by SDS-PAGE as above for Western blotting. Blots were visualized using SynGene G:Box Chemi XR5, running Genesys software, v1.4.3 (Syngene, Frederick, MD, USA).

Electrocardiography and hemodynamic analyses

Mice in 3 age groups (4, 12, and 17 mo old) were anesthetized with 2% isoflurane, and surgical anesthesia was verified by a lack of response to toe pinch. The standard limb lead II configuration electrocardiographic system was inserted subcutaneously to limbs by needle electrodes, and electrocardiograms were recorded throughout the study. QT, RR, PR, and QRS intervals and heart rate were analyzed offline after acquisition. Corrected QT interval (QT_c) was calculated based on Mitchell’s formula specifically for mice (24): QT_c = QT/(RR/100)^1/2. For hemodynamic analysis, the right carotid artery was exposed through a cervical midline incision, and the left ventricle was catheterized via the right carotid artery using a 1.0 F Millar Micro-Tip catheter transducer (model SPR-1000) connected to a pressure transducer (Millar Instruments, Houston, TX, USA). Baseline blood pressures were recorded before advancing the catheter into the left ventricle. The real-time data were collected by Powerlab/8sp system (ADInstruments, Colorado Springs, CO, USA). LabChart 7.2.1 software (ADInstruments) was used for electrocardiographic and hemodynamic data acquisition and analysis.

RESULTS

Kcne4 transcript expression is enriched in ventricles of male sexually mature mice

We first quantified mouse cardiac Kcne4 expression by real-time qPCR using Kcne4^−/− tissue as a negative control and to further confirm genotype. In sexually mature mice, Kcne4 expression was highest in the left ventricle of male mice, next highest in the male mouse right ventricle, and then the male atria, and was comparatively low in all 4 female heart chambers (Fig. 1D).

Kcne4 deletion sex-specifically impairs ventricular and atrial myocyte repolarization in mice

We next measured K_v currents using whole-cell patch clamp of ventricular myocytes isolated from sexually mature Kcne4^+/+ and Kcne4^−/− mice. Mean current density (peak and sustained current) was unaffected by Kcne4 deletion in female ventricular myocytes isolated from the septum (Fig. 1E, F) and also those from the apex (Supplemental Fig. S1). This was consistent with the relatively low expression of Kcne4 in the female mouse heart. In contrast, Kcne4 deletion reduced peak K_v current density in male ventricular septal myocytes to match that of females but did not alter sustained currents (Fig. 1E, F).

The 3 major components of adult mouse ventricular myocyte K_v currents (I_to,f, I_to,s, and I_K,slow) can be isolated by fitting current decay with 3 exponential functions, distinguishing their 3 different inactivation rates, and permitting quantification of the amplitude of each component (7, 25). The inactivation kinetics of the Kcne4-dependent current in males were consistent with it comprising both the rapidly inactivating I_to,f (generated by K_v4 subunits in musine and human ventricles) and the more slowly inactivating I_K,slow (generated by K_v1.5 and K_v2.1 α subunits in adult mouse ventricles), but not the intermediate-inactivating I_to,s (generated by K_v1.4 in adult mouse ventricles) (Fig. 2A, B). We previously demonstrated that KCNE4 augments K_v4.2 activity (17), consistent with the reduction in I_to,f observed here on Kcne4 deletion.

Figure 2.

Kcne4 deletion impairs male mouse ventricular myocyte I_to,f and I_K,slow1. A, B) Mean amplitude (A) and decay time constant, τ (B), of current components after curve-fitting whole-cell ventricular septal cardiomyocyte K⁺ current traces from male sexually mature adult Kcne4^+/+ and Kcne4^−/− mice as in Fig. 1E, F; n = 9–26 myocytes from 3 to 6 mice per group. C) Exemplar digitally subtracted 50 µM 4-AP–sensitive K⁺ current (I_K,slow1) traces recorded from cells as in Fig. 1E, F; genotypes are as shown; n = 18–19. D) Mean digitally subtracted 50 µM 4-AP–sensitive K⁺ current (I_K,slow1) densities quantified from traces as in C; genotypes are as shown; n = 18–19. *P < 0.01 comparing current density between genotypes. E) Exemplar digitally subtracted 25 mM TEA-sensitive K⁺ current (I_K,slow2) traces recorded from cells as in Fig. 1E, F; genotypes are as shown; n = 7–9. F) Mean digitally subtracted 25 mM TEA-sensitive K⁺ current (I_K,slow2) densities quantified from traces as in E; genotypes are as shown; n = 7–9.

The ∼50% loss of amplitude in I_K,slow suggested a role for KCNE4 with K_v1.5 and/or K_v2.1 that had not been previously recognized. Indeed, pharmacologic isolation of the I_K,slow component of the Kcne4-dependent current revealed its sensitivity to 50 µM 4-AP (Fig. 2C, D) but not 25 mM TEA (Fig. 2E, F). This profile was consistent with the pharmacologic properties of I_K,slow1, generated by K_v1.5 (7).

Kcne4 deletion also reduced K_v current density in adult male mouse atrial myocytes, by >45% at +40 mV (P < 0.001; Fig. 3A, B). Curve fitting with 2 exponentials to isolate the rapid- and the slow-inactivating current components, as previously described for adult mouse atrial myocytes (26), revealed that the primary current component lost by Kcne4 deletion was I_to,f (>50% reduction at +40 mV; P = 0.03; Fig. 3C, D).

Figure 3.

Kcne4 deletion impairs male mouse atrial myocyte I_to,f. A) Representative whole-cell atrial cardiomyocyte K⁺ currents from sexually mature adult male Kcne4^+/+ and Kcne4^−/− mice (voltage protocol inset); n = 20 myocytes from 3 to 4 mice per group. B) Mean peak whole-cell atrial cardiomyocyte K⁺ current densities recorded from cells as in A; n = 20, *P < 0.001. C) Averaged K⁺ current traces elicited by a +40 mV voltage pulse from atrial myocytes isolated from sexually mature adult male Kcne4^+/+ and Kcne4^−/− mice (mean of 20 traces per genotype). D) Mean data resulting from curve fitting of +40 mV traces from recordings as in A; n = 14–19.

KCNE4 colocalizes with and augments activity of K_v1.5 in CHO cells

To validate the notion that KCNE4 directly regulates K_v1.5 channels, we studied the subunits heterologously expressed in CHO cells. KCNE4 more than doubled the density of current generated by K_v1.5 (Fig. 4A, B). KCNE4 moderately increased the slope and left-shifted the voltage dependence of activation of K_v1.5 (Fig. 4C). KCNE4 did not alter activation kinetics but moderately increased the extent of inactivation over a 1 second pulse to +40 mV of K_v1.5 (Fig. 4D, E).

Figure 4.

KCNE4 doubles K_v1.5 current density in CHO cells. A) Representative whole-cell K⁺ current traces recorded from CHO cells 48 h after transfection of K_v1.5 alone or with KCNE4 (voltage protocol inset); n = 20–21. B) Mean peak current density for cells as in A; n = 20–21, *P < 0.0001. C–E) Mean voltage dependence of activation (C), time to peak activation (D), and percent inactivation (E) for currents as in A and B; n values as in A and B.

KCNE4 colocalized in the plasma membrane of CHO cells with coexpressed K_v1.5 (Fig. 5A), and K_v1.5-KCNE4 complexes were detectable by co-IP (Fig. 5B). To quantify effects of KCNE4 on K_v1.5 surface expression, CFP-tagged K_v1.5 (K_v1.5-CFP) was expressed alone or with KCNE4 and CFP fluorescence compared with fluorescence from detection of antibody raised to an epitope on the surface of K_v1.5, using microscopy of live CHO cells. K_v1.5-CFP was expressed in puncta at the cell surface, and KCNE4 increased the number of puncta (Fig. 5C). Quantification of total and surface fluorescence intensities revealed that KCNE4 did not alter total K_v1.5-CFP expression but doubled K_v1.5-CFP surface expression, whether or not this value was normalized to total K_v1.5-CFP expression (Fig. 5D).

Figure 5.

KCNE4 forms complexes with, and doubles surface expression of, K_v1.5 in CHO cells. A) Fluorescence micrographs showing plasma membrane colocalization of KCNE4 and K_v1.5, taken 48 h after transfection of CHO cells. Scale bar, 2.5 µm. B) Left) Coimmunoprecipitation (IP) of K_v1.5 and KCNE4 from lysates isolated 48 h after CHO cell transfection. Proteins were immunoprecipitated with anti-KCNE4 antibody and immunoblotted (IB) using anti-K_v1.5 antibody. Arrow, K_v1.5 monomer. Lysate, lanes loaded with nonimmunoprecipitated lysate for control. cDNAs transfected are indicated by + and their absence by −. Right) Positive control for KCNE4 IP and IB, both using KCNE4 antibody. Arrow, KCNE4 monomer; other symbols as in upper blot. C) Fluorescence micrographs of live CHO cells expressing CFP-tagged K_v1.5 alone or with KCNE4. Fluorescence signal detected was either CFP (left) to quantify total K_v1.5-CFP expression or via antibody raised to an external epitope on K_v1.5 (right) to quantify K_v1.5-CFP surface expression, in the same cell. Scale bar, 2.5 µm. D) Quantification of mean effects of KCNE4 on total (left), surface (center), and surface/total (right) K_v1.5 expression; n = 22–23 cells per group.

Ventricular KCNE4 expression is regulated by DHT

Electrocardiographic analyses showed that, in 4-mo-old male mice, QT_c, a measure of the time for ventricular repolarization, was unaltered by Kcne4 deletion. In contrast, Kcne4 deletion increased QT_c by 24% at 12 mo of age and by 55% at 17 mo of age in male mice (Fig. 6A). This pattern was not entirely unexpected: we observed similar age-dependent Kcne-dependent QTc prolongation in both the Kcne2^−/− and Kcne3^−/− mouse lines (27, 28), suggesting that Kcne2, 3, or 4 deletion requires additional aging-related erosion of the repolarization reserve to manifest on the body surface electrocardiogram.

Figure 6.

Effects of Kcne4 deletion on ventricular repolarization are age and sex dependent. A) Left) Representative body surface electrocardiograms measured from 4- (n = 11) and 17-mo-old (n = 8–10) male Kcne4^+/+ and Kcne4^−/− mice as indicated. Right) Mean QT_c values quantified from electrocardiograms as in left and from 12-mo-old male mice (n = 5–8). B) Left) Representative body surface electrocardiograms measured from 4- (n = 9–10) and 17-mo-old (n = 10–12) female Kcne4^+/+ and Kcne4^−/− mice as indicated. Right) Mean QTc values quantified from electrocardiograms as in left and from 12-mo-old female mice (n = 3).

We also observed aging-dependent QT_c prolongation in female Kcne4^−/− mice, except that it occurred with a later onset than in males. Thus, QT_c of female mice was unaltered by Kcne4 deletion at 4 and 12 mo but increased by 47% at 17 mo (Fig. 6B), by which age female mice are postmenopausal (29). That Kcne4 deletion had an effect on postmenopausal female QT_c was surprising, given the relatively low cardiac expression of KCNE4 in young adult female mice (Fig. 1B). Other electrocardiographic parameters were not changed in either sex (Supplemental Fig. S2). Similarly, hemodynamic parameters were largely unaffected by Kcne4 deletion (Supplemental Fig. S3). Mean minimum change in pressure/time (Min dP/dt) was high in female Kcne4^−/− mice compared with Kcne4^+/+ mice (n = 7–8; P = 0.028), but the possible implications of this will require future investigation.

Given the unexpected importance of the Kcne4 subunit in ventricular repolarization of postmenopausal female mice, we quantified their Kcne4 expression and found that ventricular Kcne4 expression was >10-fold higher in postmenopausal female mice compared with young sexually mature (4–6 mo old) adult female mice and was equivalent to that of young and old adult male mice (Fig. 7A).

Figure 7.

DHT regulates ventricular Kcne4 expression and ventricular repolarization. A) Real-time qPCR quantification of Kcne4 expression in 4- and 17-mo-old mouse heart left ventricles. Kcne4^−/− tissue (open columns) was used as a negative control. Kcne4 expression levels for young adult male and female left ventricles are repeated from Fig. 1D for comparison. All values were normalized to one of the 4- to 6-mo-old male Kcne4^+/+ mouse Kcne4 expression values. B) Real-time qPCR quantification of Kcne4 expression in left ventricles from castrated mice (light gray, n = 9), and castrated/DHT pellet-implanted young adult male mice 45 (medium gray, n = 5) and 128 d (dark gray, n = 4) after implant, each quantified in triplicate. Kcne4 expression in nonpellet-implanted vs. 128 d after pellet implant, P = 0.01; all other comparisons, P > 0.05, by Tukey honest significant difference, Scheffé multiple comparison, or Bonferroni and Holm multiple comparison. All values were normalized to one of the 4- to 6-mo-old male Kcne4^+/+ mouse Kcne4 expression values from A. C) Representative body surface electrocardiograms measured from 6-mo-old castrated (n = 5) and castrated, DHT pellet-implanted (128 d) male Kcne4^+/+ mice as indicated. D) Mean QT_c values quantified from electrocardiograms as in C (n = 5).

Interestingly, in young adult mice, DHT is very high in males compared with females (a ratio of >50:1), but DHT levels rise >4-fold after menopause in female mice (29), qualitatively matching the pattern we observed for the Kcne4 transcript. To test the hypothesis that DHT regulates ventricular Kcne4 expression, we quantified the latter in young adult male mice that had been castrated at 2 mo of age and those that were castrated and simultaneously implanted with DHT pellets. At 128 d after pellet implantation, left ventricular Kcne4 expression was >3-fold higher than that of castrated, nonpellet-implanted mice (P = 0.01) and similar to that of noncastrated male mice (Fig. 7A, B). Kcne4 expression 45 d after pellet implantation was intermediate between that of castrated, nonpellet-implanted and castrated mice 128 d after DHT pellet implant (Fig. 7B). Consistent with the DHT-dependent expression of Kcne4 and the observation that Kcne4 deletion prolongs the QT interval by diminishing ventricular K_v currents generated by channels regulated by Kcne4, DHT pellet implantation shortened the QT_c interval in castrated mice (Fig. 7C, D).

DISCUSSION

Our findings reveal that DHT positively regulates Kcne4 expression in mouse ventricles and that this contributes to higher K_v current density in young adult male mice compared with their age-matched female counterparts. Postmenopausal female mice exhibit an increase in ventricular Kcne4 expression concomitant with the increased DHT known to occur on this transition (29). Interestingly, in a previous study, ventricular myocyte I_K,slow density was found to be higher in male mice than in females, with the difference linked to DHT regulation of K_v1.5 (30). In that study, as we recapitulated here (Fig. 7C, D), DHT shortened the QT_c in castrated mice. Our findings suggest that control of QT_c by DHT arises from its positive regulation not just of K_v1.5 but also of KCNE4. Also in the prior study (30), it was found that C57BL/6 male mice have relatively low DHT compared with another strain, CD-1, and that males of the latter strain also exhibit higher 4-AP–sensitive current density than do C57BL/6 male mice. It is possible that a backcross of our Kcne4^─/─ mice into the CD-1 background would yield even larger differences in Kcne4 expression between sexes than seen here.

Our data are the first to demonstrate male androgen regulation of a K⁺ channel regulatory subunit, although estrogen (and not DHT) was previously shown to positively regulate Kcne2 expression in the murine heart (31). It was suggested that such regulation provides a mechanism to counteract other factors causing QT prolongation by reducing the repolarization reserve near the end of gestation, with the surge in estrogen during this period upregulating Kcne2, which could augment I_to,f and I_K,slow1, based on our previous findings (7). Similarly, DHT up-regulation of Kcne4 expression might provide a mechanism for augmenting I_to,f and I_K,slow1 in postmenopausal female mice to counteract the QT-prolonging effects of aging that we have now observed in 3 separate Kcne^─/─ mouse colonies (27, 28).

It therefore appears that Kcne2 and Kcne4 serve, in some ways, similar roles in mouse ventricles, each augmenting both I_to and I_Kslow,1, Kcne2 being relatively more prominent in the female heart because of positive regulation by estrogen and Kcne4 more important in males because of positive regulation by DHT. Both Kcne2 and Kcne4 are expressed in multiple tissues outside the heart (32), yet hormonal regulation of these subunits in other tissues has not been studied. To fully understand the ramifications of this mode of regulation, a more comprehensive analysis is warranted. In addition, we do not yet know the mechanism by which DHT regulates Kcne4 expression. Typically, DHT binds to androgen receptors, and the complex translocates to the nucleus, where it binds to androgen-responsive elements (AREs) on specific genes. We did not find a canonical ARE within the mouse Kcne4 gene sequence, suggesting either Kcne4 harbors a noncanonical ARE or DHT increases Kcne4 expression by activating another protein (e.g., another transcription factor). DHT can also act nongenomically (e.g., its binding to androgen receptors can rapidly activate mitogen-activated protein kinases), resulting in downstream activation of transcription factors (33)—another avenue to explore in future directions.

It is possible that Kcne4 is upregulated by DHT to mirror DHT up-regulation of K_v1.5 similar to the coordinated expression of K_v4 and KChIP subunits in mice (34), presumably to avoid the deleterious effects of expression of K_v1.5 without the modifications produced by Kcne4 on channel function and trafficking that might result from an expression level mismatch. The same might apply to K_v4.2 and/or K_v4.3, the subunits generating I_to,f in mouse ventricles. K_v4.3 was previously found to be upregulated by testosterone in female canine ventricles and more highly expressed in normal male than in normal female ventricles (35).

Turning to the atria, we found that Kcne4 deletion primarily affected I_to,f, reducing current density by more than half, although we did not detect spontaneous atrial arrhythmias in Kcne4^─/─ mice. The 36% reduction in I_K,slow density did not reach the P < 0.05 significance level, but was comparable to the effect we observed for ventricular I_K,slow of male Kcne4^─/─ mice. In human heart, KCNE4 expression is reported to be 2-fold higher in the atria over the ventricles (36); this may explain the link between KCNE4 and AF (19–21), whereas ventricular arrhythmias have not been reported to date. Interestingly, unlike in the adult murine heart, K_v1.5 in the human heart is enriched in the atria compared with the ventricles (where it is difficult to detect) (37). Prior studies of the human KCNE4 G/T polymorphism at position 1057 found that the T allele (generating the KCNE4 145D vs. 145E for the G allele) is associated with increased incidence of AF (19–21). In vitro studies showed that KCNE4-145D augmented KCNQ1 current, whereas 145E inhibited it, a plausible mechanism for AF (22). In light of our findings, it will be of interest to compare the effects of the 2 human KCNE4 variants on K_v1.5 and K_v4.2/3 channel properties to determine whether these potentially contribute to, or counteract, effects on KCNQ1.

Finally, further studies are required to determine whether KCNE4 and KCNE2 are also regulated by hormones in human heart. AF is much more common in the elderly than in the young and afflicts millions within the aging population in the United States alone. As testosterone levels diminish later in life in men, it will be important to determine how the predicted reduction in cardiac KCNE4 associated with this might affect susceptibility to AF and to consider the potential safety and efficacy of correcting this with hormonal therapy. In a recent analysis of the Framingham Heart Study, testosterone deficiency in men of 80 yr of age and older was found to be strongly associated with AF risk, whereas the mechanistic basis remains incompletely understood (38). The results of mouse studies cannot be directly extrapolated to the human heart because of major differences in, for example, heart rate, chamber size, and K_v channel subunit expression. However, the more we understand about these differences, the more effectively we can use studies of the genetically highly tractable mouse to generate testable hypotheses relating to crucial aspects of human cardiac physiology and the pathogenesis of debilitating or lethal cardiac arrhythmias.

Glossary

4-AP

4-aminopyridine

AF

atrial fibrillation

ARE

androgen-responsive element

BSA

bovine serum albumin

CFP

cyan fluorescent protein

CHO

Chinese hamster ovary

co-IP

coimmunoprecipitation

DHT

5α-dihydrotestosterone

HEPES

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

IF

immunofluorescence

I_K,slow

slowly activating K⁺ current

I_to,f

fast transient outward current

I_to,s

slow transient outward current

K_v

voltage-gated potassium

qPCR

quantitative PCR

QT_c

corrected QT interval

ss

steady state

TEA

tetraethylammonium