mice carry a null mutation of the Kcna1
gene on chromosome 6 as a result of gene targeted deletion, as previously described (Smart et al., 1998
). Mice were housed at 22 °C, fed ad libitum
and submitted to a 12-hour light/dark cycle. We carried out all procedures in accordance with the guidelines of the National Institutes of Health, as approved by the Animal Care and Use Committee of Baylor College of Medicine.
We isolated genomic DNA from tail clips using DirectPCR Lysis Reagent (Viagen Biotech Inc., Los Angeles, CA). We determined the genotypes of Kcna1
mice using PCR amplification of specific alleles as done previously (Glasscock et al., 2007
). We included three primers in the PCR reaction: a mutant-specific primer (5′-CCTTCTATCGCCTTCTTGACG-3′), a wildtype-specific primer (5′-GCCTCTGACAGTGACCTCAGC-3′), and a common primer (5′-GCTTCAGGTTCGCCACTCCCC-3′). The PCR reaction yielded PCR products of about 337-bp for the wildtype allele and about 475-bp for the mutant allele.
Simultaneous video EEG-ECG recordings
Kcna1-null mice and wild-type controls between 1–2 months of age were anesthetized with an intraperitoneal injection of 0.02 mL g−1 Avertin and surgically implanted with bilateral silver wire electrodes (0.005-inch diameter) attached to a microminiature connector. EEG electrodes were inserted into the subdural space through cranial burr holes overlying the temporal cortex. For ECG, two thoracic electrodes were tunneled subcutaneously on either side and sutured in place to record cardiac activity. Mice were allowed to recover for 24 hours before measuring simultaneous EEG-ECG activity of freely moving animals using a digital EEG/video monitoring system with Harmonie software, version 6.1c (Stellate Systems; Montreal, Canada). For EEG signals, we used a sampling rate of 250 Hz and filtered using a 0.3-Hz high-pass filter, 70-Hz low-pass filter, and 60-Hz notch filter. For ECG signals, we used a sampling rate of 2 kHz with a 3-Hz high-pass filter.
ECG waveform analysis
Definitions of ECG intervals and durations are illustrated in Fig. S1
. P duration was manually measured as the time from the beginning of the upstroke of the P wave until its return to the isoelectric baseline; QRS duration was measured from the beginning of the Q wave to the peak amplitude of the downward deflection of the S wave; PR interval from the beginning of the upstroke of the P wave until the maximal amplitude of the R wave; RR interval as the time between consecutive R wave peaks; QT interval from the beginning of the Q wave until the T wave returns to the isoelectric baseline. Since the QT interval covaries with the RR interval, we calculated a rate-corrected QT interval (QTc
) using the formula: QTc
(Mitchell et al., 1998
). To calculate average ECG intervals and durations we analyzed 30-sec epochs from a 24-hr monitoring session, sampled four times daily at 00:00, 06:00, 12:00, and 18:00. Each 30-sec epoch comprised between 250–400 heart beats, depending on the heart rate, which were then averaged together using pClamp 10 software (Molecular Devices, Sunnyvale, CA) to generate a composite ECG waveform. We then measured each ECG characteristic from the average waveform, except for the RR interval which was determined for each individual RR interval during the 30-sec epoch and averaged. To calculate the average rate of interictal heart conduction blocks per hour for each genotype, we counted all non-seizure associated second degree AV blocks during the entire 24-hr recording session. We defined a second degree AV block as a non-conducted P wave in which the RR interval of the pause was at least 1.5 times the RR interval of the previously conducted P wave. To count as more than one event, we required AV blocks to be separated by at least 500 ms. To calculate the average ictal heart conduction blocks per hour, we counted second degree AV blocks occurring during seizures, divided them by the total seizure duration, and extrapolated to an hourly basis for comparison. Only mice exhibiting at least three seizures were used for ictal cardiac analysis. We defined bradycardia as ≥ 15% decrease in heart rate compared to the overall heart rate.
Cardiac magnetic resonance imaging (MRI)
Short-axis cardiac MRI images were acquired using a Bruker BioSpin MRI PharmaScan 70/16 – 7.0 T scanner (Ettlingen, Germany) at the Mouse Phenotyping Core (Baylor College of Medicine, Houston, TX). During the procedure, mice were anesthetized with isoflurane, and heart rate, respiratory rate, and body temperature were continuously monitored. Sets of eleven images were acquired for each cardiac cycle. Images were acquired with a FOV of 4 cm, slice thickness of 1 mm, and an in-plane resolution of about 313 μm. About 9–10 image sets were required to cover both ventricles. Images were analyzed off-line using ImageJ software (NIH; Bethesda, MD). Right and left ventricular endocardial areas were measured during systole and diastole and summed to calculate the overall ventricular systolic and diastolic volumes. Ejection fraction (EF) was calculated from the end diastolic volume (EDV) and the end systolic volume (ESV) according to the following formula: EF = (EDV − ESV)/EDV.
Blood pressure measurements
Systolic blood pressures were measured at the Mouse Phenotyping Core (Baylor College of Medicine, Houston, TX) using a non-invasive tail-cuff blood pressure system (IITC Life Science, Woodland Hills, CA). Two mice (2–3 months old) per genotype were allowed to acclimate to the testing conditions and then multiple blood pressure measurements were taken daily over the course of three days for each mouse. These data were pooled and averaged to calculate the average systolic blood pressure for each mouse.
To achieve complete autonomic blockade, we administered propranolol (4 mg kg−1
) and atropine (1 mg kg−1
; Sigma-Aldrich Inc., St. Louis, MO) using concentrations previously shown to be effective in mice (Shusterman et al., 2002
; Ieda et al., 2007
). For selective parasympathetic or sympathetic blockade, we administered atropine (1 mg kg−1
) or propranolol (4 mg kg−1
) alone, respectively. Drugs were dissolved in 0.9% NaCl and injected intraperitoneally at a concentration of 10 ml kg−1
. For each drug challenge experiment, Kcna1
-null mice (4–6 weeks old) were recorded by simultaneous video EEG-ECG for 2 hours immediately prior to drug administration to establish the baseline rate of second degree AV blocks, injected with drug, and then recorded for another 2 hours to determine the drug’s effect on the rate of AV blocks.
C57BL/6J mice (2–3 months old) were perfused intracardially with PBS and fixed with 4% paraformaldehyde in PBS. Vagus nerves were removed, cryoprotected for 1–2 days at 4°C in 30% sucrose in PBS, frozen in embedding medium, and cut into 10 μm sections using a cryostat maintained at −20 °C. Sections were directly mounted on slides and allowed to thaw at room temperature for about 30 minutes immediately before processing. Tissue sections were rinsed three times in PBS and incubated for 1 hour in antibody vehicle (10% BSA, 0.3% Triton X-100 in PBS). Next, the sections were incubated overnight (15–20 hours) at room temperature with rabbit polyclonal anti-Kv1.1 antibody (1:50 dilution in vehicle; antibody courtesy of Dr. J. Trimmer, University of California, Davis, CA) and/or mouse monoclonal anti-Caspr (K65/35) antibody (1:500 dilution in vehicle; UC Davis/NIH NeuroMab Facility). Subsequently, sections were washed three times in antibody vehicle and incubated for one hour in Alexa Fluor 488 goat anti-rabbit F(ab′)2 secondary antibody and/or Alexa Fluor 555 goat anti-mouse F(ab′)2 secondary antibody (1:1000 dilution in vehicle, Molecular Probes, Carlsbad, CA). Finally, sections were rinsed once in vehicle and twice in PBS and then air dried at room temperature for 30 minutes. Once dry, the slides were cover-slipped and mounted using ProLong Gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA). Images were captured using an Olympus IX71 microscope (Olympus America Inc., Center Valley, PA) and adjusted for brightness and contrast using Adobe Photoshop Elements software (Adobe Systems Incorporated, San Jose, CA). Control experiments performed by incubating slices with secondary antibodies only, as well as staining tissue sections from Kcna1-null mice with anti-Kv1.1 antibody showed an absence of background staining. For heart immunohistochemistry, mice (129 strain, age 2–3 months) were transcardially perfused with PBS and heart nodal regions dissected. Frozen heart sections (10 μm) were then cut and processed as described above, but without fixation.
Heart tissue preparation
To obtain region-specific heart tissue for transcript and protein expression analysis, wild-type mice (129 strain; 2–3 months old) mice were transcardially perfused with PBS to remove blood. Dissections were then performed in ice cold PBS using a microscope. For atria, we cut left and right atrial appendages. For ventricular tissue, we collected left and right atrial walls. For sinoatrial node, we isolated tissue including the intercaval region of the right atrium bounded by the crista terminalis, the superior and inferior vena cavae, and the atrial septum. For the atrioventricular node, we obtained tissue including Koch’s triangle bounded by the triscuspid valve, the membranous septum, and the coronary sinus. Tissue was flash frozen using liquid nitrogen or placement at −80 °C. Care was taken to remove adjoining fatty tissue as completely as possible.
Reverse transcriptase PCR (RT-PCR)
Tissue from whole heart and from regional dissections of brain (cortex, cerebellum, hippocampus, olfactory bulb, spinal cord, brain stem) and heart (atria, ventricle, SA node, AV node) was homogenized in Trizol and a phenol choloroform extraction of mRNA was performed. After treatment with DNAse, the RT-PCR was performed using SuperScriptIII™ RT-PCR System (Invitrogen, Carlsbad, CA) with polydT primers as per the provided protocol. PCR amplification of Kcna1 transcripts was performed using primers (forward, 5′-GCATCGACAACACCACAGTC-3′; reverse, 5′-CGGCGGCTGAGGTCACTGTCAGAGGCTAAGT-3′) targeting a 710-bp region in the coding exon of the mouse Kcna1 transcript (accession # NM_010595). PCR amplifications used 35 cycles with a 45 second extension time. To control for the small size of the starting heart tissues, GAPDH amplification (982 bp band; forward, 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′; reverse, 5′-ATGTAGGCCATGAGGTCCACCAC-3′) of cDNA from all tissues were performed.
Western blotting and immunoprecipitation
Whole mouse brain and heart were extracted, flash frozen in liquid nitrogen, and subsequently homogenized on ice with a Tissue Tearor in lysis buffer containing (in mM): 20 Tris pH 7.5, 138 NaCl, 3 KCl, 1% Triton X-100, 1 EGTA, 2 EDTA, 1 benzamidine, 1 phenylmethylsulfonylfluoride, 1 dithiothreitol and 5 μg mL−1 each of aprotinin, leupeptin and pepstatin A. Total protein concentration of the brain and heart tissue lysates were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). 100 or 300 μg of heart protein lysate was separated on 8% Tris-glycine-SDS polyacrylamide gels, analyzed by Western immunoblot (IB) using rabbit polyclonal anti-Kv1.1 antibody (5μg mL−1 in vehicle; antibody courtesy of Dr. J. Trimmer, University of California, Davis, CA), HRP-tagged goat anti-rabbit IgG secondary antibody (1:10000 dilution in vehicle, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) and subsequently detected using a commercial chemiluminescent substrate (SuperSignal; Pierce Chemical, Rockford, IL). For immunoprecipitation experiments, heart and brain protein lysates were diluted to 1 mg mL−1 and each 1 mL sample was precleared for 1 h with 30 μL of protein A-Sepharose (GE Healthcare, Piscataway, NJ) and incubated overnight with 5 μg of mouse monoclonal anti-Kv1.1 (K20/78) antibody (UC Davis/NIH NeuroMab Facility). All incubations were performed at 4 °C with constant agitation. Antibody-bound protein complexes were captured by the addition of 30 μL of protein A-Sepharose and incubated for another 2 h. Protein A-Sepharose was pelleted by centrifugation and the immunoprecipitated protein complexes were eluted using SDS-PAGE sample buffer prior to SDS-PAGE and Western immunoblotting as described above.
All data are presented as mean ± SEM. Statistical analysis was performed using Microsoft Excel 2007 Analysis ToolPak (Microsoft Corp., Redmond, WA). For comparisons of cardiac data between Kv1.1
-null mice and wild-type controls we used two-tailed t
-tests. To assess drug effectiveness in pharmacology experiments, we calculated the symmetrized percent change in the number of AV blocks following drug administration and then tested whether the symmetrized percent change in baseline significantly differed from zero (μ = 0) using a one-sample t
-test. This method has greater statistical power to account for small numbers at baseline (Berry and Ayers, 2006
). Pharmacology data points were excluded if no AV blocks were observed during the baseline period.