Kcne2 deletion causes early-onset nonalcoholic fatty liver disease via iron deficiency anemia.

Nonalcoholic fatty liver disease (NAFLD) is an increasing health problem worldwide, with genetic, epigenetic, and environmental components. Here, we describe the first example of NAFLD caused by genetic disruption of a mammalian potassium channel subunit. Mice with germline deletion of the KCNE2 potassium channel ß subunit exhibited NAFLD as early as postnatal day 7. Using mouse genetics, histology, liver damage assays and transcriptomics we discovered that iron deficiency arising from KCNE2-dependent achlorhydria is a major factor in early-onset NAFLD in Kcne2(─/─) mice, while two other KCNE2-dependent defects did not initiate NAFLD. The findings uncover a novel genetic basis for NAFLD and an unexpected potential factor in human KCNE2-associated cardiovascular pathologies, including atherosclerosis.

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

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 (Kv) channel ß 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, Kv current density was 25% greater in ventricular myocytes from young adult males (P < 0.05). Germ-line Kcne4 deletion eliminated the sex-specific Kv current disparity by diminishing ventricular fast transient outward current (Ito,f) and slowly activating K(+) current (IK,slow1). Kcne4 deletion also reduced Kv currents in male mouse atrial myocytes, by >45% (P < 0.001). As we previously found for Kv4.2 (which generates mouse Ito,f), heterologously expressed KCNE4 functionally regulated Kv1.5 (the Kv α subunit that generates IKslow1 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.

¹²4I-Epidepride: a PET radiotracer for extended imaging of dopamine D2/D3 receptors.

OBJECTIVES: A new radiotracer, ¹²4I-epidepride, has been developed for the imaging of dopamine D2/3 receptors (D2/3Rs). ¹²4I-Epidepride (half-life of ¹²4I=4.2 days) allows imaging over extended periods compared to (18)F-fallypride (half-life of ¹8F=0.076 days) and may maximize visualization of D2/3Rs in the brain and pancreas (allowing clearance from adjacent organs). D2/3 Rs are also present in pancreatic islets where they co-localize with insulin to produce granules and may serve as a surrogate marker for imaging diabetes. METHODS: ¹²4I-Epidepride was synthesized using N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-tributyltin-2,3-dimethoxybenzamide and ¹²4I-iodide under no carrier added condition. Rats were used for in vitro and in vivo imaging. Brain slices were incubated with (124)I-epidepride (0.75 µCi/cc) and nonspecific binding measured with 10 µM haloperidol. Autoradiograms were analyzed by OptiQuant. ¹²4I-Epidepride (0.2 to 0.3 mCi, iv) was administered to rats and brain uptake at 3 hours, 24 hours, and 48 hours post injection was evaluated. RESULTS: ¹²4I-Epidepride was obtained with 50% radiochemical yield and high radiochemical purity (>95%). (124)I-Epidepride localized in the striatum with a striatum to cerebellum ratio of 10. Binding was displaced by dopamine and haloperidol. Brain slices demonstrated localization of ¹²4I-epidepride up until 48 hours in the striatum. However, the extent of binding was reduced significantly. CONCLUSIONS: ¹²4I-Epidepride is a new radiotracer suitable for extended imaging of dopamine D2/3 receptors and may have applications in imaging of receptors in the brain and monitoring pancreatic islet cell grafting.

Kcne2 deletion creates a multisystem syndrome predisposing to sudden cardiac death.

BACKGROUND: Sudden cardiac death (SCD) is the leading global cause of mortality, exhibiting increased incidence in patients with diabetes mellitus. Ion channel gene perturbations provide a well-established ventricular arrhythmogenic substrate for SCD. However, most arrhythmia-susceptibility genes, including the KCNE2 K(+) channel ß subunit, are expressed in multiple tissues, suggesting potential multiplex SCD substrates. METHODS AND RESULTS: Using whole-transcript transcriptomics, we uncovered cardiac angiotensinogen upregulation and remodeling of cardiac angiotensinogen interaction networks in P21 Kcne2(-/-) mouse pups and adrenal remodeling consistent with metabolic syndrome in adult Kcne2(-/-) mice. This led to the discovery that Kcne2 disruption causes multiple acknowledged SCD substrates of extracardiac origin: diabetes mellitus, hypercholesterolemia, hyperkalemia, anemia, and elevated angiotensin II. Kcne2 deletion was also a prerequisite for aging-dependent QT prolongation, ventricular fibrillation and SCD immediately after transient ischemia, and fasting-dependent hypoglycemia, myocardial ischemia, and AV block. CONCLUSIONS: Disruption of a single, widely expressed arrhythmia-susceptibility gene can generate a multisystem syndrome comprising manifold electric and systemic substrates and triggers of SCD. This paradigm is expected to apply to other arrhythmia-susceptibility genes, the majority of which encode ubiquitously expressed ion channel subunits or regulatory proteins.

Kcne3 deletion initiates extracardiac arrhythmogenesis in mice.

Mutations in the human KCNE3 potassium channel ancillary subunit gene are associated with life-threatening ventricular arrhythmias. Most genes underlying inherited cardiac arrhythmias, including KCNE3, are not exclusively expressed in the heart, suggesting potentially complex disease etiologies. Here we investigated mechanisms of KCNE3-linked arrhythmogenesis in Kcne3(-/-) mice using real-time qPCR, echo- and electrocardiography, ventricular myocyte patch-clamp, coronary artery ligation/reperfusion, blood analysis, cardiac synaptosome exocytosis, microarray and pathway analysis, and multitissue histology. Kcne3 transcript was undetectable in adult mouse atria, ventricles, and adrenal glands, but Kcne3(-/-) mice exhibited 2.3-fold elevated serum aldosterone (P=0.003) and differentially expressed gene networks consistent with an adrenal-targeted autoimmune response. Furthermore, 8/8 Kcne3(-/-) mice vs. 0/8 Kcne3(+/+) mice exhibited an activated-lymphocyte adrenal infiltration (P=0.0002). Kcne3 deletion also caused aldosterone-dependent ventricular repolarization delay (19.6% mean QTc prolongation in females; P<0.05) and aldosterone-dependent predisposition to postischemia arrhythmogenesis. Thus, 5/11 Kcne3(-/-) mice vs. 0/10 Kcne3(+/+) mice exhibited sustained ventricular tachycardia during reperfusion (P<0.05). Kcne3 deletion is therefore arrhythmogenic by a novel mechanism in which secondary hyperaldosteronism, associated with an adrenal-specific lymphocyte infiltration, impairs ventricular repolarization. The findings highlight the importance of considering extracardiac pathogenesis when investigating arrhythmogenic mechanisms, even in inherited, monogenic channelopathies.

NHE isoform switching and KChIP2 upregulation in aging porcine atria.

Aging increases the risk of cardiac pathologies including atrial fibrillation and can alter myocardial responsiveness to therapeutic agents. Here, seeking molecular correlates of myocardial aging processes, we performed global "whole transcript" analysis of 25,388 genes using 572,667 probes to compare the left atrial (LA) transcriptomes of young adult (9 months old) versus elderly (10 years old) female swine. NHE2 (>9-fold) and KChIP2 (3.8-fold) exhibited the highest aging-related expression increases. Real-time qPCR recapitulated these findings and indicated a 50% decrease in LA NHE1, a twofold increase in right atrial KChIP2, but no significant changes for these transcripts in either ventricle. Notably, even in young adult pigs, NHE2 transcript was detectable and enriched in the atria over the ventricles. NHE1, the recognized cardiac isoform of the sodium hydrogen exchanger, has proven a compelling but clinically disappointing therapeutic target with respect to reperfusion arrhythmias. Our data challenge the dogma that NHE1 is alone in the myocardium and suggest that NHE2 could negatively impact the pharmacological responsiveness of atrial tissue to NHE1-specific inhibitors. KChIP2 is a cytosolic ß subunit essential for generation of I to. The increased KChIP2 expression we observed with aging substantially shortened in silico atrial myocyte action potential duration, a predisposing factor in atrial fibrillation. Consistent with this, 4/5 elderly swine sustained pacing-induced AF≥15 s after cessation of stimulation, compared to 0/3 young swine. Our findings uncover potential molecular bases for increased arrhythmogenicity and reduced pharmacologic efficacy in the aging atrium, in a large animal model of human cardiac physiology.

Synthesis and biological evaluation of 18F-Norfallypride in the rodent brain using PET imaging.

Norfallypride (N-[(2-pyrolidinyl)methyl]-2,3-dimethoxy-5-(3'-fluoropropyl)benzamide), an analog of fallypride, has been synthesized and evaluated as a potential PET imaging agent for dopamine receptors with increased subtype selectivity. In order to synthesize (18)F-Norfallypride, the substituted benzamide tosylate (S)-N-[(1-BOC-2-pyrolidinyl)methyl]-2,3-dimethoxy-5-(3'-tosyloxypropyl)-benzamide) was radiolabeled with (18)F using Kryptofix and K2CO3 in acetonitrile and deprotected with trifluoroacetic acid to yield (S)-(18)F-Norfallypride in approx. 10% radiochemical yields. Norfallypride exhibited an IC50 of 0.63µM for displacing (18)F-fallypride in rat brain slices. In vitro rat brain autoradiographic studies revealed weak binding of (18)F-norfallypride to striatal regions. PET imaging in rats showed low brain uptake of (18)F-norfallypride in the rat brain. Ex vivo brain PET analysis displayed binding of (18)F-norfallypride in several brain regions. With respect to the cerebellum, ex vivo PET ratios were: striatum>3; hypothalamus>2; hippocampus~2; cerebellar nuclei >2 while autoradiographic ratios were 14, 9, 4 and 6 respectively. (18)F-Norfallypride exhibited a unique binding profile to rat brain regions known to contain significant amounts of dopamine D3 and serotonin 5HT3 receptors. Efforts are currently under way to increase brain permeability and fully characterize the binding of (18)F-norfallypride in vivo.

Synthesis and evaluation of 3-¹²³I-iodo-5-[2-(S)-3-pyrrolinylmethoxy]-pyridine (niodene) as a potential nicotinic α4ß2 receptor imaging agent.

Nicotinic acetylcholine receptors (nAChRs) are downregulated in disease conditions such as Alzheimer's and substance abuse. Presently, (123)I-5-IA-85380 is used in human studies and requires over 6h of scanning time, thus increases patient discomfort. We have designed and synthesized 3-iodo-5-[2-(S)-3-pyrrolinylmethoxy]pyridine (niodene) with the aim to have faster binding kinetics compared to (123)I-5-IA-85380, which may reduce scanning time and help in imaging studies. Binding affinity K(i) of niodene for rat brain α4ß2 receptors in brain homogenate assays using (3)H-cytisine was 0.27 nM. Niodene, 10nM displaced >95% of (18)F-nifene bound to α4ß2 receptors in rat brain slices. By using the iododestannylation method, (123)I-niodene was obtained in high radiochemical purity (>95%) but with low radiochemical yield (<5%) and low specific activity (∼100 Ci/mmol). Autoradiograms show (123)I-niodene localized in the thalamus and cortex, which was displaced by nicotine (thalamus to cerebellum ratio=4; cortex to cerebellum ratio=1.6). Methods of radioiodination need to be further evaluated in order to obtain (123)I-niodene in higher radiochemical yields and higher specific activity of this potentially useful new SPECT imaging agent.

Transcriptomic analysis reveals atrial KCNE1 down-regulation following lung lobectomy.

Lone atrial fibrillation (AF) is associated with various ion channel gene sequence variants, notably the common S38G loss-of-function polymorphism in the KCNE1 K(+) channel ancillary subunit gene. New-onset postoperative AF (POAF) generally occurs 48-72 h after major surgery, particularly following procedures within the chest, but its molecular bases remain poorly understood. To begin to address this gap in knowledge, we analyzed molecular changes in the left atrium (LA) in relation to simultaneous changes in hemodynamics, LA effective refractory period (ERP), and the capacity to sustain electrically-induced AF following left upper lung lobectomy in swine. Relative to control pigs (no previous surgery), 3 days after lobectomy higher values for mean pulmonary artery pressure (16 ± 1 vs 22 ± 2 mmHg; P=0.045) and pulmonary vascular resistance (273 ± 47 vs 481 ± 63 dyns/cm(5); P=0.025) were evident, whereas other hemodynamic variables were unchanged. LA ERP trended toward reduction in lobectomy animals (187 ± 16 vs 170 ± 20 ms, P>0.05). None of the lobectomy pigs developed spontaneous POAF as assessed by telemetric ECG. However, all lobectomy pigs, but none of the controls, were able to sustain AF induced by a 10s burst of rapid pacing for ≥ 30 s (P=0.0079), independent of LA ERP; AF was sustained ≥ 60s in 3/5 postoperative pigs versus 0/5 controls and correlated with a shorter ERP overall (P=0.023). Transcriptomic analysis of LA tissue revealed 23 up-regulated and 10 down-regulated transcripts (≥ 1.5-fold, P<0.05) in lobectomy pigs. Strikingly, of the latter, KCNE1 down-regulation showed the statistically strongest link to surgery (2.0-fold, P=0.009), recapitulated at the protein level with Western blotting (P=0.039), suggesting KCNE1 down-regulation as a possible common mechanistic factor in POAF and lone AF. Of the up-regulated transcripts, while Teneurin-2 was the strongest linked (1.5-fold change, P=0.001), DSCR5 showed the highest induction (2.7-fold, P=0.02); this and other hits will be targeted in future functional studies.

Nicotinic acetylcholine receptors in rat forebrain that bind ¹8F-nifene: relating PET imaging, autoradiography, and behavior.

Nicotinic acetylcholine receptors (nAChRs) in the brain are important for cognitive function; however, their specific role in relevant brain regions remains unclear. In this study, we used the novel compound ¹8F-nifene to examine the distribution of nAChRs in the rat forebrain, and for individual animals related the results to behavioral performance on an auditory-cognitive task. We first show negligible binding of ¹8F-nifene in mice lacking the ß2 nAChR subunit, consistent with previous findings that ¹8F-nifene binds to α4ß2* nAChRs. We then examined the distribution of ¹8F-nifene in rat using three methods: in vivo PET, ex vivo PET and autoradiography. Generally, ¹8F-nifene labeled forebrain regions known to contain nAChRs, and the three methods produced similar relative binding among regions. Importantly, ¹8F-nifene also labeled some white matter (myelinated axon) tracts, most prominently in the temporal subcortical region that contains the auditory thalamocortical pathway. Finally, we related ¹8F-nifene binding in several forebrain regions to each animal's performance on an auditory-cued, active avoidance task. The strongest correlations with performance after 14 days training were found for ¹8F-nifene binding in the temporal subcortical white matter, subiculum, and medial frontal cortex (correlation coefficients, r > 0.8); there was no correlation with binding in the auditory thalamus or auditory cortex. These findings suggest that individual performance is linked to nicotinic functions in specific brain regions, and further support a role for nAChRs in sensory-cognitive function.

Evaluation of F-nifene binding to α4ß2 nicotinic receptors in the rat brain using microPET imaging.

MicroPET imaging studies using (18)F-nifene, a new positron emission tomography (PET) radiotracer for nicotinic acetylcholinergic receptors (nAChR) α4ß2 receptors in rats, have been carried out. Rats were imaged for 90 min after intravenous injection of (18)F-nifene (0.8 to 1 mCi), and binding potential (BP(ND)) was measured. (18)F-Nifene binding to thalamic and extrathalamic brain regions was consistent with the α4ß2 nAChR distribution in the rat brain. Using the cerebellum as a reference, the values for the thalamus varied less than 5% (BP(ND) = 1.30, n = 3), confirming reproducibility of (18)F-nifene binding. (18)F-Nifene microPET imaging was also used to evaluate effects of nicotine in a group of Sprague-Dawley rats under isoflurane anesthesia. Nicotine challenge postadministration of (18)F-nifene demonstrated reversibility of (18)F-nifene binding in vivo. For α4ß2 nAChR receptor occupancy (nAChR(OCC)), various doses of nicotine (0, 0.02, 0.1, 0.25, and 0.50 mg/kg nicotine free base) 15 min prior to (18)F-nifene were administered. Low-dose nicotine (0.02 mg) reached > 80% nAChR(OCC) while at higher doses (0.25 mg) > 90% nAChR(OCC) was measured. The small amount of (18)F-nifene binding with reference to the cerebellum affects an accurate evaluation of nAChR(OCC). Efforts are underway to identify alternate reference regions for (18)F-nifene microPET studies in rodents.

(R)-N-Methyl-3-(3'-[F]fluoropropyl)phenoxy)-3-phenylpropanamine (F-MFP3) as a potential PET imaging agent for norepinephrine transporter.

A decline of norepinephrine transporter (NET) level is associated with several psychiatric and neurological disorders. Therefore positron emission tomography (PET) imaging agents are greatly desired to study the NET pathway. We have developed a C-fluoropropyl analog of nisoxetine: (R)-N-methyl-3-(3'-[(18)F]fluoropropyl)phenoxy)-3-phenylpropanamine ((18)F-MFP3) as a new potential PET radiotracer for NET with the advantage of the longer half-life of fluorine-18 (110 min compared with carbon-11 (20 min). Synthesis of (R)-N-methyl-3-(3'-fluoropropyl)phenoxy)-3-phenylpropanamine (MFP3) was achieved in five steps starting from (S)-N-methyl-3-ol-3-phenylpropanamine in approx. 3-5% overall yields. In vitro binding affinity of nisoxetine and MFP3 in rat brain homogenates labeled with (3)H-nisoxetine gave Ki values of 8.02 nM and 23 nM, respectively. For radiosynthesis of (18)F-MFP3, fluorine-18 was incorporated into a tosylate precursor, followed by the deprotection of the N-BOC-protected amine group with a 15% decay corrected yield in 2.5 h. Reverse-phase chromatographic purification provided (18)F-MFP3 in specific activities of >2000 Ci/mmol. Fluorine-18 labeled (18)F-MFP3 has been produced in modest radiochemical yields and in high specific activities. Evaluation of (18)F-MFP3 in animal imaging studies is in progress in order to validate this new fluorine-18 radiotracer for PET imaging of NET.