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Advances in implantable radio-telemetry or diverse biologging devices capable of acquiring high-resolution ambulatory electrocardiogram (ECG) or heart rate recordings facilitate comparative physiological investigations by enabling detailed analysis of cardiopulmonary phenotypes and responses in vivo. Two priorities guiding the meaningful adoption of such technologies are: (1) automation, to streamline and standardize large dataset analysis, and (2) flexibility in quality-control. The latter is especially relevant when considering the tendency of some fully automated software solutions to significantly underestimate heart rate when raw signals contain high-amplitude noise. We present herein moving average and standard deviation thresholding (MAST), a novel, open-access algorithm developed to perform automated, accurate, and noise-robust single-channel R-wave detection from ECG obtained in chronically instrumented mice. MAST additionally and automatically excludes and annotates segments where R-wave detection is not possible due to artefact levels exceeding signal levels. Customizable settings (e.g. window width of moving average) allow for MAST to be scaled for use in non-murine species. Two expert reviewers compared MAST's performance (true/false positive and false negative detections) with that of a commercial ECG analysis program. Both approaches were applied blindly to the same random selection of 270 3-min ECG recordings from a dataset containing varying amounts of signal artefact. MAST exhibited roughly one quarter the error rate of the commercial software and accurately detected R-waves with greater consistency and virtually no false positives (sensitivity, Se: 98.48% ± 4.32% vs. 94.59% ± 17.52%, positive predictivity, +P: 99.99% ± 0.06% vs. 99.57% ± 3.91%, P 0.001 and P = 0.0274 respectively, Wilcoxon signed rank; values are mean ± SD). Our novel, open-access approach for automated single-channel R-wave detection enables investigators to study murine heart rate indices with greater accuracy and less effort. It also provides a foundational code for translation to other mammals, ectothermic vertebrates, and birds.

Significance The development of selective antagonists for muscarinic acetylcholine receptors is challenging due to high homology in orthosteric binding sites among subtypes. Starting from a single amino acid difference in the orthosteric pockets in M2 muscarinic acetylcholine receptor (M2R) and M3R, we developed an M3R-selective antagonist using molecular docking and structure-based design. The resulting M3R antagonist showed up to 100-fold selectivity over the M2R in affinity and 1,000-fold selectivity in vivo. The docking-predicted geometry was further confirmed by a 3.1 Å crystal structure of M3R in complex with the selective antagonist. The potential of structure-based design to develop selective drugs with reduced off-target effects is supported by this study., Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

The rapidly activating delayed rectifier K+ channel (IKr) is encoded by the human ether-a-go-go-related gene (hERG), which is important for the repolarization of the cardiac action potential. Mutations in hERG or drugs can impair the function or decrease the expression level of hERG channels, leading to long QT syndrome. Thus, it is important to understand hERG channel trafficking and its regulation. For this purpose, G protein-coupled receptors (GPCRs), which regulate a vast array of cellular processes, represent a useful route. The development of designer GPCRs known as designer receptors exclusively activated by designer drugs (DREADDs) has made it possible to dissect specific GPCR signaling pathways in various cellular systems. In the present study, by expressing an arrestin-biased M3 muscarinic receptor-based DREADD (M3D-arr) in stable hERG-expressing human embryonic kidney (HEK) cells, we demonstrate that β-arrestin signaling plays a role in hERG regulation. By exclusively activating M3D-arr using the otherwise inert compound, clozapine-N-oxide, we found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited β-arrestin-1 to the plasma membrane, and promoted phosphoinositide 3-kinase-dependent activation of protein kinase B (Akt). The activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase and Rab11 to facilitate hERG recycling to the plasma membrane. Potential β-arrestin signaling-mediated increases in hERG and IKr were also observed in hERG-HEK cells as well as in neonatal rat ventricular myocytes treated with the muscarinic agonist carbachol. These findings provide novel insight into hERG trafficking and regulation.

The M3 muscarinic acetylcholine receptor (M3R) is implicated in cardiopulmonary control (Fisher et al. 2004), however, the M3R is also reported to play a role in metabolism and glucose homeostasis (Gautam et al. 2006). We investigated whether activation of an M3 DREADD, selectively expressed in skeletal muscle under control of the HSA promoter, impacts cardiopulmonary function in mice maintained on a high fat diet. Heart rate (HR), body temperature (Tb) and locomotor activity (ACT) were measured in 4 conscious, chronically instrumented M3 DREADD mice and 4 wildtype controls. Circadian values of HR, BT and ACT were not different between genotypes (p>0.05). Activation of the M3 DREADD by clozapine N-oxide (0.1 mg/kg) resulted in: a significant drop in heart rate at 2 hours after injection compared with the time matched control for the same animals (460 ± 28 vs. 532 ±6, p

The pathophysiologic processes distinguishing classic asthma (CA), cough-variant asthma (CVA), and methacholine (MCh)-induced cough but normal airway sensitivity (COUGH) are inadequately understood and may be a result of differences in the ability to bronchodilate following a deep inspiration (DI). The purpose of this study was to compare the bronchodilating effect of DIs in individuals with CA, CVA, and COUGH using high-dose MCh. Individuals aged 18-65 yr with CA or suspected CVA completed high-dose MCh testing to a maximum change in forced expiratory volume in 1 s (FEV1) of 50% from baseline (MAX). Impulse oscillometry (IOS) measurements and partial and maximal-flow volume curves (used to calculate a DI index) were recorded at baseline and at each dose of MCh. Body plethysmography was performed at baseline and MAX. Twenty-eight subjects [25 women, 39.8 ± 11.9 yr (means ± SD)] were studied ( n = 11 CA, n = 10 CVA, and n = 7 COUGH). At MAX, the percent change in FEV1 was greater in subjects with CA compared with those with CVA ( P < 0.001) and COUGH ( P < 0.001), and the percent change in forced vital capacity was greater in those with CA than with COUGH ( P = 0.017). Subjects with CA and CVA developed dynamic hyperinflation and gas trapping. In subjects with CA and CVA, all IOS parameters were significantly increased from baseline to MAX, except for central respiratory resistance (R20). In individuals with COUGH, total respiratory resistance, R20, and resonant frequency were significantly increased from baseline. At MAX, the DI index was positive in all groups, suggesting preserved bronchodilation (CA, 0.67 ± 0.97; CVA, 0.51 ± 0.73; COUGH, 0.01 ± 0.36; P = 0.211). We conclude that the bronchodilating effect of DIs is preserved in individuals with CA, CVA, and borderline with COUGH; however, hyperinflation and gas trapping are avoided in subjects with COUGH alone.

The clinical relevance of cough during methacholine challenge in individuals with normal airway sensitivity is unknown. We compared responses of individuals with chronic cough who cough during high-dose methacholine bronchoprovocation and have normal versus increased airway sensitivity to healthy controls. Fifteen healthy participants (CONTROL) aged 26 ± 7 yr (mean ± SD) and 32 participants aged 42 ± 14 yr with chronic cough and suspected asthma completed high-dose methacholine challenge testing. Three participants who did not cough and had normal airway sensitivity were excluded. Spirometry and lung volumes were compared at the maximum response (MAX) among 1) ASTHMA [ n = 15, provocative concentration of methacholine causing a 20% fall in forced expiratory volume in 1 s (FEV1) from baseline (PC20) 4.71 ± 1.37 mg/ml], 2) methacholine-induced cough with normal airway sensitivity (COUGH, n = 14, PC20 41.2 ± 18.7 mg/ml for 3 participants with a measurable PC20), and 3) CONTROL ( n = 15; PC20 93.4 ± 95.4 mg/ml for 4 participants with a measurable PC20). Esophageal pressure-derived pulmonary mechanics were compared at MAX for the ASTHMA and COUGH groups. From baseline to MAX, FEV1 and forced expiratory flow between 25% and 75% of forced vital capacity decreased more in ASTHMA (−36.2 ± 3.8 %pr; −47.1 ± 6.9 %pr, respectively) than COUGH (−12.2 ± 3.0 %pr ( P < 0.001); −24.7 ± 6.5 %pr ( P < 0.001), respectively) and CONTROL (−13.7 ± 2.0 %pr ( P < 0.001); −32.8 ± 5.4 %pr ( P < 0.017), respectively). In both ASTHMA and COUGH, inspiratory capacity decreased by 500–800 ml, and functional residual capacity and residual volume increased by ~800 ml. Individuals with COUGH develop dynamic hyperinflation and gas trapping comparable to individuals with ASTHMA despite less bronchoconstriction and smaller reductions in mid-to-late expiratory flows, which leads us to believe that COUGH is a distinct phenotype. NEW & NOTEWORTHY Healthy individuals and individuals with chronic cough who demonstrate normal airway sensitivity but cough during methacholine bronchoprovocation bronchoconstrict less than individuals with mild asthma. However, those who cough and have normal airway sensitivity develop dynamic hyperinflation and gas trapping comparable to individuals with mild asthma. Thus, methacholine-induced cough with normal airway sensitivity may be clinically relevant, related to reversible small airway obstruction and preservation of the bronchodilating and/or bronchoprotective effects of deep inspirations.

The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl(-) currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway.

Purpose To assess the effect of deep inspirations (DIs) on airway behaviour in individuals with classic asthma (CA), cough variant asthma (CVA), and methacholine (MCh)-induced cough but normal airway sensitivity (COUGH) during bronchoprovocation. Methods Twenty-five adults (18 female; 44.8 ± 12.3 years (Mean ± SD); n = 9 CA, n = 9 CVA, and n = 7 COUGH) completed two single-dose MCh challenges, with and without DIs. Bronchoprotection was assessed by comparing changes in bronchoconstriction (FEV 1 , FVC, FEV 1 /FVC, FEF 50 , FEF 25-75 ), gas trapping (RV, RV/TLC) and impulse oscillometry (IOS) measurements. Results The% changes in FEV 1 with and without DIs were not significantly different within any group. Decreases in FEF 50 and FEF 25-75 were greater in CA (p = 0.041 and p = 0.029), decreases in FVC (% predicted) and FEV 1 /FVC(%) were less in CVA (p = 0.048 and p = 0.010), and increases in RV (L) and RV/TLC (% predicted) were less in COUGH (p = 0.007 and p = 0.028), respectively. No differences in IOS measurements were noted. Conclusions DIs triggered bronchoconstriction in CA, bronchoprotection in CVA, and prevented gas trapping in COUGH.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator–knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

The Read CO2 rebreathing method (Read DJ. A clinical method for assessing the ventilatory response to carbon dioxide. Australas Ann Med 16: 20–32, 1967) provides a simple and reproducible approach for studying the chemical control of breathing. It has been widely used since the modifications made by Duffin and coworkers. Our use of a rebreathing laboratory to challenge undergraduate science students to investigate the control of breathing provided 8 yr of student-generated data for comparison with the literature. Students (age: 19–22 yr, Research Ethics Board approval) rebreathed from a bag containing 5% CO2 and 95% O2 (to suppress the peripheral chemoreflex to hypoxia). Rebreathing was performed, and ventilation measured, after hyperventilation to deplete tissue CO2 stores and enable the detection of the central chemoreflex threshold. We analyzed 43 data sets, of which 10 were rejected for technical reasons. The mean threshold and ventilatory sensitivity to CO2 were 43.3 ± 3.8 mmHg and 4.60 ± 3.04 l·min−1·mmHg−1 (means ± SD), respectively. Threshold values were normally distributed, whereas sensitivity was skewed to the left. Both mean values agreed well with those in the literature. We conclude that the modified rebreathing protocol is a robust method for undergraduate investigation of the chemical control of breathing.

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (I-SC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene) glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible I-SC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin I-SC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Good vision is essential in sports, but many athletes go without correction because the solution is worse than the problem. This study shows that soft contact lenses correct more than vision.

We developed an automated, non-invasive method to detect real-time cardiac contraction in post-larval (1.1-1.7 mm length), juvenile oysters (i.e. oyster spat) via a fibre-optic trans-illumination system. The system is housed within a temperature-controlled chamber and video microscopy imaging of the heart was coupled with video edge-detection to measure cardiac contraction, inter-beat interval, and heart rate (HR). We used the method to address the hypothesis that cool acclimation (10°C vs. 22°C – Ta10 or Ta22, respectively; each n = 8) would preserve cardiac phenotype (assessed via HR variability, HRV analysis and maintained cardiac activity) during acute temperature changes. The temperature ramp (TR) protocol comprised 2°C steps (10 min/experimental temperature, Texp) from 22°C to 10°C to 22°C. HR was related to Texp in both acclimation groups. Spat became asystolic at low temperatures, particularly Ta22 spat (Ta22: 8/8 vs. Ta10: 3/8 asystolic at Texp = 10°C). The rate of HR decrease during cooling was less in Ta10 vs. Ta22 spat when asystole was included in analysis (P = 0.026). Time-domain HRV was inversely related to temperature and elevated in Ta10 vs. Ta22 spat (P < 0.001), whereas a lack of defined peaks in spectral density precluded frequency-domain analysis. Application of the method during an acute cooling challenge revealed that cool temperature acclimation preserved active cardiac contraction in oyster spat and increased time-domain HRV responses, whereas warm acclimation enhanced asystole. These physiologic changes highlight the need for studies of mechanisms, and have translational potential for oyster aquaculture practises.

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.

In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene–related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway.

Unpleasant sensory and emotional experiences originating from the respiratory system, such as dyspnea, breathlessness, unsatisfied inspiration or air hunger are not immediately associated with nociceptive neurons, although they may be detected by the same class of C-fibre afferents. Nevertheless, the conceptual parallel between pain research, nociception and dyspnea is emerging as a paradigm that has the potential to identify novel molecular mechanisms contributing to dyspnea. Recent advances in pain biology such as the cloning of the transient receptor potential vanilloid 1 (TRPV1) ion channel, exploration of the molecular mechanisms leading to TRPV1 activation and the production of a TRPV1 mutant mouse have defined its role as an integrator of noxious thermal and chemical stimuli. These observations, coupled with the biology of lung C-fibre afferents and their reflex effects, suggest that TRPV1 may contribute to the multifactorial nature of dyspnea. The present review summarizes the properties of the TRPV1 ion channel known to be expressed in lung C-fibre afferents, the possible role of TRPV1 in respiratory sensation and neurogenic inflammation, as well as the limitations and opportunities associated with current TRPV1 antagonists.

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene ( GRK5- /-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5- /-and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from β2-adrenergic receptor activation were diminished in GRK5- /-mice. These data suggest that M2muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5- /-mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

The purpose of this report is to describe the process, results, and implications in the phone recruitment of African Americans through church rosters for a survey of diet-and blood pressure-related awareness and hypertension prevalence.The survey was conducted using a non-probability sample of churches and a random selection of participants from church rosters. Recruitment strategies included frequent contact with pastors and church representatives, presentations, standard and tailored recruitment approaches, and bi-annual progress reports. Church representatives provided the rosters and assisted in arranging interviews, which were conducted at church or the participants' homes.Of 742 randomly selected, 315 (42.4%) were ineligible because of an unavailable or unreachable number, a move, discontinued church membership, death, or other reasons. Of the 344 eligible, 45.8% participated, 30.2% refused, 4.4% agreed to participate but did not, and 19.6% were incompletes (called less than three times before recruitment was terminated). Among participants, 70.4% were female, 58.2% had completed college, and the age range was 19-91 years. The survey's sample size goal of 196 was met.In this study population, over 45% who were eligible participated. Rapport established with church representatives and congregations was critical to the sampling process. Using church rosters can be a low-cost, effective recruitment tool. However, key factors to consider when recruiting African Americans in this manner include: trust, study eligibility criteria, roster accuracy, and time, and generalizability.

The influence of G-protein-coupled receptor (GPCR) signaling in lung biology has a rich history, which is also reflected by a recurring theme among Nobel laureates. For the airways and heart, the pre-GPCR observation of Wilhelm Einthoven in 1892 that electrical stimulation of the vagus nerve resulted in bradycardia (decrease in heart rate, HR;) and bronchoconstriction (airway narrowing) was a seminal observation in the journey towards cardiopulmonary GPCR therapeutics [1]. A contextual review of the 1994 Noble Prize [2] for Gilman and Rodbell concluded that ‘Elucidation of the universal role and mechanism by which G proteins transduce signals across the plasma membrane has provided a unifying view of diverse biological processes of extraordinary explanatory power.’ The 2012 Nobel prize for Lefkowitz and Kobilka for studies of GPCRs marked the cloning of a major target in asthma, the β2 adrenergic receptor (β2AR) coupled with the evolving role of intracellular second messenger amplification, signaling and structure that continues to shed new light on the therapeutics associated with GPCRs in the airways [3]. GPCR structural biology has a major impact on the understanding and design of novel therapeutic approaches that target the airways (see Kruse et al. and Rominger et al., in this issue).

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel, which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome, leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that the muscarinic receptor agonist carbachol increased the expression and function of hERG, but not ether-a-go-go or Kv1.5 channels stably expressed in human embryonic kidney cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) but not by the M2 antagonist AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one). Treatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the protein kinase C (PKC) pathway abolished the carbachol-induced enhancement of hERG channels. Our data suggest that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.

This chapter explores the development, structure, and function of the lower airways. Sections 1 and 2 describe the anatomy, morphology, and distribution of the pulmonary innervation in pre- and postnatal murine, porcine, and human lungs. Recent advances in techniques (e.g., confocal microscopy, optical projection tomography) are reviewed with respect to their use in mutant/transgenic models, which have increased our understanding of pulmonary innervation throughout the pseudoglandular, canalicular, and saccular stages of development. Significant advances in our understanding of the potential role of the neuroendocrine cell system, including neuroepithelial bodies, are examined with respect to the origins of chemo- and mechano-sensation in the lung and the impact on airway regulation. Section 3 addresses pre- and postnatal airway smooth muscle function, while Section 4 provides an overview of the role of muscarinic receptors in the lung. In summary, this chapter documents the innervation of the lung and airway smooth muscle throughout development.

Bronchoscopy is a commonly used clinical tool that provides a direct image of the bronchial lumen. However, bronchoscopy has seen little use as a quantitative measurement tool, mainly because of the wide-angle lens which distorts the image. The present authors have tested the ability of numerical algorithms and commercial software to correct for this distortion. Test objects of known size were imaged with four different bronchoscopes. Commercial image analysis software was used to measure the size of features in the images before and after applying distortion correction algorithms. The technique was then applied by measuring airway narrowing in anaesthetized pigs during vagal stimulation. Without correction, object size near the edge of the field of view is underestimated by approximately 40%. The error in measured diameter of concentric circles was dependent on the radius of the circle, increasing to 25% for circles occupying 90% of the field. Third order polynomial functions were required to correct these errors. After correction, errors were independent of object size or location in the image. Correction for lens distortion was independent of the distance between bronchoscope and object. The authors conclude that modern image processing software can correct for the distortion produced by wide-angle bronchoscope lenses.

We addressed the hypothesis that vagal C-fiber afferents and cyclooxygenase products are the mechanisms responsible for lactic acid (LA)-induced bronchoconstriction in the newborn dog. Perineural capsaicin and indomethacin were used to block conduction of vagal C fibers and production of cyclooxygenase products, respectively. Perineural capsaicin eliminated (85%) the increase in lung resistance (Rl; 45 ± 5.6%) due to capsaicin (25 μg/kg), whereas the increase in Rl (54 ± 6.9%) due to LA (0.4 mmol/kg) was only inhibited by 37 ± 4.7% ( P < 0.05). Atropine reduced LA-induced bronchoconstriction (42 ± 2.1%) by an amount similar to that obtained with perineural capsaicin. However, inhibition was significantly increased when atropine was combined with indomethacin (61 ± 2.7%; P < 0.05), implicating cyclooxygenase products in the LA-induced bronchoconstrictor response. We conclude that the mechanisms responsible for LA-induced bronchoconstriction in the newborn are 1) activation of vagal C-fibers, which, through projections to medullary respiratory centers, leads to activation of vagal cholinergic efferents; 2) production of cyclooxygenase products, which cause bronchoconstriction independent of medullary involvement; and 3) an unknown bronchoconstrictor mechanism, putatively tachykinin mediated. On the basis of our data, pharmaceutical targeting of pulmonary afferents would prevent multiple downstream mechanisms that lead to airway narrowing due to inflammatory lung disease.

Contraction and relaxation of airway smooth muscles is mediated, in part, by G protein-coupled receptors (GPCRs) and dysfunction of these receptors has been implicated in asthma. Phosphorylation of GPCRs, by G protein-coupled receptor kinase (GRK), is an important mechanism involved in the dampening of GPCR signaling. To determine whether this mechanism might play a role in airway smooth muscle physiology, we examined the airway pressure time index and heart rate (HR) responses to intravenous administration of the cholinergic agonist methacholine (MCh) in genetically altered mice lacking one copy of GRK2 (GRK2 +/−), homozygous GRK3 knockout (GRK3 −/−), and wild-type littermates. (GRK2 −/− mice die in utero.) GRK3 −/− mice demonstrated a significant enhancement in the airway response to 100 and 250 μg/kg doses of MCh compared with wild-type and GRK2 +/− mice. GRK3 −/− mice also displayed an enhanced sensitivity of the airway smooth muscle response to MCh. In addition, GRK3 −/− mice displayed an altered HR recovery from MCh-induced bradycardia. Although direct stimulation of cardiac muscarinic receptors measured as vagal stimulation-induced bradycardia was similar in GRK3 −/− and wild-type mice, the baroreflex increase in HR associated with sodium nitroprusside-induced hypotension was significantly greater in GRK3 −/− than wild-type mice. Therefore, these data demonstrate that in the mouse, GRK3 may be involved in modulating the cholinergic response of airway smooth muscle and in regulating the chronotropic component of the baroreceptor reflex.

rAAV is a widely used viral vector for the human gene therapy, but its inherently small 4.679kb genome is a significant challenge for delivering effective expression of a large gene. Our main goal is to develop rAAV vectors for delivering the cystic fibrosis transmembrane conductance regulator (CFTR) for cystic fibrosis lung disease gene therapy. To express the CFTR gene of 4.43kb coding sequence from a rAAV vector, a short promoter well functioned in airway epithelium is required. In the present study, we screened a set of 100-mer synthetic enhancer elements for their ability to augment CFTR expression from a short 83-bp synthetic promoter. With an empirical approach, screening for their effectiveness in the delivery of reporter gene expression were conducted in step-wise fashion – in plasmids, proviral vectors, and rAAV vectors. Our initial studies in monolayer (non-polarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI) in the context of AAV vector infection found that the combination of F5tg83 (183bp) is the most efficient promoter in both ALI cultures, leading to 19.6-fold and 57.5-fold increases reporter expression, respectively, over the enhancer-less counterpart. We also demonstrated that the F5tg83 promoter produced the highest level of transgene expression in the ferret airway in vivo. Since rAAV-CFTR vectors greater than 5.0kb in size are dramatically impaired with respect to vector efficacy, we utilized a shortened ferret CFTR minigene with a 159 bp deletion in the R-domain to construct a rAAV vector (AV2/2. F5tg83-fCFTRDR). This vector yielded an ≈17-fold increase in vector derived CFTR mRNA transcription and significantly improved Cl- currents in human CF ALI cultures when the vectors were administrated through basolateral infection. We also tested the F5tg83 promoter activity in the context of rAAV/HBoV1 vector, which packages an oversized rAAV genome up to 5.5kb in human bocavirus virus 1 (HBoV1) caspid and efficiently transduces HAE-ALI from apical membrane. We found that the F5tg83 promoter delivered robust and persistent expression of different reporter genes (firefly luciferase or the secreted guassia luciferase) from the HAE-ALI following apical infections of the AV2/HBoV1 chimeric vectors. The expanded package capacity of rAAV2/HBoV1 vector makes it possible to further optimize the F5tg83 promoter directed CFTR gene expression with the full-length CFTR gene. In summary, our study has identified a small enhancer/promoter combination that may have broad utility for rAAV-mediated CF gene therapy to the airway.Furthermore, the enhancer screening approach may be useful for rAAV applications in other organs that seek to deliver large transgenes that approach the packing capacity of rAAV.

In the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 micrograms, were scanned using reflectance densitometry with 127 microns step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 micrograms vs. volume were fit by a second-order regression: Volume = -0.58 x protein load2 + 16.82 x protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 micrograms and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS-PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.

We addressed the role of muscarinic receptor subtypes in neurally mediated bronchoconstriction in vivo and airway smooth muscle contraction in vitro in the newborn dog. The in vivo dosenresponse effects of iselectivei muscarinic antagonists on changes in lung resistance (RL) and heart rate (HR) in response to electrical stimulation of the vagus nerves were obtained in four groups of newborns. Each group was exposed to a different muscarinic antagonist: M1-selective pirenzepine (pir), M2-selective AF-DX 116 (11-(2-((diethylamino)methyl)-1-piperidinyl)acetyl-5,11-dihydro-6H- pyrido-(2,3-b)-(1,4)-benzodiazepine-6-one), M3-selective p-F-HHSiD (p-fluoro-hexahydro-sila-difenidol), and nonselective atropine (atr). In vitro concentrationnresponse effects of pir and AF-DX 116 were obtained for neurally induced contractions of tracheal smooth muscle, elicited by electrical field stimulation. In a separate series of experiments we measured the bronchoconstrictor response to the muscarinic agonist acetylcholine delivered by right heart injection. Muscarinic antagonists reduced RL and HR responses to vagal stimulation in a dose-dependant fashion; however, ED50 values and selectivity for airway and cardiac responses (HR/RL ED50 ratio) were significantly different between antagonists. The rank order of potencies for inhibition of the increase in RL was atr > pir, M1 > p-F-HHSiD, M3 > AF-DX 116, M2, while that for HR was atr > AF-DX 116 > pir > p-F-HHSiD. AF-DX 116 preferentially inhibited the HR response, as reflected by the lowest HR/RL ED50 ratio (p < 0.001). The remaining antagonists preferentially inhibited RL, with the highest HR/RL ED50 ratio seen for p-F-HHSiD. These data suggest that muscarinic receptor subtypes are differentiated at birth and mediate cardiac and airway responses to vagal stimulation. We did not find autoinhibitory actions of airway M2 receptors on either the in vivo bronchoconstrictor response or the in vitro contractile response to electrical field stimulation. This suggests that neonatal airway M2 receptors, but not myocardial M2 receptors, are reduced in number or weakly coupled to muscarinic signal transduction mechanisms. Direct activation of airway smooth muscle by acetylcholine caused dose-dependent increases in RL that reached a plateau at ≈200% at 100 μg, similar to values reported for vagal stimulation.

The rationale for the use of muscarinic antagonists in bronchopulmonary dysplasia (BPD) is based on the physiology and pharmacology of airway smooth muscle, the pathology of BPD, and the response of infants with BPD to bronchodilators, in vivo and in vitro studies of airway smooth muscle of newborn animals and humans indicate that vagal efferent airway innervation and/or muscarinic receptors are functional at birth, as well as early in gestation. Current concepts regarding muscarinic receptor subtypes suggest that M3 receptors mediate airway smooth muscle contraction, M2 receptors are autoinhibitory and limit vagally-mediated bronchoconstriction, and M1 receptors may play a facilitatory role in ganglionic transmission. Muscarinic receptor subtypes appear to be functionally expressed at birth but may undergo developmental regulation. Infants with BPD have an elevated pulmonary resistance that is accompanied by hypertrophy of airway smooth muscle, b2-agonists cause bronchodilation in BPD as does atropine in infants recovering from severe BPD. The synthetic congener of atropine, ipratropium bromide (IPB) causes bronchodilation in ventilator-dependent infants with BPD in a dose-dependent fashion. Nebulized IPB causes a decrease in respiratory resistance that reaches a maximum of 20% at 175 mg. The bronchodilation seen with muscarinic antagonists suggests that part of the elevated resistance associated with BPD is due to increased muscarinic tone, presumably vagal in origin. When IPB is combined with salbutamol (0.04 mg) the response is increased in magnitude and duration; reaching a slightly larger decreases in resistance (26%) that is now accompanied by an increase in compliance (20%).(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes is a common comorbidity in cystic fibrosis (CF) that worsens prognosis. The lack of an animal model for CF-related diabetes (CFRD) has made it difficult to dissect how the onset of pancreatic pathology influences the emergence of CFRD. We evaluated the structure and function of the neonatal CF endocrine pancreas using a new CFTR-knockout ferret model. Although CF kits are born with only mild exocrine pancreas disease, progressive exocrine and endocrine pancreatic loss during the first months of life was associated with pancreatic inflammation, spontaneous hyperglycemia, and glucose intolerance. Interestingly, prior to major exocrine pancreas disease, CF kits demonstrated significant abnormalities in blood glucose and insulin regulation, including diminished first-phase and accentuated peak insulin secretion in response to glucose, elevated peak glucose levels following glucose challenge, and variably elevated insulin and C-peptide levels in the nonfasted state. Although there was no difference in lobular insulin and glucagon expression between genotypes at birth, significant alterations in the frequencies of small and large islets were observed. Newborn cultured CF islets demonstrated dysregulated glucose-dependent insulin secretion in comparison to controls, suggesting intrinsic abnormalities in CF islets. These findings demonstrate that early abnormalities exist in the regulation of insulin secretion by the CF endocrine pancreas.

Although asthma is characterized as an inflammatory disease, recent reports highlight the importance of pulmonary physiology outcome measures to the clinical assessment of asthma control and risk of asthma exacerbation. Murine models of allergic inflammatory airway disease have been widely used to gain mechanistic insight into the pathogenesis of asthma; however, several aspects of murine models could benefit from improvement. This review focuses on aligning lung mechanics measures made in mice with those made in humans, with an eye toward improving the translational utility of these measures. A brief description of techniques available to measure murine lung mechanics is provided along with a methodological consideration of their utilization. How murine lung mechanics outcome measures relate to pulmonary physiology measures conducted in humans is discussed and we recommend that, like human studies, outcome measures be standardized for murine models of asthma.

Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl − permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl − transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Pulmonary neuroepithelial bodies are polymodal sensors widely distributed within the airway mucosa of mammals and other species. Neuroepithelial body cells store and most likely release serotonin and peptides as transmitters. Neuroepithelial bodies have a complex innervation that includes vagal sensory afferent fibers and dorsal root ganglion fibers. Neuroepithelial body cells respond to a number of intraluminal airway stimuli, including hypoxia, hypercarbia, and mechanical stretch. This article reviews recent findings in the cellular and molecular biology of neuroepithelial body cells and their potential role as airway sensors involved in the control of respiration, particularly during the perinatal period. Alternate hypotheses and areas of controversy regarding potential function as mechanosensory receptors involved in pulmonary reflexes are discussed.

Altered autonomic (ANS) tone in chronic respiratory disease is implicated as a factor in cardiovascular co-morbidities, yet no studies address its impact on cardiovascular function in the presence of murine allergic airway (AW) hyperresponsiveness (AHR). Since antigen (Ag)-induced AHR is used to model allergic asthma (in which ANS alterations have been reported), we performed a pilot study to assess measurement feasibility of, as well as the impact of allergic sensitization to ovalbumin (OVA) on, heart rate variability (HRV) in a murine model. Heart rate (HR), body temperature (TB) and time- and frequency-domain HRV analyses, a reflection of ANS control, were obtained in chronically instrumented mice (telemetry) before, during and for 22 h after OVA or saline aerosolization in sensitized (OVA) or Alum adjuvant control exposed animals. OVA mice diverged significantly from Alum mice with respect to change in HR during aerosol challenge (P < 0.001, two-way ANOVA; HR max change Ctrl = +80 ± 10 bpm vs. OVA = +1 ± 23 bpm, mean ± SEM), and displayed elevated HR during the subsequent dark cycle (P = 0.006). Sensitization decreased the TB during aerosol challenge (P < 0.001). Sensitized mice had decreased HRV prior to challenge (SDNN: P = 0.038; Low frequency (LF) power: P = 0.021; Low/high Frequency (HF) power: P = 0.042), and increased HRV during Ag challenge (RMSSD: P = 0.047; pNN6: P = 0.039). Sensitized mice displayed decreased HRV subsequent to OVA challenge, primarily in the dark cycle (RMSSD: P = 0.018; pNN6: P < 0.001; LF: P < 0.001; HF: P = 0.040; LF/HF: P < 0.001). We conclude that implanted telemetry technology is an effective method to assess the ANS impact of allergic sensitization. Preliminary results show mild sensitization is associated with reduced HRV and a suppression of the acute TB response to OVA challenge. This approach to assess altered ANS control in the acute OVA model may also be beneficial in chronic AHR models.

Hypoxia exposure in small mammals elicits an initial rise in ventilation followed by a reduction to levels that are often less than the normoxic value. The fall in ventilation is matched by a decrease in metabolism rate and a reduction in core body temperature (Tb). The transient receptor potential vanilloid 1 (TRPV1) ion channel has been implicated in thermoregulation (Caterina et al., Science 288:306-313, 2000) and recently shown to exert a tonic effect on Tb in human subjects (Gavva et al., Pain 136:202-210, 2008). We review herein the hypothesis that TRPV1 modulates the Tb response to hypoxia. We provide preliminary evidence that a 24 h hypoxia (FIO(2)=0.1) exposure caused an enhanced decrease in Tb in mutant TRPV1(-/-) mice compared to the TRPV1(+/+) genotype (Tb was » 1°C lower than TRPV1(+/+)). Further investigation is warranted to determine the extent of TRPV1 ion channel involvement in acute and adaptive responses to hypoxia.

Cardiac repolarization is controlled by the rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes I(Kr), whereas KCNQ1 and KCNE1 together encode I(Ks). Decreases in I(Kr) or I(Ks) cause long QT syndrome (LQTS), a cardiac disorder with a high risk of sudden death. A reduction in extracellular K(+) concentration ([K(+)](o)) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether I(Ks) compensates for the reduced I(Kr) under low K(+) conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K(+) for 6 h completely eliminated the mature hERG channel expression but had no effect on KCNQ1. When hERG and KCNQ1 were co-expressed, KCNQ1 significantly delayed 0 mM K(+)-induced hERG reduction. Also, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K(+) conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1-expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels that are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation, as well as the pathology of LQTS.