The Nasal Solitary Chemosensory Cell Signaling Pathway Triggers Mouse Avoidance Behavior to Inhaled Nebulized Irritants.

The nasal epithelium houses a population of solitary chemosensory cells (SCCs). SCCs express bitter taste receptors and taste transduction signaling components and are innervated by peptidergic trigeminal polymodal nociceptive nerve fibers. Thus, nasal SCCs respond to bitter compounds, including bacterial metabolites, and these reactions evoke protective respiratory reflexes and innate immune and inflammatory responses. We tested whether SCCs are implicated in aversive behavior to specific inhaled nebulized irritants using a custom-built dual-chamber forced-choice device. The behavior of mice was recorded and analyzed for the time spent in each chamber. Wild-type (WT) mice exhibited an aversion to 10 mm denatonium benzoate (Den) or cycloheximide and spent more time in the control (saline) chamber. The SCC-pathway knock-out (KO) mice did not exhibit such an aversion response. The bitter avoidance behavior of WT mice was positively correlated with the concentration increase of Den and the number of exposures. Bitter-ageusic P2X2/3 double KO mice similarly showed an avoidance response to nebulized Den, excluding the taste system's involvement and pointing to an SCC-mediated major contributor to the aversive response. Interestingly, SCC-pathway KO mice showed an attraction to higher Den concentrations; however, chemical ablation of the olfactory epithelium eliminated this attraction attributed to the smell of Den. These results demonstrate that activation of SCCs leads to a rapid aversive response to certain classes of irritants with olfaction, but not gustation, contributing to the avoidance behavior during subsequent irritant exposures. This SCC-mediated avoidance behavior represents an important defense mechanism against the inhalation of noxious chemicals.

Phenotypic Expression, Natural History, and Risk Stratification of Cardiomyopathy Caused by Filamin C Truncating Variants.

BACKGROUND: Filamin C truncating variants (FLNCtv) cause a form of arrhythmogenic cardiomyopathy: the mode of presentation, natural history, and risk stratification of FLNCtv remain incompletely explored. We aimed to develop a risk profile for refractory heart failure and life-threatening arrhythmias in a multicenter cohort of FLNCtv carriers. METHODS: FLNCtv carriers were identified from 10 tertiary care centers for genetic cardiomyopathies. Clinical and outcome data were compiled. Composite outcomes were all-cause mortality/heart transplantation/left ventricle assist device (D/HT/LVAD), nonarrhythmic death/HT/LVAD, and sudden cardiac death/major ventricular arrhythmias. Previously established cohorts of 46 patients with LMNA and 60 with DSP-related arrhythmogenic cardiomyopathies were used for prognostic comparison. RESULTS: Eighty-five patients carrying FLNCtv were included (42±15 years, 53% men, 45% probands). Phenotypes were heterogeneous at presentation: 49% dilated cardiomyopathy, 25% arrhythmogenic left dominant cardiomyopathy, 3% arrhythmogenic right ventricular cardiomyopathy. Left ventricular ejection fraction was <50% in 64% of carriers and 34% had right ventricular fractional area changes (RVFAC=(right ventricular end-diastolic area - right ventricular end-systolic area)/right ventricular end-diastolic area) <35%. During follow-up (median time 61 months), 19 (22%) carriers experienced D/HT/LVAD, 13 (15%) experienced nonarrhythmic death/HT/LVAD, and 23 (27%) experienced sudden cardiac death/major ventricular arrhythmias. The sudden cardiac death/major ventricular arrhythmias incidence of FLNCtv carriers did not significantly differ from LMNA carriers and DSP carriers. In FLNCtv carriers, left ventricular ejection fraction was associated with the risk of D/HT/LVAD and nonarrhythmic death/HT/LVAD. CONCLUSIONS: Among patients referred to tertiary referral centers, FLNCtv arrhythmogenic cardiomyopathy is phenotypically heterogeneous and characterized by a high risk of life-threatening arrhythmias, which does not seem to be associated with the severity of left ventricular dysfunction.

Echocardiography-fluoroscopy fusion imaging: The essential features used in congenital and structural heart disease interventional guidance.

Increased catheter-based interventions in congenital and structural heart disease require imaging modalities to be oriented in the same visual perspective. The use of echocardiography-fluoroscopy fusion (EFF) imaging has been developed for better characterization of complex anatomy and to facilitate key steps in interventional procedures. This review will detail the technology behind EFF, the differences between the two ultrasound fusion systems, and essential features of EFF imaging in congenital and structural heart disease interventions.

Three-dimensional reconstructions of mouse circumvallate taste buds using serial blockface scanning electron microscopy: I. Cell types and the apical region of the taste bud.

Taste buds comprise four types of taste cells: three mature, elongate types, Types I-III; and basally situated, immature postmitotic type, Type IV cells. We employed serial blockface scanning electron microscopy to delineate the characteristics and interrelationships of the taste cells in the circumvallate papillae of adult mice. Type I cells have an indented, elongate nucleus with invaginations, folded plasma membrane, and multiple apical microvilli in the taste pore. Type I microvilli may be either restricted to the bottom of the pore or extend outward reaching midway up into the taste pore. Type II cells (aka receptor cells) possess a large round or oval nucleus, a single apical microvillus extending through the taste pore, and specialized "atypical" mitochondria at functional points of contact with nerve fibers. Type III cells (aka "synaptic cells") are elongate with an indented nucleus, possess a single, apical microvillus extending through the taste pore, and are characterized by a small accumulation of synaptic vesicles at points of contact with nerve fibers. About one-quarter of Type III cells also exhibit an atypical mitochondrion near the presynaptic vesicle clusters at the synapse. Type IV cells (nonproliferative "basal cells") have a nucleus in the lower quarter of the taste bud and a foot process extending to the basement membrane often contacting nerve processes along the way. In murine circumvallate taste buds, Type I cells represent just over 50% of the population, whereas Types II, III, and IV (basal cells) represent 19, 15, and 14%, respectively.

Congenital and Structural Heart Disease Interventions Using Echocardiography-Fluoroscopy Fusion Imaging.

With the increasing frequency of catheter-based interventions in congenital heart disease and structural heart disease, the use of fusion imaging has become a major enhancement for understanding complex anatomy and facilitating key steps in interventional procedures. Because transesophageal echocardiography and fluoroscopy are displayed in different visual perspectives, the interventional cardiologist must mentally reregister the images from the two modalities during the procedure. Echocardiography-fluoroscopy fusion (EFF) imaging displays the x-ray and ultrasound overlay images in the same visual perspective. This new technology allows for enhanced team communication, improved visual guidance, and more efficient navigation. The purpose of this review is to describe the EFF imaging technology, current uses of EFF imaging in congenital and structural heart disease, and future directions that will enhance this unique imaging technology to guide interventional procedures.

Differential Expression of Mucins in Murine Olfactory Versus Respiratory Epithelium.

Mucins are a key component of the surface mucus overlying airway epithelium. Given the different functions of the olfactory and respiratory epithelia, we hypothesized that mucins would be differentially expressed between these 2 areas. Secondarily, we evaluated for potential changes in mucin expression with radiation exposure, given the clinical observations of nasal dryness, altered mucus rheology, and smell loss in radiated patients. Immunofluorescence staining was performed to evaluate expression of mucins 1, 2, 5AC, and 5B in nasal respiratory and olfactory epithelia of control mice and 1 week after exposure to 8 Gy of radiation. Mucins 1, 5AC, and 5B exhibited differential expression patterns between olfactory and respiratory epithelium (RE) while mucin 2 showed no difference. In the olfactory epithelium (OE), mucin 1 was located in a lattice-like pattern around gaps corresponding to dendritic knobs of olfactory sensory neurons, whereas in RE it was intermittently expressed by surface goblet cells. Mucin 5AC was expressed by subepithelial glands in both epithelial types but to a higher degree in the OE. Mucin 5B was expressed by submucosal glands in OE and by surface epithelial cells in RE. At 1-week after exposure to single-dose 8 Gy of radiation, no qualitative effects were seen on mucin expression. Our findings demonstrate that murine OE and RE express mucins differently, and characteristic patterns of mucins 1, 5AC, and 5B can be used to define the underlying epithelium. Radiation (8 Gy) does not appear to affect mucin expression at 1 week. LEVEL OF EVIDENCE: N/A (Basic Science Research).IACUC-approved study [Protocol 200065].

The acute effects of edge-to-edge percutaneous mitral valve repair on the shape and size of the mitral annulus and its relation to mitral regurgitation.

BACKGROUND: The effects of edge-to-edge percutaneous mitral valve repair on the shape and size of the mitral annulus and its relation to mitral regurgitation (MR) have not been well characterized. We evaluated acute changes in mitral annular shape and dimensions, and their effect on MR severity, in patients with functional and degenerative MR following MitraClip® . METHODS: Patients that underwent MitraClip® between January 2013 and May 2016 at our institution were retrospectively reviewed. EXCLUSIONS: inadequate images, prior mitral valve repair, and rapid atrial fibrillation. Intra-procedure TEE 3D images acquired prior to and after implantation of MitraClip® were analyzed using software to model the mitral valve apparatus. RESULTS: Of seventy-eight patients that underwent MitraClip® procedure, 60 were eligible. Mean age was 78.3 ± 11 years. Severe MR (4+) was present in 37 patients, moderately/severe MR (3+) in 23. All patients achieved MR reduction to ≤2. 3D annular circumference, bicommissural diameter, and anteroposterior diameter had a significant size reduction after MitraClip® . None of the mitral annular measures had significantly different mean change between the large and small MR change groups at the 0.05 significance level. CONCLUSIONS: In patients with functional or degenerative MR, the MitraClip® significantly affect mitral annular dimensions; however, these changes do not correlate with the immediate MR reduction.

Filamin C Truncation Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell-Cell Adhesion Structures.

OBJECTIVES: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. BACKGROUND: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. METHODS: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. RESULTS: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. CONCLUSIONS: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Endogenous Sex Steroids Dampen Neuroinflammation and Improve Outcome of Traumatic Brain Injury in Mice.

The role of biological sex in short-term and long-term outcome after traumatic brain injury (TBI) remains controversial. The observation that exogenous female sex steroids (progesterone and estrogen) reduce brain injury coupled with a small number of clinical studies showing smaller injury in women suggest that sex steroids may play a role in outcome from TBI. We used the controlled cortical impact (CCI) model of TBI in mice to test the hypothesis that after CCI, female mice would demonstrate less injury than male mice, related to the protective role of endogenous steroids. Indeed, adult females exhibit histological protection (3.7 ± 0.5 mm3) compared to adult male mice (6.8 ± 0.6 mm3), and females that lacked sex steroids (ovex) showed increased injury compared to intact females. Consistent with histology, sensorimotor deficits measured as reduced contralateral limb use were most pronounced in male mice (31.9 ± 6.9% reduced limb use) compared to a 12.7 ± 3.8% reduction in female mice. Ovex mice exhibited behavioral deficits similar to males (31.5 ± 3.9% reduced limb use). Ovex females demonstrated increased microglial activation relative to intact females in both the peri-injury cortex and the reticular thalamic nucleus. Ovex females also demonstrated increased astrogliosis in comparison to both females and males in the peri-injury cortex. These data indicate that female sex steroids reduce brain sensitivity to TBI and that reduced acute neuroinflammation may contribute to the relative protection observed in females.

ß-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional ß-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest ß-catenin plays a greater role in renewal of anterior versus posterior taste buds.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors.

Tonotopic alterations in inhibitory input to the medial nucleus of the trapezoid body in a mouse model of Fragile X syndrome.

Hyperexcitability and the imbalance of excitation/inhibition are one of the leading causes of abnormal sensory processing in Fragile X syndrome (FXS). The precise timing and distribution of excitation and inhibition is crucial for auditory processing at the level of the auditory brainstem, which is responsible for sound localization ability. Sound localization is one of the sensory abilities disrupted by loss of the Fragile X Mental Retardation 1 (Fmr1) gene. Using triple immunofluorescence staining we tested whether there were alterations in the number and size of presynaptic structures for the three primary neurotransmitters (glutamate, glycine, and GABA) in the auditory brainstem of Fmr1 knockout mice. We found decreases in either glycinergic or GABAergic inhibition to the medial nucleus of the trapezoid body (MNTB) specific to the tonotopic location within the nucleus. MNTB is one of the primary inhibitory nuclei in the auditory brainstem and participates in the sound localization process with fast and well-timed inhibition. Thus, a decrease in inhibitory afferents to MNTB neurons should lead to greater inhibitory output to the projections from this nucleus. In contrast, we did not see any other significant alterations in balance of excitation/inhibition in any of the other auditory brainstem nuclei measured, suggesting that the alterations observed in the MNTB are both nucleus and frequency specific. We furthermore show that glycinergic inhibition may be an important contributor to imbalances in excitation and inhibition in FXS and that the auditory brainstem is a useful circuit for testing these imbalances.

Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope.

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

Incidence of Atrial Fibrillation following Alcohol Septal Ablation for Hypertrophic Cardiomyopathy.

BACKGROUND: Patients with hypertrophic cardiomyopathy (HCM) are at a fourfold to sixfold higher risk of developing atrial fibrillation (AF) compared to the general population, though incidence rates among patients undergoing alcohol septal ablation (ASA) are not well characterized. The purpose of this study was to evaluate atrial fibrillation incidence following ASA. METHODS: We studied 132 consecutive HCM patients without comorbid AF that underwent 154 ASA procedures. The incidence of AF in follow-up was assessed through chart abstraction including electrocardiography. Survival free of AF was estimated using Kaplan-Meier methodology. RESULTS: Over a mean follow-up of 3.6 ± 2.7 years (maximum 11.3 years), 10 (7.6%) patients developed new-onset AF. Of those who developed AF, both resting and provoked left ventricular outflow tract (LVOT) gradients had improved significantly (difference -79.78 mm Hg, P ≤ 0.005). Severity of mitral regurgitation improved in 7 (70%) patients. Survival free of AF was estimated to be 99.1%, 93.7%, and 91.7% at 1, 3, and 5 years. CONCLUSIONS: Despite relieving LVOT obstruction and improving mitral regurgitation severity via ASA, new-onset AF remained a common complication of hypertrophic cardiomyopathy.

Arrhythmogenic Phenotype in Dilated Cardiomyopathy: Natural History and Predictors of Life-Threatening Arrhythmias.

BACKGROUND: Patients with dilated cardiomyopathy (DCM) may present with ventricular arrhythmias early in the disease course, unrelated to the severity of left ventricular dysfunction. These patients may be classified as having an arrhythmogenic DCM (AR-DCM). We investigated the phenotype and natural history of patients with AR-DCM. METHODS AND RESULTS: Two hundred eighty-five patients with a recent diagnosis of DCM (median duration of the disease 1 month, range 0 to 7 months) and who had Holter monitoring at baseline were comprehensively evaluated and followed for 107 months (range 29 to 170 months). AR-DCM was defined by the presence of ≥1 of the following: unexplained syncope, rapid nonsustained ventricular tachycardia (≥5 beats, ≥150 bpm), ≥1000 premature ventricular contractions/24 hours, and ≥50 ventricular couplets/24 hours, in the absence of overt heart failure. The primary end points were sudden cardiac death (SCD), sustained ventricular tachycardia (SVT), or ventricular fibrillation (VF). The secondary end points were death from congestive heart failure or heart transplantation. Of the 285 patients, 109 (38.2%) met criteria for AR-DCM phenotype. AR-DCM subjects had a higher incidence of SCD/SVT/VF compared with non-AR-DCM patients (30.3% vs 17.6%, P=0.022), with no difference in the secondary end points. A family history of SCD/SVT/VF and the AR-DCM phenotype were statistically significant and cumulative predictors of SCD/SVT/VF. CONCLUSIONS: One-third of DCM patients may have an arrhythmogenic phenotype associated with increased risk of arrhythmias during follow-up. A family history of ventricular arrhythmias in DCM predicts a poor prognosis and increased risk of SCD.

Cdon promotes neural crest migration by regulating N-cadherin localization.

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning.

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residue at this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant.