Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant.

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function in vivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel in vivo animal model. At baseline, young AnkBp.E1458G+/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G+/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G+/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G+/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G+/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in in vivo cardiac function as well as the impact of single AnkB loss-of-function variants in vivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity.

Altered Expression of Zonula occludens-1 Affects Cardiac Na+ Channels and Increases Susceptibility to Ventricular Arrhythmias.

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/-). ZO-1+/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

Inherited Variants in SCARB1 Cause Severe Early-Onset Coronary Artery Disease.

[Figure: see text].

Aberrant Expression of a Non-muscle RBFOX2 Isoform Triggers Cardiac Conduction Defects in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the CTG repeat expansion in the 3'-untranslated region of DMPK gene. Heart dysfunctions occur in ∼80% of DM1 patients and are the second leading cause of DM1-related deaths. Herein, we report that upregulation of a non-muscle splice isoform of RNA-binding protein RBFOX2 in DM1 heart tissue-due to altered splicing factor and microRNA activities-induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX240 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX240-driven splicing defects in voltage-gated sodium and potassium channels, which alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX240 isoform in DM1 cardiac pathogenesis.

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal ß-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and ß-catenin. A pharmacological activator of the WNT/ß-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and ß-catenin, and evidence for targeted activation of the WNT/ß-catenin pathway as a potential treatment for this disease.

Defining new mechanistic roles for αII spectrin in cardiac function.

Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin (SPTAN1) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered ß-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels NaV1.5 and KV4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte.

Protein Phosphatase 2A Regulates Cardiac Na+ Channels.

RATIONALE: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. OBJECTIVE: To define phosphatase pathways that regulate INa,L in vivo. METHODS AND RESULTS: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. CONCLUSIONS: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.

Novel Mechanistic Roles for Ankyrin-G in Cardiac Remodeling and Heart Failure.

Ankyrin polypeptides are intracellular proteins responsible for targeting cardiac membrane proteins. Here, the authors demonstrate that ankyrin-G plays an unexpected role in normal compensatory physiological remodeling in response to myocardial stress and aging; the authors implicate disruption of ankyrin-G in human heart failure. Mechanistically, the authors illustrate that ankyrin-G serves as a key nodal protein required for cardiac myofilament integration with the intercalated disc. Their data define novel in vivo mechanistic roles for ankyrin-G, implicate ankyrin-G as necessary for compensatory cardiac physiological remodeling under stress, and implicate disruption of ankyrin-G in the development and progression of human heart failure.

Novel Variant in the ANK2 Membrane-Binding Domain Is Associated With Ankyrin-B Syndrome and Structural Heart Disease in a First Nations Population With a High Rate of Long QT Syndrome.

BACKGROUND: Long QT syndrome confers susceptibility to ventricular arrhythmia, predisposing to syncope, seizures, and sudden death. While rare globally, long QT syndrome is ≈15× more common in First Nations of Northern British Columbia largely because of a known mutation in KCNQ1. However, 2 large multigenerational families were affected, but negative for the known mutation. METHODS AND RESULTS: Long QT syndrome panel testing was carried out in the index case of each family, and clinical information was collected. Cascade genotyping was performed. Biochemical and myocyte-based assays were performed to evaluate the identified gene variant for loss-of-function activity. Index cases in these 2 families harbored a novel ANK2 c.1937C>T variant (p.S646F). An additional 16 carriers were identified, including 2 with structural heart disease: one with cardiomyopathy resulting in sudden death and the other with congenital heart disease. For all carriers of this variant, the average QTc was 475 ms (±40). Although ankyrin-B p.S646F is appropriately folded and expressed in bacteria, the mutant polypeptide displays reduced expression in cultured H9c2 cells and aberrant localization in primary cardiomyocytes. Furthermore, myocytes expressing ankyrin-B p.S646F lack normal membrane targeting of the ankyrin-binding partner, the Na/Ca exchanger. Thus, ankyrin-B p.S646F is a loss-of-function variant. CONCLUSIONS: We identify the first disease-causing ANK2 variant localized to the membrane-binding domain resulting in reduced ankyrin-B expression and abnormal localization. Further study is warranted on the potential association of this variant with structural heart disease given the role of ANK2 in targeting and stabilization of key structural and signaling molecules in cardiac cells.

MRI Conditionality in Patients with Spinal Cord Stimulation Devices.

BACKGROUND: Because of the commonality of diagnostic magnetic resonance imaging (MRI), MRI conditional technology has increased throughout the device industry. It is often difficult to be aware of MRI specifications for each device. OBJECTIVES: We provide a review of the clinical experience with MRI and spinal cord stimulation (SCS) devices and develop a general reference of current device/MRI specifications. METHODS: We reviewed the available literature on the clinical experience with SCS devices and examined its specifications. RESULTS: We developed a user-friendly table of the specific compatibility of SCS devices in the USA and the European Union, and examined the existing literature on the clinical experience with MRI and SCS devices. We share our experience with obtaining spine MRI with MRI conditional SCS leads. CONCLUSION: By describing SCS device specifications and reviewing the literature, we provide a guide to implanting and treating physicians on obtaining MRIs in patients who have SCS devices.

Common human ANK2 variant confers in vivo arrhythmia phenotypes.

BACKGROUND: Human ANK2 (ankyrin-B) loss-of-function variants are directly linked with arrhythmia phenotypes. However, in atypical non-ion channel arrhythmia genes such as ANK2 that lack the same degree of robust structure/function and clinical data, it may be more difficult to assign variant disease risk based simply on variant location, minor allele frequency, and/or predictive structural algorithms. The human ankyrin-B p.L1622I variant found in arrhythmia probands displays significant diversity in minor allele frequency across populations. OBJECTIVE: The objective of this study was to directly test the in vivo impact of ankyrin-B p.L1622I on cardiac electrical phenotypes and arrhythmia risk using a new animal model. METHODS: We tested arrhythmia phenotypes in a new "knock-in" animal model harboring the human ankyrin-B p.L1622I variant. RESULTS: Ankyrin-B p.L1622I displays reduced posttranslational expression in vivo, resulting in reduced cardiac ankyrin-B expression and reduced association with binding-partner Na/Ca exchanger. Ankyrin-B(L1622I/L1622I) mice display changes in heart rate, atrioventricular and intraventricular conduction, and alterations in repolarization. Furthermore, ankyrin-B(L1622I/L1622I) mice display catecholamine-dependent arrhythmias. At the cellular level, ankyrin-B(L1622I/L1622I) myocytes display increased action potential duration and severe arrhythmogenic afterdepolarizations that provide a mechanistic rationale for the arrhythmias. CONCLUSION: Our findings support in vivo arrhythmogenic phenotypes of an ANK2 variant with unusual frequency in select populations. On the basis of our findings and current clinical data, we support classification of p.L1622I as a "mild" loss-of-function variant that may confer arrhythmia susceptibility in the context of secondary risk factors including environment, medication, and/or additional genetic variation.

SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia.

Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins.

Evaluation of Diffuse Reflection Infrared Spectrometry for End-of-Shift Measurement of α-quartz in Coal Dust Samples.

The inhalation of toxic substances is a major threat to the health of miners, and dust containing respirable crystalline silica (α-quartz) is of particular concern, due to the recent rise in cases of coal workers' pneumoconiosis and silicosis in some U.S. mining regions. Currently, there is no field-portable instrument that can measure airborne α-quartz and give miners timely feedback on their exposure. The U.S. National Institute for Occupational Safety and Health (NIOSH) is therefore conducting studies to investigate technologies capable of end-of-shift or real-time measurement of airborne quartz. The present study focuses on the potential application of Fourier transform infrared (FT-IR) spectrometry conducted in the diffuse reflection (DR) mode as a technique for measuring α-quartz in respirable mine dust. A DR accessory was used to analyze lab-generated respirable samples of Min-U-Sil 5 (which contains more than 90% α-quartz) and coal dust, at mass loadings in the ranges of 100-600 µg and 600-5300 µg, respectively. The dust samples were deposited onto three different types of filters, borosilicate fiberglass, nylon, and polyvinyl chloride (PVC). The reflectance, R, was calculated by the ratio of a blank filter and a filter with deposited mine dust. Results suggest that for coal and pure quartz dusts deposited on 37 mm PVC filters, measurements of -log R correlate linearly with known amounts of quartz on filters, with R(2) values of approximately 0.99 and 0.94, respectively, for samples loaded up to ∼4000 µg. Additional tests were conducted to measure quartz in coal dusts deposited onto the borosilicate fiberglass and nylon filter media used in the NIOSH-developed Personal Dust Monitor (PDM). The nylon filter was shown to be amenable to DR analysis, but quantification of quartz is more accurate when the filter is "free," as opposed to being mounted in the PDM filter holder. The borosilicate fiberglass filters were shown to produce excessive interference, making quartz quantification impossible. It was concluded that, while the DR/FT-IR method is potentially useful for on-filter measurement of quartz in dust samples, the use of PVC filters produced the most accurate results.

Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart.

Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate "global" PP2A function from the activities of multiple "local" holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.

Deposition Uniformity of Coal Dust on Filters and Its Effect on the Accuracy of FTIR Analyses for Silica.

Miners are exposed to silica-bearing dust which can lead to silicosis, a potentially fatal lung disease. Currently, airborne silica is measured by collecting filter samples and sending them to a laboratory for analysis. Since this may take weeks, a field method is needed to inform decisions aimed at reducing exposures. This study investigates a field-portable Fourier transform infrared (FTIR) method for end-of-shift (EOS) measurement of silica on filter samples. Since the method entails localized analyses, spatial uniformity of dust deposition can affect accuracy and repeatability. The study, therefore, assesses the influence of radial deposition uniformity on the accuracy of the method. Using laboratory-generated Minusil and coal dusts and three different types of sampling systems, multiple sets of filter samples were prepared. All samples were collected in pairs to create parallel sets for training and validation. Silica was measured by FTIR at nine locations across the face of each filter and the data analyzed using a multiple regression analysis technique that compared various models for predicting silica mass on the filters using different numbers of "analysis shots." It was shown that deposition uniformity is independent of particle type (kaolin vs. silica), which suggests the role of aerodynamic separation is negligible. Results also reflected the correlation between the location and number of shots versus the predictive accuracy of the models. The coefficient of variation (CV) for the models when predicting mass of validation samples was 4%-51% depending on the number of points analyzed and the type of sampler used, which affected the uniformity of radial deposition on the filters. It was shown that using a single shot at the center of the filter yielded predictivity adequate for a field method, (93% return, CV approximately 15%) for samples collected with 3-piece cassettes.

Evaluating portable infrared spectrometers for measuring the silica content of coal dust.

Miners face a variety of respiratory hazards while on the job, including exposure to silica dust which can lead to silicosis, a potentially fatal lung disease. Currently, field-collected filter samples of silica are sent for laboratory analysis and the results take weeks to be reported. Since the mining workplace is constantly moving into new and often different geological strata with changing silica levels, more timely data on silica levels in mining workplaces could help reduce exposures. Improvements in infrared (IR) spectroscopy open the prospect for end-of-shift silica measurements at mine sites. Two field-portable IR spectrometers were evaluated for their ability to quantify the mass of silica on filter samples loaded with known amounts of either silica or silica-bearing coal dust (silica content ranging from 10-200 µg/filter). Analyses included a scheme to correct for the presence of kaolin, which is a confounder for IR analysis of silica. IR measurements of the samples were compared to parallel measurements derived using the laboratory-based U.S. Mine Safety and Health Administration P7 analytical method. Linear correlations between Fourier transform infrared (FTIR) and P7 data yielded slopes in the range of 0.90-0.97 with minimal bias. Data from a variable filter array spectrometer did not correlate as well, mainly due to poor wavelength resolution compared to the FTIR instrument. This work has shown that FTIR spectrometry has the potential to reasonably estimate the silica exposure of miners if employed in an end-of-shift method.