Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

Comparison of the prevalence of Toxocara egg shedding by pet cats and dogs in the U.S.A., 2011-2014.

County based prevalence maps were produced using the annual data from the years 2011 through 2014 of the prevalence of Toxocara egg shedding in more than 500,000 pet cat and 2.5 million pet dog fecal samples submitted to centralized testing laboratories. Fecal examination results were obtained at these centers through examination of the samples by centrifugal floatation and microscopy, and were previously reported as annual data on the Companion Animal Parasite Council (CAPC) website. The county maps were generated with mapping and spatial analysis software, and statistical comparisons made using two data analysis packages. The national prevalence of eggs in the feces of pet cats and dogs during this four-year period was 4.6-5.1% and 1.8-2.0%, respectively. Thus, Toxocara cati and Toxocara canis remain considerably prevalent and geographically distributed in our pet populations in spite of the availability of effective and safe treatments. Furthermore, pet cats are found to be shedding Toxocara eggs more commonly than pet dogs. This trend was especially evident in the Northeastern, Midwestern and Southern regions of the U.S.A. when prevalence rates of fecal shedding for cats and dogs in different regions were compared using general linear modeling. In spite of this, fecal endoparasite examination tests for cats comprise only 16-17.6% of the total number of samples annually requested in this data set. This high prevalence of egg shedding poses a significant public health risk, as emphasized by the recent naming of toxocariasis to the list of the top five neglected parasitic infections of Americans. Therefore, it is essential for veterinarians to continue to stress to owners the importance of routine anthelmintic treatment for pets of all ages, and to place greater emphasis on the importance of testing and treatment of parasitic infections in cats.

Ranolazine effectively suppresses atrial fibrillation in the setting of heart failure.

BACKGROUND: There is a critical need for safer and more effective pharmacological management of atrial fibrillation (AF) in the setting of heart failure (HF). METHODS AND RESULTS: This study investigates the electrophysiological, antiarrhythmic, and proarrhythmic effects of a clinically relevant concentration of ranolazine (5 µmol/L) in coronary-perfused right atrial and left ventricular preparations isolated from the hearts of HF dogs. HF was induced by ventricular tachypacing (2-6 weeks at 200-240 beats per minute; n=17). Transmembrane action potentials were recorded using standard microelectrode techniques. In atria, ranolazine slightly prolonged action potential duration but significantly depressed sodium channel current-dependent parameters causing a reduction of maximum rate of rise of the action potential upstroke, a prolongation of the effective refractory period secondary to the development of postrepolarization refractoriness, and an increase in diastolic threshold of excitation and atrial conduction time. Ranolazine did not significantly alter these parameters or promote arrhythmias in the ventricles. Ranolazine produced greater inhibition of peak sodium channel current in atrial cells isolated from HF versus normal dogs. A single premature beat reproducibly induced self-terminating AF in 10 of 17 atria. Ranolazine (5 µmol/L) suppressed induction of AF in 7 of 10 (70%) atria. In the remaining 3 atria, ranolazine reduced frequency and duration of AF. CONCLUSIONS: Our results demonstrate more potent suppression of AF by ranolazine in the setting of HF than previously demonstrated in nonfailing hearts and absence of ventricular proarrhythmia. The data suggest that ranolazine may be of benefit as an alternative to amiodarone and dofetilide in the management of AF in patients with HF.

A temporal window of vulnerability for development of atrial fibrillation with advancing heart failure.

AIMS: Heart failure (HF) is associated with development of AF and life-threatening ventricular tachycardia and fibrillation (VT/VF). Vulnerability to development of AF and VT/VF at different stages of HF and the underlying pathophysiological mechanisms are poorly defined. The present study was designed to determine the time-course of development of electrical and structural remodelling of the atria and ventricles, and their contribution to induction of AF and VT/VF in a canine model of HF. METHODS AND RESULTS: Dogs were ventricular tachypaced (VTP) for 2-3 weeks or 5-6 weeks ('early' and 'late' HF, respectively). Electrophysiological studies were performed in isolated atrial and ventricular preparations and correlated with cardiac dimensions and haemodynamic parameters recorded in vivo. Vulnerability to programmed electrical stimulation-induced AF was greater in early vs. late stages of HF (78% vs. 38%). In contrast, VT/VF was inducible in late but not in early stages of HF (38% vs. 0%). The temporal distinction in atrial and ventricular arrhythmia susceptibility was associated with a much more rapid development of electrical and structural remodelling in atria. Vulnerability to AF developed following moderate electro-structural remodelling and waned with further progression to severe remodelling, which averted rapid atrial activation. CONCLUSIONS: A temporal window of vulnerability for AF appears relatively early during development of VTP-induced HF in dogs, whereas VT/VF vulnerability is observed at more advanced stages of HF. These findings, if confirmed in humans, may have clinical implications with regard to prognosis and approach to therapy of patients with HF.

Cardiomyocyte calcium cycling in a naturally occurring German shepherd dog model of inherited ventricular arrhythmia and sudden cardiac death.

OBJECTIVE: To further characterize arrhythmic mechanisms in German shepherd dogs (GSDs) affected with inherited ventricular arrhythmias by evaluating intracellular calcium cycling and expression of calcium handling genes. ANIMALS: Twenty five GSDs, 9 backcross dogs, and 6 normal mongrel dogs (controls) were studied. The GSDs and backcross dogs were from a research colony of inherited ventricular arrhythmias. The control research dogs were purchased. METHODS: Action potentials (APs) and pseudo-electrocardiograms (ECG) were recorded from left ventricular (LV) wedge preparations of GSDs and normal dogs. Midmyocardial (Mid) LV cells from GSDs and normal mongrels were isolated by enzymatic digestion. Cells were either field stimulated or voltage clamped and calcium transients were measured by confocal microscopy using the indicator Fluo-3AM. Expression of calcium handling genes was measured by quantitative RT-PCR. RESULTS: Mean calcium transient decay (tau) was not different between affected GSDs and control dogs, but striking cell-to-cell variability for tau was observed within affected GSDs and between affected GSDs and controls (P < 0.0001 each); within-dog variability accounted for 75% of total variability. Calcium sparks and afterdepolarizations occurred in GSD but not control cells. ATP2A2/SERCA2a expression was significantly reduced (P = 0.0063) in affected GSDs and inversely correlated (P = 0.0006) with severity of ventricular arrhythmias. CONCLUSIONS: German shepherd dogs with inherited ventricular arrhythmias have electrophysiologic abnormalities in calcium cycling associated with reduced ATP2A2/SERCA2a expression. These animals provide a unique opportunity to study calcium remodeling at the genetic and molecular level in familial ventricular arrhythmias.

Physiological consequences of transient outward K+ current activation during heart failure in the canine left ventricle.

BACKGROUND: Remodeling of ion channel expression is well established in heart failure (HF). We determined the extent to which I(to) is reduced in tachypacing-induced HF and assessed the ability of an I(to) activator (NS5806) to recover this current. METHOD AND RESULTS: Whole-cell patch clamp was used to record I(to) in epicardial (Epi) ventricular myocytes. Epi- and endocardial action potentials were recorded from left ventricular wedge preparations. Right ventricular tachypacing-induced heart failure reduced I(to) density in Epi myocytes (Control=22.1±1.9pA/pF vs 16.1±1.4 after 2weeks and 10.7±1.4pA/pF after 5 weeks, +50mV). Current decay as well as recovery of I(to) from inactivation progressively slowed with the development of heart failure. Reduction of I(to) density was paralleled by a reduction in phase 1 magnitude, epicardial action potential notch and J wave amplitude recorded from coronary-perfused left ventricular wedge preparations. NS5806 increased I(to) (at +50mV) from 16.1±1.4 to 23.9±2.1pA/pF (p<0.05) at 2weeks and from 10.7±1.4 to 14.4±1.9pA/pF (p<0.05) in 5 weeks tachypaced dogs. NS5806 increased both fast and slow phases of I(to) recovery in 2 and 5-week HF cells and restored the action potential notch and J wave in wedge preparations from HF dogs. CONCLUSIONS: The I(to) agonist NS5806 increases the rate of recovery and density of I(to), thus reversing the HF-induced reduction in these parameters. In wedge preparations from HF dogs, NS5806 restored the spike-and-dome morphology of the Epi action potential providing proof of principal that some aspects of electrical remodelling during HF can be pharmacologically reversed.

Interactions between TGFß1 and cyclic strain in modulation of myofibroblastic differentiation of canine mitral valve interstitial cells in 3D culture.

OBJECTIVES: The mechanisms of myxomatous valve degeneration (MVD) are poorly understood. Transforming growth factor-beta1 (TGFß1) induces myofibroblastic activation in mitral valve interstitial cells (MVIC) in static 2D culture, but the roles of more physiological 3D matrix and cyclic mechanical strain are unclear. In this paper, we test the hypothesis that cyclic strain and TGFß1 interact to modify MVIC phenotype in 3D culture. ANIMALS, MATERIALS AND METHODS: MVIC were isolated from dogs with and without MVD and cultured for 7 days in type 1 collagen hydrogels with and without 5 ng/ml TGFß1. MVIC with MVD were subjected to 15% cyclic equibiaxial strain with static cultures serving as controls. Myofibroblastic phenotype was assessed via 3D matrix compaction, cell morphology, and expression of myofibroblastic (TGFß3, alpha-smooth muscle actin - αSMA) and fibroblastic (vimentin) markers. RESULTS: Exogenous TGFß1 increased matrix compaction by canine MVIC with and without MVD, which correlated with increased cell spreading and elongation. TGFß1 increased αSMA and TGFß3 gene expression, but not vimentin expression, in 15% cyclically stretched MVIC. Conversely, 15% cyclic strain significantly increased vimentin protein and gene expression, but not αSMA or TGFß3. 15% cyclic strain however was unable to counteract the effects of TGFß1 stimulation on MVIC. CONCLUSIONS: These results suggest that TGFß1 induces myofibroblastic differentiation (MVD phenotype) of canine MVIC in 3D culture, while 15% cyclic strain promotes a more fibroblastic phenotype. Mechanical and biochemical interactions likely regulate MVIC phenotype with dose dependence. 3D culture systems can systematically investigate these phenomena and identify their underlying molecular mechanisms.

Use of computed tomography and silicon endocasts to identify pulmonary veins with echocardiography.

The pulmonary veins were identified from the silicone endocast heart models of 19 dogs. Although variation in the number of the more peripheral veins on each specimen existed, all of the casts had a consistency with regards to the most proximal coalescence of the pulmonary veins as they entered the body of the left atrium. That is, the confluence of the veins formed three ostia at the atrial entry point that consisted of 1) right cranial and right middle pulmonary lobe veins; 2) right caudal, accessory, and left caudal pulmonary lobe veins; and 3) both the left cranial and left caudal pulmonary lobe veins of the left cranial lung lobe. The location of these structures identified by the 3-dimensional endocasts were then used to assist in the identification of the pulmonary veins using computed tomography of 2 dogs. Slices were made that approximated those commonly performed during echocardiographic examination. Understanding which pulmonary veins are seen by echocardiography in the different imaging planes will permit prospective evaluations of pulmonary vein size and abnormal flow patterns.

The mechanobiology of mitral valve function, degeneration, and repair.

In degenerative valve disease, the highly organized mitral valve leaflet matrix stratification is progressively destroyed and replaced with proteoglycan rich, mechanically inadequate tissue. This is driven by the actions of originally quiescent valve interstitial cells that become active contractile and migratory myofibroblasts. While treatment for myxomatous mitral valve disease in humans ranges from repair to total replacement, therapies in dogs focus on treating the consequences of the resulting mitral regurgitation. The fundamental gap in our understanding is how the resident valve cells respond to altered mechanical signals to drive tissue remodeling. Despite the pathological similarities and high clinical occurrence, surprisingly little mechanistic insight has been gleaned from the dog. This review presents what is known about mitral valve mechanobiology from clinical, in vivo, and in vitro data. There are a number of experimental strategies already available to pursue this significant opportunity, but success requires the collaboration between veterinary clinicians, scientists, and engineers.

Low-energy control of electrical turbulence in the heart.

Controlling the complex spatio-temporal dynamics underlying life-threatening cardiac arrhythmias such as fibrillation is extremely difficult, because of the nonlinear interaction of excitation waves in a heterogeneous anatomical substrate. In the absence of a better strategy, strong, globally resetting electrical shocks remain the only reliable treatment for cardiac fibrillation. Here we establish the relationship between the response of the tissue to an electric field and the spatial distribution of heterogeneities in the scale-free coronary vascular structure. We show that in response to a pulsed electric field, E, these heterogeneities serve as nucleation sites for the generation of intramural electrical waves with a source density ρ(E) and a characteristic time, τ, for tissue depolarization that obeys the power law τ ∝ E(α). These intramural wave sources permit targeting of electrical turbulence near the cores of the vortices of electrical activity that drive complex fibrillatory dynamics. We show in vitro that simultaneous and direct access to multiple vortex cores results in rapid synchronization of cardiac tissue and therefore, efficient termination of fibrillation. Using this control strategy, we demonstrate low-energy termination of fibrillation in vivo. Our results give new insights into the mechanisms and dynamics underlying the control of spatio-temporal chaos in heterogeneous excitable media and provide new research perspectives towards alternative, life-saving low-energy defibrillation techniques.

Ultrastructural changes in cardiac myocytes from Boxer dogs with arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVES: We sought to quantify the number and length of desmosomes, gap junctions, and adherens junctions in arrhythmogenic right ventricular cardiomyopathy (ARVC) and non-ARVC dogs, and to determine if ultrastructural changes existed. ANIMALS: Hearts from 8 Boxer dogs afflicted with histopathologically confirmed ARVC and 6 dogs without ARVC were studied. METHODS: Quantitative transmission electron microscopy (TEM) and Western blot semi-quantification of α-actinin were used to study the intercalated disc and sarcomere of the right and left ventricles. RESULTS: When ARVC dogs were compared to non-ARVC dogs reductions in the number of desmosomes (P = 0.04), adherens junctions (P = 0.04) and gap junctions (P = 0.02) were found. The number of gap junctions (P = 0.04) and adherens junctions (P = 0.04) also were reduced in the left ventricle, while the number of desmosomes was not (P = 0.88). A decrease in the length of desmosomal complexes within LV samples (P = 0.04) was found. These findings suggested disruption of proteins providing attachment of the cytoskeleton to the intercalated disc. Immunoblotting did not demonstrate a quantitative reduction in the amount of α-actinin in ARVC afflicted samples. All Boxers with ARVC demonstrated the presence of electron dense material originating from the Z band and extending into the sarcomere, apparently at the expense of the cytoskeletal structure. CONCLUSIONS: These results emphasize the importance of structural integrity of the intercalated disc in the pathogenesis of ARVC. In addition, observed abnormalities in sarcomeric structure suggest a novel link between ARVC and the actin-myosin contractile apparatus.

Termination of equine atrial fibrillation by quinidine: an optical mapping study.

OBJECTIVE: To perform the first optical mapping studies of equine atrium to assess the spatiotemporal dynamics of atrial fibrillation (AF) and of its termination by quinidine. ANIMALS: Intact, perfused atrial preparations obtained from four horses with normal cardiovascular examinations. MATERIALS AND METHODS: AF was induced by a rapid pacing protocol with or without acetylcholine perfusion, and optical mapping was used to determine spatial dominant frequency distributions, electrical activity maps, and single-pixel optical signals. Following induction of AF, quinidine gluconate was perfused into the preparation and these parameters were monitored during quinidine-induced termination of AF. RESULTS: Equine AF develops in the context of spatial gradients in action potential duration (APD) and diastolic interval (DI) that produce alternans, conduction block, and Wenckebach conduction in different regions at fast pacing rates. Quinidine terminates AF and prevents subsequent reinduction by reducing the maximal frequency and increasing frequency homogeneity. CONCLUSIONS: Heterogeneity of APD and DI promote alternans and conduction block at fast pacing rates in the equine atrium, predisposing to the development of AF. Quinidine terminates AF by reducing maximum frequency and increasing frequency homogeneity. Our results are consistent with the hypothesis that quinidine increases effective refractory period, thereby decreasing frequency.

The patch clamp technique: principles and technical considerations.

The development of techniques that allow the high fidelity measurement of small scale ionic currents ushered in a new era of investigation into the role of ion channels in the physiologic and pathophysiologic function of excitable tissue. Based upon the formation of a high resistance (gigaohm) seal with the membrane of the cell being studied, these "patch clamp" techniques have improved our understanding of a wide variety of cardiac disease states with respect to diagnosis, treatment and prognosis. This review outlines the basic principles underlying the patch clamp technique, including the properties of biological membranes and ion channels, and provides an elementary summary of its application to the recording of cardiac ionic currents, with a particular focus on issues related to myocyte isolation, electrode manufacturing and the voltage clamp configuration.

Pulmonary thromboembolism in cats.

Pulmonary thromboembolism (PTE) is rarely diagnosed in cats, and the clinical features of the disease are not well known. PTE was diagnosed at postmortem examination in 17 cats, a prevalence of 0.06% over a 24-year period. The age of affected cats ranged from 10 months to 18 years, although young (<4 years) and old (>10 years) cats were more commonly affected than were middle-aged cats. Males and females were equally affected. The majority of cats with PTE (n = 16) had concurrent disease, which was often severe. The most common diseases identified in association with PTE were neoplasia, anemia of unidentified cause, and pancreatitis. Cats with glomerulonephritis, encephalitis, pneumonia, heart disease, and hepatic lipidosis were also represented in this study. Most cats with PTE demonstrated dyspnea and respiratory distress before death or euthanasia, but PTE was not recognized ante mortem in any cat studied. In conclusion, PTE can affect cats of any age and is associated with a variety of systemic and inflammatory disorders. It is recommended that the same clinical criteria used to increase the suspicion of PTE in dogs should also be applied to cats.