Use of machine learning and Poincaré density grid in the diagnosis of sinus node dysfunction caused by sinoatrial conduction block in dogs.

BACKGROUND: Sinus node dysfunction because of abnormal impulse generation or sinoatrial conduction block causes bradycardia that can be difficult to differentiate from high parasympathetic/low sympathetic modulation (HP/LSM). HYPOTHESIS: Beat-to-beat relationships of sinus node dysfunction are quantifiably distinguishable by Poincaré plots, machine learning, and 3-dimensional density grid analysis. Moreover, computer modeling establishes sinoatrial conduction block as a mechanism. ANIMALS: Three groups of dogs were studied with a diagnosis of: (1) balanced autonomic modulation (n = 26), (2) HP/LSM (n = 26), and (3) sinus node dysfunction (n = 21). METHODS: Heart rate parameters and Poincaré plot data were determined [median (25%-75%)]. Recordings were randomly assigned to training or testing. Supervised machine learning of the training data was evaluated with the testing data. The computer model included impulse rate, exit block probability, and HP/LSM. RESULTS: Confusion matrices illustrated the effectiveness in diagnosing by both machine learning and Poincaré density grid. Sinus pauses >2 s differentiated (P < .0001) HP/LSM (2340; 583-3947 s) from sinus node dysfunction (8503; 7078-10 050 s), but average heart rate did not. The shortest linear intervals were longer with sinus node dysfunction (315; 278-323 ms) vs HP/LSM (260; 251-292 ms; P = .008), but the longest linear intervals were shorter with sinus node dysfunction (620; 565-698 ms) vs HP/LSM (843; 799-888 ms; P < .0001). CONCLUSIONS: Number and duration of pauses, not heart rate, differentiated sinus node dysfunction from HP/LSM. Machine learning and Poincaré density grid can accurately identify sinus node dysfunction. Computer modeling supports sinoatrial conduction block as a mechanism of sinus node dysfunction.

Electrocardiographic patterns of ventricular pre-excitation in dogs with right-sided accessory pathways.

INTRODUCTION: The aim of the study was to describe the electrocardiographic features of ventricular pre-excitation (VPE) patterns characterized by the presence of delta (δ) wave, short P-δQRS interval, wide δQRS complexes in dogs with right-sided accessory pathways. ANIMALS, MATERIALS AND METHODS: Twenty-six dogs with a confirmed accessory pathways (AP) via electrophysiological mapping were included. All dogs underwent a complete physical examination, 12-lead ECG, thoracic radiography, echocardiographic examination and electrophysiologic mapping. The AP were located in the following regions: right anterior, right posteroseptal, right posterior. The following parameters were determined: P-δQRS interval, δQRS duration, δQRS axis, δQRS morphology, δ-wave polarity, Q-wave, R-wave, R'-wave, S-wave amplitude, and R/S ratio. RESULTS: In lead II, the median δQRS complex duration was 82.4 (IQR 7.2) and the median P-δQRS interval duration was 54.6 (IQR 4.2) msec. The median δQRS complex axis in the frontal plane was: + 68° (IQR 52.5) for right anterior APs, - 24 ° (IQR 24) for right postero-septal APs, - 43.5 ° (IQR 27.25) for right posterior APs (P = 0.007). In lead II, the polarity of the δ wave was positive in 5/5 right anterior APs and negative in 7/11 postero-septal APs and 8/10 in right posterior APs. In precordial leads of all dogs, R/S was ≤ 1 in V1 and > 1 in all leads from V2 to V6. CONCLUSION: Surface electrocardiogram can be used to distinguish right anterior APs from right posterior and right postero-septal ahead of an invasive electrophysiological study.

Optimizing single-chamber pacing in dogs. Part 2: Rate adaptive pacing.

Proper programming of pacemakers for dogs in the rate adaptive mode requires an understanding of the rate requirements for each individual and the interplay of programmable features. The specific advantages and disadvantages of the rate adaptive mode should be considered on a case by case basis. Fundamentally, two components are linked in the implementation of rate adaptive pacing: (1) sensing the need for a change in rate and (2) responding with the appropriate alteration in pacing rate. The programming interaction of these two components are interdependent and affected by the rates programmed. These features may be adjusted manually or automatically. In this review (Part 2 of a two-part review) the considerations required to program each aspect that optimizes the pacing rate profile are reviewed. These include the lower rate, upper sensor rate, activities of daily life rate, sensor threshold, acceleration and deceleration, slope, activities of daily life zone, exertion zone, automatic versus manual adjustments and closed loop stimulation. The programming features of pacemakers manufactured by three companies are summarized (Abbott, formerly St. Jude; Biotronik/Dextronix; Medtronic). Means of assessing the success of pacemaker programing is examined through examples of pacemaker data, Holter analysis, Poincaré plots and tachograms. Finally, the questions and considerations for rate adaptive pacing in dogs that demand investigation are proposed.

Optimizing single-chamber pacing in dogs Part 1: Rate determinations, rate interventions and hysteresis.

Determining ideal pacing rates to meet physiological needs and optimizing programming to prevent unnecessary right ventricular pacing in dogs requires an understanding of heart rate profiles and applicable pacing technology. The heart rate and rhythm of the dog is complex necessitating investigation of rate requirements of activity and circadian influences. Overlaying this information are a multiplicity of other factors such as age, breed, temperament, cardiovascular disease and underlining rhythm disorders that contribute to the difficulty in making general conclusions. However, all such information permits better implementation of programming options with the goal of better outcomes. In this review (Part 1 of a two-part review) instantaneous heart rate, rolling average heart rate, simple average heart rate, heart rate tachograms, RR interval tachograms (2D, 3D and dynamic), and Poincaré plots (2D, 3D and dynamic) are discussed as they apply to decisions in the determination and examination of pacing rates for dogs programmed in the VVI pacing mode (Ventricular paced, Ventricular sensed, Inhibited pacing). The applicable pacing operations available for three pacemaker companies are reviewed (Abbott, Biotronik/Dextronix, and Medtronic). The programmable options considered include: slowest pacing rate without additional features to extend the pacing interval, sleep/rest rate preferences, hysteresis to lengthen pacing interval following intrinsic beats, and intermittent increases in pacing following abrupt loss of intrinsic rhythm. Recommendations are suggested for follow-up of individual dogs with examination of pacing statistics and Holter monitoring.

Temporal changes in clinical and radiographic variables in dogs with preclinical myxomatous mitral valve disease: The EPIC study.

BACKGROUND: The Evaluation of pimobendan in dogs with cardiomegaly caused by preclinical myxomatous mitral valve disease (EPIC) study monitored dogs with myxomatous mitral valve disease (MMVD) as they developed congestive heart failure (CHF). OBJECTIVES: To describe the changes in clinical and radiographic variables occurring as dogs with MMVD and cardiomegaly develop CHF, compared to similar dogs that do not develop CHF. ANIMALS: One hundred and thirty-five, and 73 dogs that did or did not develop CHF, respectively. MATERIALS AND METHODS: The following variables were evaluated in 2 groups of dogs (dogs that did or did not develop CHF): Heart rate (HR), clinic respiratory rate (RR), home-measured resting respiratory rate (RRR), rectal temperature (RT), body weight (BW), and vertebral heart sum (VHS). Absolute value and rate of change of each variable were calculated for each day a dog was in study. Daily means were calculated and plotted against time. The onset of CHF or last visit before leaving the study were set as reference time points. RESULTS: The most extreme values and rate of change occurred in variables immediately before onset of CHF. Vertebral heart sum increased earliest. Heart rate, RR, and RRR also increased. Rectal temperature and BW decreased. Increases in RR and RRR were most extreme and occurred immediately before CHF. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with MMVD and cardiomegaly experience increases in HR, RR, RRR, and VHS, and decreases in BW and RT as they develop CHF. The variables with highest absolute change and rate of change were RR and RRR. These findings reinforce the value of RR and RRR as indicators of impending or incipient CHF.

Long-term incidence and risk of noncardiovascular and all-cause mortality in apparently healthy cats and cats with preclinical hypertrophic cardiomyopathy.

BACKGROUND: Epidemiologic knowledge regarding noncardiovascular and all-cause mortality in apparently healthy cats (AH) and cats with preclinical hypertrophic cardiomyopathy (pHCM) is limited, hindering development of evidence-based healthcare guidelines. OBJECTIVES: To characterize/compare incidence rates, risk, and survival associated with noncardiovascular and all-cause mortality in AH and pHCM cats. ANIMALS: A total of 1730 client-owned cats (722 AH, 1008 pHCM) from 21 countries. METHODS: Retrospective, multicenter, longitudinal, cohort study. Long-term health data were extracted by medical record review and owner/referring veterinarian interviews. RESULTS: Noncardiovascular death occurred in 534 (30.9%) of 1730 cats observed up to 15.2 years. Proportion of noncardiovascular death did not differ significantly between cats that at study enrollment were AH or had pHCM (P = .48). Cancer, chronic kidney disease, and conditions characterized by chronic weight-loss-vomiting-diarrhea-anorexia were the most frequently recorded noncardiovascular causes of death. Incidence rates/risk of noncardiac death increased with age in AH and pHCM. All-cause death proportions were greater in pHCM than AH (65% versus 40%, respectively; P < .001) because of higher cardiovascular mortality in pHCM cats. Comparing AH with pHCM, median survival (study entry to noncardiovascular death) did not differ (AH, 9.8 years; pHCM, 8.6 years; P = .10), but all-cause survival was significantly shorter in pHCM (P = .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: All-cause mortality was significantly greater in pHCM cats due to disease burden contributed by increased cardiovascular death superimposed upon noncardiovascular death.

Beat-to-Beat Patterning of Sinus Rhythm Reveals Non-linear Rhythm in the Dog Compared to the Human.

The human and dog have sinus arrhythmia; however, the beat-to-beat interval changes were hypothesized to be different. Geometric analyses (R-R interval tachograms, dynamic Poincaré plots) to examine rate changes on a beat-to-beat basis were analyzed along with time and frequency domain heart rate variability from 40 human and 130 canine 24-h electrocardiographic recordings. Humans had bell-shaped beat-interval distributions, narrow interval bands across time with continuous interval change and linear changes in rate. In contrast, dogs had skewed non-singular beat distributions, wide interval bands {despite faster average heart rate of dogs [mean (range); 81 (64-119)] bpm compared to humans [74.5 (59-103) p = 0.005]} with regions displaying a paucity of intervals (zone of avoidance) and linear plus non-linear rate changes. In the dog, dynamic Poincaré plots showed linear rate changes as intervals prolonged until a point of divergence from the line of identity at a mean interval of 598.5 (95% CI: 583.5-613.5) ms (bifurcation interval). The dog had bimodal beat distribution during sleep with slower rates and greater variability than during active hours that showed singular interval distributions, higher rates and less variability. During sleep, Poincaré plots of the dog had clustered or branched patterns of intervals. A slower rate supported greater parasympathetic modulation with a branched compared to the clustered distribution. Treatment with atropine eliminated the non-linear patterns, while hydromorphone shifted the bifurcated branching and beat clustering to longer intervals. These results demonstrate the unique non-linear nature of beat-to-beat variability in the dog compared to humans with increases in interval duration (decrease heart rate). These results provoke the possibility that changes are linear with a dominant sympathetic modulation and non-linear with a dominant parasympathetic modulation. The abrupt bifurcation, zone of avoidance and beat-to-beat patterning are concordant with other studies demonstrating the development of exit block from the sinus node with parasympathetic modulation influencing not only the oscillation of the pacing cells, but conduction to the atria. Studies are required to associate the in vivo sinus node beat patterns identified in this study to the mapping of sinus impulse origin and exit from the sinus node.

International collaborative study to assess cardiovascular risk and evaluate long-term health in cats with preclinical hypertrophic cardiomyopathy and apparently healthy cats: The REVEAL Study.

BACKGROUND: Hypertrophic cardiomyopathy is the most prevalent heart disorder in cats and principal cause of cardiovascular morbidity and mortality. Yet, the impact of preclinical disease is unresolved. HYPOTHESIS/OBJECTIVES: Observational study to characterize cardiovascular morbidity and survival in cats with preclinical nonobstructive (HCM) and obstructive (HOCM) hypertrophic cardiomyopathy and in apparently healthy cats (AH). ANIMALS: One thousand seven hundred and thirty client-owned cats (430 preclinical HCM; 578 preclinical HOCM; 722 AH). METHODS: Retrospective multicenter, longitudinal, cohort study. Cats from 21 countries were followed through medical record review and owner or referring veterinarian interviews. Data were analyzed to compare long-term outcomes, incidence, and risk for congestive heart failure (CHF), arterial thromboembolism (ATE), and cardiovascular death. RESULTS: During the study period, CHF, ATE, or both occurred in 30.5% and cardiovascular death in 27.9% of 1008 HCM/HOCM cats. Risk assessed at 1, 5, and 10 years after study entry was 7.0%/3.5%, 19.9%/9.7%, and 23.9%/11.3% for CHF/ATE, and 6.7%, 22.8%, and 28.3% for cardiovascular death, respectively. There were no statistically significant differences between HOCM compared with HCM for cardiovascular morbidity or mortality, time from diagnosis to development of morbidity, or cardiovascular survival. Cats that developed cardiovascular morbidity had short survival (mean ± standard deviation, 1.3 ± 1.7 years). Overall, prolonged longevity was recorded in a minority of preclinical HCM/HOCM cats with 10% reaching 9-15 years. CONCLUSIONS AND CLINICAL IMPORTANCE: Preclinical HCM/HOCM is a global health problem of cats that carries substantial risk for CHF, ATE, and cardiovascular death. This finding underscores the need to identify therapies and monitoring strategies that decrease morbidity and mortality.

Clinical and electrocardiographic presentations of transient trifascicular block in three cats.

This report describes transient trifascicular block in three cats presented with lethargy and inappetence, and elevated cardiac troponin I concentrations. The electrocardiogram (ECG) of cat 1 showed a sinus rhythm with pronounced first-degree atrioventricular (AV) block, right bundle branch block, and left anterior fascicular block. The ECG of cat 2 showed truncular left bundle branch block alternating with left anterior fascicular block coupled with prolonged PR intervals, second-degree heart block, and paroxysmal third-degree AV block. The ECG of cat 3 showed first-degree AV block with concomitant right bundle branch block. The diagnosis of trifascicular block was made when paroxysmal third-degree AV block was documented. All cats recovered with medical management within weeks. Each cat resumed a sinus rhythm. Elevated cardiac troponin I concentrations suggested myocarditis that improved.

Cardiovascular-renal axis disorders in the domestic dog and cat: a veterinary consensus statement.

OBJECTIVES: There is a growing understanding of the complexity of interplay between renal and cardiovascular systems in both health and disease. The medical profession has adopted the term "cardiorenal syndrome" (CRS) to describe the pathophysiological relationship between the kidney and heart in disease. CRS has yet to be formally defined and described by the veterinary profession and its existence and importance in dogs and cats warrant investigation. The CRS Consensus Group, comprising nine veterinary cardiologists and seven nephrologists from Europe and North America, sought to achieve consensus around the definition, pathophysiology, diagnosis and management of dogs and cats with "cardiovascular-renal disorders" (CvRD). To this end, the Delphi formal methodology for defining/building consensus and defining guidelines was utilised. METHODS: Following a literature review, 13 candidate statements regarding CvRD in dogs and cats were tested for consensus, using a modified Delphi method. As a new area of interest, well-designed studies, specific to CRS/CvRD, are lacking, particularly in dogs and cats. Hence, while scientific justification of all the recommendations was sought and used when available, recommendations were largely reliant on theory, expert opinion, small clinical studies and extrapolation from data derived from other species. RESULTS: Of the 13 statements, 11 achieved consensus and 2 did not. The modified Delphi approach worked well to achieve consensus in an objective manner and to develop initial guidelines for CvRD. DISCUSSION: The resultant manuscript describes consensus statements for the definition, classification, diagnosis and management strategies for veterinary patients with CvRD, with an emphasis on the pathological interplay between the two organ systems. By formulating consensus statements regarding CvRD in veterinary medicine, the authors hope to stimulate interest in and advancement of the understanding and management of CvRD in dogs and cats. The use of a formalised method for consensus and guideline development should be considered for other topics in veterinary medicine.

Atrial septal pacing in small dogs: a pilot study.

OBJECTIVE: To determine the feasibility of atrial septal pacing via a delivery catheter-guided small non-retracting helix pacing lead. ANIMALS: Six healthy beagles (8.3-12.9 kg). METHODS: Using single plane fluoroscopic guidance, Medtronic(®) 3830 SelectSecure leads were connected to the atrial septum via Medtronic® Attain Select® II standard 90 Left Heart delivery catheter. Pacing threshold and lead impedance were measured at implantation. The Wenckebach point was tested via atrial pacing up to 220 paced pulses per minute (ppm). Thoracic radiographs were performed following implantation to identify the lead position, and repeated at 24 h, 1 month, and 3 months post-operatively. RESULTS: Macro-lead dislodgement occurred in two dogs at 24 h and in three dogs at one-month post-implantation. Lead impedance, measured at the time of implantation, ranged from 583 to 1421 Ω. The Wenckebach point was >220 ppm in four of the six dogs. The remaining two dogs had Wenckebach points of 120 and 190 ppm. CONCLUSIONS: This pilot study suggests the selected implantation technique and lead system were inadequate for secure placement in the atrial septum of these dogs. The possible reasons for inadequate stability include unsuitable lead design for this location, inadequate lead slack at the time of implantation and inadequate seating of the lead as evidenced by low impedance at the time of implantation. Other implantation techniques and/or pacing leads should be investigated to determine the optimal way of pacing the atria in small breed dogs that are prone to sinus node dysfunction.

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.

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.

Transthoracic echocardiographically-guided interventional cardiac procedures in the dog.

OBJECTIVES: Interventional cardiac procedures are traditionally performed using fluoroscopy, or, more recently, transesophageal echocardiography (TEE). Neither modality is widely available to practicing cardiologists worldwide. We examined whether balloon valvuloplasty of pulmonic stenosis (PS) and transarterial occlusion of patent ductus arteriosus (PDA) in dogs could be performed safely with transthoracic echocardiography (TTE). ANIMALS: A prospective consecutive case series of 26 client-owned dogs with PS (n = 10) and PDA (n = 16). METHODS: The cardiovascular procedures were performed using TTE. Each dog was positioned on a standard echocardiography table in right lateral recumbency (dogs with PS) or left lateral recumbency (dogs with PDA). Guide wires, balloon catheters, Amplatz(®) Canine Ductal Occluder (ACDO) delivery sheaths, and ACDO were imaged by standard echocardiographic views optimized to allow visualization of the defects and devices. RESULTS: Procedures were performed successfully without major complications in 20 dogs. In 2 dogs (German shepherds) with Type III PDA, ACDO placement was unsuccessful; 2 other German Shepherds were excluded from the procedure because their ductal diameters, measured echocardiographically, exceeded the limits of the maximal ACDO size. Two dogs weighing ≤3.5 kg had suboptimal echocardiographic visualization of the PDA and were considered too small for safe ACDO deployment. All intravascular devices at the level of the heart and great vessels appeared hyperechoic on TTE image and could be clearly monitored and guided in real-time. CONCLUSIONS: We have demonstrated that TTE monitoring can guide each step of pulmonic balloon valvuloplasty and PDA occlusion without fluoroscopy.

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.

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.