Isometric myography is a feasible method to identify and quantify endothelial dysfunction in dogs with myxomatous mitral valve disease.

OBJECTIVE: To assess the feasibility of isometric myography in pet dogs with myxomatous mitral valve disease (MMVD) to determine its use in quantifying endothelial dysfunction. ANIMALS: 9 dogs euthanized for medical reasons. METHODS: Femoral, renal, and mesenteric arteries were collected postmortem and stored in physiological saline solution at 4 °C for myography. Mitral valves were scored for myxomatous degeneration (grades 1 to 4). Sections of arteries were mounted in wells, immersed in physiological saline solution perfused with 95% O2 and 5% CO2 at 37 °C, and stretched to an internal circumference (IC) that generated the maximal difference between active and passive wall tension (IC1). Normalization factors were calculated by dividing the IC1 by the IC at which the passive wall tension was 100 mm Hg (IC100). Vasoconstriction to phenylephrine and vasodilation to acetylcholine (endothelial dependent) and sodium nitroprusside (endothelial independent) were assessed by cumulative dose-response curves. RESULTS: Median MMVD grade was 3. Mean values of normalization factors were 1.00 ± 0.14 (renal, n = 15), 1.00 ± 0.10 (femoral, 8), and 1.05 ± 0.12 (mesenteric, 6). Responses to phenylephrine were similar between dogs (P = .14). Reduced responses to acetylcholine compared with sodium nitroprusside were identified in 15 arteries, suggesting endothelial dysfunction. CLINICAL RELEVANCE: Isometric myography of arteries from pet dogs is feasible and can identify loss of endothelial-dependent relaxation in dogs with MMVD postmortem. Its use in further research can lead to a better understanding of the pathophysiology mechanisms of this disease.

Retrospective evaluation of systemic hypertension in dogs with nonassociative (primary) immune-mediated hemolytic anemia (December 2016 to April 2019): 26 cases.

OBJECTIVE: To report the prevalence of arterial hypertension in a population of dogs with nonassociative immune-mediated hemolytic anemia (IMHA) on presentation and during hospitalization. To determine the relationships of systolic blood pressure (SBP) with mortality and a prognostic indicator, the canine hemolytic anemia objective score. DESIGN: Retrospective observational study (December 2016 to April 2019). SETTING: University teaching hospital. ANIMALS: Twenty-six clinical dogs presenting to the ICU with nonassociative (primary) IMHA and a control group of 23 clinical dogs with idiopathic epilepsy hospitalized in the ICU for seizure treatment or monitoring. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Hypertension was defined as SBP ≥ 160 mm Hg and severe hypertension as SBP ≥ 180 mm Hg. Mean SBP was significantly increased in IMHA dogs (161 mm Hg, SD = 21) compared to ICU control dogs (138 mm Hg, SD = 14; P < 0.005). Hypertension was present in 13 of 26 (50.0%) dogs across the period of hospitalization and was severe in three of 26 (11.5%). During at least 1 day of hospitalization, 18 of 26 (69.2%) dogs were hypertensive and eight of 26 (34.6%) were severely hypertensive. Hypertension was not associated with short-term mortality or canine hemolytic anemia objective score. CONCLUSIONS: In this retrospective study, hypertension was more prevalent in dogs with nonassociative IMHA than a control population of ICU-hospitalized dogs. An association between autoimmune conditions and hypertension has been previously reported in people but not within a canine population. Hypertension in dogs may have an inflammatory or autoimmune etiology. SBP should be monitored closely in canine IMHA, in case antihypertensive treatment is required.

Branham sign in dogs undergoing interventional patent ductus arteriosus occlusion or surgical ligation: A retrospective study.

BACKGROUND: The Branham sign is a baroreceptor response that follows patent ductus arteriosus (PDA) closure. Although described in dogs following both interventional and surgical ductal closure, a direct comparison of the Branham sign elicited by these two techniques has not been made. AIM: Since closure with an Amplatz canine ductal occluder (ACDO) occurs over 10 minutes and surgical ligation (SL) is more rapid, we hypothesized that the Branham sign following occlusion of a PDA with an ACDO would be less severe than following SL. METHODS: Clinical records of dogs diagnosed with left-to-right shunting PDA between 2008 and 2018 were retrospectively reviewed. Of 139 dogs undergoing PDA occlusion, only 41 dogs (ACDO n = 32, SL n = 9) were included after applying exclusion criteria. Heart rate (HR) and blood pressure (BP) from occlusion time (T 0) until 30 minutes post occlusion (T 30) were recorded. Signalment and anesthetic protocol were also recorded. The influence of age and weight on the hemodynamic variations was assessed. Hemodynamic variables and calculations were compared between and within groups using a repeated measures general linear model, and post hoc tests were applied if significance was identified. RESULTS: A mild Branham sign was present in both groups, and hemodynamic changes were not significantly different between groups. In both groups, there was a significant decrease in HR (11 bpm, 5.3-16.3; p < 0.001) (10.4%, 5.4-15.5; p < 0.001) and increase in diastolic BP (9.5 mmHg, 3-16; p = 0.002) (23.5%, 7.1-39.9; p = 0.002), but systolic BP did not change significantly (p = 0.824). Age and weight did not influence Branham sign. CONCLUSION: The Branham sign in dogs is mild in both groups, lasts for at least 30 minutes, and is independent of the method of PDA closure.

Association of diet with left ventricular wall thickness, troponin I and IGF-1 in cats with subclinical hypertrophic cardiomyopathy.

BACKGROUND: Cats with subclinical hypertrophic cardiomyopathy (sHCM) have elevated serum insulin and serum amyloid A concentrations correlating with the degree of cardiac hypertrophy. Diet might affect these and other cardiac variables. OBJECTIVE: Evaluate the effect of a complete, balanced diet with restricted starch and supplemented with eicosapentaenoic acid + docosahexaenoic acid (EPA + DHA) on echocardiographic variables and cardiac biomarkers in cats with sHCM. ANIMALS: Forty-four client-owned cats with sHCM. METHODS: A prospective, randomized, double-blind, multicenter study enrolled cats with end-diastole interventricular septum thickness (IVSd) or left ventricular wall thickness (LVWd) ≥6 mm, or both. Nonsedated, fasted cats were examined at baseline and after 6 and 12 months of Test (restricted starch and EPA + DHA supplements) (n = 23) or Control (unrestricted starch without EPA + DHA supplementation) (n = 21) diet. Assessments included auscultation, body weight, body condition score, echocardiography and blood analysis. Linear and generalized mixed models analyzed diet, time and diet * time interactions (5% significance level). RESULTS: No differences between diet groups were significant for any variable at any timepoint. There were significant decreases in the Test but not Control group in maximum IVSd (P = .03), maximum LVWd (P = .02) and insulin-like growth factor-1 levels (P = .04) after 12 months, and in ultrasensitive cardiac troponin I (cTnI) (P = .001) after 6 months; effect sizes (95% confidence interval) were 0.53 (0.09; 0.99), 0.63 (0.18; 1.09), 0.61 (0.16; 1.07), and 0.37 (-0.06; 0.8), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Cats with sHCM fed Test diet had significant decreases in echocardiographic variables of sHCM and in cTnI and IGF-1.

Standardisation of electrocardiographic examination in corn snakes (Pantherophis guttatus).

INTRODUCTION: Corn snakes are a very common pet reptile species, yet there is an absence of evidence-based literature standardising collection of ECG or detailing ECG deflection morphology in the normal animal. The authors describe a well-tolerated, reproducible technique and detail the cardiac cycle in terms of lead 2 equivalent waveforms and intervals. ANIMALS: 29 adult corn snakes. MATERIALS AND METHODS: This prospective study evaluated, under species-appropriate, standardised conditions, a technique for producing standard six-lead ECG tracings. Lead 2 equivalent cardiac cycles were described in detail and statistically analysed for sex, weight, length, heart rate and mean electrical axis. RESULTS: High-quality tracings demonstrated common ECG characteristics for this species, including no Q, S or SV waves, prolonged PR and RT intervals, rhythmic oscillation of the baseline, short TP segments, and a right displaced mean electrical axis. An influence of sex, weight or length on heart rate and mean electrical axis was not identified. CONCLUSIONS: To the authors' knowledge, this is the first study to describe a standardised technique for recording ECG in significant numbers of normal corn snakes. Ranges have been provided that may be of diagnostic value or form the basis for future development of reference intervals for this species.

Associations among echocardiography, cardiac biomarkers, insulin metabolism, morphology, and inflammation in cats with asymptomatic hypertrophic cardiomyopathy.

BACKGROUND: Insulin, insulin-like growth factor-1 (IGF-1), and inflammation possibly are involved in cats with asymptomatic hypertrophic cardiomyopathy (aHCM). OBJECTIVES: To evaluate echocardiography, morphology, cardiac and inflammatory markers, insulin and IGF-1 in cats with aHCM. ANIMALS: Fifty-one client-owned cats with aHCM. METHODS: Observational descriptive study. Variables (body weight [BW], body condition score [BCS], echocardiography, and serum concentrations of N-terminal pro-B-type natriuretic peptide [NT-proBNP], ultra-sensitive troponin-I [c-TnI], serum amyloid A [SAA], insulin, glucose and IGF-1) were evaluated for significant increases above echocardiography cutoff values and laboratory reference ranges, associations and effect of left atrial (LA) remodeling and generalized hypertrophy. RESULTS: Cats with aHCM had BCS ≥6/9 (P = .01) and insulin (P < .001), NT-proBNP (P = .001) and cTn-I (P < .001) above laboratory reference ranges. Associations were present between NT-proBNP and maximum end-diastolic interventricular septum thickness (IVSd; ρ = .32; P = .05), maximum end-diastolic left ventricular free wall thickness;(ρ = .41; P = .01), LA/Aorta (ρ = .52; P = .001) and LA diameter (LA-max; ρ = .32; P = .05); c-TnI and LA/Aorta (ρ = .49; P = .003) and LA-max (ρ = .28; P = .05); and SAA and number of IVSd regions ≥6 mm thickness (ρ = .28; P = .05). Body weight and BCS were associated with IGF-1 (r = 0.44; P = .001), and insulin (ρ = .33; P = .02), glucose (ρ = .29; P = .04) and IGF-1 (ρ = .32; P = .02), respectively. Concentrations of NT-proBNP (P = .02) and c-TnI (P = .01), and SAA (P = .02), were higher in cats with LA remodeling, and generalized hypertrophy, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest potential implications of insulin, IGF-1, and inflammation in cats with aHCM, but it remains to be confirmed whether these findings represent a physiological process or a part of the pathogenesis and development of disease.

Survival of activated myofibroblasts in canine myxomatous mitral valve disease and the role of apoptosis.

Myxomatous mitral valve disease (MMVD) is the single most important acquired cardiovascular disease of the dog. Much is known about the cellular changes and the contribution of activated myofibroblasts (valve interstitial cells (aVICs) to the valve extra-cellular matrix remodelling characteristic of the disease. However, little is known on how aVIC survival might contribute to disease pathogenesis. This study examined the temporal (disease severity-dependent) and spatial distribution of aVICs in MMVD valves, the expression of a range of apoptosis-related genes in cultured VICs from both normal (quiescent VIC (qVIC) and diseased (aVIC) valves, and the differential effects of doxorubicin treatment, as a trigger of apoptosis, on expression of the same genes. Activated myofibroblasts were identified in normal valves at the valve base only (the area closest to the annulus), and then became more numerous and apparent along the valve length as the disease progressed, with evidence of cell survival at the valve base. There were no significant differences in basal gene expression comparing qVICs and aVICs for CASP3, FAS, BID, BAX, BCL2, CASP8, DDIAS, XIAP and BIRC5. After doxorubicin treatment (2 mM) for 8 h there was significant difference (P < .05) in the expression of BID, BCL2, DDIAS, and CASP8, but when assessed for interactions using a mixed model ANOVA only CASP8 was significantly different because of treatment (P < .05). These data suggest aVIC survival in MMVD valves may be a consequence of heightened resistance of aVICs to apoptosis, but would require confirmation examining expression of the relevant proteins.

Vasovagal tonus index (VVTI) as an indirect assessment of remission status in canine multicentric lymphoma undergoing multi-drug chemotherapy.

Vasovagal tonus index (VVTI) is an indirect measure of heart rate variability and may serve as a marker of disease severity. Higher heart rate variability has predicted lower tumour burden and improved survival in humans with various tumour types. The purpose of this pilot study was to evaluate VVTI as a biomarker of remission status in canine lymphoma. The primary hypothesis was that VVTI would be increased in dogs in remission compared to dogs out of remission. Twenty-seven dogs were prospectively enrolled if they had a diagnosis of intermediate to high-grade lymphoma and underwent multidrug chemotherapy. Serial electrocardiogram data were collected under standard conditions and relationships between VVTI, remission status and other clinical variables were evaluated. VVTI from dogs in remission (partial or complete) did not differ from dogs with fulminant lymphoma (naive or at time of relapse). Dogs in partial remission had higher VVTI than dogs in complete remission (p = 0.021). Higher baseline VVTI was associated with higher subsequent scores (p < 0.001). VVTI also correlated with anxiety level (p = 0.03). Based on this pilot study, VVTI did not hold any obvious promise as a useful clinical biomarker of remission status. Further investigation may better elucidate the clinical and prognostic utility of VVTI in dogs with lymphoma.

Tricuspid valve dysplasia: A retrospective study of clinical features and outcome in dogs in the UK.

The objective of this study was to determine the demographic, clinical and survival characteristics and to identify risk factors for mortality due to tricuspid valve dysplasia in UK dogs. Records of client-owned dogs diagnosed with tricuspid valve dysplasia at a referral centre were retrospectively reviewed. Only dogs diagnosed with tricuspid valve dysplasia based on the presence of a right-sided heart murmur identified prior to one year of age, and confirmed with Doppler echocardiography, were included. Dogs with concomitant cardiac diseases, pulmonary hypertension and/or trivial tricuspid regurgitation were excluded. Analysed data included signalment, reason for presentation, clinical signs, electrocardiographic and echocardiographic features, survival status and cause of death. Survival times and risk factors for mortality were evaluated using Kaplan-Meier curves and Cox regression. Eighteen dogs met inclusion criteria. Border collies were over-represented (p= 0.014). Dogs were most frequently referred for investigation of heart murmur. The most common arrhythmia was atrial fibrillation (n=3). Median survival time from diagnosis of tricuspid valve dysplasia was 2775 days (range 1-3696 days; 95% CI 1542.41-4007.59) and from onset of right-sided congestive heart failure was 181 days (range 1-2130 days; 95% CI 0-455.59). Syncope was the sole risk factor for cardiac death. In this population of UK dogs, tricuspid valve dysplasia was uncommon but, when severe, frequently led to right-sided congestive heart failure. Prognosis was favourable for mild and moderate tricuspid dysplasia. Survival time was reduced with right-sided congestive heart failure but varied widely. Risk of cardiac death was significantly increased if syncope had occurred.

Developmental pathways and endothelial to mesenchymal transition in canine myxomatous mitral valve disease.

Epithelial to mesenchymal transition (EMT) and the cardiovascular equivalent, endothelial to mesenchymal transition (EndoMT), contribute to a range of chronic degenerative diseases and cancer metastasis. Chronic valvulopathies exhibit some features of EndoMT and activation of developmental signalling pathways, such as osteogenesis and chondrogenesis, expression of cell differentiation markers, basement membrane damage and endothelial transformation. The aim of the present study was to investigate the potential role of developmental mechanisms in canine myxomatous mitral valve disease (MMVD) using a combination of transcriptomic array technology, RT-PCR and immunohistochemistry. There was significant differential expression for genes typically associated with valvulogenesis and EndoMT, including markers of inflammation (IL6, IL18 and TLR4), basement membrane disarray (NID1, LAMA2 and CTSS), mesenchymal and endothelial cell differentiation (MYH11 and TAGLN) and EndoMT (ACTA2, SNAI1, CTNNB1, HAS2, CDH5, and NOTCH1), with fold changes from +15.35 (ACTA2) to -5.52 (LAMA2). These changes in gene expression were confirmed using RT-PCR, except for HAS2. In silico analysis identified important gene networks and canonical pathways in MMVD that have associations with development and organogenesis, including inflammation, valve morphogenesis and EMT, as well as components of the basement membrane and extra-cellular matrix. Immunohistochemistry identified changes in the expression of hyaluronic acid synthase (Has2), Snai1, α-smooth muscle actin (α-SMA) and VE-cadherin (CDH5), and co-expression of Has2 with α-SMA. These research findings strongly suggest involvement of developmental signalling pathways and mechanisms, including EndoMT, in the pathogenesis of canine MMVD.

Endothelin in nondiabetic chronic kidney disease: preclinical and clinical studies.

The incidence and prevalence of chronic kidney disease (CKD) is increasing. Despite current therapies, many patients with CKD have suboptimal blood pressure, ongoing proteinuria, and develop progressive renal dysfunction. Further therapeutic options therefore are required. Over the past 20 years the endothelin (ET) system has become a prime target. Experimental models have shown that ET-1, acting primarily via the endothelin-A receptor, plays an important role in the development of proteinuria, glomerular injury, fibrosis, and inflammation. Subsequent animal and early clinical studies using ET-receptor antagonists have suggested that theses therapies may slow renal disease progression primarily through blood pressure and proteinuria reduction. This review examines the current literature regarding the ET system in nondiabetic CKD.

Viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species.

Non-primate hepacivirus (NPHV), equine pegivirus (EPgV) and Theiler's disease associated virus (TDAV) are newly discovered members of two genera in the Flaviviridae family, Hepacivirus and Pegivirus respectively, that include human hepatitis C virus (HCV) and human pegivirus (HPgV). To investigate their epidemiology, persistence and clinical features of infection, large cohorts of horses and other mammalian species were screened for NPHV, EPgV and TDAV viraemia and for past exposure through serological assays for NPHV and EPgV-specific antibodies. NPHV antibodies were detected in 43% of 328 horses screened for antibodies to NS3 and core antibodies, of which three were viraemic by PCR. All five horses that were stablemates of a viraemic horse were seropositive, as was a dog on the same farm. With this single exception, all other species were negative for NPHV antibodies and viraemia: donkeys (n=100), dogs (n=112), cats (n=131), non-human primates (n=164) and humans (n=362). EPgV antibodies to NS3 were detected in 66.5% of horses, including 10 of the 12 horses that had EPgV viraemia. All donkey samples were negative for EPgV antibody and RNA. All horse and donkey samples were negative for TDAV RNA. By comparing viraemia frequencies in horses with and without liver disease, no evidence was obtained that supported an association between active NPHV and EPgV infections with hepatopathy. The study demonstrates that NPHV and EPgV infections are widespread and enzootic in the study horse population and confirms that NPHV and potentially EPgV have higher frequencies of viral clearance than HCV and HPgV infections in humans.

Nonprimate hepaciviruses in domestic horses, United kingdom.

Although the origin of hepatitis C virus infections in humans remains undetermined, a close homolog of this virus, termed canine hepacivirus (CHV) and found in respiratory secretions of dogs, provides evidence for a wider distribution of hepaciviruses in mammals. We determined frequencies of active infection among dogs and other mammals in the United Kingdom. Samples from dogs (46 respiratory, 99 plasma, 45 autopsy samples) were CHV negative by PCR. Screening of 362 samples from cats, horses, donkeys, rodents, and pigs identified 3 (2%) positive samples from 142 horses. These samples were genetically divergent from CHV and nonprimate hepaciviruses that horses were infected with during 2012 in New York state, USA. Investigation of infected horses demonstrated nonprimate hepacivirus persistence, high viral loads in plasma (10(5)-10(7) RNA copies/mL), and liver function test results usually within reference ranges, although several values ranged from high normal to mildly elevated. Disease associations and host range of nonprimate hepaciviruses warrant further investigation.

Evaluation of innervation of the mitral valves and the effects of myxomatous degeneration in dogs.

OBJECTIVE: To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs. SAMPLE POPULATION: Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years. PROCEDURES: Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically. RESULTS: MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation. CONCLUSIONS AND CLINICAL RELEVANCE: Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Distribution of myofibroblasts, smooth muscle-like cells, macrophages, and mast cells in mitral valve leaflets of dogs with myxomatous mitral valve disease.

OBJECTIVE: To map the cellular distribution and phenotypic alteration of the predominant stromal cell population throughout the entire valve length of dogs with myxomatous mitral valve disease (MMVD). SAMPLE POPULATION: 31 mitral valve complexes (ie, mitral valve leaflets) collected from 4 clinically normal dogs and 27 dogs with MMVD of varying severity. PROCEDURES: A combination of standard histologic and immunohistochemical techniques was used to identify pathologic changes, the presence of mast cells, and the density and distribution of cells expressing vimentin, desmin, alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin, and the macrophage marker MAC387. RESULTS: Vimentin-positive cells predominated in the mitral valve leaflets from clinically normal dogs and were located throughout the leaflet, but cell density was appreciably decreased with disease progression, and minimal cell numbers were found in distinct myxomatous areas. Cells that were positive for alpha-SMA were uncommon in the mitral valve leaflets from clinically normal dogs and only seen in appreciable numbers in mitral valves of dogs with severe late-stage disease, in which cells were typically located close to the ventricularis valve surface. A slight increase in mast cell numbers was observed in the distal zone of affected leaflets. CONCLUSIONS AND CLINICAL RELEVANCE: Activated-myofibroblasts (alpha-SMA-positive cells) were increased and inactive-myofibroblasts (vimentin-positive cells) were reduced in mitral valve leaflets of dogs with MMVD, compared with that of clinically normal dogs. Impact on Human Medicine-This is the first description of spatial and temporal alterations in mitral valve cells of any species with MMVD and has clinical importance in the understanding of disease development in dogs and humans.