Antioxidant status and biomarkers of oxidative stress in dogs with congestive heart failure

Alterations in antioxidant status and oxidative stress have been documented in dogs with dilated cardiomyopathy (DCM). The purpose of this study was to more broadly assess this relationship in dogs with congestive heart failure (CHF). Malondialdehyde (MDA), 8-F(2alpha)-isoprostane, protein carbonyls, reduced (GSH) and oxidized (GSSG) glutathione, vitamins A, C, and E, and oxygen radical absorbance capacity (ORAC) were measured from a single venous blood sample from dogs with CHF secondary to DCM or chronic valvular disease (CVD) and in healthy controls. Nineteen dogs with CHF (14 CVD and 5 DCM) and 12 healthy controls were enrolled in the study. Concentrations of 8-F(2alpha)-isoprostane (CHF: 44.6 pg/mL [range, 27.1-98.0 pg/mL], controls: 25.3 pg/ mL [range, 11.1-80.4 pg/mL]) but not MDA (CHF: 4.11 microM [range, 1.89-6.39 microM], controls: 3.88 microM [range, 2.14-4.72 microM]) or protein carbonyls (0.69 nmol/mg protein [range, 0.37-1.67 nmol/mg protein], controls: 0.80 nmol/mg protein [range, 0.40-1.14 nmol/mg protein]) were significantly higher in the dogs with CHF than in the controls. Vitamin E concentration (CHF: 2,215 microg/ dL [range, 916-3,499 microg/dL], controls: 2,820 microg/dL [range, 1,738-3,775 microg/dL]) and GSH:GSSG (CHF: 12.0 [range, 3.69-30.1], controls: 22.7 [range, 12.5-227]) were significantly lower, whereas ORAC (CHF: 824 micromol Trolox equivalent/L [range, 304-984], controls: 497 micromol Trolox equivalent/L [range, 258-759]) and vitamin C (CHF: 0.90 mg/dL [range, 0.55-2.02 mg/dL], controls: 0.72 mg/dL [range, 0.43-0.85 mg/dL]) concentrations were higher in dogs with CHF than in controls. Vitamin A concentrations were not different between dogs with CHF and controls. No differences in any of the parameters were detected between dogs with DCM versus those with CVD. Some antioxidant defenses are decreased in dogs with CHF, and some biomarkers of oxidative stress are increased in dogs with CHF. The effect of dietary interventions to correct this imbalance in antioxidant defenses warrants further study.     

Lymphocyte beta -adrenoceptor downregulation in great danes with occult dilated cardiomyopathy (DCM) and with DCM and heart failure.

The importance of the adrenergic nervous system in supporting the failing heart has long been known. The adrenergic drive on cardiac structure and function has however some adverse effects, which include myocardial beta-adrenoceptor (beta-AR) downregulation and decreased beta-adrenergic responsiveness to cathecolamines. In dog lymphocytes, beta(1)-AR and beta(2)-AR populations are almost equally represented (with a slight prevalence of beta(2)) and a significant correlation between cardiac and lymphocytic adrenoceptors has been found. The aim of the present study was to investigate possible differences between the concentration of lymphocytic beta-AR in healthy dogs, dogs with dilated cardiomyopathy (DCM) and dogs with occult DCM. Three groups of great danes were considered: a control group (n =10), dogs with DCM (n =9) and dogs with occult DCM (n =4). Lymphocytic beta-AR populations were determined in all dogs. A substantial and significant decrease (P<0.05) in total-AR, beta(1)-AR and beta(2)-AR concentrations in the lymphocytes of dogs with symptomatic DCM and occult DCM compared to the control group was found. Although the mean value of the lymphocyte beta(1)-AR number in the asymptomatic group was double compared to the DCM group, this difference was not statistically significant. We conclude that in dogs beta-AR downregulation occurs early in the course of dilated cardiomyopathy. This finding may suggest the value of early use of a beta-blocker in the therapeutic regimen. Moreover, the continuous monitoring of lymphocytic beta-AR may represent a useful tool for the development of a more effective individual therapy.     

Effects of dilated cardiomyopathy on the renin-angiotensin-aldosterone system, atrial natriuretic peptide activity, and thyroid hormone concentrations in dogs

OBJECTIVE: To evaluate the effect of dilated cardiomyopathy (DCM) on activity of the renin-angiotensin-aldosterone system (RAAS), the N-terminal fragment of proatrial natriuretic peptide (NT-proANP), and thyroid hormone concentrations in dogs. ANIMALS: 15 dogs with clinical signs of DCM, 15 dogs without clinical signs of DCM, and 15 age-, breed-, and sex-matched control dogs. PROCEDURE: Physical examinations, thoracic radiography, ECG, and echocardiography were performed on all dogs, and blood and urine samples were collected. RESULTS: Plasma renin activity (PRA), plasma aldosterone concentration (PAC), urine aldosterone-to-creatinine ratio, and NT-proANP concentrations were significantly increased in dogs with clinical signs of DCM, compared with dogs without clinical signs and control dogs. Thyroid-stimulating hormone and total thyroxine concentrations did not differ significantly among groups; however, free thyroxine (FT4) concentrations were significantly decreased in dogs with clinical signs of DCM, compared with control dogs and DCM-dogs without clinical signs. Concentrations of PRA, PAC, FT4, and urine aldosterone-to-creatinine ratio were significantly correlated, whereas plasma concentrations of NT-proANP only correlated with FT4 concentration. CONCLUSION AND CLINICAL RELEVANCE: In dogs with clinical signs of DCM, increased concentrations of components of the RAAS were associated with increased concentrations of NT-proANP Analysis of the neurohormonal system may aid in identification of clinical stages of DCM for groups of dogs, but the range is too great and there are too many dogs that have neurohormonal concentrations within reference ranges to assess dogs on an individual basis.     

Effect of thyroid hormone supplementation on survival of euthyroid dogs with congestive heart failure due to systolic myocardial dysfunction: a double-blind, placebo-controlled trial

Nineteen euthyroid dogs of 12 breeds with echocardiographic signs of dilated cardiomyopathy (DCM) and radiographic and clinical signs of congestive heart failure (CHF) were evaluated in a randomised, double-blind, and placebo-controlled study. The dogs received either thyroxine or placebo as an adjunct to digoxin, furosemide and propranolol. The group assignment of individual dogs and serum concentrations of thyroid hormones remained unknown to owners and investigators during the entire study period. Dogs were evaluated clinically and with electrocardiography (ECG), thoracic radiography, echocardiography and measurement of total thyroxine (tT4) and thyroid stimulating hormone (TSH) before beginning of the trial, and then one week, 2 months, 6 months and yearly after initial examination, and, when applicable, at the time of euthanasia. End-point of the study was euthanasia (n = 17) due to severe congestive heart failure or sudden death (n = 2). Survival times ranged from 17 to 1030 days (median 187 days) in the placebo group, and from 18 to 1000 days (median 73 days) in the treatment group. There was no statistically significant difference in survival times between the treatment group and the placebo group (p = 0.46). Post mortem and histopathologic examinations revealed the attenuated wavy fiber type of DCM in 11 dogs, and myocardial infarcts, arteriosclerosis and chronic valvular disease in one dog. In conclusion, there was a wide range in survival times of dogs treated with digoxin, furosemide and propranolol. Adding thyroid hormones to the treatment did not significantly influence survival.     

Correlation between activation of the sympathetic nervous system estimated by plasma concentrations of norepinephrine and Doppler echocardiographic variables in dogs with acquired heart disease

OBJECTIVE: To evaluate correlations between plasma concentrations of norepinephrine and Doppler echocardiographic variables for dogs with degenerative mitral valve disease (DMVD) or dilatative cardiomyopathy (DCM) to better understand the time course and magnitude of sympathetic activation in dogs with heart failure (HF). ANIMALS: 15 healthy dogs, 15 dogs with DMVD, and 15 dogs with DCM. PROCEDURES: Dogs were positioned in lateral recumbency with minimal restraint for at least 20 minutes. Plasma samples were obtained and assayed by use of high-performance liquid chromatography. Concentrations were correlated with HF classification and with the main Doppler echocardiographic variables for each group. RESULTS: Mean +/- SD norepinephrine concentration was significantly higher in dogs with DMVD (494.4 +/- 204.8 pg/mL) or DCM (655.7 +/- 652.5 pg/mL) than in healthy dogs (205.8 +/- 78.9 pg/mL), but concentrations did not differ significantly between the 2 groups with HF. Correlations were not detected between norepinephrine and heart rate or any M-mode echocardiographic variables evaluated, except for fractional shortening (FS) in DCM dogs. In that group, norepinephrine was inversely correlated with FS values. In DMVD dogs, no significant correlation was found between norepinephrine and the left atrium-to-aortic root ratio or mitral regurgitation. CONCLUSIONS AND CLINICAL RELEVANCE: A proportional inverse correlation exists between norepinephrine and FS values in dogs with DCM. However, norepinephrine concentration was not correlated with the evaluated echocardiographic variables in dogs with DMVD. Sympathetic antagonists should be evaluated as a treatment option because of the increased plasma concentrations of norepinephrine detected in dogs with HF.     

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B.The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM.     

Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy

OBJECTIVE: To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). SAMPLE POPULATION: Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. PROCEDURES: Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. RESULTS: Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. CONCLUSIONS AND CLINICAL RELEVANCE: Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.     

Canine dilated cardiomyopathy: lymphocyte and cardiac alpha(1)- and beta-adrenoceptor concentrations in normal and affected great danes.

Serum catecholamine levels and myocardial and lymphocyte adrenergic receptor (AR) concentrations were measured in adult great danes affected by canine dilated cardiomyopathy (DCM) and compared to those of healthy animals. A non-homogeneous population of beta -AR, consisting of beta(1)-AR and beta(2)-AR, was observed in healthy (41 and 59%, respectively) and affected (17 and 83%, respectively) dog lymphocytes. Binding assays revealed that total beta -AR, beta(1)-AR and alpha(1)-AR were significantly downregulated (P<0.05;P<0.01;P<0. 001), both in lymphocyte and myocardial cell membranes of affected dogs. beta(2)-Adrenergic receptor concentrations were significantly reduced only in lymphocyte and right atrium cell membranes (P<0.05). Downregulation was not associated with alterations in receptor binding characteristics, as no significant differences in K(d)values were found. Mean plasma catecholamine levels were significantly higher (P<0.01) in DCM dogs (939+/-41) than in normal subjects (348+/-32), thus suggesting a sympathetic activation. The present study indicates a condition similar to that observed in human patients affected by DCM and that adrenergic receptors in canine lymphocytes reflect the fluctuation of adrenergic receptor concentrations in the myocardium.     

Azotemia and glomerular filtration rate in dogs with chronic valvular disease

BACKGROUND: Little information is available about the prevalence of renal dysfunction in dogs with chronic valvular heart disease (CVD). HYPOTHESIS: Azotemia and a decrease in glomerular filtration rate (GFR) are more severe with increased severity of CVD. ANIMALS: 124 (study No. 1) and 24 (study No. 2) client-owned dogs with CVD. METHODS: A retrospective study (study No. 1) was performed to assess the prevalence of azotemia in the New York Heart Association (NYHA) classes of heart failure in dogs with CVD. A prospective study (study No. 2) was then designed to determine GFR in dogs with different degrees of CVD severity. Complete physical examination, electrocardiography, blood pressure measurement, thoracic radiographs, echocardiography, and plasma and urine analyses were also performed. RESULTS: In study No. 1, 50% of the dogs were azotemic and the percentage of azotemic dogs increased with functional class (up to 70% in NYHA class IV patients). In study No. 2, 8/24 dogs were azotemic. Plasma urea and creatinine were higher in NYHA class III-IV dogs compared with class I-II dogs. The GFR was lower (P < .001) in NYHA class III-IV dogs (1.7 +/- 0.7 mL/min/kg) than in class I to II dogs (3.1 +/- 0.8 mL/min/kg). Only 1 dog in class I-II had a GFR below 2 mL/min/kg and only 2/9 class III-IV dogs had a GFR above 2 mL/min/kg. CONCLUSION AND CLINICAL RELEVANCE: Azotemia and renal impairment increase with the severity of congestive heart failure and are frequent findings in dogs with CVD. It remains to be shown if deterioration of renal function is a direct result of progression of the heart disease.     

Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy

OBJECTIVE: To evaluate the role of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy (DCM). ANIMALS: 6 dogs with DCM, including 2 Doberman Pinschers, 2 Newfoundlands, and 2 Great Danes. PROCEDURE: All dogs had clinical signs of congestive heart failure, and a diagnosis of DCM was made on the basis of echocardiographic findings. Blood samples were collected from each dog, and genomic DNA was isolated by a salt extraction method. Specific oligonucleotides were designed to amplify the promoter, exon 1, the 5'-part of exon 2 including the complete coding region, and part of intron 1 of the canine phospholamban gene via polymerase chain reaction procedures. These regions were screened for mutations in DNA obtained from the 6 dogs with DCM. RESULTS: No mutations were identified in the promoter, 5' untranslated region, part of intron 1, part of the 3' untranslated region, and the complete coding region of the phospholamban gene in dogs with DCM. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that mutations in the phospholamban gene are not a frequent cause of DCM in Doberman Pinschers, Newfoundlands, and Great Danes.     

Thyroid function in dogs with spontaneous and induced congestive heart failure.

The effects of spontaneous and experimentally induced congestive heart failure on serum thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3'5'-triiodothyronine (reverse T3), free T4, free T3 concentrations, and the serum T4 and T3 concentrations in response to administration of thyrotropin were studied. Serum thyroid hormone concentrations were not different between eight dogs with spontaneous congestive heart failure and normal age matched control dogs. Seven dogs with experimental heart failure were tested before and after induction of congestive heart failure by rapid ventricular pacing. Mean serum T4 and free T3 concentrations were decreased and mean serum reverse T3 concentration was increased following induction of heart failure. The serum T4 and T3 responses to thyrotropin were not altered. Thyroid gland morphology appeared normal in dogs with experimental heart failure. Experimental congestive heart failure, similar to some other nonthyroidal illnesses, alters thyroid hormone secretion and metabolism in dogs.     

Plasma endothelin-1 immunoreactivity in normal dogs and dogs with acquired heart disease

We sought to measure plasma endothelin-1 (ET-1) concentrations in normal dogs and to compare them with those measured in dogs with acquired heart disease with or without pulmonary edema. A sandwich enzyme-linked immunosorbent assay kit was validated and used to measure ET-1 immunoreactivity in plasma samples obtained from 32 normal dogs and 46 dogs with either dilated cardiomyopathy (DCM, n = 27) or degenerative valvular disease (CDVD, n = 19) with (n = 30) or without (n = 16) overt congestive heart failure (CHF). Plasma ET-1 concentrations (geometric mean, 95% confidence interval of geometric mean) were 1.17 (1.04-1.32) fmol/mL in the 32 normal control dogs, 1.25 (0.981-1.60) fmol/mL in 16 dogs with DCM (n = 9) or CDVD (n = 7) without CHF, and 2.51 (2.10-3.01) fmol/mL in 30 dogs with DCM (n = 18) and CDVD (n = 12) with CHE Plasma immunoreactivity of ET-1 was significantly higher in dogs with CHF in comparison with normal dogs (P < .001) and dogs with heart disease without CHF (P < .001). No significant difference was found between normal dogs and dogs with heart disease but without CHF (P > .05). Significant correlations were between plasma ET-I concentrations and left atrial:aortic ratio (P < .0001, r2 = .39), left ventricular internal dimension at end-diastole indexed to aortic diameter (P < .0001, r2 = .30) or body surface area (BSA) (P = .0071, r2 = .10), and left ventricular internal dimension at end-systole indexed to aortic diameter (P = .0003, r- = .17) or BSA (P = .0008, r2 = .15).     

The immunohistochemical evaluation of selected markers in the left atrium of dogs with end-stage dilated cardiomyopathy and myxomatous mitral valve disease – a preliminary study

Background

Dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD) are the most common diseases noted in dogs. Although their pathogenesis varies, both include a significant enlargement of the left atrium.The study was carried out on left atrial specimens obtained from 56 dogs, including those from 34 dogs with clinically diagnosed MMVD, 15 dogs with DCM and 7 dogs without heart disease (control group). Dogs in the MMVD and the DCM groups presented with left atrial enlargement and stage D heart failure. The specimens underwent immunohistochemical examination using desmin, vimentin, periostin and caspase-3 antibodies.

Results

There were alterations in the expression of the studied proteins in the study groups compared to the control group. The changes included: irregularity of desmin cross-striation and desmosomes, a higher amount of vimentin-positive cells, a change in the periostin expression pattern from cytoplasmic to extracellular, and a lower expression of caspase-3. The alterations were more pronounced in the DCM group than in the MMVD group.

Conclusions

During heart failure, the pattern of desmin, vimentin, periostin and caspase-3 expression alters in the left atrium, regardless of the cause. The changes are more pronounced in dogs with DCM than in dogs with MMVD and similar left atrial enlargement, suggesting that volume overload may not be the only cause of myocardial changes in DCM.

     

The prevalence of cardiomyopathy in the Irish wolfhound: a clinical study of 500 dogs

The prevalence of cardiomyopathy in Irish wolfhounds was evaluated by retrospective review of the results of cardiovascular examinations carried out in 500 dogs presented for veterinary services at the author's practice. Abnormalities were found in 209 (41.8%) of the dogs examined. Dilated cardiomyopathy (DCM) was diagnosed in 121 (24.2%) of the dogs and was accompanied by atrial fibrillation in 106 dogs. Seventeen dogs were suffering from advanced congestive heart failure (CHF), and 55 dogs were suffering from mild to moderate CHF as a result of DCM. Congestive heart failure was most commonly characterized by mild to severe pleural effusion due to right-sided heart failure in addition to pulmonary edema. Rhythm disturbances without evidence of DCM were detected in 48 dogs. Forty dogs had echocardiographic abnormalities without signs of DCM. Soft to moderate mitral regurgitations were diagnosed in 13 (2.6%) of these 40 dogs examined. In 39 dogs that died as a result of DCM, the median survival time from the time of diagnosis was 5.1 months, and in 59 dogs with DCM that are still alive, the median survival time is 15.7 months.     

The vasovagal tonus index as a prognostic indicator in dogs with dilated cardiomyopathy

Objectives : To investigate the prognostic and diagnostic value of heart rate variability (HRV) using the vasovagal tonus index (VVTI) in dogs suffering from idiopathic dilated cardiomyopathy (DCM). Methods : Electrocardiographic (ECG) recordings of 369 patients presented to a referral centre between 1993 and 2006 were reviewed. Results : VVTI values were calculated from 132 dogs. Lower VVTI values were found in patients in International Small Animal Cardiac Health Council (ISACHC) heart failure (HF) class 2 and 3 compared with class 1. VVTI was found to be positively correlated with survival time (ST) in class 2 and 3 patients. When a cut‐off value of 7·59 for VVTI was used, the test could differentiate patients in ISACHC HF class 1 versus 2 and 3 with a sensitivity of 89 per cent and a specificity of 62·5 per cent. The ST for patients with VVTI values less than 7·59 was significantly lower. Clinical Significance : The VVTI is a useful index, obtained from a standard ECG recording that estimates HRV in dogs and does not require any specific equipment for its calculation. It can be useful as a diagnostic tool to assess the severity of HF and is a useful prognostic tool in dogs with DCM.     

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis

OBJECTIVE: To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. SAMPLE POPULATION: Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. PROCEDURE: Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. RESULTS: Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.