Caryn Reynolds

Utility of plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) to distinguish between congestive heart failure and non-cardiac causes of acute dyspnea in cats.

BACKGROUND Circulating plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration facilitates emergency diagnosis of congestive heart failure (CHF) in people. Its utility to discriminate between dyspneic cats with CHF vs. primary respiratory disease requires further assessment. Our objectives were to determine if NT-proBNP (1) differentiates dyspneic cats with CHF vs. primary respiratory disease; (2) increases with renal insufficiency; (3) correlates with left atrial dimension, radiographic cardiomegaly, and estimated left ventricular filling pressure (E/E(a)). METHODS NT-proBNP was measured in 167 dyspneic cats (66 primary respiratory disease, 101 CHF) to evaluate (1) relationship with clinical parameters; (2) ability to distinguish CHF from primary respiratory disease; (3) optimal cut-off values using receiver operating characteristic (ROC) curve analysis. RESULTS NT-proBNP (1) was higher (median and inter-quartile [25th-75th] percentile) in CHF (754 pmol/L; 437, 1035 pmol/L) vs. primary respiratory disease (76.5 pmol/L; 24, 180 pmol/L) cohorts (P<0.001); (2) positively correlated in CHF cats with increased inter-ventricular septal end-diastolic thickness (rho=0.266; P=0.007) and LV free wall thickness (rho=0.218; P=0.027), but not with radiographic heart size, left atrial size, left ventricular dimensions, E/E(a) ratio, BUN, creatinine, or thyroxine; (3) distinguished dyspneic CHF cats from primary respiratory disease at 265 pmol/L cut-off value with 90.2% sensitivity, 87.9% specificity, 92% positive predictive value, and 85.3% negative predictive value (area under ROC curve, 0.94). CONCLUSIONS NT-proBNP accurately discriminated CHF from respiratory disease causes of dyspnea.

Percutaneous Transendocardial Delivery of Self-complementary Adeno-associated Virus 6 Achieves Global Cardiac Gene Transfer in Canines

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated-ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6-so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated--ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6--so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.

Prediction of first onset of congestive heart failure in dogs with degenerative mitral valve disease: the PREDICT cohort study.

OBJECTIVE To identify risk factors for first-onset congestive heart failure (CHF) in dogs with degenerative mitral valve disease (DMVD). ANIMALS Eighty-two dogs with and without CHF secondary to DMVD were retrospectively assigned to a derivation cohort. Sixty-five dogs with asymptomatic DMVD were recruited into a prospective validation cohort. METHODS Variables associated with risk of CHF in dogs were identified in a derivation cohort and used to construct a predictive model, which was then prospectively tested through longitudinal examination of a validation cohort. RESULTS Logistic regression analysis of the derivation cohort yielded a predictive model that included the left atrial to aortic root dimension ratio (LA:Ao) and plasma concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP). When this model was prospectively applied to the validation cohort, it correctly predicted first-onset of CHF in 69.2% of cases. Analysis of the validation cohort revealed that plasma NT-proBNP concentration and indexed left ventricular end-diastolic diameter (LVIDd:Ao) were independent risk factors for development of first-onset CHF in dogs with DMVD (NT-proBNP ≥ 1500 pmol/L, odds ratio (OR), 5.76, 95% confidence interval (CI), 1.37-24.28, P = 0.017; LVIDd:Ao ≥ 3, OR, 6.11, 95% CI, 1.09-34.05, P = 0.039). CONCLUSIONS Measures of left heart size and plasma NT-proBNP concentration independently estimate risk of first-onset of CHF in dogs with DMVD. These parameters can contribute to the management of dogs with DMVD.

Long-term Restoration of Cardiac Dystrophin Expression in Golden Retriever Muscular Dystrophy Following rAAV6-mediated Exon Skipping

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Multicenter Evaluation of Plasma N-Terminal Probrain Natriuretic Peptide (NT-pro BNP) as a Biochemical Screening Test for Asymptomatic (occult) Cardiomyopathy in Cats

BACKGROUND B-type natriuretic peptide concentrations reliably distinguish between cardiac and respiratory causes of dyspnea, but its utility to detect asymptomatic cats with occult cardiomyopathy (OCM) is unresolved. HYPOTHESIS/OBJECTIVES Determine whether plasma N terminal probrain natriuretic peptide (NT-proBNP) concentration can discriminate asymptomatic cats with OCM from normal cats, and whether NT-proBNP concentration correlates with clinical, biochemical, and echocardiographic parameters. ANIMALS One hundred and fourteen normal, healthy cats; 113 OCM cats. METHODS Prospective, multicenter, case-controlled study. NT-proBNP was prospectively measured and cardiac status was determined from history, physical examination, and M-mode/2D/Doppler echocardiography. Optimal cut-off values were derived using receiver operating characteristic (ROC) curve analysis. RESULTS NT-proBNP was higher (median, interquartile range [25th and 75th percentiles]) in (1) OCM (186 pmol/L; 79, 478 pmol/L) versus normal (24 pmol/L; 24, 32 pmol/L) (P < .001); and (2) hypertrophic obstructive cardiomyopathy (396 pmol/L; 205, 685 pmol/L) versus hypertrophic cardiomyopathy (112 pmol/L; 48, 318 pmol/L) (P < .001). In OCM, NT-proBNP correlated (1) positively with LVPWd (ρ = 0.23; P = .01), LA/Ao ratio (ρ = 0.31; P < .001), LVs (ρ = 0.33; P < .001), and troponin-I (ρ = 0.64; P < .001), and (2) negatively with %FS (ρ = -0.27; P = .004). Area under ROC curve was 0.92; >46 pmol/L cut-off distinguished normal from OCM (91.2% specificity, 85.8% sensitivity); >99 pmol/L cut-off was 100% specific, 70.8% sensitive. CONCLUSIONS AND CLINICAL IMPORTANCE Plasma NT-proBNP concentration reliably discriminated normal from OCM cats, and was associated with several echocardiographic markers of disease severity. Further studies are needed to assess test performance in unselected, general feline populations, and evaluate relationships between NT-proBNP concentrations and disease progression.

Assessment of serum N-terminal pro-B-type natriuretic peptide concentration for differentiation of congestive heart failure from primary respiratory tract disease as the cause of respiratory signs in dogs

OBJECTIVE To determine whether serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentration is useful in discriminating between cardiac and noncardiac (ie, primary respiratory tract disease) causes of respiratory signs (ie, coughing, stertor, stridor, excessive panting, increased respiratory effort, tachypnea, or overt respiratory distress) in dogs. DESIGN Multicenter cross-sectional study. ANIMALS P 115 dogs with respiratory signs. PROCEDURES Dogs with respiratory signs were solicited for study. Physical examination, thoracic radiography, and echocardiography were used to determine whether respiratory signs were the result of cardiac (ie, congestive heart failure) or noncardiac (ie, primary respiratory tract disease) causes. Serum samples for NT-proBNP assay were obtained at time of admission for each dog. Receiver-operating characteristic curves were constructed to determine the ability of serum NT-proBNP concentration to discriminate between cardiac and noncardiac causes of respiratory signs. RESULTS Serum NT-proBNP concentration was significantly higher in dogs with cardiac versus noncardiac causes of respiratory signs. In dogs with primary respiratory tract disease, serum NT-proBNP concentration was significantly higher in those with concurrent pulmonary hypertension than in those without. A serum NT-proBNP cutoff concentration > 1,158 pmol/L discriminated between dogs with congestive heart failure and dogs with primary respiratory tract disease with a sensitivity of 85.5% and a specificity of 81.3%. CONCLUSIONS AND CLINICAL RELEVANCE Measuring serum NT-proBNP concentration in dogs with respiratory signs helps to differentiate between congestive heart failure and primary respiratory tract disease as an underlying cause.

Weekly variability of plasma and serum NT-proBNP measurements in normal dogs

OBJECTIVES To determine the weekly variability of serum and plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations in healthy dogs. ANIMALS, MATERIALS AND METHODS Fifty-three normal dogs were examined prospectively. Serum (n=25) or plasma (n=28) samples were obtained for NT-proBNP assay at one week interval for 3 consecutive weeks. RESULTS Median serum or plasma NT-proBNP concentration did not change over 3 consecutive weeks. Twenty-two of 53 dogs (42%) had at least one NT-proBNP value >500 pmol/L, including 14 dogs with at least one serum NT-proBNP concentration >500 pmol/L and 8 dogs with at least one plasma NT-proBNP concentration >500 pmol/L during the 3-week sampling period. The difference between the maximum and minimum NT-proBNP value obtained over the 3-week sampling period was <100 pmol/L in 40% of dogs, between 100 and 200 pmol/L in 40% of dogs, and >200 pmol/L in 20% of dogs. Of the 19 dogs with a value >500 pmol/L on either week 1 or 2, 11 dogs (58%) had a subsequent NT-proBNP value <500 pmol/L on either week 2 or 3. CONCLUSIONS There is a high degree of variability in weekly serum and plasma NT-proBNP values in healthy dogs. Individual variability should be considered when interpreting NT-proBNP results in dogs.

Tricuspid Annular Plane Systolic Excursion (TAPSE) in Dogs: Reference Values and Impact of Pulmonary Hypertension

BACKGROUND The impact of pulmonary hypertension (PH) on right ventricular systolic function is difficult to assess. Tricuspid annular plane systolic excursion (TAPSE) is an echocardiographic measurement of right ventricular systolic function and a strong predictor of outcome in human PH patients. HYPOTHESIS/OBJECTIVES Determine a reference range for TAPSE in healthy dogs, and quantify TAPSE in dogs with PH. It is hypothesized that TAPSE is lower in dogs with PH compared with a reference group, and decreases as PH worsens. ANIMALS Fifty normal dogs and 30 dogs with PH. METHODS TAPSE was measured by 2-dimensional echocardiography-guided M-mode from the left apical 4-chamber view. Peak systolic tricuspid valve regurgitation jet velocity was measured by continuous-wave Doppler to estimate right ventricular-to-right atrial pressure gradient. PH was subjectively classified as mild, moderate, and severe. RESULTS There was a curvilinear correlation between TAPSE and body weight. The upper and lower limits of the 95% reference interval were determined by quantile regression. Interobserver and intraobserver agreement was adequate with a coefficient of variation <10%. There were significant differences when comparing dogs with PH and the healthy group, as well as between the PH subgroups (P < .01), except between dogs with mild and moderate PH (P = .99). Only dogs in the severe PH group had TAPSE values that were mostly below the lower limit of the reference interval. CONCLUSIONS AND CLINICAL IMPORTANCE TAPSE is easily obtainable with acceptable inter and intraobserver agreement. TAPSE is decreased in PH and below the reference interval in most dogs with severe PH.

Effect of azotemia on serum N-terminal proBNP concentration in dogs with normal cardiac function: A pilot study

OBJECTIVES To evaluate amino-terminal pro-B type natriuretic peptide (NT-proBNP) concentration in dogs with renal dysfunction and normal cardiac structure and function. ANIMALS Eight dogs with renal disease, 23 healthy control dogs. METHODS Serum NT-proBNP concentration was measured in healthy dogs and dogs with renal disease using an ELISA validated for use in dogs. Affected dogs were eligible for inclusion if renal dysfunction was diagnosed based on urinalysis and serum chemistry, and if they were free of cardiovascular disease based on physical exam, systolic blood pressure, and echocardiography. RESULTS The geometric mean serum NT-proBNP concentration was significantly higher in dogs with renal disease (617 pmol/L; 95% CI, 260-1467 pmol/L) than in healthy control dogs (261 pmol/L; 95% CI, 225-303 pmol/L; P=0.0014). There was a modest positive correlation between NT-proBNP and BUN and creatinine. Median NT-proBNP concentration was not significantly different between groups when indexed to BUN (median NT-proBNP:BUN ratio; renal, 14.2, IQR, 3.93-17.7 vs. control, 16.3, IQR, 9.94-21.2; P=0.29) or creatinine (median NT-proBNP:creatinine ratio; renal, 204, IQR, 72.6-448 vs. control, 227, IQR, 179-308; P=0.67). CONCLUSION Dogs with renal disease had significantly higher mean serum concentration of NT-proBNP than control dogs. Renal function should be considered when interpreting NT-proBNP results as concentrations may be falsely elevated in dogs with renal dysfunction and normal cardiac function. The effect of renal disease was lessened by indexing NT-proBNP to BUN or creatinine. Future studies in dogs with both renal and heart disease are warranted.

Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.