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Background N-terminal prohormone of brain natriuretic peptide (NT-proBNP) concentrations may be increased in cats with various cardiac disorders. The point-of-care (POC) ELISA assay uses the same biologic reagents as the quantitative NT-proBNP ELISA. Previous studies have evaluated the sensitivity and specificity of the POC ELISA in cats with cardiac disease. Objectives To prospectively evaluate the diagnostic utility of the POC ELISA in a select population of cats. Animals Thirty-eight client-owned cats presented to the University of Florida Cardiology Service for cardiac evaluation. Fifteen apparently healthy cats recruited as part of another study. Methods Physical examination and echocardiography were performed in all cats. The POC ELISA was assessed visually as either positive or negative by a reader blinded to the echocardiographic findings, and results were analyzed relative to quantitative assay results. Results Twenty-six cats were diagnosed with underlying cardiac disease, and 27 cats were considered free of cardiac disease. Cats with cardiac disease included: 21 with hypertrophic cardiomyopathy, 2 with unclassified cardiomyopathy, 2 with restrictive cardiomyopathy, and 1 with 3rd degree atrioventricular (AV) block. The POC ELISA differentiated cats with cardiac disease with a sensitivity of 65.4% and specificity of 100%. Conclusions and Clinical Importance The POC NT-proBNP ELISA performed moderately well in a selected population of cats. A negative test result cannot exclude the presence of underlying cardiac disease, and a positive test result indicates that cardiac disease likely is present, but further diagnostic investigation would be indicated for a definitive diagnosis.

OBJECTIVE To compare mitochondrial oxygen consumption rate (OCR) of fibroblasts from Doberman Pinschers with and without dilated cardiomyopathy (DCM) and mutation of the gene for pyruvate dehydrogenase kinase isozyme 4 (PDK4) and to evaluate in vitro whether treatment with adeno-associated virus (AAV) vector (ie, gene therapy) would alter metabolic efficiency. ANIMALS 10 Doberman Pinschers screened for DCM and PDK4 mutation. PROCEDURES Fibroblasts were harvested from skin biopsy specimens obtained from Doberman Pinschers, and dogs were classified as without DCM or PDK4 mutation (n = 3) or with occult DCM and heterozygous (4) or homozygous (3) for PDK4 mutation. Fibroblasts were or were not treated with tyrosine mutant AAV type 2 vector containing PDK4 at multiplicities of infection of 1,000. Mitochondrial OCR was measured to evaluate mitochondrial metabolism. The OCR was compared among dog groups and between untreated and treated fibroblasts within groups. RESULTS Mean ± SD basal OCR of fibroblasts from heterozygous (74 ± 8 pmol of O2/min) and homozygous (58 ± 12 pmol of O2/min) dogs was significantly lower than that for dogs without PDK4 mutation (115 ± 9 pmol of O2/min). After AAV transduction, OCR did not increase significantly in any group (mutation-free group, 121 ± 26 pmol of O2/min; heterozygous group, 88 ± 6 pmol of O2/min; homozygous group, 59 ± 3 pmol of O2/min). CONCLUSIONS AND CLINICAL RELEVANCE Mitochondrial function was altered in skin fibroblasts of Doberman Pinschers with DCM and PDK4 mutation. Change in mitochondrial function after in vitro gene therapy at the multiplicities of infection used in this study was not significant. (Am J Vet Res 2016;77:156–161)

Objectives The biologic variability of N-terminal pro-brain natriuretic peptide (NT-proBNP) and its impact on diagnostic utility is unknown in healthy cats and those with cardiac disease. The purpose of this study was to determine the biologic variation of NT-proBNP within-day and week-to-week in healthy adult cats. Methods Adult cats were prospectively evaluated by complete blood count (CBC), biochemistry, total thyroxine, echocardiography, electrocardiography and blood pressure, to exclude underlying systemic or cardiac disease. Adult healthy cats were enrolled and blood samples were obtained at 11 time points over a 6 week period (0, 2 h, 4 h, 6 h, 8 h, 10 h and at weeks 2, 3, 4, 5 and 6). The intra-individual (coefficient of variation [CVI]) biologic variation along with index of individuality and reference change values (RCVs) were calculated. Univariate models were analyzed and included comparison of the six different time points for both daily and weekly samples. This was followed by a Tukey’s post-hoc adjustment, with a P value of Results The median daily and weekly CVI for the population were 13.1% (range 0–28.7%) and 21.2% (range 3.9–68.1%), respectively. The index of individuality was 0.99 and 1 for daily and weekly samples, respectively. The median daily and weekly RCVs for the population were 39.8% (range 17.0–80.5%) and 60.5% (range 20.1–187.8%), respectively. Conclusions and relevance This study demonstrates high individual variability for NT-proBNP concentrations in a population of adult healthy cats. Further research is warranted to evaluate NT-proBNP variability, particularly how serial measurements of NT-proBNP may be used in the diagnosis and management of cats with cardiac disease.

To determine the relationship between aortoseptal angle (AoSA) and the short- and long-term systolic pressure gradient (PG) reduction following combined cutting and high-pressure balloon valvuloplasty (CB/HPBV) in dogs with severe subaortic stenosis.Retrospective study of 22 client-owned dogs of various breeds with severe subaortic stenosis (mean left ventricular to aortic PG = 143 mmHg; range = 80-322 mmHg) that underwent CB/HPBV.Initial angiographic and left apical and right-sided parasternal long-axis view echocardiographic video loops were used for measuring the angle between the plane of the interventricular septum and the longitudinal axis of the ascending aorta. The PG reduction ratio immediately after CB/HPBV and 6 and 12 months later were compared with AoSA.Weak correlations were observed for all instances of PG reduction ratio and AoSA type. Significantly greater mean differences of PG reduction ratio were observed for angles160° than for angles160° at 24 h (160° mean: 54.45, standard error [SE]: ±3.8;160° mean: 39.88, SE: ±2.09), 6 months (160° mean: 57.73, SE: ±10.9;160° mean: 28.22, SE: ±3.42), and 12 months (160° mean: 76.11, SE: ±17.5;160° mean: 27.61, SE: ±6.44; p=0.003).Dogs with AoSA160° on right-sided parasternal long-axis view echocardiograms responded with a greater PG reduction following CB/HPBV than did dogs with AoSA160°. This suggests that AoSA is associated with long-term outcomes of CB/HPBV, and measurement could help in the evaluation of dogs that are candidates for CB/HPBV.

Dilated cardiomyopathy (DCM) is estimated to be the third most common inherited type of heart disease in humans reported to affect 35.6 and 0.57 in 100,000 adults and children, respectively. The Doberman Pinscher (DP) is a canine breed affected with idiopathic, non-ischemic DCM that closely resembles the human counterpart acting as a clinically relevant human model. Recently, a 16 base pair deletion in the 5’ donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 (PDK4) gene, encoding for a mitochondrial protein involved in energy metabolism, has been identified but the relationship between the PDK4 mutation, mitochondrial function and phenotypic development of DCM is largely unknown. Our research has focused on understanding the relationship between the consequences of the PDK4 mutation on mitochondrial function and phenotypic development of DCM as well as the potential of cardiac gene therapy to correct the phenotype. A total of 64 DP were tested for the PDK4 mutation and screened for DCM. Healthy dogs and dogs diagnosed with DCM were further divided into 3 groups based on the presence of the genetic mutation: Wild-type (PDK4wt/wt), Heterozygous (PDK4wt/del), and Homozygous (PDK4del/del). Preliminary analyses using isolated mitochondria from skin fibroblasts of these dogs demonstrated a lack of detectable PDK4 protein expression in PDK4del/del, and a significant decrease in PDK4wt/del (48% of normal). Phase contrast microscopy and immunofluorescence studies revealed significantly different morpho-phenotypic characteristics and reduced capacity to adapt to unfavorable metabolic conditions as compared to healthy dogs. Assessment of mitochondrial metabolic potential and function (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) were performed using an extracellular flux analyzer. Cells were stressed using serum glucose starvation. Compared to healthy dogs, PDK4wt/del and PDK4del/del revealed a much lower basal respiration rate (OCR reduction of 46% and 68% of normal, respectively) and decreased metabolic potential to meet energy demands (85% and 72% of normal, respectively). Maximal mitochondrial respiration and spare respiratory capacity were all significantly lower in the affected dogs (PDK4wt/del 42% and 14%, and PDK4del/del 40% and 1.4% of normal OCR, respectively). Our results indicate that the mitochondria of affected dogs have a lower metabolic capacity and thus exhaust the two major energy-producing pathways, aerobic and glycolytic metabolism, at a much faster rate when under stress compared to healthy control dogs. Although we detected a significant decrease in PDK4 protein levels, PDK4 transcription levels have yet to be determined in these samples. Our immediate future goals include 1) quantitative PCR to compare PDK4 transcript levels, 2) PCR and sequencing to identify abnormalities in PDK4 mRNA splicing in affected dogs, 3) assessments of mitochondria ATP production and PDK4 protein kinase function. Our long-term goal is to perform in vitro experiments to assess if mitochondrial abnormalities can be improved with gene therapy. To this end, we have cloned canine healthy PDK4 into an adeno-associated virus construct. Ultimately, our efforts will lead us to a cardiac gene therapy clinical trial for Doberman PDK4-DCM.